Investigating Alternative and Emerging Pasteurization ......to use a method known as flash-heating...

Investigating Alternative and Emerging Pasteurization Techniques for Human Milk Preservation

by

Michael Anthony Pitino

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Department of Nutritional Sciences

University of Toronto

© Copyright by Michael Anthony Pitino 2018

ii

Investigating Alternative and Emerging Pasteurization Techniques for Human Milk Preservation

Michael Anthony Pitino

Master of Science Department of Nutritional Sciences

University of Toronto 2018

ABSTRACT The effect of Holder (62.5oC, 30 minutes), flash-heating (brought to boil), UV-C

(250nm, 25 minutes) and high hydrostatic pressure (HHP, 500MPa, 8 minutes) pasteurization on

human milk composition was studied. Seventeen pools of human milk from different women

underwent each of the 4 pasteurization techniques. Macronutrient, vitamin C and folate and

bioactive components (bile salt-stimulated lipase (BSSL), lysozyme, and lactoferrin) were

measured in raw and pasteurized milk to determine any losses. Bacteriology was carried out to

assess the ability of each technique to yield a negative culture (<1000 CFU/L); all methods were

found equally effective. Holder, flash-heating, and UV-C resulted in significant reductions in

vitamin C, folate and BSSL, lysozyme (not Holder) and lactoferrin concentrations. Except for

vitamin C which was equally affected, HHP was the least impactful on these nutrients and

bioactive components. The feasibility of HHP for large-scale human milk pasteurization should

be further explored.

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ACKNOWLEDGEMENTS

Over the course of this Master’s degree, I have truly acquired a deeper understanding of the

scientific research process, how to think critically, and most notably, learning how to confront

and solve the many hurdles which sometimes impede progress. Many thanks are due to Dr.

Deborah O’Connor, who, in spite of busy schedules, deadlines and teaching, always made time

to chat, and offer guidance, support and wisdom. I sincerely appreciated all your motivational

talks, your life lessons and your ability to help me approach difficulties from different

perspectives. You have made me a more critical thinker, a more detail-oriented scientist, and

most importantly, a better person. Producing this document would not have been possible

without my family, Angelo, Rose, Alexandra, and Jonathan. I truly thank all of you for enduring

my rants (of triumphs and tribulations) and for being a stable part of my life over the course of

this degree. I would also like to thank my lab family—your support, social interaction and

copious amounts of laughter truly helped me maintain good mental health and helped to lighten

the mood after spending long hours running experiments.

I would like to take this opportunity to thank my advisory and examination committee, Drs.

Sharon Unger, Yves Pouliot, Harvey Anderson, and Mary L’Abbé. Your expertise, advice, and

contributions to this thesis are invaluable. I appreciate all your insightful comments, and critique

towards the completion of this important research. I also thank Dr. Anthony Hanley and Beatrice

Boucher, who both were excellent course instructors and taught me how to properly appraise and

analyze literature involving nutritional epidemiology.

Finally, I would like to thank all the mothers who donated their milk for research to the Rogers

Hixon Ontario Human Milk Bank. This research would not be possible without your generosity.

To all milk bank staff, especially Debbie Stone, Sheena Ragoo, and Carleigh Jenkins, thank you

for all your assistance over the past couple of years.

iv

TABLE OF CONTENTS Abstract ........................................................................................................................................... ii

Acknowledgements ........................................................................................................................ iii

Table of Contents ........................................................................................................................... iv

List of Tables ................................................................................................................................. vi

List of Figures ............................................................................................................................... vii

List of Abbreviations ................................................................................................................... viii

List of Appendices ......................................................................................................................... ix

CHAPTER 1 INTRODUCTION .................................................................................................... 1

CHAPTER 2 LITERATURE REVIEW ......................................................................................... 4 2.1 Human milk for infant feeding ........................................................................................................... 4

2.1.1 Donor human milk for preterm infants ........................................................................................ 4 2.1.2 Maternal barriers to breastfeeding ............................................................................................... 4 2.1.3 Use of heat-treated milk as a supplement to mother’s milk ........................................................ 5 2.1.4 Human donor milk banking ......................................................................................................... 7

2.2 Composition of human milk ............................................................................................................... 8 2.2.1 Factors affecting composition ..................................................................................................... 8 2.2.2 Macronutrients ............................................................................................................................ 8 2.2.3 Micronutrients ........................................................................................................................... 10 2.2.4 Bioactive proteins ...................................................................................................................... 10

2.3 Pasteurization methods ..................................................................................................................... 13 2.3.1 Holder ........................................................................................................................................ 13 2.3.2 Flash-heating pasteurization ...................................................................................................... 14 2.3.3 Alternative pasteurization techniques ....................................................................................... 14 2.3.4 Comparison of conventional and alternative pasteurization techniques ................................... 17

CHAPTER 3 INVESTIGATION OF ALTERNATIVE AND EMERGING PASTEURIZATION TECHNIQUES FOR HUMAN MILK PRESERVATION .......................................................... 30

3.1 Introduction ...................................................................................................................................... 30 3.2 Methods ............................................................................................................................................ 32

3.2.1 Study Design ............................................................................................................................. 32 3.2.2 Analysis of Nutrients, Bioactive Components and Bacteriology .............................................. 40 3.2.3 Statistical Analyses ................................................................................................................... 45

3.3 Results .............................................................................................................................................. 46 3.3.1 Nutrients and Energy ................................................................................................................. 46 3.3.2 Bacteriology .............................................................................................................................. 55 3.3.3 Exploratory ................................................................................................................................ 56

3.4 Discussion ......................................................................................................................................... 57 CHAPTER 4 CONCLUSIONS AND FUTURE DIRECTIONS ................................................. 67

References ..................................................................................................................................... 69 Appendix A. Research Ethics Board Approval Letter .................................................................. 82

v

Appendix B. Standard Operation Procedure for the Determination of Total Folate in Human Milk....................................................................................................................................................... 83

Appendix C. Standard Operation Procedure for the Determination of Total Vitamin C, Ascorbic Acid and Dehydroascorbic Acid in Human Milk ......................................................................... 85

Appendix D. Standard Operation Procedure for the Determination of Lactoferrin in Human Milk....................................................................................................................................................... 87

Appendix E. Standard Operation Procedure for the Determination of Lysozyme Activity in Human Milk .................................................................................................................................. 89

Appendix F. Microbiology protocol used for analysis of samples at the Rogers Hixon Ontario Human Milk Bank......................................................................................................................... 90

Appendix G. Standard Operation Procedure for the Determination of Fatty Acid Composition . 96

Appendix H. Standard Operation Procedure for the Determination of Available Lysine in Human Milk ............................................................................................................................................... 98

Appendix I. Representative Chromatograms of Total Vitamin C Content in Human Milk ......... 99

Appendix J. Representative Chromatograms of Lactoferrin Content in Human Milk ............... 100

Appendix K. Student's Contribution ........................................................................................... 101

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LIST OF TABLES Table 1. The effects of both thermal and non-thermal pasteurization techniques on energy and

nutrient content of human milk ............................................................................................. 19 Table 2. The effects of both thermal and non-thermal pasteurization techniques on select

bioactive components of human milk ................................................................................... 25 Table 3. Optimization Experiments for HHP pasteurization of human milk ............................... 39 Table 4. Mid-infrared analysis of macronutrients and energy using the Miris human milk

analyzer 172 ............................................................................................................................ 40 Table 5. Nutrients and bioactive levels following pre- and post-pasteurization of human donor

milk ....................................................................................................................................... 54 Table 6. Summary of microbiology results assessing pasteurization effectiveness ..................... 55 Table 7. The impact of Holder and UV-C pasteurization on the fatty acid composition (mg/mL)

of human milk (N=2): a pilot study ...................................................................................... 56 Table 8. The impact of Holder and High Hydrostatic Pressure Processing on the free lysine

content of human milk (N=5): a pilot study ......................................................................... 56

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LIST OF FIGURES Figure 1. Operation of a HHP unit for food pasteurization .......................................................... 16 Figure 2. Experimental design and overview of study ................................................................. 33 Figure 3. Experimental design of UV-C pasteurization adapted from Christen et al. .................. 36 Figure 4. The relationship between milk solids and effective UV-C pasteurization dose to

achieve 5-log reduction. ........................................................................................................ 37 Figure 5. Changes in macronutrient composition following pasteurization ................................. 46 Figure 6. The impact of different pasteurization methods on the folate concentration of human

milk ....................................................................................................................................... 47 Figure 7. The impact of different pasteurization methods on the total vitamin C concentration of

human milk ........................................................................................................................... 48 Figure 8. The impact of different pasteurization methods on the bile salt stimulated lipase

activity of human milk .......................................................................................................... 50 Figure 9. The impact of different pasteurization methods on the lactoferrin concentration in

human milk ........................................................................................................................... 52 Figure 10. The impact of different pasteurization methods on the lysozyme activity in human

milk ....................................................................................................................................... 53

viii

LIST OF ABBREVIATIONS

HHP: High Hydrostatic Pressure Processing UV-C: Ultraviolet-C HMBANA: Human Milk Banking Association of North America HPLC: High Performance Liquid Chromatography NICU: Neonatal Intensive Care Unit NEC: Necrotizing enterocolitis WHO: World Health Organization AAP: American Academy of Pediatrics BSSL: Bile Salt-Stimulated Lipase

ix

LIST OF APPENDICES Appendix A. Research Ethics Board Approval Letter .................................................................. 82 Appendix B. Standard Operation Procedure for the Determination of Total Folate in Human Milk

.............................................................................................................................................. 83 Appendix C. Standard Operation Procedure for the Determination of Total Vitamin C, Ascorbic

Acid and Dehydroascorbic Acid in Human Milk ................................................................. 85 Appendix D. Standard Operation Procedure for the Determination of Lactoferrin in Human Milk

.............................................................................................................................................. 87 Appendix E. Standard Operation Procedure for the Determination of Lysozyme Activity in

Human Milk .......................................................................................................................... 89 Appendix F. Microbiology protocol used for analysis of samples at the Rogers Hixon Ontario

Human Milk Bank ................................................................................................................ 90 Appendix G. Standard Operation Procedure for the Determination of Fatty Acid Composition . 96 Appendix H. Standard Operation Procedure for the Determination of Available Lysine in Human

Milk ....................................................................................................................................... 98 Appendix I. Representative Chromatograms of Total Vitamin C Content in Human Milk ......... 99 Appendix J. Representative Chromatograms of Lactoferrin Content in Human Milk ............... 100 Appendix K. Student's Contribution ........................................................................................... 101

1

CHAPTER 1 INTRODUCTION

1.0 Introduction

When human milk is shared, pasteurization is recommended to remove pathogens (e.g. bacteria

and viruses). This is necessary in certain populations of vulnerable infants, including those born

very preterm or to mothers with pre-existing HIV infection. There is frequently insufficient

mother’s milk for preterm infants to adequately meet their nutritional requirements and

therefore, Holder pasteurized (62.5°C for 30 min) human donor milk is used to supplement

feeding as an alternative to preterm formula1,2. Similarly, pasteurization of raw mother’s milk is

recommended to HIV+ mothers under certain circumstances in resource poor conditions, to

decrease the likelihood of maternal-to-child transmission3.

Although pasteurization is believed necessary to ensure the safety of milk consumed by these

vulnerable groups of infants, thermal processing has been shown to alter the nutritional

composition of human milk. Further, thermal processing has been shown to negatively affect

many of the biologically active proteins in human milk that are essential for nutrient absorption,

growth and protection against infection4. For example, human milk contains many enzymes,

such as bile salt-stimulated lipase (BSSL). It ensures adequate fat absorption which provides

energy for growth5. Human milk also contains many immune proteins, including lactoferrin and

lysozyme, which confer protection to the developing infant during a period of time when their

native immune system (innate and acquired) is still underdeveloped6,7. Specifically, in the

preterm population, it is thought that the activity of both lactoferrin and lysozyme work

synergistically to assist in the prevention of necrotizing enterocolitis (NEC), a serious, and

possibly fatal bowel disease8. These immune factors have also been implicated in infants at risk

of HIV infection in low-resource countries where they are thought to be the biological factors

contributing to the reduction in diarrhea and infection observed when human milk is consumed

instead of infant formula9.

Holder pasteurization (62.5°C for 30 min) is the current standard of practice for the treatment of

human milk in many high-income countries. In low-resource countries, mothers are encouraged

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to use a method known as flash-heating (~73°C for 15 min) to pasteurize their milk and

inactivate HIV3,10. While the negative effects of Holder pasteurization on human milk

composition are well documented in the literature, there has been very little work on the impact

of flash-heating11. Since flash-heating requires a shorter exposure to heat, it may result in fewer

changes to the composition of human milk. Given recent advances in food science and

technology, investigation into alternative, non-thermal methods is warranted so as to reduce the

loss of nutrients and bioactives in human milk as a result of processing.

In the literature review that follows, the use of pasteurized human milk for feeding preterm

infants and those born to mothers with pre-existing HIV will be discussed. The first section

emphasizes the importance of donor milk for preterm infants and the various maternal barriers

to breastfeeding which exist. An overview of the benefits of donor milk as opposed to preterm

formula when mother’s milk is insufficient in volume is discussed, including the potential risks.

The first section also introduces human milk banking in high-resource countries, in addition to

the current breastfeeding guidelines for low-income countries where HIV is endemic, including

the use of flash-heating to pasteurize milk.

The second section of the literature review summarizes what is known regarding the

composition of human milk, including maternal factors that affect composition. Some of these

factors are modifiable, such as nutritional status and intake, and others are not modifiable, such

as stage of lactation and gestational age of the infant at birth. The macronutrient and

micronutrient composition of human milk are examined in detail, in addition to the many

biologically active proteins found therein.

The final section of the literature review focuses on various pasteurization techniques, thermal

and non-thermal. Their ability to reduce bacterial load and their effects on nutrients and

bioactives are discussed and systematically reviewed. In addition to Holder and flash-heating,

UV-C irradiation, a novel technique which utilizes light in the UV spectrum to remove

potentially pathogenic bacteria from human milk is reviewed. Similarly, high hydrostatic

pressure processing (HHP) which subjects milk to extremely high pressures to effectively lyse

and destabilize bacteria found in human milk is examined. In spite of the many gaps, the current

3

literature on the use of these novel techniques with application to human milk is summarized,

including their effect on human milk composition, especially folate.

Chapter three of this thesis is the final report for my thesis research. The purpose of the present

study was to explore potential alternative, non-thermal techniques to pasteurize human milk and

compare them to the thermal techniques of Holder and flash-heating. Specifically, the research

objectives were to determine the nutrient losses from pasteurizing raw human milk via Holder,

flash-heat and other techniques (including UV-C irradiation and HHP). The nutrients assessed

include those that are both essential for growth and development, and those potentially affected

by heat including macronutrients (fat, carbohydrate, protein), micronutrients (folate, vitamin C),

as well as biologically active enzymes and immune proteins (bile salt –stimulated lipase

[BSSL], lactoferrin, lysozyme). The ability of each method to reduce the bacterial load to a

negative culture (<1000 CFU/L) was also assessed. It was hypothesized that non-thermal

techniques would result in fewer changes in milk composition, while still being able to reduce

the bacterial load to a negative culture.

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CHAPTER 2 LITERATURE REVIEW

2.1 Human milk for infant feeding

Human milk is the gold standard for feeding all infants as it provides essential nutrients to the

developing newborn. Reduced infant mortality is recognized as an attributable consequence to

breastfeeding, and evidence indicates that populations who artificially feed their young have

poorer survival12. The current recommendation as outlined by the World Health Organization

(WHO), American Academy of Pediatrics (AAP), and Health Canada states that all infants

should be exclusively breastfed until 6 months of age, after which, continued breastfeeding

should be complimented by the introduction of solid foods13-15. For the purposes of this thesis,

breastfeeding is defined as provision of human milk at the breast, by bottle or feeding tube.

Consequently, community- and government-based initiatives have aimed to make breastfeeding

the normative standard for feeding infants. Through the provision of health-promoting bioactive

proteins and small molecules that are exclusive to human milk, the benefits of breastfeeding go

beyond fulfilling basic macro-and micro-nutrient nutritional requirements.

2.1.1 Donor human milk for preterm infants

Following the recommendations of the AAP and the WHO, breastfeeding in North America, and

Canada in particular, is increasingly being promoted as the optimal nutrition for infants, as

scientific research continues to point at the benefits of human milk for long-term infant growth

and development. Over the past 50 years, growing interest into providing human milk to all

infants has become a focus as it is thought that the early environment during pregnancy and

especially lactation, can determine physiological, structural, immune, metabolic and behavioural

development: a component of the classical theory of the developmental origins of health and

disease16. At present, this is especially relevant given the dramatic rise in chronic non-

communicable diseases that human milk may play a role in preventing16.

2.1.2 Maternal barriers to breastfeeding

While the majority of mothers choose to initiate breastfeeding, under certain circumstances,

difficulties may arise which may cause mothers to deviate from providing an exclusive human

5

milk diet to their newborn. In the case of mothers of preterm infants, several factors may

synergistically decrease milk production and subsequent breastfeeding. Maternal psychological

stress, anxiety and post-partum depression have been observed to be elevated as a consequence

of giving birth preterm, given that it is relatively unexpected to the mother17. This is in addition

to the demand placed on both mother and father to make important medical decisions for their

infant very early during the antenatal period. Many studies have concluded that maternal stress

and poor psychological health may reduce or impede milk production18-20.

Physiological barriers to breastfeeding also affects a mother’s ability to provide milk to their

preterm infant. Deficits in lactation ability have been attributed to pre-pregnancy diabetes

mellitus, severe preeclampsia, and cesarean-section, all factors associated with preterm birth21-

23. Delayed lactogenesis II due to inadequate mammary secretory cell development is also

apparent in this population of mothers and contributes to overall low milk volumes. In addition

to physiological factors, many mothers face physical barriers which lead to poor lactation.

Preterm infants are often unable to suckle at the breast and additionally have a long-term

maternal-infant separation in the neonatal intensive care unit (NICU), which can, in and of

itself, reduce or delay lactogenesis24. A lack of manual expression or electric pumping is also a

factor in poor lactogenesis.

2.1.3 Use of heat-treated milk as a supplement to mother’s milk

In many developed countries, such as Canada, pasteurized donor milk is often prescribed as a

supplement to mother’s milk for preterm infants in the NICU, as an alternative to using preterm

formula 25,26. A recent systematic review and meta-analysis by Quigley and McGuire in 2014

found that feeding pasteurized donor human milk compared to preterm formula resulted in a

lower risk of developing NEC; however, donor milk also resulted in poorer growth outcomes

(e.g. weight, length, and head circumference)27. Many of the studies included in the

aforementioned systematic review and meta-analysis used unfortified donor milk, which may

have accounted for the poorer growth observed. Most recently, in a double-blind, randomized

6

controlled trial, fortified donor milk reduced the incidence of NEC and showed no difference in

growth outcomes as compared to preterm formula28.

