Accepted Manuscript

Ex vivo digestion of raw, pasteurised and homogenised milk - Effects on lipolysis and
proteolysis
Mohammad Ashiqul Islam, Hanne Devle, Irene Comi, Ellen Kathrine Ulleberg, EllingOlav Rukke, Gerd Elisabeth Vegarud, Dag Ekeberg
PII:

S0958-6946(16)30300-4

DOI:

10.1016/j.idairyj.2016.09.008

Reference:

INDA 4080

To appear in:

International Dairy Journal

Received Date: 27 June 2016

Revised Date:

7 September 2016

Accepted Date: 10 September 2016

Please cite this article as: Islam, M.A., Devle, H., Comi, I., Ulleberg, E.K., Rukke, E.-O., Vegarud, G.E.,
Ekeberg, D., Ex vivo digestion of raw, pasteurised and homogenised milk - Effects on lipolysis and
proteolysis, International Dairy Journal (2016), doi: 10.1016/j.idairyj.2016.09.008.
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Ex vivo digestion of raw, pasteurised and homogenised milk - Effects on lipolysis and proteolysis

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Mohammad Ashiqul Islam*a,b, Hanne Devleb, Irene Comib, Ellen Kathrine Ullebergb, Elling-Olav Rukkeb,

Gerd Elisabeth Vegarudb, Dag Ekebergb

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Department of Dairy Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

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Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences,

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P.O. Box: 5003, NO-1432 s, Norway

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*Corresponding author. Tel.: +88 091 61510

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Email address: m.a.islam@bau.edu.bd (M. A. Islam)

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ABSTRACT

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This study investigated the impact of pasteurisation and homogenisation on ex vivo gastrointestinal

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digestion of proteins and fat in bovine milk. Milk that was homogenised at 150 bar showed the

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highest degree of lipolysis (78%) after 120 min duodenal digestion. In comparison, milk homogenised

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at 50 bar showed 56% release of fatty acids whereas raw and pasteurised milk showed 49% and 44%

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hydrolysis, respectively. Pasteurisation had very much less effect upon lipolysis and proteolysis.

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Homogenisation increased the gastric proteolysis of specifically -lactoglobulin, but also of -

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lactalbumin. However, after 5 min duodenal digestion all the proteins were degraded in all milks

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tested. Thus, both lipolysis and proteolysis were increased by homogenisation of the milk, making

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fatty acids from lipids and peptides from -lactoglobulin more readily available to the human body.

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1.

Introduction

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Milk and dairy products are important and complex but highly nutritious food sources. Since
the shelf-life of these products is short, heat treatment and homogenisation are used to preserve milk

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quality. However, an increasing number of people purchase raw milk, motivated by claims that it is

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more easily digested and healthier (Waser et al., 2006).

In modern commercial dairy processing, pasteurisation and homogenisation are standard

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techniques performed to ensure longer shelf-life of milk due to removal of spoilage and potentially

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pathogenic bacteria, and to avoid separation of the fat phase in the milk, respectively. A pressure of

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100 to 200 bar is commonly used to reduce the size of the native milk fat globules (MFG). The native

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MFG ranges from < 0.2 m to 20 m diameter, whereas the newly formed lipid droplets in

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homogenised milk range from 0.5 m to 1.0 m diameter (Huppertz & Kelly, 2006; Michalski & Januel,

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2006). Homogenisation increases the number of lipid droplets as well as the interface to the aqueous

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medium by 410 fold (Michalski & Januel, 2006). The native milk fat globule membrane (MFGM) is

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unable to cover all newly formed interfaces. Therefore, native MFGM fragments and some whey

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proteins, along with casein micelles or fragments thereof, cover the new, smaller lipid droplets

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(Huppertz & Kelly, 2006). With the increase of homogenisation pressure, the protein load at the

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surface area increases, but the composition of the surface proteins remains almost the same (Cano-

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Ruiz & Richter, 1997). Thus, homogenisation alters the membrane surrounding the milk fat, and

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around 68% of the milk casein becomes associated with the newly formed membrane (Fox, 2002).

