ECA for Farm Milking Systems

Author's personal copy
Optimization and modeling of an electrolyzed oxidizing water based
Clean-In-Place technique for farm milking systems using a pilot-scale
milking system
Satyanarayan R.S. Dev, Ali Demirci ⇑
, Robert E. Graves, Virendra M. Puri
Department of Agricultural & Biological Engineering, Pennsylvania State University, University Park, PA 16802, United States
a r t i c l e i n f o
Article history:
Received 19 July 2013
Received in revised form 30 December 2013
Accepted 22 February 2014
Available online 12 March 2014
Keywords:
Electrolyzed oxidizing water
CIP
Milk
Milking system
Milk safety
a b s t r a c t
Electrolyzed oxidizing (EO) water has been recommended to be used as a cleaning and sanitizing agent
for Clean-In-Place (CIP) of on-farm milking systems. The CIP process for milking system with EO water
was optimized using a pilot-scale pipeline milking system. The milking system was soiled using raw milk
inoculated with four common microorganisms found in milk and cleaned using EO water. The cleaning
effectiveness of the EO water treatment was evaluated by ATP bioluminescence test and microbiological
analysis through enrichment culture. The effect of different temperatures of the alkaline EO water (45–
75 °C) and acidic EO water (25–45 °C) were investigated. A generalized mathematical model was built
and validated for the pilot scale milking system as well as compared with the conventional CIP technique.
This study indicated that the EO water CIP outperformed the conventional CIP, indicating that it has a
potential to be used in commercial dairy farms.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Most US dairy farms use a pipeline milking system to harvest
milk from cows’ udders and transport it to an on-farm milk cooling
and storage system/tank in a sanitary and efﬁcient manner. Proto-
cols and equipment involved with milk handling are regulated by
the US Food and Drug Administration and can be found in the
Grade ‘A’ Pasteurized Milk Ordinance (PHS/FDA, 2011). According
to this, the maximum bacteria count of raw milk intended for pas-
teurization must be below 100,000 CFU/ml. Whereas, Dairy Prac-
tices Council (DPC, 1997) recommends less than 5000 CFU/ml.
Moreover milk processors pay premium for higher quality milk
(milk containing less than 10,000 CFU/ml). Inadequate cleaning
practices at the farm level result in loss of thousands of dollars
per month for milk producers in reduced milk prices (Reinemann
et al., 1997).
In order to reduce microbial contamination of the raw milk,
milking systems are commonly cleaned with a four-step or ﬁve-
step including an optional clear rinse Clean-In-Place (CIP) process,
which involves: a warm water rinse, washing with a highly alka-
line solution, a clear rinse with warm water, a rinse with an acidic
solution, and then sanitizing with an EPA-registered sanitizing
agent immediately prior to the next milking time. These chemicals
are often purchased in large quantities in highly concentrated form
and stored on the farm and diluted on demand as per the instruc-
tions provided by the manufacturer for CIP purposes. Storage and
handling of these chemicals is a potential hazard for workers and
visitors, especially children. Oftentimes, children experience inad-
vertent contact with these chemicals through external contact or
internal consumption. There are approximately 100,000 children
injured every year in farm accidents in the United States alone
resulting in nearly 300 deaths and about 10% of those accidents
are due to hazardous chemicals stored at the farm (HICAHS,
2012). Therefore, an alternative to the use of these chemicals on
the farm would be highly advantageous to the farmers.
Electrolyzed oxidizing (EO) water can be produced by passing
an electric current between two electrodes immersed in a dilute
(0.1%) sodium chloride solution and separated by a semi-perme-
able membrane. The positively charged sodium and negatively
charged chloride ions migrate towards the opposite electrodes,
yielding an alkaline solution and an acidic solution at the cathode
and anode, respectively. EO water is a novel cleaning and sanitizing
agent. A semipermeable membrane separates the solutions thus
produced. The alkaline solution has a pH of 11.5 and an oxida-
tion–reduction potential (ORP) of À850 mV, while the acidic water
has a pH of 2.6, an ORP of 1150 mV, and contains about 80 ppm of
free chlorine (Sharma and Demirci, 2003; Kim et al., 2000).
http://dx.doi.org/10.1016/j.jfoodeng.2014.02.019
0260-8774/Ó 2014 Elsevier Ltd. All rights reserved.
⇑ Corresponding author. Tel.: +1 814 863 1098; fax: +1 814 863 1031.
E-mail address: demirci@psu.edu (A. Demirci).
Journal of Food Engineering 135 (2014) 1–10
Contents lists available at ScienceDirect
Journal of Food Engineering
journal homepage: www.elsevier.com/locate/jfoodeng

Using EO water for CIP cleaning of milking systems eliminates
many of the dangers associated with storing and using costly
cleaning chemicals. EO water is not harmful to human skin for
short exposure times (Cheng et al., 2012). Since EO water has both
alkaline and acidic components, it is compatible with the four-step
washing process for CIP cleaning of milking systems by replacing
the alkaline wash and acid rinse solutions with EO water solutions.
EO water also has the potential to be more cost effective than con-
ventional cleaning agents. Once the initial capital investment is
made to purchase an EO water generator, the only operating ex-
penses are water, sodium chloride (ordinary table salt), water heat-
ing cost and electricity to run the unit. However, the actual cost
advantage needs to be determined.
