The Pennsylvania State University
The Graduate School
College of Agricultural Sciences
EVALUATION OF ELECTROLYZED WATER FOR CLEAN-IN-PLACE OF
DAIRY PROCESSING EQUIPMENT
A Thesis in
Food Science
by
Yun Yu
2014 Yun Yu
Submitted in Partial Fulfillment
of the Requirements
for the Degree of
Master of Science
May 2014
ii
The thesis of Yun Yu was reviewed and approved* by the following:
Robert F. Roberts
Professor of Food Science
Head of the Department of Food Science
Thesis Advisor
Catherine N. Cutter
Professor of Food Science
Gregory R. Ziegler
Professor of Food Science
Ali Demirci
Professor of Agricultural and Biological Engineering
*Signatures are on file in the Graduate School
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ABSTRACT
Good cleaning and sanitation practices in the dairy industry are essential to
maintaining public health and increasing profitability for producers. Dairy processing
equipment is commonly cleaned using a four-step clean-in-place (CIP) procedure: rinse
with water, wash with an alkaline solution, rinse with water again to remove alkaline
residue, and then rinse with an acid sanitizer. The chemicals used in CIP procedures of
dairy processing equipment are usually handled and stored in concentrated forms, and
may have adverse effects on the environment, and can cause skin or eye burns on contact.
Electrolyzed water is produced via electrolysis of a dilute sodium chloride solution,
which results in a sodium hydroxide solution, called electrolyzed reducing (ER) water
(pH ca. 11.0 and ORP ca. -850 mV), and an acidic solution, called electrolyzed oxidizing
(EO) water (pH ca. 2.5, ORP ca. 1168 mV and 80-100 ppm of chlorine). Thus,
electrolyzed water has the potential to serve as an alternative to CIP chemicals. The
antimicrobial efficacy of acid EO water has been demonstrated. The efficacy of
electrolyzed water used as cleaning and disinfecting agent for on-farm milking systems
also has been demonstrated. The use of acid EO water in CIP applications for dairy
processing systems, especially those involving heat treatment of milk has not been
evaluated.
The purpose of this research was to investigate the efficacy of electrolyzed water
for CIP procedure of dairy processing equipment, specifically a refrigerated milk storage
tank and a tank used for thermal processing of milk. In this work, a pilot scale test
system composed of a 15 liter (4 gallon) stainless steel test vessel was constructed and
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characterized to allow evaluation and optimization of electrolyzed water as CIP agent for
dairy processing equipment. Use of the test system was validated by pilot trials of CIP
cleaning with conventional CIP detergent and sanitizer. Using the same CIP procedure,
electrolyzed water was successfully employed to clean the test vessel after soiling with
milk at refrigerated temperatures (2-4°C). The effectiveness of cleaning was assessed
using a microbiological enrichment method, as well as ATP bioluminescence and
residual protein detection assays. Finally, use electrolyzed water for CIP procedures of
the test vessel soiled by heating milk was evaluated using a response surface model to
optimize temperatures and times for both alkaline ER water and acid EO water
treatments. Parameters for 4-step CIP procedures using electrolyzed water were: wash
with alkaline ER water at 54.6°C for 20.5 min and sanitize with acid EO water at 25°C
for 10 min. The validation study demonstrated that a complete CIP procedure using
electrolyzed water with optimal operational temperatures and time was capable of
returning the surface of the test vessel to a satisfactory clean condition with non-
detectable residual ATP and protein. The study demonstrated the cleaning efficacy and
potential application of using electrolyzed water for CIP procedures in dairy plant. In
contrast to conventional CIP chemicals that are usually prepared by diluting of
concentrated chemicals, electrolyzed water has the advantage of on-site generation, dairy
processing plants, especially small dairy foods manufactures, could benefit by reducing
the risk and cost of storing and handling of concentrated CIP chemicals.
v
TABLE OF CONTENTS
List of Figures ............................................................................................................. viii
List of Tables ............................................................................................................... x
List of Abbreviations ................................................................................................... xi
Acknowledgements ...................................................................................................... xiii
LITERATURE REVIEW ............................................................................ 1 Chapter 1
1.1 MICROORGANISMS IN MILK ................................................................... 2 1.1.1 Pseudomonas fluorescens ..................................................................... 3 1.1.2 Escherichia coli .................................................................................... 3
1.1.3 Enterococcus faecalis ........................................................................... 4 1.2 TANKS USED IN DAIRY PLANT ............................................................... 4
1.2.1 Raw milk silo ........................................................................................ 4
1.2.2 Processing tanks ................................................................................... 5 1.2.3 Fouling in milk process equipment ...................................................... 6
1.3 CLEANING AND SANITATION IN THE DAIRY INDUSTRY ................ 7 1.3.1 Importance of cleaning and sanitation .................................................. 7 1.3.2 Clean-out-of-place (COP) .................................................................... 8
1.3.3 Clean-in-place (CIP) ............................................................................. 9
1.3.4 Key factors influencing cleaning efficiency ......................................... 11 1.4 ASSESSMENT OF CLEANLINESS ............................................................. 17
1.4.1 Visual inspection .................................................................................. 17
1.4.2 Microbiological analysis ...................................................................... 17 1.4.3 ATP bioluminescence method .............................................................. 18
1.4.4 Protein residue detection ...................................................................... 21 1.5 ELECTROLYZED WATER .......................................................................... 22
1.5.1 History .................................................................................................. 22 1.5.2 Generation and Properties .................................................................... 22
1.5.3 Advantages and disadvantages of electrolyzed water: ......................... 24 1.5.4 Application of electrolyzed water in food industry .............................. 26
HYPOTHESIS AND OBJECTIVES .......................................................... 34 Chapter 2
CONSTRUCTION, CHARACTERIZATION AND VALIDATION OF Chapter 3
TEST SYSTEM .................................................................................................... 35
ABSTRACT ......................................................................................................... 35 3.1 INTRODUCTION .......................................................................................... 36
3.2 MATERIALS AND METHODS ................................................................... 38 3.2.1 Construction of test system .................................................................. 38 3.2.2 Characterization of the test system ....................................................... 41
vi
3.2.3 Performance validation of the test system used for CIP ....................... 42 3.2.4 Test for swab sampling variability ....................................................... 46
3.2.5 Statistical analysis ................................................................................ 47 3.3 RESULTS AND DISCUSSION ..................................................................... 49
3.3.1 Characterization of test system ............................................................. 49 3.3.2 Performance validation of the test system used for CIP ....................... 52 3.3.3 Test for swab sampling variability ....................................................... 54
3.4 CONCLUSION............................................................................................... 56
CIP USING ELECTROLYZED WATER FOR A REFRIGERATED Chapter 4
MILK STORAGE TANK ..................................................................................... 58
ABSTRACT ......................................................................................................... 58 4.1 INTRODUCTION .......................................................................................... 59 4.2 MATERIALS AND METHODS ................................................................... 62
4.2.1 Bacterial cultures verification and characterization ............................. 62 4.2.2 Preparation of inoculated milk ............................................................. 64
4.2.3 Generation and characterization of electrolyzed water ........................ 65 4.2.4 Preparation of commercial CIP chemicals ........................................... 66 4.2.5 Preparation of test system ..................................................................... 67
4.2.6 Soiling the system ................................................................................. 67 4.2.7 CIP procedure of electrolyzed water treatment .................................... 68
4.2.8 CIP control treatments .......................................................................... 69 4.2.9 Assessments of cleanliness and data collection .................................... 70
4.2.10 Statistical design and analysis ............................................................ 72 4.3 RESULTS AND DISCUSSION ..................................................................... 72
4.3.1 Inoculum bacterial cultures verification and characterization .............. 72 4.3.2 Chemical properties of electrolyzed water ........................................... 73 4.3.3 Cleanliness assessments ....................................................................... 74
4.3.4 CONCLUSION .................................................................................... 79
CIP USING ELECTROLYZED WATER FOR A HEATED MILK Chapter 5
PROCESSING TANK .......................................................................................... 80
ABSTRACT ......................................................................................................... 80
5.1 INTRODUCTION .......................................................................................... 81 5.2 MATERIALS AND METHODS ................................................................... 83
5.2.1 Preparation of electrolyzed water ......................................................... 83 5.2.2 Experimental design -- response surface model ................................... 84 5.2.3 Soiling and CIP treatments ................................................................... 86 5.2.4 Cleanliness assessments ....................................................................... 87 5.2.5 Other potential factors (Nuisance factor) ............................................. 89
5.2.6 Data analyses and modeling ................................................................. 92 5.2.7 Validation ............................................................................................. 96
5.3 RESULTS AND DISCUSSION ..................................................................... 96
vii
5.3.1 Assessment the importance of nuisance factors ................................... 96 5.3.2 Regression model of RLU3 data after wash and post-rinse ................. 98
5.3.3 Regression model of RLU4 data after sanitizing ................................. 102 5.3.4 Regression model of Protein data ......................................................... 104 5.3.5 Validation ............................................................................................. 106
5.4 CONCLUSION............................................................................................... 109
CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE Chapter 6
RESEARCH ......................................................................................................... 111
REFERENCES: ........................................................................................................... 112
APPENDIX: ................................................................................................................. 120
viii
LIST OF FIGURES
Figure 1-1. Schematic of electrolyzed water generator and produced compounds
(Huang et al., 2008). ............................................................................................. 24
Figure 3-1. Schematic of pilot scale dairy processing test system. ............................. 38
Figure 3-2. Test system. (A) Test vessel (center of the system) with agitator in
place. (B) A modified stainless steel milk can served as reservoir for water
used for CIP cleaning. (C) CIP solution reservoir with coil heat exchanger
connected to a circulating water bath. (D) 360° static spray ball. ....................... 40
Figure 3-3. Flow chart of CIP performance evaluation of test system. CIP
cleaning was conducted at flow rate of 8.3 L/min. ............................................... 43
Figure 3-4. Schematic of test for swab sampling variability. Each rectangle area
represents 50 cm2 (5 cm × 10 cm) inner surface of test vessel. Different
colors represent the sampling for ATP bioluminescence assays after different
steps. Red marked areas were swabbed “after soiling”, green areas were
swabbed “after pre-rinse”, and blue areas were swabed “after sanitizing”. For
one trial, each assessments of ATP bioluminescence assays was conducted in
three replicates. ..................................................................................................... 48
Figure 3-5. Mean volumetric flow rates (L/min) at different VFD pump settings.
The regression equation is: Flow Rate (L/min) = 0.1844 + 0.1918 Pump
Setting. .................................................................................................................. 49
Figure 3-6. Riboflavin coverage test. (A) Riboflavin solution sprayed on test
vessel before rinse. (B) Residual riboflavin remaining after rinsing test
vessel with water at flow rate of 1.8 L/min (pump setting of 10) for 1 min.
(C) Residual riboflavin remaining after rinsing test vessel with water at flow
rate of 3.9 L/min (pump setting of 20) for 7 min. (D) Residual riboflavin
remaining after rinsing test vessel with water at flow rate of 6.0 L/min (pump
setting of 30) for 17 min. (E) Residual riboflavin remaining after rinsing test
vessel with water at flow rate of 8.3 L/min (pump setting of 40) for 1 min. ........ 51
Figure 3-7. Log10 RLU values of after soiling, after pre-rinse, and after sanitizing.
Error bars represent standard deviation of the evaluations of three locations.
Tukey’s comparison was conducted between 9 sets of data. Means that do
not share a letter are significantly different (α = 0.05). ........................................ 55
Figure 4-1. Electrolyzed water generator used in this research.(Model ROX20,
Hoshizaki America Inc.) ....................................................................................... 65
Figure 4-2. Flow chart of CIP procedures of cold milk storage tank using
electrolyzed water. CIP procedures were conducted at flow rate of 8.3 L/min. .. 71
ix
Figure 4-3. Mean populations of cultures during overnight incubation. Error bars
represent standard deviation of the log10 CFU/ml of three replicates.
Tukey’s comparisons were conducted between three sets of data at each
sampling point. Means that do not share a letter are significantly different (α
= 0.05). .................................................................................................................. 73
Figure 5-1. Flow chart for CIP procedure of hot milk processing tank using
electrolyzed water. ................................................................................................ 87
Figure 5-2. Contour plot of versus treatment time and
temperature of ER water wash, generated based on regression model
(Equation 6). ......................................................................................................... 99
Figure 5-3. Surface plot of versus treatment time and
temperature of ER water wash, generated based on regression model
(Equation 6). ......................................................................................................... 100
Figure 5-4. Optimization plot for ln((RLU2-RLU3)/RLU2) versus treatment time
and temperature of ER water wash. The plot suggested the highest
desirability was 0.98, when setting ER water wash temperature at 54.6°C and
time at 20.5 min, and the predicted ln((RLU2-RLU3)/RLU2) = -0.0092,
indicating 99.08% of RLU reduction. ................................................................... 101
Figure 5-5. Optimization plot for “RLU2-RLU4” and “ln((RLU2-RLU3)/RLU2)”.
When setting ER water wash parameters at 53.7°C for 21.6min, setting EO
water sanitizing parameters at 25°C for 10 min, it can be predicted that RLU
reduction achieved 99.08% after ER water treatment, 1.99 × 106 after EO
water treatment, respectively. ............................................................................... 104
Figure 5-6. Means of RLU values comparison between treatments. Error bar
indicates standard deviation of triplicate analysis. Tukey’s comparisons were
conducted between all RLU values. Means that do not share a letter are
significantly different (α = 0.05). (Pos. Ctrl = positive control; EW validation
= electrolyzed water treatment with optimal parameters; Neg. Ctrl = negative
control). ................................................................................................................. 108
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LIST OF TABLES
Table 3-1. Cleanliness assessments made using of ATP and protein measurements
after different steps of standard CIP procedure using commercial chemicals
(*RLU values of zero were assigned as 0.5 for base-10 logarithm
calculation). .......................................................................................................... 53
Table 4-1. Operation temperature and time of four treatments. .................................. 69
Table 4-2. Chemical properties of electrolyzed water of each trial. P-values were
obtained from Tukey’s comparison between treatments. ..................................... 74
Table 4-3. Microbiological data of inoculated milk after soiling and of swab
enrichment test. ..................................................................................................... 75
Table 4-4. Protein residue levels. ................................................................................. 78
Table 5-1. Levels of four independent variables for CIP using electrolyzed water. ... 84
Table 5-2. Box-Behnken response surface design with 27 trails, including 3 center
points (in bold). WashTemp = temperature for ER water wash; WashTime =
time for ER water wash; SaniTemp = Temperature for EO water sanitizing;
SaniTime = Time for EO water sanitizing. ........................................................... 85
Table 5-3. Summary of cleanliness assessments at different sampling points. ........... 88
Table 5-4. P-values of Pearson correlations between variables. Small p-values (<
0.05) indicated the corresponding pair of variables may be related. .................... 97
Table 5-5. Response surface regression: “ln((RLU2-RLU3)/RLU2)” as a function
of ER water treatment temperature and time (WashTemp = temperature of
ER water treatment; WashTime = treatment time of ER water wash). ................ 99
Table 5-6. Regression of “RLU2-RLU4” versus ER water and EO water treatment
factors (WashTemp = temperature of ER water treatment; WashTime =
treatment time of ER water wash; SaniTemp = temperature of EO water
treatment; SaniTime = treatment time of EO water sanitizing). ........................... 103
Table 5-7. Protein detection of validation experiment and control treatments
(where Pos. Ctrl = positive control; EW validation = electrolyzed water
treatment with optimal parameters; Neg. Ctrl = negative control). ...................... 108
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LIST OF ABBREVIATIONS
⁰C degree Celsius
ANOVA analysis of variance
APC aerobic plate count
ATP adenosine triphosphate
BCA bicinchoninic acid
BLAST basic local alignment search tool
CFU colony forming unit
CIP clean in place
COP clean out of place
CPC calcium phosphate-citrate
DNA deoxyribonucleic acid
EO water electrolyzed oxidizing water
ER water electrolyzed reducing water
EW electrolyzed water
GMP good manufacturing practice
HACCP hazard analysis critical control point
HTST high-temperature short-time
ml milliliter
mM millimolar
ORP oxidation reduction potential
PI pre-incubation
PMO pasteurized milk ordinance
ppm parts per million
RLU relative light unit
rRNA ribosomal ribonucleic acid
SPC standard plate count
spp. species
xii
St. Dev. standard deviation
TSA trypticase soy agar
TSB trypticase soy broth
UV ultraviolet
VFD variable-frequency drive
μl microliter
μM micromolar
xiii
ACKNOWLEDGEMENTS
This work was carried out at the Department of Food Science, the Pennsylvania
State University, and funded by a USDA milk safety grant. I would like to express my
appreciation to the department and funding agency for giving me the opportunity and
support needed to pursue a Master of Science degree. This thesis could not have been
completed without the help and support of many people. I would like to take this time to
thank those people.
I owe my deepest gratitude to my advisor Dr. Robert Roberts for giving me the
opportunity to study Food Science under his guidance. Dr. Roberts is a great mentor,
who is always encouraging, supportive, and patient. It was a wonderful learning
experience working with Dr. Roberts. The knowledge, skills, and confidence that I
gained in Dr. Roberts’ lab will be invaluable in my career development. I look forward
to continuing to pursue a Ph.D. degree under Dr. Roberts’ guidance.
I would like to express my appreciation to my committee members Dr. Gregory
Ziegler, Dr. Catherine Cutter, and Dr. Ali Demirci. I would like to thank Dr. Ziegler for
the suggestions on experimental design and statistical analyses. His support helped me
broaden my knowledge in engineering and statistics. I would like to thank Dr. Cutter for
her knowledge and experiences, and for caring about this project. I would also like to
thank Dr. Demirci for bringing up the project, and for his enthusiasm, experiences and
support of this project.
I would like to thank my lab mates, Dr. Emily Furumoto, Dr. Joe Loquasto,
Zhaoyong Ba, and Sarah Brown for the help, suggestions, and support. I would
xiv
especially thank Dr. Emily Furumoto for helping with ordering suppliers for the project.
I also would like to thank undergraduate helpers, especially Jeff Amos, for the help with
my experiments.
I would like to thank faculty, staff and my fellow graduate students. Without their
help, encouragement, and friendship, I would not have been able to complete the graduate
courses and my research, and stay cheered during graduate school.
I would like to thank staff in Berkey Creamery, especially thank to Tom Palchak
for his support and caring, to David Long for helping me with modifying the pilot scale
test system, to Bonnie Ford, Bill Kurtz, and Bob Rosenberry for teaching me the
procedures and tests of cleaning. I also would like to thank Mark Ivkovich from the
Ecolab Inc. and Vivian Saunders from the Charm Inc. for their technique support.
Lastly, I would like to thank my husband Kan Shen, my friends in State College
and my Family in China, for their love and support during the graduate school.
1
Chapter 1
LITERATURE REVIEW
America’s dairy industry is a key economic component of agricultural business
and an important contributor to the nation’s overall economy. This industry includes
small and large dairy farms, dairy foods processors and those corporations involved with
marketing, selling and transporting dairy products. Dairy products are produced in all 50
states and more than 130,000 jobs are created by the dairy industry, including those on
dairy farms, in dairy processing, marketing and transportation, retail stores and other
companies related to the dairy industry (USDA, 2004). Consumers in the United States
spend over 11% of their food expenses on dairy products (Markets, 2006). In
Pennsylvania, according to the Center for Dairy Excellence, the dairy industry generates
more cash receipts than any other agricultural industry in the state. Approximately 40%
of the $3.8 billion in agricultural cash receipts comes from the dairy industry. In
addition, the dairy industry is also a vital economic stimulus to the state and supports
about 40,000 job opportunities (Wolff, 2010).
Dairy products are an ideal growth medium for many microorganisms. The
Centers for Disease Control (CDC) reported 29 outbreaks associated with pasteurized
dairy products between 1998 and 2012 in U.S. (CDC, 2013). Thus, good practices of
cleaning and sanitizing in the dairy industry are essential to decreasing the risk of
outbreaks for public health and increasing profitability for producers.
2
1.1 MICROORGANISMS IN MILK
Milk is a highly perishable food that supports the growth of microorganisms,
which can lead to chemical changes during storage. The nutrients contained in milk, such
as lactose, milk fat, whey proteins, casein, other trace elements and vitamins are
favorable for microbial growth. In addition, the neutral pH value (pH = 6.8) and high
water activity (aw = 0.97 to 1.00) of milk also favor bacterial multiplication (Mostert and
Buys, 2008). Furthermore, some enzymes, produced by bacteria or which originate in the
cow’s udder, can cause chemical degradation of milk components (proteolysis, lipolysis,
etc.) (Bylund, 2003; Robinson, 2002).
Although milk is considered sterile when secreted from healthy cows, it can be
contaminated by microorganisms during the milking process from a variety of different
sources. One of the major sources of bacterial contamination is insufficiently cleaned
and sanitized processing equipment (Robinson, 2002). Microorganisms can also come
from water supplies on the dairy farm, from soil or vegetation near the dairy farm (Rice
and Johnson, 2000), from the exterior of teats or udders, and from the udder canal, in the
case of mastitis (Bylund, 2003; Robinson, 2002).
The numbers and types of microorganisms present in milk differ between farms.