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The lipid globules of homogenised milk are important from a technological point of view,

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especially, in relation to acid coagulation and rennet coagulation (Huppertz & Kelly, 2006). However,

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more data are needed to determine if there are any nutritional consequences of milk
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homogenisation. Of particular interest is whether the many smaller lipid droplets with their newly

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formed membranes are digested differently from the native MFG. An in vitro digestion study showed

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that triacylglycerols in homogenised milk underwent 2-fold higher lipolysis compared with those in

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unhomogenised milk (Berton et al., 2012). Another recent study also showed that homogenisation

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significantly increased the release of free fatty acids (FFAs) compared with that from raw milk (Tunick

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et al., 2016). The changed composition of the lipid droplet membrane during homogenisation has

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been revealed to be important for lipolysis and absorption of the milk fat, as well as for postprandial

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hypertriglyceridaemia (Michalski, 2009). Proteolysis could also be affected by milk processing, as

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Tunick et al. (2016) showed that -lactoglobulin (-lg) was digested more slowly in pasteurised and in

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both pasteurised and homogenised milk than in raw milk.

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The present study investigated the impact of pasteurisation and different homogenisation

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pressures on ex vivo lipolysis and proteolysis of milk.

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2.

Materials and methods

2.1.

Milk samples

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Raw milk was collected from the bulk tank supply of the dairy pilot plant of Norwegian

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University of Life Sciences, S-1432, Norway. The amount of raw milk required was kept separate; the

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rest was pasteurised at a temperature of 72 C for 15 s in the dairy pilot plant. Parts of the

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pasteurised milk were subjected to a Rannie Homogenizer Model Lab (Rannie AS, Copenhagen,

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Denmark). Homogenisation of a portion of pasteurised milk was done at 50 bar (single stage) and the

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rest was homogenised at 150 bar (single stage). Milk samples were collected and one Bronopol tablet
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(D & F Control Systems Inc., Norwood, MA, USA) was added per 40 mL of milk. Depending on the

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treatment, the various milk samples were divided into the following 4 groups: (i) raw untreated (R),

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(ii) pasteurised (P), (iii) pasteurised + homogenised at 50 bar (PH50), and (iv) pasteurised +

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homogenised at 150 bar (PH150). Aliquots of samples needed for different steps of ex vivo digestion

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were made and kept at 46 C until digested and analysed. The composition of the milk was analysed

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with the MilkoScan FT1 (Foss, Hillerd, Denmark). Mastersizer 3000 (Malvern, WR, UK) was used to

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measure the milk fat globule size.

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2.2.

Human digestive enzymes

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The human digestive juices were aspirated from 25 volunteers aged between 25 to 40 years
old using the method described by Ulleberg et al. (2011). The responsible hospital for aspiration was

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Lovisenberg Diakonale Hospital, Oslo; the protocol was approved by the Regional Committees for

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Medical and Health Research Ethics (2011). Pooled batches of human digestive juice from all of the

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volunteers were stored at -80 C until use. The pepsin activity in the gastric juice (HGJ), and trypsin

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and lipase activity in the duodenal juice (HDJ) were assayed with the method described by Minekus et

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al. (2014). One unit of pepsin activity (U mL-1) in the HGJ was defined as A280 of 0.001 per min at 37

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C, pH 2. One unit of trypsin activity (U mL-1) in the HDJ was defined as hydrolysis of 1 mol of p-

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toluene-sulfonyl-L-arginine methyl ester per min at pH 8 and 25 C. One unit of pancreatic lipase (U

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mL-1) was defined as 1 mol free fatty acid (FFA) per min generated from tributyrin at 37 C, pH 8 in

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the presence of 4 mM sodium taurodeoxycholate, excess colipase and 2 mM calcium ions. The

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simulated gastric and intestinal digestion fluids (SGF and SIF, respectively) were composed according

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to Minekus et al. (2014) and Islam, Ekeberg, Rukke, and Vegarud (2015a) with some modifications.
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The SGF contained 360 L gastric electrolyte stock solution (1.25), 400 L HGJ, 5 L 0.3 M CaCl2, 94

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L 1 M HCl and 101 L water. In comparison, 100 L intestinal electrolyte solution (1.25), 1830 L

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HDJ, 5 L 0.3 M CaCl2 and 135 L 1 M NaOH constituted the SIF.