The main motivation behind the use of donor milk stems from its NEC protective properties.

The onset of NEC is rapid and is associated with high morbidity and mortality29. It is believed

that human milk, whether maternal or donor, confers protection against NEC, most likely owing

to its composition rich in immunological proteins and bioactive components30-32. Although the

pathogenesis of NEC is not completely understood, a perturbation of the developing intestinal

microbiome and a hyper-inflammatory response have been implicated in its onset29. Additional

research is warranted to fully understand the mechanism by which human milk protects against

NEC.

In low and middle-income countries, the WHO recommends that HIV+ mothers should

breastfeed as much as possible, regardless of duration, until an adequate and safe diet without

human milk is achievable33. It is recommended that HIV+ mothers breastfeed for 12-24 months,

provided they are undergoing antiretroviral therapy to reduce their risk of HIV transmission and

have healthcare support3,34. Although breastfeeding is universally recognized as the optimal

strategy for the improvement of maternal and child health, exclusive breastfeeding up to 6

months of age is rare in countries where HIV is endemic—partly because breastfeeding

substantially increases the risk of HIV transmission from mother to child35,36 . This is especially

true in countries such as South Africa, where health workers often lack skills needed to offer

breastfeeding support and advice to mothers. In resource poor countries, despite the increased

risk of HIV transmission from mother to child, the use of commercial human milk substitutes

and replacement feeds increase morbidity and mortality in HIV+ populations9,37-41. Therefore,

the benefits of human milk strongly outweigh the risk, and as such, breastfeeding is extremely

encouraged for the prevention of diarrhea, pneumonia and undernutrition42. Difficulties arise in

infants and mothers which may cause breastfeeding to cease, including: being born low-birth-

weight, infants who are unable to breastfeed, maternal illness or unavailability of antiretroviral

drugs. The WHO recommends that mothers heat treat their expressed milk which can decrease

the transmission of HIV through inactivation.

7

2.1.4 Human donor milk banking

The establishment and operation of regulated human milk banks in high-resource countries

ensures donor milk is safely prepared for vulnerable preterm infants in the NICU. The idea of

human milk banking was conceived in Vienna, Austria, with the opening of the first human milk

bank in 190943. Boston in the United States followed suit and opened a human milk bank in

191943. Thereafter, human milk banks were steadily established globally; however, many ceased

operations by the mid-1980’s as a result of the ongoing HIV epidemic because of concerns it

would be transmitted to infants through donor milk. In the years that followed, advanced donor

screening protocols were established to reduce the risk of HIV transmission, in a manner similar

to that of blood donors. This is in addition to the heat treatment of milk that has been shown to

destroy the HIV virus44. As a result, human milk banking is making a dramatic re-emergence so

that the provision of donor milk to very preterm infants as a supplement to maternal milk is

feasible. In Canada, there are currently 4 operational milk banks including the BC Women’s

Provincial Milk Bank in Vancouver BC, the NorthernStar Mothers Milk Bank in Calgary, AB,

the Rogers Hixon Ontario Human Milk Bank in Toronto, ON, and Hema Quebec’s milk bank in

Montreal, QC, which receive and process donated milk, and dispense milk to Canadian

NICUs45.

All non-profit milk banks in North America must abide by guidelines put forth by the Human

Milk Banking Association of North America (HMBANA)45. HMBANA guidelines specifically

cover details regarding the establishment and operation of a donor human milk bank. Milk

banks must also abide by regulations outlined by local public health authorities, who ensure

milk is safe and adequately screened before it is sent to hospitals for feeding. Specifically, at the

Rogers Hixon Ontario Human Milk Bank (opened 2013), potential donors are screen

serologically for blood borne diseases. Donated milk from approved donors is first pooled from

multiple donors to improve nutritional homogeneity. It is then pasteurized by the Holder method

(62.5°C for 30 min) to ensure that it is safe for infant consumption. Donated milk is cultured

prior to pooling and pasteurization so as to ensure that high bacteria load milk exits the supply

chain and is not pooled with other milks. After pasteurization, batches of pasteurized milk are

also cultured to ensure the milk is culture negative. Although beneficial bacteria are inherently

found in human milk, environmental contaminants and spoilage bacteria pose a threat to very

vulnerable infants who receive the processed milk.

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2.2 Composition of human milk

The main rationale as to why feeding infants human milk is highly recommended, as opposed to

other forms of nutrition, is because it contains many important macro-and micro-nutrients which

help support growth, development, and neuromaturation. Moreover, it contains many bioactive

proteins and immunological factors which contribute to overall health and help protect against

infection.

2.2.1 Factors affecting composition

The composition of human milk is dynamic; it shows considerable variability, and is affected by

many factors including: term or preterm birth, stage of lactation, and maternal dietary intake and

status46. Many nutrients in human milk are sensitive to maternal status while others are secreted

into human milk consistently, regardless of maternal levels. For example, vitamins, such as

vitamin A, thiamin, riboflavin, vitamin B6, vitamin B12 and choline are particularly sensitive to

maternal status; however, other nutrients such as folate, trace elements and minerals are not 47-49.

2.2.2 Macronutrients

There have been two recent systematic reviews published which have examined the

macronutrient composition in both term and preterm milk. In the first review by Gidrewicz et al.

postnatal age and gestational age were the best predictors of human milk composition50. The

macronutrient concentration of human milk, and therefore its energy content, has also been

shown to be dependent on several factors including maternal diet, timing of milk collection,

nursing frequency and volume of milk produced51-53. For example, the volume of milk produced

and protein concentration were found to be negatively correlated with nursing frequency.

Maternal protein intake was also positively correlated with the lipid concentration of human

milk only after 16 weeks post-partum. In a review from Boyce et al., reference ranges of

preterm milk (<37 weeks gestation) were proposed given its distinct differences from term

milk54. Preterm milk has high temporal and inter-mother variability in the macronutrient

composition and is unlikely to meet the nutritional requirement of preterm neonates without

appropriate fortification53,55.

9

Protein intake from human milk is currently a focus of infant nutrition research given its

importance for lean mass accretion56. Protein is required by all infants, and is especially

important for preterm infants to support their growth, as well as maintain and synthesize

tissues57. There is currently no one concentration that can be used for the protein content of

human milk because levels of protein have been shown to vary significantly58. Giving birth

preterm or term, being at a certain stage of lactation (early or late), and duration of previous

breastfeeding/pumping, can yield milk that is intrinsically different in peptide signature and total

protein levels59,60. Preterm milk is higher in true protein than term milk; however, in both term

and preterm milk, colostrum is higher in protein than mature milk for protein, but has lower

levels of energy fat, and lactose50. Average total protein levels can range from 0.9 to 1.2g/dL,

however, this does not account for differences in amino acid proportion or composition51.

Specifically of importance in human milk is the limiting essential amino acid lysine, required

for protein synthesis and needed for the biosynthesis of carnitine, a molecule involved in fatty

acid metabolism61,62. Lysine is found in human milk at an average concentration of 6.7g/100g

protein, as established by the European Directive EEC91/3263. When heated, lysine side-chains

are particularly susceptible to reactions with lactose which can render them unavailable to the

infant64. A decrease in the synthesis of carnitine due to lysine bio-unavailability may also lead to

altered fatty acid metabolism; an important consideration for the preterm population, where

energy requirements are elevated and fat reserves are limited65.

The amount of fat in human milk is critical for infant growth and development as it is the prime

determinant of total energy in human milk. Although fat varies between mothers (genetics, age)

and across lactation, the average concentration in human milk is 3.2 to 3.6 g/dL51. However,

variation also exists in the proportion of fatty acids including saturated, monounsaturated,

omega-3 and omega-6 – affected by maternal age, time of day of lactation, and stage of

lactation66-69. Omega-3 and omega-6 fatty acids found in human milk are essential for the

growth and proper functioning of the nervous tissue70-72. The most abundant fatty acids in

human milk are the monounsaturated fatty acids; they contribute to approximately 40% of all

fatty acids73. Similarly, as a percentage of total fatty acids in human milk, saturated, omega-3

and omega-6 fatty acids constitute 38%, 15% and 13%, respectively73.

10

2.2.3 Micronutrients

The micronutrient composition of human milk, in addition to body stores laid down in utero, are

thought to be sufficient for optimal growth for the first 6 months of life for the healthy term

infant 74. Due to low body stores as a result of interruption of maternal to fetal transfer of

nutrients and elevated nutritional requirements, the nutritional needs of hospitalized very

preterm infants cannot be met by human milk alone to achieve current recommendations75. This

is true for many of the heat-sensitive, water-soluble vitamins, including vitamin C and folate.

Vitamin C is an antioxidant in the body which helps to neutralize free radicals76. It is important

for the developing preterm infant as it is required for the cross-linking of collagen in the growth

of cartilage and bone76. The Institute of Medicine, in establishing the adequate intake of term-

born infants for vitamin C, reviewed 8 studies with over 500 mothers which assessed the

vitamin C content of human milk. They determined the mean vitamin C concentration in human

milk from well-nourished women was 50mg/L human milk74,77. However, the mean vitamin C

content of human milk is highly variable as its secretion is dependent on maternal status, storage

time, and analytical technique. In a sample of Canadian women, the mean vitamin C

concentration in human milk was 22mg/L, compared to human milk from Bangladeshi women

where the mean concentration was approximately 30mg/L48,78,79. In a sample of American

women who delivered preterm, the mean concentration of vitamin C in milk was approximately

41mg/L80.

Compared to vitamin C, the concentration of folate in human milk remains relatively consistent

regardless of dietary intake, except in cases of very severe maternal folate deficiency81. In 1998,

the Institute of Medicine developed the folate dietary reference intakes for infants 0-6 months of

age using a concentration of 85µg/L (190 nmol/L) as the average levels in human milk as the

standard, based on work by Lim et al 1997, Brown et al. 1986 and O’Connor et al. 199182-85.

This is comparable to what was recently observed in a sample of Swedish mothers where the

mean folate level in human milk was as 150nmol/L86.

2.2.4 Bioactive proteins

In addition to macro-and micro-nutrients, human milk contains hundreds to thousands of

bioactive components which when consumed, contribute to improved immunity, microbial

11

colonization and overall health of the infant51. The bioactives found within human milk

originate from (1) the mammary epithelium proper, (2) are transferred across the mammary

epithelium by receptor-mediated transcytosis or (3) are produced by cells within the milk51. The

levels of such bioactive factors also show variability between mothers and change dramatically

over lactation87. Specifically of importance, is the secretion of acquired and innate immune

factors in human milk6. These function to bolster the infant’s host defence early in life during a

period of time when the immune system is immature and not fully developed7. The immune

properties of human milk are even more significant for vulnerable infants, such as those born

preterm or born to mothers with a pre-existing infection, such as HIV. Both of these populations

of infants are susceptible to infection, either due to their immature immune systems or being

exposed to viral agents present their milk7.

Discussion of all the bioactive components in human milk is beyond the scope of this literature

review. Detailed discussion of these can be found in the review of Ballard et al51. A brief

discussion follows of three bioactive components found in human milk which have been shown

to be important in infant health and have been reported to be affected by thermal processing,

including lactoferrin, lysozyme and bile salt-stimulated lipase (BSSL). Although all three

bioactives are not found in infant formula, lactoferrin and lysozyme are present in cow’s milk.

The most abundant levels of immune bioactives are found in human colostrum, milk early in

lactation, which then eventually decrease over time51. One protein in human milk which has

been observed to be key in the early host defense of the infant is lactoferrin.

Lactoferrin

Lactoferrin is an iron-chelating protein of the transferrin family88,89. Its antibacterial properties

are attributable to its ability to sequester iron: required for bacterial growth and colonization90.

Once ingested, lactoferrin undergoes enzymatic proteolysis by pepsin to yield lactoferricin, a

peptide which encompasses a large portion of the functional domain of the intact protein but

possesses stronger antimicrobial properties91. Lactoferricin can also act directly to increase

bacterial membrane permeability90. This is owing to lactoferricin’s ability to bind the lipid A

portion of lipopolysaccharide found on the cell surface of gram negative bacteria, including

those from the Klebsiella, Acinetobacter, Pseudomonas and E. coli genera90. As such, many

studies have shown that lactoferrin in human milk prevents necrotizing enterocolitis (NEC) and

12

may reduce the incidence of neonatal sepsis. Results from a recent systematic review and meta-

analysis suggest that lactoferrin supplementation of enteral feeds decreases late-onset sepsis and

NEC stage II or III in preterm infants without adverse effects; however, this evidence was

derived from low quality studies and additional research is required to determine whether

lactoferrin is wholly responsible for the NEC preventive qualities of an exclusive human milk-

based diet8,92-94.

Aside from its antimicrobial properties, lactoferrin may also be involved in neuro-protection and

development, owing to its structure rich in sialic acid glycosylations. Sialic acid is a key

monosaccharide used for the synthesis of brain gangliosides and sialyted glycoproteins 95.

Evidence, primarily animal studies in rats has demonstrated that bovine lactoferrin

supplementation positively affects brain lipid metabolism, provides neuronal protection and

increases cognitive performance, especially during stress 96,97.

Lysozyme

Lactoferrin has been shown to act synergistically with lysozyme to kill gram-negative bacteria;

lactoferrin binds and removes the lipopolysaccharide from the cell wall, exposing the cell wall

to lysozyme upon which it will act 98. Lysozyme, an antimicrobial enzyme that is able to

degrade the bacterial cell walls, also confers innate immunity to the human milk fed infant. This

is accomplished through enzymatic degradation of ß-1,4 linkages of N-acetylmuramic acid and

2-acetylamino-2-deoxy-D-glucose reside of bacterial cell walls99. Secretion of lysozyme in

human milk varies widely. The concentration of lysozyme is higher in preterm milk than term

milk100. Lysozyme secretion has also been shown to decrease during the first month postpartum,

but increase between 11 to 17 months post-partum 101. Despite its presence in human milk, its

biological activity is highly dependent on its native 3-dimensional conformational folding. Its

biological activity may be lost due to protein aggregation as a result of thermal denaturation and

reversibility is highly dependent on pH and ionic strength 102.

Bile salt-stimulated lipase

Recent investigations have demonstrated that BSSL may also play a role in the infants’ innate

immunity. BSSL has been found to bind to dendritic cells of the immune system, in vitro,

preventing HIV-trans infection of CD4+ T-cells103,104. BSSL has also been shown to inhibit the

13

binding of Norwalk viral capsids to their carbohydrate ligands105. Apart from a role in

immunity, bioactive components in human milk may also play a role in nutrition. For example,

BSSL is a highly glycosylated bioactive protein that functions to enzymatically cleave

triglycerides, releasing bound fatty acids106. This facilitates absorption of fats, and thus,

improves extraction of energy from human milk to support growth and development. The

bioactive region of BSSL is highly influenced by the specific glycans present, dependent on

maternal genetics and stage of lactation107. BSSL in human milk is essential for all infants and

especially important for preterm infants who are unable to produce a sufficient amount of

pancreatic lipase 108-110. Many studies have demonstrated that pancreatic lipase only accounts for

a fraction of the fat digestion in the neonate, while human milk provides the rest.

2.3 Pasteurization methods

Pasteurization is used to reduce the risk of introducing pathogenic bacteria to vulnerable infants

fed human milk. More classical pasteurization methods for liquids involve heat. These include

high-temperature-short-time, higher-heat-shorter-time, ultra-pasteurization, low-temperature-

long-time (Holder), and flash-pasteurization. Alternative techniques exist which do not require

heat including microfiltration, UV-C irradiation and HHP. In the next section, the following

techniques will be discussed: Holder, given its widespread-use in milk banks worldwide; flash-

heating, as it is recommended by the WHO for use by HIV+ women in the absence of anti-

retroviral therapy, UV-C and HHP, as they are alternative techniques which do not require heat.

2.3.1 Holder

Holder pasteurization involves heating milk up to 62.5°C where it must be kept for 30 minutes.

Although the main aim of human milk banks is to provide safe donor milk to preterm infants

during a period of time when there is insufficient maternal supply, pasteurized donor milk

cannot simply be seen as a replacement to raw mother’s milk. There are many steps in its

processing (freezing and thawing) and especially thermal pasteurization that alter the biological

composition of raw mother’s milk111,112. The effects of Holder pasteurization on human milk

constituents are well documented in the literature and are summarized in Table 1 and Table 2.

At this time, Holder pasteurization is the only high-throughput technique that can reliably

pasteurize milk. While its negative effects on human milk have been extensively researched,

14

current human milk banking practices compromise on lower concentration of nutrients and

bioactive components to ensure safety. The Holder method has been used for over 100 years.

As such, there is an ongoing push to explore other ways to pasteurize human milk for vulnerable

infants in developed countries as well as in low resource countries, to improve post-processing

nutrient retention and to ensure safety from potentially pathogenic bacteria and viruses.

2.3.2 Flash-heating pasteurization

For the past decade, heat-treatment of human milk is and has been recommended by the WHO

when mother’s milk needs to be provided and a mother is not receiving anti-retroviral therapy

for HIV113. Research into flash-heating of human milk from HIV-infected mothers began in

Zimbabwe by Israel-Ballard in 2007 to determine whether HIV+ milk could be made safe for

infant consumption by pasteurization, while preserving its nutritional and bioactive

properties114. Flash-heating is a low-tech method developed to pasteurize human milk using

household items in a low-resource setting. The method mimics commercial flash pasteurization,

also known as high-temperature short-time, whereby expressed human milk is placed into a jar

that can withstand heat, and the jar is placed into a pot with water. The water and jar are

simultaneously heated rapidly—the jar is removed once the water visibly begins to boil to

prevent degradation of important nutrients. Although research into this technique for human

milk pasteurization is limited, this method has been shown to inactivate HIV114. The effects of

flash-heating on select human milk constituents are summarized in Table 1 and Table 2.

2.3.3 Alternative pasteurization techniques

Although thermal techniques have been shown to be effective at pasteurizing human milk, heat-

sensitive nutrients and bioactive molecules are often impacted and their levels reduced as a

result of processing. To mitigate these losses, investigation into alternative, non-thermal

techniques is warranted, including the need to reduce bacterial load. A recent systematic review

by Peila et al. 2017 on human milk processing concluded that currently, the Holder technique

remains the best option to ensure microbiological safety while limiting the losses of bioactive

components in human milk11. It is mentioned that although data on the microbiological safety

are scare for novel methods, Ultraviolet C (UV-C) irradiation has been identified as a possible

alternative method that does not require heat.

