Ex Vivo Digestion of Proteins and Fat in Buffalo Milk

Accepted Manuscript
Ex vivo digestion of proteins and fat in buffalo milk

Mohammad Ashiqul Islam, Dag Ekeberg, Elling-Olav Rukke, Gerd Elisabeth Vegarud
PII:
S0958-6946(15)00163-6
DOI:
10.1016/j.idairyj.2015.08.008
Reference:
INDA 3872
To appear in:
International Dairy Journal
Received Date: 18 February 2014

Revised Date:
15 August 2015
Accepted Date: 17 August 2015
Please cite this article as: Islam, M.A., Ekeberg, D., Rukke, E.-O., Vegarud, G.E., Ex vivo digestion of
proteins and fat in buffalo milk, International Dairy Journal (2015), doi: 10.1016/j.idairyj.2015.08.008.
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ACCEPTED MANUSCRIPT
Ex vivo digestion of proteins and fat in buffalo milk
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Mohammad Ashiqul Islam*, Dag Ekeberg, Elling-Olav Rukke, Gerd Elisabeth Vegarud
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Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life
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Sciences, P. O. Box 5003, NO-1432 Aas, Norway
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*Corresponding author. Tel.: +47 6496 5900
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Email address: mohammad.islam0@nmbu.no (M. A. Islam)
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Mymensingh-2202, Bangladesh. E-mail address: m.a.islam@bau.edu.bd
Permanent address: Department of Dairy Science, Bangladesh Agricultural University,
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ABSTRACT
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Ex vivo digestion of proteins and fat in whole buffalo milk (WBM) and skimmed buffalo milk
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(SBM) was studied. Proteolysis, generation of peptides, lipolysis and generation of fatty acids
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were monitored. In both types of milk, - and -caseins were hydrolysed almost completely after
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20 min of gastric digestion. Residual S-caseins were digested completely on 5 min of duodenal
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digestion. Of the main whey proteins, -lactalbumin was only partially hydrolysed and -
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lactoglobulin seemed to be resistant during 40 min of gastric digestion. However, by 5 min of
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duodenal digestion, both -lactalbumin and -lactoglobulin were fully hydrolysed in both WBM
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and SBM. Only small variations between WBM and SBM were found for the release of peptides.
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For the WBM, 30% lipolysis was observed after 30 min of duodenal digestion. Different extents
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of lipolysis of fatty acids among/within different groups, based on carbon number and double
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bond(s), were observed.
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1.
Introduction
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The casein (CN) ratios in buffalo and cows milk is somewhat different, with a ratio of
2.8:1.0:3.1:1.2 of S1-, S2-, - and -CN in buffalo milk (Islam et al., 2014) and 2.7:1.0:2.7:0.9
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in cows milk (Miranda, Mahe, Leroux, & Martin, 2004). The differences in protein content and
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ratio may affect the digestibility of the proteins (Almaas et al., 2006). The homology of buffalo
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and cow CNs ranges from ~93% to ~98% (Abd El-Salam & El-Shibiny, 2011). This variation
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may also lead to the variability in the formation and content of peptides after proteolysis
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(Ulleberg, 2011).
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Milk is a natural emulsion in which the lipids are present in the form of a colloidal
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assembly, the milk fat globule. Meena, Ram, and Rasool (2007) reported that the total fat content
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of buffalo milk ranges from 3.37% to 14.42%. Milk fat mainly contains triglycerides (98%) with
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small fractions of diglycerides (0.3%), monoglycerides (0.03%), free fatty acids (FFA, 0.1%),
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phospholipids (0.8%), and sterols (0.32%) (Walstra, Wouters, & Geurts, 2006). The
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understanding of the fate and kinetics of dietary lipid digestion is important because of the
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implication for lipid consumption on human health and in the development of new products.
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Gastrointestinal (GI) digestion of dietary lipids is influenced by the physicochemical
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characteristics of lipid droplets, i.e., size, fatty acid distribution on the triglyceride backbone, and
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the organization and composition of the interface (Berton et al., 2012). The milk fat globule size
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in buffalo milk is larger than that in cows milk. Buffalo milk fat has been reported to have less
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milk fat globule membrane (1.40 to 1.57 g per 100 g fat) than that of cows milk (1.47 to 1.97 g
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per 100 g fat), but almost all the membrane components were found at higher concentrations in
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the buffalo milk fat globule, although this depends upon several factors such as breed, season,
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and stage of lactation (Abd El-Salam & El-Shibiny, 2011).