EO water is proven to be an effective antimicrobial agent for
decontamination of foods and food preparation/equipment sur-
faces (Cheng et al., 2012). Studies conducted by Koseki et al.
(2001), Park et al. (2002b), Russell (2003), Sharma and Demirci
(2003) and Bialka et al. (2004) indicated that the EO water is a
highly effective sanitizer for various food decontamination applica-
tions and they reckoned the antimicrobial activity of the EO water.
Significant amount of research has gone into evaluating the effec-
tiveness of EO water as a cleaning and sanitizing agent for the CIP
of food processing and handling equipment. In almost all the stud-
ies, EO water was found to be very effective both as a cleaning agent
(alkaline EO water) and as a sanitizing agent (acidic EO water)
(Walker et al., 2005a,b; Morita et al., 2002; Venkitanarayanan
et al., 1999; Park et al., 2002a,b).
Walker et al. (2005a,b) had conducted trials to assess the feasi-
bility of using EO water for CIP of milking systems. The efficacy of
the EO treatment was evaluated for both short term (single routine
of milking and cleaning) and long term (10 consecutive routines of
milking and cleaning). Despite being conclusive of the effective-
ness of the EO water CIP for milking systems, Walker et al.
(2005a,b) study did not take into account the fact that the acidic
EO water need not be heated to a temperature as high as the
alkaline EO water, as it will result in significant loss of chlorine
(as confirmed by preliminary trials in our laboratory) and thereby
compromising its potential as an effective sanitizer. Moreover, the
cleaning effectiveness of the alkaline EO water could be improved
at temperatures higher than the temperature (60 °C) investigated
in their study, besides the fact that the temperature does not re-
main constant throughout the wash solution circulation time due
to heat lost to the surroundings. Therefore the temperatures of
the wash solutions used for CIP of milking systems need to be
optimized.
Therefore, this study was undertaken to perform optimization
of the temperature of the alkaline and acidic EO water to attain
maximum cleaning of the milking system and to build a general-
ized mathematical model.
2. Materials and methods
2.1. Construction of the pilot-scale milking system
As described in Walker et al. (2005b), a pilot-scale milking sys-
tem with all the major components of a typical stainless steel pipe-
line milking system was constructed (Fig. 1), with some additions
including a pulsator and a vacuum gauge in order to mimic the ac-
tual farm milking system. A milking unit (DeLaval, Kansas City,
MO) was connected to a 3.05 m (10 feet) PVC milk hose and to a
wash manifold (or) a 16 mm (5/800
) milk inlet, depending on
whether it was the milking (or) the cleaning routine respectively.
This in turn was connected to eight 3.05 m (10 feet) long segments
and a 0.9 m (3 feet) pipe segment of 38.1 mm (1.500
) diameter food
grade 304L stainless steel sanitary pipeline. The sections of
stainless steel pipe were joined with rubber gaskets and tri-clamps
(Tri-CloverÒ
-DeLaval). The pipeline was mounted on a 6.1 m
(20 feet) long steel frame, wrapping around the frame in a spiral
fashion. There were two 90° elbows connecting every 6.1 m
(20 feet) length of straight pipeline, making a 180° turn in the
direction of flow. Thus eight 90° elbows were used to join the pipe
lengths at the ends of the frame resulting in four 180° turns which
finally discharged into a glass receiving jar assembly.
Therefore, the pilot scale milking system consisted of four loops
of stainless steel pipeline with four 180° bends resulting in a total
of 26.5 m (87 feet) length of pipe from the milk inlet to the receiv-
ing jar. Additional 1 m (3.5 feet) piping was located at the begin-
ning to accommodate the wash inlet manifold and air injector.
The pipelines were sloped at 0.8% to facilitate draining. A 38.1 L
(10 gallons) glass receiver jar and milk pump (DeLaval) fitted with
an industrial motor (1.5 hp) (Baldor, Fort Smith, AR) was also
mounted on the steel frame. An automated liquid level controller
(DeLaval) was installed to operate the milk pump based on the
amount of milk in the receiving jar. The receiver jar was connected
to a regulated vacuum source via a stainless steel sanitary trap,
which was supported using a scissor jack platform. A dual scale
vacuum gauge (±1% precision) (McMaster-Carr, Cleveland, NJ)
and a vacuum controller (DeLaval) were used to setup and monitor
a vacuum of 50 kPa (1500
of Mercury) in the system. A pulsator (60/
40) (DeLaval) was attached to the PVC vacuum line and connected
to the claw with a twin hose in order to the simulate milking a cow
during the soiling and to facilitate the free entry of the cleaning
water during CIP by flexing the rubber liner. A solenoid valve con-
trolled air injector (DeLaval) was installed for periodic air injection
into the pipeline for the formation of slugs during cleaning.
Before the experiments, the system was adjusted to provide
acceptable flow dynamics for CIP cleaning by adjusting the on/off
timing and admission rate of the air injector to achieve slug flow
with a mean slug velocity of 9.1 ± 1 m/s (30 ± 3 ft/s) and 3 slugs
per min with an air admission rate of 0.12 m3
/min (30 gallons
per min). Fig. 1 gives the schematic of the milking system configu-
ration with the pipelines and all the components.
