An ASABE – CSBE/ASABE Joint

Meeting Presentation

Paper Number: 141896778

Optimization of a blended electrolyzed oxidizing water-

based Cleaning-In-Place technique using a pilot scale
milking system

Xinmiao Wang, Ali Demirci, Virendra M. Puri, and Robert E. Graves

Department of Agricultural and Biological Engineering, Pennsylvania State University, University
Park, Pennsylvania, USA, 16802

Written for presentation at the

2014 ASABE and CSBE/SCGAB Annual International Meeting
Sponsored by ASABE
Montreal, Quebec Canada
July 13 – 16, 2014

Abstract. Milk safety is a major food safety concern in the United States. The cleanliness of on-farm milking
systems directly affects raw milk quality. The generally-accepted four-step procedure for milking system
Cleaning-in-Place (CIP) comprises: (1) warm water rinse, (2) alkaline wash, (3) acid wash, and (4) sanitizing
rinse prior to the next milking. Electrolyzed oxidizing (EO) water is emerging technology, which generates
acidic electrolyzed oxidizing (EO) water and alkaline EO water by electrodialysis of dilute salt solution. Previous
studies in our laboratory had shown that EO water can be an alternative for conventional milking system CIP,
both on a lab scale milking system and on a commercial dairy farm. Recently, a one-step cleaning has been
adopted on an increasing number of dairy farms; it combines the alkaline wash and acid wash cycle together;
and this saves chemical expenditure and water usage, energy cost, and time. By mixing acidic EO water with
alkaline EO water, a less corrosive but with a high oxidation reduction potential (ORP) blended near neutral EO
water can be produced. Therefore, it was proposed in this study that the blended EO water solution can be
applied to a lab scale pilot milking system as one-step CIP and the cleaning and sanitization performance
between using blended EO water solution and commercial one-step cleaning chemicals are comparable. A
response surface method was applied to determine the optimal condition of the cleaning time, the starting
temperature of the blended EO water solution and the acidic EO water percentage in the blended EO water
solution. The CIP performance comparison among the optimal blended EO water solution and two commercial
one-step cleaning chemicals were performed. Results showed that cleaning time of 17 min, a starting
temperature of 59°C and an acidic EO water percentage of 60% in the blended EO water solution (pH of 2.66,
ORP of 1109 and chlorine concentration of 48.0 ppm) could achieve the required 100% CIP performance. The
CIP performance of using optimal blended EO water solution and the commercial one-step cleaning chemicals
showed that the optimal blended EO water solution is comparable to those of the commercial one-step
cleaning chemicals. Overall, this study showed that the blended EO water had the potential to be adapted as
an alternative for one-step CIP for pilot milking system.

Keywords. Blended electrolyzed oxidizing water, Cleaning-In-Place, Pilot milking system, Milk safety.
Introduction
According to the 2011 data from the Food and Agriculture Organization of the United Nations, the United
States produced 90,865,000 metric tons of cow milk, ranking first in the world (FAOSTAT, 2012). The annual
average consumption of dairy products per capita in the United States has increased from 244.5 kg in 1975 to
277.6 kg in 2012(USDA, 2012). Quality and safety of consumed milk is always important, therefore, the Food
and Drug Administration has issued several regulations to assure the raw milk quality and safety in the United
States (FDA, 2012). It is of great importance to ensure the safety and quality of raw milk and other dairy
products and provide the public with safe milk to consume.
The milking system cleaning-in-place (CIP) is performed right after the milking is completed, which consists of
four steps: warm water rinse, alkaline wash, acid wash, and a sanitizing rinse prior to the next milking event
(Table 1). During the alkaline and acid wash cycles, concentrated chemicals are usually used; but they
possess potential hazard to the workers and the environment after use. Therefore, previous studies in our
laboratory have contributed to addressing this issue by using a potential alternative method – electrolyzed
oxidizing (EO) water, for the milking system CIP.
Table 1. CIP Recommendations for parlor milking systems (DPC, 2010)
Cleaning Cycle Conventional CIP
Warm water rinse 2 min; 43.3-48.9°C
Alkaline wash 8-10 min; start:71.1°C-76.7°C; finish:48.9°C; pH >12.0; 120 ppm chlorine; 1100 ppm alkalinity;
>20 slugs
Acid wash 3-5 min; pH~3.0
Sanitizing rinse EPA registered dairy sanitizer solution

EO water is an emerging technology developed in Japan. Using electric current in an electrodialysis chamber,
two types of EO water solutions are generated simultaneously from dilute sodium chloride solution. Alkaline EO
water is generated from cathode with a pH as high as 11.6 and oxidation reduction potential (ORP) up to -795
mV; acidic EO water is generated simultaneously from anode with a pH as low as 2.6 and ORP up to 1150 mV
(Figure 1).