The fatty acid (FA) composition of cows milk fat is highly complex (Reklewska et al.,
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2002); however, C4:0-C24:0 FAs are more common and dominant in the form of saturated (S,
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70%) or unsaturated (US, 30%) FAs. Considerable variations in the FA composition of
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buffalo and cow milk fat have been reported (Abd El-Salam & El-Shibiny, 2011; El-Shibiny,
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Fontecha, Juarez, & Abd Rabou, 2005; Islam et al., 2014; Medhammar et al., 2012; Menard et
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al., 2010). The variation in the cow and buffalo milk fatty acid composition may result from
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differences in the fatty acid distribution in the triglyceride, and thus also contributes to the
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variation of the molecular size of the triglycerides. All these will affect the physiological fate of
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the dietary lipids, including digestion, absorption, and subsequent metabolism.
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A complex combination of mechanical, physiochemical and physiological processes are
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involved in the GI digestion of proteins and lipids in humans. A number of factors, such as food
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composition, buffering capacity of the food, pH, concentration and activities of the enzymes
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secreted, peristaltic movements, emptying of the stomach, and duration of the digestion may
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influence digestion. To understand better how food is being digested, in vivo studies provide the
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most accurate data but are time consuming, costly, and have ethical aspects. As a compromise
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between accuracy and ease of utilisation, interest in ex vivo digestion model is increasing;
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however, simulation of human digestion is challenging because of the inherent complexity of the
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process.
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In a review by Hur, Lim, Decker, and McClements (2011), wide variability was shown in
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the existing in vitro and ex vivo models, especially regarding enzymes used, pH, duration, and
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steps of digestion. These authors also emphasised the importance of using physiologically
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relevant enzymes and other gut-relevant components (such as bile acids) when designing
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digestive fluids. By using aspirates and GI juices from human volunteers in the ex vivo model, a
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digestion model may be considered as a good approach to mimic the in vivo physiological
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conditions.
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Almaas et al. (2006) developed a digestion model using human GI enzymes, and
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observed that goat milk proteins were digested faster than the cow milk proteins. In another
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study, Inglingstad et al. (2010) reported species variability in the in vitro digestion of horse, cow,
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goat, and human milk proteins using human GI enzymes. Devle et al. (2014) showed the
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reciprocal interacting effects of proteins and lipids during ex vivo digestion of cows milk. The
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absence of milk fat favours the digestion of -Lg, but the absence of milk proteins reduces the
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amount of lipolysis. To best of our knowledge, no such ex vivo study has been done on digestion
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of buffalo milk proteins and lipids. In addition, the data on milk peptides derived from whole and
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skimmed buffalo milk by using human GI enzymes are also very scant.
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The aim of the present study was to investigate the impact of digestion on buffalo milk
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proteins and lipids using human gastric and duodenal juices. Degradation of the immunogenic
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proteins, S1-casein and -Lg, generation of peptides, lipolysis of milk fat in whole milk, and
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release of free fatty acids and the effect of lipids on proteolysis were examined.
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2.
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2.1.
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Materials and methods
Milk samples
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Pooled, whole, raw fluid milk was collected from nine lactating buffaloes of the
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Bangladesh Livestock Research Institute (BLRI) buffalo farm (Savar, Dhaka, Bangladesh). The
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sampling was carried out on the morning milk. The milk samples were preserved by adding
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Bronopol tablet (one tablet per 40 mL of milk; D & F Control Systems, Inc. Norwood, MA,
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USA) immediately after milking of the animals, followed by freezing (-20 C), transported to the
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Norwegian University of Life Sciences (Aas, Norway). The milk samples were kept at -20 C
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until used. Before use, it was thawed in ice-water overnight, and then tempered to 37 C in a
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water bath. Skimmed milk was prepared by removing lipids by centrifugation at 850 g, at 4 C
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for 20 min (Allegra 25R centrifuge with TS-5.1-500 rotor head: Beckman Coulter, Brea, CA,
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USA). The true protein (3.5%) and fat (5.84%) contents were measured by micro-Kjeldahl (Foss
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Analytical Lab., Hoganas, Sweden) and Auto Milk Analyzer (Lactostar, Funke-Gerber, Berlin,
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Germany), respectively, as previously reported by Islam et al. (2014).