15

Ultraviolet-C pasteurization

UV-C irradiation was first introduced as an alternative pasteurization technique for human milk

in 2013 by Christen and colleagues115. The shorter wavelength of UV-C (200-280nm), as

opposed to UV-B (280-315nm) or UV-A(315-400nm), dimerizes pyrimidine residues found in

bacterial DNA and inhibits replication116. The use of UV-C for reducing bacterial load is not

novel; it is widely used in the food industry for surface sterilization117. It is not widely used to

pasteurize liquids, other than water, given the difficulty for UV-C to penetrate the liquid. This is

especially challenging for human milk applications because of the associated turbidity of

suspended milk solids and fat globules. As described, to utilize UV-C as a technique to

pasteurize human milk, continuous mixing of the sample is required to ensure adequate

penetration and exposure to the germicidal lamp115. It was also noted that the amount of UV

required to achieve a 5-log reduction in bacterial load is dependent on the concentration of milk

solid. A higher dose is required to penetrate samples of milk with higher concentration of milk

solids.

Since investigation of UV-C is in its early stages, there has been very limited research assessing

the effects of UV-C pasteurization in terms of its effectiveness in reducing the bacterial load of

donor milk, as well as how it impacts fragile micronutrients and dissolved bioactive

components. A recent promising finding demonstrated the ability of UV-C to ablate

cytomegalovirus, a virus commonly excreted into breastmilk and a common cause of infection

in vulnerable infants in the NICU118,119. The effects of UV-C on human milk constituents are

summarized in Table 1 and Table 2. Further research is fundamental in order to thoroughly

understand (1) how UV-C can be best optimized for safe pasteurization, (2) how to

commercialize UV-C use for large quantities in the context of milk banking applications, (3)

what effect UV-C has on the nutritional aspect of human milk and (4) whether UV-C

pasteurization is feasible for use in countries with low resources.

High hydrostatic pressure processing

A solution to the poor penetrability of UV-C irradiation to eliminate bacterial contamination in

human milk is the use of high hydrostatic pressure processing (HHP). HHP is a non-thermal

pasteurization technique that can be used to inactivate bacteria while minimizing chemical

16

reactions in food; limiting the losses of nutrients that are susceptible to heat damage and

maintaining the organoleptic properties (colour, flavour etc.) of the milk. HHP was first

introduced into the food industry in the early 1990s as a way to extend the shelf life of various

fruit products including jams and juices without the use of additives, and is currently used

for a wide range of food products, including prepackaged foods such as hams, sausages, etc120.In

Canada, HHP is recognized as an acceptable form of food processing, and as of December 22nd

2016, Health Canada published a position demonstrating that there exists sufficient knowledge

and data on the safe use of HHP to remove the novel status121. In spite of its widespread use in

the food industry, HHP remains a novel process with application to human milk. There has

been limited research into the effects of HHP of various human milk components, and only 2

studies that have assessed the effectiveness of HHP—its ability to reduce the bacterial load of

human milk to below detectable levels. One study found that 400 megapascals (MPa) held for

30 min is required for inactivation for most common resistant strains of bacteria122.

Figure 1. Operation of a HHP unit for food pasteurization

Food product in container placed in vessel

On the other hand, Permanyer and colleagues found that pressures ranging from 400-600MPa

held for 5 minutes are adequate for reducing bacteria to below detectable limits123. It is

important to note that both studies assessing pasteurization effectiveness were conducted in

Water Tank

Pressurization via compression of water

Processed food product (elimination of bacteria)

Figure 1. A schematic of the operation of a HHP machine for the elimination of bacteria. The food product is placed in a container within the HHP vessel. The vessel is then filled via a low-pressure line with water. The level of compression of water within the vessel generates varying degrees of high hydrostatic pressure. After a set amount of time depending on the food product, the product is removed and can safely be consumed.

17

inoculated human milk samples and were not tested using native bacteria found in human milk,

inherent or contaminant.

Using the current literature, it is difficult to assess whether HHP is as a viable alternative to the

Holder technique to pasteurize human milk, as inconsistencies among studies exist in

experimental design and optimization of conditions (pressure, holding time). Studies showing

reproducibility of results are required to fully understand the effect of HHP on human milk. The

effects of HHP on select human milk components are summarized in Table 1 and Table 2.

Given the sparse nature of this literature, there still exists large gaps in knowledge and

discrepancies in the results.

2.3.4 Comparison of conventional and alternative pasteurization techniques

Reduction of bacterial load

The ability of Holder pasteurization to reduce the bacterial load of human milk to below

detectable levels is well established in the literature and is the main rationale as to why the

Holder technique is used in milk banks globally124,125. It has been observed that Holder

pasteurization can be ineffective at reducing the bacterial load if it is contaminated with Bacillus

cereus: a spore-forming bacterium resistant to thermal treatment126. Despite limited

investigation into the bacterial safety of flash-heating human milk, one study reported

reductions in bacterial colonies after inoculation with specific pathogens (S. aureus, E. coli)10;

however, levels were <2000 CFU/L, well above the culture-negative cut-off recommended by

HMBANA (<1000 CFU/L).

Similarly, research into the bacteriological safety of alternative pasteurization techniques, such

as UV-C and HHP, is lacking. The effectiveness of UV-C was assessed in one study using

inoculated bacterial species115. Although 5-log reductions were reported, colony counts above 1

CFU/mL or 1x103 CFU/L persisted. More research is needed to fully understand whether the

use of UV-C is effective enough to be feasibly implemented. As discussed previously, HHP is

relatively understudied in the context of human milk. The efficacy of some pressure/time

combinations have been reported to successfully reduce the bacterial load; however,

18

optimization of experimental conditions is required to fully understand the implications of this

technique.

Losses of nutrients and bioactives

As summarized in tables 1 and 2, Holder pasteurization appears to have no significant influence

on levels of lactose and total carbohydrates. Limited reductions were observed in protein

content following Holder, with the exception of one study that reported high reductions in

available lysine. In spite of the various analytical techniques utilized, 12 studies investigated the

effect of Holder on fat content. Of the 12 studies reviewed, 5 saw reductions ranging from 3.5%

to 18%; the remainder reported null findings. Fat was shown to not be impacted by HHP or

UV-C, excluding two studies that observed changes in individual fatty acids.

Overall, folate appears to be significantly impacted by the thermal pasteurization. The Holder

technique was shown to reduce the folate concentration of milk, while flash-heating results in

increases in milk folate concentration. To our knowledge, the effects of UV-C and HHP on

folate in human milk has not been assessed. Vitamin C shows sensitivity to Holder

pasteurization and is reduced, while unaffected by flash-heating and HHP. The majority of

studies assessing lysozyme report significant reductions following Holder and flash-heating,

with the exception of HHP which appears to have no effect. Likewise, BSSL has only been

assessed following Holder and UV-C pasteurization; its activity is completely destroyed after

Holder and fully retained after UV-C. Studies have yet to determine the effect of flash-heating

and HHP on BSSL activity.

Lactoferrin shows vulnerability to both thermal and alternative pasteurization techniques and is

shown in the literature to be consistently and significantly reduced. Research into the

thermodynamic properties of human lactoferrin suggests the maximum peak temperature of

lactoferrin denaturation is 67°C, which increases to 91°C if fully saturated with iron127.

Similarly, in human lactoferrin expressed in rice, research shows the maximum temperature of

thermal denaturation is reported at 72°C, and increases to 93°C when fully saturated with

iron128.

19

Table 1. The effects of both thermal and non-thermal pasteurization techniques on energy and nutrient content of human milk

Human Milk Constituent

Pasteurization Experimental Conditions Analytical Method Sample

Type Sample Size

Main Finding Study Country

Lactose & Total Carbohydrate

Holder 62.5°C, 30 min

Mid-infrared Mature HM

28 NS Garcia-Lara et al. 2013 129

Spain

Gas Chromatography

Mature HM Colostrum

18 NS Espinosa-Martos et al. 2013 130

Spain

Gas Chromatography

Mature HM

21 NS de Segura et al. 2012 126

Spain


57 NS Vieira et al. 2011 131

Brazil

Colorimetric Mature HM

12 NS Braga and Palhares 2007 132

Brazil

Picric Acid Method Mature HM

60 NS Goés et al. 2002 133

Brazil

Protein Holder 62.5°C, 30 min


28 NS (Total Nitrogen)

Garcia-Lara et al. 2013 129

Spain


57 3.9% reduction

Vieira et al. 2011 131

Brazil

19

20



Type Sample Size


Protein Holder 62.5°C, 30 min

Bicinchoninic acid method

Mature HM

17 NS Ley et al. 2011 134

Canada

OPA method Mature HM

-- NS (Available Lysine)

Baro et al. 2011 135

Italy

Spectrophotometric Mature HM

12 NS Braga and Palhares 2007

Brazil

Fluorimetric Mature HM

30 NS (30% reduction available Lysine )

Silvestre et al. 2006 136

Spain

Refractive index Colostrum 101 13% reduction

Koenig et al. 2005 137

Brazil

Standard procedures Mature HM

4 NS Hamprecht et al. 2004 138

Germany

Folin Phenol reagent Mature HM

60 NS Goés et al. 2002 133

Brazil

Fat Holder 62.5°C, 30 min

Chromatography, Infrared-Spectroscopy, NMR

Mature HM

1 NS (Fat & Fatty Acids)

Borgo et al. 2015 139

Brazil

21



Type Sample Size



Crematocrit Mature HM

44 18% reduction

Vazquez-Roman et al. 2014 140

Spain


28 3.5% reduction


Spain

Gas Chromatography

Mature HM

6 NS Delgado et al. 2014 141

Spain


17 9% reduction Ley et al. 2011 134

Canada


57 5.5% reduction

Vieira et al. 2011131

Brazil

Gas Chromatography

Mature HM


Molto-Puigmartí et al. 2011142

Spain

Spectroscopy Mature HM

12 NS Braga and Palhares 2007 132

Brazil


60 NS Goés et al. 2002 133

Brazil

Gravimetric Mature HM


Fidler et al. 2001 143

Germany



Type Sample Size



Folch/Capillary Gas LC

Mature HM

6 NS (Fatty Acids)

Henderson et al. 1998 144

USA

Folch Mature HM

16 6% reduction Lepri et al. 1997 145

Italy

UV Multiple doses Gas Chromatography

Mature HM

10 18% increase 8:0

Christen et al. 2013 115

Australia

HHP 400/600 MPa, 3/6 min

Gas Chromatography

Mature HM

6 Some reductions in n-3/n-6

Delgado et al. 2014 141

Spain

400/500/600 MPa, 5 min

Gas Chromatography

Mature HM

11 NS Molto-Puigmartí et al. 2011 142

Spain

Energy Holder 62.5°C, 30 min


28 2.8% reduction


Spain

Bomb calorimetry Mature HM

17 NS Ley et al. 2011 134

Canada

Folate Holder 62.5°C, 30 min

HPLC Mature HM

38 22% reduction

Buttner et al.201486

Sweden

22

23



Type Sample Size


Folate Holder 62.5°C, 30 min

Chemiluminescent immunoassay

Mature HM

4 NS Hamprecht et al. 2004 138

Germany

Microbiological assay

Mature HM

10 16% reduction

Donnelly-Vanderloo et al. 1994 146

Canada

Radioassay Mature HM

9 31% reduction

van Zoeren-Grobben et al. 1987 147

Netherlands

Microbiological assay

Mature HM

3 35% reduction

Goldsmith et al. 1983 148

USA

Isotope-labelled gel filtration

Mature HM

Not reported

10% reduction (folate binding)

Ford et al. 1977 149

UK

Flash Rapid heating Chemiluminescent immunoassay

Mature HM

5 40% increase Israel-Ballard et al. 200510

USA

Chemiluminescent immunoassay

Mature HM

50 34% increase Israel-Ballard et al. 2008 150

USA

Vitamin C Holder 62.5°C, 30 min

HPLC Mature HM

11 20% reduction

Molto-Puigmartí et al. 2011142

Spain

24



Type Sample Size


Vitamin C Holder 62.5°C, 30 min

HPLC Mature HM

10 26% reduction

Romeu-Nadal et al. 2008 151

Spain

HPLC Mature HM

9 36% reduction

van Zoeren-Grobben et al. 1987147

Netherlands

Fluorometric determination

Mature HM

3 NS Goldsmith et al. 1983 148

USA

Flash Rapid heating HPLC Mature HM

5 NS Israel-Ballard et al. 200510

USA

HPLC Mature HM

50 NS Israel-Ballard et al. 2008

USA

HHP 400, 500,600 MPa, 5 min

HPLC Mature HM

11 NS Molto-Puigmartí et al. 2011 142

Spain

25

Table 2. The effects of both thermal and non-thermal pasteurization techniques on select bioactive components of human milk



Type Sample Size


Bile-salt Stimulated Lipase

Holder 62.5°C, 30 min

Endpoint Reaction- P-nitrophenol

Mature HM

Not Reported

>99%reduction

Baro et al. 2011135

Italy

Standard Procedures Mature HM

4 >99%reduction

Hamprecht et al. 2004 138

Germany

Stable Isotope Triglyceride Emulsion

Mature HM

6 >99%reduction

Henderson et al. 1998 144

USA

UV-C Multiple doses QuantiChrom Lipase Assay Kit

Mature HM

10 NS Christen et al. 2013 115

Australia

Lysozyme Holder 62.5°C, 30 min

Lysoplate-M. Lysodeikticus

Mature HM

50 NS Daniels et al. 2017 152

South Africa

Turbidimetric- M. Lysodeikticus

Colostrum 11 44% reduction

Sousa et al. 2014 153

Portugal

Enzyme Immune Assay Kit

Mature HM

10 59% reduction


Australia

ELISA Kit Mature HM

31 NS Chang et al. 2013 155

Taiwan

26



Type Sample Size


Lysozyme Holder 62.5°C, 30 min

Immunological Mature HM

33 60% reduction

Akinbi et al. 2010 156

USA

Enzyme Immune Assay Kit

Mature HM

22 60% reduction

Czank et al. 2009 157

Australia


Mature HM

1 (10 pooled)

48% reduction

Viazis et al. 2007 158

USA


Colostrum 101 NS Koenig et al. 2005 137

Brazil

Radial immunodiffusion

Mature HM

4 35% reduction

Hamprecht et al. 2004 138

Germany

Electroimmunoassay Mature HM

25 24% reduction

Evans et al. 1978 159

UK


Mature HM

Not reported

NS Ford et al. 1977 149

UK

Lysoplate-M. Lysodeikticus

Mature HM

1 (49 pooled)

36% reduction

Gibbs et al. 1977 160

UK

Flash Rapid heating Turbidimetric- M. Lysodeikticus

Mature HM

50 22% reduction

Daniels et al. 2017 152

South Africa

27



Type Sample Size


Lysozyme Flash Rapid heating M. Luteus activityassay

Mature HM

50 45% reduction

Chantry et al. 2011 161

South Africa

SDS-PAGE/ Western Blot

Mature HM

9 NS Israel-Ballard et al. 2005 10

USA

UV-C Multiple doses Enzyme Immune Assay Kit

Mature HM

10 25% reduction


Australia

HHP 200,400,600 MPa (2.5,15,30 min)


Colostrum 11 NS Sousa et al. 2014 153

Portugal


Mature HM

1 (10 pooled)

NS Viazis et al. 2007 158

USA

Lactoferrin Holder 62.5°C, 30 min

Bacterial inhibition assay

Mature HM

50 29% reduction


South Africa

ELISA kit Mature HM

10 91% reduction

Christen et al. 2013154

Australia

ELISA kit Mature HM

31 66% reduction

Chang et al. 2013 155

Taiwan

SDS-PAGE/Western Blot

Mature HM

28 94% reduction

Reeves et al. 2013 162

USA

28



Type Sample Size


Lactoferrin Holder 65°C, 30 min

SDS-PAGE/Western Blot

Mature HM

Not reported

Reduction Baro et al. 2011 135

Italy

Sandwich ELISA kit Mature HM

22 78% reduction

Czank et al. 2009 157

Australia

Immunological Mature HM

33 44% reduction

Akinbi et al. 2009 156

USA

Sandwich ELISA kit Mature HM (skim)

10 80% reduction

Mayayo et al. 2008 163

Spain

Electroimmunoassay Mature HM

25 57% reduction

Evans et al. 1987 159

UK

Radial immunodiffusion

Mature HM

Not reported

61% reduction

Ford et al. 1977 149

UK

Flash Rapid heating Bacterial inhibition assay

Mature HM

50 61% reduction


South Africa

SDS-PAGE/ Western Blot

Mature HM

50 20% reduction

Chantry et al. 2011 161

South Africa

ELISA kit Mature HM

10 84% reduction

Israel-Ballard et al. 2005 10

USA

29



Type Sample Size


Lactoferrin UV-C Multiple doses ELISA kit Mature HM

10 13% reduction

Christen et al. 2013154

Australia

HHP 300,400,500,600 MPa, 15 min

Sandwich ELISA kit Mature HM (skim)

10 9,23,34,48% reduction respectively

Mayayo et al. 2008 163

Spain

30

CHAPTER 3 INVESTIGATION OF ALTERNATIVE AND EMERGING PASTEURIZATION TECHNIQUES FOR HUMAN MILK PRESERVATION

3.1 Introduction

Mother’s milk is the optimal nutrition for all infants, including vulnerable infants. This includes

both preterm infants in the NICU and infants born to HIV+ mothers who benefit from the

provision of a well-tolerated and reliable source of nutrients, immunological proteins and

bioactives. The advantages of mother’s milk are well established and have recently been

summarized in a systematic review and meta-analysis highlighting both the short-term reduction

in mortality and morbidity, as well as long-term protection against obesity, and non-

communicable diseases42. The main cause for concern is that mothers of these vulnerable infants

from these two distinct populations often experience barriers to breastfeeding. Mothers of

preterm infants often have difficulty expressing a sufficient amount of their own milk, which

could be a result of various factors including their own health status, physiological deficits in

lactogenesis and physical barriers. Infant breastfeeding difficulties also exist for mothers who

are HIV+. The WHO recommends HIV+ mothers to breastfeed for as long as possible, provided

they are undergoing antiretroviral therapy, reducing the risk for HIV transinfection3,33.

In North American NICU’s, the advent of human milk banking has allowed preterm infants

whose mothers may not be producing a sufficient amount of milk, to receive human milk.

Donors who provide this milk are serologically screened for blood borne diseases, including

HIV/AIDS, Hepatitis B and C, Human T-lymphotropic virus I and II and syphilis. Donated milk

is then pasteurized using the Holder method (62.5°C for 30 min) to ensure it is safe for infant

consumption and free from potentially pathogenic bacteria which are commonly found in human

milk. Despite its proven effectiveness to pasteurize human milk, the thermal processing

involved with the Holder method has been shown to negatively impact many heat-sensitive

vitamins and bioactive components124,164. The extra freeze-thaw cycle, container changes and

thermal processing associated with Holder pasteurization have been shown to reduce the levels

of energy-containing macronutrients found in human milk and decrease the functionality of

lactoferrin and lysozyme. The latter antimicrobial proteins are thought to be protective against

31

NEC8,165. Previous research has demonstrated that heat- and light-labile micronutrients,

including vitamin C and folate, are sensitive to Holder pasteurization, and are essential nutrients

for bone and cartilage growth, as well as DNA/RNA synthesis147,151,166 .