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2.3.

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Ex vivo digestion

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The digestion method comprised a gastric (G) phase followed by a duodenal (D) phase. In the

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gastric phase, the ratio of milk to SGF was 1:1. The constituents of SGF were added separately to 1 mL

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of milk in a 50 mL tube and was then digested at pH 3.0 for 60 min (G 60) in a 37 C water bath. The

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duodenal digestion was performed at pH 7.0 for 5 min (D 5), 30 min (D 30) and 120 min (D 120). All

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the samples for duodenal digestion were first went through G 60 and during the duodenal phase, the

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ratio of gastric digesta to SIF was 1:1.

The activities of pepsin, trypsin and pancreatic lipase in the HGJ and HDJ were measured

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according to the method reported by Minekus et al. (2014) and were found to be 909 U mL-1, 21.9 U

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mL-1and 584 U mL-1, respectively. Hence, 1183 U of pepsin activity and 119 U of trypsin activity per

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gram of milk protein and 171 U of lipase activity per gram of lipid were used. The bile salt content of

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the HDJ was 1.1 mM, which was measured according to Ulleberg et al. (2011).

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The digested samples for the protein analysis were placed in ice water and transferred to the

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freezer at -20 C until sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE)

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analysis. For lipid analysis, the tubes with whole gastric or intestinal digesta were added 20 mL of

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chloroform:methanol (2:1, v/v) and kept at -20 C until analysed by gas chromatography-mass

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spectrometry (GC-MS). Duplicate digestion experiments were performed (n=2).

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2.4

Protein analysis

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All the milk samples were diluted to the final volume of the duodenal phase; they were then

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mixed with 2 SDS sample buffer (0.125 M Tris-Cl, pH 6.8, 4% SDS, 20% glycerol, 3.1% DTT and 0.03

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mM-bromophenol blue) in a ratio of 1:1 and boiled for 5 min.

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Separating gels, electrophoresis equipment, the molecular weight marker and image analysis

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software used were from Bio-Rad Laboratories (California, USA). The diluted digesta and undigested

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samples were applied to precast Stain-Free 12% gels (Mini-PROTEAN TGX) and SDS-PAGE system

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(Mini-PROTEAN Tetra Cell) was run at 200 V for 30 min (Laemmli, 1970). A low molecular weight

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marker (SDS-PAGE molecular weight standard, low range) was used. A minimum of three runs per

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samples were performed.

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Coomassie blue staining was used to visualise the protein separation. The degradation profile

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for -lactoglobulin (-lg) was evaluated with the help of Imagine Lab Software version 5. In the

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undigested samples, the quantity of -lg was defined as 100% and a relative percentage of the

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remaining amount of the -lg in the digested samples was calculated.

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2.5.

Lipid analysis

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The method described by Islam, Ekeberg, Rukke, and Vegarud (2016), with modifications, was

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used to analyse the lipids. Briefly, it involved lipid extraction followed by fractionation into neutral

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lipids (NLs) and FFAs by solid phase extraction followed by transesterification and esterification,

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respectively, to form the fatty acid methyl esters (FAMEs) that were analysed by GC-MS. Separate

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internal standards of NL and FFA in chloroform were added to the sample tubes containing the
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digesta and the chloroform:methanol mixture. After horizontal shaking of the sample tubes at 350

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rpm for 20 min, 4 mL of 0.9% NaCl was added to each tube and vortexed, followed by centrifugation

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for 10 min at 850 g at 20 C. The organic phase was collected and dried by N2 gas at 37 C and the

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samples were dissolved in 1 mL chloroform.