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2.2.
Human gastric and duodenal juices
The human gastric juice (HGJ) and duodenal juice (HDJ) were collected by following the
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methods described by Holm, Hanssen, Krogdahl, and Florholmen (1988) and Ulleberg et al.
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(2011). The aspiration of juices were carried out on six volunteer adults (20 to 37 years old),
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healthy and fasted (for 8 h), from Lovisenberg Diakonale Hospital (Oslo, Norway). Aspiration
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was approved by the Norwegian Research Ethics Committee. In brief, the aspirates were
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collected in a tube placed on ice by using a triple lumen tube (Maxters catheters, Marseille,
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France). After collection, the cell debris and mucus were removed by centrifugation and the
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aspirates stored at -20 C until used. The volume, pH, and enzyme activities of HGJ and HDJ
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were measured. Then a pooled batch of HGJ and HDJ were prepared for use in the ex vivo
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digestion model. A method described by Ulleberg et al. (2011) was used to assay the pepsin
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activity in HGJ, lipase and total proteolytic activity, and total bile salts in HDJ. The pooled
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batches of HGJ and HDJ were stored at -80 C until used. Before use, the juices were thawed by
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keeping them overnight on ice, in a cold room at 4-6 C.
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2.3.
Ex vivo digestion
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A method described by Devle et al. (2014) with some modifications was used for the
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digestion of whole and skimmed buffalo milk. A continuous two phase digestion was carried out
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in a water bath at 37 C; first a gastric phase (by HGJ), then a duodenal phase (by HDJ). In the
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gastric phase, the pH of milk (1 mL sample) was reduced to 5.0 by adding 2 M HCL before
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adding the HGJ and digested for 20 min (G20), then the pH was further reduced to 2.5 and
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digestion continued for another 20 min (G40). Thereafter, pH was adjusted to 7.0 by adding 2 M
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NaOH, then HDJ added and samples taken after 5 min (D05), 30 min (D30), 60 min (D60) and
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120 min (D120) of duodenal digestion. The samples, acid and/or alkali and juices were kept
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mixed by magnetic stirring (200300 rpm). The volume of HGJ and HDJ added to 1 mL of milk
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was 800 L (711 units pepsin activity per g milk protein) and 1150 L (558 units total
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proteolytic activity per g milk protein), respectively.
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Enzyme activity was measured according to Ulleberg et al. (2011) and the amount of
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HGJ and HDJ for per mL of milk was calculated from Devle et al. (2011) considering the pepsin
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and total proteolytic activity, respectively. The HDJ contained 889 units of lipase activity and 2.4
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mM bile acids as well.

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For protein and peptide analyses, samples (1 mL) were placed on ice immediately after
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digestion and stored at -20 C with minimum delay. To the lipid samples (1 mL), 20 mL
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chloroform:methanol (2:1) was added immediately after the digestion and stored at -20 C. The
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digestion was carried out three times. The gastric digested samples were cloudy in appearance.
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G40 was more heterogeneous than that of the G20 digest, and the duodenal digested samples
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were homogeneous clear solutions with a yellow colour.
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2.4.
Protein and peptide analysis
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2.4.1. Milk protein degradation profiles by SDS-PAGE
To visualise protein hydrolysis in whole and skimmed buffalo milk after the different
gastric and duodenal steps of digestion, SDS-PAGE was carried out using the method of Devle et
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al. (2014) with some modifications. The gastric samples (1 mL) were homogenised using an
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Ultra Turrax (Yellow Line DI 18 basic, IKA-Werke GmbH & Co. KG, Staufen, Germany) at
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speed 3 for few seconds. The sample was mixed with SDS-PAGE sample buffer at a ratio of 1:2.