Although Holder pasteurization is widely used globally, poorer countries, such as those in

Africa, do not have adequate resources to conduct Holder pasteurization on a large scale.

Rather, the WHO and UNICEF jointly recommend using a flash-heating procedure of human

milk as an interim feeding strategy for mothers who are HIV+ if antiretroviral drugs are

unavailable or if the mother is unwell and temporarily unable to breastfeed3. Flash-heating is

being endorsed as a cost-effective technique to pasteurize human milk, which only requires the

use of easily attainable household items (glass jar, pot and a heat source [fire, hot plate etc.])

While very little research has been done on flash-heating, this approach appears to inactivate

HIV and other viruses; scant data are available on the impact of flash-heating on heat-sensitive

nutrients and bioactive components4,10,150.

Fundamentally, thermal pasteurization techniques affect many of the heat-sensitive vitamins and

may result in the denaturing and/or inactivation of many biologically active proteins and

enzymes4. As such, feeding vulnerable infants pasteurized milk may result in a reduction in the

observed benefits seen with feeding raw, unpasteurized milk. To overcome the limitations of

thermal pasteurization, novel, non-thermal alternatives are currently being investigated. One

such alternative technology is UV-C irradiation which is a disinfection technology that employs

electromagnetic irradiation against bacteria, viruses, protozoa, and yeasts167. UV-C irradiation

with application to human milk has been shown to inactivate cytomegalovirus and can lead to

better retention of secretory immunoglobulin A, lactoferrin and lysozyme while still proving

effective at achieving a 5-log reduction in bacterial load118,154. The successful reproducibility of

this pasteurization technique has yet to be investigated and the effects on other human milk

components remain unknown.

Another non-thermal technique under investigation is HHP, in which human milk is exposed to

high pressures (400-600 MPa)168. This disrupts non-covalent bonds, keeps covalent bonds intact,

while being temperature controlled. Few studies have investigated the use of HHP with

application to human milk, with only two studies assessing its effectiveness at reducing the

32

bacterial load to safe levels. Although HHP was found to be effective at reducing various strains

of inoculated bacteria, there is no general consensus on which time and pressure combinations

lead to the most effective pasteurization122,123. Moreover, what consequence the most effective

HHP pasteurization conditions has on the various human milk nutrients and bioactives is

unknown.

The aim of this study was to assess the effectiveness of reducing the bacterial load of human

milk using emerging and alternative pasteurization techniques to Holder pasteurization, as well

as the impact of each pasteurization technique on human milk macronutrients, micronutrients

and bioactive components. The primary research objective was to determine whether alternative

pasteurization techniques for human donor milk result in less of a reduction in the concentration

of folate, a proxy measure of the water-soluble and heat sensitive vitamins. Secondary

objectives include the assessment of the levels of macronutrients and micronutrients (vitamin C)

following conventional (Holder) and alternative pasteurization (Flash-heating, UV-C, and

HHP). Additionally, secondary objectives include determination of whether the alternative

techniques are more effective than Holder at reducing the bacterial load to below detectable

levels. Changes in the concentration of bioavailable lysine, in addition to fatty acid composition

were pre-planned exploratory outcomes. It was hypothesized that alternative pasteurization

techniques would result in reduced changes in levels of folate, as well as micronutrients and

bioactive components. It was also hypothesized that alternative, non-thermal pasteurization

techniques would be more effective at reducing the bacterial load in human milk to a level that

yields culture negative bacteriology.

3.2 Methods 3.2.1 Study Design

This project was part of a larger Canadian Institute for Health Research funded program of

research entitled, MaxiMOM, maximizing mother’s own milk (FDN#143233). The overarching

goal of this research is to improve the health and development of very low birth-weight infants

through improved nutrition, achieved by maximizing nutrients and bioactives found in human

milk. Prior to collection of human donor milk, research ethics board approval was obtained from

The Hospital for Sick Children and the Human Milk Banking Association of North America

(HMBANA) (Appendix A). Consent was obtained from each mother at the time of milk

donation to use the milk for research. The experimental design is summarized in (Figure 2).

33

Figure 2. Experimental design and overview of study

Sample collection

Donor human milk that contained a total bacterial load >5 x 107 CFU/L was collected from the

Rogers Hixon Ontario Human Milk Bank. Donated milk above this pre-pasteurization threshold

would not be processed or dispensed for use according to the Rogers Hixon Ontario Human

A total of 17 raw samples of donor milk was collected frozen from the Rogers Hixon Ontario Human Milk Bank. After undergoing baseline culture and composition analysis (macronutrients, micronutrients [vitamin C, folate], and bioactive components (BSSL, lysozyme), each of the 17 samples underwent each of the 4 pasteurization techniques. Post-pasteurization culture and composition analyses were conducted for each sample for each technique. Comparisons between pre-and post- pasteurization were used to assess the impact of pasteurization while the difference in bacterial cultures pre- and post- gave an indication of the ability of the method of yielding culture-negative bacteriology.

34

Milk Bank policy and procedures and would have been otherwise discarded. Seventeen samples

of 2L each were collected. Each of the 17 samples of human milk originated from 17 distinct

donors. Mother’s milk was usually frozen in aliquots of 4-6 ounces in plastic milk collection

bags and transported from each mother’s home in insulated boxes containing 5L of frozen milk.

Milk samples were kept frozen at -20°C until the multiple milk collection bags from each donor

were thawed and pooled. Each pool was prepared by thawing milk overnight in a refrigerator at

4°C and then transferring the contents of each collection bag into a large 4L beaker. The beaker

was then placed in a shaker waterbath set at 37°C and agitated using a glass rod and a magnetic

stirring plate every half an hour until the milk reached 37°C. This technique was carried out to

be certain that milk allocated for each pasteurization was homogenous. Four 400mL aliquots for

each pasteurization method were transferred into 500mL containers and frozen immediately at -

20°C until pasteurization. Further, raw, pre-pasteurization aliquots of each sample of human

donor milk were then taken and frozen and subsequently transferred to -80°C long-term storage

until microbiology and nutrient analyses.

Pasteurization techniques

Samples for all 4 pasteurization techniques that were previously frozen at -20°C were thawed

overnight in a refrigerator at 4°C and heated in a shaker waterbath as described above to ensure

the milk fat globules and other milk constituents were evenly distributed. After each

pasteurization, aliquots for bacteriology and milk composition were collected and frozen

immediately on dry ice and then stored at -80°C until analysis.

One hundred and twenty mL from each of the 17 milk samples were gently poured into

specialized bottles (Sterifeed, Medicare Colagate Ltd., UK), capped, and were heat sealed.

Holder pasteurization was carried out using a Sterifeed T30 tabletop pasteurizer (Medicare

Colagate Ltd., UK), equipped with a temperature probe and data logger to monitor the process.

Milk samples were then heated to 62.5°C for 30 min, and then cooled to 4°C.

Flash-heating was carried out according to a previously published protocol with some

modification10,114. This method of human milk pasteurization is similar to what is being

recommended for HIV+ women to carry out in Africa and low-resource countries to limit viral

transmission to their infants. Homogenous samples were gently poured into a 250mL media jar

35

that could withstand heat, and covered with the lid. The jars were then placed in a beaker filled

with 1L of water (room temperature). The beaker and media bottle were then simultaneously

heated rapidly on a hot plate until the water in the beaker began to boil. Once removed, the jar

containing the milk sample was cooled to 4°C and aliquots were taken for analyses.

UV-C irradiation of human milk was adapted from the experimental design protocol published

by Christen et al. with modification, as shown in Figure 3115. A UV germicidal lamp (Phillps

TUV PL-L, 2.3 Watt) was used as the source of UV in the experiments. The procedure consisted

of having a turbulent flow of milk around a UV-C emitting lamp for a specified dosage which

was dependent on the quantity of milk solids. This was to ensure that a sufficient amount of

UV-C irradiation penetrated the opacity of human milk. The calculation of dosage (J/L) was

determined by what was observed to be effective (5 log reduction in bacterial load) in the

experiments of Christen et al. 2011 using 120g/L as an approximate average of milk solids in

human milk169 (Figure 4). The calculated UV-C dose (J/s or watt) was converted into time based

on the UV-C wattage rating of the lamp used. The calculated time to achieve 5-log reduction

was 15 minutes. As a precaution, aliquots for bacteriology and nutrient analyses were taken at

both 15 minutes and at 25 minutes to ensure adequate pasteurization.

36

Figure 3. Experimental design of UV-C pasteurization adapted from Christen et al.

The experimental design of UV-C pasteurization as adapted from Christen et al 2011. Magnetic stirring induces turbulent flow in human milk, allowing penetration of UV irradiation. The entire apparatus is covered in aluminum foil prior to use.

37

Figure 4. The relationship between milk solids and effective UV-C pasteurization dose to achieve 5-log reduction.

HHP is a technology used in the food industry to preserve food using very high pressure that can

inactivate most microorganisms. It is commercially used in Canada for a variety of food

applications including ready-to-eat meats, raw meats, fruit and vegetable-based

juices/smoothies, eggs and spreads121. For this work, we collaborated with Drs. Yves Pouliot

and Alain Doyen from the Université Laval who brought expertise in HHP. Dr. Pouliot is a

Canada Research Chair on the efficiency of milk processing methods in the dairy industry.

To effectively pasteurize human milk using HHP, experimental conditions were first optimized

in a series of experiments to reduce bacterial load below 1000 CFU/L. HHP can be optimized by

manipulating the pressure of the vessel, the temperature at which pressurization occurs, and the

length of time pressure is applied. In these experiments, HHP was applied in a discontinuous

hydrostatic pressurization (Hiperbaric 135, Burgos, Spain) with water as the pressure

y = 68.407e0.029x

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

80 90 100 110 120 130 140 150

Joul

es/L

Milk Solids (g/L)

Effective UV-C Dosage Increases with Milk Solids

Adapted from Christen et al. 2013. Data shown demonstrate the relationship between milk solids and the UV-C dose to achieve a 5-log reduction in bacterial load.

38

transmission medium and temperature of 4°C. The stainless-steel pressure vessel measured 0.30

m in diameter and 2.20 m in length with a working volume of 135 L.

Using literature suggestions, various pressure, temperature and holding time combinations were

studied and assessed for efficacy (Table 3). After the first experiment, it was observed that a

pressure of 500MPa at 4°C for either 8 or 10 min reduced the bacterial load to <1000 CFU/L.

The conditions in experiment 1 were repeated in experiment 2. Double cycles were tested in

experiments 3 and 4. It was hypothesized that the first cycle of HHP functions to germinate any

dormant spores, while a second cycle would eliminate any germinated bacteria. There was no

observed benefit of a double cycle of HHP. Overall, these results confirmed that a pressure of

either 400 or 500 MPa applied once for 8 min would be sufficient to pasteurize human milk,

however, as a precaution, the higher pressure of 500MPa was used to account for the presence

of potentially resistant bacteria.

39

Table 3. Optimization Experiments for HHP pasteurization of human milk

Experiment Pressure (MPa)

Temperature (°C)

Holding Time (min)

CFU/L Double Cycle

1

500

4

3 >1000

No

4 >1000 5 >1000 8 0 10 0

600

1 >1000 2 >1000 3 >1000 4 >1000 5 >1000

2 400 8 0 10 0 500 8 0 10 0 3

400

3,3

>50 000

Yes

0 0

500 2000

0 0

600 1000 0 4

200,600* 20, 4† 4000

0 0 *Cycle 1: 200 MPa, Cycle 2: 600MPa † Cycle 1: Temperature = 20°C was used, Cycle 2: Temperature = 4°C In the first experiment, various holding times were tested at 500 and 600 MPa, the most effective being 8 and 10 minutes of 500MPa. In the second experiment, the holding time of 8 and 10 min was re-tested at 400 and 500MPa. In the third experiment, pressures of 400MPa and 500MPa were tested in triplicate in a double cycle (3 min cycle, 24h incubation at 4°C, 3 min cycle). 600MPa was tested in duplicate. In the fourth experiment, a double cycle was tested at two different pressures and temperatures.

40

3.2.2 Analysis of Nutrients, Bioactive Components and Bacteriology Macronutrients and energy

The macronutrient content was determined using a mid-infrared human milk analyzer (Miris

Human Milk Analyzer, Uppsala, Sweden), calibrated to measure fat, protein, carbohydrate and

total solids simultaneously. Energy content was calculated from macronutrient data.

Methodological details of the mid-infrared analysis are summarized in Table 4. Mid-infrared

human milk analysis using Miris instrumentation has been validated against wet-chemistry

techniques170,171. As an assessment of the precision of the instrument for measurement of

macronutrient content, aliquots of a homogenous pool of human donor milk was run daily in our

laboratory, the results are summarized in Table 4.

Table 4. Mid-infrared analysis of macronutrients and energy using the Miris human milk analyzer 172 Analyte Infrared

Determination Absorbance (µm)

Wet-Chemistry Validation

Reported Imprecision

Observed CV (%)*

Crude Protein Secondary amide (II) groups of peptide bonds

6.5 Kjeldahl 173

<0.2 g/100mL 4.5

Carbohydrate

OH- groups of lactose and mono-oligosaccharides

9.6 Proximate analysis 174,175

<0.4 g/100mL 2.1

Fat Carbonyl group of ester bonds of glycerides

5.7 Röse-Gottlieb 176

<0.2 g/100mL 2.7

Energy Calculated from Macronutrients 177

- - - 2.1

Total Solids Calculated from fat, crude protein, carbohydrate †

- - - 2.0

CV: coefficient of variation *Calculated from repeated analysis of a homogenous pool of human donor milk (n=9) † Assuming an approximate 0.2g/100mL mineral content

41

Folate Aliquots of raw and pasteurized milk for folate analyses were frozen in 1% w/v sodium

ascorbate (Sigma-Aldrich, St. Louis, MO) to prevent degradation of folates during prolonged

storage at -80°C178. At all times, samples were shielded from UV light to prevent any

degradation of folate, given its inherent sensitivity to light and heat. All chemicals were

purchased from Sigma-Aldrich, St. Louis, MO and analysis was conducted as per O’Connor et

al. 1991, with some modification (See Appendix B)85. Folates from each sample were extracted

using freshly prepared Wilson-Horne buffer that contained 2% w/v sodium ascorbate solution in

50mM HEPES 50mM CHES and 0.2M 2-mercaptoetahnol at pH 7.85. Once extracted, samples

underwent a tri-enzyme digestion using ⍺-amylase, protease and rat conjugase, as described

previously179

The microbiological assay was used to quantify amounts of extracted folates as described by

Molloy and Scott, using Lactobacillus rhamnosus (ATCC #7469) as the test organisms, and 5-

methyltetrahydrofolate to produce the standard curve178. A homogenous pool of human milk

and a certified reference material, BCR-487 (European Commission, Joint Research Centre)

which underwent both extraction and trienzyme digestion, were run with the samples daily to

assess the precision and accuracy, respectively, of study procedures. The resulting coefficient of

variation (CV) following the analysis of the human milk samples (N=11) and the pig liver (n=7)

was 5.5% and 10% respectively. All milk and quality control samples were assayed in triplicate.

Vitamin C

Raw and pasteurized human milk samples were diluted 1:1 with 10% metaphosphoric acid

(ACS reagent 79613, Sigma-Aldrich, St. Louis MO) and 1% oxalic acid (Anhydrous, 75688,

Sigma-Aldrich, St. Louis MO, USA) to inhibit degradation and were mixed by inversion prior to

freezing at -80 °C180. The aliquots of human milk were thawed at room temperature (22°C)

protected from light and mixed gently using a vortex to ensure homogeneity of sampling. Total

vitamin C content (ascorbic acid and dehydroascorbic acid), and ascorbic acid were determined

separately (Appendix C). Sample preparation and HPLC analysis was carried out using the

previously published method of Romeu-Nadal et al with some modification (See Appendix

C)181. L-Ascorbic acid (ACS reagent, 255564, Sigma-Aldrich, St. Louis) was used to generate

42

the standard curve and an Agilent 1260 Infinity II HPLC (Agilent Technologies, Santa Clara,

CA) was utilized.

Standard addition experiments were conducted to verify the accuracy of the HPLC method for

vitamin C. Specifically, the highest ascorbic acid standard (80mg/L) was added to 4 separate

samples of homogenous donor milk. Ascorbic acid was then determined by HPLC (Appendix

C). The average recovery of ascorbic acid was 101.1% with a coefficient of variation of 2.4%.

To assess the precision of the method, multiple samples of the homogenous pool of donor milk

were run on different days. The inter-assay CV of the measurements (n=5 days) was 14%.

Bile salt-stimulated lipase

Bile-salt stimulated lipase (BSSL) activity was determined using a commercially available kit

(QuantiChrom Lipase Assay Kit, BioAssay Systems, Hayward, CA, USA) validated for use in

human milk and able to detect lipase activity from 40-1600U/mL115,182. Samples frozen at -80°C

were gently thawed on ice and were centrifuged at 6500xg for 10 min at 4°C to separate the

milk into its solid and aqueous fractions. The aqueous fraction of each sample was removed and

diluted by a factor of 80 with deionized water. Samples, including a pool of homogenous donor

milk were then plated on a 96-well microplate. The absorbance was measured by

spectrophotometer at 405nm after 10 and 20 min of incubation at room temperature. The lipase

activity was calculated according to kit instructions (Figure 4). One unit of enzyme was defined

as the amount able to cleave 1μmol of dimercaptopropanol tributyrate per minute under the

assay conditions (pH 8.5). Precision was assessed by repeated measure of BSSL activity in

aliquots of a homogenous pool of donor milk measured on different days. Measurements

yielded an inter-assay CV of 7.4% (n=9).

Figure 4. Calculation of Bile Salt-Stimulated Lipase Activity using the QuantiChrom Lipase Assay Kit

Lipase Activity = 𝑶𝑶𝑶𝑶𝟐𝟐𝟐𝟐 𝒎𝒎𝒎𝒎𝒎𝒎− 𝑶𝑶𝑶𝑶𝟏𝟏𝟐𝟐 𝒎𝒎𝒎𝒎𝒎𝒎

𝑶𝑶𝑶𝑶𝑪𝑪𝑪𝑪𝑪𝑪𝒎𝒎𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪− 𝑶𝑶𝑶𝑶𝑯𝑯𝟐𝟐𝑶𝑶 x 735 (U/L)

Lipase activity is calculated using the optical density (405 nm) at 10 and 20 min of the test sample and is compared to the calibrator at 20 min. One unit of enzyme catalyzes the cleavage of 1 µmol of dimercaptopropanol tributyrate per minute under the assay conditions (pH 8.5)

43

Lactoferrin The concentration of lactoferrin was determined by HPLC using the method established by Yao

et al. with modification for application to human milk (Appendix D). Briefly, samples were

thawed at room temperature (22°C), gently mixed by vortex and defatted using 0.45µm

centrifugal filters. Lactoferrin from human milk (Sigma-Aldrich, St. Louis MO, USA) was used

to generate the standard curve. Modifications to the method were conducted to ensure optimal

detection of lactoferrin (Appendix D). A homogenous pool of milk was used to determine the

precision of the method, while standard addition experiments were used to determine accuracy

and yield. Repeated measures of lactoferrin in the homogenous pool of donor milk (N=6)

yielded a mean ± standard deviation of 0.52 ± 0.04 g/L (CV= 7%). When assayed in triplicate,

inter-assay variability was determined to be <5%. Multiple standard addition experiments

conducted yielded a percent recovery of approximately 88%.