In the United States, the maximum allowable standard plate count (SPC) for Grade A raw
milk is < 100,000 CFU/ml before commingling, and < 300,000 CFU/ml for commingled
milk prior to pasteurization (USDHHS, PHS, & FDA, 2011). Typical groups of
microorganisms present in raw milk include psychrotrophic, mesophilic aerobic, and
thermoduric bacteria (Robinson, 2002).
3
1.1.1 Pseudomonas fluorescens
In milk, psychrotrophic microorganisms, which can multiply at refrigerated
temperatures (< 7°C), are often Gram-negative rods. Pseudomonas fluorescens is the
most common psychrotroph occurring in raw milk, and also occurs typically in processed
dairy products as a result of post-pasteurization contamination (Robinson, 2002; Dogan
and Boor, 2003). Contamination with Pseudomonas spp. is a major contributor to shelf-
life reduction. Pseudomonas spp. are known to produce enzymes, such as lipases and
proteases, at refrigerated temperatures, which are heat stable and remain active after
thermal processing. Such enzymes reduce the shelf life of dairy products due to
degradation of milk compounds, leading to off-flavors and odors (Sørhaug and Stepaniak,
1997; Dogan and Boor, 2003).
1.1.2 Escherichia coli
Coliforms are another group of Gram-negative, rod-shaped bacteria often present
in raw milk. Escherichia coli is one specie contained in this group. Although E. coli is
recognized as a fecal-associated microorganism, the presence of E. coli does not provide
direct evidence of fecal contamination (WHO, 1993). Rather, the presence of E. coli
suggests inadequate cleaning of udders prior to milking or unsatisfactory sanitizing
practices. Although most E. coli are non-pathogenic isolates, some pathogenic strains of
E. coli, such as E. coli O157:H7 can cause severe disease in humans.
4
1.1.3 Enterococcus faecalis
Enterococcus spp. including Enterococcus faecalis are a genus of mesophilic
streptococci, which are Gram-positive, non-spore-forming, lactic acid bacteria (Robinson,
2002). E. faecalis is frequently isolated from dairy food products (Gaspar et al., 2009).
Enterococcus spp. is a controversial genus. Some species of Enterococcus spp. are used
as starter cultures or occur as non-starter cultures in food fermentations, such as during
the production of artisan cheeses in Europe. Specific strains of E. faecalis and E. faecium
have been shown to produce bacteriocins that inhibit spoilage or pathogenic bacteria, and
some are known to develop positive sensory characteristics (Giraffa, 2003). However, it
has been shown that some isolates of E. faecalis are potentially pathogenic to humans
(Giraffa et al., 1997).
1.2 TANKS USED IN DAIRY PLANT
A variety of tanks are used in dairy processing facilities. Two major categories
are refrigerated storage tanks (silos) and tanks used to thermally process milk.
1.2.1 Raw milk silo
Since milk is a good growth medium for microorganisms, development of
refrigerated storage was important for maintaining high quality and extending shelf-life
of milk (Robinson, 2002). The milk silo often serves as a storage vessel for received raw
milk, which is held at refrigerated temperature in dairy processing plants. Capacities of
5
raw milk silos range from 3,000 gallons to 70,000 gallons; many processing facilities
have multiple silos, depending upon the capacity of manufacture and the schedule of raw
milk delivery (Bylund, 2003). Most milk silos are located outdoors with double-walled
construction for better insulation. The inside walls usually are made of stainless steel
(grade ss304 or ss316) to reduce fouling and enhance cleanability (Mostert and Buys,
2008). Milk silos are usually equipped with temperature and level indicators. Agitators
are installed near the bottom of silos. After emptying a silo, a thin film of milk may be
adhered to the wall. As such, cleaning should be applied as soon as possible after
emptying because it is more difficult to remove milk residue when it has dried onto the
surface (Bylund, 2003). Because of the size of silos, clean-in-place (CIP) methods
usually are applied.
1.2.2 Processing tanks
Processing tanks, such as yogurt fermentation tanks, tanks for starter culture
preparation, and batch pasteurizers, are used commonly in the dairy industry. Processing
tanks are also made of stainless steel. Although the properties and configurations of
processing tanks vary, most processing tanks are equipped with agitators and temperature
controling and monitoring systems. Heat treatment of milk results in more difficult soil
removal (Bylund, 2003). When milk is heated to 60°C or above, milk proteins,
especially whey proteins, begin to denature. The denatured proteins may bind to the
equipment surface and form aggregates that are difficult to remove (Robinson, 2002).
Milk salts, such as calcium, magnesium, sodium, potassium etc., are equilibrated between
6
a soluble and colloidal phase (Walstra et al., 2006a). When milk is heated above 60°C,
milk salts become less soluble and transform into a colloidal phase. In other words, the
proportion of insoluble calcium phosphate increases (Marriott and Gravani, 2006). In
addition, by combining with denatured proteins, “milk stone” can deposit quickly on
heated surfaces (Walstra et al., 2006a; Marriott and Gravani, 2006).
1.2.3 Fouling in milk process equipment
Soiling in dairy plants occurs when undesired materials deposit on equipment
surfaces, including liquid milk or other dairy product residues,and other foreign matters
such as lubricants or water scale. Dairy soil is a complex material consisting of inorganic
(minerals) and organic compounds (lactose, proteins, and fat). Some compounds, such as
lactose and sodium, are soluble in water and are easy to remove using a water rinse.
Other compounds, including proteins, fat and some mineral salts are less soluble in water.
To remove this type of soil, alkaline detergents or surface-active agents are generally
required (Mostert and Buys, 2008; Bylund, 2003).
The composition of soil complexes deposited on different dairy processing
equipment depends upon product compositions and specific operational conditions.
Stanga (2010) summarized studies that were published by Harper (1972) and Belitz
(1987), and compared the composition of cold milk fouling and hot milk fouling. The
average composition of cold milk soil was 26.6% protein, 38.1% sugar, 29.95% fat and
5.3% minerals; the average composition of hot milk soil was 30.3% protein, trace level of
sugar, 23.1% fat and 46.6% mineral (Stanga, 2010). On cold surfaces, such as raw milk
7
pipelines or raw milk silos, fouling was light and less viscous, and could be cleaned
easily with water (Stanga, 2010). On heated surfaces, the mechanisms of fouling were
different, when compared with cold surfaces. Researchers proposed three fouling
mechanisms that could occur on dairy processing equipment surfaces associated with
heat treatment, including reaction fouling (e.g. whey protein denaturation); crystallization
or precipitation fouling (e.g. the formation of milk stone from proteins and calcium
phosphate); and biological fouling (e.g. attachment of a single microorganism to growth
of biofilms) (Fryer et al., 2011; Fryer and Asteriadou, 2009).
1.3 CLEANING AND SANITATION IN THE DAIRY INDUSTRY
1.3.1 Importance of cleaning and sanitation
Good practices of cleaning and sanitation are important in food manufacturing.
When soil is deposited on the surface of equipment, the performance of processing, and
efficiency of heat exchangers, is reduced. In addition, food residue deposits favor
microbial growth and may lead to food quality and safety issues. Furthermore, cross
contamination between batches can affect the quality of food, or result in contamination
with food allergens. Fryer and Asteriadou (2009) proposed a five-stage model of fouling:
initiation, transportation, attachment, removal, and aging. They indicated that drying or
hardening of the food deposit (aging stage) changes the nature of the deposit and
increases difficulty in cleaning (Fryer and Asteriadou, 2009). Thus, once processing has
8
been completed, cleaning should be begun as soon as possible (Bylund, 2003). There are
two main strategies for cleaning: clean-out-of place (COP) and clean-in-place (CIP).
1.3.2 Clean-out-of-place (COP)
There are some pieces of dairy processing equipment (e.g. parts of separators,
homogenizers, and fillers) need to be cleaned manually using clean-out-of-place (COP)
technology (Mostert and Buys, 2008). Another situation, when heavy deposits have
occurred, COP operation, such as manual scrubbing, may also be required (Mostert and
Buys, 2008).
The procedure for COP is described as follows: a) disconnect or dismantle parts;
b) pre-rinse dismantled parts with warm water (20-40°C) to remove loose fouling; c)
prepare detergent solution with correct concentration (alkaline chlorinated cleansers are
usually used); d) wash pre-rinsed parts with warm detergent (40-60°C) and by manually
brushing or by submerging in COP tanks, in which alkaline cleaning solution is circulated
(i.e. dishwasher); e) post-rinse with water to remove detergent residue; and f) sanitize
parts with hot water or chemical sanitizer (e.g. chlorine sanitizer) by spraying or dipping
(Tamime and Robinson, 1999; Mostert and Buys, 2008; Marriott and Gravani, 2006).
Some limitations must be considered when comparing COP with CIP. Compared
with CIP operations, COP cleaning is more laborious. With manual operations, only
moderate temperatures and mild cleaning solutions can be applied to avoid skin irritation
or burning of employees (Marriott and Gravani, 2006). To assure efficacy and
appropriate methods for COP, well developed standard operating procedures (SOPs) and
9
training, as well as implementation of good manufacturing practices (GMP) are essential
to efficient COP operations (Mostert and Buys, 2008).
1.3.3 Clean-in-place (CIP)
Clean-in-place (CIP) was first developed in the 1960s and is employed frequently
in the dairy industry (Tamime and Robinson, 1999). CIP is defined as cleaning and
sanitizing the entire pipe line, vessel, or other food processing system without
dismantling or opening; by circulating or spraying water or cleaning solutions throughout
the system (Tamime, 2008).
Although the specific CIP program will vary, depending upon the type of
equipment and the nature of the soil, the basic procedures are similar and can be
summarized as follows (Bylund, 2003; Mostert and Buys, 2008; Walstra et al., 2006b):
Recovery of product residues: During this step, the product is drained from the
equipment to facilitate cleaning. To minimize product losses and reduce the load of
sewage, some plants use high quality water to “chase” product out of the equipment.
Pre-rinse with water: After draining the equipment, the next step is to rinse with
water. The purpose of the pre-rinse step is to remove bulk fouling and to loosen any soil
attached to equipment surfaces. Softened water is preferred for the pre-rinsing step to
prevent formation of water scale. A cold pre-rinse is sufficient for removal of light
fouling, such as that deposited in cold milk tanks. A warm temperature is recommended
for removal of thick and fatty fouling such as cream, yogurt or ice cream mix, since a
temperature of at least 32.2°C is needed to melt the fat (Lloyd, 2008). However, it is
10
recommended the temperature not surpass 55°C to avoid denaturation of proteins, which
will make cleaning more difficult. The pre-rinsing step should be conducted until the
water runs clear. An efficient pre-rinsing should remove 90~99% of bulk soil (Bylund,
2003).
Detergent wash cycles and intermediate rinsing with water: Following the pre-
rising step, a wash cycle is applied. This step usually involves circulating alkaline
detergent for a period of time. The alkaline detergent removes most of the attached soil,
including proteins and milk fat. In some systems, after an intermediate rinse with water
to remove all traces of alkaline cleanser, another wash step, employing an acid detergent
is performed. This acid wash step is optional and usually applied once a week for
removal of mineral based soil (e.g. milk stone) from equipment, such as heat exchangers
and other thermal processing equipment.
Sanitizing or disinfection: After post-rinsing with water to remove residual
detergent, the sanitizing step is conducted. The process of sanitizing reduces microbial
contamination to levels considered safe from a public health perspective, by destroying
vegetative cells. There were two methods used for sanitation or disinfection: thermal
disinfection and chemical disinfection. Thermal treatments, such as running hot water
(~85°C) or steam within systems, are applied normally for disinfection of high-
temperature short-time (HTST) pasteurizers. A minimum exposure of equipment surface
to a temperature at 77°C for 5 minutes is required for hot water sanitizing
(USDHS/PHS/FDA, 2011). Chemical sanitizers, such as chlorine, iodophors, quaternary
ammonium compounds and peroxy acid compounds are also commonly used in dairy
plants (Boufford, 2003). Sanitizing could be applied immediately before processing, or
11
at the end of CIP cycles. If a processing system is shut down for more than 4 hours
between shifts, re-sanitizing before manufacturing is recommended (Mauck et al., 2001).
1.3.4 Key factors influencing cleaning efficiency
Sinner (1960) proposed four factors that influence cleaning efficiency: operating
temperature, action or mechanical force, chemistry of detergent and sanitizer, and contact
time (TACT) (Packman et al., 2008). Those four factors interact and must be considered
together. Each of the four factors is described in following sections.
1.3.4.1 Temperature
During cleaning, suitable operating temperatures must be high enough to melt
milk fat to ease removal, but low enough to prevent protein denaturation, which would
make cleaning more difficult (Tamime and Robinson, 1999). In general, the
effectiveness of cleaning is improved as the temperature increases. However, there are
limitations to maximum operating temperatures. COP cleaning or manually cleaning is
usually carried out at less than 60°C for safety reasons (to avoid human injury). When
using enzyme-based detergents, temperature is usually limited to less than 55°C to assure
the enzyme remains active. For heavily fouled surfaces, such as yogurt processing
equipment, CIP procedures might be conducted at higher temperature (85-90°C). When
using chlorinated sanitizer, the operating temperature for sanitizing should be less than
40°C to avoid chlorine volatilization (Tamime and Robinson, 1999).
12
1.3.4.2 Action (mechanical force)
Mechanical forces in CIP procedures include flow rate and flow pressure, which
provide the energy needed for lifting fouling from surface.
For cleaning pipe lines, a sufficient flow rate is essential to provide turbulent flow
for better efficiency of soil removal. Centers for Disease Control and Prevention (CDC)
recommend 1.5 m/s (5 feet/sec) as the minimum flow rate. This means 75 L/min (20
gallon/min) for a 3.8 cm-diameter (1.5 inch) pipe, and 568 L/min (150 gallon/min) for a
10 cm-diameter (4 inch) pipe (CDC, 2012). Bylund (2003) also indicates that flow
velocities of 1.5~3.0 m/s in the pipes provide a turbulent flow.
For CIP cleaning of dairy tanks, simple distribution devices are often applied.
Static spray balls are used to provide a high flow rate and low pressure cleaning liquid;
while rotating devices, such as rotating spray heads and rotating jet heads, enhance the
impact of cleaning liquid by providing higher pressure (Moerman, 2005). In general,
when cleaning a tank, liquid is sprayed on the upper part of the tank, and then falls down
the tank wall in the form of falling film (Bylund, 2003). Coverage is another important
parameter when considering mechanical force. Coverage is classified as direct coverage
or indirect coverage. Direct coverage occurs when the cleaning liquid contacts the
surface to be cleaned directly from the spray device. Indirect coverage results from either
a splash-back effect or falling film effect on tank wall. When cleaning larger tanks,
multiple spray devices might be installed in order to achieve full coverage.
A method used to assess completeness of CIP coverage, the riboflavin-
fluorescence test is commonly used in the dairy and pharmaceutical industries (Tamime,
13
2008). Riboflavin is a water soluble dye that presents bright yellow/green fluorescence
under UV/black light with wave length of 365 nm. To perform this test, a riboflavin
water solution (200 ppm) is sprayed on the surface to be inspected. After delivering
water through a spray device under the flow rate of the CIP cycle for a certain period of
time, the riboflavin residue is inspected with a black light. Any surface that is not
sufficiently rinsed presents fluorescence.
1.3.4.3 Chemicals used in cleaning and sanitizing
In addition to water, which is the solvent and makes up the bulk of the cleaning
compounds, various detergents and sanitizers are employed.
1.3.4.3.1 Alkaline detergents
Sodium hydroxide (NaOH, caustic soda), is typically used in alkaline detergent.
Sodium hydroxide saponifies fat, and converts fat to soap (fatty acid salt), which helps
remove other organic contamination, such as proteins. This is called saponification. In
addition to saponification, alkaline cleansers provide negative ions, which disrupt the
structure, swell, break the soil, and disperse small soil particles into the cleaning solution
due to electrostatic repulsion. Other alkali compounds, including potassium hydroxide
(caustic potash), sodium carbonate (soda ash), sodium silicates, and trisodium phosphate
(TSP), also can serve as ingredients in alkaline detergent (Mauck et al., 2001).
In addition to alkali compounds, surfactants are often employed in alkaline
detergents, and serve as wetting, emulsification and suspension functions, to promote
removal of deposits. Wetting agents function by reducing surface tension and promote
14
penetration of detergent into the soil (Bylund, 2003). Anionic surfactants, such as teepol
(alkyl aryl sulphonate), are high foamers and usually are used as a wetting agent. Other
anionic surfactants, such as sodium triphosphate and complex phosphate compounds,
promote emulsification by absorbing at the oil-water interface, to promote removal of soil
from the surface and maintain soils suspended in cleaning solutions, without re-
deposition or flocculation.
Unlike anionic surfactants, nonionic surfactants are added usually into CIP
cleansers as defoamers, which are usually used at higher temperatures (>40°C). To
prevent precipitation of hard water scale, sequestrants, such as phosphates and EDTA, are
often added to detergent formulations. Sequestrants also help in removal of fouling that
may complex with metallic ions (calcium and magnesium) (Watkinson, 2008).
1.3.4.3.2 Acid detergents
Acid detergents are used for removal of mineral deposits, and to prevent milk
stone formation on dairy processing equipment. Commonly used acids include
phosphoric, nitric, sulfamic and hydrochloric. Sometimes, organic acids, such as
hydroxyacetic acid, citric acid and gluconic acid are also used. An acid rinse is usually
applied once a week, and an “override” cleaning strategy may be employed. In this
strategy, the acid rinse is applied first and then the alkaline detergent is added to
“override” the acid. Override systems save time, water and energy.
1.3.4.3.3 Sanitizers/disinfectants
Chlorine is commonly used in the dairy industry as a sanitizing agent. Chlorine
has a broad antimicrobial activity against bacteria, fungi, and bacteriophage. The main
15
functional compound in chlorine sanitizer solutions is hypochlorous acid (HClO). The
hypochlorite ions kill microorganisms by attacking lipids in cell walls and destroying the
cell membrane structure and enzymes inside the cells. Chlorine compounds also have a
cleaning function and peptize proteins thus enhancing their solubility (Mauck et al.,
2001). Chlorine is stable at higher pH (pH >7), but releases chlorine gas and becomes
toxic and corrosive when the pH falls below 4.0 (Marriott, 1997).
In addition to chlorine, idophores and quaternary ammonium compounds (QACs)
also are used as disinfectants in the dairy industry (Tamime and Robinson, 1999).
Iodophors contain iodine, surfactants and acids, which are more stable and less corrosive
than chlorine. QACs are effective against bacteria, yeasts and molds, and can provide
residual antimicrobial activity in no-rinse applications. Neither iodophors or QACs are
used commonly as CIP sanitizers due to foaming problems (Marriott, 1997).
In addition to a sanitizing function, acid sanitizers also can prevent mineral or
milk stone build up. Those sanitizers include acid-anionic sanitizers, carboxylic acid
sanitizers, and peroxy acid sanitizers.
Acid-anionic sanitizers, containing anionic surfactants and acids, are used as no-
rinse food contact surface sanitizers, which are non-staining and noncorrosive to stainless
steel. This class of sanitizers is less effective against yeasts and molds, when compared
to chlorine. Use of acid-anionic sanitizers is limited in CIP applications due to foaming
issues (Boufford, 2003).
In contrast, carboxylic acid sanitizers, such as sulfonated fatty acids, have lower
foaming characteristics (Boufford, 2003). Carboxylic acid sanitizers are also non-
corrosive to stainless steel. However, carboxylic acid sanitizers may damage plastics and
16
rubber materials at temperatures above 38°C. Both acid-anionic sanitizers and carboxylic
acid sanitizers are active at low pH (pH 2-3, and pH <4, respectively), and may cause
corrosion to stainless steel, when the water is high in chlorine.
When compared with acid-anionic and carboxylic acid sanitizers, peroxy acid
sanitizers have low foam characteristics and a broad range of active pH, up to pH 7.5
(Boufford, 2003). Peroxy acid sanitizers also are less corrosive than chlorine and
idophores. It has been found that peroxy sanitizers are one of the most effective
sanitizers against Listeria and Salmonella (Marriott, 1997). When compared with
organic acids, the antimicrobial effectiveness of peroxy acid sanitizers is improved,
making them more effective against various yeasts and molds.
1.3.4.4 Contact time
Sufficient contact time is essential for effective cleaning. The contact time
required is dependent on the type of equipment, the nature of the soil, and the cleaning
solutions applied. For example, cleaning a pasteurizer requires longer contact time than
cleaning a refrigerated milk storage tank, because the deposit is heavier and includes
heat-denatured protein on the surface of the heat exchanger surfaces of the pasteurizer.
However, the costs of energy for heating and pumping, water and labor also need to be
considered when determining contact time of cleaning programs (Romney, 1990).
17
1.4 ASSESSMENT OF CLEANLINESS
Various methods have been used to evaluate cleanliness in dairy processing
plants, to monitor hygienic conditions, to evaluate cleaning efficiency, and to adjust or
optimize cleaning procedures. Methods for assessments of surface cleanliness include:
visual inspection, microbiological analysis, ATP bioluminescence or protein residue
detection (Asteriadou and Fryer, 2008).