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Solid phase extraction was carried out using a liquid handling robot (Gilson, GX-274 ASPEC,
Middleton, WI, USA) with a flow of 1 mL min-1. The columns used were Chromabond NH2-

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polypropylene (Macherey-Nagel, 500 mg, 3 mL, Dren, Germany) and were conditioned with 7.5 mL

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hexane. The NL and FFA fractions were eluted separately by 5 mL of chloroform and 5 mL of diethyl

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ether:acetic acid (98:2, v/v), respectively. The solvents were evaporated under N2 gas at 37 C. For the

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formation of FAMEs from the NL fraction, 1.5 mL sodium methanolate (3.3 mg mL-1) in methanol was

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added to the NL. After 30 min incubation at room temperature on a shaker set at 350 rpm, the

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analyte was extracted with 2 mL hexane. The organic phase was collected in the GC-vials after leaving

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the tubes in a vertical position for 10 min. Formation of FAMEs from the FFA fraction was achieved by

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adding 1 mL boron trifluoride-methanol complex (14% BF3 in CH3OH, Sigma-Aldrich, Steinheim,

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Germany) and incubated at 70 C for 5 min. Subsequently, 1 mL hexane was added and the hexane

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phase was collected in GC-vials and stored at -20 C until analysed.

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FAMEs were analysed using an Autospec Ultima MS (Micromass Ltd., Manchester, UK), as

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described by Devle et al. (2012). The ion source was electron ionisation (70 eV) and the mass range

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was 40600 m/z. The MS was coupled with Agilent 6890 series gas chromatograph (Agilent

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Technology, Wilmington, DE, USA). The separation of the analytes was obtained with a 60 m Rtx-2330

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capillary column (i.d. 0.25 mm and 0.20 m film thickness; Restek, Bellefonte, PA, USA). MS library

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search and comparison of retention time with standards were used for identification of the FAMEs.

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Peak area in relation to internal standard was used for quantification.

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3.

Results and discussion

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Initial analysis revealed that the total protein and fat content of the milk were 3.4% and 4.6%,

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respectively. The average size of milk fat globules (D4,3) were 3.89, 1.45 and 0.58 m in P, PH50 and

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PH150 milk, respectively.

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3.1.

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Proteolysis

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Protein separation by gel electrophoresis revealed that no intact milk proteins remained after
only 5 min of duodenal digestion, irrespective of the milk sample (data not shown). The main

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differences between the milk samples seemed to be how they were digested during the gastric step.

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Fig. 1a shows that no intact caseins remain after 60 min of gastric digestion for any of the milk

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samples. This is in accordance with the recent results of Tunick et al. (2016). Gastric digestion of the

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pasteurised and homogenised samples resulted in smaller fragments compared with digested raw

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milk. Although -lg is usually considered resistant to gastric digestion (Eriksen et al., 2010; Mandalari

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et al., 2009), we observed an increased digestion of -lg in the homogenised milk compared with that

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in raw and pasteurised milk (Fig. 1b). More than 75% -lg remained in the latter samples while only

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49% and 31% remained in PH50 and PH150, respectively. This is contrary to the study by Tunick et al.

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(2016), where homogenisation did not affect digestion of -lg. However, the observed difference in

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the digestion of -lg could be caused by a difference in both processing conditions of the milk sample

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and the digestion model applied. In the present study we used human gastric juice, which likely

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contains a combination of different pepsin isoforms (Jones et al., 1993) compared with pure pepsin

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used in the study by Tunick et al. (2016). Homogenisation also affects the gastric digestion of -

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lactalbumin (-la).
Whether the observed differences in protein digestion may influence human health is not

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possible to say. Nutritionally the milk proteins all seem to be well digested; however, differences in

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peptide sequences and amino acids produced could not be ruled out.

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3.2.

Lipolysis

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The results of lipolysis of milk lipids in raw, pasteurised and homogenised milk after gastric (60

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min) and duodenal digestion (5, 30 and 120 min) are shown in Fig. 2. The FFA contents in raw, P, PH50

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and PH150 milk after 60 min of gastric and 5, 30 and 120 min of duodenal digestion are presented in

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Table 1. It also includes the release rate (%) of each of the individual fatty acids from milk lipids. No

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significant gastric lipolysis was observed in any of the milk types. These results are in agreement with

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earlier findings on ex vivo digestion of cow and buffalo milk (Devle et al., 2014; Islam et al., 2015a,

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2016).