Figure 1. Schematics of electrolyzed water generator and produced compounds (Huang et al., 2008).
The similarity in properties between the EO water with that of the conventional milking system CIP chemicals
motivated the consideration of implementing EO water solutions to the milking system CIP. Several studies had
been conducted in our laboratory during the past decade. The feasibility of utilizing EO water for the CIP of
milking systems was first assessed by Walker et al. (2005 a&b). After the promising results from cleaning of the
milking system materials in the lab, a response surface model was developed for optimizing the cleaning
parameters by using a pilot-scale milking system. Their results showed that EO water cleaning solutions at
temperatures of 60°C or lower were able to remove all detectable bacteria successfully. These optimized
cleaning temperatures are lower than the temperatures recommended (70 to 75°C) for the conventional dairy
CIP process (DPC, 2010), which indicates that using EO water for milking system CIP could be an energy
effective process. Later study in our laboratory suggested a starting temperature of 70°C for the alkaline EO

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2014 ASABE – CSBE/SCGAB Annual International Meeting Paper Page 1
water and 45°C for the acidic EO water were enough to achieve a 100% cleaning effectiveness on a pilot
milking system (Dev et al., 2014). However, the pilot-scale milking set-up is a scaled-down system and does
not fully and sufficiently represent a commercial-scale dairy farm CIP; therefore, a real-world study was also
conducted on a commercial dairy farm to evaluate the EO water performance for CIP of the milking system
(Wang et al., 2013). Based on a four-month trial on a mid-size commercial dairy farm in Pennsylvania, it was
concluded that the EO water achieved same or better cleaning effectiveness compared with the conventional
CIP method for most sampling locations and milking system components. In addition, based on the cost
analyses, the estimated operational cost of using the EO water CIP is lower than using the conventional CIP by
approximately 25%, provided an EO water generator unit and other associated accessories are already in
place. EO water is shown to be a promising technology for CIP of milking systems, both from
cleaning/sanitizing performance and economic aspects.
Recently, a new CIP approach has received attention on an increasing number of dairy farms (Parr, 2013).
This new CIP approach combines the alkaline wash and the acid wash cycles together as one, to conduct a
“one-step” CIP for the milking system. In order to do this, several commercially available one-step CIP
chemicals are on the market and their CIP performance is claimed to be comparable to the conventional
alkaline wash and acid wash of milking system. It was claimed that using one-step CIP can be cost saving
through eliminating the separate alkaline and acid wash cycles, decreasing the usage of water, chemicals,
energy, and time. In addition, the manufactures claimed that their products are capable of keeping the bacterial
count low for standard tests. However, the chemical compositions of these products are not available, nor are
any published experimental results for the milking system CIP.
There have been some commercially available EO water generators that produce a mixed EO water solution,
named as “near neutral EO water” or “mixed oxidant”. The mixing of acidic EO water into alkaline EO water can
result in less corrosive pH EO water. This near neutral EO water contains the components of chlorine found in
acidic EO water. Comparing to acidic EO water, near neutral EO water possesses a relatively less corrosive pH
while having a high ORP to function as a disinfecting agent (Guentzel et al., 2008). Researchers assessed the
disinfecting effectiveness of neutral EO water on the inactivation of five microorganisms (Escherichia coli,
Salmonella Typhimurium, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis) on the
surfaces of spinach and lettuce, and results showed a 4.0-5.0 log10 CFU/mL bacterial reduction for all five
microorganisms (Guentzel et al., 2008). In addition, compared to a water rinse, the near neutral EO water
treatment of spinach and lettuce surfaces does not leave any significant total residual chlorine. Furthermore,
the effective utilization of near neutral EO water on peach and grape surfaces postharvest sanitation to prevent
incidence of microorganism infection (Botrytis cinerea and Monilinia fructicola) was demonstrated (Guentzel et
al., 2010). These results expand the application of near neutral EO water from simple sanitization onsite to the
enhancement of fruit shelf life in commercial markets. When comparing to other fungicide like “Captan” to
conduct the disinfecting experiments on strawberry plants, study showed twice a week using near neutral EO
water with 100 ppm chlorine concentration treatment is significantly more effective than the once per week; at
the same time, the safety advantage of using near neutral EO water with 100 ppm chlorine concentration for
the strawberry plant spray is that this method did not leave significant phytotoxicity compared to the water
treatment (Guentzel et al., 2011). Mechanisms of the fungicidal efficiencies of near neutral EO water might be
attributed to more •OH radical compared to acidic EO water at the same chlorine concentration. Study showed
that the •OH radical plays an important role in destroying the cellular structures of microorganism (Aspergillus
flavus) conidia (Xiong et al., 2010). Given the rapid development of near neutral EO water applications and the
desired one-step milking system CIP approach, this study is undertaken to evaluate and optimize the blended
EO water CIP process for a lab scale pilot milking system as a one-step milking system CIP alternative.