1.4.1 Visual inspection
The Dairy Practices Council (2001) recommends application of visual cleanliness
inspection to surfaces that are difficult to clean in dairy processing systems. Areas
specifically mentioned included “dead ends” in pipelines, valves, and gaskets. A clean
stainless steel surface should be bright, with no residual moisture, scum, or loose
deposits. In addition to visual examination, sour or stale odors can indicate inadequate
cleaning (Asteriadou and Fryer, 2008).
1.4.2 Microbiological analysis
Microbiological analysis to assess surface hygiene can be conducted using
different methods, including the contact plate method, swab-based sampling methods,
and the “rinse-count” methods. The contact plate method samples equipment surface by
pressing an appropriate agar against the surface, followed by incubation to allow bacteria
present on the surface to grow and produce visible colonies that can be counted
18
(Robinson, 2002). The contact plate method is most suitable for sampling flat or slightly
curved surfaces that are smooth and non-porous. Swab-based sampling methods collect
cells by rubbing equipment surfaces with a moistened swab or sponge. Swabbing
methods may not be applied to some areas that are hard to reach by hand. Rinse count
methods can be applied to any surface, and are especially useful on hard-to-reach areas.
In this method, small pieces of equipment can be rinsed with a sterile liquid. The rinse
solution is examined using viable count procedures for general aerobic microorganisms,
coliforms, or other specific groups. Rinse count methods are less sensitive, when
compared to the contact plate methods or swab sampling methods because the sample has
been diluted. In addition to surface sampling, finished product evaluations such as the
standard plate count (SPC) and ongoing shelf-life assessments, also indicate hygienic
conditions of dairy processing equipment.
1.4.3 ATP bioluminescence method
1.4.3.1 ATP in milk
Adenosine-5’-triphosphate (ATP) is a molecule found in all living organisms. In
bovine milk, ATP molecules are associated with bacterial cells, somatic cells, and there
are also free-ATP molecules associated with colloidal calcium phosphate-citrate (CPC)
complex of casein micelles (Richardson et al., 1980). Richardson et al. (1980) indicated
that the primary source of ATP in bovine milk was free ATP. In addition, the
concentration of ATP in bovine milk was not closely related to the bacterial population
19
and somatic cell levels (Richardson et al., 1980; Poulis et al., 1993). Although ATP
molecules present on dairy processing equipment may indicate bacterial contamination.
ATP also indicates the presence of food residues.
1.4.3.2 Mechanism of ATP bioluminescence assay
The ATP bioluminescence assay, based on the luciferin-luciferase system, is
widely used in the food industry for monitoring hygiene (Griffiths, 1996). The luciferin-
luciferase system is a substrate-enzyme complex found in the firefly, Photinus pyralis
(Griffiths, 1993, 1996). In this reaction, luciferin binds with ATP, forming a dianion of
luciferyl-adenylate-AMP, and then the luciferyl-adenylate-AMP complex is oxidized by
O2 and converted to oxyluciferyl-adenylate-AMP, a cyclic dioxetanone. The oxidation
reaction is followed by decarboxylation, loss of AMP, and formation of the biradical
monoanion of oxyluciferin in an excited state. The excited state molecule then rapidly
returns to ground status by releasing energy as a photon of light. The intensity of light
released during the reaction is found to be proportional to the level of ATP. In other
words, the greater the level of ATP presented in a sample, the greater the intensity of
light emitted. Luciferase is the enzyme required for both oxidation and decarboxylation
steps (Campbell, 1988; Rhodes and McElroy, 1958). In this project, a novaLUM
luminometer (Charm Science, Inc. Lawrence, MA) was employed for ATP
bioluminescence detection, which is based on photomultiplier tube technology (Van
Dyke et al., 2002).
20
1.4.3.3 Applications in Food /Dairy Industry
The ATP bioluminescence assay has been applied in the food industry for safety
and quality management. For monitoring hygiene, several researchers have used the ATP
bioluminescence assay to detect overall contamination on a variety of food processing
equipment: meat slicers (Seeger and Griffiths, 1994), breweries (Ogden, 1993), fruit juice
operations (Bautista et al., 1992), milking equipment (Vilar et al., 2008), and milk
transport tankers (Bell et al., 1994). The ATP bioluminescence assay was found to be a
rapid method for monitoring hygienic conditions, providing real-time results when
compared to traditional microbiological methods. Generally, there was a 70% agreement
between passed or failed results when assessing system by the ATP assay and traditional
plate count (Griffiths, 1996). However, several authors also suggested that such a high
correlation was not always expected between ATP bioluminescence and microbiological
methods, because ATP bioluminescence measurements indicate contamination of both
food residues and bacterial cells (Ogden, 1993; Bautista et al., 1992; Bell et al., 1994;
Griffiths, 1993).
1.4.3.4 Advantages and limitations
The advantages of the ATP bioluminescence method, to provide rapid and reliable
assessment of cleanliness to assist in ensuring good manufacturing practice (GMP), has
been discussed extensively and is not controversial (Griffiths, 1993; Robinson, 2002).
However, the limitations of the ATP bioluminescence assay should be considered when it
is used for monitoring hygiene. Temperature can affect the accuracy of the test. The
21
optimum temperature for the ATP bioluminescence assay was found to be 25°C
(Robinson, 2002). The pH of the reaction also was found to be an important factor
(Campbell, 1988, Chollet and Ribault, 2012). In addition, cleansers and sanitizers may
cause enhancement or quenching of the bioluminescence signal. Cleansers and sanitizers
that were examined by Velazquez and Feirtag presented enhancement effects at lower
concentration but quenching effects at higher concentration (Velazquez and Feirtag,
1997).
1.4.4 Protein residue detection
Protein residue detection also can be used to assess cleanliness. One common test
is based on an oxidation reduction reaction (Sapan et al., 1999). After swabbing a
surface, the reagents are released onto the swab. In the reagent mix, cupric ions (Cu2+
)
are converted to cuprous ions (Cu+), after forming a complex with peptide bonds under
alkaline condition. Then the cuprous ions (Cu+) react with bicinchoninic acid (BCA),
forming a purple complex. The higher the level of protein present, the darker the purple
color that develops. Similar to the ATP bioluminescence assay, protein residue tests are
able to be used in the plant and provide results rapidly without incubation. However,
commercially available protein residue tests provide only semi-quantitative
measurements of surface cleanliness. When compared to the ATP bioluminescence
assays, protein residue tests are subject to more error, because of the requirement to
visually estimate color. Although there are limitations to this test, protein residue
22
assessment is used for cleanliness evaluation, especially for detection of proteins that
commonly cause food allergies.
1.5 ELECTROLYZED WATER
1.5.1 History
The phenomenon of water electrolysis was firstly observed in 1789 by a Dutch
merchant, named Adriaan Paets van Troostwijk and his friend, a medical doctor, Johan
Rudolph Deiman (de Levie, 1999; Trasatti, 1999). Troostwijk and Deiman immersed
two thin golden wires that were connected to an electrostatic generator into a glass tube
of water, and observed gas evolution on both wires (de Levie, 1999; Trasatti, 1999). The
idea of using electrolyzed water for decontamination was originally developed in Russian
medical institutions to disinfect surgeon’s hands (Nikitin and Vinnik, 1965). Since the
1980s, electrolyzed water has been approved in Japan for use in disinfecting medical and
dental equipment and for treating wounds (Harada et al., 1983; Shimizu and Hurusawa,
1992). More recently, electrolyzed water has been utilized as a disinfectant in
agriculture, livestock management and food processing (Kondo and Mieno, 1989).
1.5.2 Generation and Properties
Electrolyzed water (EW), also known as electro-chemical activation (ECA) water
is produced via electrolysis of a diluted sodium chloride solution (Huang et al., 2008).
To produce EW/ECA, a sodium chloride (NaCl) solution is pumped into an electrolysis
23
chamber, and then dissociated into sodium (Na+) and chlorine ions (Cl
-); hydrogen (H
+)
and hydroxyl (OH-) ions are also formed at this time (Figure 1-1). In the electrolysis
chamber, the anode and cathode are separated by a semi-permeable membrane. Passing a
current through the electrodes causes the negatively charged ions, including Cl- and OH
-
to migrate towards the anode, where chlorine gas (Cl2), hypochlorite ion (OCl-),
hypochlorous acid (HOCl), hydrochloric acid (HCl) and oxygen gas (O2) are formed.
Meanwhile, positively charged ions, such as Na+ and H
+ migrate to the cathode,
producing sodium hydroxide (NaOH) and hydrogen gas (H2). The acidic solution
produced at the anode side is called anolyte, acidic electrolyzed water (AEW), or
electrolyzed oxidizing (EO) water. The alkaline solution produced on the cathode side, is
called catholyte, alkaline electrolyzed water, basic electrolyzed water (BEW), or
electrolyzed reducing (ER) water (Shimizu and Hurusawa, 1992). The acidic solution
has a low pH (2.3-2.7), is high in oxidation-reduction potential (ORP, above 1100 mV),
contains dissolved oxygen gas and has an available chlorine concentration of 10 to 100
ppm, depending on the type and settings of the electrolyzed water generator.
Characteristics of the alkaline solution include a high pH (10.0-11.5), an ORP of -800 to -
900 mV, and the presence of dissolved hydrogen gas and sodium hydroxide.
24
Figure 1-1. Schematic of electrolyzed water generator and produced compounds (Huang
et al., 2008).
1.5.3 Advantages and disadvantages of electrolyzed water:
One of the advantages of using electrolyzed water as a CIP reagent is its safety.
In contrast with conventional cleansers and sanitizers, usually prepared by diluting from
concentrated chemicals, electrolyzed water can be produced on site, avoiding the need to
store, transport and handle concentrated chemicals. Although the acid EO water has a
low pH, it was not found to cause irritation of skin (Al-Haq et al., 2005). In addition,
using electrolyzed water for CIP procedures also has the potential to reduce the cost of
CIP cleaning (Wang et al., 2012; Al-Haq et al., 2005). Wang et al. (2012) did an on-farm
study and compared the costs of CIP for a milking system using electrolyzed water and
conventional chemicals, and concluded the operational expense of CIP cleaning using
electrolyzed water was 25% less than conventional method.
Anode:
2H2O → 4H+
+ O2 ↑+ 4e-
2NaCl→ Cl2 ↑+ 2 Na+
+ 2e-
Cl2 + H2O → HCl + HOCl
Cathode:
2H2O + 2e- → 2OH
-+ H2↑
2NaCl + 2OH-
→ 2NaOH + Cl-
25
The main disadvantage reported for electrolyzed water is the short shelf life. The
antimicrobial activity of acidic electrolyzed water is rapidly lost due to decomposition of
hypochlorous acid (HOCl) and volatilization of chlorine gas (Cl2). Len et al. (2000)
reported the stability of electrolyzed water could be affected by light, storage
temperature, agitation, packaging conditions (open or closed system), and pH. There was
equilibration of Cl2, HOCl and OCl- dependent on pH. HOCl is the primary bactericide
form of chlorine. When pH was 4.0~5.0, HOCl was at a higher concentration in solution.
HOCl decomposed to H+ and OCl
- when the pH increased, and released Cl2 gas when the
pH decreased (Len et al., 2000). Fabrizio and Cutter (2003) investigated the stability of
electrolyzed water and found the ORP of alkaline ER water was not consistent and
increased after 1-day of storage in sterile Pyrex bottles at both 4°C and 25°C. Thus, the
electrolyzed water used for all experiments in this research, was generated freshly and
stored in polypropylene carboys that closed with lids, at room temperature for no more
than 3 hours.
In addition, acid EO water has been shown to be corrosive to some materials used
in food processing equipment. Work conducted by Ayebah and Hung (2005)
demonstrated stainless steel presented “outstanding corrosion resistance” when immersed
in acid EO water (pH of 2.42, ORP of 1077 mV, and 48.66 ppm of chlorine) at room
temperature (22°C), whereas other materials such as carbon steel, copper, and aluminum
were less resistant to acid EO water. Compared with acid EO water, chlorine water (pH
of 8.72, ORP of 656 mV, and 49.16 ppm of chlorine) and modified EO water by
increasing its pH (pH of 6.12, ORP of 774 mV, and 50.39 ppm of chlorine) were less
26
corrosive. The results indicated that the corrosiveness of acid EO water was due to its
low pH and high ORP.
1.5.4 Application of electrolyzed water in food industry
1.5.4.1 Pure bacterial cultures
The effectiveness of acid EO water has been evaluated in many studies, and
revealed that acid EO water could inactivate various microorganisms, including
Escherichia coli O157:H7 (Fabrizio & Cutter, 2003; Issa-Zacharia, et al. 2010; Kim, et
al. 2000a; Rahman, et al. 2010; Stevenson, et al. 2004; Venkitanarayanan, et al. 1999a),
Listeria monoctogenes (Fabrizio and Cutter, 2003; Kim et al., 2001, 2000b; Rahman et
al., 2010; Venkitanarayanan et al., 1999b), Salmonella Typhimurium (Rahman et al.,
2010; Fabrizio and Cutter, 2003), Salmonella Enteritidis (Venkitanarayanan et al.,
1999b), Staphylococcus aureus (Issa-Zacharia et al., 2010; Rahman et al., 2010), Bacillus
sp. spores (Kim et al., 2000b; Kiura et al., 2002), Clostridium perfringens spores, and
Cryptosporidium parvum oocysts (Venczel et al., 1997).
Venkitanarayanan et al. (1999a) inoculated E. coli O157:H7, S. Enteritidis, and L.
monocytogenes into EO water (~ 80 ppm of free chlorine) at initial populations of 8 log10
CFU/ml. A 5-min treatment at 4°C or 23°C resulted in a reduction of 7 log10 CFU/ml,
and a 10-min treatment at both temperature reduced the population of all three strains to
undetectable levels (Venkitanarayanan et al., 1999a). Kim et al. (2001) investigated the
efficacy of acid EO water in inactivating biofilm forming bacteria on equipment surfaces.
27
A 300-second treatment using acid EO water (pH of 2.6, ORP of 1160 mV and 56 ppm
free chlorine) on stainless steel significantly reduced the number of biofilm-forming
bacteria from 10 log10 CFU/82.5 cm2 to below detectable levels (Kim et al., 2001). When
compared to chlorinated water containing the same concentration of chlorine, acid EO
water was found to be more effective in inactivating bacterial spores or spore formers,
such as Clostridium perfringens spores, Cryptosporidium parvum oocysts (Venczel et al.,
1997), and Bacillus cereus (Kim et al., 2000b).
Neutral EO water has been studied because it is less corrosive than acid EO water.
The disinfectant efficacy of neutral EO water has been examined (Rahman et al., 2010;
Issa-Zacharia et al., 2010). The work conducted by Issa-Zacharia et al. (2010) indicated
neutral EO water (pH of 5.8, ORP of 948 mV, and 21 ppm of free chlorine) was less
efficient as a sanitizer than acid EO water (pH of 2.6, ORP of 1140 mV, and 45 ppm of
free chlorine), but was capable of causing a 5 log10 CFU/ml reduction in pure culture of
Staphylococcus aureus and Escherichia coli after 90s of exposure. The reduced
efficiency of neutral EO water in this study was caused by not only neutral pH but also
lower concentration of free chlorine. In another study, Rahman et al. (2010) reported that
in spite of being lower in chlorine concentration, at least 95% of chlorine in neutral EO
water was in the form of hypochlorous acid, which is effective as a sanitizer.
1.5.4.2 Shell eggs
The application of electrolyzed water in reducing pathogens on egg shells has
been investigated. EO water was found to have the potential to reduce the population of
28
pathogens, including Salmonella spp., E. coli O157 and K12, L. monocytogenes, S.
aureus and Campylobacter jejuni. The efficacy was found to be dependent on the
method of application (Bialka et al., 2004; Park et al., 2005; Russell, 2003). In a study
conducted by Park et al. (2005), inoculated shell eggs were submerged into alkaline ER
water (pH of 11.2, ORP of -940 mV) for 1 min followed by immersion into acid EO (pH
of 2.5, ORP of 1117 mV, 41 ppm total chlorine) water for 1 min. This sequential
treatment achieved 4.39 log10 CFU/egg reductions of L. monocytogenes and 3.66 log10
CFU/egg reductions of S. Enteritidis, which was equivalent to a chlorinated water (200
ppm) wash. The effect of this sequential alkaline-acidic electrolyzed water treatment on
egg quality was also examined by Bialka et al. (2004). Results indicated that acid EO
water treatment did not significantly change albumen height or eggshell strength, but
could affect the cuticle presence and turn eggshells “spotty”. It was found that using an
electrostatic spraying system to apply acid EO water (pH of 2.1, ORP of 1150 mV and
free chlorine of 8 ppm) on inoculated egg shells improved the antimicrobial efficacy. In
this way, pathogenic bacteria, such as S. Typhimurium, S. aureus, L. moncytogenes, and
E.coli were completely eliminated (Russell, 2003).
1.5.4.3 Poultry
Different methods (spraying, immersing, or a combination of both methods) using
acid EO water for reducing pathogenic bacteria on chicken carcasses have been
investigated (Kim et al., 2005; Park et al., 2002; Fabrizio et al., 2002). Park and Huang
(2002) soaked inoculated chicken wings in EO water (pH of 2.57, ORP of 1082 mV, and
29
51.6 ppm free chlorine) with agitation (100 rpm) for 10 min or 30 min at 4°C or 23°C,
and the population of C. jejuni was reduced by ca. 3 log10 CFU/ml by the four treatments.
Fabrizio et al. (2002) reported that acid EO water (pH of 2.6, ORP of 1150 mV, and 39.6
ppm free chlorine) was more effective in reducing bacterial load than chlorine water
(30.9 ppm free chlorine) using an immersion method. These researches revealed that
treatment by immersion of inoculated chicken carcasses into acid EO water was effective
in reducing the population of pathogenic bacteria and preventing recovery of viable cells.
However, treatment with acid EO water by spraying were not effective. Spraying acid
EO water failed to provide a significant reduction in bacterial load, likely due to
insufficient contact time (Kim et al., 2005; Park et al., 2002; Fabrizio et al., 2002). The
work conducted by Kim et al. (2005) indicated that pre-spraying with alkaline ER water
was more effective in removing fecal contamination from chicken carcasses as compared
to 10% trisodium phosphate (TSP), which is used during defeathering to remove a thin
layer of lipids and protect against bacterial growth.
1.5.4.4 Meat
Fabrizio and Cutter (2004 & 2005) examined the effectiveness of using acid EO
water (pH of 2.4, ORP of 1160 mV and 50 ppm free chlorine) as an intervention to
enhance safety of meat products. By spraying acid EO water on fresh pork bellies for
15s, the populations of S. Typhimurium, C. coli, E. coli, and total coliforms were reduced
by 1.67 log10 CFU/cm2, 1.81 log10 CFU/cm
2, 1.13 log10 CFU/cm
2, and about 1 log10
CFU/cm2, respectively. The level of L. monocytogenes was not significantly reduced by
30
the EO water spray treatment. Although the treatment reduced some levels of undesired
bacteria, the authors suggested increased contact time was necessary to improve the
disinfection effectiveness (Fabrizio and Cutter, 2004). In terms of ready-to-eat (RTE)
meat products, frankfurters and ham inoculated with L. monocytogenes (5 log CFU/ml)
were dipped or sprayed with acid EO water (pH of 2.3, ORP of 1154 mV, and 45 ppm
free chlorine) or alkaline ER water (pH of 11.2, ORP of -795 mV) followed by acid EO
water. None of the treatments was able to reduce the bacterial load greater than 1 log10
CFU/g. Although the EO water treatments did not result in bleaching of RTE meat, the
disinfectant strength was not sufficient to meet regulatory requirements (Fabrizio and
Cutter, 2005).
1.5.4.5 Seafood
Acid EO water use in seafood processing has been reported, and the disinfectant
efficacy was not sufficient. Huang et al. (2006a) soaked pathogen-inoculated tilapia in
acid EO water (pH of 2.47, ORP of 1159 mV, and free chlorine of 120 ppm) for 10 min,
and populations of Vibrio parahaemolyticus and E. coli were reduced by 2.6 log10
CFU/cm2 and 0.76 log10 CFU/cm
2, respectively. There were no bacterial cells detected in
acid EO water after soaking, indicating using acid EO water could prevent cross-
contamination during tilapia processing. Ozer and Demirci (2006) investigated EO water
treatment under different conditions (temperature and time), and found that treatment by
soaking inoculated salmon fillet in acid EO water (pH of 2.6, ORP of 1150 mV and 76-90
ppm of free chlorine) at 35 for 64 min reduced populations of L. monocytogenes (1.12
31
log10 CFU/g) and E. coli (1.07 log10 CFU/g). Treatment of salmon fillet with alkaline ER
water prior to this acid EO water treatment did not improve the decontamination rate.
Experiments conducted by Huang et al. (2006b) demonstrated a treatment combining CO
gas with acid EO water containing more than 50 ppm chlorine could extend the shelf life
of tuna fillet from 6 days to 8 days during refrigerated storage.
