1

AN ALTERNATIVE METHOD AND DESIGN TO INACTIVATE BIOLOGICAL CONTAMINANTS IN

MILK BY PULSED ELECTRIC FIELD TREATMENT FOR SMALL-SCALE MILK OPERATIONS

Department of Bioresource Engineering, Macdonald Campus, McGill University, Ste.-Anne-de-Bellevue,

Quebec

C. Jang, I. Han, E. Riskulov, T. Rajchgot, D. Stanger

*16 Pages Before the Appendix

ABSTRACT

Presented is an alternative method and design to conventional heat treatment of milk for a small scale

milk operation. The literature review aimed to understand the limits of standard pasteurization and to

explore better treatment methods. Several treatment methods were researched and analyzed to

ascertain an optimal treatment for milk. The PEF method was found to be optimal on the grounds that it

optimally addressed objective of treating raw milk for a small-scale operation without altering the

desired natural qualities of the product, while at the same time mitigating the associated health risks

and ensuring the safety of consumers. A design section is provided which describes the flow design, PEF

design parameters, and pump design.

CONTENTS

Abstract .................................................................................................................................................................... 1

Contents ................................................................................................................................................................... 1

1. Introduction.......................................................................................................................................................... 2

2.0 an Overview of Raw and Pasteurized Milk .......................................................................................................... 3

Pasteurization Definition ...................................................................................................................................... 4

3.0 Literature Review of Alternative Technologies .................................................................................................... 4

3.1 Batch Pasteurization ....................................................................................................................................... 4

3.2 Microwave Treatment .................................................................................................................................... 5

Introduction ...................................................................................................................................................... 5

Mechanism of Inactivation ................................................................................................................................ 5

Food Safety ....................................................................................................................................................... 5

Food Quality ..................................................................................................................................................... 6

3.3 Pulsed Electric Field (PEF) Processing .............................................................................................................. 6

2

Introduction ...................................................................................................................................................... 6

Mechanism of inactivation ................................................................................................................................ 6

Food Safety ....................................................................................................................................................... 7

Food Quality ..................................................................................................................................................... 8

3.4 High Pressure Processing (HPP) ....................................................................................................................... 8

Introduction ...................................................................................................................................................... 8

Food Safety ..................................................................................................................................................... 10

Food Quality ................................................................................................................................................... 11

3.6 Ultraviolet Processing ................................................................................................................................... 11

Introduction .................................................................................................................................................... 11

Mechanism of Operation ................................................................................................................................ 12

Efficiency ........................................................................................................................................................ 13

Food Safety ..................................................................................................................................................... 13

Food Quality ................................................................................................................................................... 13

4. Design................................................................................................................................................................. 14

Final Design ........................................................................................................................................................ 14

Process ........................................................................................................................................................... 15

Flow Design .................................................................................................................................................... 15

Pump Design ................................................................................................................................................... 16

PEF Design ...................................................................................................................................................... 16

5. Conclusion .......................................................................................................................................................... 16

6. APPENDIX ........................................................................................................................................................... 18

7. Works Cited ........................................................................................................................................................ 20

1. INTRODUCTION

Dairy is and has been a large, if not the primary, source

of nutrition for mammals throughout history. Raw milk

was the principal form of milk before the development

of extensive processing systems in the 1930s (Potter,

3

1984). Raw milk and its derivatives are still highly

popular in several European countries, such as France,

with growing popularity in North America due to a

growing “consuming natural and/or local” trend (Claeys

et al., 2012). Raw milk has been linked to several

outbreaks of disease stemming from microbial sources

throughout history, and as a result dairy is

conventionally treated with heat to kill pathogenic

microbes. Despite the inherent health risks associated

with the ingestion of raw milk, it is advocated that raw

milk has superior nutritional value, in addition to other

benefits, when compared to its processed counterpart

(Claeys et al., 2012). Processed milk is milk that has

been altered from its natural (raw) state by heating and

various unit processes. This is, in part, due to

denaturation of beneficial enzymes, a process that

occurs during heat treatment and homogenization

amongst other processes. Some argue that pasteurizing

milk also alters the taste and consistency of the product

(Walstra, 1999). It is therefore important to investigate

methods by which to treat raw milk without altering the

desired natural qualities of the product, while at the

same time mitigating the associate health risks and

ensuring the safety of consumers. Some methods of

treatment, such as vat pasteurization, microwave

treatment, high pressure-processing (HPP), ultraviolet

light treatment (UV treatment) and pulsed electric field

(PEF) treatment are said by some to conserve the

properties of raw milk better than conventional

pasteurization. This paper and accompanying design will

explore these possible alternatives in a small dairy farm

setting by comparing and contrasting the associated

risks and benefits of each treatment.

2.0 AN OVERVIEW OF RAW AND PASTEURIZED

MILK

Milk and dairy products are staples of the North

American diet; consumers consistently demand vast

quantities of high-quality product. In recent years,

pasteurized milk has started to be viewed with some

skepticism from consumers of health food, as well as

gourmet foodies. For these consumers, raw,

unpasteurized milk and its derivatives represent a

healthier, tastier alternative to the market standard.

However, there are inherent risks involved in

consumption of such products; if consumed

unpasteurized, these foods can present a health hazard

due to possible contamination with pathogenic

bacteria. These bacteria can originate even from

clinically healthy animals from which milk is derived or

from environmental contamination occurring during

collection and storage of milk. The decreased frequency

of bovine carriage of certain zoonotic pathogens and

improved milking hygiene have contributed

considerably to decreased contamination of milk but

have not, and cannot, fully eliminate the risk of milk

borne disease (Angulo et al., 2009).

Pathogenic milk bacteria, such as species of E. coli,

Campylobacter, and Salmonella, all present challenges

in milk safety. Milk can be contaminated through

collection and processing in the following ways:

1. Cow feces coming into direct contact with the

milk

2. Infection of the cow's udder (mastitis)

3. Cow diseases (e.g., bovine tuberculosis)

4. Bacteria that live on the skin of cows

5. Environment (e.g., feces, dirt, processing

equipment)

6. Insects, rodents, and other animal vectors

7. Humans, for example, by cross-contamination

from soiled clothing and boots (CDC)

At greatest risk for harmful symptoms of milk borne

illness are infants, young children, pregnant women, the

elderly, and those with compromised immune systems.

As a result of several highly-publicized deaths, Canada

has effectively banned the sale of raw milk. This is in

contrast to the European Union’s regulation and

protection of the raw milk market, as it holds historical

and commercial significance in terms of traditional

European dairy products such as raw milk cheese.

French Roquefort, for example, is required by law to be

produced from raw sheep’s milk; pasteurized milk

cheeses are widely panned in the French market. Thus,

what is the difference between these two schools of

thought? In essence, it appears that Canadian dairy

4

hygiene standards are not up to par with their European

counterparts; in addition, the risk involved in

consumption of dairy products, minimal as it may be

with proper treatment, is not considered acceptable by

North American food regulating agencies.

PASTEURIZATION DEFINITION

Pasteurization is the heat treatment of milk in the

service of killing or denaturing harmful pathogens and

degradative enzymes, thus increasing the shelf life of

milk. There are two main types of pasteurization: The

first is High Temperature Short Time (HTST), which is

the treatment of milk at 72oC for 20 seconds, thus

inactivating pathogenic agents present in the liquid.