2.2. Generation and heating of EO water
Electrolyzed oxidizing water was generated with an EO water
generator (Model ROX60SA) (Hoshizaki Electric Co. Ltd., Sakae, Ja-
pan). Fig. 2 provides a conceptual schematic of the mechanism of
EO water generation in a typical EO water generator (Huang
et al., 2008).
A 16.6 kW tankless water heater (Model EX1608TC, Eemax Inc.,
Oxford, CT) was used to heat the EO water solutions to the required
temperature into a spray foam insulated double-bowl stainless
steel wash sink with 76.2 L (20 gallons) capacity in each bowl. A
system of valves was used to control the flow of plain water and
EO water solutions into the tankless heater. Two 1 kW immersion
heaters (Model 712, VWR International, Radnor, PA) were used to
heat the wash solutions above the 65 °C provided by the tankless
heater.
Based on the dimensions of the pilot scale milking equipment,
the total volume of CIP solution or rinse water required for CIP
was calculated using the formulas published by Reinemann
(1995). Though the minimum water required was calculated to
be 45.8 L (12 gallons), the experiments were conducted with
57.2 L (15 gallons) per wash solution for circulation, considering
a safety factor of 1.25.
2.3. Experimental design
Based on the recommendations of DPC (2010) and Reinemann
et al. (1995, 1997), by eliminating the variables of time and slug
2 S.R.S. Dev et al. / Journal of Food Engineering 135 (2014) 1–10

velocity, the following Response Surface Methodology (RSM) face
centered central composite design with 3 central point replicates
(Table 1) was implemented to assess the effect of the temperature
of the alkaline EO water (45–75 °C) and the acidic EO water
(25–45 °C) on the cleaning effectiveness.
It is not recommended to increase the temperature of the acidic
EO water over 45 °C as significant amount of chlorine is lost at high
temperatures and this could be hazardous to the operating person-
nel as well as result in a loss of effectiveness of the solution. Preli-
minary trials in our laboratory indicated that during the time taken
to heat the acidic EO water solution from ambient temperature to
60 °C resulted in significant loss of chlorine.
After conducting the experiments, a response surface model
was developed from the experimental data using MATLAB Soft-
ware (Version R2011a, MathWorks, Natick, MA).
2.4. Preparation of milk
Fresh raw milk was collected from the Dairy Farm at The Penn-
sylvania State University (University Park, PA) by directly milking
the cow into a 38.1 L (10 gal) stainless steel milk can. Milk loses
its heat in the stainless steel can during its transportation to the
lab and cools down by 3–5 °C. Therefore, the raw milk was heated
to 40 °C (104 °F) using the hot water from the laboratory faucet at
45 °C (113 °F) with the help of a copper tube heat exchanger
(Fig. 3). Therefore, after mixing the inoculum milk, it would mimic
the temperature of milk coming from a cow, which is 38 °C.
A cocktail of bacteria containing Pseudomonas fluorescens B2,
Micrococcus luteus (ATCC 10240), Enterococcus faecalis (ATCC
51299), and Escherichia coli (ATCC 25922) was used as the inocu-
lum in this study to increase microbial population of raw milk.
Fig. 1. Schematic of the milking system.
Fig. 2. Schematic of the mechanism EO water generation (Huang et al., 2008).
Table 1
Response surface methodology experimental design.
Trial
No
Alkaline EO water temperature
(°C)
Acidic EO water temperature
(°C)
1 45 25
2 45 45
3 75 25
4 75 45
5 45 35
6 75 35
7 60 25
8 60 45
9 60 35
10 60 35
11 60 35
Hot water
inlet
Cold water
outlet
Helical copper
tube
Raw milk
Fig. 3. Copper tube heat exchanger setup for heating of raw milk.
S.R.S. Dev et al. / Journal of Food Engineering 135 (2014) 1–10 3

The microbial cultures were obtained from the Culture Collection
in the Food Science Department at Pennsylvania State University.
Each bacterial culture was grown in 500 ml of tryptic soy broth
(TSB, Difco, Sparks, MD) for 24 h at its optimum temperature:
30 °C for P. fluorescens and M. luteus, and 37 °C for E. faecalis and
E. coli. The total population in each culture was found to be above
109
CFU/ml.
After the incubation step, each culture broth was centrifuged at
4000g for 40 min (Model Sorvall T12, Thermo Fisher Scientific Inc.,
Waltham, MA) and the supernatant was decanted. Then the bacte-
rial cells from each culture were re-suspended in about 250 ml
(0.05 gal) of raw milk in four individual containers. Finally, these
cultures were then added to the remaining 37.1 L (9.8 gal) of raw
milk at 40 °C (104 °F), which brought down the temperature of
the milk to 38 °C (100.4 °F), which is the body temperature of the
cow.
2.5. Preparation of the milking system for CIP process
2.5.1. Shock cleaning
Before each experiment, the system was cleaned to return the
system to a clean condition, which is the baseline for the experi-
ments. This was done using a strong cleaning protocol also referred
to as ‘‘shock cleaning’’ in the dairy industry that consisted of:
(1) A wash with 68.14 L (18 gal) of commercial sodium hydrox-
ide-based cleaning solution (Dairy Cycle 3, Chemland Inc.,
Kansas City, Mo.) supplemented with 100 g of lye (sodium
hydroxide). This cleaning solution was heated to 80 °C and
circulated through the system for 10 min.