Using acid EO water as disinfectant in seafood processing facilities has been
reported. To investigate the disinfectant effect of acid EO water on seafood processing
surfaces, Liu and Su (2006) reported that treatment with acid EO water containing 50
ppm of free chlorine for 5 min significantly reduced the load of L. monocytogenes on
various seafood processing surfaces, including a stainless steel sheet (reduction of 3.73
log10 CFU/25cm2, 2.33 log10 CFU/25cm
2 with food residue), ceramic tile (reduction of
4.24 log10 CFU/25cm2, 2.33 log10 CFU/25cm
2with food residue), and floor tile (reduction
of 5.12 log10 CFU/25cm2, 1.52 log10 CFU/25cm
2with food residue). Treatment by
immersion in acid EO water containing 40 ppm free chlorine reduced L. monocytogenes
on seafood processing gloves as well, providing 1.60 to 2.41 log10 CFU/cm2 reduction on
reusable gloves, and 2.54 to 3.87 log10 CFU/cm2 reductions on disposable.
1.5.4.6 Milking system
The feasibility of using electrolyzed water as cleaning and sanitizing agent for
CIP of milking system has been investigated. Walker et al. (2005a) conducted trials
using response surface design to evaluate the cleaning efficacy of electrolyzed water in
cleaning for coupons of five materials commonly used in milking systems: stainless steel
32
sanitary pipe, PVC milk hose, rubber liners, rubber gasket, and polysulfone plastic. The
cleanliness of material coupons was evaluated using ATP bioluminescence assays and
aerobic plate counts. Operational parameters, namely time and temperature combinations
of alkaline ER water and acid EO water, were determined based on liner regression
analyses. The results indicated the potential of electrolyzed water as a cleaning and
sanitizing agent for CIP cleaning of milking system. Furthermore, the effectiveness of
using electrolyzed water for CIP cleaning of a pilot scale milking system after soiling
with inoculated raw milk was also investigated (Walker et al., 2005b). The cleaning
efficacy of electrolyzed water treatments was evaluated using ATP bioluminescence
assays and a microbial enrichment method. Treatment with electrolyzed water for 7.5
min for both the alkaline wash and acid sanitize steps at a starting solution temperature of
60°C showed equivalent cleaning efficacy, when compared with a conventional cleaning
treatment.
However, both work done by Walker et. al. used the same temperature for both
steps of the CIP process, i.e. the alkaline wash with ER water and acid sanitizing with
acid EO water, which is not typical of CIP operations in the dairy industry. Alkaline
wash steps are usually conducted at higher temperature (50~72°C) for better efficacy of
cleaning; while the acid sanitizing step using chlorine sanitizer is usually conducted at
temperatures less than 40°C to avoid chlorine volatilization (Mauck et al., 2001; Tamime
and Robinson, 1999).
The operating temperatures for CIP cleaning with electrolyzed water for a pilot
scale milking system were further optimized by Dev et al. (2014), using response surface
modeling. In this work, cleanliness was assessed using the ATP bioluminescence
33
method. A set of operational temperatures was optimized: 58.8°C and 37.9°C for
alkaline ER water and acid EO water treatments respectively. This optimal CIP
procedure using electrolyzed water for cleaning of the pilot scale milking system was
able result in a clean surface with 100% RLU reduction
Another on-farm study conducted by Wang and Demirci compared cleaning
performance of conventional CIP solutions and electrolyzed water (Wang, et al., 2012).
In this on-farm study, after cleaning the milking system using electrolyzed water, 9 areas
of pipeline inner surface near tri-clamps along with gaskets were sampled for cleanliness
evaluation, using ATP bioluminescence assays and microbial enrichment method. The
authors suggested that electrolyzed water achieved the same or better cleaning efficacy in
CIP cleaning of cold milking system, when compared to conventional CIP solutions.
Although a few studies have been done using electrolyzed water as cleaning and
sanitizing solutions for CIP of system soiled with cold milk, the cleaning efficacy of
electrolyzed water for CIP of dairy processing system in dairy plant, especially when the
soil has been heated has not been investigated. Heat treatment during manufacturing
increases the difficult of cleaning, due to protein denaturation and mineral deposition
(milk stone formation).
In this study, the potential of using alkaline ER water and acid EO water as CIP
reagents for cleaning, by CIP, of surfaces soiled with cold milk or surfaces soiled during
heating of milk was evaluated. Comparison of the results of this work with conventional
CIP procedures using commercial cleanser and sanitizer will provide valuable in future
about the potential application of electrolyzed water for CIP procedures in dairy plant.
34
Chapter 2
HYPOTHESIS AND OBJECTIVES
Hypothesis:
Electrolyzed water can be used as an effective cleanser and sanitizer for CIP cleaning of
milk processing equipment.
To address this hypothesis, research was undertaken with the following objectives:
1. Construction of a pilot scale test system to allow evaluation and optimization of
electrolyzed water as CIP agent for dairy processing equipment (Chapter 3).
2. Determination of the efficacy of electrolyzed water as a CIP solution for cleaning a
refrigerated milk storage tank using a pilot scale test system (Chapter 4).
3. Optimization, using response surface model, of a CIP procedure with electrolyzed
water, for cleaning a processing equipment used to heat milk (Chapter 5).
35
Chapter 3
CONSTRUCTION, CHARACTERIZATION AND VALIDATION
OF TEST SYSTEM
ABSTRACT
Clean-in-place (CIP) is commonly used in the dairy industry for cleaning and
sanitation of dairy processing equipment. Optimization of CIP procedures has gained
importance, because of a desire to reducing operational costs and environmental impact.
Electrolyzed water is a set of alkaline and acidic solutions, which have been considered
as potential alternatives for CIP detergent and sanitizer. The goal of this research was to
develop a pilot scale test system to mimic soiling of tanks used to store refrigerated milk
and to thermally process milk, to evaluate efficacy of CIP procedures, and to further
optimize CIP procedures using electrolyzed water. The primary tank in the pilot scale
test system was a 15 liter (4 gallon) stainless steel vessel, equipped with a 360° static
spray ball delivering of CIP liquid. The test system was characterized, in terms of flow
rate, and the minimal flow rate providing full tank coverage was determined. Preliminary
trials using a standard CIP procedure and conventional detergent and sanitizer were
conducted, for cleaning the test vessel after being used to heat milk. The efficacy of the
standard CIP procedure was assessed with ATP bioluminescence assays and protein
residue detection assays. Results showed demonstrated the test vessel was completely
cleaned by conventional CIP procedure, which validated the suitability of the system for
evaluation and optimization of CIP procedures with other cleaning compounds.
36
3.1 INTRODUCTION
Dairy processing equipment is complex and requires frequent cleaning and
sanitizing. Cleaning and sanitizing play an essential role in the dairy industry in ensuring
quality products by preventing microbial contamination and by avoiding reduced
processing equipment performance due to fouling. Most dairy processing equipment is
cleaned using a highly automated technique, namely clean-in-place (CIP), which is a
method of cleaning the internal surface of pipe lines or processing equipment by jetting,
spraying or circulating cleaning solutions without opening or disassembly of the
equipment (Romney, 1990). CIP cleaning includes four steps: rinsing with water to
remove bulk dairy food residue; washing with alkaline detergents to remove proteins and
fat more firmly attached to equipment surfaces; rinsing with water again to remove
residue cleanser and dissolved soil; and finally rinsing with sanitizer to reduce bacterial
contamination (Lloyd, 2008).
Electrolyzed water is a set of alkaline and acid solutions, produced via electrolysis
of a dilute (0.1%) sodium chloride solution. This process results in an alkaline
electrolyzed reducing (ER) water containing sodium hydroxide (pH ca. 11.0 and ORP ca.
-850 mV) and acid electrolyzed oxidizing (EO) water containing 10-100 ppm chlorine
(pH ca. 2.5, ORP ca. 1168 mV) (Huang et al., 2008; Al-Haq et al., 2005). In contrast to
conventional CIP chemicals, which are usual stored and handled in concentrated forms,
electrolyzed water can be produced onsite, and offers an attractive alternative to
conventional cleaning and sanitizing solutions. As described in Chapter 1, using
electrolyzed water for CIP cleaning provides the possibility of reducing manufacturing
37
costs by reducing the cost of handling and storing concentrated cleaning and sanitizing
chemicals.
CIP procedures are well established and widely used not only in the dairy industry
but also in the beverage and pharmaceutical industries. Optimization of CIP procedures
and improving efficiency of cleaning have become more important in recent years (Fryer
et al., 2011). Cleaning and sanitizing generates large amounts of water containing
cleaning chemicals, which presents an environmental impact issue (Vourch et al., 2008).
In addition, costs of manufacturing could be reduced by reducing cleaning costs related to
energy, labor and purchase of cleaning chemicals. However, cleaning protocols are
commonly overdesigned and semi-empirical, because: (1) manufacturers need to ensure
the safety and quality of food rather than take a risk to narrow the margin of cleanliness
criteria; (2) assessments of cleanliness are normally conducted at the end of cleaning, and
online measurements during cleaning processing are usually missing; (3) it is difficult to
compare cleaning efficiency of removal of different types of soils (Fryer and Asteriadou,
2009; Fryer et al., 2011).
In this work, a pilot scale test system was constructed: (i) to mimic soiling in
different types of tanks used in dairy processing plants, (ii) to allow the assessment of
cleanliness at any time during CIP cleaning, and (iii) to evaluate cleaning efficiency and
to optimize CIP procedures using electrolyzed water.
38
3.2 MATERIALS AND METHODS
3.2.1 Construction of test system
A pilot-scale dairy processing system was designed and constructed for testing the
efficacy of cleaning and sanitizing (Figure 3-1).
Figure 3-1. Schematic of pilot scale dairy processing test system.
Test vessel. A 15-liter (4-gallon) stainless steel vessel equipped with a
heating/cooling jacket, which allowed temperature control during soiling, served as the
base for the test system (Figure 3-2A). For mimicking a refrigerated milk silo, chilled
water (4°C) was circulated within the jacket to maintain the refrigerated temperatures
during soiling. For mimicking thermal processing, hot water (75°C to 80 °C) was
circulated through the jacket during soiling. The bottom of the test vessel has a cone
shape to allow complete drainage. To measure temperature of test vessel during soiling
39
and cleaning, a temperature probe was installed at the bottom of the 15-liter (4-gallon)
test vessel. The temperature probe was also connected to an AJ-310 microprocessor-
based circular chart recorder (Anderson Instrument Co., Fultonville, NY) for temperature
monitoring and recording. A customized stainless steel lid equipped with a gasket to seal
the tank was held in place by four clamp fasteners during soiling and cleaning. To allow
agitation during soiling, a 1.5-inch fitting welded on to the lid served as the entrance for
the shaft of the agitator. The fitting was covered when the agitator was not in use. Thus,
the test vessel could be sealed to maintain temperature, to prevent against evaporation
during heated soiling, and to avoid leakage during cleaning.
Water tank and CIP tank. A stainless steel milk can welded with a 0.5-inch fitting
served as water reservoir for storing water for the pre-rinse and post-rinse steps during
CIP processes (Figure 3-2B). Similarly, an 11.4 L (12-quart) stainless steel pot welded
with a 0.5-inch fitting served as the reservoir for CIP solutions during washing and
sanitizing operations (Figure 3-2C). In order to maintain temperatures of CIP solutions
during cleaning, a coil heat exchanger with external diameter of 0.64 cm (¼ inch)
(constructed by Swagelok Co., Pittsburgh, PA) connected to a circulating water bath
(Thermo Fisher Scientific Inc., Pittsburgh, PA) was immersed into CIP solution reservoir.
During CIP cleaning, the water bath was pre-set at a temperature of about 10% higher
than the specified operating temperature to compensate for heat loss due to CIP fluid
circulation.
40
Figure 3-2. Test system. (A) Test vessel (center of the system) with agitator in place. (B)
A modified stainless steel milk can served as reservoir for water used for CIP cleaning.
(C) CIP solution reservoir with coil heat exchanger connected to a circulating water bath.
(D) 360° static spray ball.
Liquid delivery system. A 360° static spray ball (Model 149588, Sani-Matic Inc.,
Madison, WI) was installed at the top of the 15-liter (4-gallon) test vessel, attached in the
center of the lid, was used to deliver CIP liquids (Figure 3-2D). Liquid used for CIP
cleaning was delivered to the 15-liter (4-gallon) test vessel by a variable frequency drive
B A
C
D A
41
(VFD) pump (Fluid-o-Tech, Plantsville, CT), which allowed control of flow rate during
cleaning. The test vessel, CIP tanks and VFD pump (Model AT13-18-56CB, World
Wide Electric Corp., Pittsford, NY) were connected via 3-way or 2-way valves, and
sanitary stainless steel pipes with diameter of ½" (1.27 cm), which provided different
flow paths and allowed circulation or rinse-to-drain.
3.2.2 Characterization of the test system
3.2.2.1 Volumetric flow rate measurement
To characterize the performance of the VFD pump, the flow rate was evaluated at
each pump speed setting. Water was pumped from the rinse tank, and collected for 1 min
at each pump speed setting. The volume of collected water was measured using a
graduated cylinder. The flow rate (L/min) of each pump speed setting was measured in
triplicate.
3.2.2.2 Evaluation of spray ball coverage
Coverage tests were conducted using a riboflavin solution to assure the system
was capable of delivering water or CIP solutions to the entire internal surface of the
vessel. Riboflavin emits fluorescence under UV light at a wavelength of 365 nm
(Bowser, 2005). For the coverage tests, a riboflavin in water solution (200 ppm, Sigma-
Aldrich Co. LLC, Saint Louis, MO) was sprayed on the internal surface of the test vessel
using a spray bottle. The test vessel was then rinsed with room temperature (ca. 22°C)
42
tap water using the 360° static spray ball at different flow rates (established using the
VFD pump). Tests were conducted at flow rates of 1.8, 3.9, 6.0, 8.3, and 10.0 L/min.
The residual riboflavin remaining on the surface of the test vessel was evaluated visually
using a UV light (model 8CC-MIG-BLB, K&H Industries, Inc., Hamburg, NY). The
first inspection of residual riboflavin was conducted after rinse for 1 min at each pre-set
flow rate. After the first inspection, the residual riboflavin was inspected visually under
the UV light after every 2- min of rinsing.
3.2.3 Performance validation of the test system used for CIP
To validate the suitability of the test system for evaluation and optimization of
CIP procedures, a standard CIP procedure using commercial cleanser and sanitizer was
performed (Figure 3-3).
3.2.3.1 Pre-cleaning
Before each experiment, the test system was cleaned manually and thoroughly,
according to “Sanitation Standard Operating Procedure” used by Penn State Berkey
Creamery (Votano et al., 2007), using a commercial detergent and sanitizer to return the
vessel to a presumably clean condition. To perform pre-cleaning, the internal surface of
the test vessel was sprayed with tap water at ambient temperature (ca. 22°C), followed by
brushing with warm (45~50°C) chlorinated alkaline detergent, HC-10®
solution (ca. 16
g/l, Ecolab USA Inc., St. Paul, MN). This step was followed by another rinse with tap
43
water at room temperature (ca. 22°C) to remove the residue of alkaline solution and cool
down the test vessel. Finally, the test vessel was brushed with a chlorinated sanitizer,
XY-12® solution (ca. 100 ppm of available chlorine, Ecolab USA Inc.) at room
temperature (ca. 22°C).
Figure 3-3. Flow chart of CIP performance evaluation of test system. CIP cleaning was
conducted at flow rate of 8.3 L/min.
3.2.3.2 Soiling
After returning the test vessel to a presumably clean condition, 11.4 liters (3
gallons) of HTST pasteurized whole milk, purchased from Penn State Berkey Creamery,
was poured into the vessel. Hot water at ca. 77°C was circulated through the jacket for
soiling the test vessel under heated conditions. Milk was heated from 4°C to 74°C, which
44
took ca. 15 min, and then the temperature of the milk was kept at 74 ± 1°C for another 15
min. Thus, the total soiling time was about 30 min. An agitator (Stir-Pak, Laboratory
Stirrer, Model 4554, Cole-Parmer Co., Vernon Hills, IL) with a 316 stainless steel 3-
blade propeller (8.9 cm diameter, Cole-Parmer Co.) and a 316 stainless steel shaft (1.0
cm diameter × ca. 60 cm length, Cole-Parmer Co.) was employed to ensure the tank was
heated and soiled evenly.
3.2.3.3 Four-step conventional CIP
A CIP procedure was conducted at a flow rate of 8.3 L/min, as determined by the
coverage tests. Before CIP cleaning, milk was drained from the test vessel. Next, the test
vessel was sprayed with tap water via the spray ball for 3 min at ambient temperature (ca.
22°C) using the rinse-to-drain flow path as a pre-rinse step. After pre-rinsing a
commercial chlorinated alkaline detergent, namely Principal® (3200-4000 ppm, Ecolab
USA Inc.), was circulated within the system for 15 min at 63°C. Following the alkaline
wash, the system was post-rinsed with tap water for 3 min at ambient temperature (ca.
22°C) using the rinse-to-drain flow path as well. Finally, the test vessel was sanitized
with the commercial sanitizer, XY-12® (ca. 100 ppm of available chlorine, Ecolab USA
Inc.), by circulating through the system for 3 min at room temperature (ca. 22°C). Tap
water for both pre-rinse and post-rinse steps was not reused. Therefore, for each rinse
step, ca. 24.9 L water was needed. In contrast, about 11 L detergent or sanitizer was used
for each circulating step.
45
3.2.3.4 Assessment of cleanliness
Cleanliness of the inner surface of the test vessel was assessed at several points
during the procedure: after pre-cleaning as a background check; after soiling to determine
the initial condition of the vessel; after pre-rinse; after post-rinse to evaluate the effect of
the alkaline wash; and after sanitizing to evaluate the effect of acidic sanitizing. Direct
surface sampling using two swabbing methods was applied for cleanliness assessments:
an ATP bioluminescence assay and a protein residue detection assay.
3.2.3.4.1 Detection of residual ATP
For each ATP bioluminescence analysis, 5 cm × 10 cm of the tank surface was
swabbed using PocketSwab Plus swabs (Charm Science, Inc. Lawrence, MA) for ca. 15
sec, vertically and horizontally, in an overlapping pattern according to manufacturer’s
instructions. After the sample was collected, the swab was twisted down to break the
metal foil seal, releasing the buffering agent and to dissolve the luciferin/luciferase tablet.
The buffering agent also contains compounds that help releasing ATP from cells. Once
the ATP was exposed to the luciferein and luciferease complex, light was produced
(Griffiths, 1996). The intensity of light given off was detected using a novaLUM palm-
sized luminometer (Charm Science, Inc.), which provides a measure of relative light units
(RLU). According to the manufacturer, the amount of ATP is proportional to RLU. An
RLU of zero (0) indicates an acceptably clean stainless steel surface (Griffiths, 1993).
3.2.3.4.2 Detection of residual protein
Protein is a major component of milk soil. To detect residual protein, an area of 5
cm × 10 cm of the inner surface was swabbed for ca. 15 sec using a Pro-Clean Rapid
46
Protein Residue Test Swabs (Hygiena llc., Camarillo, CA), vertically and horizontally in
an overlapping pattern. After swabbing, the reagent contained in the snap vial was
released to react with protein on the swab, which causes a color change from green to a
purplish-violet color (Sapan et al., 1999). Color was evaluated 10 min after releasing the
reagent. Cleanliness assessment using this protein detection method provides only
categorical data. A green color after reaction indicates an acceptable cleanliness with
protein residue of less than 20 µg. Colors of gray, light purple and dark purple indicate
protein residue levels of 20~40 µg, 40~100 µg and > 100 µg.
3.2.4 Test for swab sampling variability
For the swab-sampling methods, each 5 cm × 10 cm inner surface could be
swabbed only once during any given trial. Thus, cleanliness evaluations after different
steps had to be assessed at different locations. Consequently, the locations for swab-
sampling might be a “nuisance factor” leading to variability in evaluations of cleanliness,
specifically for the ATP bioluminescence assay. To avoid this situation, swab-sampling
were assessed at the same height of the inner surface area of the test vessel. In addition, a
separate experiment was conducted to assess the variability of cleanliness at various
positions in the test vessel.
To conduct this experiment, the test vessel was pre-cleaned and soiled with milk
at high temperature (ca. 74°C) for 15 min using the same procedures as described above.
After draining the milk out of the test vessel, in order to enhance the difficulty of
cleaning, the system was allowed to dry for 15 min before sampling for ATP
47
bioluminescence analyses, and the RLU data was marked as “after soiling”. Three
randomly picked areas of 50 cm2
(5 cm × 10 cm) at different positions were swabbed for
each cleanliness assessment. Sampling and assessment of cleanliness took ca. 15 min.
Thus, after 30 min following draining, the test vessel was rinsed with tap water at flow
rate of 8.3 L/min for 3 min at room temperature (ca. 22°C). Then, another cleanliness
assessment was performed again using the ATP bioluminescence method, and marked as
“after pre-rinsing”. Lastly, instead of using a commercial sanitizer, sanitizing with acid
electrolyzed oxidizing (EO) water was applied for 2 min at room temperature.
Cleanliness of three areas was determined using the same procedure for ATP
bioluminescence analyses and marked as “after sanitizing” (Figure 3-4). This experiment
was replicated in three times.
3.2.5 Statistical analysis
The ATP bioluminescence data (RLU values) were converted to base 10
logarithms. Since log10 (0) is undefined, the RLU values of zero were assigned a value of
0.5 for logarithmic calculation. Statistical analyses were performed using Minitab
software version 16 (Minitab Inc., State College, PA). Analysis of variance (ANOVA)
test using Tukey’s comparison was performed for comparing the means of log10 RLU
after different CIP steps. Means were consider significantly different at a level of 0.05 (α
= 0.05).