Lysozyme

Lysozyme activity was determined using the Micrococcus lysodeikticus turbidimetric assay

adapted from Shugar et al.183 using a commercially available kit (Sigma-Aldrich, Lysozyme

detection kit, St. Louis MO, USA). The test organism, M. lysodeikticus, is a gram-positive

bacterium. Human milk samples stored at -80°C were thawed on ice and defatted. Previous

studies in lysozyme isolation of human milk have shown that de-fatting is essential as lipid

globules interfere with assay detection184. De-fatted samples were then mixed gently using a

vortex and diluted appropriately (Appendix E).

Lysozyme can hydrolyze the β 1,4 glycosidic linkages between N-acetylmuramic acid and N-

acetylglucosamine found in the mucopeptide cell wall structure of M. lysodeikticus. The

presence of lysozyme in a cell suspension solution of M. lysodeikticus results in cleavage of the

cells and a reduction in turbidity over time. Lyophilized lysozyme isolated from chicken egg

white (L6876, batch SLBQ0509V, Sigma-Aldrich, St. Louis MO) was used as a certified

reference to validate the assay. We modified the protocol from a cuvette method to a high-

throughput 96 well microplate method modified from previous work by Lee and Yang and

validated in our lab (Appendix E)185.

44

Given the inherent differences in path length and volumes, we conducted a validation

experiment to determine a correction coefficient for use in the microplate format. For this

validation, two separate experiments were conducted concurrently and the same volume of

prepared lysozyme was assayed in both cuvette and microplate. The ratio of the cuvette

measured rate as compared with the microplate measured rate was 4.94, and was used as a

correction coefficient for microplate utilization. Once calculated, the results were then corrected

using the 96-well plate coefficient as determined by our validation experiments. Accuracy and

precision were determined via the analysis of a standard in each experiment from lyophilized

lysozyme isolated from chicken egg white. Results in these experiments showed that the mean

activity of the standard which was expected to be 306 U/mL was 499U/mL corrected with an

inter-assay CV of 9%.

Bacteriology

Total bacterial counts on raw and pasteurized samples were analyzed at the Hospital for Sick

Children core microbiology lab following the same protocol used at the Rogers Hixon Ontario

Human Milk Bank (Appendix F). Total bacterial count was determined for each sample using

both blood agar and MacConkey agar (incubated for 48 hours at 37°C) and referenced against

cut-offs used by the Rogers Hixon Ontario Human Milk Bank. Colonies were not counted above

50 (>50 000 CFU/L). Although not a requirement of all milk banks, The Rogers Hixon Ontario

Human Milk Bank uses a pre-pasteurization bacteriological screen to determine if the

contamination of the milk is sufficiently low (<5x107 CFU/L) to be pasteurized and used for

feeding infants. To ensure consistency between bacteriology conducted for the milk bank at

Sinai Health System and those conducted for this study at The Hospital for Sick Children, we

cross-validated a subset of samples by analysing the 2 separate aliquots of the sample at each

lab. We observed excellent agreement between the two labs. A total of 9 out of 11 test samples

yielded the same result from the two labs.

Exploratory analyses

Exploratory analyses were conducted to assess the feasibility and value of assessing changes in

fatty acid composition and available lysine following pasteurization. The fatty acid composition

of two randomly selected samples was quantified after each pasteurization technique. The

available lysine was measured in 5 samples, chosen at random, following each pasteurization

45

technique. The number of samples analyzed in each of the pilot studies were chosen based on

cost and expected variability in human milk.

Lipids were extracted from human milk following methodologies adapted from Folch, Lees, and

Sloane Stanley186. Briefly, samples were homogenized and extraction was carried out in 2:1:0.8

chloroform: methanol: potassium chloride (0.88%) with a known amount of internal standard

(NuChek-Prep, Elysian, MN, USA) as described previously187. Extracted samples were then

dried down under nitrogen gas, and methylated by heating at 100 °C in 0.3:1 hexane: boron

trifluoride-methanol. As previously described, the fatty acid methyl esters in hexane were

removed by adding deionized water and analyzed via gas chromatography flame ionization

detection on a Varian-430 gas chromatograph (Varian, Lake Forest, CA, USA) with some

modification (Appendix G)188.

Analysis of available lysine was carried out using a BASi 460 Microbore HPLC system with

electrochemical detection (Bio-analytical Systems Inc., West Lafayette, IN, USA) using a

Uniget C-18 reverse phase microbore column (BASi, 8912; analytical-1 x 150 mm, 5 μm ODS)

as the stationary phase as described previously with some modification 189,190 (Appendix H).

3.2.3 Statistical Analyses

Statistical analyses were conducted using SAS v. 9.4 (SAS Institute, Cary, NC, USA).

The distribution of the outcome variables (nutrient and bioactive composition) was verified

using PROC UNIVARIATE. Mean nutrient and bioactive concentrations were compared across

groups (i.e raw, Holder, flash-heat, UV-C and HHP) by analysis of variance (ANOVA) using

mixed models (PROC MIXED). When a statistically significant result was found, pair-wise

comparisons were conducted using LS-MEANS. Using this procedure, levels of nutrients and

bioactives after each pasteurization technique were compared to raw, unpasteurized milk, and

were compared to each other to determine method superiority and establish a hierarchy of least

to greatest changes in composition for the pasteurization. The proportion of samples with

negative cultures (pass) for each pasteurization technique was assessed using logistic regression

(PROC GENMOD). Paired t-tests were used to determine mean differences in levels of

available lysine between Holder and HHP as part of the pilot.

46

3.3 Results 3.3.1 Nutrients and Energy

Overall, there were minimal changes in the macronutrient content of the donor milk following

all pasteurization procedures, where only a statistically significant reduction in total

carbohydrates following HHP (ß=-0.24g/100mL, p=0.04) was observed (Figure 5).

In terms of micronutrients, there were significant reductions in levels of folate in donor milk

following Holder (ß= -52 nmol/L, p=0.01), Flash-heat (ß=-46 nmol/L, p=0.03) and UV-C (ß=

-48 nmol/L, p= 0.02) pasteurization, however there was no statistically significant loss of folate

after HHP (Figure 6). There were no significant differences related to loss of folate when

individual methods were compared to one another (Figure 6).

Figure 5. Changes in macronutrient composition following pasteurization

Macronutrient composition as determined by mid-infrared analysis. The macronutrient concentration post-pasteurization is not significantly altered as compared to raw milk (all p>0.05). A statistically significant reduction in carbohydrate was observed post-HHP (p=0.04).

47

Figure 6. The impact of different pasteurization methods on the folate concentration of human milk

Similarly, Holder, flash-heating, UV-C and HHP all resulted in a significant losses of vitamin C

(ß= -9.4 mg/L, -8.8 mg/L, -10.5 mg/L and -11.1 mg/L for each pasteurization respectively) in

addition to ascorbic acid alone (ß= -8.6 mg/L, -8.5 mg/L, -11.1 mg/L and -10.7 mg/L for each

pasteurization method respectively) (Figure 7). All p-values were <0.001. There were no

significant differences when comparisons were made among methods. Representative

chromatograms are appended (Appendix I).

Data are presented as medians (bold horizontal line) and first and third quartiles (horizontal lines). Whiskers were calculated as far as the data extended to a maximum of 1.5 times the interquartile range. Differences in levels of folate between both raw and pasteurized samples were analyzed using ANOVA with mixed modelling. Overall, there was a significant effect of pasteurization (p<0.0001). The concentration of folate was significantly reduced following Holder (p=0.01), flash heat (p=0.03) and UV-C (p=0.02) as compared to raw milk. No significant difference was found between raw milk and high hydrostatic pressure (HHP) (p=0.5). Different letters indicate statistical significance for post-hoc least squares means analysis (p<0.05).

a b b b ab

48

Figure 7. The impact of different pasteurization methods on the total vitamin C concentration of human milk

a b b b b

Data are presented as medians (bold horizontal line) and first and third quartiles (horizontal lines). Whiskers were calculated as far as the data extended to a maximum of 1.5 times the interquartile range. Differences in levels of vitamin C between both raw and pasteurized samples were analyzed using ANOVA with mixed modelling. Overall, there was a significant effect of pasteurization (p<0.0001). Different letters indicate statistical significance for post-hoc least squares means analysis (p<0.05). The concentration of total vitamin C was significantly altered after every pasteurization method as compared to raw milk. There were no differences observed between methods.

49

In terms of bioactive components, a complete abolishment of BSSL (<1% of original activity),

was observed following thermal pasteurization, Holder (ß= -52 U/mL, p <0.0001) and Flash-

heating (ß=-53 U/mL, p <0.0001) (Figure 8).

To a lesser degree, there was a significant reduction in BSSL activity following UV-C

irradiation (ß=-26 U/mL, p<0.0001), and no reduction in activity following HHP. BSSL activity

was less diminished following UV-C when compared to Holder (ß=-27 U/mL, p<0.0001) and

Flash-heating (ß=-24 U/mL, p<0.0001).

There was no loss of BSSL activity following HHP compared to raw milk, accordingly, levels

of BSSL activity were significantly lower (p<0.0001) with Holder (-48 U/mL), Flash-heating (-

48 U/mL) and UV-C (-20 U/mL) methods.

The concentration of lactoferrin was also impacted by pasteurization (Figure 9). There was a

statistically significant reduction in levels following all pasteurization techniques compared to

raw milk, including Holder (ß=-0.43 g/L, p<0.0001), flash-heating (ß= - 0.66 g/L, p<0.0001),

UV-C (ß=-0.43 g/L, p<0.0001) and HHP (ß=-0.22 g/L, p=0.02). Among methods, there were no

difference observed between levels of lactoferrin following Holder and UV-C. Flash-heating

resulted in the greatest reduction in lactoferrin as compared to Holder (ß=-0.23 g/L, p=0.01),

UV-C (ß=-0.23 g/L, p=0.01) and HHP (ß=-0.44 g/L, p<0.0001). HHP reduced the level of

lactoferrin the least compared to the other methods and had significantly higher lactoferrin as

compared to Holder (ß=0.21 g/L), flash-heating (ß=0.44 g/L, p<0.0001), and UV-C (ß=0.21 g/L,

p=0.02). Representative chromatograms are appended (Appendix J).

50

Figure 8. The impact of different pasteurization methods on the bile salt stimulated lipase activity of human milk

Lysozyme activity was also significantly impacted by some pasteurization techniques (Figure

10). There was a significant loss of activity following Flash-heating (ß= -11.3 U/L, p=0.004)

and UV-C (ß= -19.0 U/L, p<0.0001); however, there was no statistically significant loss of

activity following Holder or HHP. As such, lysozyme activity following Holder and HHP was

significantly higher than flash-heating (ß=16.4 U/L, p<0.0001 and ß=8.5, p=0.03 respectively).

a c c b a

Data are presented as medians (bold horizontal line) and first and third quartiles (horizontal lines). Whiskers were calculated as far as the data extended to a maximum of 1.5 times the interquartile range. Differences in levels of bile salt stimulated lipase activity between both raw and pasteurized samples were analyzed using ANOVA with mixed modelling. Overall, there was a significant effect of pasteurization (p<0.0001). Different letters indicate statistical significance for post-hoc least squares means analysis (p<0.05). The bile salt stimulated lipase activity was significantly reduced compared to raw milk after Holder, flash-heating and UV-C (p<0.0001) but not HHP (p=0.18). There was significantly higher activity after UV-C than Holder and flash-heat (p<0.0001)

51

HHP resulted in a 7.8 U/L higher lysozyme activity when compared to Holder (p=0.04). The

level of lysozyme activity was significantly lower after UV-C pasteurization by 16.3 U/L, 7.7

U/L, and 24 U/L when compared to Holder, flash-Heating and HHP.

The changes in nutrients and bioactives following the 4 pasteurizations are summarized in

Table 5.

52

Figure 9. The impact of different pasteurization methods on the lactoferrin concentration in human milk

Data are presented as medians (bold horizontal line) and first and third quartiles (horizontal lines). Whiskers were calculated as far as the data extended to a maximum of 1.5 times the interquartile range. Differences in lactoferrin between both raw and pasteurized samples were analyzed using ANOVA with mixed modelling. Overall, there was a significant effect of pasteurization (p<0.0001). Different letters indicate statistical significance for post-hoc least squares means analysis (p<0.05). The concentration of lactoferrin was significantly reduced compared to raw milk after Holder, flash-heating, UV-C (all p<0.0001) and HHP (p=0.02). The concentration of lactoferrin was significantly lower following flash-heating compared to Holder (p=0.01), UV (p=0.01) and HHP (p<0.0001). The level of lactoferrin was not significantly different between Holder and UV-C. HHP resulted in a higher level of lactoferrin compared to Holder (p=0.02), flash-heating (p<0.0001) and UV-C (p=0.02) methods.

a c d c b

53

Figure 10. The impact of different pasteurization methods on the lysozyme activity in human milk

ab a c d b

Data are presented as medians (bold horizontal line) and first and third quartiles (horizontal lines). Whiskers were calculated as far as the data extended to a maximum of 1.5 times the interquartile range. Differences in levels of lysozyme activity between both raw and pasteurized samples were analyzed using ANOVA with mixed modelling. Overall, there was a significant effect of pasteurization (p<0.0001). Different letters indicate statistical significance for post-hoc least squares means analysis (p<0.05). The concentration of lysozyme activity is significantly reduced compared to raw milk after flash-heating (p=0.004) and UV-C (p<0.0001) but not HHP (p=0.19) or Holder (p=0.47). There was significantly higher activity after HHP than Holder (p=0.04).

54

Analyte Raw Post-Holder Post-Flash Heat Post UV-C Post-HHP

Carbohydrate (g/100mL) 7.0 (6.8, 7.2) a 6.9 (6.6, 7.2) a 6.9 (6.8, 7.1) a 6.9 (6.7, 7.1) a 6.9 (6.5, 7.0) b

Fat (g/100mL) 3.4 (2.7, 3.8) a 3.3 (2.8, 3.7) a 3.3 (2.9, 3.6) a 3.3 (2.6, 3.6) a 3.2 (2.7, 3.4) a

Crude protein (g/100mL) 1.0 (0.95, 1.1) a 1.0 (0.95, 1.1) a 1.0 (1.0, 1.1) a 1.0 (0.90, 1.1) a 1.0 (0.90, 1.1) a

Energy (kcal/100mL) 63 (60,66) a 63 (60, 66) a 63 (58,66) a 62 (57, 66) a 62 (56, 63) a

Folate (nmol/L) 161 (149, 192) a 138 (119, 156) b 133 (111, 178) b 149 (121, 168) b 173 (148, 184) ab

Total Vitamin C (mg/L) 15 (5.0, 22) a 3.3 (0.5, 5.7) b 3.4 (0.1, 6.3) b 2.7 (1.1, 6.6) b 2.6 (1.8, 5.7) b

Ascorbic Acid (mg/L) 14 (5.0, 21) a 3.5 (0.87, 6.7) b 3.6 (0.87, 6.5) b 2.7 (1.1, 6.6) b 2.6 (0.23, 5.6) b

Bile salt-stimulated lipase (U/mL) 55 (45, 67) a 0.3 (0.2, 1.0) c 0.6 (0.1, 0.8) c 24 (21, 36) b 51 (37, 60) a

Lysozyme (U/L) 22 (14, 34) ab 21 (11, 34) a 12 (9.0, 14) c 7 (4.0, 9.0) d 27 (17, 45) b

Lactoferrin (g/L) 0.89 (0.72, 0.95) a 0.46 (0.31, 0.55) c 0.20 (0.17, 0.27) d 0.40 (0.35, 0.48) c 0.64 (0.48, 0.72) b

Values as medians and IQR- Interquartile range; Statistical comparisons made using ANOVA. Statistical significance indicated by different letters; Statistical significance was reached following HHP (p=0.04) however, reduction not clinically relevant; Significant reductions observed following Holder, flash-heating and UV-C, (p=0.01, 0.03, 0.03 respectively). Significant reductions in Total Vitamin C and in ascorbic acid alone were observed after all pasteurization methods (All p < 0.001). All reductions in bile salt-stimulated lipase significant p<0.0001

Table 5. Nutrients and bioactive levels following pre- and post-pasteurization of human donor milk

55

3.3.2 Bacteriology

The microbiology results assessing the effectiveness of the pasteurization techniques are

summarized in Table 6. Out of a total of 17 samples pasteurized, HHP resulted in the greatest

number of samples that were below the acceptable microbiological threshold for safe infant

consumption. UV-C was observed to be the most ineffective mode of pasteurization out of those

tested, having the fewest number of samples that met the HMBANA criteria for milk safe for

infant consumption (i.e <1000 CFU/L). Results from a X2 test indicate that there is a difference

in all techniques, compared to raw milk, in the number of passes and fails (p<0.001); however,

there were no significant differences between methods (p=0.17). A post-hoc power calculation

determined that given the observed effect size (0.27) and the sample size (17), the study was

underpowered to detect differences between methods. Total bacterial counts in samples that

failed to meet HMBANA criteria, were greatest and most variable with HHP and UV-C followed

by Holder and flash-heating.

Table 6. Summary of microbiology results assessing pasteurization effectiveness Pasteurization Technique

Pass (<1000 CFU/L) †

Fail (≥1000CFU/L) †

Colony Count (CFU/L) ‡

Samples N (%) Samples N (%) Holder

9 (53) 8 (47) 5000 (3000-10 000)

Flash Heat

7 (41) 10 (59) 2000 (1000, 11 000)

UV-C 25 min 6 (35) 11 (65) 8000 (4000, 50 000) 15 min§ 3 (27) 8 (73) 27 500 (5500, 50 000) High Hydrostatic Pressure Processing

12 (71)

5 (29)

10 000 (2000, 50 000)

† Post-pasteurization cut-off as defined by the Human Milk Banking Association of North America guidelines. ‡Colony count for samples which failed the post-pasteurization bacteriology screen expressed as median (interquartile range) § Bacteriology was only conducted on 11/17 samples A “pass” is defined as a total bacterial count being <1000 CFU/L. Samples that passed bacteriological screening were assumed to contain zero colonies for the purpose of statistical comparisons. There were significant differences when each method was compared to raw milk (p<0.001); however, no differences were observed between methods (p>0.2)

56

3.3.3 Exploratory

The results of the pilot studies are summarized in Table 7 and Table 8. Although there was

variability observed between the two samples analyzed for fatty acid composition, there does not

seem to be any observed effect of pasteurization. Similarly, there were no statistically significant

changes observed in available lysine concentration following Holder and HHP. As a result, it

was determined that further investigation was not warranted.