Materials and Methods

Microorganisms and medium
A cocktail of four types of common microorganisms in raw milk, including Pseudomonas fluorescens (B2,
obtained from the culture collection of Department of Food Science at Pennsylvania State University),
Micrococcus luteus (ATCC 10240), Enterococcus faecalis (ATCC 51299), and E. coli (ATCC 25922) was
prepared as the initial inoculum to mimic the worst case scenario and increase the microbial population of the
raw milk. Each bacterium was grown in 500 ml of Tryptic Soy Broth (TSB) for 24 hr at their optimal
temperatures. For P. fluorescens and M. luteus, the incubate temperature was set at 30°C, and for E. faecalis
and E. coli at 37°C.

2014 ASABE – CSBE/SCGAB Annual International Meeting Paper Page 2

Description of the pilot milking system for one-step cleaning trial
A 114 L (30 gallon) single bowl sink was used for the one-step pilot milking system CIP (Figure 2). Other
components that are essential to the milking system are included in the pilot system as previously described
(Dev et al., 2014). The test portion of the milking system pipeline consisted of a set of straight pipes with a total
length of 24.4 m (80 ft) and eight 90° elbows. The CIP process of the pilot milking system is powered by
vacuum, set at a 50 kPa constant level. A 38 L (10 gallon) receiver jar was installed at the end of the milking
system pipeline for temporary storage then either redirect the cleaning solution back to the sink or drain directly
using a electrically powered CIP motor pump. The slope of the pipeline in the system was set to 0.8% to
facilitate water draining. When conducting CIP, the solution fluid dynamic properties were adjusted to be at 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
gal per min).
pH and ORP sensors along with several thermocouples were used for monitoring the pH, ORP and
temperature during the CIP process. Two thermocouples were placed at each end close to the bottom of the
sink, as well as at the outlets of the alkaline EO water, acidic EO water, and tap water faucet, one inserted at
the end of the return line to monitor the returning solution temperature.

Figure 2. Pilot plant milking system (revised from Dev et al., 2014; not to scale).

Preparation of milk
Fresh raw milk was collected from the Penn State dairy barn by directing the milk from the cow to a 38 L (10
gallon) stainless steel milk can. During the transportation of milk from the dairy barn to the pilot plant, the
temperature of the milk, 38°C, dropped by approximately 5-6°C. The milk was reheated to about 38°C by using
a custom-made hot water copper tube heat exchanger, to simulate the milk temperature from a cow.
The cocktail of the four types of microorganisms incubated for 24 hr at their optimal temperatures in 500 ml
TSB before use. After incubation, the culture broth was centrifuged at 4,000 g for 40 min and supernatant
decanted. Each bacterium was re-suspended using 500 ml of the heated fresh raw milk and then mixed with
the rest of the heated fresh raw milk. The total bacterial population for the initial “contaminated” milk for soiling
was about 108 CFU/ml.
Preparation of blended EO water solutions
Alkaline EO water was heated before use by a 305 L (80 gal) capacity tank water heater (Model RUE PRO-80-
2, Ruud Manufacturing Co., Atlanta, GA) and acidic EO water was heated using a tankless heater (Model
EX1608TC, Eemax Inc., Oxford, CT). Both EO water solutions were freshly generated before use and the
properties tested, both for heated and unheated solutions. For the unheated EO water solutions, the acidic EO
water had pH of about 2.6, ORP of about 1150 mV, and free chlorine content of about 80 ppm; whereas for the
unheated alkaline EO water had pH of about 11.5 and ORP of about -850 mV. The chlorine content of the
acidic EO water was measured by titrating against an N, N-diethyl-p-phenylenediamine-ferrous ethylene
2014 ASABE – CSBE/SCGAB Annual International Meeting Paper Page 3
diammonium sulfate (DPD-FEAS) solution prepared using a test kit (Hach, Inc., Loveland, CO). When heated,
the acidic EO water solution had pH of about 2.8, ORP of about 1150 mV, and free chlorine content around 65
ppm, which depends on the heated temperature, whereas the alkaline EO water solution had pH of about 10.5
and ORP of about -800mV.
Preparation of the milking system for CIP process

i) Shock cleaning

Shock cleaning of the pilot milking system was conducted every time before each experiment to create a
consistent base line. The term “shock cleaning” comes from a more powerful, namely more concentrated,
cleaning solution for the milking system CIP:

a. Alkaline wash: 710 ml (24 oz) of Dairy Cycle 3 (Chemland Inc., Kansas City, MO) diluted into 68 L (18
gal) of alkaline wash solution with a starting temperature of 80°C and a circulation time of 10 min;

b. Water rinse: 38 L (10 gal) of tap water heated to 40°C for rinsing the system without recirculation;

c. Acid wash: 107 ml (3.6 oz) of Dairy M. S. R. 50 (Chemland Inc., Kansas City, MO) diluted into 68 L (18
gal) of acid wash solution with a starting temperature of 80°C and a circulation time of 10 min;

d. Sanitizing rinse: right before each experiment, 27 ml (0.9 oz) of LCS (Classic Technologies., Kansas
City, MO) diluted into 68 L (18 gal) of sanitizing solution with a starting temperature of 40°C and no
recirculation.