This typically allows for a refrigerated shelf life of two to

three weeks. The second type, which is less popular in

North America, is Ultra Heat Treatment (UHT), which is

the treatment of milk for 1-2 seconds at 135 oC, which

combined with sterile processing and aseptic packaging,

can extend the shelf life of milk to 6-9 months

unrefrigerated. Unfortunately, this process caramelizes

some of the lactose sugars present in milk, leading to an

oddly sweet taste that North American consumers

dislike.

3.0 LITERATURE REVIEW OF ALTERNATIVE

TECHNOLOGIES

Alternative methods to HTST and UHT are described in

the following literature review. Our team focused on:

batch pasteurization,

3.1 BATCH PASTEURIZATION

The legal definition of pasteurization is the process of

heating every particle of milk and milk products to the

minimum required temperature (for that specific milk

or milk product), and holding it continuously for the

minimum required time in equipment that is properly

designed and operated (WSDA, 2010). Vat

pasteurization (also referred to as batch pasteurization

by some sources) still involves the heating of raw milk

to meet this objective; however, during this process the

heating occurs at lower temperatures than UHP and

HTST, and over an extended period of time (Gao et al.,

2002). The milk is held at a moderate temperature

(usually between 60 and 70 °C) for half an hour and

then quickly cooled. Milk processed by vat

pasteurization retains a higher percentage of the milks

natural enzymes and beneficial bacteria that would be

denatured or killed at higher temperatures. This is said

to contribute to a stronger flavour and added health

benefits of vat pasteurized milk when compared to

HTST and UHP processed milks (Buffa et al., 2001).

Vat pasteurization involves several unit processes. A

typical system involves a stainless steel vat that has

been fitted with water and steam to the jacket liner,

in which the product is heated. Time and temperature

requirements, methods of operation, properly

designed valves, thermometers to monitor and record

product temperatures and some means of agitation to

assure uniformity in temperature distribution should

also be included in a vat pasteurizer system (WSDA,

2010). During this method all of the components (such

as sugar or sweeteners etc.) are added before

pasteurization. The milk and/or other liquid

ingredients are then heated in the pasteurization vat

for 30 minutes at the specified temperature. The

process is monitored and the results are collected and

recorded. Vat pasteurization was the original method

of pasteurization and is now primarily used in the

dairy industry for preparing milk to be used in the

production of cheese, yogurt, buttermilk and some ice

cream mixes (IDFA, 2009).

The Canadian standards for vat pasteurization of dairy

call for heating of products to temperatures between

60 and 70 °C that are maintained over a half-hour

time period. The following are the generally accepted

pasteurization schedules for dairy products produced

by batch pasteurization in Canada: (1) Milk based

products below 10% milk fat (fluid milk, goat milk,

whey) are to be heated to 63°C for 30 minutes, (2)

Milk based products of 10% milk fat or higher, or

added sugar (fluid cream, cream for butter, chocolate

milk, flavoured milk, etc.) are to heated to 66°C for 30

minutes (CFIA, 2013). Standards for vat or batch

pasteurization are also regulated provincially and may

vary from these national standards. If there is variance

5

in the provincial and federal standards, the more strict

standard must be met (CFIA, 2013).

Raw milk is difficult to find in commercial groceries.

This is due to the fact that sale of raw milk for human

consumption is illegal in Canada (Guelph, 2013). In the

United States the sale of raw goat and sheep’s milk is

legal in some states and not others, however it is

technically illegal to produce raw cow’s milk to sell for

human consumption (Fund, 2013). Where raw milk is

available for purchase, consumers will find that the

cost of this product is significantly higher than that of

milk processed through HTST. Vat pasteurization has

higher energy requirements than the notably shorter

process of HTST pasteurization. The lengthier heating

time involved with vat pasteurization has a higher

operating cost than HTST, which is ultimately reflected

in the market price of vat pasteurized products

(Systems, 2013). Therefore many consumers who wish

to reap the benefits of raw milk, without the

associated health risk, must accept the additional cost

when turning to vat pasteurized milk alternative.

3.2 MICROWAVE TREATMENT

INTRODUCTION

Microwave treatment is the intense heat treatment of a

product in a microwave at high frequency. Although it

has the same basic principle as normal pasteurization,

the microwave treated milk can be found to have the

majority of its nutrients to be more intact than in

normal conventional pasteurization. This is due to the

fact that in microwave treatment takes a less amount of

time and the milk is exposed to lower amount of heat.

This reduction in time spent heating up a product

means the loss of much of the vitamins and other

important enzymes are reduced (Özilgen and Özilgen,

1991).

The cost of this process can be assumed to be relatively

low, as there have been research done where

microwave treatment could be done using a

conventional home microwave. A study conducted by

(Sieber et al., 1996) attempted to pasteurize milk using

a conventional microwave. In their experiment, their

milk was able to attain temperatures from 50˚C to 78˚C,

with a microwave using 2450 MHz frequency. They

were able to conclude that this method does not differ

from conventional pasteurization in terms nutritional

loss. However, the uneven temperature distribution by

microwave have shown to have some negative side

effects (Seiber et al., 1995). Unfortunately, the short

lifespan of a microwave raises doubts on whether it is a

good economic alternative for milk pasteurization

(Bansal and Chen, 2006).

MECHANISM OF INACTIVATION

The process of treating milk through microwaves is a

very simple task. In Seiber et al.’s (1995), a traditional

household microwave was used, so it can be assumed

that equipment depending on scale is easy to obtain. In

that process, milk was placed in a vessel and heated in

the microwave at a frequency of 2450 MHz. The high

penetration power of the microwave heats the milk to a

high enough temperature for the harmful bacteria to be

destroyed. At that point, the same basic heat principles

of normal pasteurization is taking place. The enzymes

and proteins lost in normal pasteurization and

microwave treatment is nearly the same, except for the

amount of amino acids and vitamins that were affected

by either treatments (Albert et al., 2009). Overall this

process and equipment needed is incredibly simple, and

the results are very favorable.

FOOD SAFETY

The reason microwave treatment is not used more

widely as a method of pasteurizing milk is due to the

uneven temperature distribution across the sample. The

uneven heat transfer across samples in a microwave

would cause for some bacteria to remain in the milk.

However, if a method for microwave treatment that

evenly distributes heat can be achieved, the quality and

safety of the microwave treated milk would be better

than that of the conventional heat pasteurized milk.

Escherichia coli and Bacillus subtilis, are bacteria that

can be found in raw milk that cause many foodborne

6

diseases. Microwave treatment can inactivate these

cells efficiently at high enough temperatures. In an

experiment (Woo Is, 2000) at 80C, both these cells have

been found to have a fairly good log reduction. When

temperature is raised from 50 to 60C, both E. coli and B.

Subtilis cells have higher log-reductions at 3 and 2

respectively. Therefore, in microwave treatment,

temperatures higher than 60C causes less efficient in

inactivating these bacteria. At higher temperatures, E.

coli is seen to be more efficiently inactivated, but at

lower temperatures, from 40 to 50C, B. Subtilis

inactivates better with a log-reduction of 3.66 while E.

coli has 3.23-log reduction. It was found that when

temperature increases between 40 to 60C, B. Subtilis, is

more sensitive. Therefore it can be concluded that, in

microwave treatment, bacterial inactivation depends on

the increases of temperature and different temperature

ranges. (Woo, 2000).