*Data shown as sample 1, sample 2

Table 7. The impact of Holder and UV-C pasteurization on the fatty acid composition (mg/mL) of human milk (N=2): a pilot study

Pasteurization Technique

Saturated Fatty Acids

Monounsaturated Fatty Acids

Omega 6 Fatty Acids

Omega 3 Fatty Acids

n-6: n-3 ratio

Raw*

14.3, 26.8

10.8, 18.4

3.5, 6.0

0.62, 0.73

5.6, 8.3

Holder*

15.3, 28.5 11.6, 19.5 3.6, 6.4 0.64, 0.77 5.6, 8.3

UV-C*

14.5, 29.2 11.1, 20.2 3.4, 6.5 0.60, 0.78 5.6, 8.3

Table 8. The impact of Holder and High Hydrostatic Pressure Processing on the free lysine content of human milk (N=5): a pilot study

Pasteurization Technique Free Lysine (µmol/dL) * Raw

2.1 (1.3, 2.9)

Holder

2.2 (1.2, 2.8)

High Hydrostatic Pressure Processing

1.7 (1.2, 2.4)

*Data shown as medians and first and third quartiles. Statistical significance was denoted at p<0.05. Paired t-tests were conducted to determine changes in available lysine. There were no statistically significant differences observed.

57

3.4 Discussion Although it is the optimal nutrition for all infants, there are many challenges which exist in

providing human milk to populations of vulnerable infants, including those born preterm at low

birth weight and those born to mothers with HIV infection42. Common to both populations of

infants is the requirement to pasteurize human milk before consumption (for donor milk for

preterm infants or for mother’s milk for the HIV positive mother who are not receiving

antiretroviral therapy). Pasteurization is required to ablate potentially pathogenic bacteria and

viruses, ensuring milk can be safely consumed without the risk of infection to already fragile and

immunocompromised infants7,35. Infants born very preterm in countries with adequate resources,

such as Canada, often receive medically-prescribed pasteurized donor milk as a supplement to

mother’s own milk. This has been shown to decrease the incidence of NEC, while increasing the

risk of poor growth27. Infants born to mothers with pre-existing HIV infection are instructed to

pasteurize their milk to decrease the likelihood of vertical transmission if antiretroviral drugs are

not available. Pasteurization of mother’s milk is done as an alternative to formula feeding which

has been shown to increase morbidity and mortality9. Although pasteurization is necessary,

heating of milk can negatively impact the nutritional composition of human milk, and may

reduce levels of important bioactives in human milk, which are thought to carry much of the

benefit attributable to its consumption. Improvements to pasteurization techniques, including

moving towards non-thermal techniques is warranted so as to delicately balance safety with

retention of nutritional and bioactive components.

To our knowledge, this is the first study to systematically assess both conventional and novel

techniques of pasteurizing human milk together in one experiment and taking into account safety

(microbiological) and nutritional composition. In addition to comparing changes in nutrient

composition pre- and post-pasteurization, we compared novel techniques to more classical

thermal pasteurization, notably, Holder, given that it is the current standard of practice. Overall,

it appears that alternative techniques, specifically, HHP, can reduce the bacterial load to below

detectable levels while maintaining most of the important nutrients and bioactives included in

this analysis.

58

In terms of our primary outcome, significant reductions of approximately 50nmol/L of folate

were observed post-Holder, -Flash-heat, -and UV-C, a reasonable conclusion given its sensitivity

to light and heat191. There were no significant reductions in folate following HHP. An

overwhelming majority of studies that have assessed folate post-Holder have reported significant

reductions, irrespective of the analytical technique utilized. Contrary to the findings reported

here, Israel-Ballard et al 2005 and 2008 found between a 30% and 40% increase in folate levels

post-Flash-heating; however, the pasteurization was carried out in a jar without a lid10,150.

Evaporative loss of the water-component of human milk could have concentrated the folate,

leading to a pseudo-increase. It is important to note that there was significantly higher variability

in the levels of folate post-Flash-heating; a likely cause being the variation in pasteurization time

as the method is based on subjective assessment of boiling. Moreover, folate may be released

from folate binding protein during pasteurization as a result of thermal denaturation; this may

account for the higher levels and increased variability observed192. Finally, this study is

noteworthy as it is the first to provide an assessment of folate levels following HHP and UV-C

pasteurizations.

In line with previous research, there were no statistically significant differences in the levels of

protein following all pasteurizations compared to raw milk, and no differences between

pasteurizations techniques. Although there is currently no literature on Flash-heating, UV-C and

HHP pasteurizations, research into Holder pasteurization assessing protein also reported non-

significant changes129,132,134-136,138; except for one study in human colostrum which saw a 13%

reduction in total protein and one study in mature human milk which only reported a 3.9%

reduction131,137. Similarly, with the exception of HHP, we found no statistically significant

differences in total carbohydrate following pasteurization, in agreement with the current

literature surrounding Holder where no differences have been reported126,129-133. The

concentration of total carbohydrate was marginally, but significantly reduced following HHP;

however, this reduction was estimated to only be 0.24g/100mL and not clinically relevant. A

probable cause of this discrepancy could be explained by the reported imprecision of the mid-

infrared analyzer, of approximately 0.4g/100mL, used to measure total carbohydrate. Likewise,

there were no differences in levels of fat following any of the pasteurization methods: an

59

interesting finding given that some studies in Holder pasteurization have found reductions in

fat129,131,134, and others have concluded that fat concentrations do not change132,133,141-144. Often,

adherence of fat to the container walls during pasteurization is thought to be responsible for fat

losses4, however, in this study, special care was given to ensuring adequate mixing of milk at

37°C to avoid attachment. This could explain why fat levels were consistently unaffected post-

pasteurization. Due to the complex matrix of human milk, is often difficult for fat globules to be

homogenous and evenly distributed; a lack of homogeneity in either the process of pasteurization

or in sampling and measurement may explain why some reductions are observed in the literature.

In support of this study, Molto-Puigmarti et al. 2011 found no significant reductions in the

concentration of fat post-HHP142.

Similar to folate, we also observed significant reductions in the concentration of vitamin C as a

result of pasteurization; the level of vitamin C decreased by both thermal and alternative

processing alike. In terms of Holder pasteurization, these results are consistent with the majority

of previous studies which report between a 20%-36% reduction in vitamin C as assessed by

HPLC142,147,151, except for one study which used a fluorometric method and found no significant

reductions148. The results of this study do not fully agree with what has been reported in the

literature for flash-heating and HHP, which found non-significant reductions in vitamin C. We

suspect, similar to folate, that because no lid was used in the experiments for flash-heating, any

losses of vitamin C due to heat were counterbalanced by the concentrating effect of evaporation

leading to loss of liquid.

In terms of HHP, one study carried out in sub-zero temperatures found that pressurization of

200MPa resulted in only a 6% non-significant reduction in total vitamin C, while ascorbic acid

was significantly reduced by 11%193. The experimental design of the former study differed

significantly from the methods used in our experiment presented here; moreover, the sample size

consisted of only 7 mothers193. Furthermore, in a study of 11 milk samples, HHP did not

significantly reduce levels of vitamin C142. The HHP protocol reported pressurizations of

between 400-600MPa for only 5 minutes—different from the protocol used in this study.

Outside of the human milk literature, ascorbic acid was reduced following the HHP treatment of

tomato puree at 400MPa for 15 min at 25°C by 40% while HHP treatment of blueberry juice

60

followed by refrigerated storage caused a 31% reduction in ascorbic acid194,195. Overall, we

suspect that the loss of vitamin C observed is most likely due to a combination of adiabatic

heating and oxidation as opposed to the pressurization, as proposed by Sukhmanov et al., who

reported through kinetic modeling that heating as a result of pressurization affected vitamin C

greater than the pressurization itself196. During its characterization following HHP, Oey et al.

report that vitamin C degradation at both elevated and atmospheric pressures is directly

proportional to the soluble oxygen concentration; this is evidence that vitamin C degradation

during this process is a product of oxidation. In support of this hypothesis is the contour plot of

vitamin C residual content following HHP which shows the greatest degradation of vitamin C

(30%-40%) occurs at pressures 400-550 MPa (10-20 min)—similar to the conditions in our

study197. Nonetheless, additional studies are required to fully understand the effect of HHP on

vitamin C in the complex matrix of human milk so as to determine whether loss of vitamin C is

occurring as a result of processing, pressurization or as an artefact of analysis.

Significant reductions in the vitamin C concentration were also observed following UV-C

irradiation, comparable to those losses post-thermal treatment; this is a novel finding given this is

the first study to address the effect of UV-C irradiation on the vitamin C content of human milk.

This reduction is reasonable given reports of vitamin sensitivity to UV light in bovine milk198;

however, we cannot discount the potential oxidative degradation occurring due to the inherent

thermal energy emitted by the UV lamp.

This study found that BSSL was significantly impacted by pasteurization. Human milk that

underwent both Holder and flash-heating completely lost all activity of endogenous BSSL,

important for the removal of fatty acids from triglycerides. Although this is the first report of

BSSL activity post-Flash-heating, this finding is consistent with previous studies of Holder

pasteurization showing complete abolishment of lipase activity, in spite of the various analytical

methods utilized135,138,144. BSSL activity was also significantly reduced following UV-C

pasteurization, however, not to the extent that was observed post-Holder and Flash-heating. This

is in contrast to what was reported by Christen et al. 2013 who found that UV-C did not

significantly affect BSSL activity115. A probable cause for this may be the increased UV-C dose

which we administered as required to achieve negative bacteriology cultures. We chose not to

61

assess the BSSL activity using the same UV-C dosage as Christen et al. given results from a pilot

experiment showing the dose to be consistently ineffective at pasteurization. It has been

demonstrated that aromatic amino acids, tryptophan, tyrosine and phenylalanine are sensitive to

UV- light given that they capture energy in the range of 250-290nm199. In doing so, they become

energized, which effectively alters tertiary and quaternary protein structure199. Given that

aromatic residues comprise approximately 10% of the BSSL protein, this may explain why

reductions in BSSL activity are observed post-UV-C pasteurization200. Finally, we observed that

there we no statistically significant differences in BSSL activity following HHP; HHP had higher

levels of BSSL than milk treated with Holder, flash-heating and UV-C. Although there is no past

literature that has reported on this, this supports our hypothesis that alternative, non-thermal

methods are superior in terms of mitigating nutrient and bioactive losses.

Varying changes in the activity of lysozyme were observed. Despite the current discordant

literature surrounding the effects of Holder pasteurization and lysozyme, we found that there was

no significant impact post-Holder on its activity. Many studies report significant reductions in

lysozyme activity, which range from 24%159 to 60%156,157. Our results are consistent with some

studies in human milk, including a more recent report from 2017 which found no significant

reduction in lysozyme activity152. With regard to Flash-heating, a 45% reduction in lysozyme

activity was observed, in line with similar findings from Chantry et al., who likewise found a

45% reduction161. This result is also compatible with Daniels et al. who found a smaller, yet

significant, 22% reduction in activity152. Moreover, our results contrast a study with null finding,

however, lysozyme in that study was quantified as concentration by gel electrophoresis as

opposed to enzymatic activity10. Thus, lysozyme activity was more severely impacted by flash-

heating than Holder, despite both being thermal methods. It is possible that more intense heating

and a higher peak temperature reported (73ºC), may increase the susceptibility of lysozyme

inactivation10. When investigating the non-thermal pasteurization techniques, UV-C irradiation

resulted in a significant 68% reduction in lysozyme activity, similar to a less severe, 25%

significant reduction found by Christen et al.154. Similar to BSSL, the protein sequence of

lysozyme contains approximately 10% aromatic amino acids, discussed previously in terms of

increasing susceptibility to UV irradiation201. No significant reductions in lysozyme activity

post-HHP were observed, similar to previous reports in the literature158. In fact, lysozyme

62

activity was significantly higher post-HHP compared to raw milk, potentially due to a stabilizing

effect to the quaternary structure. An increase in activity following HHP has been previously

observed in bovine milk; it has been hypothesized that partial unfolding of the lysozyme protein

induced by HHP may increase activity as a result of greater surface area and increased

probability of contact with its cellular targets202,203. Both HHP and Holder did not decrease

lysozyme activity, and were superior to flash-heating and UV-C which impacted its activity.

All pasteurization methods resulted in significant reductions in the level of lactoferrin compared

to raw milk. We observed that flash-heating had the greatest reduction in lactoferrin

concentration, HHP resulted in the least reduction, while the reduced level of lactoferrin

following Holder and UV-C were not statistically different from each other. A reduction of

approximately 66% was determined when aggregating the findings of 10 studies assessing the

effect of Holder pasteurization on human lactoferrin (Table 2). Consistent with the literature,

results from our study demonstrated that lactoferrin was reduced by approximately 48%

(Interquartile Range: 38% - 65%). In terms of flash-heating, there have only been 3 studies

conducted that have reported on its effect on lactoferrin. The average reduction reported was

approximately 55%; however, it was observed to be as high as 84% in one study10. Our results

are consistent as we report a reduction in lactoferrin by flash-heating of approximately 78%. This

is potentially of concern as it is a key bioactive protein that can help reduce infection, often

endemic to low-resource countries where flash-heating is practiced. Large reductions in

lactoferrin were expected following thermal pasteurization given the susceptibility of lactoferrin

to heat. In particular, similar to bovine milk, we suspect that heat-induced changes in casein-

bound calcium phosphate, increases casein amorphicity and its interaction with lactoferrin204.

Increased interactions with lactoferrin have been shown to increase its sensitivity to heat and

denaturation potential.

We found that UV-C pasteurization resulted in approximately a 55% reduction in lactoferrin

concentration, which is significantly higher than the 13% reduction that was reported in one

other previous study assessing UV-C and lactoferrin. The increased reduction of lactoferrin was

likely a consequence of the increased dose of UV-C that was administered to our milk samples as

a requirement to achieve significant reductions in bacterial load. Similar to Holder and flash-

63

heating, we believe that generation of heat during UV-C irradiation may account for some losses

observed. As previously discussed, UV-C targets aromatic amino acids such as tyrosine,

tryptophan and phenylalanine, which comprise approximately 10% of all amino acids in human

lactoferrin by weight205. As such, UV-C irradiation may itself cause some denaturation of

lactoferrin observed; however, it is difficult to disentangle the various potential sources leading

to lactoferrin degradation.

Following HHP, we found that there was a small, but significant reduction in lactoferrin

concentration compared to raw milk. In only 1 other study where the effect of HHP on

lactoferrin was assessed, a 34% reduction was observed using a similar pressure and holding

time as our study163. Consistent with this study, we observed a 28% reduction in lactoferrin.

This finding has also been concluded in studies in bovine milk where it has been demonstrated

that a relationship exists between lactoferrin denaturation and the pressure and holding time of

HHP treatments. This is in line with our hypothesis which stated that alternative, non-thermal

methods would result in less of a reduction, as seen here with HHP and lactoferrin.

The paired design of our study allowed us to concurrently assess the ability of each

pasteurization technique to reduce bacterial load. The current study used donated human milk

that had a bacterial load >5x107 CFU/L, which is above the screening criteria used by the milk

bank and therefore, would not be pasteurized in actual practice. The results of the study showed

that although HHP had the greatest number of samples with a bacterial load <1000 CFU/L, the

study was underpowered to see a statistical difference among pasteurization methods. The use of

milk with a pre-pasteurization bacterial load >5x107 CFU/L may have also resulted in us

underestimating the true ability of the methods yielding culture-negative results, specifically,

what would happen if this technology was used with milk representative of all donations

received at the milk bank. This would account for the discrepancies that are observed when

comparing the Holder pass rate reported in the literature and what is reported in this study124.

Although the use of milk with a bacterial load >5x107 CFU/L is a limitation of the study, our

aims were not to determine the true effectiveness, but the ability of each method to yield culture-

negative bacteriology in comparison to current practice, Holder technique. In doing so, we

observed that HHP successfully pasteurized more samples and therefore, would likely have an

64

even higher pass rate if less contaminated milk was tested. In contrast to HHP, flash-heating and

UV-C pasteurizations resulted in greater culture positive tests than Holder—these two methods

are starkly more unreliable and individually, may not be suitable alternatives to pasteurize milk

in a safe and consistent manner.

A strength of this study is that it is the first to assess the effectiveness of novel pasteurization

techniques (Flash-heating, UV-C, HHP) by challenging native bacteria (commensal and

pathogenic), as well as contaminants, that would normally be found in donated milk at the milk

bank. To our knowledge, there have been only 2 studies that have systematically assessed the

ability of HHP to pasteurize human milk using specific inoculated species; however, many

inconsistencies existed in terms of the pressure and holding time combinations used that were

proven to be effective. For example, in one study by Viazis et al. 2008, 400MPa for 30 minutes

was required to pasteurize the samples, different from a study by Permanyer et al. 2010, where

between 400 and 600 MPa for 5 minutes was necessary122,123. All other studies investigating

HHP tested various pressure and holding time combinations with the primary outcome being a

change in nutritional composition, not taking into account safety, being the limiting factor. In our

methods development, we established novel HHP conditions; 500MPa held for 8 minutes is

required for consistently effective pasteurization of native bacteria in milk. This result is

consistent to Permanyer et al. using the test organism Enterobacteriaceae123. When evaluating

the use of UV-C as a pasteurization medium, we found that our results contradicted the literature,

in terms of its ability to yield culture-negative bacteriology. In the study by Christen et al. ,

dosing inoculated human milk with a UV-C lamp was found to cause a 5-log reduction in

resistant bacteria including Bacillus cereus, a common contaminant in human milk banks115.

Despite increasing the time of pasteurization, and effectively the dose of UV, we still observed

that the effectiveness of UV-C remained poor for complete pasteurization of the 17 samples in

this study. There are several factors to consider. Although turbulent flow was generated in the

container to allow for UV-C penetration, the opacity of the human milk samples may have

attenuated irradiation exposure of the sample. Secondly, a greater dose of UV-C may have been

needed (longer pasteurization time) in order to fully destroy all the bacteria found within the

highly contaminated milk sample. Similarly, inconsistencies were observed in bacteriology

following flash-heating where only 41% (7/17) of samples had a culture negative reading post-

65

pasteurization; in contrast to other studies evaluating native bacteria where the pass rate was

nearly 99%206,207. Likewise, a study that inoculated specific pathogens found that flash-heating

was 99% effective at eliminating all bacteria161. However, it is important to note that in all 3

studies that found flash-heating to be nearly 100% effective, only approximately 40-44% of

samples were found to contain bacteria pre-pasteurization; 100% of the raw samples in this study

all contained levels of bacterial contamination greater than 5x107 CFU/L161,206,207. Differential

concentrations of pre-pasteurization bacteria could explain the disparity observed.