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Prepared samples (10 L) were applied to the respective wells of Any kDTM polyacrylamide
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separating gels (6.5200 kDa; mini PROTEAN TGXTM precast gels, Tris glycine extended,
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Bio-Rad Laboratories, Inc., Hercules, CA, USA). A low molecular mass marker (LMW-SDS
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Marker Kit; GE Healthcare, Little Chalfont, Bucks, UK) was used. The gel was run for 35 min at
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200 V and fixed, stained with Coomassie Brilliant Blue and destained.
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2.4.2. In-gel digestion of protein bands and identification by ultra-performance liquid
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chromatography and Q-exactive mass spectrometry

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For checking the trace(s) of hydrolysed -Lg, the bands around -Lg and -LA on the
SDS-PAGE gels (lane D05) were cut and in-gel digested according to Devle et al. (2014).
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Subsequently, the tryptic peptides were dried, and loading solution (0.05% trifluoroacetic acid,
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2% acetonitrile in water) was added. The samples were loaded onto a nano ultra-performance
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liquid chromatography (UPLC; RSLC3000, Dionex/Thermo Fisher Scientific, Bremen,
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Germany) equipped with a trap column (Acclaim PepMap100, C18, 5 m, 100 , 300 m i.d.
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5 mm, Thermo Fisher Scientific), back flushed onto a 50 cm 75 m analytical column
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(Acclaim PepMap RSLC C18, 2 m, 100 , 75 m i.d. 50 cm, nanoViper; Thermo Fisher
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Scientific). For the separation of the peptides, a 45 min gradient from 4 to 40% solution B (80%
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acetonitrile, 0.1% formic acid) at a flow rate of 300 nL min-1 was used. The set-up of the Q-
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Exactive MS (Thermo Fisher Scientific) was a full scan (300-1600 m/z) at R=70,000, followed
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by (up to) five MS2 scans at R=35,000, using an NCE setting of 28. For MS/MS, singly charged
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precursors were excluded, as were precursors with z > 5. The dynamic exclusion was set to 30 s.
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The Masconvert module of ProteoWizard (http://proteowizard.sourceforge.net/) was used to
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convert the raw files to mgf format. The files were then submitted to database search (Swissprot,
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taxonomy - other mammals) on an in-house Mascot (v.2.4) server. Mass tolerance was 10 ppm
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and 20 mamu for MS and MS/MS, respectively, and allowed for up to two missed proteolytic
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sites. The selected fixed and variable modifications were carbamidomethylated cysteine and
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oxidised methionine, respectively.
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2.4.3. Peptide identification by UPLC and quadrapole-time of flight mass spectrometry
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Peptides from the G20, G40, D05 and D120 samples were desalted and concentrated
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according to Furlund et al. (2013). Identification of peptides was achieved by the method of
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Qureshi, Vegarud, Abrahamsen, and Skeie (2012) with some modifications. In brief, peptide
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mixtures (containing 0.5% formic acid) were applied to a nanoACQUITYTM UPLC (Waters,
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Milford, MA, USA), equipped with 5-m symmetry C18 trap column (180 m 20 mm;
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Waters) in front of a 1.7-m BEH C18 analytical column (75 m 100 mm; Waters). Each
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sample was run in MS and data dependent tandem MS mode into a Q-TOF Ultima MS
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(Micromass Ltd., Manchester, UK). A non-redundant protein sequence database version was
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employed NCBInr 20130131 (22749596 sequences; 7819872540 residues).
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2.4.4. Multiple sequence alignment of peptides
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A method described by Furlund et al. (2013), with some modifications, was used to carry
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out the sequence alignment of peptides. At first, peptides were selected with minimal overlap for
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every sample. Next, a pool of G peptides from G20 and G40 samples, and also for D peptides
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from D05 and D120 samples, were prepared. G and D peptides of whole and skimmed buffalo
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milk were compared using Clustal omega (version: CLUSTAL O (1.2.0 and 1.2.1);
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http://www.ebi.ac.uk/Tools/msa/clustalo/), and for consensus sequence, MS editor Jalview 2.8
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and 2.8.2 (http://www.jalview.org/) was used. The whole sequences of S1-, S2-, - and -CN
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and -Lg, and other relevant information, were obtained from http://www.ncbi.nlm.nih.gov/ and
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http://www.uniprot.org/. The multiple sequence alignment technique was also used to localize
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the proteolytic sites of a particular protein by aligning the peptides from the whole sequence of a
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specific protein.
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2.5.