Table 2. Operation process comparison between the blended EO water and commercial one-
step chemicals for pilot milking system CIP.
Operating cycle
Operating parameter
Sanitize Soiling Warm water rinse Blended EO water CIP Commercial one-step CIP

Starting temperature (°C) 40 38 40 Variable 70

Quantity (L) 68 38 (12.7×3 parts) 38 57 57

Air injection No No No Yes Yes

Recirculation No No No Yes Yes

32 (<1 min/part and

Operation time (minute) 3.5 2 Variable 10
10 min break/part)

ii) Soiling the system:

The contaminated milk was used to soil the pilot milking system. Under the vacuum, milk was drawn into
the system pipeline and accumulated in the receiver then drained out without recirculation. The milk drawn
into the system was in turbulent state with a Reynolds number to the order of magnitude of 105. The 38 L
(10 gal) milk was introduced into the system pipeline in three equal portions of approximately 13 L (3.3 gal)
each time with a 10-min air dry between the soiling processes. The air drying was conducted with the
vacuum on, namely the claw was drawing ambient air instead of milk in to the system. The milk was not
recirculated in the system pipeline to minimize churning, cream separation, and other physio-chemical
changes in the milk.

Experimental design and blended EO water CIP process

Three factors were studied in the CIP process, namely the cleaning time (10 – 20 min), the starting
temperature (50 – 70°C) of the blended EO water solution, and the acidic EO water percentage (25 – 60%) in
the blended EO water solution. Preliminary experiments were conducted to determine the near neutral acidic
EO water percentage range in the blended EO water solution by testing the CIP performance for all sampling
locations using different acidic EO water percentages with a fixed mid-point for the other two factors’ respective
ranges.
2014 ASABE – CSBE/SCGAB Annual International Meeting Paper Page 4
The optimization process used Box-Behnken three-factor response surface method (Table 3), and the total
quantity of the blended EO water solution was fixed at 57 L (15 gal) based on the calculation from the length of
the system pipeline and other accessories (Reinemann, 1995). When the optimal condition of each factor was
achieved, three validation runs were conducted at this optimal condition to verify the optimization result.

Table 3. Three factor Box-Behnken experimental design for CIP optimization process.

Run Order Time (min) Temperature (°C) Acid percentage (%)

1 10 70 42.5
2 15 70 60
3 20 60 25
4 15 50 60
5 15 60 42.5
6 20 70 42.5
7 20 60 60
8 15 60 42.5
9 10 50 42.5
10 10 60 25
11 20 50 42.5
12 15 70 25
13 10 60 60
14 15 50 25
15 15 60 42.5

Evaluation
Sampling locations are categorized into pipes, elbows, and other materials which include gaskets, liners, and a
milk hose (Figure 3). Eight straight pipe sampling locations and eight elbow sampling locations were selected
along the milking system pipeline (Figure 3) (Dassault Systèmes SolidWorks Co., Waltham, MA).
Disassembled and cut-away schematics illustrating the swabbing areas of sampling locations of pipes and
elbows for soil and microorganisms are shown in Figure 4. For each of the sampling location, two types of
evaluation methods were used: Adenosine Triphosphate (ATP) bioluminescence for the presence of soils and
calcium alginate-tipped applicators swabbing for microbial cells presence. Samples were collected from each
sampling location by disassembling the connections (also shown in Figure 3) and swabbing the inner surfaces
of elbows and straight pipes (Figure 4). With reference to the cleaning solution flow direction, the right half of
the connections of sampling locations were sampled using ATP swabs for bioluminescence analyses and the
left half using sterile calcium alginate-tipped applicators for analyzing the bacterial presence. Other system
components of gaskets, liners and milk hose were swabbed for ATP bioluminescence and bacterial presence
as well, but the only difference is the sample was taken by swabbing over the entire inner circle instead of half
inner circle due to the limited availability of milk contact surfaces for swabbing in these components.

Analysis
Inner surfaces of all sampling sites were evaluated using ATP bioluminescence PocketSwab Plus swabs
(Charm Science, Inc., Lawrence, MA) for soils and sterile calcium alginate-tipped applicators (Puritan Medical
Production Co. LLC, Guilford, ME) for analyses of bacterial cell presence.