Although heating milk can inactive bacteria, additional concerns arise; the reactive side chains of

the amino acid lysine can react with the carbonyl group of lactose (reducing sugar) as part of the

Maillard reaction (non-enzymatic glycation) to produce a conjugate making lysine biologically

unavailable. We observed no significant changes in levels of bioavailable lysine following

Holder pasteurization as part of a pilot experiment. This is consistent with the previous findings

of Baro et al. 2011 where levels of bioavailable lysine were not significantly altered compared to

raw milk135. This is inconsistent with the findings of Silvestre et al. 2006 that observed a 30%

reduction in available lysine following pasteurization136. The disparity could be explained by the

analytical technique used to quantify available lysine; an HPLC method was employed in this

study, whereas Silvestre et al. used a fluorimetic technique. The rate, extent, and course of the

Maillard reaction is influenced by several factors including the type of reactants, the temperature,

pH and water activity208,209. Theoretically, since pressure is not a known factor in the Maillard

reaction, it should not result in conjugation of lysine; this is congruous with the findings of this

study where no differences were observed following HHP. However, a possible rationale as to

why there were no changes following Holder could be because the Maillard reaction takes places

most readily at temperatures above 100°C, as compared to 62.5°C in Holder210. Previous studies

in bovine milk report that direct heating at 120°C to 130°C for 5 to 6 minutes resulted in an

increase in blocked or biounavailable lysine by 3.6%-6.8%, whereas heating at 115°C for 10 to

40 minutes increased blocked lysine from 11% to 13%211.

The fatty acid profile (saturated fatty acids, monounsaturated fatty acids, omega-3/6) tested in

the pilot resulted in no statistically significant differences between raw, Holder and UV-C. This

finding is consistent with the current literature where there were also no statistically significant

66

differences in the fatty acid profile following Holder pasteurization139,142-144. Only one study has

assessed the fatty acid profile post UV-C and found that there was a significant augmentation in

levels of 8:0 saturated fatty acids115. There are two main rationales as to why human milk

appears to be resilient to changes in fatty acid profile. Firstly, it has been hypothesized that due

to the thermal inactivation of human milk lipases, endogenous enzymatic hydrolysis of

triglycerides is dramatically reduced; triglycerides are more resistant to oxidation than free fatty

acids, and therefore would help preserve levels of all fatty acids. We also speculate that fatty

acids in the milk samples subjected to either Holder or UV-C are protected against oxidation due

to the high antioxidant activity of human milk212,213. Although free radicals are often produced

by heating fatty acids, antioxidants, such as tocopherols, act to neutralize free radicals and

prevent any further oxidation reactions142.

67

CHAPTER 4 CONCLUSIONS AND FUTURE DIRECTIONS

Overall, this study systematically assessed 4 different pasteurization procedures in the context of

human milk. This study has many strengths, one being that each pasteurization technique was

compared to the milk banking criteria of culture-negative bacteriology—the technique was not

used unless it was proven to result in some degree of consistency of attaining culture-negative

bacteriology. Since this is what occurs in the majority of milk banks, we can generalize our

findings to what operations might encounter. A limitation of our study is that the milk used for

analysis had a bacterial load >5x107 CFU/L and is not representative of the majority of milk

donations received at the milk bank. It is unethical to use milk that could otherwise be fed to

vulnerable infants in the NICU for research purposes. Our study design and inclusion of milk

with a bacterial count >5x107 CFU/L was adequate for assessing changes in milk composition

due to the pasteurization technique. Additional studies are warranted to assess what effect these

procedures have on milk with less bacterial contamination. Another strength of our study is our

use of paired study design which allowed us to make direct comparisons of the method and

increased our statistical power to detect differences. Although we only assessed the effect of

pasteurization in 17 samples, a potential limitation, we were adequately powered to detect

differences in composition.

Overall, none of the pasteurization methods affected the macronutrient composition of milk,

differences arose when assessing micronutrients, as in folate and vitamin C levels, as well as in

bioactive proteins. HHP was observed to yield similar culture-negative bacteriology as Holder

and was shown to affect the composition of human milk the least. In fact, it did not affect the

concentration of lactoferrin, it was shown to increase activity of lysozyme and fully retain the

activity of BSSL. These findings are consistent with our hypothesis that non-thermal techniques,

including HHP, resulted in the least changes to human milk composition and bioactivity. Given

that donor milk fed to infants is already fortified so as to allow them to meet their nutritional

requirements, the loss of vitamin C observed post-HHP is more easily adjusted for by enrichment

than replacing the bioactives degraded post-Holder/Flash/UV-C, only found secreted in human

milk. The implications for this research are widespread. Firstly, it should inform policy for

68

breastfeeding in mothers with HIV. Flash-heating, the method proposed by the WHO, was

found to be somewhat effective at pasteurizing milk relative to Holder, however, significant

changes to the micronutrient profile, including BSSL, lysozyme and lactoferrin should be noted

when making future recommendations. This is especially important as this milk is eventually fed

to very vulnerable infants at risk of contracting HIV and other endemic infectious agents. The

use of a vitamin supplementation for infants consuming flash-heated milk long-term may help

compensate for any losses during pasteurization.

Second, in the attempt to find a replacement technology for Holder, future directions should

include further investigation of HHP in terms of its effect on protein digestibility and efficiency,

cost feasibility and method optimization, especially for future application to milk banking

operations. Other technologies such as UV-C are highly unreliable and are destructive to many

essential bioactive components. Although additional research is required to fully implement

change in either the milk bank or in low-resource countries, it is the hope that this research may

assist in the optimization of technologies used to feed human milk to very vulnerable infants.

69

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139. Borgo LA, Coelho Araujo WM, Conceicao MH, Sabioni Resck I, Mendonca MA. Are fat acids of human milk impacted by pasteurization and freezing? Nutr Hosp. 2014;31(3):1386-1393.

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149. Ford JE, Marshall VME, Reiter B. Influence of the heat treatment of human milk on some of its protective constituents J Pediatr 1977;90(1):29-35.

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151. Romeu-Nadal M, Castellote aI, Gayà a, López-Sabater MC. Effect of pasteurisation on ascorbic acid, dehydroascorbic acid, tocopherols and fatty acids in pooled mature human milk. Food Chem 2008;107:434-438.

152. Daniels B, Schmidt S, King T, Israel-Ballard K, Amundson Mansen K, Coutsoudis A. The Effect of Simulated Flash-Heat Pasteurization on Immune Components of Human Milk. Nutrients. 2017;9(2).

153. Sousa SG, Delgadillo I, Saraiva JA. Effect of thermal pasteurisation and high-pressure processing on immunoglobulin content and lysozyme and lactoperoxidase activity in human colostrum. Food Chem. 2014;151:79-85.

154. Christen L, Lai CT, Hartmann B, Hartmann PE, Geddes DT. The Effect of UV-C Pasteurization on Bacteriostatic Properties and Immunological Proteins of Donor Human Milk. PLoS ONE. 2013;8:e85867.

155. Chang JC, Chen CH, Fang LJ, Tsai CR, Chang YC, Wang TM. Influence of prolonged storage process, pasteurization, and heat treatment on biologically-active human milk proteins. Pediatr Neonatol. 2013;54(6):360-366.

156. Akinbi H, Meinzen-Derr J, Auer C, et al. Alterations in the host defense properties of human milk following prolonged storage or pasteurization. J Pediatr Gastroenterol Nutr. 2010;51:347-352.

157. Czank C, Prime DK, Hartmann B, Simmer K, Hartmann PE. Retention of the immunological proteins of pasteurized human milk in relation to pasteurizer design and practice. Pediatric Research. 2009;66:374-379.

158. Viazis S, Farkas BE, Allen JC. Effects of High-Pressure Processing on Immunoglobulin A and Lysozyme Activity in Human Milk J Hum Lact. 2007;23(3).

159. Evans TJ, Ryley HC, Neale LM, Dodge JA, Lewarne VM. Effect of storage and heat on antimicrobial proteins in human milk. Arch Dis Child. 1978;53(3):239-241.

160. Gibbs JH, Fisher C, Bhattacharya S, Goddard P, Baum JD. Drip breast milk: it's composition, collection and pasteurization. Early Hum Dev. 1977;1(3):227-245.

161. Chantry CJ, Wiedeman J, Buehring G, et al. Effect of flash-heat treatment on antimicrobial activity of breastmilk. Breastfeed Med. 2011;6(3):111-116.

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162. Reeves AA, Johnson MC, Vasquez MM, Maheshwari A, Blanco CL. TGF-beta2, a protective intestinal cytokine, is abundant in maternal human milk and human-derived fortifiers but not in donor human milk. Breastfeed Med. 2013;8(6):496-502.

163. Mayayo C, Montserrat M, Ramos SJ, et al. Kinetic parameters for high-pressure-induced denaturation of lactoferrin in human milk. International Dairy Journal. 2014;39:246-252.

164. American Academy of Pediatrics. Breastfeeding and the use of Human Milk. Pediatrics. 2005 115:495-506. .

165. Underwood MA. Human Milk for the Premature Infant. Pediatric Clinics of North America. 2013;60:189-207.

166. Chin K-Y, Ima-Nirwana S. Vitamin C and Bone Health: Evidence from Cell, Animal and Human Studies. Curr Drug Targets. 2015.

167. Yin R, Dai T, Avci P, et al. Light based anti-infectives: ultraviolet C irradiation, photodynamic therapy, blue light, and beyond. Curr Opin Pharmacol. 2013;13(5).

168. Brown A. Understanding Food: Principles and Preparation. 2007:696. 169. Michaelsen KF, Skafte L, Badsberg JH, Jørgensen M. Variation in macronutrients in

human bank milk: influencing factors and implications for human milk banking. J Pediatr Gastroenterol Nutr. 1990;11:229-239.

170. Casadio YS, Williams TM, Lai CT, Olsson SE, Hepworth AR, Hartmann PE. Evaluation of a mid-infrared analyzer for the determination of the macronutrient composition of human milk. J Hum Lact. 2010;26:376-383.

171. Menjo A, Mizuno K, Murase M, et al. Bedside analysis of human milk for adjustable nutrition strategy. Acta Paediatr. 2009;98(2):380-384.

172. Human Milk Analyzer User Manual- M&S0003-12a. Version 0.92. Miris AB, Uppsala, Sweden. .

173. International Organization for Standardization. ISO, 8968-1:2014 Milk and milk products- Determination of nitrogen content- Part 1: Kjeldhl principle and crude protein calculation. 2014. Geneva, Switzerland. .

174. Standardization IOf. ISO, "6731:2010 MIlk, cream, and evaporated milk- Determination of total solids content (reference method). Geneva, Switzerland 2010.

175. Merrill, A., Watt, B., USDA. Energy value of foods, basis and derivation (Agriculture Handbook No 74 Slightly Revised 1973).

176. International Organization for Standardization. 1211:2010 Milk-Determination of fat content-Gravimetric method (reference method). 2010. Geneva, Switzerland. .

177. Polberger S, Lönnerdal B. Simple and rapid macronutrient analysis of human milk for individualized nutrition: Basis for improved nutritional management of very-low-birth-weight infants? J Pediatr Gastroenterol Nutr. 1993;17:283-290.

178. Molloy AM, Scott JM. Microbiological assay for serum, plasma, and red cell folate using cryopreserved, microtiter plate method. Methods in Enzymology. 1997;281:43-53.

179. Hyun TH, Tamura T. Trienzyme extraction in combination with microbiologic assay in food folate analysis: an updated review. Exp Biol Med 2005;230:444-454.

180. Elisia I, Kitts DD. Differences in vitamin E and C profile between infant formula and human milk and relative susceptibility to lipid oxidation. International journal for vitamin and nutrition research 2013;83:311-319.

181. Romeu-Nadal M, Morera-Pons S, Castellote AI, Lopez-Sabater MC. Rapid high-performance liquid chromatographic method for Vitamin C determination in human milk

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versus an enzymatic method. J Chromatogr B Analyt Technol Biomed Life Sci. 2006;830(1):41-46.

182. Molinari CE, Casadio YS, Arthur P, Hartmann PE. The effect of storage at 25 °C on proteins in human milk. International Dairy Journal 2011;21(4 ):286-293.

183. Shugar D. The measurement of lysozyme activity and the ultra-violet inactivation of lysozyme. Biochimica et Biophysica Acta. 1952;8:302-309.

184. Kimura M, Otaki N, Murayama K, Ogawa H, Kobayashi S. Isolation and Characterization of Human Milk Lysozyme. Ind Health 1970;8:22-30

185. Lee YC, Yang D. Determination of lysozyme activities in a microplate format. Analytical Biochemistry. 2002;310:223-224.

186. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957;226(1):497-509.

187. Chen CT, Liu Z, Bazinet RP. Rapid de-esterification and loss of eicosapentaenoic acid from rat brain phospholipids: an intracerebroventricular study. J Neurochem. 2011;116(3):363-373.

188. Lin LE, Chen CT, Hildebrand KD, Liu Z, Hopperton KE, Bazinet RP. Chronic dietary n-6 PUFA deprivation leads to conservation of arachidonic acid and more rapid loss of DHA in rat brain phospholipids. J Lipid Res. 2015;56(2):390-402.

189. Pan Y, Chatterjee D, Gerlai R. Strain dependent gene expression and neurochemical levels in the brain of zebrafish: focus on a few alcohol related targets. Physiol Behav. 2012;107(5):773-780.

190. Chatterjee D, Shams S, Gerlai R. Chronic and acute alcohol administration induced neurochemical changes in the brain: comparison of distinct zebrafish populations. Amino Acids 2014;46(4):921-930.

191. Fortification Basics https://www.dsm.com/content/dam/dsm/nip/en_US/documents/stability.pdf. Accessed November 11th, 2017

192. Gregory JF, 3rd. Denaturation of the folacin-binding protein in pasteurized milk products. J Nutr. 1982;112(7):1329-1338.

193. Martysiak-Zurowska D, Puta M, Barczak N, et al. Effect of High Pressure and Sub-Zero Temperature on Total Antioxidant Capacity and the Content of Vitamin C, Fatty Acids and Secondary Products of LIpid OXidation in Human Milk Polish Journal of Food and Nutrition Sciences 2017;67(2):117-122.

194. Sanchez-Moreno C, Plaza L, de Ancos B, Cano M. Impact of high-pressure and traditional thermal processing of tomato puree on carotenoids, vitamin C and antioxidant activity Journal of Science of Food and Agriculture 2006;86(2):171-179

195. Barba F, Jager H, Meneses N, Esteve M, Frigola A, Knorr D. Evaluation of quality changes of blueberry juice during refrigerated storage after high-pressure and pulsed electric fields processing Innovative Food Science and Emerging Technologies 2012;14:18-24.

196. Sukkhmanov V, Shatalov V, Petrova J, Birca A, Gaceu L. The influence of high pressure on bio-system reaction kinetics and the preservation of vitamin C. LWT-Food Science and Technology 2014;58(2):375=380

197. Koutchma T, Popović V, Ros-Polski V, Popielarz A. Effects of Ultraviolet Light and High-Pressure Processing on Quality and Health-Related Constituents of Fresh Juice Products Comp Revi Food Sci Saf 2016;15(5):844-867.

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198. Guneser O, Karagul Yuceer Y. Effect of ultraviolet light on water- and fat-soluble

vitamins in cow and goat milk. J Dairy Sci. 2012;95:6230-6241. 199. Neves-Petersen, MT., Petersen, S., Gajula, GP. (2012). UV Light Effects on Proteins:

From Photochemistry to Nanomedicine, Molecular Photochemistry - Various Aspects, Dr. Satyen Saha (Ed.), ISBN: 978-953-51-0446-9, InTech, Available from: http://www.intechopen.com/books/molecular- photochemistry-various-aspects/uv-light-effects-on-proteins-from-photochemistry-to-nanomedicine.

200. Blackberg L, Hernell O. The bile-salt-stimulated lipase in human milk. Purification and characterization. Eur J Biochem. 1981;116(2):221-225.

201. Lysozyme-UniProtKB. http://www.uniprot.org/uniprot/B2R4C5. 202. Iucci L, Patrignani F, Vallicelli M, Guerzoni ME, Lanciotti R. Effects of high pressure

homogenization on the activity of lysozyme and lactoferrin against Listeria monocytogenes Food Control. 2007;18:558-565.

203. Vannini L, Lanciotti R, Baldi D, Guerzoni ME. Interactions between high pressure homogenization and antimicrobial activity of lysozyme and lactoperoxidase. International Journal of Food Microbiology. 2004;94:123-135.

204. Sanchez L, Peiro JM, Castillo M, Perez MD, Ena JM, Calvo M. Kinetic Parameters for Denaturation of Bovine Milk Lactoferrin J Food Sci 1992;57(4):873-879

205. Querinjean P, Masson PL, Heremans JF. Molecular weight, single-chain structure and amino acid composition of human lactoferrin. Eur J Biochem. 1971;20(3):420-425.

206. Chantry CJ, Young SL, Rennie W, et al. Feasibility of using flash-heated breastmilk as an infant feeding option for HIV-exposed, uninfected infants after 6 months of age in urban Tanzania. JAIDS 2012;60(1):43-50.

207. Naicker M, Coutsoudis A, Israel-Ballard K, Chaudhri R, Perin N, Mlisana K. Demonstrating the efficacy of the FoneAstra pasteurization monitor for human milk pasteurization in resource-limited settings. Breastfeed Med. 2015;10(2):107-112.

208. Ames JM. Control of the Maillard reactions in food systems Trends Food Sci Technol 1990;1 150-143.

209. van Boekel MA. Kinetic aspects of the Maillard reaction: a critical review Nahrung 2001;45(3 ):150-159

210. Shimamura T, Ukeda H. Maillard reaction in milk-effect of heat treatment In: Hurley WL, ed. Biochemistry, Genetics and Molecular Biology "Milk Protein" 2012.

211. Finot PA, Deutsch R, Bujard E. The extent of the Maillard reaction during the processing of milk Prog Ed Nutri Sci 2013;5.

212. Buescher ES, McIlheran SM. Colostral antioxidants: separation and characterization of two activities in human colostrum. J Pediatr Gastroenterol Nutr. 1992;14(1):47-56.

213. Goldblum RM, Dill CW, Albrecht TB, Alford ES, Garza C, Goldman AS. Rapid high-temperature treatment of human milk. J Pediatr. 1984;104(3):380-385.