48
Figure 3-4. Schematic of test for swab sampling variability. Each rectangle area
represents 50 cm2 (5 cm × 10 cm) inner surface of test vessel. Different colors represent
the sampling for ATP bioluminescence assays after different steps. Red marked areas
were swabbed “after soiling”, green areas were swabbed “after pre-rinse”, and blue areas
were swabed “after sanitizing”. For one trial, each assessments of ATP bioluminescence
assays was conducted in three replicates.
49
3.3 RESULTS AND DISCUSSION
3.3.1 Characterization of test system
3.3.1.1 Volumetric flow rate measurement
The volumetric flow rates at 9 pump speed settings were measured. Figure 3-5
shows volumetric flow rate as a function of pump speed setting. Standard deviation was
obtained from three replicates. The volumetric flow rate increased as the speed of VFD
pump was increased. The regression equation indicates that an increase of 10 unit of
VFD pump speed setting lead to an increase of 1.9 (L/min) in flow rate (R2 = 99.8%).
0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0
0
5
1 0
1 5
2 0
V F D p u m p se t tin g
me
an
flo
w r
ate
(L
/min
)
Figure 3-5. Mean volumetric flow rates (L/min) at different VFD pump settings. The
regression equation is: Flow Rate (L/min) = 0.1844 + 0.1918 Pump Setting.
50
3.3.1.2 Coverage test
Spraying riboflavin solution on the inner surface of the test vessel resulted in
uniform fluorescence under UV light. After rinsing with water at each pump speed
setting, the pattern of residual riboflavin revealed the coverage of the 360° static spray
ball (Figure 3-6). At a pump speed setting of 10 (1.8 L/min), ca. half of the inner wall of
the test vessel was rinsed. The spray ball failed to provide full coverage of the test-tank
at pump speed settings of 10 (1.8 L/min), 20 (3.9 L/min), or 30 (6.0 L/min). In fact, even
after rinsing the test vessel with water for 17 min at a setting of 30 (6.0 L/min), there was
still some riboflavin remaining on the upper wall of the test vessel. However, at pump
speed settings of 40 (8.3 L/min) and 50 (10.0 L/min), water was able to contact and wet
an internal surfaces thoroughly after a 3 min rinsing. The coverage test along with
volumetric flow rate measurements revealed the lowest flow rate providing full coverage
was 8.3 L/min (pump speed setting of 40). Hence, a flow rate of 8.3 L/min was chosen
for CIP procedures for the entire project.
51
Figure 3-6. Riboflavin coverage test. (A) Riboflavin solution sprayed on test vessel
before rinse. (B) Residual riboflavin remaining after rinsing test vessel with water at
flow rate of 1.8 L/min (pump setting of 10) for 1 min. (C) Residual riboflavin remaining
after rinsing test vessel with water at flow rate of 3.9 L/min (pump setting of 20) for 7
min. (D) Residual riboflavin remaining after rinsing test vessel with water at flow rate of
6.0 L/min (pump setting of 30) for 17 min. (E) Residual riboflavin remaining after
rinsing test vessel with water at flow rate of 8.3 L/min (pump setting of 40) for 1 min.
The European Hygienic Engineering & Design Group (EHEDG) recommends the
flow rates for tank cleaning using static spray ball should be 30-50 liters per minute per
meter of tank circumference (Fliessbach, 2013). The circumference of the test vessel
used in this study was 0.718 meter, with diameter of 0.229 meter (9 inch). To meet the
EHEDG recommendation, the flow rates should achieve 21.5-35.9 liter per minute,
A B
C D E
52
beyond the capacity of the VFD pump. Thus, according to EHEDG, the flow rate was
insufficient. However, it has been reported that this situation is not uncommon when
cleaning tanks using a static spray ball (Packman et al., 2008). Since complete coverage
of the tank surface was demonstrated, the system was considered suitable for this study.
3.3.2 Performance validation of the test system used for CIP
The purpose of evaluating the effectiveness of conventional CIP procedure using
commercial chemicals of the test vessel after soiling with whole milk after heating was to
validate the test system was suitable for evaluation and optimization of CIP procedures
using electrolyzed water.
Results of ATP bioluminescence assays, the RLU values and log10 RLU are
presented in Table 3-1. The ATP levels, which are proportioned to RLU values, are
significantly higher “after soiling” and “after pre-rinse” than the residual ATP levels of
“after post-rinse” and “after sanitizing” (p-value = 0.000). The ATP levels detected
“after soiling” were not statistically different from those detected “after pre-rinsing” (p-
value = 0.461). The slightly increase of ATP level observed is likely due to loosening of
the soil by water rinsing. These results demonstrated the test vessel was returned to a
satisfactorily clean condition after alkaline wash with commercial chemicals and post-
rinse with water, indicating the standard procedure using conventional CIP chemicals
succeeded in cleaning for the test vessel after soiling with hot milk.
53
Table 3-1. Cleanliness assessments made using ATP and protein measurements after
different steps of standard CIP procedure using commercial chemicals (*RLU values of
zero were assigned as 0.5 for base-10 logarithm calculation).
Step Replicate RLU*1
Log10
RLU Protein (μg) 2
Cleanliness
assessment
After soiling
1 328,785 5.52 > 100 Fail
2 239,806 5.38 40-100 Fail
3 450,775 5.65 > 100 Fail
After pre-rinse
1 455,831 5.66 > 100 Fail
2 596,649 5.78 > 100 Fail
3 667,585 5.82 > 100 Fail
After post-rinse
1 0 -0.30 < 20 Pass
2 0 -0.30 < 20 Pass
3 0 -0.30 < 20 Pass
After sanitizing
1 0 -0.30 < 20 Pass
2 0 -0.30 < 20 Pass
3 0 -0.30 < 20 Pass
1 RLU = relative light units. An RLU of 0 is considered clean.
2 A residue protein level of < 20 μg is considered clean.
Protein detection results are also summarized in Table 3-1. Cleanliness
assessments in terms of residual protein failed “after soiling” and “after pre-rinse”. In
contrast, “after post-rinse” and “after sanitizing”, the protein levels were less than 20 μg
per 50 cm2 sampling area, which indicated an acceptable cleanliness. Results revealed
pre-rinse with only water was insufficient to remove most residual proteins from the
surface of the test vessel soiled with milk at high temperature (ca. 74°C). After heat-
treatment, the milk proteins were likely denatured, making them more difficult to remove
(Stanga, 2010). Washing the test vessel with commercial alkaline chlorinated cleanser
was able to remove residual protein and denatured proteins to an undetectable level (<20
μg). Results of residual protein analyses agreed with the cleanliness assessments made
54
using the ATP bioluminescence assay. This observation again verified the CIP
procedure, using a commercial cleanser and sanitizer, was able to return the test vessel to
a cleaning condition after soiling with whole milk at high temperature. This experiment
confirmed the test system was suitable for further study of CIP procedures.
3.3.3 Test for swab sampling variability
The purposes of this experiment were to determine whether swab sampling
location was a factor leading to variance in cleanliness, as measured by RLU values, and
to determine if this approach could led to systematic errors between trials.
To determine the impact of sampling location on variance of RLU readings, three
areas of the test vessel internal surface were swabbed for ATP bioluminescence analyses
at each cleanliness assessment (after soiling, after pre-rinse or after sanitizing). Means
and standard deviations of RLU values are presented in Figure 3-7. The small standard
deviations obtained indicated the variability of cleanliness at different positions of the test
vessel was small, and the test vessel was evenly soiled and rinsed. Thus, location of
sampling was not a factor leading to variance in cleanliness assessment.
This experiment was conducted in triplicate. ANOVA using Tukey’s comparison
revealed that log10 RLU values were not significantly different between trials. Results
revealed the repeatability of trials using the test system was acceptable (Figure 3-7).
55
Figure 3-7. Log10 RLU values obtained after soiling, after pre-rinse, and after sanitizing.
Error bars represent standard deviation of the evaluations of three locations. Tukey’s
comparison was conducted between 9 sets of data. Means that do not share a letter are
significantly different (α = 0.05).
When comparing the log10 RLU values between steps (after soiling, after pre-
rinse, and after sanitizing), there were no statistically significant differences in general.
The log10 RLU values of “after pre-rinse” were slightly higher than the log10 RLU values
of “after soiling”. This observation may be attributed to the loosening of attached soil
deposits by pre-rinsing with water. The mechanism proposed by Bird and Fryer (1991)
showed that the deposit swelled and formed a gel by a chemical reaction and mass
transfer process, making it more removable.
AB AB AB A A AB AB B AB
0.000
1.000
2.000
3.000
4.000
5.000
6.000
7.000
Trial #1 Trial #2 Trial #3
Log1
0 R
LU
After soiling
After pre-rinse
After sanitizing
56
Comparing the cleanliness “after soiling” with “after sanitizing” (Figure 3-7), the
log10 RLU values were slightly, but not significantly decreased. This result also indicated
that the ATP analysis was not quenched in the presence of acid electrolyzed water (EW).
3.4 CONCLUSION
In this work, a pilot scale test system was constructed for soiling and CIP cleaning
of dairy processing equipment. The test system was designed for optimization of CIP
procedures by controlling soiling conditions, such as soiling temperature; and controlling
CIP parameters, such as flow rate, temperature and time. The pilot scale test system was
characterized with regards to flow rate and coverage. A flow rate of 8.3 L/min was
determined to assure adequate coverage of the test vessel and was used throughout the
thesis project.
Additional tests showed the test vessel was able to be soiled and sprayed evenly;
indicating sampling location was not a factor leading to variance of RLU values. The
repeatability of cleanliness assessments after soiling or CIP procedure also was
determined as acceptable. Results of this experiment also suggested rinsing with acid EO
water did not significantly decrease ATP load on the inner surface of the test vessel. In
other words, acid EO water did not have a quenching effect on ATP in the
bioluminescence assay.
In addition, use of the test system was validated by successful cleaning using a
standard CIP procedure with conventional CIP detergent and sanitizer for the test vessel
57
after soiling with whole milk heated to high temperature (77°C). The results indicate that
the test system was suitable for evaluation and optimization of CIP procedures.
58
Chapter 4
CIP USING ELECTROLYZED WATER FOR A REFRIGERATED
MILK STORAGE TANK
ABSTRACT
Electrolyzed water (EW) is a set of alkaline and acid solutions, namely
electrolyzed reducing (ER) water (pH ca 11.0 and ORP ca. -850 mV) and electrolyzed
oxidizing (EO) water (pH ca. 2.5, ORP ca. 1168 mV and 80-100 ppm of chlorine).
Electrolyzed water has the potential to serve as an alternative to clean-in-place (CIP)
chemicals. Compared to conventional CIP detergents and sanitizers, which are prepared
by dilution of concentrated chemicals, electrolyzed water is safer and more
environmental friendly. In this research, CIP procedures using electrolyzed water for
cleaning of a pilot sale cold milk storage tank were evaluated. Cleanliness was assessed
using ATP bioluminescence analysis, protein residue detection and a microbiological
enrichment assay. After soiling with milk inoculated with Pseudomonas fluorescens,
Enterococcus faecalis and Escherichia coli at refrigerated temperature, to mimic a raw
milk silo, the stainless steel vessel was successfully cleaned using a 4-step CIP procedure
employing electrolyzed water. The procedure included a wash step of using alkaline ER
water for 15 min at 40 °C and sanitizing step of using acid EO water for 1 min at 25 °C.
59
4.1 INTRODUCTION
After being received from the dairy farm, milk is often stored in a refrigerated raw
milk silo at the dairy plant to maintain high milk quality and extend the shelf-life, by
delaying multiplication of microorganisms and inhibiting enzyme activity (Robinson,
2002). The U.S. Department of Health and Human Services (USDHS) Grade “A”
Pasteurized Milk Ordinance (2011) requires that raw milk silos or other storage tanks
should be cleaned immediately after being emptied. Regardless, raw milk silos or storage
tanks must be emptied and cleaned at least every 72 hours.
Because of the size of many raw milk storage tanks (3,000 to 70,000 gallons), CIP
technology is commonly used for cleaning and sanitation. CIP procedures in the dairy
industry are commonly conducted in 4 steps, rinse – wash – rinse – sanitize. The CIP
procedure for cleaning of raw milk silos or storage tanks suggested by the Dairy Practices
Councils (DPC) are summarized as follow: pre-rinse with water at temperature range
from ambient to 43.3°C to remove and loosen milk fouling; wash with chlorinated
alkaline detergent for 15 min at 62.8°C to remove attached milk fouling, such as fat
globules and proteins; post-rinse with cold water to remove residual detergent; and finally
rinse with a sanitizer for destruction of microorganisms and to retard bacterial growth
(Mauck et al., 2001).
Use of electrolyzed water (EW) as a cleaning and sanitizing agent has gained the
attention of the food industry (Huang et al., 2008; Al-Haq et al., 2005). Electrolyzed
water is produced via electrolysis of a dilute sodium chloride solution into sodium and
chlorine ions. A semi-permeable membrane installed between the anode and cathode
60
electrodes separates ions with different charges. An acidic solution is generated on the
anode side. This material is called acidic electrolyzed water (AEW), or electrolyzed
oxidizing (EO) water, and is characterized by having a low pH (2.3-2.7), a high
oxidation-reduction potential (ORP, >1100 mV), and a free chlorine content of 10 to 100
ppm, depending on the types and settings of the electrolyzed water generator. An
alkaline solution is generated on the cathode side. This solution is called basic
electrolyzed water (BEW) or electrolyzed reducing (ER) water, and is characterized
by a high pH (10-13), and low ORP (-800 to – 900 mV). The major component of ER
water is sodium hydroxide (Len et al., 2000; Hricova et al., 2008).
Use of acid EO water as a disinfectant for food processing, for example to reduce
microbial contamination on processing utensils (cutting board, gloves) or food products
(sea food, meat, poultry, shell eggs, fruit and vegetables), has been well investigated
(Monnin et al., 2012; Liu and Su, 2006; Ozer and Demirci, 2006; Fabrizio and Cutter,
2004; Fabrizio et al., 2002; Bialka et al., 2004; Izumi, 1999). In addition, since sodium
hydroxide (caustic soda) has been a typical ingredient in alkaline detergents, it seems
feasible to use ER water as an alkaline detergent for cleaning. Compared with
conventional CIP cleaning, where cleaning and sanitizing solutions are commonly
prepared by dilution of concentrated chemicals, using electrolyzed water for CIP reagents
could avoid purchase, storage and handling of concentrated chemicals. This advantage
improves the operating environment in terms of safety and may also reduce the cost of
production.
Walker et al. (2005a) evaluated the efficacy of alkaline ER water and acid EO
water in cleaning of coupons of five materials used in milking systems. Furthermore, the
61
effectiveness of using both electrolyzed water solutions for removal of milk residue (ATP
reduction) and reducing bacterial level of a pilot scale milking system were also
investigated (Walker et al., 2005b). Both the laboratory and pilot studies suggested that
electrolyzed water had potential as a cleanser and disinfectant for CIP cleaning of
milking systems. The operating temperatures of CIP procedures using electrolyzed water
for a pilot scale milking system were further optimized (Dev et al., 2014). A set of wash
temperature (58.8°C) and sanitizing temperature (37.9°C) were determined and achieved
100% ATP reduction. An on-farm study using electrolyzed water for CIP of a milking
system revealed that electrolyzed water achieved the same or better cleaning efficacy as a
conventional CIP procedure using commercial chemicals of a milking system using
(Wang et al., 2012).
To our knowledge, application of electrolyzed water for CIP procedures of
processing equipment in dairy plants has not been investigated. The purpose of this work
was to evaluate cleaning efficacy of electrolyzed water as a cleanser and sanitizer in CIP
procedures for refrigerated milk storage tanks, using a pilot scale test system.
62
4.2 MATERIALS AND METHODS
4.2.1 Bacterial cultures verification and characterization
In order to mimic raw milk silo soiling, milk used for soiling was inoculated with
a cocktail of three common raw milk cultures (Pseudomonas fluorescens, Enterococcus
faecalis ATCC 51299, and Escherichia coli ATCC 25922). These cultures were obtained
as stock cultures from the Department of Agricultural and Biological Engineering, The
Pennsylvania State University. To activate bacterial cells, each culture was streaked on
trypticase soy agar (TSA), and incubated aerobically for 24 hours at their optimum
growth temperatures: P. fluorescens was incubated at 32°C, while E. faecalis and E. coli
were incubated at 37°C. Individual colonies of each culture were picked and inoculated
into 10 ml of sterile trypticase soy broth (TSB) (Difco; Becton Dickinson and Company,
Sparks, MD), then incubated for 24 h at their optimum temperatures prior to stock culture
preparation.
In order to verify the cultures were the actual species, 16S ribosomal RNA gene
sequence analysis was conducted. To obtain DNA for sequencing, bacteria liquid PCR
(polymerase chain reaction) was conducted, which involved longer initial denaturation
step (5 min) comparing to regular PCR (2 min denaturation), to allow amplification of the
target DNA fragment by PCR directly from liquid culture. Two universal primers for
16S rRNA, which were adopted from sequencing primers developed by the 454 Life
Sciences (Roche Titanium-compatible), were employed for the PCR reactions (Integrated
DNA Technologies, Inc., Coralville, IA):
Test8F (5’-CCAATCCCCTGTGTGCCTTCGCAGTC-3’)
63
Test518R (5’-CCATCTCATCCCTGCGTGTCTCCGAC-3’)
The 25-μl PCR amplification mixtures consisted of 5 μl culture broth (~109
CFU/ml), 5 μl of 5× Colorless GoTaq Reaction Buffer (Promega, Madison, WI) with
MgCl2 (1.5 mM of final concentration), 200 μM of deoxynucleotide triphosphate
(dNTPs, Promega), 0.5 units of GoTaq DNA Polymarase (Promega), and 5 μM of each
primer (Integrated DNA technologies Coralville, IA). The PCR reactions were
performed with a Master-cycler gradient machine (Eppendorf, Hamburg, Germany). The
cycling consisted of an initial denaturation step for 5 min at 95°C, followed by 32
amplification cycles (denaturation for 30 sec at 94°C, annealing for 45 sec at 58°C, and
extension for 1 min at 72°C), and then a final extension step for 5 min at 72°C.
Electrophoresis of PCR amplicons was performed using a horizontal 1% agarose
gel at 110 V for 90 min. Bands were visualized on a UV trans-illuminator after staining
in an ethidium bromide solution (10 mg/ml) for 1 h. The target DNA bands were
extracted from the agarose gel and purified using a QIAquick gel extraction kit (Qiagen,
Valencia, CA) following the manufacturer’s instructions.
Purified PCR amplicons were sequenced in both the forward and reverse
directions using universal 16S rRNA PCR primers mentioned above. Sequencing was
conducted at the Huck Institute Genomic Core Facility of The Pennsylvania State
University. Sequencing was accomplished on an ABI 3730XL DNA analyzer with 3’
BigDye-labeled dideoxynucleotide triphosphate (v 3.1 dye terminators; Applied
Biosystems, Foster City, CA) and ABI sequence analysis software (version 5.1.1). The
16s rRNA gene sequences were identified using nucleotide BLAST (Basic Local
Alignment Search Tool, http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al., 1990).
64
To determine the incubation time required to obtain cell concentrations of ca. 109
CFU/ml, the population of viable bacterial cells were assessed using aerobic plate count
(APC) after incubation for 16 h, 20 h, and 24 h, respectively. These assessments were
repeated three times. To compare the difference of cell concentrations at each incubation
point, one-way ANOVA and Tukey’s comparison were applied (Minitab 16, Minitab
Inc., State College, PA, USA). Incubation for 24 h was determined to yield about 109
CFU/ml cells for each culture. For long-term storage, stock culture of each organism
were prepared by mixing 0.5 ml of each cultures with 0.5 ml of 20% glycerol in sterile
screw cap microcentrifuge tube, and freezing at -80°C.
4.2.2 Preparation of inoculated milk
Three bacterial cultures were grown separately as described above. For each
culture, cells from 10 ml of culture were harvested by centrifugation (Model Sorval ST
16 centrifuge, Thermo Scientific, Ashville, NC) at 5,000 g for 10 min at room
temperature. After discarding the supernatant, the bacterial cell pellet was re-suspended
in ca. 2 ml pasteurized whole milk by vortexing. To mimic raw milk, this suspension
was used to inoculate 11.4 liters (3 gallons) of HTST pasteurized whole milk. The final
concentration of bacterial cells was ca. 3×106 CFU/ml. Note that all three test organisms
were added to the milk.
65
4.2.3 Generation and characterization of electrolyzed water
Electrolyzed water generator (Model ROX20, Hoshizaki America Inc., Peachtree
City, GA) was employed to generate electrolyzed water (Figure 4-1). A continuous
stream of deionized water and a 12% sodium chloride solution were fed into the
electrolytic chamber at room temperature to yield a dilute sodium chloride solution with a
concentration of approximate 0.1%. A pressure-reducing valve was installed on the
deionized water supply line to ensure the water pressure was ca. 17 psi, resulting in a
flow rate of 1.9 L/min (0.5 gallon/min) of each output as recommended by the
manufacture (Hoshizaki America Inc.). The operating amperage and voltage were set at
18 amp and 10 volts; respectively. To stabilize the quality of electrolyzed water, the
water electrolyzer was allowed to operate for 10 min before beginning collection. The
alkaline and acid electrolyzed water were prepared prior to each use, and stored
separately in 5-gallon polypropylene carboys that closed with lids, at room temperature
for no more than 3 h.