i) ATP bioluminescence

The ATP swabs’ firefly enzyme luciferase gave a quantitative measurement in terms of Relative Light Unit
(RLU) by using a novaLUM palm-sized luminometer (Charm Science, Inc., Lawrence, MA); the RLU
readings served as an indicator of the surface cleanliness. The RLU reading of zero represents the surface
to be clean and higher RLU readings represented “dirtier” surfaces. For further discussion purposes, RLU
2014 ASABE – CSBE/SCGAB Annual International Meeting Paper Page 5
 reduction percentage and RLU log reduction were used for CIP performance comparison. The RLU
reduction percentage is the after CIP RLU reading compared to the before CIP RLU reading; and higher
RLU reduction percentage represented a more effective CIP process. The RLU log reduction is difference
between the natural logarithm of before CIP RLU reading and the natural logarithm of after CIP RLU
reading. The equations used for calculations of RLU reduction percentage and RLU log reduction are
presented as follows:

(Equation 1)

(Equation 2)

In equation (2), the numeral 1 was added to avoid indeterminacy of log when the measured RLU is zero.
Since the RLU values are in the order of 106, the error introduced is negligible. By definition of the RLU
reduction percentage, a 100% RLU reduction represents an after CIP RLU reading of 0.

ii) Calcium alginate sampling

Bacterial samples were collected with sterile calcium alginate-tipped applicators (Puritan Medical
Production Co. LLC, Guilford, ME), which were placed in a TSB medium, and then incubated at 37°C for
48 hr for possible microbial growth. The clear medium observed visually was accepted as “negative” while
the opaque (turbid) ones as “positive.” For further discussion purposes, negative enrichment percentage
was calculated by percentage of the number of negative samples with respect to the total number of
samples (number of negative samples plus positive samples) for all three sampling categories. By this
definition, higher negative enrichment percentage represents a more sanitized surface condition.

(Equation 3)

iii) Statistical analysis

Response surface method was generated and results analyzed using Minitab 16.2 (MINITAB Inc, State
College, PA). Three replication runs were conducted at the optimal blended EO water conditions. The
significant differences in mean values were determined using Tukey’s method at the 95% confidence
interval in Minitab 16.2.

Figure 3. Sampling location schematic of the pipes and elbows (P1 – P8 are pipe sampling
locations and E1 – E8 are elbow sampling locations, to scale).

2014 ASABE – CSBE/SCGAB Annual International Meeting Paper Page 6

 Figure 4. Sampling schematic detail: (a) elbow sampling location; (b) pipe sampling location.
Based on the cleaning flow direction, the right half sampled using ATP swabs and the left half
for bacterial presence (Dev et al., 2014).

Results and Discussions

This study investigated the use of blended EO water as an alternative for a lab-scale pilot milking system one-
step CIP. An optimization process was conducted firstly to determine the optimal condition of three affecting
parameters: the CIP time, the starting temperature of the blended EO water solution, and the acidic EO water
percentage in the blended EO water solution using a response surface method. The CIP performance was
compared among using the optimized blended EO water and two commercial one-step CIP chemicals by
evaluating the ATP bioluminescence and the bacterial presence on the sampling locations of pipes, elbows and
other accessories.

Determining the range of parameters evaluated for one-step CIP.

The cleaning time of the blended EO water solution was evaluated in the range from 10 to 20 min. The
recommendation was “a minimum of 24 slugs for each CIP process” (DPC, 2010), therefore based on the
configuration of the pilot milking system, a 10 min wash circulation for one cycle was used in our previous
studies(Dev et al., 2014). Therefore, the cleaning time of the blended EO water solution varies from one
complete CIP cycle (10 min) to two complete CIP cycles (20 min). The starting temperature of the blended EO
water solution was set between 50 to 70°C, based on the following reasons: i) The previously optimized
alkaline solution temperature was 70°C (highest) and acid solution was 45°C (lowest) for the pilot milking
system which provided a rough range of the solution starting temperature, and ii) The one-step CIP needs to
remove of both organic (mostly by alkaline wash cycle) and mineral materials (mostly by acid wash cycle) from
the milk-contact surfaces. Therefore, 50 to 70°C temperature range was selected to find out the optimal
temperature for the combined steps. There were no previous published studies on the range of the acidic EO
water percentage for the milking system one-step cleaning, and neither were any published recommendations
available to refer to. Therefore, an acidic EO water percentage in the blended EO water solution ranging from