214. Pfeiffer C, Rogers L, Gregory J. Determination of Folate in Cereal-Grain Food Products Using Trienzyme Extraction and Combined Affinity and Reversed-Phase Liquid Chromatography. J Agric Food Chem. 1997;45(2):407-413.

215. Yao X, Bunt C, Cornish J, Quek SY, Wen J. Improved RP-HPLC method for determination of bovine lactoferrin and its proteolytic degradation in simulated gastrointestinal fluids. Biomed Chromatogr. 2013;27(2):197-202.

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216. Morrison WR, Smith LM. Preparation of fatty acid methyl esters and dimethylacetyls from lipids with boron fluoride-methanol. J Lipid Res. 1964;5:600-608.

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APPENDIX A. RESEARCH ETHICS BOARD APPROVAL LETTER

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APPENDIX B. STANDARD OPERATION PROCEDURE FOR THE DETERMINATION OF TOTAL FOLATE IN HUMAN MILK Determination of total folates in human milk Version No.1 SOP#1 Original Date: Feb 1, 2016 Author: M Pitino Version Date: March 13, 2016 Approved by: D.L O’Connor

1. Preservation of folates during freezer storage prior to analyses:

Prior to freezing in 1mL aliquots, add 10mg of sodium ascorbate (1% w/v) to each vial to

prevent oxidation of folates.

2. Extraction:

Gently thaw frozen samples on ice. Take 100 µL of each sample and mix well with 900 µL

of Wilson-Horne buffer. Freeze on dry ice immediately.

3. Preparation of amylase, protease and rat conjugase:

Based on Hyun and Tamura 3U/mg milk ⍺-amylase, and 0.4 U/mg milk protease is required

for digestion179. Using the enzyme preparations, 0.3mL of ⍺-amylase and 0.5mL of protease

is required per sample. A total of 0.25mL of rat conjugase per sample is also required based

on previously published work by Pfeiffer et al214. To begin, combine 3g of ⍺-amylase

(Sigma-Aldrich, A-6211, 39.3 U/mg) with 150 mL of deionized water, and combine 1g of

protease (Sigma-Aldrich, P5147, 6.2.U/mg) with 100mL of deionized water. To obtain

conjugase, obtain rat blood from normal, otherwise healthy rats by cardiac puncture with a

syringe and 16-gauge needle without EDTA. Centrifuge whole blood at 900xg for 20 min at

6°C to separate the hematocrit from the plasma. Once separated, add 5 g activated charcoal

(Sigma-Aldrich, St. Louis, MO, USA) 100mL plasma and stir on ice for 1 hour to remove

most rat folates present in the whole blood.

4. Trienzyme digestion:

To carry out trienzyme digestion, combine 0.5mL of the extracted sample with 0.5mL of

protease and 1mL of 1% w/v sodium ascorbate in 0.1M phosphate buffer at pH. 4.1 and

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incubate in a light-shielded water-bath at 37°C for two hours. Boil samples at 100°C for 10

min to inactive the protease. Once cooled, add 0.3 mL of ⍺-amylase and 0.25mL of rat

conjugase to the vial, in addition to 1mL of 1% w/v sodium ascorbate in 0.1M phosphate

buffer at pH. 6.8. Incubate samples at 37°C for 2 hours, centrifuge at 5000xg for 10 min and

store at -80°C until folate microbiological assay.

5. Microbiological assay

The folate microbiological assay is conducted using Lactobacillus rhamnosus (ATCC 7469) as

the test organism and 5-methyltetrahydrofolate for the standard curve. Dilute samples (x20 in

sodium ascorbate) samples and pig liver reference (x100 in sodium ascorbate) and plate in

triplicate on a 96 well plate. Add 200 µL of Lactobacillus casei media, inoculated with L.

rhamnosus, to each well. After 42h incubation at 37°C, measure the absorbance using a plate

reader at 405nm and 590 as reference.

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APPENDIX C. STANDARD OPERATION PROCEDURE FOR THE DETERMINATION OF TOTAL VITAMIN C, ASCORBIC ACID AND DEHYDROASCORBIC ACID IN HUMAN MILK Determination of total vitamin C, ascorbic acid and dehydroascorbic acid in human milk

Version No.2 SOP#1 Original Date: April 1st,2017 Authors: M Pitino Version Date: June 1st, 2017 Approved by: D.L O’Connor

1. Preservation of vitamin C during freezer storage prior to analysis:

Dilute human milk samples 1:1 with 10% metaphosphoric acid (ACS reagent 79613, Sigma-

Aldrich, St. Louis MO) and 1% oxalic acid (Anhydrous, 75688, Sigma-Aldrich, St. Louis

MO, USA) to inhibit degradation and mix by inversion prior to freezing at -80 °C.

2. Sample preparation for the determination of total vitamin C and ascorbic acid:

To determine total vitamin C content in human milk samples, DL-dithiotreitol (DL-DTT)

(BP172, Fisher Scientific, Fair Lawn, NJ, USA), add to reduce dehydroascorbic acid to

ascorbic acid. Specifically, combine 300 µL of homogenized human milk and 300 µL of 0.56

% w/v metaphosphoric acid with 800 µL of 100mM DL-dithiotreitol in a centrifugal

filtration tube (Microsep Advance Centrifugal Device 0.45µm Supor Membrane, Pall

Corporation, Port Washington, NY). Simultaneously, combine 600µL of homogenized

human milk with 600µL of 0.56% w/v metaphosphoric acid in a similar centrifugal filtration

tube to analyze ascorbic acid. Mix tubes gently using a vortex to ensure adequate

homogeneity and incubate at room temperature for 15 min. Centrifuge tubes at 6500 x g at

4°C (Eppendorf Centrifuge 5430-R) to de-fat the sample and remove extraneous proteins

prior to chromatography. Once centrifuged, samples syringe-filter in amber vials (Agilent

Technologies, Santa Clara, CA, USA) using a 0.2 µm PVDF membrane filter (LC 13mm

Syringe Filter, Pall Corporation, Port Washington, NY, USA).

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3. Preparation of the L-Ascorbic acid standards:

Prepare, by serial dilution, fresh standards of L-Ascorbic acid (ACS reagent, 255564, Sigma-

Aldrich, St. Louis, MO) in deionized water on the day of analysis (80mg/L, 40mg/L,

20mg/L, 10mg/L, 5mg/L, 2.5mg/L) in addition to one blank. Once prepared, similar to the

samples, syringe-filter standards into amber vials.

4. HPLC analysis:

For chromatography, use the Agilent 1260 Infinity II HPLC (Agilent Technologies, Santa

Clara, CA) fitted with an InfinityLab Poroshell 120-EC-C18 column, 4.6x100mm, 2.7µm

(G7112B093, Agilent Technologies, Santa Clara, CA), a UHPLC InfinityLab Poroshell 120-

EC-C18 guard column ,4.6x5mm, 2.7µm, (820750-911, Agilent Technologies, Santa Clara,

CA) and diode array detector (G7115A, Agilent Technologies, Santa Clara, CA).

Conduct an isocratic separation using a mobile phase of 0.1% v/v acetic acid (ACS grade,

320099, Sigma-Aldrich, St. Louis, MO, USA) in deionized water and methanol (HPLC-

grade, 154903, Sigma-Aldrich, St. Louis, MO) in a relative proportion of 97.5:2.5 (v/v).

Filter both mobile phase solutions using a 0.45µm HNWP filter (Millipore, HNWP044700).

Set the column temperature to 25°C and the eluent flow rate to 1.3 mL/min. Inject 30µL of

each prepared sample or standard into the column and adjust UV detection wavelength to

254nm. Identify ascorbic acid peaks in samples by comparing retention times to previously

run standards. Use the Agilent Open Lab CDS Chem Station to quantify levels of ascorbic

acid by standard curve analysis.

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APPENDIX D. STANDARD OPERATION PROCEDURE FOR THE DETERMINATION OF LACTOFERRIN IN HUMAN MILK Determination of lactoferrin in human milk Version No.1 SOP#1 Original Date: November 1st, 2017 Authors: M Pitino Version Date: November 1st, 2017 Approved by: D.L O’Connor

The determination of lactoferrin from human milk is based on the procedure outlined by Yao et

al. 2012 with modification for human milk215.

1. Sample Preparation

Thaw samples gently at room temperature (22°C) and mix gently by vortex to ensure

homogeneity. Defat milk samples using a 0.45µm centrifugal filter (Microsep Advance

Centrifugal Device, Supor Membrane, Pall Corporation, Port Washington, NY) for 15 min at

4°C at 7000xg. Syringe filter the de-fatted sample using 0.2 µm HPLC certified PVDF

membrane filter into autosampler vials (LC 13mm Syringe Filter, Pall Corporation, Port

Washington, NY, USA).

2. Standard Preparation

Prepare standards by serial dilution using 0.2 µm HPLC certified PVDF filtered deionized water.

Use lactoferrin from human milk (Sigma-Aldrich, St. Louis, MO, USA) to make the standards

from a 4g/L stock solution. Make 5 standards: 4g/L, 2g/L, 1g/L, 0.5g/L, 0.25g/L.

3. HPLC separation

For chromatography, use the Agilent 1260 Infinity II HPLC (Agilent Technologies, Santa Clara,

CA) fitted with an InfinityLab Poroshell 120-EC-C18 column, 4.6x100mm, 2.7µm (G7112B093,

Agilent Technologies, Santa Clara, CA), a UHPLC InfinityLab Poroshell 120-EC-C18 guard

column ,4.6x5mm, 2.7µm, (820750-911, Agilent Technologies, Santa Clara, CA) and diode

array detector (G7115A, Agilent Technologies, Santa Clara, CA). Monitor lactoferrin elution at

3 wavelengths: 210 nm, 260 nm, 280 nm.

88

Solution A is <0.1% trifluorotriacetic acid in water and HPLC grade acetonitrile in a ratio of

95:5. Solution B is <0.1% trifluorotriacetic acid in water and HPLC grade acetonitrile in a ratio

of 5:95. Set the column temperature to 37°C, the flow rate to 0.850 mL/min and injection volume

to 2µL.

Begin isocratic separation at 30% B for 1 min, followed by a gradient from 30% to 60% B over 7

minutes. Re-equilibrate the column to 30% B for a minimum of 4 minutes. Identify lactoferrin

peaks in samples by comparing retention times to previously run standards. Use the Agilent

Open Lab CDS Chem Station to quantify levels of ascorbic acid by standard curve analysis.

89

APPENDIX E. STANDARD OPERATION PROCEDURE FOR THE DETERMINATION OF LYSOZYME ACTIVITY IN HUMAN MILK Determination of lysozyme activity in human milk

Version No.1 SOP#1 Original Date: January 2017 Authors: M Pitino Version Date: January 2017 Approved by: D.L O’Connor

1. Sample preparation:

Gently thaw frozen samples on ice and centrifuge at 10000 rpm at 4°C for 10 minutes to

allow for the separation of the fat from the aqueous fraction of human milk. Mix de-fatted

samples by using a vortex which will ensure homogeneity. Dilute samples by a factor of 150

in deionized water and keep on ice.

2. Preparation of lysozyme standards

Use lyophilized lysozyme isolated from chicken egg white as a certified reference to validate

the assay. Prepare the reference standard by combining 10mg of lyophilized lysozyme isolate

and 10mL of cold, deionized water. Carry out two serial dilutions to achieve a solution of

lysozyme of approximately 300U/mL (dependant on the certified value of lysozyme

standard)

3. Preparation of Micrococcus lysodeikticus turbidimetric assay

The assay is used determine active lysozyme activity is the Micrococcus lysodeikticus

turbidimetric assay adapted from Shugar et al. 1952183. To make the cell suspension

solution, combine 18mg of M. lysodeikticus with 30mL of lysozyme reaction buffer (Sigma-

Aldrich, L9295). A 96-well microplate modified by Lee and Yang will be used185. Add 100

µL of de-fatted and diluted samples to a 96-well plate in triplicate. Add 150 µL of M.

lysodeikticus (18mg/30mL) to each well. Measure the absorbance using a microplate reader

(Opsys MR Dynex, Dynex Technologies Inc., Chantilly, VA, USA)) at 450nm with 590nm

as reference. One unit of Lysozyme is defined as a change in absorbance of 0.001/min at

450nm (pH 6.24, 25°C), using a suspension of M. lysodeikticus as a substrate in a 2.6 mL

reaction mixture. Multiply lysozyme by 4.94 to correct for path-length differences between

the cuvette method and the modifications for 96 well plate assay.

90

APPENDIX F. MICROBIOLOGY PROTOCOL USED FOR ANALYSIS OF SAMPLES AT THE ROGERS HIXON ONTARIO HUMAN MILK BANK

96

APPENDIX G. STANDARD OPERATION PROCEDURE FOR THE DETERMINATION OF FATTY ACID COMPOSITION Determination of fatty acid composition in human milk

Version No.1 SOP#1 Original Date: June 2017 Authors: M Pitino Version Date: June 2017 Approved by: D.L O’Connor

Fatty acid extraction, by Folch extraction:

1. Thaw, homogenize and then vortex samples of a given mass in 6 mL of 2:1 chloroform:

methanol (by vol.) in the presence of known quantities of heptadecanoic acid (17:0) and

docosatrienoic ethyl ester (22:3n-3) as internal standards.

2. Incubate for 24-h at -20°C. Afterwards, bring samples to room temperature and add 1.6

mL of 0.88% potassium chloride aqueous buffer and centrifuge at 1460 rpm for 10 min at

4°C to impart organic phase separation.

3. Transfer the lower organic phase containing chloroform and dissolved lipids to a clean

labelled glass tube, and wash the remaining aqueous phase with 4 mL of chloroform.

Proceed to vortex and centrifuge.

4. Transfer the lower chloroform phase and combine with the previously extracted phase.

Transesterification adapted from Morrison and Smith216

5. Evaporate a portion of the total lipid extract under nitrogen.

6. Dissolve dried lipids in 300 μL of hexane, add 1 mL of 14% boron trifluoride in

methanol samples and heat at 100 °C for 1 hour.

7. Take samples out of the oven and allow to cool, add 2mL of deionized water and

centrifuge at 1360 rpm to separate the phases, prior to collecting the top phase.

8. Extract the resulting fatty acid methyl esters into 1 mL of hexane, evaporate under

nitrogen, and transfer into auto-sampler vials.

9. Dilute samples in hexane to a final concentration of approximately 0.5 mg of lipid/ml of

solvent.

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10. Quantify fatty acid methyl esters on a Varian 430 gas chromatograph (Bruker, Billerica,

MA, USA) equipped with a SP-2560 100 m × 0.25 mm i.d. × 0.20 μm df, non-bonded poly

(biscyanopropyl siloxane), capillary column (Supelco by Sigma-Aldrich, Bellefonte, PA,

USA) with helium carrier gas at a constant flow rate of 3.0 mL/min.

11. Inject Samples (1μl) in splitless injection mode into a heated injection port held at 250°C

via a Varian CP-8400 autosampler.

12. Elute fatty acid methyl esters using an oven temperature program initially set at 60°C and

hold for 2 min, increasing at 10°C/min to 170°C and hold for 4 min, then at 6.5°C/min to

reach 175°C, then 2.6°C/min to reach 185°C, then 1.3°C/min to reach 190°C, and then

8.0°C/min to 240°C and hold for 11 min.

13. Use a flame ionization detector at 300°C to detect the fatty acid methyl esters using a

sampling frequency of 20 Hz. Identify peaks by retention time in comparison to an

external reference standard mixture (GLC – 569, Nu-Chek Prep Inc).

14. Quantify by dividing peak area under the curve by the 22:3n-3 internal standard area

under the curve, then correcting for extracted sample weight.

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APPENDIX H. STANDARD OPERATION PROCEDURE FOR THE DETERMINATION OF AVAILABLE LYSINE IN HUMAN MILK Determination of available lysine in human milk

Version No.1 SOP#1 Original Date: June 2017 Authors: M Pitino Version Date: June 2017 Approved by: D.L O’Connor

1. Add 10 μL of 2.5M cold perchloric acid to 500 μL of both raw and pasteurized samples.

2. Gently mix the samples using a vortex, incubate on ice for 10 min and then centrifuge at

15,000 rpm for 10 min at 4°C.

3. Collect 10 μL of the supernatant for ophthalaldehayde (OPA) derivatization.

4. Add 5 μL of homo-serine (125 pmol in artificial cerebral spinal fluid) as the internal

standard to all samples during OPA derivatization,

5. Inject 10 μL of the derivatized solution into the column.

6. Use a mobile phase of 0.15M sodium acetate buffer, 1mM EDTA, pH 5.4 and 50%

acetonitrile. Use a flow rate of 0.8mL/min.

7. Set the working electrode (Uniget 3mm glassy carbon, BAS MF-1003) to 750 mV vs.

Ag/Ag/Cl reference electrode.

8. Set the detection gain at 1.0 nA, the filter to 0.2 Hz and detection limit to 100 nA.

9. Use the standard L-lysine (Sigma-Aldrich, St. Louis, MO, USA) to identify and quantify

peaks on the chromatogram.

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APPENDIX I. REPRESENTATIVE CHROMATOGRAMS OF TOTAL VITAMIN C CONTENT IN HUMAN MILK

Raw Human Milk

Post-Holder Pasteurization

Post-Flash Heat Pasteurization

Post-UV-C Pasteurization

Post-HHP Pasteurization

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APPENDIX J. REPRESENTATIVE CHROMATOGRAMS OF LACTOFERRIN CONTENT IN HUMAN MILK

Raw Human Milk

Post-Holder Pasteurization

Post-Flash Heat Pasteurization

Post-UV-C Pasteurization

Post-HHP Pasteurization

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APPENDIX K. STUDENT'S CONTRIBUTION

• Designed research study and prepared a detailed research proposal

• Submitted study to Research Ethics Committee at The Hospital for Sick Children and The Human Milk Banking Association of North America

• Obtained ethics approval from both The Hospital for Sick Children and The Human Milk

Banking Association of North America

• Collected samples of donor human milk, optimized and carried out pasteurization techniques

• Coordinated collaboration with Dr. Yves Pouliot (Canada Research Chair in Dairy

Science) at Université Laval for High Hydrostatic Pressure Processing

• Processed milk samples and carried out laboratory analyses (macronutrients, folate, vitamin C, bile salt-stimulated lipase, lactoferrin, lysozyme)

• Developed and validated laboratory protocols for the analysis of vitamin C and lactoferrin in human milk by high performance liquid chromatography

• Performed laboratory data analysis and statistical analysis

• Wrote thesis

• Wrote, submitted and presented an abstract at the Canadian Nutrition Society Annual

Conference 2017

• Wrote, submitted, and orally presented an abstract at the University of Toronto’s Department of Pediatrics Research Day / Neonatal Research Day 2017

• Wrote and submitted an abstract for Pediatric Academic Societies Annual Conference

(2018)

Investigating Alternative and Emerging Pasteurization ......to use a method known as flash-heating...

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