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expected due to the intact triple layer membrane of phospholipids and proteins that exist in native

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MFGs. After 5 min of duodenal digestion, we observed a significant degree of lipolysis in all milk

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samples (Fig. 2). The highest release of FFAs was from the milk that was pasteurised and homogenised

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at the highest pressure (PH150, 58%). Raw milk, pasteurised milk and milk that was homogenised at

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the lower pressure (PH50), demonstrated a degree of lipolysis of 30%, 26% and 46%, respectively.

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After 30 min of duodenal digestion, PH150 had lipolysis of 75%, whereas the other milk samples had

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lipolysis of 3746%. After an additional 90 min exposure to duodenal enzymes, the PH150 milk only

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showed 3% further lipolysis and ended at a total of 78% released FFAs. This means that for PH150 an

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approximate steady state was reached after only 30 min of duodenal digestion.

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The observations of steady state is probably due to the use of a static digestion model where
the products are not continuously removed from the system, and are in agreement with earlier

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findings (Devle et al., 2014; Islam, Ekeberg, Rukke & Vegarud, 2015b; Islam et al., 2015a, 2016). After

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120 min of duodenal digestion, raw, pasteurised and PH50 milk had still not reached a steady state.

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The degree of lipolysis in these milk samples was 49%, 44% and 56%, respectively. These results can

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be explained by the homogenisation reducing the size of MFGs, and a subsequent increase in

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available surface for lipolytic enzymes. The average size of the lipid droplets in homogenised milk

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(PH150) showed 85% reduction (0.58 m) compared with that of the lipid droplets in pasteurised milk

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(3.89 m). However, a lower homogenisation pressure (PH50) did not reduce the droplet size that

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efficiently (1.45 m) and only 56% lipolysis was observed. PH50 milk lipids showed 22% less lipolysis

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than that of the PH150 milk lipid, which was still 712% more than the lipolysis of unhomogenised (R

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and P) milk lipids. These results are in contrast to those of Berton et al. (2012), where the extent of

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lipid hydrolysis did not correspond to the extra-large available surface after homogenisation.

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However, the study by Berton et al. (2012) was performed with only recombinant human pancreatic

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lipase and no other pancreatic enzymes. In the present study, the digestive duodenal juice contains all

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intestinal enzymes, proteases and lipases that could assist the removal of the proteins barrier in the

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newly formed membrane surrounding the lipid droplets. This might result in more intensive lipolysis

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by the lipases.

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As mentioned earlier, the variation in the amounts of the individual FFAs after 5, 30 and 120

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min of duodenal digestion are also shown in Table 1. No results regarding C4:0 are present. According

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to Islam et al. (2016), accurate measurement of C4:0C8:0 is difficult since the volatility of fatty acids
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increases with the reduction of carbon number. The overall release rate (%) of individual fatty acids

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from the milk lipids is also presented in Table 1. All the fatty acids showed significantly higher release

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rate (%) during duodenal digestion of PH150 milk compared with the other milk types. That

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corresponds well with the results presented on lipolysis in Fig. 2 and results on amount of free fatty

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acids in different milk after different steps of digestion in Table 1.