2014 ASABE – CSBE/SCGAB Annual International Meeting Paper Page 7

0% to 100% was evaluated for the pilot milking system CIP during preliminary experiments. By keeping the CIP
time and solution starting temperature at mid-point of their respective ranges, namely at 15 min for CIP time
and 60°C for solution starting temperature, preliminary experiments were conducted to determine the acidic EO
water percentage range (Figures 5 and 6). Results from the preliminary experiments showed that the CIP
performance was not desirable when the blended EO water solution approaches near neutral pH range; and
the more acidic solutions had relative better CIP performance compared to the more basic solutions. RLU
reduction percentage and negative enrichment percentage was used for analysis. When the acidic EO water
percentage reached or beyond 46.7%, the RLU reduction percentages for sampling locations of pipes and
elbows reached 100%, and the negative enrichment percentages for pipes and elbows also reached higher
than 60%. On the other hand, when the acidic EO water percentage went down to 26.7% or lower, the RLU
reduction percentages for sampling locations of pipes and elbows were above 99.90% until when using a pure
alkaline EO water solution dropped down to around 99.8%. The negative enrichment percentages of the more
basic blended EO water solutions also showed a relative higher result compared to near neutral conditions;
even not as high as the more acidic blended EO water solutions. Upon review, it was determined that the more
acidic blended EO water solutions did result in a better CIP performance when holding the rest two factors
constant, both from the ATP bioluminescence method and the bacterial presence data. A more important
reason to choose more acidic solution over more alkaline solution is that, based on the final RLU results from
the preliminary experiments and a set of cut-off values presented in previous studies of system materials of
rubber (cut-off RLU of 4,500) and stainless steel (cut-off RLU of 1,000) (Wang et al., 2013), the more acidic
CIP trials resulted in a below the cut-off value RLU reading while the more basic CIP trial could not. Therefore,
it was determined that the acidic EO water percentage for the response surface method to be from 25% to
60%.

Figure 5. RLU reduction percentages at different acidic EO water percentages for sampling
categories of pipes, elbows and other accessories.

2014 ASABE – CSBE/SCGAB Annual International Meeting Paper Page 8

 Figure 6. Negative enrichment percentages at different acidic EO water percentages for
sampling categories of pipes, elbows and other accessories.

Optimization of the blended EO water CIP process

Based on the response surface experimental design in Table 1, altogether 15 runs were conducted to get the
optimal condition of each factor. Table 4 shows the results of the response surface design, including the RLU
reduction percentages for the three sampling categories; the RLU log reductions for the three sampling
categories; and the negative enrichment percentages for the three sampling categories.
Table 4 shows the complete set of results from the response surface method. Run # 5, 8 and 15 are
experiments at the center point of each factor, which showed a relative consistency for RLU reduction
percentage. The RLU log reduction category showed how much ATP was removed for each experiment. The
log removal was different for each experiment, which resulted from i) the CIP performance being different for
each solution, and ii) the initial RLU reading differed from experiment to experiment due to the quality of the
raw milk acquired. Any conclusions solely based on the RLU log reduction are not sufficiently reliable;
therefore, the term RLU reduction percentage, which is a relative number, was used for remainder of the
analyses in this study. However, despite the differences caused by the raw milk quality for every experiment,
the CIP process was able to remove at least 2.5 log of residue ATP on the surfaces.
When using RLU reduction percentage as a response for the optimization process, the regression model for
the pipes, elbows and other materials are found as follows from Minitab (T stands for time, AP stands for acid
percentage, and TEMP stands for temperature):

Pipe RLU Reduction Percentage (%) = 99.980 + 0.060×T + 0.016×TEMP + 0.089×AP – 0.040×T2 –
0.023×TEMP2 – 0.048×AP2 – 0.008×T×TEMP – 0.043×AP×T – 0.030×AP×TEMP (R2=97.72%)

Elbow RLU Reduction Percentage (%) = 99.977 + 0.056×T + 0.015×TEMP + 0.084×AP – 0.028×T2 –
0.021×TEMP2 – 0.038×AP2 – 0.013×T×TEMP – 0.045×AP×T – 0.018×AP×TEMP (R2=98.04%)

Other RLU Reduction Percentage (%) = 99.670 – 0.116×T - 0.019×TEMP + 0.514×AP + 0.018×T2 +
0.075×TEMP2 – 0.363×AP2 – 0.063×T×TEMP – 0.400×AP×T – 0.163×AP×TEMP (R2=88.70%)

2014 ASABE – CSBE/SCGAB Annual International Meeting Paper Page 9

 Table 4. Response surface method result for each sampling category.
Run RLU Reduction Percentage (%) RLU Log Reduction Negative Enrichment Percentage (%)
Order
Pipe Elbow Other Pipe Elbow Other Pipe Elbow Other

1 99.86 99.88 99.49 2.88 2.94 2.44 25 25 33

2 99.99 100 99.71 3.84 4.36 2.77 63 50 33
3 99.89 99.91 97.99 2.95 3.07 1.70 25 25 33
4 99.99 99.99 99.90 4.24 4.16 3.12 75 63 33
5 99.99 99.97 99.72 4.00 3.56 2.45 50 38 33
6 99.98 99.99 99.81 3.79 3.98 2.71 50 63 33
7 100 100 100 6.34 6.34 6.25 75 75 68
8 99.97 99.99 99.69 3.59 3.92 2.69 38 50 33
9 99.84 99.84 99.84 2.80 2.81 2.87 13 25 33
10 99.70 99.73 99.45 2.54 2.61 2.35 13 13 33
11 99.99 100 99.91 4.11 4.47 3.03 63 75 33
12 99.89 99.88 98.54 2.97 2.91 1.93 25 25 33
13 99.98 100 99.86 3.73 6.54 2.88 75 50 33
14 99.77 99.80 99.38 2.68 2.71 2.30 13 25 33
15 99.98 99.97 99.49 3.68 3.53 2.79 50 38 33