FOOD QUALITY

The quality change of milk after microwave treatment

was determined to be minimal according to the

experiment done by Seiber et al. Majority of the

vitamins, proteins and enzymes found in raw milk, were

left relatively untouched and shelf life was found to

have increased. A survey was also carried out to test the

flavor and texture of the microwaved milk, and around

half claimed the quality to be equivalent to that of

normally heated milk (Seiber et al., 1995). In another

experiment performed by Cs Albert et al in 2009, it was

found that the amount of enzymes and proteins lost in

microwave heated milk were much lower than that of

milk pasteurized by conventional methods. The

difference found in amino acids and vitamins losses

were even larger. Microwave treated milk were found

to have a much higher concentration of vitamins and

amino acids than that of normal heat convention

(Albert et al, 2009).

3.3 PULSED ELECTRIC FIELD (PEF) PROCESSING

INTRODUCTION

In a study, (Qin et al., 1995) describes Pulsed Electric

Field (PEF) processing as a method which utilizes quick

successive applications of voltage upon food placed

between two electrodes. Often PEF systems include a

high-voltage power source, capacitors to store energy, a

resistor to limit the current (Ortega-Rivas, 2010). A

charging current limiting resistor, a switch which

discharges energy from the capacitor across the food,

and a treatment chamber. In other systems, other

accessories are used. In continuous systems, a pump

transports the food to and through the treatment

chamber. Additionally, a cooling systems, such as a heat

exchanger, or water bath, might be necessary to reduce

the heating effect of the PEF treatment (Figure 1 and 2).

Generally, two waveforms are utilized in PEF treatment:

exponential and square. Square has been shown to be

more effective in inactivation and in efficiency, but

requires a more advanced circuit assembly. The circuit

designs for exponential and square are shown in Figure

3.

MECHANISM OF INACTIVATION

Membrane breakdown occurs when the

transmembrane potential exceeds a critical voltage (Ec)

of 1 V in most cells (Martín-Belloso and Soliva-Fortuny,

2010). For example, this potential corresponds to an

external electric field of about 10 kV/cm for Escherichia

coli (Castro et al., 1993).

Electrical breakdown theory regards the cell membrane

as a capacitor filled with dielectric material. There is a

natural build up of 10-mV transmembrane potential

(Zimmermann, 1986). Applying an external electrical

field of 1V induces a stronger polarization of the

membrane, a larger transmembrane potential, resulting

in a reduction of the membrane thickness. When Ec is

exceeded the membrane breaks down occurs. The

breakdown causes the formation of membrane pores.

These pores allow the electrical discharge to travel from

the medium into the cytoplasm and expedite the the

membrane decomposition. If the amount and size of

pores is large enough compared to the cell’s surface

area, than the membrane will break down irreversibly,

7

as the membrane can not reseal the pore openings.

When the field is removed, electroporation occurs,

during which the proteins in the phospholipid bilayer

temporarily destabilized from the application of PEF.

The altered proteins in the membrane effectively

changes the cell becomes semi permeable to small

molecules, leading to the cell to swell and eventually

rupture.

Advantage with this technology is the rapid inactivation

rate (quasi-instantaneous) which can have large

potential on production scale. Limitations include

design of generator sufficient enough to provide

enough field strength and power at a reasonable capital

cost. Components such as cooling systems and anti-

bubble formation units are subjected to additional

maintenance costs. Furthermore, high energy

intensities are required for microbial inactivation in

liquid foods. Consequently, higher energy and upkeep

costs can limit PEF implementation (Martín-Belloso and

Soliva-Fortuny, 2010). Estimated investment cost can be

2-3 million US for production rate of 5t/hr.

FOOD SAFETY

In general, PEF reaches high levels of microorganismal

inactivation. Maximum inactivation of 9log10 reduction

was achieved on E. coli by Qin et al. in 1995. However,

inactivation depends on the microorganism, treatment

media, processing atmosphere, and equipment

(Sampedro et al., 2005). PEF seems to be weaker

against gram negative bacteria than gram positive ones

(Ortega-Rivas, 2010). A study (Dutreux et al., 2000)

concluded that conductivity was the most important

parameter in inactivating microorganisms with PEF

(Sampedro et al., 2005): Gupta and Murray observed

that bacteria were better protected against PEF when

milk fat content was higher, and that critical treatment

time (tc) was lowest for low fat milk. Qin et al., in 1994

concluded similarly with regards to spores of the

Bacillus genus. This conclusion seems to indicate that

PEF is more apt to be applied to low fat rather than high

fat milk.

A study performed by (Sensoy et al., 1997) found that

inactivation increased when the conductivity of the

product decreased, as there was an increase of osmotic

forces induced pressure on the membrane, making it

more sensitive to pulses. Later, the researchers

increased the field intensity from 25 to 40 kV/cm,

treatment time from 25 μs to 100 μs, and temperature

from 10°C to 50°C. Under these conditions, the

inactivation increased, following a logarithmic trend.

They developed a new inactivation kinetic model,

combining the effect of time and field intensity or

temperature of the product. Several authors have

suggested the use of alternative technologies combined

with PEF treatment such as addition of nisin or minor

thermal processing to achieve optimal inactivation

(Sampedro et al., 2005). Other authors found a

beneficial synergy between thermal and PEF treatments

(Ortega-Rivas, 2010). It was suggested that the

significant increase of PEF inactivation efficacy at 55°C

may be due to the phase transition of the cell

membrane at this temperature. The membrane

transitions from a gel-like consistency to a crystalline

liquid one, making the bacterial cells more susceptible

to PEF (Jayaram et al., 1992).

(Dutreux et al., 2000) achieved 4.7 to 5.7 log10

reductions of Escherichia coli ATCC 11775 in pasteurized

skim milk in 10 pulses. Nearly complete inactivation was

achieved after 35 pulses. Mañas et al. (2001) studied

inactivation of Escherichia coli NTCC 9001 inoculated in

sterile dairy cream and achieved a 5 log10 reduction with

a treatment time of 261 μs (Sampedro et al., 2005).

Gupta and Murray, in 1988, achieved 4.5 log reductions

in milk inoculated with Pseudomonas frag and later, in

1996, studied inactivation of Pseudomonas fluorescens

in UHT milk (Sampedro et al., 2005). They established Ec

(critical field intensity) and the tc (critical treatment

time), which they found to be 22.4 kV/cm and 19.8 μs

respectively for 4 log reductions (Sampedro et al.,

2005).

While studying inactivation in the Salmonella genus,

Dunn and Pearlman, in 1987, obtained 4 log reductions

8

when used Salmonella dublin (3800 CFU/mL) in

pasteurized, homogenized milk (Sampedro et al., 2005).

While studying inactivation of the Listeria genus, Dunn,

1995 used L. innocua in treated raw whole milk, at

process temperature above 55°C and achieved over 6

log reductions (Sampedro et al., 2005).

Gongora-Nieto et al. (2003) reported the problematic

effect bubbles present in the treatment chamber,

finding that field strength experiences a significant drop

along the boundary regions of the bubbles, causing food

safety problems (Martín-Belloso and Soliva-Fortuny,

2010). In the treatment of milk, this issue could be to

ensuring a laminar flow. The Available Net Positive

Suction Head (NPSHA) must exceed Required Net

Positive Suction Head (NPSHR) or else cavitation will

occur resulting in bubble formation. Our team’s design

would guarantee this relationship to reduce bubble

formation.

FOOD QUALITY

Pulsed electric fields have little or very insignificant

impacts on sensory, textural and nutritional qualities of

raw milk. Maintaining quality with as little impact as

possible has been shown to be ineffective at high ends

of field strengths. A study conducted by Michalac et al.