(2) A clear rinse was done with 10 gal (38.1 L) of warm water at
40 °C without recirculation to rinse off any leftover alkaline
solution in the system before passing the acid through the
system.
(3) This was followed by a 10 min phosphoric and sulfuric acid
wash (Dairy M.S.R. 50, Chemland Inc., Kansas City, Mo.)
starting at 80 °C with 18 gal (68.14 L) of acid wash solution.
(4) Moreover, 30 min before every soiling, the system was
sanitized by rinsing with a sodium hypochlorite sanitizing
solution (LCS, Classic Technologies, Kansas City, MO.) diluted
to 50 ppm concentration in 18 gal (68.14 L) of water.
2.5.2. Soiling the milking system
The inoculated (i.e., bacteriologically enriched) raw milk was
heated to the cow’s body temperature 38 °C (100.4 °F) using the
hot water from the laboratory faucet with assist from a copper tube
heat exchanger (Fig. 3). With the suction produced by the vacuum
pump, the heated/inoculated milk was introduced into the system
by inserting the milking unit into the container of milk. The milk
was drawn through the entire length of the pipeline to the receiver,
ensuring contact with all inside surfaces due to the highly turbulent
flow (Re % 3 Â 105
). Ten gallons of milk were introduced into the
system in three equal portions of approximately 13 L (3.3 gallons),
with a 10 min break between each portion. During this time, the
vacuum pump and the air injector were kept onto allow the milk
to dry and adhere on all the pipelines and milk contact surfaces
to maximize and harden the deposits making it tougher to clean,
thereby simulating the worst case scenario. The milk was not re-cir-
culated. It was pumped from the receiver and discarded in order to
minimize churning, cream separation, and other physio-chemical
changes in the milk that was used for soiling the system.
2.6. CIP Process using EO water
There has been extensive research on optimizing the wall stres-
ses during CIP conducted by Reinemann (1995) and Lelievre et al.
(2002) and it was concluded that a minimum of 20 slugs is re-
quired for effective cleaning of the system with a slug velocity in
the range of 8–10 m/s (27–33 ft/s). Dairy Practices Council (DPC)
recommends a minimum of 24 slugs for each wash solution during
its circulation time for effective cleaning of the pipelines (DPC,
2010). In the case of the pilot scale milking system, with the above
mentioned slug parameter calculation, 24 slugs require 9.25 min to
pass through, thus conforming to the recommended circulation
time of 10 min as the wash solution circulation time. Moreover,
in general, the wash solution circulation time of more than
10 min renders the wash fluids much less effective due to the
resulting low temperatures (<45 °C) in that duration. Besides being
not effective, lower temperatures also result in re-deposition (or)
sedimentation of the removed soil back onto the pipeline surfaces,
compromising the performance of CIP. Therefore, the wash solu-
tion circulation time should be 10 min.
In order to perform CIP effectively with the EO water, the fol-
lowing protocol was followed.
(1) A pre-rinse with 38.1 L (10 gal) of tap water heated to 40 °C
without recirculation was done to rinse off all the milk par-
ticles that do not require any cleaning agents to be removed
off the inner surface of the pipelines.
(2) A wash with 68.14 L (18 gal) of alkaline EO water. This clean-
ing solution was heated to a temperature of 75, 60, and 45 °C
for different experimental trials and circulated through the
system for 10 min.
(3) A clear rinse was done with 38.1 L (10 gal) of warm water at
40 °C without recirculation to rinse off any leftover alkaline
solution in the system before passing the acid through the
milking system.
(4) This was followed by a 10 min acidic EO water wash, starting
at 45, 35, and 25 °C for different experimental trials, using
68.14 L (18 gal) of acidic EO water.
While using this protocol, it was ensured that the acidic EO
water was never heated for longer than 10 min thereby preserving
the total/free chlorine content in the solution. Table 2 summarizes
the soiling and CIP protocols used for the experiments.
Table 2
Soiling and CIP protocols (all the fluids flow at 3.21 Â 10À4
m3
/s (5.1 GPM) into the milking system).
Unit/operating parameter Operating cycle
Soiling (milking) Pre-rinse Alkali wash Clear rinse Acid wash
Initial temperature (°C) 35 20 20 20 20
Inlet temperature (°C) 38 40 75, 60, 45 40 45, 35, 25
Heating time (min) Approx. 8 3.5 18, 12.5, 8 3.5 8, 5, 0
Quantity (l) 38.1 (12.7 Â 3 parts) 38.1 68.6 38.1 68.8
Air injection Off Off On On On
Recirculation No No Yes No Yes
Operating time (min) 32 (<1 min/part and 10 min break/part) 2 10 2 10

2.7. Evaluation
The tri-clamp connections are the only possible locations for
sampling in the stainless steel pipeline. The entire length of the
pipeline was divided into: straight pipeline segments and elbows;
the presence of elbows caused the flow pattern to be different vs.
straight segments. As mentioned earlier in the construction of
the milking system, each straight pipeline section is 3.05 m
(10 feet) long hence the exit (outlet) ends of the straight pipeline
segments for the eight straight pipeline sections were used as sam-
pling locations.
The exit (outlet) end of each elbow for the eight elbows con-
necting the four 20 feet straight pipeline segments were also used
as sampling locations. Apart from the pipelines, other parts of the
milking system including the rubber liners of the teet cups, milk
hose, rubber gaskets used at the connections of the pipeline, the in-
ner surface of the claw and the inlet and outlet of the glass jar were
sampled to assess the overall cleanliness of the system by biolumi-
nescence ATP and microbiological tests.