Figure 4-1. Electrolyzed water generator used in this research.(Model ROX20, Hoshizaki
America Inc.)
66
The characteristics of alkaline and acid solutions were determined before each
use. Characterization included determination of the pH of both alkaline and acidic
solutions using a pH meter (SevenEasy pH meter S20, Mettler Toledo, Columbus, OH)
along with a pH probe (Model InLab 413, Mettler Toledo); ORP of both solutions were
measured using with a combination redox/ORP electrode (Model 9678BNWP, Thermo
Scientific Inc., Beverly, MA) connecting with a pH/ORP meter (Model InLab 413,
Mettler Toledo); the total and free chlorine concentrations of the acidic solution were
evaluated using a digital titrator (Model 16900, Hach Inc., Loveland, CO), equipped with
a N,N- diethyl-p-phenylenediamine-ferrous ethylene diammonium sulfate (DPD-FEAS)
titration cartridge (Hach, Inc.); the OH- concentration of the alkaline solution was
calculated from the pH values. The electrolyzed water was heated in a stainless steel pot
over a gas range prior to use to 42°C, which was slightly higher than the treatment
temperature to compensate for heat loss during liquid transfer.
4.2.4 Preparation of commercial CIP chemicals
Conventional CIP procedures using a commercial cleanser and sanitizer were
employed as a positive control. For the alkaline wash step, Principal®
(3.2~4 ml/L,
Ecolab USA Inc., St. Paul, MN) was used as cleanser. Principal® is a chlorinated alkaline
detergent commonly used for dairy processing equipment. For the sanitizing step, XY-
12®, a sodium hypochlorite sanitizer was used (1.2 ml/L, Ecolab USA Inc.). The
concentrations of both solutions were determined using a titration kit (Ecolab USA Inc.)
following the manufacture’s instruction, to ensure that the concentrations of working
67
solutions were 3200~4000 ppm for Principal® solution, and 100 ppm available chlorine
contained in XY-12® solution. The Principal
® working solution was heated in a stainless
steel pot over a gas range prior to use to 67°C, which was also slightly higher than the
temperature used for positive treatment to compensate for heat loss during liquid transfer.
4.2.5 Preparation of test system
Before each experiment, the test system was cleaned manually and thoroughly,
according to “Sanitation Standard Operating Procedure” used by the Penn State Berkey
Creamery (Votano et al., 2007), as described in Chapter 3.
4.2.6 Soiling the system
To mimic soiling of a raw milk silo, the test vessel was soiled by holding 3
gallons of inoculated milk for 18 h at refrigerated temperatures (2 ~ 4 °C). The cooling
media, sweet water, was circulated through the heating/cooling jacket of the test system
during soiling to maintain the vessel temperature at 1.4 ~ 2.2°C. After soiling, inoculated
milk was drained from the system and 10 ml of milk sample was collected during
draining for aerobic plate count (APC) analyses to determine the population of viable
bacterial cells after soiling.
68
4.2.7 CIP procedure using electrolyzed water treatment
Operating parameters for both treatments are summarized in Table 4-1. For the 4-step
CIP procedure for cleaning of the test system soiled with cold milk, the test vessel was
first pre-rinsed with water for 1 min at ambient temperature (ca. 22°C). After pre-rinsing
with water, the test vessel was treated with alkaline ER water. Two treatments using
electrolyzed water were evaluated: “EW short” with a 5-min ER water wash and “EW
long” with a 15-min ER water wash. Pre-heated (40°C) alkaline electrolyzed water was
circulated on the inner surface of the test vessel by spraying. The temperature of the
alkaline electrolyzed water (40±1°C) was maintained by a coil heat exchanger connected
with a circulating water bath (Thermo Fisher Scientific Inc.). The alkaline electrolyzed
water was drained from the test vessel at the end of the alkaline wash step. The next step
was to post-rinse with tap water for 1 min at ambient temperature (ca. 22°C). Finally, the
test vessel was sanitized by circulating acidic electrolyzed water for 1 min at 25°C, using
the same flow path as that for the alkaline wash step described above. The system was
drained at the end of the sanitizing step. The flow rate was set at 8.3 L/min for the entire
CIP procedure. Each treatment was conducted in triplicate.
69
Table 4-1. Operation temperature and time of four treatments.
Treatments
Wash Sanitizing
Chemical Temp.
(°C)
Time
(min)
Chemical Temp.
(°C)
Time
(min)
Pos Ctrl1 Principal
® 63 15 XY-12
® 25 1
EW Long2 ER water 40 15 EO water 25 1
EW Short3
ER water 40 5 EO water 25 1
Neg Ctrl4
water 40 15 water 25 1
1 Pos Ctrl = Positive control (As recommended by the Berkey Creamery)
2 EW Long = electrolyzed water treatment with longer wash time (15 min, chosen
according to preliminary experiments that remove most soil after rinsing with water).
3 EW Short = electrolyzed water treatment with shorter wash time (5 min)
4 Neg. Ctrl = negative control
4.2.8 CIP control treatments
As a negative control, the system was “cleaned and sanitized” using only water.
As a positive control, the system was cleaned using commercial chemicals following a
conventional CIP procedure. Both negative and positive controls were replicated three
times. The test vessel was soiled, cleaned and sampled for cleanliness assessment by the
same strategies as those for the electrolyzed water.
For the negative control treatment, the test vessel was pre-rinsed with water for 1
min at room temperature (ca. 22°C) after soiling. Then, a “wash step” was conducted by
circulating pre-heated water through the system for 15 min at 40°C. After a 1-minute
post-rinse with water at room temperature (ca. 22°C), the water was circulated through
the system for 1 min at 25°C as the sanitizing step of negative control.
70
For the positive control treatment, the vessel was pre-rinsed with water as
described above for the electrolyzed water. Then, a chlorinated alkaline detergent,
Principal® (3200-4000 ppm, Ecolab USA Inc.), was circulated through the system for 15
min at 63°C, as prescribed by manufacturer. After the Principal®
solution was drained
out of test vessel; another post-rinse with water was conducted for 1 min at room
temperature. Finally, the test system was sanitized by circulating a chlorine sanitizer,
XY-12® (ca. 100 ppm of available chlorine, Ecolab USA Inc.), through the system for 1
min at room temperature. The system was drained before evaluation of cleanliness.
4.2.9 Assessments of cleanliness and data collection
Cleanliness of the inner surface of the test vessel was inspected at several steps
during the cleaning cycle. To ensure the experiment started with a clean test vessel with
RLU = 0 and less than 20 µg per 50 cm2 of protein, cleanliness was assessed after manual
pre-cleaning. During each trial, assessments of cleanliness were conducted after soiling
and draining, after pre-rinse, after alkaline wash and post-rinse, and then after sanitizing
(Figure 4-2). Direct surface sampling using three swabbing methods was employed: ATP
bioluminescence analysis, protein residue detection, and microbiological enrichment
assay. The ATP bioluminescence analysis and protein residue detection were the same as
the methods described in Chapter 3.
71
Figure 4-2. Flow chart of CIP procedures of cold milk storage tank using electrolyzed
water. CIP procedures were conducted at flow rate of 8.3 L/min.
To evaluate the cleanliness in terms of microbial load, 50 cm2 (5 cm × 10 cm) of
surface was carefully swabbed vertically and horizontally in an overlapping pattern using
a sterile calcium alginate swab (Amd Ritmed Inc., Tonawanda, NY) pre-moisturized with
sterile TSB (Difco; Becton Dickinson and Company). After swabbing, the alginate swab
was placed in 10 ml of sterile TSB (Difco; Becton Dickinson and Company) and incubated
for 48 hours at 30°C. The presence of turbidity in the enrichment broth was considered
evidence of viable microorganisms.
72
4.2.10 Statistical design and analysis
One-way ANOVA and Tukey’s comparison were employed using Minitab 16
(Minitab Inc., State College, PA, USA), to compare means of RLU value of each
assessment between treatments. Statistical significance was set at 0.05 (α = 0.05).
4.3 RESULTS AND DISCUSSION
4.3.1 Inoculum bacterial cultures verification and characterization
The identity of the cultures (P. fluorescens, E. faecalis ATCC 51299, and E. coli
ATCC 25922) was verified to the species level using BLAST of 16s rRNA gene
sequence. BLAST results yield 99%~100% identities to their corresponding strains with
sequence lengths of 437~518 bases and 0% gaps (Figure A-1 to A-3).
After growth in 10 ml of TSB for 24 hours at optimum temperatures, viable cell
counts of P. fluorescens, E. faecalis, and E.coli were 9.04 ± 0.07 log10 CFU/ml, 9.46 ±
0.09 log10 CFU/ml, and 9.11 ± 0.03 log10 CFU/ml, respectively (Figure 4-3). Cell
concentrations of E. faecalis were higher than the other two organisms during the
incubation. P. fluorescens had the lowest concentrations. Differences in cell
concentrations of different cultures were likely due to the different initial inoculation
levels and different growth rates. However, the population of all three organisms was ca.
9 log10 CFU/ml after 24-hour of incubation.
73
Figure 4-3. Mean populations of cultures during overnight incubation. Error bars
represent standard deviation of the log10 CFU/ml of three replicates. Tukey’s
comparisons were conducted between three sets of data at each sampling point. Means
that do not share a letter are significantly different (α = 0.05).
4.3.2 Chemical properties of electrolyzed water
Chemical properties of electrolyzed water are listed in Table 4-2. There was no
significant difference in the chemical properties between treatments of EW short and EW
long (p-values are also listed in Table 4-2). Correlations between RLU values and
corresponding chemical concentrations also were evaluated. The results suggested no
correlation between RLU values “after post-rinse” and OH- concentration; or between
RLU values “after sanitizing” and chlorine concentration. Statistical analyses of the
chemical properties of the electrolyzed water analyzed indicated a consistent
composition.
B B' B" A
A' A"
C
C'
B"
7.5
8
8.5
9
9.5
10
16 20 24
log 1
0 C
FU/m
l
Incubation time (h)
Escherichia coli
Enterococcus faecalis
Pseudomonas fluorescens
74
Table 4-2. Chemical properties of electrolyzed water of each trial. P-values were
obtained from Tukey’s comparison between treatments.
Treatment Trials
EO Water1 ER water
2
pH
ORP
(mV)
Total
[Cl-]
(ppm)
Free
[Cl-]
(ppm)
pH
ORP
(mV)
[OH-]
(ppm)
EW Short3
1 2.45 1157 76.7 70.6 11.58 -851 64.7
2 2.35 1178 93.7 90.0 11.76 -880 97.9
3 2.40 1173 83.3 78.6 11.61 -877 69.3
Average 2.40 1169 84.6 79.7 11.65 -869 77.3
St.Dev. 0.04 8.96 7.00 7.96 0.08 13.02 14.69
EW Long4
1 2.33 1170 94.5 93.6 11.65 -877 76.0
2 2.48 1159 68.7 67.1 11.63 -874 72.6
3 2.33 1170 84.7 83.9 11.69 -885 83.3
Average 2.38 1166 82.6 81.5 11.66 -879 77.3
St.Dev. 0.07 5.19 10.63 10.95 0.02 4.64 4.48
p-value 0.75 0.70 0.84 0.86 0.92 0.39 1.00
1 EO Water = electrolyzed oxidizing water
2 ER Water = electrolyzed reducing water
3 EW Short = electrolyzed water treatment with shorter wash time (5 min)
4 EW Long = electrolyzed water treatment with longer wash time (15 min)
4.3.3 Cleanliness assessments
4.3.3.1 Microbiological analyses results
Results of microbiological analyses are presented in Table 4-3. The viable cell
population of the bacterial cocktail in milk was 6.5 ± 0.09 log CFU/ml, after soiling for
18 h at refrigerated temperature (1.4~2.2°C). There was no significant difference in
viable cell population between the treatments (p-value = 0.238). In terms of the surface
swab enrichment tests, only one swab tested negative after “pre-rinse” with water. In
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general, swabs tested positive “after soiling” and “after pre-rinse”. In contrast, no
positive enrichment results were observed “after wash and post-rinse” or “after
sanitizing”. Regarding microbiological data, rinsing the test system soiled with cold milk
with only water was able to reduce the bacterial population to undetectable levels. In
other words, the soiling of cold milk storage tank, such as that of a milk silo, was
considered light soiling that is easy to remove (Robinson, 2002).
Table 4-3. Microbiological data of inoculated milk after soiling and of swab
enrichment test.
Treatment Trials
APC1
(log
CFU/ml)
After
Soiling2
After
Pre-rinse2
After
Post-rinse2
After
Sanitizing2
Pos. Ctrl3
1 6.453 + + - -
2 6.452 + - - -
3 6.434 + + - -
EW Long4
1 6.491 + + - -
2 6.562 + + - -
3 6.412 + + - -
EW Short5
1 6.574 + + - -
2 6.686 + + - -
3 6.472 + + - -
Neg. Ctrl6
1 6.658 + + - -
2 6.547 + + - -
3 6.472 + + - -
1 Aerobic plate count of inoculated milk after soiling at refrigerated
temperature overnight
2 Indicates turbidity (+) or no turbidity (-) after the enrichment step.
3 Pos. Ctrl = positive control
4 EW Long = electrolyzed water treatment with longer wash time (15 min)
5 EW Short = electrolyzed water treatment with shorter wash time (5 min)
6 Neg. Ctrl = negative control
76
4.3.3.2 ATP bioluminescence assay results
Results of cleanliness assessments based on ATP bioluminescence assays are
presented in Figure 4-4. After soiling, ANOVA using Tukey’s comparison showed no
significant difference for RLU values between the four treatments (p-value = 0.452),
indicating the soiling procedure was repeatable. The RLU values for each of the four
treatments are significantly higher after soiling than the RLU values of the other
conditions (p-value = 0.000). In other words, after a pre-rinse with water, most of the
fouling was removed, as was expected. It has been suggested that a sufficient pre-rinse
should remove 90~99% of fouling (Bylund, 2003). If, as suggested by the manufacturer
of the ATP swabs (Charm Sciences, Inc.), an RLU=0 indicates a clean surface, both the
EW long treatment and positive control returned the test vessel to clean conditions after
alkaline wash and post-rinse. Although the efficacy of the EW long treatment or positive
control was better, the EW short treatment returned the test vessel to a clean state after
the complete four-step CIP cleaning. In contrast, the negative control, cleaned only with
water, did not return the tank surface to acceptable conditions, eliminating the effect of
mechanical force or flow rate.
77
Figure 4-4. Means of RLU values comparison between treatments. Error bar
indicates standard deviation of triplicate analysis. Tukey’s comparisons were conducted
between all RLU values. Means that do not share a letter are significantly different (α =
0.05). (Pos. Ctrl = positive control; EW Long = electrolyzed water treatment with 15-
min wash time; EW Short = electrolyzed water treatment with 5-min wash time; Neg.
Ctrl = negative control)
4.3.3.3 Protein detection results
Results of cleanliness based on protein residue levels are presented in Table 4-4.
The protein residue detection verified fouling on the surface of the test vessel after
soiling. Results revealed that most residual protein on the surface of test system was
removed by the pre-rinse step. After an alkaline wash with ER water or a commercial
detergent, the protein level on the inner surface of the test vessel was so low as to be
a
b
d d
a
b
d d
a
b
c
d
a
b b
b
1
10
100
1000
10000
100000
1000000
After Soiling After Pre-rinse After Post-rinse After Sanitizing
RLU
Pos ctrl
EW long
EW short
Neg ctrl
78
undetectable (< 20 μg per 50 cm2). In contrast, the negative control treatment was less
effective, exhibiting detectable protein residue after both the wash and post-rinse steps.
As expected the results also indicated that swabbing for protein detection was less
sensitive than ATP bioluminescence assay.
Table 4-4. Protein residue levels.
Protein
(ug/ 50 cm2)
Trials After
Soiling
After
Pre-rinse
After
Post-rinse
After
Sanitizing
Pos. Ctrl1
1 > 100 20 ~ 40 0 ~ 20 0 ~ 20
2 > 100 20 ~ 40 0 ~ 20 0 ~ 20
3 > 100 20 ~ 40 0 ~ 20 0 ~ 20
EW Long2
1 > 100 20 ~ 40 0 ~ 20 0 ~ 20
2 > 100 20 ~ 40 0 ~ 20 0 ~ 20
3 > 100 20 ~ 40 0 ~ 20 0 ~ 20
EW Short3
1 > 100 20 ~ 40 0 ~ 20 0 ~ 20
2 > 100 20 ~ 40 0 ~ 20 0 ~ 20
3 > 100 20 ~ 40 0 ~ 20 0 ~ 20
Neg. Ctrl4
1 > 100 20 ~ 40 20 ~ 40 0 ~ 20
2 > 100 20 ~ 40 20 ~ 40 0 ~ 20
3 > 100 20 ~ 40 20 ~ 40 0 ~ 20
1 Pos. Ctrl = positive control
2 EW Long = electrolyzed water treatment with longer wash time (15 min)
3 EW Short = electrolyzed water treatment with shorter wash time (5 min)
4 Neg. Ctrl = negative control
79
4.3.4 CONCLUSION
In this research, two treatments, “EW long” (15-min wash) and “EW short” (5-
min wash), using electrolyzed water as cleanser and sanitizer for CIP of refrigerated milk
storage tank were evaluated. Both treatments were able to return the test vessel to
acceptably clean conditions after the complete 4-step CIP procedure. The effectiveness
of EW long treatment (15-min wash) was comparable to conventional CIP procedure
using a commercial cleanser and sanitizer. Furthermore, the alkaline wash step using ER
water was conducted at lower temperature (40°C) than the conventional operating
temperature (63°C), which indicated that using electrolyzed water as CIP solutions has
the potential to save energy cost and reduce production costs, although the efficacy of
conventional CIP treatment at low temperature was not measured.
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Chapter 5
CIP USING ELECTROLYZED WATER FOR A HEATED MILK
PROCESSING TANK
ABSTRACT
Electrolyzed water is produce by electrolysis of a weak sodium chloride solution
into alkaline electrolyzed reduction (ER) water and acid electrolyzed oxidization (EO)
water. Previous studies revealed the disinfectant effect of acid EO water on various food
products and cleaning. The potential application in CIP procedure of using alkaline ER
water and acid EO water for cleaning of milking system was also demonstrated. The CIP
application of electrolyzed water on dairy processing plants has not been reported in
literature. In this research, CIP procedures using electrolyzed water were optimized for
cleaning a pilot scale test vessel soiled by thermally processing milk. Cleanliness was
assessed using an ATP bioluminescence assays and protein residue detection. A set of
optimal CIP operational parameters was obtained from regression analyses of a response
surface model. The optimal conditions were alkaline wash with ER water at 54.6°C for
20.5 min and acid sanitize with EO water at 25°C for 10 min. The complete 4-step CIP
procedure using electrolyzed water with optimal parameter combination was validated
and was able to return the test vessel into clean conditions.
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5.1 INTRODUCTION
Cleaning and sanitizing plays a very important role in dairy foods production.
Not only because of concern about food safety and public health, but also because the
fouling deposits on equipment surface can reduce processing, for example by decreasing
the heat transfer rate of heat exchangers. Design of cleaning procedures depends on
several factors, including characteristics of soil, types of materials to be cleaned,
limitation of cleaning settings in terms of maximum flow rate, temperature, time, and the
degree of cleanliness demanded (Tamime and Robinson, 1999). Soil on dairy processing
equipment used in heat treatment is relatively difficult to remove, requiring more
aggressive cleaning procedures and/or cleaning reagents than is required to clean other
types of dairy processing equipment. A well designed and optimized cleaning procedure
should be able to return equipment to a satisfactory state with a minimum cost, energy
consumption and environmental impact.
Clean-in-Place (CIP) techniques are widely used in the dairy industry. A typical
CIP cycle for cleaning of dairy processing equipment used for heat treatment usually
includes four steps: pre-rinse with cold or warm (43.3°C) water to remove the bulk of the
dairy food residue; then wash with alkaline cleanser at high temperature (62.8°C);
followed by a cold water rinse; and finally by sanitizing, generally with an acid sanitizer
to disinfect the equipment surface (Mauck et al., 2001). The chemicals used to prepare
CIP techniques and sanitizing solutions are usually diluted from concentrated stock
chemicals.
82
Use of electrolyzed water (EW) as a cleaning and sanitizing agent has recently
gained the attention of the food industry (Huang et al., 2008; Al-Haq et al., 2005).
Electrolyzed water is generated by passing a 0.1% sodium chloride solution stream into
an electrolyzer equipped with two electrodes, separated by a semi-permeable membrane.
Positively and negatively charged ions move towards the opposite electrodes, producing
alkaline electrolyzed reducing (ER) water and acid electrolyzed oxidizing (EO) water.
