The dynamics of milk emulsion structure during in vitro neonatal gastric digestion : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand
Efficient fat digestion is an essential part of neonatal development. In this respect, it
is noteworthy that the process by which infants digest fat differs from that in adults;
key differences include the immaturity of the pancreatic function and elevated
gastric pH. The digestion of emulsified lipids may accordingly be rendered less
efficient in ambient conditions in the infant gastric lumen. For example, it may be
postulated that covariation in optimal conditions of proteolytic and lipolytic
digestion may differently affect the digestion and disruption of the droplet
membrane, the interfacial accessibility of lipase and the subsequent fatty acid
Differences between formulated emulsion structures may therefore influence the rate
of digestion; previous human studies have indicated that infants digest formula feeds
more slowly than they do breast milk (Splinter and Schreiner, 1999). To further
explore this observation, the lipid digestion of native biological milk (human breast
milk), commercial infant formulae (liquid and powder), and model emulsions
(Intralipid containing lactoferrin) were investigated in an in vitro gastric system.
The aim was to gain a better understanding on the changes in emulsion structure and
fat digestibility with various interfacial layers and pH environments under simulated
The introduction and a rationale for the focus of this thesis are shown in Chapter 1.
Chapter 2 gives a critical overview and review of the literature pertaining to this
thesis, and presents possible explanations of how the properties of milk fat globules
and their membranes are related to the digestion outcome in the digestive system of
infants. The review also examines the effects of physicochemical factors on
emulsion stability. Then, Chapter 3 presents the general materials and methods used
in the experimental work.
The first experimental design is described in Chapter 4. This chapter compares the
characteristics and physicochemical properties of different types of milks. Infant
formulae are prepared from cow’s milk and designed to mimic human milk as much
as possible. However, even with the advances of technology, there are still
differences observed between the breast milk and commercial infant formulae.
Therefore the microstructure, droplet size and droplet charge of these different types
of milk (human milk, raw cow’s milk, commercial liquid formulae and commercial
powder formulae) were examined before studying the emulsion digestibility under
simulated infant physiological conditions.
Chapter 5 gives a description on how digestion affects emulsion structure of a
typical formula emulsion at different pH levels (25.5) in an in vitro system that
replicates the shear rates that would normally be encountered in the infant stomach.
The system is designed to simulate infant gastric conditions using different
combinations of porcine pepsin and fungal lipase (Rhizopus oryzae). Thus, digestion
in the presence and absence of proteolytic and lipolytic enzymes was evaluated by
observing changes in microstructure, particle size and surface charge.
In liquid infant formulae, droplet size increased progressively by coalescence during
in vitro digestion at pHs between 3.5 and 4.5 when both lipase and protease were
present, but not when either enzyme was omitted or when pH levels were outside
this range. Coalescence was augmented by shear, notably at rates above the normal
physiological range. The fidelity of in vitro systems did not appear to be
compromised by the use of fungal lipases but compromised by the use of
inappropriately high stirring rates. The stability and structural properties of formula
emulsions appeared to be influenced by disruption of the proteinaceous oil/water
interface during digestion, being most susceptible to the concerted activity of pepsin
and gastric lipase over a limited range of pH. Given that the onset of secretion of
pepsin, lipase and hydrochloric acid does not occur synchronously in the developing
infant stomach, inappropriately formulated milks may lower digestive efficiency.
Chapter 6 progresses the findings from chapter 5, employing a model
phospholipidstabilised emulsion which was digested alone, and in combination
with the milk protein lactoferrin. It was postulated that the lactoferrin would form an
electrostatic layer-on-layer complex with the phospholipid allowing comparison to
be made between digestion of the phospholipidstabilised emulsion and the
emulsion stabilised by lactoferrinphospholipid complex.
Lipolysis of untreated Intralipid, as evidenced by the increase in droplet size i.e. d43
and by confocal microscopy, took place at pH levels between 3.5 and 5.5.
Coalescence was evident with lipase alone and with mixtures of pepsin and lipase at
pH 3.5, but did not occur in the presence of pepsin alone. Conversely, no
coalescence was evident on digestion of Intralipid treated with lactoferrin, with
lipase alone at pH levels below 5.5. However, coalescence of droplets in treated
Intralipid did take place at pH levels above 2 when both pepsin and lipase were
present. Changes in surface potential indicated that interfacial proteolysis was
required for lipase-mediated coalescence to occur. Findings indicated that the
interaction of lactoferrin with the oil/water interface of soybean oil droplets may
have inhibited the action of lipase pending digestion by pepsin.
The findings of Chapter 5 and 6 demonstrate the co-dependent role of proteolytic
and lipolytic enzymes on the stability of emulsions during digestion, and the
contribution of pH on enzymatic function. This knowledge should be a key factor for
the design of emulsion structures in infant formula emulsions.
Chapter 7 describes how digestion affects the structure of human breast milk. Fat
droplets showed no significant propensity towards flocculation and aggregation
during incubation both with and without either enzyme at all pH. Additionally, the
breast milk emulsion was seen to be resistant to coalescence across all pH’s and
enzymatic conditions studied. The difference in structural behaviour is attributed to
variance in lipid composition of the MFG relative to the emulsion systems studied in
chapters 5 and 6. Accordingly, it is suggested that the by-products of lipolysis of the
breast milk emulsion may serve to stabilise droplets rather than cause instability.
Thus the MFGM of maternal milk is not considered inhibitory to the action of either
of these two enzymes (porcine pepsin and fungal lipase) under in vitro simulation of
infant gastric conditions.
Chapter 8 describes the overall conclusions and addresses the major findings and
recommendations for future work.