2.7.1. Bioluminescence ATP test
In order to determine the effect of alkaline and acidic EO water
temperatures on the cleanliness, bioluminescence ATP test was
used, which provided the data in terms of RLU (Relative Light
Units) for bioluminescence using Pocket Swab Plus (Charm Science,
Lawrence, MA). Various surfaces on the milking system were
swabbed with a Pocket Swab Plus after the first warm water rinse
following milking. The system was swabbed only after the first
warm water rinse for the following reasons.
(1) The first warm water rinse is a common step for both the EO
water CIP as well as the conventional CIP and therefore eval-
uating the status of the contamination level of milking sys-
tem after this step would be appropriate to assess the
effectiveness of the CIP process.
(2) The contamination levels (RLU counts) were between 105
and 108
times higher before the rinse and the variability of
data was too high for the same sampling location, whereas
the contamination levels were consistent for repeated
experiments after the first warm water rinse.
After completing the entire CIP routine, surfaces were swabbed
again with a Pocket Swab Plus. The following were the swabbing
locations: (a) At the end of every 10 feet of the straight section (8
locations), (b) At the end of every elbow in the system (8 locations),
(c) Inside two claw rubber liners, (d) Inlet of the glass receiver jar,
(e) The manifold end of the PVC milk hose and (f) Two representa-
tive rubber gaskets one at 40 feet and one at 80 feet of the pipeline.
The RLU measured is a combination of the left over milk soil and
bacteria in the system at the time of swabbing. However, biolumi-
nescence ATP data cannot be used directly to determine the sani-
tation (microbiological cleanliness) of the system.
Bioluminescence ATP test is a sensitive test; the RLU readings
vary from zero to millions depending on the status of the surfaces.
RLU reading of zero represents the surface to be clean and the
higher the RLU reading the ‘‘dirtier’’ the surface is. A practical
cut-off of RLU reading is 1000 for stainless steel (namely elbow,
straight pipeline, and milk inlet in this study) and 4500 for porous
rubber material (liner and milk hose in this study). As both the
conventional CIP technique as well as the EO water CIP resulted
in at least one log reduction in the RLU numbers and for a com-
pletely clean surface the RLU reading will be zero and log zero is
infinity, in order to statistically evaluate the cleaning performance
and to build a valid model the Percentage RLU Reduction (PRR) was
used.
2.7.2. Microbiological test
Swabbing using sterile calcium alginate swabs (VWR interna-
tional, Radnor, PA) were performed to determine the microbiolog-
ical cleanliness of the system. As the initial rinse removes a large
amount of bacteria, the count falls below the threshold for stan-
dard plate count techniques (Walker et al., 2005b). Therefore,
enrichment in tryptic soy broth was done to assess the presence/
absence of bacteria before and after the CIP process. Similar to
the bioluminescence ATP test, the various surfaces on the milking
system were swabbed with a Pocket Swab Plus after the first warm
water rinse following milking to assess the status of the contami-
nation level of milking system before the CIP process. After com-
pleting the entire CIP wash routine, surfaces were swabbed again
with a Pocket Swab Plus.
The following were the swabbing locations for the microbiolog-
ical test. (a) At the beginning of every 10 feet of the straight section
(8 locations), (b) At the beginning of every elbow in the system (8
locations), (c) Inside two claw rubber liners, (d) Outlet of the glass
jar, (e) The claw end of the PVC milk hose and (f) Two representa-
tive rubber gaskets one at 20 feet and one at 60 feet of the pipeline.
2.7.3. Issue of Inaccuracy due to repeated swabbing and its
remediation
Swabbing an area results in mechanical cleaning of the sam-
pling location and hence, the same area cannot be swabbed twice
in the same trial. Therefore, the pipe circumference was divided
into two halves along the vertical axis. This resulted in four swab-
bing areas on either side of a connection at each sampling point
(Fig. 4). For the RLU measurements, two halves of the cross section
of each pipe segment on one side (i.e., the beginning of the pipe
segment or in other words, the upstream end) were used for swab-
bing before and after the CIP treatment. For microbiological analy-
sis, the two halves on the other side (i.e., the end of the pipe
segment or in other words, the downstream end) were used for
swabbing before and after the CIP treatment. The samples, which
developed visual turbidity in the enrichment cultures following
48-h incubation, were considered as the evidence of presence of
viable microorganisms.
2.8. Data analyses and modeling
Table 1 shows only the starting temperatures for the wash solu-
tions. Practically, throughout the CIP process, there is significant
heat loss to the surroundings, mainly through the stainless steel
pipes leading to a continuous decrease in the temperature of the
solutions during the circulation time of 10 min. Therefore, there
is a temperature gradient throughout the system during CIP. Based
on the Newton’s law of cooling, the temperature does not vary lin-
early with time, an arithmetic mean would not represent the actual
effective mean temperature; whereas a logarithmic mean would
precisely represent the mean temperature of the treatment (Stolar-
sky, 1975). Therefore, the actual effective temperature for both the
cleaning solutions has to be determined based on the logarithmic
mean temperature (TLMT) for each solution during CIP using Eq.