(1999) demonstrated that there existed no variation in

color, pH, moisture and particle size of raw milk

subjected to field strength of 35kV/cm and pulse time

and width of 90 and 3 microseconds, respectively,

before and after treatment. In cheese production, PEF

attracts large attention due to the retention of sensory

and textural attributes similar to that of raw milk. In a

study by (Sepulveda-Ahumada et al., 2000), further

comparative evaluation of textural parameters such as

hardness springiness, adhesiveness and sensory

qualities in Cheddar cheese was conducted. This study

supported the enhanced aroma and textural qualities of

PEF treated milk in comparison to thermal processed

milk. In another study by Qin et al (1995),

physicochemical and sensory attributes were evaluated

and conducted in comparison to conventional thermal

pasteurization which resulted in little or insignificant

changes of given attributes for 2% fat milk (40kV/cm)

for an extended shelf life of 2 weeks at 4C. Shelf life was

later evaluated by Fernandez-Molina (1999) for PEF

treatment (30-50kV/cm) and PEF in addition to heat

process (80 C, 6s), which concluded shelf-life time of

over 14 days and 22 days respectively, at refrigerated

conditions (Sampedro et al., 2005).

Furthermore, Dunn and Pearlman (1987) observed in

PEF pasteurized and homogenized milk that, after

storage for 8 d at 7 to 9°C, no significant changes were

evaluated in nutritional attributes including “enzyme

activity, fat integrity, starter growth, rennet clotting

yield, cheese production, calcium distribution, casein

structure, and protein integrity” in raw milk treated

with PEF at lower field strength ranges (Sampedro et al.,

2005). In contrast, fat particles seem to protect the

microorganisms against electric pulses (Ortega-Rivas,

2010). Additionally, certain enzymes are found to be

more resistant to PEF, such as protease, and further

exhibit larger resistances in batch processes compared

to continuous flow (Bendicho et al., 2002). Substantial

amounts of energy are required to inactivate particular

enzymes which greatly limits the application of PEF on

raw milk (Ortega-Rivas, 2010). Inactivation of enzymes

further varies with field parameters (strength/pulse

number), type of enzyme (size/shape) and media.

Therefore, the retention of organoleptic characteristics

similar to raw milk would further enhance the qualities

and process making of cheese, butter, and ice-cream.

No destruction of vitamins, such as riboflavin and

thiamine, were observed by Bendicho et al. (1999) at

field strengths of 16-33 kV/cm. However, slight

destruction was observed at pulse numbers greater

than 100 (Sampedro et al., 2005).

3.4 HIGH PRESSURE PROCESSING (HPP)

INTRODUCTION

High pressure processing (HPP) in milk is the application

of high hydrostatic pressure (HHP) on medium and

consists of three essential components: pressure vessel,

delivery system, process fluid. Pressure vessel must be

9

properly designed to withstand high magnitudes of

pressure and fatigue. Furthermore, delivery systems can

consist of continuous or batch processing. Factors

impacting bacterial inactivation include: the type of

microorganism, food composition, pH, and water

activity (Nguyen and Balasubramaniam, 2011). Gram-

positive organisms are more resistant than gram-

negatives. Water activity also has a major influence on

the microbial inactivation rate.

Examples of high-pressure pasteurized products

available in the United States, Europe, and Japan

include smoothies, guacamole, deli meat slices, ready to

eat (RTE) food ready-meal components, poultry,

oysters, ham, fruit juices, and salsa (Dunne, 2005).

From Le Chatelier's principle, which states the

equilibrium of system shifting towards the smallest

volume, pressure will be the reaction force that results

in decrease of volume (microorganism). It is possible to

alter the mechanism of cell and further inactivate it

provided that sufficient energy is applied to the system.

In pressure process, mass is an independent variable

and is not altered by the objects shape or size because

pressure forces are equally distributed on the medium.

PACKAGING

Semicontinuous systems are used for liquid products,

which are then aseptically packaged after treatment

(Nguyen and Balasubramaniam, 2011). Flexible or

semirigid packaging (packaging with at least one flexible

interface) is well suited for batch processing. As the high

moisture foods compress by 15-20% during pressure

treatment of 600 MPa, HPP packaging must be able to

accommodate the reduction in size and return to the

original volume without losing its seal. For this reason,

cans are not suited for HPP.

WATER

Water has also emerged as a the optimal pressure

producing fluid due to its “availability, nontoxicity, and

low cost” (Nguyen and Balasubramaniam, 2011).

Furthermore, water produces a greater energy buffer,

improving the safety and efficacy of the processing.

OPERATION

Pressure levels necessary for effective inactivation

depend on types of microorganisms constituting in milk.

Increase of pressure affects the temperature

equilibrium, and likely pH levels (slightly) and viscous

properties of medium in the system at high levels of

pressure. Inactivation kinetics are altered when heat is

introduced to the medium. Pressure heat treatment can

be synergistic and antagonistic in inactivating for safety

of product and quality of food. Proper modeling for

conservation of mass and energy is often required to

keep the uniformity of pressure treatment. Designing

an effective HPP system requires proper design of

equipments (vessel, delivery system, safety

mechanisms) Often, it is required to minimize pressure

come-up, holding and decompression time (1 cycle) in

order to increase productivity and potentially save costs

in required energy delivered.

PRESSURE PASTEURIZATION

Pressure pasteurization treatment typically fall within

the pressure range of 600 MPa (87,000 psi) and

temperature range around room temperature for a

specified treatment time (Chef-tel, 1995; Farkas and

Hoover, 2000; Anon 2006). High-pressure pasteurization

have successfully inactivated pathogenic and spoilage

bacteria, yeasts, and molds, but produced limited

inactivation against spores and enzymes.

OSCILLATORY PRESSURE TREATMENT OR PRESSURE

PULSING

(Meyeret al., 2000) found that applying two or more

pressure pulses (referred to as “pressure pulsing” or

“oscillatory pressure treatment”) at an equivalent

holding time is more effective at inactivation. Pressure

pulsing can be used for both pasteurization and

sterilization (Nguyen and Balasubramaniam, 2011).

However, the measure of improved inactivation due to

10

pulsed pressure treatment must be compared to the

“design capabilities of the pressure unit, the added

compression costs, added wear on the pressure unit,

possible detrimental effects on the sensory quality of

the product, and the additional time required for

cycling.”

Oscillatory high pressure systems can be applied for

enhanced inactivation results. For instance, use of

dynamic high pressure (DHP) batch system has been

shown to be very effective in destruction of Listeria

monocytogenes, Escherichia coli and Salmonella enteric

(Vachon et al., 2002). Listeria monocytogenes is a gram-

positive, non-spore forming facultatively anaerobic

bacteria. Listeria monocytogenes can grow in a pH

range of 4.1–9.6 and a temperature range of 0–45 °C

(Chen and Hoover, 2003). Its ability to grow in

refrigerated temperatures and anaerobic conditions

makes it a food safety threat, especially since it can

cause serious illnesses and death (Jay, 1996; Ryser and

Marth, 1999).

PRESSURE ASSISTED THERMAL TREATMENT

Naturally, higher operating pressure levels are more

effective in the inactivation of microorganisms.

However, it is shown that similar effectiveness can be

achieved by treating milk at higher pressure-shorter

time, and at lower pressure-longer time (Mussa and

Ramaswamy, 1997) (also refer to Figure 4).

COST

Capital costs of high pressure equipment increase

exponentially with a linear increase in operating

pressures. Therfore, it is economically viable to utilize

low pressure processing in conjunction with other

processing methods to obtain the desired inactivation,

and, at the same time, maintain the sensory and

nutrient characteristics of the processing (Chen and

Hoover, 2003).