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For all the milk types, the short-chain FAs (C6:0 C10:0) showed the highest degree of release

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(5091%) compared with that from the medium- (C12:0C16:0, 475%) and long-chain (>C17:0) FAs

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(4883%). The higher release of the short- and long-chain FAs in milk can be explained by their

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abundance in sn-1 and sn-3 positions in the triglycerides, compared with medium-chain FAs (Angers,

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Tousignant, Boudreau & Arul, 1998; Blasi et al., 2008). The preference of the pancreatic lipase to

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attack the ester bond at sn-1 and sn-3 of the glycerides (Armand, 2007; Rogalska, Ransak & Verger,

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1990). The unsaturated fatty acids showed 40%, 39%, 52% and 66% release in raw, P, PH50 and

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PH150 milk, respectively. This was 14%, 11%, 10% and 16% less than that of the saturated fatty acids

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in the same milk samples. This is in agreement with the findings of Islam et al. (2016), where it was

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reported that the saturated fatty acids in buffalo milk (5.84% fat, unhomogenised) showed 9% more

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release than that of the unsaturated fatty acids. On the contrary, in bovine milk (3.5% fat,

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homogenised) Devle et al. (2014) reported 10% more release of unsaturated fatty acids than of

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saturated fatty acids.

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Conclusion

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This study showed that pasteurisation had minor effect upon lipolysis and proteolysis of
bovine milk. However, homogenisation affected both lipolysis and proteolysis of the milk. A higher
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degree of lipolysis was observed in the milk homogenised at 150 bar compared with that

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homogenised at 50 bar. No significant lipolysis was found after the gastric stage, but the lipolysis

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proceeded during duodenal digestion. After 120 min of gastrointestinal digestion the lipolysis seemed

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to reach equilibrium only for the milk that was pasteurised and homogenised at 150 bar.

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Homogenisation affected the gastric digestion of -lactoglobulin and -lactalbumin compared

with raw and pasteurised milk. However, after intestinal digestion all the milk proteins were degraded

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within 5 min. Fast release of free fatty acids, peptides and amino acids during digestion may affect the

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bioavailability of these components in the gut.

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Acknowledgements

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The authors would like to thank the Norwegian University of Life Sciences for financial support

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for the visiting research grant, project number 15/00122. We also thank Dr. Arne Rseth, Head of the

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gastro-policlinic at Lovisenberg Diakonale Hospital, Oslo for aspiration and collection of human juices.

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Figure legends

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Fig. 1. Ex vivo digestion of milk proteins with human gastric juice for 60 min (G60) in raw milk,
pasteurised milk (P), and milk that was both pasteurised and homogenised at 50 bar (PH50) or
150 bar (PH150). Panel A, SDS-PAGE protein profiles of digested milk; STD, molecular mass

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standard; start, undigested milk; G60, 60 min of gastric digestion at pH 3.0 and 37 C; -la, lactalbumin; -lg, -lactoglobulin. Panel B, amount of intact -lg remaining after gastric

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digestion of milk; standard deviation bars indicate probable extent of variation in the data set,
n=2.

Fig. 2. Proportions (%) of free fatty acids (
) and neutral lipids (
) in the total lipids of raw,

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pasteurised and homogenised milk during ex vivo gastrointestinal digestion with human gastric,
followed by duodenal juices: P, pasteurised; PH50, pasteurised and homogenised at 50 bar;
PH150, pasteurised and homogenised at 150 bar; G60, gastric digestion for 60 min at pH 3.0

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and 37 C; D5/D30/D120, duodenal digestion for 5, 30 or 120 min, respectively, at pH 7.0 and

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37 C. Standard deviation bars indicate probable extent of variation in the data set, n=2.

ACCEPTED MANUSCRIPT

Table 1
Free fatty acid content (g mL-1) of raw, pasteurised, and both pasteurised and homogenised milk after gastric and duodenal digestion. a
Conditions