(1) and it is this TLMT that must be optimized in this study in order
to generalize the results of this study.
TLMT ¼ ðTi À Tf Þ= lnðTi=Tf Þ ð1Þ
In Eq. (1), Ti is the initial temperature of the wash solution at the
beginning of the wash solution circulation time and Tf is the final
temperature of the wash solution at the end of the wash solution
circulation time. Therefore, experimental design for statistical
analysis as shown in Table 1 must be augmented with the TLMT
instead of the starting temperatures for both the alkaline (basic)
as well as the acidic EO water solutions. The response surface

optimization was performed over this augmented experimental
design to determine the most effective logarithmic mean temper-
atures of the alkaline/basic EO water (LMTB) and the acidic EO
water (LMTA) using MATLAB, that resulted in the maximum per-
centage RLU reduction (PRR) of 100.
2.8.1. Calculation of starting temperatures of the EO water cleaning
solutions based on the optimized TLMT obtained from this study
If the initial and final temperatures of the cleaning solution are
Ti and Tf, respectively, the logarithmic mean temperature of the
cleaning solution in the pipeline is TLMT, the ambient temperature
is T, all in °C and the total time of circulation is t in seconds, then
the total energy lost by wash solution (Q) in joules given by Eq.
(2) must be equal to the total energy lost to the surrounding (Q)
in joules over t seconds given by Eq. (3) with the assumption that
heat loss from the insulated stainless steel wash sinks and other
non-stainless steel parts of the milking system is negligible and
therefore the total surface area for heat transfer is the total surface
area of the stainless steel pipeline.
Q ¼ mCpðTi À Tf Þ ð2Þ
Q ¼ UAðTLMT À TÞt ð3Þ
where m is the total mass of the cleaning solution used, Cp is the
specific heat capacity of wash solution, U is the overall heat transfer
coefficient and A is the total surface area of the pipeline.
From Eqs. (2) and (3)
Ti À Tf ¼ UA=mCpðTLMT À TÞt ð4Þ
Also from Eq. (1)
Ti À Tf eððTiÀTf Þ=TLMTÞ
¼ 0 ð5Þ
Once the effective LMT is optimized using the response surface
modeling approach, the temperatures Ti and Tf can be solved using
Eqs. (4) and (5).
2.8.2. Validation and comparison
The optimized mathematical model parameters (LMTB and
LMTA) were used to calculate the starting temperatures of the
cleaning solutions for the pilot scale milking system by solving
for Ti for each solution using Eqs. (4) and (5). The overall heat trans-
fer coefficient (U) required for Eq. (4) was determined using the
experimental temperature data obtained from the optimization tri-
als. The starting temperatures thus calculated, were adjusted for a
3% safety factor to account for the error in the precision of the ther-
mocouples. These adjusted starting temperatures were evaluated
for effectiveness. The evaluation was done by implementing the
set optimal temperatures for three soiling and cleaning routines
and swabbing the surface for both the RLU and total plate count
in the same set of locations as specified in the sampling protocol
before and after every cleaning. For comparison of the effectiveness
of the EO water with that of the conventional cleaning method,
experiments were conducted following the DPC recommended
CIP protocol after performing a strong cleaning using the shock
cleaning protocol explained in the section ‘‘Preparation of the milk-
ing system for CIP process’’. For the conventional CIP starting tem-
perature of the alkaline cleaning solution was 75 °C and that of
the acid was 40 °C. Except for the different temperatures men-
tioned above, the same operating protocol as presented in Table 2
was followed for soiling and cleaning the system.
3. Results and discussion
Based on the above-mentioned protocols, results were analyzed
and compared to assess the feasibility of using the EO system gen-
erated alkaline and acidic waters in place of the conventional
chemicals as cleaning and sanitizing agents. As a first step towards
this, the temperatures of the wash solutions were optimized to
maximize the cleaning effectiveness while minimizing energy con-
sumption for the process.
3.1. Optimization
Based on the response surface experimental design presented in
Table 1, trials were conducted to identify the optimal temperatures
of the alkaline and acidic EO waters for achieving 100% RLU reduc-
tion. Fig. 5 shows the PRR for different locations of the milking sys-
tem and temperature combinations of the alkaline and acidic EO
waters investigated. The data show that the lower temperatures
of alkaline EO water (below 60 °C) are inadequate to effectively
clean any sections of milking system, irrespective of the acidic
EO water temperature (Fig. 5). This is evident from the straight
pipelines section of Fig. 5, where all the straight pipelines were
100% cleaned at 60 °C and above for all acidic EO water tempera-
tures. Thus, the straight pipeline sections were the easiest to clean
and were effectively cleaned at alkaline EO water temperatures as
low as 60 °C irrespective of the acidic EO water temperature. On
the other hand, the elbows section of Fig. 5 indicates that the el-
bows required higher acidic EO water (35 °C and above) tempera-
ture even when the alkaline EO water was at 60 °C. Moreover,
elbows are 100% cleaned at alkaline EO water temperature of
75 °C irrespective of the acidic EO water temperature. Similarly,
the other parts comprising mainly the porous materials such as
the liners, gaskets and the milk hose require a higher alkaline EO
water temperature (60 °C or above) as well as higher acidic EO
water temperature (45 °C). But, one common inference from all
the parts in terms of PRR is that all the parts are completely clean
irrespective of the acidic EO water temperature as long as the alka-
line EO water is as hot as 75 °C. Any reduction in the alkaline EO
water temperature requires an increase in the acidic EO water
temperature to achieve 100% cleaning effectiveness. Moreover
Fig. 4. Illustration of the swabbing for RLU and microbiological analysis in straight
pipeline sections and elbows.