FOOD SAFETY

Dogan assumed that pressure inactivation of

microorganisms followed the first-order rate kinetics

(Dogan and Erkmen, 2004):

(1)

(2)

(Chen and Hoover, 2003)

Where,

N = number of surviving microorganisms after a

pressure treatment for time t (min),N0 = initial number

of microorganisms, and k = inactivation rate constant

(min−1).

At higher pressures of 600 and 700 MPa, D values

obtained compared well with those for thermal

inactivation of Listeria (Dogan and Erkmen, 2004). HPP

treatment of L. monocytogenes in milk was not as

effective as was in borth and fruit juices. Dogan and

Erkman (2004) claimed that fat and protein content in

whole milk buffered the bacteria to HPP. As in UV

treatment, perhaps we should use HPP for low fat milk

if it is chosen as method for our final design.

HPP has the potential to commercially sterilize heat

sensitive products such as milk, soups, tea and others

using pressure assisted thermal processing (PATP).

Commercial sterilization is the inactivation of all

pathogens to render them incapable of growth and

reproduction under non refrigerated conditions. Spores

and prions are known to be very resistant even under

high temperature (Nguyen and Balasubramaniam,

2011). Furthermore, the application of pressure pulsing

much like UV or PEF pulsing have been shown to be

more effective than non-pulsed pressurizing at

equivalent holding times The rate of microbial

destruction was much more rapid than enzyme

inactivation or color and viscosity changes (Mussa and

Ramaswamy, 1997). Studies have reported that PATP

reduces the process time of thermal inactivation

thereby preserving food quality, especially texture,

color and flavor.

11

Several studies have shown the effectiveness of

temperature assisted pressure processing (Chen and

Hoover, 2003). Temperature assisted pressure process

inactivation of L. monocytogenes was obtained at four

temperatures (22, 40, 45 and 50 °C) and two pressure

levels (400 and 500 MPa) in UHT whole milk. “A 5-min

treatment of 500 MPa at 50 °C resulted in a more than

8-log10 reduction of L. monocytogenes, while at 22 °C a

35-min treatment was needed to obtain the same level

of inactivation.” CHEN Temperature greatly impacted

the level of inactivation in pressure treatment of L.

monocytogenes. As temperature increased, inactivation

did as well. A 12-min treatment of 400 MPa at 50 °C

resulted in an roughly 7-log10 reduction of L.

monocytogenes in UHT whole milk, while at 22 °C more

than 120 min was needed to obtain the same

inactivation level. A 5-min treatment of 500 MPa at 50

°C resulted in a more than 8-log10 reduction of L.

monocytogenes in UHT whole milk, while at 22 °C a 35-

min treatment was needed to obtain the same level of

inactivation. In both pressure levels in this study,

increasing the temperature levels from 40 to 45 °C or

from 45 to 50 °C decreased the time to reach the same

level of inactivation by more than half. Chen found that

the log-logistic and Weibull models provide the most

accurate models for inactivation.Patterson and

Kilpatrick (1998) found that applying high pressure and

mild heat was more effective in inactivating Escherichia

coli O157:H7 and Staphylococcus aureus than either

treatment alone. A 5-min treatment of 500 MPa at 50

°C obtained a 6.0-log10 reduction of S. aureus in UHT

milk, while less than a <1.0-log10 reduction was

achieved with either treatment alone. Ponce, Pla,

Capellas, Guamis and Mor-Mur (1998) and Ponce, Pla,

Sendra, Guamis and Mor-Mur (1999) studied the effect

of temperature on the pressure inactivation of

Salmonella enterica serovar Enteritidis and E. coli. They

found that 50 °C to be the most effective temperature

in inactivation of the two gram-negative bacteria among

four temperature levels studied (−15, 2, 20 and 50 °C)

(also refer to Figure 5 and 6).

FOOD QUALITY

For milk and other liquid foods, the effect of adiabatic

heating can alter the sensory and nutritional changes if

pressure is not strictly controlled or high pressure

inputs are delivered to the system. At high pressure

levels (above 700 MPa), denaturation of proteins, lipids

and starches can potentially occur from additional

thermal stress. However, the effect of pressure alone

has no sensory changes preserving food freshness

because HPP has no effects on conformation of

macromolecules (does not break covalent bonds)

(Nguyen and Balasubramaniam, 2011) Furthermore,

viscosity affects the resultant texture of cheese. At high

pressures where proteins begin to denature, particular

enzymes in milk begin to coagulate thereby changing its

viscous effects (Mussa and Ramaswamy, 1997).

Nonetheless, HHP processing did not substantially

change the viscosity of milk (Mussa and Ramaswamy,

1997). High lipidic milk (3.5% fat milk) can accelerate

the heating due to its increase heat of compression

(Nguyen and Balasubramaniam, 2011). Additionally, it

can be advantageous in achieving temperature to

refrigeration level more effectively due to accelerated

heating rate and heat loss to the medium during

pressure come-down phase. Color and smell of the milk

was not affected by HHP processing to a large extent

(Mussa and Ramaswamy, 1997). Overall, HPP

processing is known to retain most of sensory and

nutritional values of milk, thereby retaining most of milk

freshness, by effectively inactivating pathogens through

the control of heat-pressure parameters for fresh and

quality cheese production.

3.6 ULTRAVIOLET PROCESSING

INTRODUCTION

Pulsed UV is a recent technology which offers an

alternative to inactivating pathogens in fruit juices,

apple cider, syrup, water and milk. It has been

successfuly applied to disinfecting surfaces of fresh

fruits and lettuces (Lu et al., 2011). The quick succession

of UV pulses of Pulsed UV outperforms the inactivation

effect of continuous UV. Pulsed UV treatment differs

from unpulsed UV. Pulsed UV requires a very simple set

12

up, a xenon inert gas lamp is placed to apply the UV

rays to the product (from lecture slides). In study

(Reinemann et al., 2006), germicidal UV lamp (UV-C

range) of 30 watts (1.4KJ/L) at standard-254 nm field

strength was used. (Rossitto et al., 2012)’s technology

used continuous turbulent flow and low-power UV

reactor at 254 nm at 1.5KJ/L to attain a maximum of 3-

log reductions. (Lu et al.) pumped raw milk through a

helical tubing set up around a pulsed UV lamp (Figure

7). This set up illustrates the ability for lamps to be

shaped to fit any application.

Ultraviolet processing is comprised of two methods:

conventional method and pulsed ultraviolet (PUV)

method. Conventional method uses regular mercury

lamp (range 100-400 nm) as a flashhead source for

inactivation whereas pulsed UV uses Xenon flash head.

In pulsed more, energy is stored and released from a

capacitor in form of pulses. Ultraviolet range from 200-

280 (UV-C) is germicidal. Other components of a typical

UV system includes a control module, which controls

the exposure variables, and high voltage supply, capable

to generate high pulsed optical energy. It can also

include additional components such as blower or

cooling unit that removes any volatile gases, such as

ozone, that can be detrimental to environment. PUV

can generate wideband generation in broad spectrum

range (100-1100 nm) which makes it more favorable

over continuous conventional ultraviolet processing. For

this particular processing, we will be focusing more on

PUV technology, its advantages and its disadvantages

(from lecture slides.

Factors affecting the efficaciousness of PUV have been

identified to include: UV wavelength,intensity and dose

rate, thickness of the radiation path, andflow

turbulence (Rossitto et al., 2012).