The 3D surface contour plot of sampling categories of pipes and elbows are shown in Figures 7 and 8. From
the trend of the surface plots and also the statistical analyses for each category, the cleaning time and acidic
EO water percentage significantly affected the RLU reduction percentage for sampling locations of pipes and
elbows (P<0.05) – higher starting temperature and longer cleaning time are more favorable to achieve a
satisfactory CIP performance. Take the RLU reduction percentage of elbow as an example, with a shorter time
(10 min) and a lower acidic EO water percentage (25%), and a median temperature of 60°C, the RLU reduction
percentage was about 99.70%, but it went up to almost 100% if the cleaning time is longer (20 min) with a
higher acidic EO water percentage (60%) without changing the temperature setting. The blended EO water
solution starting temperature, on the other hand, generated a mixed set of results –the increased temperature
would increase the reaction rate thus enhancing the cleaning performance, but the increased temperature also
decreased the chlorine concentration in the blended EO water solution, which resulted in a reduced soil
removal power. From the statistical analysis, the temperature effect was not significant for all three sampling
locations of pipes (P=0.109), elbows (P=0.090) and other accessories (P=0.153).

2014 ASABE – CSBE/SCGAB Annual International Meeting Paper Page 10

 SURFACE PLOTS OF PIPE RLU REDUCTION

100.00% 100.00%

eduction 99.95% reduction 99

99.90%
90%
99.90%
1 99.80% 1
99.85%
0 99.70% 0
temperature acid percentage
-1 -1
0 -1 0 -1
1 1
time time

(15min)
(60°C)
Hold Values (42.5%)
100.00% time 0
reduction
99.90% temperature 0
99.80%
1 acid percentage 0
0
acid percentage
-1
0 -1
temperature 1

Figure 7. Surface plots of RLU reduction percentage for pipes.

SURFACE PLOTS OF ELBOW RLU REDUCTION

100.00% 100.00%
eduction 99.95% reduction 99
99.90%
90%
99.90% 99.80%
1 1
99.85%
0 99.70% 0
temperature acid percentage
-1 -1
0 -1 0 -1
time 1 time 1

100.00%
Hold Values (15min)
reduction time 0(60°C)
(42.5%)
99.90%
1
temperature 0
99.80% 0
acid percentage 0
acid percentage
-1
0 -1
temperature 1

Figure8. Surface plots of RLU reduction percentage for elbows.

When comparing the results among different sampling categories, it was observed that the stainless steel
materials such as pipes and elbows are more easily cleaned compared to other materials such as rubber and
Polyvinyl chloride (PVC); the average RLU reduction percentage of sampling location of pipes was as high as
99.92% and elbows 99.93% while for liners, hose and gaskets, the average RLU reduction percentage was
only about 99.52%. Rubbers had been shown to have “caverns” and crevices over the surfaces and from a
scanning electron microscopy analysis of a rubber liner sample surface collected directly from an operating

2014 ASABE – CSBE/SCGAB Annual International Meeting Paper Page 11

dairy farm, and results showed the presence of cracks and of areas with other materials on the surface (Latorre
et al., 2010). One study demonstrated a greater number of Pseudomonas fragi and Listeria monocytogenes
and Bacillus cereus attachment onto buna-N-rubber and teflon (Gaspar-Rolle, 1991). The highly developed
porosity on the rubber surfaces made a desired CIP performance harder to achieve compared to smoother
stainless steel surfaces. It is recommended that the rubber goods should be inspected and replaced on a
regular basis on a dairy farm, twice a year (recommended), to prevent the rubber from aging and making more
porous (DPC, 2010).
The optimizer suggested a CIP time of 17 min, a starting temperature of 59°C and an acidic EO water
percentage of 60% to be the optimal conditions for the blended EO water solution. Three validation runs were
conducted at this optimal condition and results showed good reproducibility (Figure 9). At this condition, 100%
RLU reduction percentages were achieved for both sampling locations of pipes and elbows experimentally, as
expected from Minitab desirability calculation; and the other accessories resulted in a 99.95% RLU reduction
percentage experimentally, which is much higher compared to the other accessory average of 99.52% during
the 15 runs of the optimization process. The negative enrichment percentages of sampling locations of pipes
and elbows reached around 80% on average, and as stated above of the material property differences, no
improvement in negative enrichment percentage was observed (33.3%) for the other accessories including
liners, hose, and gaskets.