Fatty acid
C6:0

C8:0

C10:0

C12:0

C14:0

C15:0

C16:0

C16:1,
9cis
2.00.2
nd
3.82.2
4.30.2

C17:0

C18:0

C18:1, 9cis

1.20.8
5.62.4
4.12.1
2.50.6

64.20.8
437102
1594.1
16920

72.223.4
15310.4
4249.9
51349.1

21.04.2
50.84.5
1881.8
24310.2

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Digestion

Total

42.93.8
49.59.5
58.513.3
63.45.8

C18:2, 9,
12cis
2.90.9
3.40.5
2.40.2
4.91.1

125482
188085
313394
4403339

1870183
259197
5621121
63561111

2074.6
32824.0
45014.8
44190.4

8724
10676
20767
28064

R
P
PH50
PH150

nd
2.90.7
2.10.6
2.40.3

0.50.1
2.50.5
2.10.6
2.70.2

3.10.7
7.20.7
6.41.7
8.01.1

10.51.3
13.33.9
9.72.8
11.11.8

23.61.2
43.110.8
39.210.5
44.05.6

1.30.1
4.11.3
3.41.0
4.52.1

1421.3
35599.6
20427.3
21734.4

D5

R
P
PH50
PH150

26.20.2
14.02.1
26.31.2
43.30.5

60.72.9
60.80.7
92.75.2
1594.2

18029
2161.8
4080.6
6078.1

24964
2658
54310
77010

904329
94236.3
226157.5
3317248

58.215.6
88.43.7
2371.4
33613.9

3821663
408771
7385168
108761194

D30

R
P
PH50
PH150

38.75.6
20.21.3
51.21.8
61.12.1

78.011.8
72.210.3
1572.8
2047.0

21914
39566
47127
7720.1

29759
565106
52139
98011

1097373
2405439
180519
4316137

75.618.8
23834.1
2055.6
42615.9

4605796
77391336
732953
14037539

96.025.8
36247.6
32231.0
63317.6

29.54.5
18334.3
1740.2
32015.0

15827.9
2714340
420527
5977117

2299111
4743681
5856701
8148129

22413.9
41315.5
39131.8
48312.2

10640
19848
21485
36356

D120

R
P
PH50
PH150

59.33.9
19.32.7
57.64.4
69.012.3

971.8
10114
20122
2379.9

27525
4114.7
607112
84521

37360
5692.7
67795
103429

1381408
23972
2288296
440084

10318.2
25415.5
26920.9
45078.7

5837841
7873258
87461296
14470709

13324.3
39126.6
43434.6
65228.2

47.74.6
19326.2
2158.3
3341.7

236026
2944273
4761558
6421197

3121127
5119269
7309842
9260387

24610.2
4368.6
43118.1
52014.6

14032
20707
25989
38691

Release
rate (%)

R
P
PH50
PH150

78.00.1
53.15.4
87.73.6
98.01.5

73.50.4
54.714.7
87.71.4
94.20.5

49.84.1
46.81.9
60.82.0
80.04.5

45.51.9
48.71.6
47.76.5
71.36.9

37.94.6
43.66.0
41.40.02
72.32.2

47.81.4
56.810.1
52.20.6
74.73.1

46.01.0
48.00.2
52.21.5
77.11.4

42.61.2
40.97.7
51.02.2
65.72.5

53.89.8
41.50.9
54.80.4
84.43.9

57.82.1
49.80.8
76.53.6
88.51.7

47.24.5
41.70.2
60.61.9
69.56.4

31.31.4
33.40.9
45.15.7
63.61.3

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G60

294
923
494
534

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Milk was raw (R), pasteurised (P), and both pasteurised and homogenised at either 50 bar (PH50) or 150 bar (PH150). Gastric digestion (G60) was
for 60 min at pH 3.0 and 37 C; duodenal digestion for 5, 30 and 120 min (D5, D30 and D120, respectively) at pH 7.0 and 37 C. All the duodenal
samples were first digested under gastric conditions as for G60. The release rate (%) of individual fatty acids from the neutral lipids (NL), where the
release rate = [(individual fatty acid in the neutral lipids after G60 individual fatty acid in the neutral lipids after D120) individual fatty acid in the
neutral lipids after G60] 100. Data from duplicate digestions are presented; standard deviation indicates probable extent of variation in the data
set.

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A)

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kDa

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B)

Figure 1

ACCEPTED MANUSCRIPT

110

RI
PT

100
90

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80

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% Lipolysis

70
60
50

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40
30

EP

20

0
Raw

PH50

G60

Figure 2

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10

PH150

Raw

PH50

D5

PH150

Raw

PH50

D30

PH150

Raw

PH50

D120

PH150