the exegetic value of the water at 75 °C is much higher than that of
water at 45 °C. Therefore, it is ideal to determine the lowest alka-
line EO water temperature that can provide 100% cleaning effec-
tiveness in combination with the appropriate acidic EO water
temperature to minimize energy losses and economize the process.
It can be noted in Fig. 5 that 99.99% cleaning can be attained overall
at an alkaline EO water temperature of 60 °C and an acidic EO
water temperature of 45 °C. Therefore it can be concluded that
the optimal temperature to attain 100% cleaning lies between 60
and 75 °C for the alkaline EO water and between 35 and 45 °C for
the acidic EO water.
Moreover, Fig. 6 shows the maximum bacterial inactivation in
terms of the effectiveness of the different temperature combina-
tions of the alkaline and acidic EO waters. All the samples (i.e.,
100%) were positive for the lower temperature combinations. Even
with alkaline EO water at 60 °C there were 50% (4 out of 8) posi-
tives for the porous parts of the milking system, if the acidic EO
water is at 25 °C. It is also worth noting that it is these porous parts
have at least 25% (2 out of 8) positives for all temperatures of the
acidic EO water when the alkaline EO water temperature is 60 °C,
indicating that achieving 100% microbial destruction in these part
require a higher alkaline EO water temperature.
Walker et al. (2005b) found similar trends in RLU reduction and
microbiological destruction. But the highest alkaline EO water
temperature used by them for a similar pilot scale study was only
60 °C and it is clear from Fig. 6 that this alkaline EO water temper-
ature is not enough to achieve either 100% PRR or 100% microbial
destruction. Moreover, analyzing the results in terms PRR, intro-
duced in this study, can help identify the lowest logarithmic mean
temperatures of the alkaline and acidic EO water solutions to
achieve 100% cleanliness of the system.
Venkitanarayanan et al. (1999) evaluated the effects of acidic
EO water at four different temperatures: 4, 23, 35, and 45 °C on
suspensions of E. coli O157:H7, Salmonella Enteritidis, and Listeria
monocytogenes. They found a signiﬁcant increase in the microbici-
dal effect of the acidic EO water with the increase in temperature,
similar to the results found in this study for CIP. They also pre-
scribed acidic EO water at 45 °C for maximum effectiveness of
the acidic EO water, because at that temperature, a 1-min exposure
to acidic EO water was sufﬁcient to inactivate E. coli O157:H7 as
compared to requirement of 10 min to achieve the same at 4 °C.
Bari et al. (2003) evaluated the effect of EO water in conjunction
with dry heat and sonication on E. coli O157:H7 on a variety of
seeds: alfalfa, radish, and mung bean. When combined with dry
heat and a solution temperature of 45 °C, a maximum reduction
of 3.42 CFU/g was reached.
3.2. PRR model using logarithmic mean temperatures for EO water CIP
The log mean temperatures (LMT) were determined using Eq.
(1) from the temperature variation data during the alkaline and
acidic EO water circulation times as presented in Fig. 7(a) and
Fig. 5. Percentage RLU Reduction (PRR) for different temperature combinations of alkaline and acidic EO water CIP for different parts of the milking system.
Fig. 6. Microbiological analysis of different temperature combinations of alkaline
and acidic EO water CIP.

(b), respectively. However, there was an exception to this. In the
case of the 25 °C acidic EO water treatment, there was no change
in temperature during the entire circulation time of 10 min. Hence
for this temperature alone, the LMT was not calculated and the
mean temperature was taken as 25 °C.
Using the data presented in Fig. 7, the LMTB and LMTA were cal-
culated using Eq. (1) and an augmented experimental design was
developed for the purpose of modeling as shown in Table 3. Eq.
(6) gives the generic form of the model equation showing the full
quadratic model. Table 4 gives the values for the coefficients along
with their corresponding R2
values. These model equations were
used to the optimize LMTB and LMTA values in order to achieve
100% PRR (Percentage RLU Reduction), with the help of the optimi-
zation toolbox in MATLAB.
PRR ¼ a Ã LMTB þ b Ã LMTA þ c Ã LMTB Ã LMTA À d Ã LMTB2
À e Ã LMTA2
À f ð6Þ
The coefficients (a, b, c, d, e, and f), variables (LMTA and LMTB), for
different milking system parts (straight pipelines, elbows, and non-
metallic parts) are presented in Table 4. The optimum values of log-
arithmic mean temperatures (58.8 and 39.3 °C for the alkaline and
acidic EO water, respectively) determined by response surface opti-
mization for the overall 100% effective cleaning of the milking sys-
tem, reiterate the results presented in Figs. 5 and 6, that the elbows
and the porous parts of the milking system require higher acidic EO
water temperatures. Additionally, the porous parts specifically re-
quire a higher alkaline EO water temperature as well. The smaller
values of the coefficients for the log mean temperature of acidic
EO water, indicate a relatively smaller impact of the acidic EO water
temperature on the overall effectiveness of the CIP. Nonetheless, the
advantage of the increased acidic EO water temperatures has been
established through extensive studies (Walker et al., 2005a,b; Ven-
kitanarayanan et al., 1999; Bari et al., 2003). Therefore, increasing
the EO water temperature is important to be able to achieve 100%
cleaning effectiveness while simultaneously being able to decrease
the temperature of the alkaline EO water used.