MECHANISM OF OPERATION

Mercury UV treatment has its complications in terms of

long exposure time on food medium which results in

heat generation, loss of energy and can reduce product

quality due to surface browning. On the other hand,

PUV sends high potential bursts of pulses in short

amount of expsure time and at low energy costs. Unlike

conventional method, it can achieve sterility because it

destroys the double and primary bonds in the DNA and

RNA bonds thereby inactivating microorganisms by

rendering their cell repair mechanism incapable of

development and reproduction. It is hypothesized that

thermal stress from UV leads to rupture of cell

membrane. However, the disadvantage of certain

pathogens having to remain resistant to UV radiation,

where only DNA sequencing is disrupted, can lead to

abnormal cellular development. Microorganisms can

potentially form pyrimidine dimers which can result in

damages, and due to repair mechanism of cell, it can

lead to cell mutation. As a society, we like to see the

food we eat is high quality and remains pathogen-free

and especially “mutated” pathogen free remaining safe

to eat (from lecture notes).

Low cost, efficient and low maintenance are main

advantages which consequently brings to better

product profit for small scale farm production of dairy

products. Product is potentially minimally processed, it

is non-thermal thereby retaining sensory and nutritional

attributes. It can be used to effectively disinfect

surfaces of products and solids in particular (fruits,

veggies, frozen meats, etc) and can be used as sanitizers

in public areas. Furthermore, This particular application

has very good advantages in water treatment for

potable water used as tertiary treatment method to

sterilize water. Good thing about PUV treatment is that

it follows a similar inactivation rate, first order kinetics,

as thermal inactivation so both effects can be hurdled

together for enhanced log reductions. Main

disadvantages include the insufficient log reduction

capacity of most pathogens for milk (12-D). This further

extends the limitation of UV processing due to low

productivity. In order to increase productivity, high

capital costs must be invested, making it more suitable

for small scale production. For non transparent liquids,

it is difficult or almost impractical to treat. For instance

in milk, calcium, fat/protein, and other constituents that

interfere with MO inactivation). Furthermore, shoulder

and tail effects occur in the inactivation curve of first

order kinetics. Shoulder effect is due to delayed

inactivation of cell injury (takes time to damage DNA).

13

Tail effect is the result of shielding of external particles

(foodstuffs) which ends up protecting pathogens. This

can be potentially eliminated by mechanical mixture so

that all surfaces come in contact with UV radiation. PUV

has several applications in cold treatment of raw milk

(Reinemann et al., 2006), reduction of thermally

resistant bacteria, inactivation in refrigerated milk in

long term storage. Additionally, PUV can reduce

pathogenic populations and improve milk quality in

areas which lack a supply of reliable and cheap energy.

EFFICIENCY

(Lu et al., 2011)’s PUV apparatus potentially may not

only “improve the safety and extend the shelf life of

milk”, but also has simpler processing procedures and

require less energy and space than does conventional

thermal pasteurization. G. Lu’s apparatus ran at a

processing rate of.194.2 kJ/kg. Krishnamurthy specified

that PUV is applicale for small-scale milk processing

operations (Krishnamurthy et al., 2007).

FOOD SAFETY

On a small scale cheese processing facility, it is possible

to achieve commercial sterility using UV treatment.

Small scale facilities have low production levels and

produced normally in batches for effectiveness.

Drawback of UV treatment of milk, but a potential

advantage for small scale production, is the small depth

of milk medium required for efficient inactivation

(Ngadi et al., 2003). However, in terms of safety or

public concern using PUV processing is the potential to

modify microbiological activity in the food medium. UV

exposure can lead to formation of dimers in

microorganisms from DNA damage. These damages can

result in mutations by impairing the replication of cell

mechanism and gene transcription sequencing. This

would normally lead to cell death. However, cells have

evolved evolutionary mechanism for repairing DNA

damage which allows survival of a mutated cell.

G. Lu et al. (2011) found that PUV could be used a viable

alternative for thermal pasteurization as PUV achieved

a greater than 6 log10 reduction in many cultures of milk

bacteria. . Krishnamurthy, applied PUV on

Staphylococcus aureus reducing the microorganism

which experienced complete inactivation in two cases

(7.23 and 7.26 log10 reductions). Matak et al. (2005)

achieved a 5log10 reduction in viable numbers of Listeria

monocytogenes with a low PUV dose of 15.8 ± 1·6 mJ

cm-1 (Donaghy et al., 2009). However, Donaghy et al.

(2009) found inactivating Mycobacterium avium ssp.

paratuberculosis (Map) with PUV difficult. At UV dose

rate of 1000 mJ ml-1 log10 reductions were all less than

2.5. “Map shows similar UV resistance to spore-forming

micro-organisms and is much more resistant than the

major microflora associated with milk.” Map, under

certain conditions, will survive pasteurization, making it

important to find viable secondary treatment. If PUV

unsuccessfully inactivates Map, another method must

be considered. Solids content in milk presents PUV a

second treatment challenge as it limits the penetration

of UV light into the liquid (Rossitto et al., 2012).

FOOD QUALITY

In terms of product quality, most studies carried out in

the last decade demonstrated significant effects on

sensory and nutritional values of UV treated milk. Off-

odours, foul taste, and nutritional imbalances were

observed.

A particular study by Rossitto et al. (2012) was carried

out to evaluate safety and quality changes of UV

treated milk. This study was based on a continuous

production system and was carried out to analyze

treatment for bovine milk destined for further

processing (cheesemaking, fermentation, etc). The

results showed a significant change in odour and

especially in taste, attaining 3-log reduction between

control and applied dose of 1.76 KJ/L. For doses below

0.88 KJ/L, there were little or no significant changes in

the quality of taste or aroma. Lactic acid levels following

waiting period remained unchanged compared to

control or non-treated milk.) Chemical imbalance at

dose of 1.76 KJ/L showed decrease change in fat,

protein, and lactose concentrations in 2% and 3.5% fat

milk. This can be explained by denaturation of enzymes

14

and breakdown of amino acids present in milk.

Additionally, oxidation of proteins occurred at high UV

doses affecting the proteolysis reaction contributing in

cheese making. UV light can also degrade vitamins by

photodegradation (especially A, C, and B2) (Demirci et

al., 2011; Scheidegger et al., 2010).

In another comparative study, conducted by Reinemann

et al. (2006) was carried out to evaluate the sensory

quality changes between HTST and UV treated milk. At

3-log reductions, milk was observed to have changes in

flavour. Results showed that excessive doses (1.4 KJ/L)

had effect on flavor being unclean with a “flat” off-

flavor. At this dose, 92% of panelists had a preference

for HTST than UV treated mlk. Whereas, at a dose of

0.47 KJ/L, sensory qualities of UV treated milk were

favored over HTST throughout the 14 day shelf-life.

Little difference was found between 1-log and 2-log

reduction of UV treated milk for storage time of 21

days. Hence, higher inactivation values can be reached

at the cost of sensory qualities of milk, which might not

be ideal for customer or production purposes.