The alkaline ER water contains sodium hydroxide and has a pH of about 11.5 and an
oxidation-reduction potential (ORP) of – 850 mV. The acid EO water has a low pH (2.3-
2.7), high ORP (>1100 mV), and a free chlorine content of 80 to 100 ppm, depending on
the type and settings of the electrolyzer. Because of its chemical properties, electrolyzed
water may be suitable for serving as a cleaning and sanitizing reagent (Huang et al.,
2008; Al-Haq et al., 2005). Using electrolyzed water for CIP procedures generated on
site for cleaning would avoid the need to purchase, store and handle concentrated
chemicals, resulting in potential cost savings and a safer manufactory environment.
The efficacy of acid EO water in reducing microbial contamination has been
investigated (Monnin et al., 2012; Liu and Su, 2006; Ozer and Demirci, 2006; Fabrizio
and Cutter, 2004; Fabrizio et al., 2002; Bialka et al., 2004; Izumi, 1999). Acid EO water
was able to achieve 1 to 7 log reduction of bacterial load on food products or food
processing surfaces (Hricova et al., 2008). Use of alkaline ER water and acid EO water
as CIP reagents on dairy farms has also been investigated. Walker et al. (2005a; b)
evaluated the effectiveness of using both electrolyzed water solutions for removal of milk
residue and bacterial contamination in laboratory and pilot scale studies. Results
suggested that electrolyzed water could serve as a CIP reagent. Dev et al. (2014)
83
optimized the CIP procedure using electrolyzed water for cleaning of pilot scale milking
system in terms of temperatures of the ER water wash and EO water sanitizing steps
regard to achieve 100% ATP reduction. In addition, the on-farm study conducted by
Wang et al. (2012) indicated that electrolyzed water resulted in the same or better
cleaning efficacy then a conventional CIP procedure employing conventional detergents
and sanitizers.
The application of electrolyzed water for CIP procedures of processing equipment
in dairy foods plant has not been reported in the literature. No information was found
related to cleaning of equipment used to heat-treat milk and milk products, soil which is
relatively difficult to remove due to protein denaturation and mineral deposition. The
objective of this work was to investigate the cleaning capability of electrolyzed water for
CIP cleaning and sanitizing of a pilot-scale stainless steel test vessel used to heat-treat
milk. Response surface modeling was employed to optimize the settings of a CIP process
using electrolyzed water. Variables included temperatures and treatment times for both
alkaline ER water and acid EO water treatments.
5.2 MATERIALS AND METHODS
5.2.1 Preparation of electrolyzed water
Electrolyzed water (EW) was generated immediately prior to each trial using an
ROX20 water electrolyzer (Hoshizaki America Inc., Peachtree City, GA) as described in
Chapter 4. Chemical properties of electrolyzed water, such as pH, ORP and chlorine
84
concentration were determined as described in Chapter 4. Concentration of hydroxide
ions [OH-] was calculated from the pH value of alkaline ER water as well using the
equation-1 below:
∵ [OH-] [H
+] =
∴ [OH-] = – -- Equation 1
5.2.2 Experimental design -- response surface model
A Box-Behnken response surface model was employed to determine the optimal
combination of four operational parameters. Specifically the temperatures and times for
both the ERW wash and the EOW sanitizing steps (Table 5-1) were evaluated. A Box-
Behnken design was chosen to minimize the number of trials, and also to avoid using
extreme parameters. The Box-Behnken design with four variables consisted of 27 trials
including 3 replicates of the center points (Table 5-2). The parameter range for each
factor was selected according to previous studies and conventional CIP procedures. The
design, including a randomized running order, was generated using Minitab 16 (Minitab
Inc., State College, PA).
Table 5-1. Levels of four independent variables for CIP using electrolyzed water.
Coded levels
Independent variables Unit -1 0 +1
Temperature for ER water wash °C 40 55 70
Treatment time for ER water wash min 5 15 25
Temperature for EO water sanitizing °C 25 35 45
Treatment time for EO water sanitizing min 1 5.5 10
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Table 5-2. Box-Behnken response surface design with 27 trails, including 3 center points
(in bold). WashTemp = temperature for ER water wash; WashTime = time for ER water
wash; SaniTemp = Temperature for EO water sanitizing; SaniTime = Time for EO water
sanitizing.
Standard
Order
Run
Order
WashTemp
(°C)
WashTime
(min)
SaniTemp
(°C)
SaniTime
(min)
1 2 40 5 35 5.5
2 10 70 5 35 5.5
3 26 40 25 35 5.5
4 6 70 25 35 5.5
5 18 55 15 25 1
6 13 55 15 45 1
7 7 55 15 25 10
8 21 55 15 45 10
9 3 40 15 35 1
10 17 70 15 35 1
11 20 40 15 35 10
12 14 70 15 35 10
13 15 55 5 25 5.5
14 19 55 25 25 5.5
15 5 55 5 45 5.5
16 11 55 25 45 5.5
17 4 40 15 25 5.5
18 12 70 15 25 5.5
19 24 40 15 45 5.5
20 16 70 15 45 5.5
21 22 55 5 35 1
22 1 55 25 35 1
23 9 55 5 35 10
24 25 55 25 35 10
25 27 55 15 35 5.5
26 8 55 15 35 5.5
27 23 55 15 35 5.5
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5.2.3 Soiling and CIP treatments
This study was conducted using the pilot scale test system described in Chapter 3.
To ensure a presumably clean condition before soiling, the test vessel was pre-cleaned
manually using HC-10®
and XY-12® (Ecolab USA Inc., St. Paul, MN, USA) as the
detergent and sanitizer as described in Chapter 3 and Chapter 4. Then, the test vessel was
soiled with pasteurized whole milk (Penn State Berkey Creamery, University Park, PA)
and heated as described in Chapter 3. The milk was heated to 74°C under agitation and
then held at 74 ± 1°C for 15 min. After soiling, the heated milk was drained from the
system.
After draining, a four-step CIP cleaning protocol was conducted at flow rate of
8.3 L/min. Steps in the process were pre-rinse, alkaline wash, post-rinse and sanitizing.
The pre-rinse and post-rinse steps were conducted for 3 min at ambient temperature (~
22°C) using tap water. The alkaline wash and sanitizing steps using alkaline ER water
and acid EO water were conducted using the different temperature and time combinations
specified by the response surface model.
Two control treatments also were evaluated in triplicate. These controls included
the same pre-rinse and post-rinse procedures as those used in the electrolyzed water
treatments. For the negative control treatment, tap water was used instead of detergent
and sanitizer. The operating parameters for the negative control were these parameters of
the center point, to be specific; the test vessel was washed with water for 15 min at 55°C,
and sanitized for 5.5 min at 35°C with water. For the positive control treatment, a
commercial detergent and sanitizer were employed. The operating parameters for the
87
positive control were determined from the manufacturer’s suggestion; specifically, wash
with Principal® (3200-4000 ppm, Ecolab USA Inc.) for 15 min at 62.8°C, and sanitize
with XY-12® (ca. 100 ppm of available chlorine, Ecolab USA Inc.) for 3 min at 25°C.
5.2.4 Cleanliness assessments
The cleanliness of the system was assessed after draining, and after different CIP
steps: after pre-rinse, after alkaline wash and post-rinse, and after sanitizing as described
in chapter 3 and 4 (Figure 5-1).
Figure 5-1. Flow chart for CIP procedure of hot milk processing tank using electrolyzed
water.
88
For each trial, cleanliness of the inner surface of the test vessel was assessed using
ATP bioluminescence method and the protein residue detection method. An area of 50
cm2 (5 cm × 10 cm) was swabbed for each cleanliness assessment. The detailed
procedures for swabbing sampling and cleanliness assessment were elaborated in Chapter
3. Table 5-3 contains a summary of the RLU value and protein residue level determined
after each assessment. “RLU0” and “Protein0” represent the responses of cleanliness after
pre-cleaning. These assessments were taken to ensure each trial started with a clean tank
i.e. an RLU = 0 and undetectable protein residues. The measurements for “RLU1” and
“Protein1” were taken immediately after soiling and draining; those for “RLU2” and
“Protein2” were taken immediately after pre-rinse with water and prior to the ER wash
step; those for “RLU3” and “Protein3” were taken after ER water wash and post-rinse
step; those for “RLU4” and “Protein4” were taken after the EO water sanitizing step (at
the end of CIP cleaning).
Table 5-3. Summary of cleanliness assessments at different sampling points.
Sampling point Name of responses
After pre-clean Assessment0 RLU0 Protein0
After soiling Assessment1 RLU1 Protein1
After pre-rinse Assessment2 RLU2 Protein2
After alkaline wash and post-rinse Assessment3 RLU3 Protein3
After sanitizing Assessment4 RLU4 Protein4
89
5.2.5 Other potential factors (Nuisance factor)
Besides the four factors of primary interest (wash temperature, wash time,
sanitizing temperature, and sanitizing time), there were several other factors that may
affect the measured responses as well. Those factors were termed nuisance factors. Data
for potential nuisance factors were monitored and recorded. The nuisance factors are
described below:
5.2.5.1 Different batches of electrolyzed water
Since the shelf-life of electrolyzed water is short, fresh electrolyzed water was
generated prior to each trial. Although electrolyzed water was generated using the same
unit as mentioned in Chapter 3 and Chapter 4 (ROX20, Hoshizaki America Inc.,
Peachtree City, GA). The chemical properties, such as pH, ORP, and hydroxide ions or
chlorine concentrations, varied slightly, within certain ranges. As mentioned above, the
chemical properties of both alkaline ER water and acid EO water were tested and
recorded for further statistical analyses.
5.2.5.2 Day effect
It took 2.5 to 3 hours to complete one trial, so the 27 trials were conducted on
different days. In this case, day was a potential nuisance factor. To be specific, the
temperature and humidity of the pilot plant environment varied on different days. After
soiling and before cleaning, the test vessel was opened for assessing cleanliness. During
90
the time required for assessment, drying and hardening of milk deposits could occur
because the inner surface of the test vessel was hot. This drying and hardening process
was described by Fryer (2009) as “ageing”, the last stage of the fouling processes, in
which the properties of deposit might change. The ambient temperature and humidity
might also influence the ageing process. In an attempt to account for this issue, the
ambient temperature and humidity for each test day were monitored and recorded.
5.2.5.3 Different batches of milk used for soiling
Because the course of the study was longer than the shelf-life of milk used for
soiling, use of more than one batch of milk could not be avoided. Thus, differences in
milk composition between batches, such as fat content and total solids content, might
influence fouling. To address this nuisance factor, a record of code date of each batch of
milk was kept for further tracking.
Even when using the same batch of milk, trials conducted on different days were
actually using milk of different ages. Thus, quality of milk could change slightly during
the storage. To determine the influence of age of milk, the days between trial running
day and milk expiration day (code date) was calculated and recorded for further statistical
analyses.
91
5.2.5.4 Heating conditions during soiling
As described above, soiling of the test vessel with milk under heated conditions
could be divided into two stages. In the 1st stage - whole milk was added to the test
vessel and heated from 4°C to 74°C, by circulating hot water through the jacket of the
test vessel. During the 2nd
stage, once a temperature of 74°C was achieved, injection of
hot water was stopped, and the test vessel was held for another 15 min at 73~75°C. The
steam status varied on different days, which influenced the rate of temperature increase
and thus the time of soiling. In other words, it took a longer time for heating milk to
74°C when the steam pressure was lower, so the test vessel was soiled for a longer time.
To deal with this variation, the heating time was recorded for further analyses. Thus, the
actual soiling time should be heat time (1st stage) + 15 min (2
nd stage).
During the 2nd
stage of soiling, the temperature of milk dropped slightly due to
loss of heat. The highest temperature during soiling (Th), and the final temperature at the
end point of soiling (Tf) were recorded. To deal with this temperature gradient, a
logarithmic mean temperature (TLMT), was considered the actual effective temperature of
2nd
stage soiling, and was calculated using the following equation (Dev et al., 2014):
TLMT =
-- Equation 2
To summarize, several nuisance factors were considered; namely the chemical
properties of electrolyzed water (pH, ORP, concentration of hydroxide ions,
concentration of total and free chlorine), ambient temperature, ambient humidity, milk
code date, days before milk expiration date, heating time, and TLMT. In order to
92
determine which nuisance factors were important enough to be included in the models for
statistical analyses, correlations between nuisance factors and measured responses were
evaluated using Minitab 16 (Minitab Inc., State College, PA).
5.2.6 Data analyses and modeling
Although the operating temperatures of CIP procedures could be controlled using
a coil heat exchanger and monitored by temperature probe, the actual measured operating
temperatures diverged slightly from the target temperatures called for in the response
surface design. For data analyses and modeling, the actual temperature and time of the
ER water wash and EO water sanitizing were used, rather than the target values set by the
response surface design.
5.2.6.1 Regression modeling and optimization of RLU3 data after wash and
post-rinse
Measurements of RLU3 (assessment of cleanliness) were taken immediately after
the alkaline ER water wash and post-rinse with water. Hence, only the temperature and
time of the alkaline ER water wash step was included in the model associated with RLU3.
Thus the predict variables of the model were “WashTemp” and “WashTime”. In order to
determine the effect of ER water and eliminate variance due to levels of fouling, the
percent reduction in RLU from before the alkaline wash step to after the wash and post-
rinse steps was calculated using the equation:
-- Equation 3
93
Percentage reduction RLU would take on a value between 0 and 100%,
representing the proportion of ATP residue removed by the ER wash and post-rinse steps.
In order to improve the residual behavior of response surface regression, natural
logarithms transformations were made. Thus, the response used for regression modeling
and optimization was
-- Equation 4
The goal of the optimization was to determine a temperature and time
combination of the ER water treatment required to achieve a 100% RLU reduction or to
achieve
= 0. Regression modeling and optimization were conducted
using the response surface tools in Minitab 16 (Minitab Inc., State College, PA, USA).
5.2.6.2 Regression modeling and optimization of RLU4 data after sanitizing
Measurements of RLU4 (assessment following complete the CIP protocol) were
taken immediately after acid EO water sanitizing. There were four predict variables in
this model: “WashTemp” represented temperature of the ER water wash step;
“WashTime” represented the treatment time of the ER water wash; “SaniTemp”
represented the temperature used for EO water sanitizing; and “SaniTime” represented
treatment time used for EO water sanitizing. Considering the initial RLU variation,
reduction in RLU from after pre-rinse (RLU2) to after acid EO water sanitizing (RLU4)
was employed as the model response (RLU2-RLU4).
94
According to preliminary experiments, RLU4 readings were likely to be 0 after
the sanitizing step, which was desired. However, not all RLU4 values of 0 represented
equal cleaning efficacy, because (1) the RLU values at the beginning of the process were
different among trials; (2) of limitations in by the sensitivity of ATP analyzer, the actual
RLU values might slightly vary but result in an RLU reading of 0. Thus, the RLU
reduction (RLU2-RLU4) can only be observed to be as high as the initial RLU values,
although a certain combination of the factor levels may have the capability to remove
more milk residue than was present. To address this situation, data were analyzed using a
normal response surface model, along with censoring-type regression model.
In the censoring-type regression model, if the observed “RLU4” value is larger
than zero, which means the observed “RLU2 - RLU4” value were actual RLU reduction,
the designated censor indicator is “0” indicating uncensored. If the observed “RLU4”
value equals zero, which means the observed response of “RLU2 – RLU4” may be less
than the actual RLU reduction, the designated censor indicator is “1” to indicate the
response was censored.
Regression modeling applied to evaluate the effects of the 4 CIP factors described
earlier (“WashTemp”, “WashTime”, “SaniTemp”, and “SaniTime”) on the response RLU
reduction (“RLU2 – RLU4). The response optimization was conducted using predicted
“RLU2 – RLU4” values obtained from the regression model. Both analyses were
conducted using Minitab 16 (Minitab Inc., State College, PA, USA).
95
5.2.6.3 Regression modeling of protein data
Assessment of milk residue in terms of protein was treated as a categorical
variable, because the protein residue was measured by visual inspection of color changes
in protein swabs. As described in Chapter 2, after sampling and testing, swab colors of
green (score 1), gray (score 2), light purple (score 3) and dark purple (score 4) indicated
protein residue levels of < 20 µg, 20~40 µg, 40~100 µg and > 100 µg, respectively.
Since protein measurements actually represent the amount of protein residue, which is
continuous variable, the protein measurements (scores of 1~4) could be considered as
ordinal variables. The goal of CIP with electrolyzed water was to reduce the milk soil
in terms of protein residue to undetectable level (< 20 µg or green color of swab). A
cumulative logistic regression model was employed to estimate the probability of
obtaining a certain level of protein residue. The response of the model was
= αj + β𝑥i -- Equation 5
where π stands for the probability of obtaining protein detection score at or below value j,
e.g. when j =1, π stands for the probability of protein detection score of 1 with the
amount of protein < 20 µg; when j =2, π stands for the probability of protein detection
scores of 1 and 2, with the amount of protein < 40 µg; The data analyses and regression
modeling were conducted using Minitab 16 (Minitab Inc., State College, PA, USA).
96
5.2.7 Validation
The CIP procedure using electrolyzed water with optimal parameters, namely
temperature and time for wash and sanitizing steps was evaluated. The evaluation was
conducted following the same soiling and cleaning steps elaborated in Figure 5-1.
Cleanliness was assessed using ATP bioluminescence assay and protein residue
detection. The cleaning efficacy of optimal CIP procedures was compared with the
treatment using commercial CIP chemicals (positive control) and the treatment using
only water (negative control). Each treatment was conducted in triplicate. RLU values
obtained from ATP bioluminescence assays were converted in logarithms. To compared
log10(RLU) values between treatments, one-way ANOVA and Tukey’s comparison were
conducted using Minitab 16 (Minitab Inc., State College, PA, USA) at confidence level
of 95% (α = 0.05).
5.3 RESULTS AND DISCUSSION
5.3.1 Assessment the importance of nuisance factors
To determine if the nuisance factors were important enough to be included in
regression models, any possible correlations between variables including nuisance factors
and response variables were identified. The outputs of analyses for correlation are
displayed in Appendix (Figure A-4 to A-7). Matrix plots of variables were also
constructed to confirm the information about correlation between variables. The
presence of a linear pattern in a plot suggested the corresponding pair of variables are
97
correlated (Figure A-8 to A-11). Table 5-4 listed the pairs of variables that had relatively
high correlations (p-value = 0.000 ~ 0.052). The Pearson correlation (r) with a range of -
1 to 1 indicates the correlation between a pair of variables: Pearson correlation value
close to -1 or 1 indicates strong linear relationship between variables. Although some
nuisance factors were found to be related to each other with Pearson correlations close to
1 or -1, correlation analyses suggested that none of the nuisance factors are correlated
with the responses (RLU values or protein scores). Thus, it was not necessary to include
any nuisance factors within the regression models.
Table 5-4. P-values of Pearson correlations between variables. Pearson correlation value
close to -1 or 1 indicated the corresponding pair of variables may be related.
Variable 1 Variable 2
Pearson
correlation (r)
p-value of
correlation
RLU1 RLU2 0.600 0.001
ambient temperature code date 0.591 0.002
TLMT* ambient temperature 0.537 0.007
TLMT ambient humidity 0.410 0.052
ORP of acid EO water pH of acid EO water 0.289 0.000
[Total Cl] of EO water pH of EO water -0.885 0.000
[Total Cl] of EO water pH of ER water 0.491 0.009
[Free Cl] of EO water pH of EO water -0.848 0.000
[OH-] of ER water pH of ER water 0.995 0.000
[Free Cl] of EO water [Total Cl] of EO water 0.974 0.000
[Total Cl] of EO water ORP of acid EO water 0.657 0.000
[OH-] of ER water [Total Cl] of EO water 0.503 0.008
* TLMT refers to logarithmic mean temperature during soiling.
Additional data in Appendix A4-A11.
98
5.3.2 Regression model of RLU3 data after wash and post-rinse
Response surface regression analysis for the response
is presented
in Table 5-5. The ER water treatment time was significant at a 95% confidence level (p-
value < 0.05). The ER water treatment temperature and interaction of ER water treatment
time and temperature were significant at the confidence level of 90% (p-value < 0.1).
The coefficient of ER water treatment temperature and time are positive, which indicated
that at higher temperatures and longer times ER water treatment was more effective at
removing ATP contained in the milk residue. To better visualize the model, contour plot
and surface plot were employed (Figure 5-2 and 5-3). The contour plot indicated that,
with certain settings of temperature and time of ER water treatment, a greater than 98.5%
RLU reduction could be achieved (
> -0.02). The regression
model of
is shown in Equation 6:
[ ]
[ ] [
]
-- Equation 6
The low R2 of the model, 48.12%, might be due to the variation of initial RLU values,
however the lack-of-fit test statistic (p-value = 0.721) suggested the model fits well.
99
Table 5-5. Response surface regression: “
” as a function of ER water
treatment temperature and time (WashTemp = temperature of ER water treatment;
WashTime = treatment time of ER water wash).