3.3. Validation and comparison of EO water CIP with conventional CIP
The optimal parameters set forth by the model (LMTB = 58.8 °C,
LMTA = 39.3 °C) for achieving an overall cleaning effectiveness of
100% PRR were used to calculate the starting temperature by solv-
ing Eqs. (4) and (5) for the initial temperature (Ti) for each solution.
The resulting parameters were adjusted for a 3% safety factor, thus
suggesting a starting temperature of 70 °C for the alkaline EO
water and 45 °C for the acidic EO water.
Fig. 8(a) and (b) presents the comparison of PRR and microbio-
logical destruction obtained by the EO water CIP vs. the conven-
tional CIP recommended by DPC, respectively, obtained in the
validation study. In terms of PRR, though the conventional CIP
did not result in a consistent 100% PRR, the resulting RLU values
after CIP were well below the cut off values indicating satisfactory
cleanliness obtained by the conventional CIP techniques. Whereas,
the EO water CIP consistently resulted in 100% PRR.
It is worth reiterating the fact that these experiments were con-
ducted to replicate a worst case scenario, by allowing the milk to
dry in the milking system for a total period of 30 min (10 min after
allowing approx. 13 L (3.4 gallons) of milk to pass through the
milking system), which will not be the case in an everyday farm
Fig. 7. Temperature variation with time during the wash solution circulation time for different starting temperatures of the wash solutions as measured in the spray foam
insulated double-bowl stainless steel wash sink (a) alkaline EO water and (b) acidic EO water. (LMT – Logarithmic Mean Temperature).
Table 3
Augmented experimental design.
Trial. No LMTBa
(°C) LMTAb
(°C)
1 42 25
2 42 42
3 65 25
4 65 42
5 42 33.5
6 65 33.5
7 53.5 25
8 53.5 45
9 53.5 33.5
10 53.5 33.5
11 53.5 33.5
a
LMTB: Logarithmic Mean Temperature of Basic (alkaline) EO
water.
b
LMTA: Logarithmic Mean Temperature of Acidic EO water.

milking scenario. Another important thing to note is that the acidic
EO water itself is an effective sanitizer as established by the previ-
ous studies (Walker et al., 2005a,b; Izumi, 1999; Morita et al.,
2002; Venkitanarayanan et al., 1999; Park et al., 2002a,b; Koseki
et al., 2001; Russell, 2003; Sharma and Demirci, 2003; Bialka
et al., 2004). In the case of EO water CIP, the acidic EO water is cir-
culated as a last step in the CIP protocol and it, has nearly 80 ppm
of free chlorine. Therefore, it was not necessary to circulate a san-
itizer before milking in case of the EO water CIP. Whereas the
acidic solution used in the conventional CIP does not contain any
free chlorine, and hence the circulation of a sanitizer before milk-
ing is recommended by the DPC. But in order to identify and estab-
lish the effectiveness of the EO water as cleaning as well as
sanitizing agent and maintain consistency in the operating proto-
col for both the CIP techniques compared in this study, the circula-
tion of sanitizer was not done. This evidently has resulted in
insufficient cleaning by the DPC recommended conventional CIP
protocol as presented in Fig. 8(a) and (b).
4. Conclusion
All the variables affecting the effectiveness of any CIP technique
except the temperature, were eliminated based on the information
available in the literature. Then the temperature of the alkaline and
acidic EO water was optimized experimentally with the aid of sta-
tistical (response surface) modeling. Mathematical modeling using
conventional heat transfer calculation was done to determine the
optimal logarithmic mean temperatures of 58.8 and 39.3 °C for
the alkaline and acidic EO water, respectively, for effective CIP of
the milking system. Using conventional heat transfer calculations,
appropriate optimal starting temperature for the alkaline and
acidic EO water can be determined based on the total surface area
of the stainless steel pipelines and the ambient temperature of the
immediate surroundings of the milking system. Thus the CIP wash
control system can be programmed to perform CIP effectively
using the EO water based on the pipeline configuration (diameter
and length) of the milking system assuring maximum cleanliness
under all circumstances. The parameters optimized in this study
were proven to be optimal for the pilot scale milking system in
the laboratory conditions. However, a universal CIP model needs
be developed to simplify the adaptation to the EO water CIP at
the farm level without compromising its effectiveness using ad-
vanced computation fluid dynamic techniques. In conclusion, from
the results of this study, the EO water at its optimized tempera-
tures outperfomed the conventional CIP technique both in terms
of PRR and bacterial destruction. The next step is to do the valida-
tion of EO water CIP on a commercial dairy farm.
Acknowledgements
This project is funded in part by the USDA – Special Milk Safety
Grant (No. 2010-34163-21179) and Pennsylvania Experiment Sta-
tion. The technical advice provided by Dr. Stephen Spencer of Penn
State University is gratefully appreciated. The instrumentation
support provided by Fisher & Thompson, Martinsburg, PA, and
Dr. Roderick Thomas of the Penn State University is gratefully
acknowledged.
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