4. DESIGN

To determine the best technology, a Pugh chart is presented:

Table 1

Criteria Weight Technology

HTST Batch Pasteurization HPP PEF PUV Microwave

Inactivation Potential 3 0 -2 3 2 1 -2

Nutritional Retention (ie. enzyme, vitamin) 3 0 2 2 2 1 0

Sensory retention (taste, smell, color) 3 0 2 3 3 -2 0

Shelf Life 1 0 -3 3 2 1 0

Cost 2 0 -2 -3 2 3 1

Production Scale-Flexible 2 0 3 -3 2 3 2

Treatment time 2 0 -3 -2 3 3 1

Total Score

0 -1 11 37 19 2

The inactivation potential, nutritional and sensory

retention were given the highest weights in the Pugh

chart to emphasize the objective of treating raw milk

without altering the desired natural qualities of the

product, while at the same time mitigating the

associated health risks and ensuring the safety of

consumers. Technologies that received a weight of 3 for

shelf life would produce a shelf-stable product.

Efficiency, capital cost and operational costs were all

accounted for when considering the weight of cost and

production scale-flexibility. These parameters were

given a weight of two because the objective of the

design works within the context small-scale, farm

operations.

When evaluating different technologies in Pugh chart,

Pulsed Electric Field was deemed superior with a score

of 37. PEF best fit the aforementioned criteria for a

small scale farming operation, and thus is technological

basis of the following design.

FINAL DESIGN

The typical small-scale dairy farm is comprised of 30

cows or less (Bagg, 2013). The typical cow produces 30

litres per day, on a two-times-a-day milking schedule

(Onario, 2013). This means that 900 litres of milk would

be produced per day, assuming that all cows were

milked. The flow rate of the system will be assumed to

be 254 litres per hour based on available pipe

diameters. Based on experimental viscosities from

University of Copenhagen the viscosity was assumed to

15

be roughly 1.55 mP*s for warmed raw milk entering the

system (Hougaard et al., 2009).

PROCESS

Milk is initially contained inside a stainless steel holding

tank (450 m^3) to allow initial bubbles to rise to

surface. As mentioned, bubbles present in the PEF

treatment chamber can adversely affect the effective

inactivation as well as lead to equipment failure from

accelerated corrosion. A centrifugal pump with an open

impeller is used to deliver controlled laminar flow at a

mean velocity of 0.1 m/s of raw milk. The treatment

chamber has a volume of 25.7 mL, utilizing a bipolar

square wave of 35 kV/cm The treatment chamber is

specified further in the design section. Using batch

treatment, it is possible to effectively process the

necessary daily flow rate using the proposed PEF

chamber design. Figure 8 below represents the

proposed general process design for a particular PEF

system for the given conditions applied.

Figure 8

Process flow diagram involved in the design.

FLOW DESIGN

The design requires that flows remain laminar.

Turbulent flows might produce bubbles which Gongora-

Nieto et al. (2003a) indicates significantly reduces the

field strength of PEF along the boundaries of the

bubbles.

(3)

Where,

), 1030

Determining the Reynold’s number for raw milk with

full fat. After holding tank has released milk,

the flow rate calculated for laminar flow is:

(4)

16

PUMP DESIGN

The Net Positive Suction Head (NPSH) is used to determine the selection of an appropriate pump for the particular laminar flow system. Bubbles should not be formed at suction and discharge. It is expressed as follows:

(5)

Where,

NPSH = Net Positive Suction Head (m)

= pressure at atmospheric sea level (101.3

kPa)

p = density of raw milk (kg/m^3)

g = gravitational constant (9.81 m/s^2)

u = mean velocity through pipe (m/s)

Pv = suction vapour pressure of raw milk at

impeller (kPa)

Givens

p = 1030kg/m^3 (JEK, 2009)

u = 0.1 m/s

Pv = 2.4 kPa (Obtained value from literature review)

NPSHA must be higher than NPSHR which can be obtained from pump manufacturers as requested and must be appropriately chosen to avoid cavitation.

PEF DESIGN

Our team found the pulse duration and energy density

needed for our PEF system.

(6)

Where,

(Mohamed and Eissa, 2012) offered standard

capacitance and resistance values for PEF:

0.12 uF for the capacitor and resister 6 MΩ.

Thus,

(7)

Where,

(Floury et al., 2006) found that to reduce E. coli by 5 log

reductions a square wave form, 64 pulses, 36 kv/cm, 2

μs per pulse, and entire treatment time was 90 μs was

necessary.

(Huang and Wang, 2009) used a gap between the electrode and volume of chamber as 0.95 cm and 25.7 ml.

That per batch, is 8.7 kJ.

5. CONCLUSION

Our team successfully addressed the objective to find a treatment option for a small-scale milk operation which

inactivates most pathogenic bacteria without significantly altering the beneficial nutritional properties of the product.

17

Based on an extensive literature review and Pugh Chart evaluation, our team found Pulsed Electric Field to best address

the problem statement. The design presented can be further expanded to further specify the design parameters and

propel the project into the prototyping phase design cycle.

Acknowledgments: Our team would like to that Professor Michael Ngadi for his sound counsel on this project

and for the comprehensive material from his course in Food Safety Engineering. DA

18

6. APPENDIX

Figure 1

Schematic diagram of PEF operation

(Martín-Belloso and Soliva-Fortuny, 2010)

Figure 2

PEF System

(from lecture notes)

Figure 3

Simplified circuitry for the generation of exponential decay and square wave pulses.

(Martin-Beloso nd Soliviva-Fortuny, 2010)

19

Figure 4

Temperature Assisted Pressure Treatment

(Nguyen and Balasubramaniam, 2011)

Figure 5

Figure 6

20

Figure 7

Experimental Set displaying versatile shaping of PUV.

(Lu et al., 2011)

7. WORKS CITED

Albert, C., Z. Mandoki, Z. Csapo-Kiss, and J. Csapó. 2009. The effect of microwave pasteurization on the composition of milk. Acta Universitatis Sapientiae Alimentaria 2(2):153-165. Angulo, F. J., J. T. LeJeune, and P. J. Rajala-Schultz. 2009. Unpasteurized milk: a continued public health threat. Clinical Infectious Diseases 48(1):93-100. Bansal, B., and X. D. Chen. 2006. A Critical Review of Milk Fouling in Heat Exchangers. Comprehensive Reviews in Food Science and Food Safety 5(2):27-33. endicho, S. l., . V. arbosa-C novas, and O. Mar n. 2002. Milk processing by high intensity pulsed electric fields. Trends in Food Science & Technology 13(6):195-204. Buffa, M. n. N., A. J. Trujillo, M. Pavia, and B. Guamis. 2001. Changes in textural, microstructural, and colour characteristics during ripening of cheeses made from raw, pasteurized or high-pressure-treated goats’ milk. International Dairy Journal 11(11):927-934. Castro, A. J., . V. AR OSA‐CÁNOVAS, and . . Swanson. 1993. Microbial inactivation of foods by pulsed electric fields. Journal of Food Processing and Preservation 17(1):47-73. CFIA. 2013. C. F. I. Agency, ed: Government of Danada. Chen, H., and D. G. Hoover. 2003. Modeling the combined effect of high hydrostatic pressure and mild heat on the inactivation kinetics of Listeria monocytogenes Scott A in whole milk. Innovative Food Science & Emerging Technologies 4(1):25-34. Demirci, A., K. Krishnamurthy, H. Zhang, G. Barbosa-Cánovas, V. Balasubramaniam, C. Dunne, D. Farkas, and J. Yuan. 2011. Pulsed Ultraviolet Light. Nonthermal processing technologies for food:249-261. Dogan, C., and O. Erkmen. 2004. High pressure inactivation kinetics of Listeria monocytogenes inactivation in broth, milk, and peach and orange juices. Journal of Food Engineering 62(1):47-52. Donaghy, J., M. Keyser, J. Johnston, F. P. Cilliers, P. A. Gouws, and M. T. Rowe. 2009. Inactivation of Mycobacterium avium ssp. paratuberculosis in milk by UV treatment. Letters in Applied Microbiology 49(2):217-221. Dunne, C. 2005. Killing pathogens: high-pressure processing keeps food safe.