Term Coefficient P-value
constant -0.603766 0.011
WashTemp 0.013945 0.068
WashTime 0.020466 0.011
WashTemp × WashTemp -0.000085 0.195
WashTime × WashTime -0.000199 0.128
WashTemp × WashTime -0.000222 0.068
WashTemp (°C)
Wa
sh
Tim
e (
min
)
656055504540
25
20
15
10
5
>
–
–
–
–
–
–
–
–
–
–
<
-0.039 -0.027
-0.027 -0.015
-0.015
-0.135
-0.135 -0.123
-0.123 -0.111
-0.111 -0.099
-0.099 -0.087
-0.087 -0.075
-0.075 -0.063
-0.063 -0.051
-0.051 -0.039
ln((RLU2-RLU3)/RLU2)
Figure 5-2. Contour plot of
versus treatment time and temperature of ER
water wash, generated based on regression model (Equation 6).
100
24
18-0.12
-0.08
12
-0.04
40
0.00
50 660
70
ln((RLU2-RLU3)/RLU2)
WashTime (min)
WashTemp (°C)
Figure 5-3. Surface plot of
versus treatment time and temperature of ER
water wash, generated based on regression model (Equation 6).
Figure 5-4 presents the optimization of ER water treatment temperature and time
to achieve a
target of 0. Results of optimization indicate that, when the
wash temperature was 54.6°C and the time was 20.5 min, the response
was predicted to be -0.0092, which was the maximum, but was still lower than the target
of 0. The corresponding RLU percentage reduction
would be 99.08%. The
absolute RLU3 value was predicted for better interpretation of the cleanliness. To predict
the RLU3 value, the mean RLU2 value of 33 trials (27 trials of response surface model, 6
trials of control treatments) was used, which was 462,105. Thus, when CIP cleaning with
ER water for 20.5 min at 54.6°C, after the post-rinse steps, 99.08% of ATP was removed
101
and the predicted RLU3 value is 4251, which indicated the surface was not clean.
Therefore, it can be concluded that the additional rinse with acid EO water step was
necessary to completely clean the system using electrolyzed water.
Figure 5-4. Optimization plot for
versus treatment time and temperature of
ER water wash. The plot suggested the highest desirability was 0.98, when setting ER
water wash temperature at 54.6°C and time at 20.5 min, and the predicted
= -0.0092, indicating 99.08% ( of RLU reduction.
102
5.3.3 Regression model of RLU4 data after sanitizing
Table 5-6 presents results of regression analysis of “RLU2-RLU4” (representing
ATP reduction after cleaning and sanitizing). The factors of temperature and time of ER
water treatment, and the factor of time for acid EO water treatment were significant at a
90% confidence level (p-value < 0.1). Acid EO water treatment temperature was not
significant (p-value = 0.24). The positive coefficients indicated that the cleaning
procedure had better performance at higher temperatures and longer treatment times. The
regression model used for calculating predicted “RLU2 – RLU4” values was:
-- Equation 7
Equation 7 was used for predict value. In the response optimizer
function of Minitab 16 (Minitab Inc., State College, PA, USA), the variables of
“
” and predicted “ ”, were selected to determine an
optimum combination of parameters for best efficacy of CIP cleaning using electrolyzed
water for the test vessel soiled with heated milk. An optimum combination of four
factors was determined to achieve the target value of 0 for “
” and to
achieve a maximum value of “RLU2 – RLU4”. The optimum condition is presented in
Figure 5-5, which suggested that for CIP procedure using electrolyzed water -washing the
103
test vessel with ER water for 22 min at 53.7°C, and then sanitizing the with acid EO
water for 10 min at 25°C after rinse with water-could achieve a desired RLU reduction of
2×106, which is larger than any observed RLU reductions between RLU2 and RLU4. It
may be concluded that it is possible to remove all ATP residue by the CIP procedure
using this optimum combination of electrolyzed water treatment parameters.
Converting the predicted “
” value (–0.0092) to percentage
reduction of RLU3, predicts that 99.08% of ATP residue could be removed by treatment
with ER water for 22 min at 53.1°C. This observation agreed with the conclusion of
response regression modeling and optimization of “
” in section 5.3.2.
Table 5-6. Regression of “RLU2-RLU4” versus ER water and EO water treatment
factors (WashTemp = temperature of ER water treatment; WashTime = treatment
time of ER water wash; SaniTemp = temperature of EO water treatment; SaniTime =
treatment time of EO water sanitizing).
Term Coefficient P-value
constant -2743012 0.062
WashTemp 33653.6 0.085
WashTime 155015 0.047
SaniTemp 31156.4 0.243
SaniTime 381579 0.035
WashTemp*WashTime -2309.31 0.101
SaniTemp*SaniTime -9372.32 0.066
104
Figure 5-5. Optimization plot for “RLU2-RLU4” and “ln((RLU2-RLU3)/RLU2)”. When
setting ER water wash parameters at 53.7°C for 21.6min, setting EO water sanitizing
parameters at 25°C for 10 min, it can be predicted that RLU reduction achieved 99.08%
( after ER water treatment, 1.99 × 106 after EO water treatment, respectively.
5.3.4 Regression model of Protein data
Protein residue detection provided additional confirmation for the response
modeling results. Protein3 was measured after ER water wash and post-rinse. The
regression model presented below:
105
[ ] [ ]
-- Equation 8
In this model, is the constant coefficient of the regression model used to predict the
probability of observing residual protein levels at or below value j (j = 1, 2, 3, or 4):
If j=1, then , which is the constant coefficient for predicting the
probability of observing green in protein residue detecting (residue protein level ≤ 20 μg
per 50 cm2 of surface);
, which is the constant coefficient for predicting the
probability of observing green and gray in protein residue detecting (residue protein
level ≤ 40 μg per 50 cm2 of surface).
When using Equation 8 to predict the probability of observing green in residual
protein assay after cleaning with ER water at 54.6°C for 20.5 min, which are optimal
parameters obtained according RLU data as described above, plug in parameters
( , WashTemp = 54.6°C, WashTime = 20.5 min) into the Equation 8 above.
This leaded to a
-1.707. Thus, the probability of observing green in protein
residue detection would be 15.35%, obtained from the equations below:
P(Y = 1) = π =
= 15.35%
-- Equation 9
106
The analysis of Protein3 indicated that, after rinsing with cold water for 3 min,
wash with ER water for 20.5 min at 54.6°C, and rinse with cold water again for 3 min,
the probability of reduce protein residue of inner tank surface less than 20 µg per 50 cm2
was low (15.35%). This observation was further confirmation that the cleaning with only
ER water was not sufficient to return the test vessel to an acceptably clean condition, and
that the acid EO water sanitizing step was necessary.
Protein4 was measured after sanitizing. There were 25 observations of green out
of 27 trials. A similar cumulative logistic regression was attempted, but the regression
model was found not to be reliable. However, the high frequency (92.6%) observations
of green in protein detection test, suggests that CIP procedures conducted with
electrolyzed water at the optimum combinations of parameters obtained according RLU
response modeling was able reduce residual proteins to an acceptable cleaning condition.
5.3.5 Validation
The CIP procedure using electrolyzed water with optimal parameters was
validated, and its cleaning efficacy was compared to treatments using commercial CIP
chemicals (positive control) and using tap water (negative control). The RLU values and
protein residue measurements are presented in Figure 5-6 and Tables 5-7. The negative
control procedure using water failed to remove all milk soil, in terms of both ATP and
protein residues. The results eliminated the effect of mechanical force or flow rate, and
further indicated the effectiveness of electrolyzed water as CIP reagents. Both
electrolyzed water and positive treatments returned the test vessel to clean conditions
107
after the complete four-step CIP procedures. However, after the alkaline wash step with
ER water at 53.3°C for 21 min and post-rinse with water for 3 min at room temperature,
the RLU reduction achieved 97.5%, and low level of protein residue was detected. While
the positive control, using conventional CIP procedure with commercial cleaning
chemicals, achieved a 100% reduction of RLU values after alkaline wash and post-rinse
step, and the protein residue was reduced to undetectable levels. Thus, the commercial
chemicals employed worked better than the electrolyzed water. This may result from the
low alkalinity (12-216 ppm) (Abramowitz and Arnold, 2002) of ER water with <100 ppm
of hydroxide ions concentration ([OH-]). In contrast, the working solution of the
commercial CIP detergent (Principal®, Ecolab USA Inc.) contains ca. 585 ppm of sodium
hydroxide and 117 ppm of sodium hypochlorite. In addition, The Dairy Practice Council
(DPC, 2001) recommended an alkalinity of 1500~1800 ppm of alkaline detergent that
used for CIP procedures of storage tank. However, both a complete 4-step electrolyzed
water treatment and the positive control treatment were able to return the inner surface of
the test vessel to acceptable cleanliness with 0 RLU value and undetectable protein
residue levels.
108
Figure 5-6. Means of RLU values comparison between validation experiment and control
treatments. Error bar indicates standard deviation of triplicate analysis. Tukey’s
comparisons were conducted between all RLU values. Means that do not share a letter
are significantly different (α = 0.05). (Pos. Ctrl = positive control; EW validation =
electrolyzed water treatment with optimal parameters; Neg. Ctrl = negative control).
Table 5-7. Protein detection of validation experiment and control treatments (where Pos.
Ctrl = positive control; EW validation = electrolyzed water treatment with optimal
parameters; Neg. Ctrl = negative control).
Treatment Trials Protein1
(ug/ 50 cm2)
Protein2
(ug/ 50 cm2)
Protein3
(ug/ 50 cm2)
Protein4
(ug/ 50 cm2)
Pos. Ctrl. 1 > 100 > 100 0 ~ 20 0 ~ 20
2 40~60 > 100 0 ~ 20 0 ~ 20
3 > 100 > 100 0 ~ 20 0 ~ 20
EW
validation 1 > 100 > 100 0 ~ 20 0 ~ 20
2 > 100 > 100 20 ~ 40 0 ~ 20
3 > 100 > 100 20 ~ 40 0 ~ 20
Neg. Ctrl. 1 > 100 > 100 > 100 40~60
2 40~60 > 100 40~60 20 ~ 40
3 40~60 > 100 > 100 20 ~ 40
a a
d d
a a
c
d
a a
ab b
1
10
100
1,000
10,000
100,000
1,000,000
After Soiling After Pre-rinse After Post-rinse After Sanitizing
RLU
Pos ctrl
EW validation
Neg ctrl
109
5.4 CONCLUSION
Optimum parameters, in terms of temperature and time of alkaline ER water and
acid EO water within a 4-step CIP procedure for the pilot scale dairy processing tank,
were determined using response surface modeling.
The response surface regression model was applied to the RLU data both before
and after an EO water sanitizing step (RLU3 and RLU4). Using the RLU3 data, several
regression models of the response surface design were employed. The model with
response of
exhibited the best fit of the models that were evaluated. The
low R2 (48.12%) and adjusted R
2 (35.76%) might due to the variance of RLU
measurements. The model indicated that both ER water treatment temperature and time
were statistically significant at 90% confidence level (p-valueWashTemp = 0.011, p-
valueWashTime = 0.068). The optimum setting of ER water treatment obtained from the
model (54.6°C for 20.5 min), was expected to reduce 99.17% of ATP during first three
steps of CIP (pre-rinse with water – ER water wash – post-rinse with water).
The regression model of RLU4 data suggested that the ER water treatment
temperature and time, and EO water treatment time were statistically significant at a
confidence level of 90% (p-valueWashTemp = 0.085, p-valueWashTime = 0.047, p-valueSaniTime
= 0.035). The optimization result suggested similar settings for ER water treatment
(53.1°C for 22 min), and the settings for EO water treatment (25°C for 10 min). CIP
procedure using electrolyzed water with the optimum settings was predicted to reduce
RLU values by 2 ×106.
110
A cumulative logistic regression model was applied for protein data. The
regression model suggested that ER water treatment alone was not sufficient to remove
all protein residues. The 4-step CIP procedure using electrolyzed water at optimum
settings-wash with ER water at 54.6°C for 20.5 min and sanitize with EO water at 25°C
for 10 min was capable of reducing the protein residue to undetectable level.
A four step CIP procedure using electrolyzed water provided similar cleaning
capability to a conventional CIP procedure. However, direct comparison between CIP
procedures using electrolyzed water and conventional cleaning and sanitizing reagent
suggested that the ER water was less effective than commercial alkaline detergent in
removal of soil from surface fouled by heat treatment. The cleaning efficacy of ER water
might be improved by increasing sodium hydroxide concentration or adding other
cleaning ingredients such as surfactants as wetting/dispersing agents.
111
Chapter 6
CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE
RESEARCH
This study demonstrated that using electrolyzed water (EW) was successful in
removal of soil from the surface of a test vessel soiled with cold milk i.e. mimicking milk
refrigerated storage tanks. By using optimum temperatures and times of a CIP procedure,
electrolyzed water was also found to be capable of cleaning the surface of a test vessel
that was used to heat milk, but was less effective when compared to commercial CIP
detergent and sanitizers. For future research, it would be interesting to improve the
cleaning efficacy of electrolyzed water, especially alkaline ER water, by adding some
surfactant, such as teepol (alkyl aryl sulphonate) or sodium triphosphate, which can serve
as wetting agent.
During the study, corrosion was observed on some surfaces when using acid EO
water as a sanitizer. This observation may occur because chlorine is not stable at low pH
(2.3~2.7) and chlorine gas (Cl2) is released, which is corrosive to many metals, including
stainless steel. To address this drawback, two potential solutions could be considered: (1)
using neutral electrolyzed water with less corrosive characteristics (pH = 7 to 8, ORP =
750 mV, 50-500 ppm of free chlorine, depending on water electrolyzer), which can be
produced by mixing acid EO water with alkaline ER water after electrolysis, or by using
a single electrolysis chamber without semi-permeate membrane; (2) adding corrosion
inhibitors, such as silicates to the acid EO water to prevent corrosion.
112
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APPENDIX:
Figure A-1: 16s rRNA Gene sequence alignment of Pseudomonas fluorescens using BLAST
121
Figure A-2: 16s rRNA Gene sequence alignment of Enterococcus faecalis using BLAST
Figure A-3:16s rRNA Gene sequence alignment of Escherichia coli using BLAST
122
Correlations: RLU3, RLU4, pH (H), pH(OH), ORP (H) (mV), ORP (OH) (mV, ... RLU3 RLU4 pH (H) pH(OH)
RLU4 0.157
0.433
pH (H) 0.016 -0.011
0.937 0.958
pH(OH) 0.080 -0.245 -0.322
0.690 0.217 0.102
ORP (H) (mV) 0.123 -0.181 -0.679 0.523
0.540 0.366 0.000 0.005
ORP (OH) (mV) -0.086 -0.096 0.289 -0.216
0.671 0.634 0.144 0.278
Total Cl (ppm) 0.047 0.010 -0.885 0.491
0.815 0.961 0.000 0.009
Free Cl (ppm) 0.064 0.035 -0.848 0.437
0.752 0.862 0.000 0.023
[OH] (ppm) 0.069 -0.231 -0.333 0.995
0.731 0.247 0.089 0.000
ORP (H) (mV) ORP (OH) (mV) Total Cl (ppm) Free Cl (ppm)
ORP (OH) (mV) -0.130
0.517
Total Cl (ppm) 0.657 -0.279
0.000 0.158
Free Cl (ppm) 0.618 -0.203 0.974
0.001 0.310 0.000
[OH] (ppm) 0.517 -0.221 0.503 0.444
0.006 0.269 0.008 0.020
Cell Contents: Pearson correlation
P-Value
Figure A-4: Correlations between RLU3, RLU4, and chemistry properties of electrolyzed water (pH
values, ORP of both solutions, total and free chlorine concentration of EO water, hydroxide ion
concentration of ER water). The highlighted Pearson correlations that were closed to 1 or -1 indicated
relatively strong correlation of the corresponding pair of variables.
123
Correlations: RLU1, RLU2, RLU3, RLU4, code date, days before , amb-temp, ... RLU1 RLU2 RLU3
RLU2 0.600
0.001
RLU3 0.150 -0.098
0.454 0.628
RLU4 0.159 -0.250 0.157
0.429 0.208 0.433
code date 0.107 -0.072 -0.210
0.595 0.723 0.293
days before expi -0.055 0.238 -0.028
0.785 0.232 0.890
amb-temp 0.053 -0.212 0.277
0.807 0.320 0.190
amb_hum -0.053 0.145 0.346
0.811 0.510 0.106
HtTime (min) 0.015 -0.155 0.050
0.959 0.598 0.866
HtTemp(LMT) 0.002 -0.152 0.159
0.994 0.449 0.427
RLU4 code date days before expi
code date 0.173
0.387
days before expi -0.030 -0.088
0.880 0.662
amb-temp 0.161 0.591 -0.087
0.453 0.002 0.687
amb_hum -0.215 -0.144 0.484
0.324 0.511 0.019
HtTime (min) -0.079 -0.168 -0.144
0.789 0.565 0.624
HtTemp(LMT) -0.058 0.181 -0.189
0.773 0.367 0.346
amb-temp amb_hum HtTime (min)
amb_hum 0.149
0.531
HtTime (min) 0.347 0.207
0.245 0.478
HtTemp(LMT) 0.537 0.410 0.411
0.007 0.052 0.144
Cell Contents: Pearson correlation
P-Value
Figure A-5: Correlations between RLU1, RLU2, RLU3, RLU4 and other nuisance factors. The data
showed no strong correlations between responses and nuisance factors.
124
Correlations: Protein3, Protein4, pH (H), pH(OH), ORP (H) (mV), ... Protein3 Protein4 pH (H) pH(OH)
Protein4 0.347
0.077
pH (H) -0.181 0.308
0.367 0.118
pH(OH) 0.104 -0.156 -0.322
0.605 0.436 0.102
ORP (H) (mV) 0.116 -0.473 -0.679 0.523
0.566 0.013 0.000 0.005
ORP (OH) (mV) -0.296 -0.075 0.289 -0.216
0.134 0.710 0.144 0.278
Total Cl (ppm) 0.138 -0.183 -0.885 0.491
0.493 0.360 0.000 0.009
Free Cl (ppm) 0.136 -0.204 -0.848 0.437
0.498 0.307 0.000 0.023
[OH] (ppm) 0.104 -0.166 -0.333 0.995
0.606 0.409 0.089 0.000
ORP (H) (mV) ORP (OH) (mV) Total Cl (ppm) Free Cl (ppm)
ORP (OH) (mV) -0.130
0.517
Total Cl (ppm) 0.657 -0.279
0.000 0.158
Free Cl (ppm) 0.618 -0.203 0.974
0.001 0.310 0.000
[OH] (ppm) 0.517 -0.221 0.503 0.444
0.006 0.269 0.008 0.020
Cell Contents: Pearson correlation
P-Value
Figure A-7: Correlations between Protein3, Protein4, and chemistry properties of electrolyzed water
(pH values, ORP of both solutions, total and free chlorine concentration of EO water, hydroxide ion
concentration of ER water). The highlighted Pearson correlations that were closed to 1 or -1
indicated relatively strong correlation of the corresponding pair of variables.
125
Correlations: Protein1, Protein2, Protein3, Protein4, code date, ... Protein1 Protein2 Protein3
Protein2 *
*
Protein3 0.060 *
0.767 *
Protein4 0.143 * 0.347
0.477 * 0.077
code date -0.372 * -0.210
0.056 * 0.293
days before expi 0.055 * 0.108
0.786 * 0.592
amb-temp -0.354 * 0.144
0.090 * 0.501
amb_hum -0.234 * 0.029
0.282 * 0.894
HtTime (min) -0.269 * 0.279
0.352 * 0.334
HtTemp(LMT) -0.327 * 0.156
0.096 * 0.438
Protein4 code date days before expi
code date -0.289
0.144
days before expi 0.383 -0.088
0.049 0.662
amb-temp -0.128 0.591 -0.087
0.552 0.002 0.687
amb_hum 0.219 -0.144 0.484
0.315 0.511 0.019
HtTime (min) -0.250 -0.168 -0.144
0.388 0.565 0.624
HtTemp(LMT) -0.174 0.181 -0.189
0.385 0.367 0.346
amb-temp amb_hum HtTime (min)
amb_hum 0.149
0.531
HtTime (min) 0.347 0.207
0.245 0.478
HtTemp(LMT) 0.537 0.410 0.411
0.007 0.052 0.144
Cell Contents: Pearson correlation
P-Value
* NOTE * All values in column are identical.
Figure A-8: Correlations between Protein1, Protein2, Protein3, Protein4, and other nuisance
factors. The data showed no strong correlations between responses and nuisance factors.
126
Figure A-9: Matrix plot of RLU3, RLU4, and chemistry properties of electrolyzed water (pH values,
ORP of both solutions, total and free chlorine concentration of EO water, hydroxide ion concentration
of ER water). Linear pattern of plot that was circled indicated that the corresponding pair of variables
was correlated.
127
Figure A-10: Matrix plot of RLU1, RLU2, RLU3, RLU4 and other nuisance factors. Linear pattern
of plot indicated that the corresponding pair of variables was correlated.
128
Figure A-11: Matrix plot of Protein3, Protein4, and chemistry properties of electrolyzed water (pH
values, ORP of both solutions, total and free chlorine concentration of EO water, hydroxide ion
concentration of ER water). Linear pattern of plot that was circled indicated that the corresponding
pair of variables was correlated.
129
Figure A-12: Matrix plot of Protein1, Protein2, Protein3, Protein4, and other nuisance factors.
Linear pattern of plot that was circled indicated that the corresponding pair of variables was
correlated.