Comparison between optimal EO water CIP and commercial one-step CIP

Two types of commercial one-step CIP chemicals were used as comparisons; they were from different
companies and hereby denoted as product 1 (P1) and product 2 (P2). Both of them have a blend of organic
and inorganic acids and a claimed good wetting capability and effective removal capability of lipid, protein and
mineral within one CIP cycle. Based on the instructions of the product labels, parameters were set at a starting
temperature of 70°C, a cleaning duration of 10 min, and a solution concentration of 29.6 ml (1 oz) chemical per
3.79 L (1 gallon) water for the commercial one-step CIP. For the blended EO water solution, the optimal
condition (CIP time of 17 min, starting temperature of 59°C and an acidic EO water percentage of 60%) was
used for comparison purpose. Results are shown in Figure 9.
The RLU reduction percentage was not significantly different among the two commercial products and the
optimal blended EO water solution (p>0.05). The RLU reduction percentage is a relative number compared to
the initial RLU reading from the soiling process; therefore, given the high initial soiling RLU readings (on the
order of magnitude of 107), it is not easy to differentiate from the RLU reduction percentage among different
methods. The average RLU reduction percentage of optimal blended EO water was 100% while the two
commercial one-step chemicals were 99.96 and 99.97%, respectively, for sampling locations of pipes, and the
average RLU reduction percentage of optimal blended EO water was 100% while the two commercial one-step
chemicals were 100 and 99.95%, respectively, for sampling locations for elbows. However, when comparing
the bacterial enrichment data, the difference between blended EO water and the two commercial products are
noticeable. For the negative enrichment percentage, blended EO water performed better compared to the
commercial one-step chemicals for the stainless steel surfaces including sampling locations of pipes and
elbows on average. Take the sampling location of pipes as an example; the average negative enrichment
percentage was 83.3% for the optimal blended EO water but only 50.0 and 54.2% for the two commercial one-
step CIP chemicals, respectively. This is not surprising given the past studies of using blended EO water
solution as a disinfectant for the produce and processing plants. Other accessory materials, including gaskets,
liners and milk hose, however, did not have a significant difference (p>0.05) among CIP methods. The natural
surface condition makes them hard to be cleaned and the crevices provided the bacteria a habitat for growth;
therefore, the non-significant differences in results were not surprising. Generally, all of these CIP methods
demonstrated satisfactory CIP performance results, indicating that they are capable of removing the organic
and inorganic soils as well as reducing the bacteria load and keep the milk contact surfaces clean and
sanitized without bringing in potential hazard.
There have been studies trying to explain how blended EO water solution worked as a disinfectant, but not as a
cleaning agent. Most of the studies demonstrated the effect of both chlorine and ORP on killing the
microorganisms – high ORP of the solution disrupts the outer membrane, thus facilitating the transfer of HOCl
across the cell membrane, resulting in further oxidation of intracellular reactions and respiratory pathways such
as the GSSH/2GSH cellular redox couple (Liao et al., 2007). For the cleaning process mechanism using
blended EO water solution, on the other hand, none is found in the current literature review; the “wetting power”
which is a necessity of detergent was not extensively studied for EO water. More studies are needed to clearly
indicate how blended EO water functions as a cleaning agent.

2014 ASABE – CSBE/SCGAB Annual International Meeting Paper Page 12

 Figure 9. CIP performance comparison among two commercial one-step cleaning chemicals
and blended EO water at its optimal conditions (P1 represents product 1 and P2 for product 2).

Conclusion
In conclusion, an optimal condition of the blended EO water solution for the pilot milking system CIP is
achieved in this study; they are: a CIP time of 17 min, a blended EO water of starting temperature of 59°C, and
an acidic EO water percentage of 60%. Three validations at this optimal condition showed 100% RLU reduction
percentages for both sampling locations of pipes and elbows experimentally, which fitted the model estimation
of CIP performance well. When comparing the CIP performance of the optimal blended EO water with two
commercial one-step CIP chemicals, the RLU reduction percentage results showed that the optimal blended
EO water was doing as good as the two commercial one-step CIP chemicals; for example, 100% on average
when using the optimal blended EO water compared to 100% on average when using one of the commercial
chemicals for sampling locations of elbows. However, the blended EO water did result in a higher negative
enrichment percentage on average compared to the commercial one-step CIP chemicals, indicating the
blended EO water at its optimal condition possessed more disinfecting capability; for example, 83.3% on
average when using the optimal blended EO water compared to 50.0% on average when using one of the
commercial products for sampling locations of pipes. Based on this research, it is concluded that the blended
EO water at its optimal condition has the potential to be adapted as an alternative for one-step milking system
CIP.

Acknowledgement
Funding for this project was provided in-part by a USDA Special Research Grant (No. 2010-34163-21179) and
the Pennsylvania Agricultural Experiment Station. We are also thankful to Hoshizaki Electric Co. Ltd. (Sakae,
Toyoake, Aichi, Japan) for the technical support for the EO water generator used in this study. We also would
like to acknowledge Roderick Thomas, Randall Bock, and all Penn State dairy barn personnel for their help in
the project.

2014 ASABE – CSBE/SCGAB Annual International Meeting Paper Page 13

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