21

Dutreux, N., S. Notermans, T. Wijtzes, M. Gongora-Nieto, G. Barbosa-Canovas, and B. Swanson. 2000. Pulsed electric fields inactivation of attached and free-living Escherichia coli and Listeria innocua under several conditions. International Journal of Food Microbiology 54(1):91-98. Floury, J., N. Grosset, N. Leconte, M. Pasco, M.-N. Madec, and R. Jeantet. 2006. Continuous raw skim milk processing by pulsed electric field at non-lethal temperature: effect on microbial inactivation and functional properties. Le Lait 86(1):43-57. Fund, F.-t.-C. L. D. 2013. Legality of Waw Milk Distribution. Verginia, US: Farm-to-Consumer Legal Defense Fund. Available at: http://www.farmtoconsumer.org/raw_milk_map.htm. Gao, A., L. Mutharia, S. Chen, K. Rahn, and J. Odumeru. 2002. Effect of Pasteurization on Survival of Mycobacterium paratuberculosis in Milk. J Dairy Sci 85(12):3198-3205. Guelph, U. o. 2013. Raw Milk. University of Guelph. Available at: https://www.uoguelph.ca/foodsafetynetwork/raw-milk. Hougaard, A. B., M. HammershØJ, J. S. Vestergaard, O. L. E. Poulsen, and R. H. Ipsen. 2009. Instant infusion pasteurisation of bovine milk. I. Effects on bacterial inactivation and physical–chemical properties. International Journal of Dairy Technology 62(4):484-492. Huang, K., and J. Wang. 2009. Designs of pulsed electric fields treatment chambers for liquid foods pasteurization process: A review. Journal of Food Engineering 95(2):227-239. IDFA. 2009. Pasteurization: Definition and Methods International Dairy Foods Association. Available at: http://www.idfa.org/files/249_Pasteurization%20Definition%20and%20Methods.pdf. Jay, J. M. 1996. Modern food microbiology. No. Ed. 5. Chapman & Hall. Jayaram, S., G. S. P. Castle, and A. Margaritis. 1992. Kinetics of sterilization of Lactobacillus brevis cells by the application of high voltage pulses. Biotechnology and Bioengineering 40(11):1412-1420. JEK.2009.Density Measurement in Dairy Industry. Ashland, VA. Krishnamurthy, K., A. Demirci, and J. M. Irudayaraj. 2007. Inactivation of Staphylococcus aureus in Milk Using Flow-Through Pulsed UV-Light Treatment System. Journal of Food Science 72(7):M233-M239. Lu, G., C. Li, and P. Liu. 2011. UV inactivation of milk-related microorganisms with a novel electrodeless lamp apparatus. European Food Research and Technology 233(1):79-87. Martín-Belloso, O., and R. Soliva-Fortuny. 2010. Pulsed Electric Fields Processing Basics. In Nonthermal processing technologies for food, 155-175. Wiley-Blackwell. Mohamed, M. E., and A. H. A. Eissa. 2012. Pulsed Electric Fields for Food Processing Technology. Mussa, D. M., and H. S. Ramaswamy. 1997. Ultra High Pressure Pasteurization of Milk: Kinetics of Microbial Destruction and Changes in Physico-chemical Characteristics. LWT - Food Science and Technology 30(6):551-557. Ngadi, M., J. P. Smith, and B. Cayouette. 2003. Kinetics of ultraviolet light inactivation of Escherichia coli O157: H7 in liquid foods. Journal of the Science of Food and Agriculture 83(15):1551-1555. Nguyen, L. T., and V. M. Balasubramaniam. 2011. Fundamentals of Food Processing Using High Pressure. In Nonthermal processing technologies for food, 1-19. Wiley-Blackwell. Onario, D. F. o. 2013. FAQ-Dairy farming. Available at: http://www.milk.org/corporate/view.aspx?content=faq/dairycattle. Ortega-Rivas, E. 2010. Processing Effects on Safety and Quality of Foods. CRC Press.

22

Özilgen, S., and M. Özilgen. 1991. A model for pasteurization with microwaves in a tubular flow reactor. Enzyme and Microbial Technology 13(5):419-423. Qin, B.-L., F.-J. Chang, G. V. Barbosa-Cánovas, and B. G. Swanson. 1995. Nonthermal inactivation of< i> Saccharomyces cerevisiae</i> in apple juice using pulsed electric fields. LWT-Food Science and Technology 28(6):564-568. Reinemann, D., P. Gouws, T. Cilliers, K. Houck, and J. Bishop. 2006. New methods for UV treatment of milk for improved food safety and product quality. ASABE paper(066088). Rossitto, P. V., J. S. Cullor, J. Crook, J. Parko, P. Sechi, and B. T. Cenci-Goga. 2012. Effects of UV Irradiation in a Continuous Turbulent Flow UV Reactor on Microbiological and Sensory Characteristics of Cow's Milk. Journal of Food Protection 75(12):2197-2207. Ryser, E. T., and E. H. Marth. 1999. Listeria: Listeriosis, and Food Safety. CRC Press. Sampedro, F., M. Rodrigo, A. Martínez, D. Rodrigo, and G. V. Barbosa-Cánovas. 2005. Quality and Safety Aspects of PEF Application in Milk and Milk Products. Critical Reviews in Food Science and Nutrition 45(1):25-47. Scheidegger, D., R. P. Pecora, P. M. Radici, and S. C. Kivatinitz. 2010. Protein oxidative changes in whole and skim milk after ultraviolet or fluorescent light exposure. J Dairy Sci 93(11):5101-5109. Sensoy, I., Q. H. Zhang, and S. K. Sastry. 1997. Inactivation kinetics of Salmonella dublin by pulsed electric field. Journal of Food Process Engineering 20(5):367-381. Sepulveda-Ahumada, D., E. Ortega-Rivas, and G. Barbosa-Cánovas. 2000. Quality aspects of cheddar cheese obtained with milk pasteurized by pulsed electric fields. Food and bioproducts processing 78(2):65-71. Sieber, R., P. Eberhard, and P. U. Gallmann. 1996. Heat treatment of milk in domestic microwave ovens. International Dairy Journal 6(3):231-246. Systems, A. B. P. 2013. Pasteurization and HTST Process. Stratford, WI: A & B Process Systems. Available at: http://www.abprocess.com/media/resources/files/Pasturization%20&%20the%20HTST%20Process.pdf. Vachon, J. F., E. E. Kheadr, J. Giasson, P. Paquin, and I. Fliss. 2002. Inactivation of Foodborne Pathogens in Milk Using Dynamic High Pressure. Journal of Food Protection 65(2):345-352. Woo Is, R. I. K. P. H. D. 2000. Differential damage in bacterial cells by microwave radiation on the basis of cell wall structure. Applied and environmental microbiology 66(5):2243-2247. WSDA. 2010. PASTEURIZER OPERATOR STUDY GUIDE: Reference and study guide for the HTST and Vat Pasteurizer Operator. W. S. D. o. Agriculture, ed. Washington, State: WSDA. Zimmermann, U. 1986. Electrical breakdown, electropermeabilization and electrofusion. In Reviews of Physiology, Biochemistry and Pharmacology, Volume 105, 175-256. Springer Berlin Heidelberg.