Lipid analysis
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Total lipid extraction, solid phase extraction (SPE) of neutral lipids (NL) and free fatty
acids (FFA), formation of fatty acid methyl esters (FAMEs) and gas chromatography-mass
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spectrometry (GC-MS) analysis of FAMEs were carried out by following the method described
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by Devle et al. (2014), with a few modifications. Briefly, for total lipid extraction, 20 mL
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chloroform:methanol (2:1, added immediately after digestion) and internal standards in
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chloroform were added. The tubes were shaken horizontally for 20 min at 350 rpm. NaCl (0.9%
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in water; 4 mL) was added and vortexed, followed by centrifugation at 850 g for 10 min at 20
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C. The organic phase was collected, dried under N2 gas at 37 C and re-dissolved in 2 mL of
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chloroform.
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A liquid handling robot (Gilson, GX-274 ASPEC, Middleton, WI, USA) was used for
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SPE of 1 mL re-dissolved lipids. The NL and FFA fraction were eluted with 5 mL chloroform
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and 5 mL diethyl ether:acetic acid (98:2), respectively. Both fractions were again dried under N2
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gas at 37 C. The NL fraction was re-dissolved by adding 2 mL hexane and methylated by
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adding 1.5 mL sodium methanolate (3.3 mg mL-1) followed by horizontal shaking for 30 min at
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350 rpm. The tubes were left for 10 min in a vertical position and the hexane phase was
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transferred to the GC-vials. The FFA fraction were mixed with 1 mL boron trifluoride-methanol
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complex (14% BF3 in CH3OH, Sigma-Aldrich, Steinheim, Germany) and heated at 70 C for 5
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min. Hexane (1 mL) was added and the hexane phase was transferred to the GC-vials. Both the
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NL and FFA FAMEs were stored at -20 C until analysed by GC-MS.
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An Autospec Ultima MS (Micromass Ltd. Manchester, UK) equiped with an electron
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ionization ion source (mass range 40-600 m/z), coupled with a gas chromatograph (Agilent 6890
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series, Agilent Technology, Wilmington, DE, USA) was used for FAME analysis. The type of
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column used was 50 m CP-Sil 88 capillary column with ID 0.25 mm and 0.20 m thickness
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(Varian, Middelburgh, The Netherlands).
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3.
Results and discussion
3.1.
Protein digestion
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The protein hydrolysis profiles of whole and skimmed buffalo milk are presented in Fig.
1. In both types of milk, - and -CNs were seen to degrade almost completely after 20 min of
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gastric digestion at pH 5.0. At the same time S-CNs were partially hydrolysed, with further
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hydrolysis along with the other two CNs during 40 min of gastric digestion at pH 2.5. The
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pattern of CN hydrolysis appeared similar in both whole and skimmed buffalo milk.
The -LA was partially hydrolysed and the -Lg was fully resistant to gastric digestion,
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whereas serum albumin was hydrolysed completely during 40 min of gastric digestion. However,
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after 5 min of duodenal digestion, both -LA and -Lg were readily digested. In both of the
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gastric steps, the -Lg band appeared more intense compared with that of the untreated milk
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(lane 0). The more dense -Lg band in the gastric step lanes may be due to the presence of
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digested caseins fragment of similar molecular weight. In whole buffalo milk, traces of -Lg as a
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single band was identified by UPLC-Q Exactive MS that was not observed in skimmed buffalo
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milk (Fig. 1A, D05, band 1). Traces of -LA with degradation products of -Lg were also
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observed both in whole milk (Fig. 1A, D05, band 2) and skimmed milk (Fig. 1B, D05, band 1
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and 2) after 5 min of duodenal digestion, which was completely degraded after 30 min (D30).
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The -LA was of bovine origin, as per database (Swissprot, taxonomy - other mammals). Buffalo
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and cow milk -LA differs only by a single amino acid, buffalo (Asp17) cow (Gly17)
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(DAmbrosio et al., 2008) reflecting homology between them.
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The clear bands observed on the SDS-PAGE (a, b, c and d in Fig. 1A and 1B) in the
region 6030 kDa during duodenal digestion were identified as the digestive enzymes in the HDJ
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(Devle et al., 2014). Some differences in the gastric digestion after 40 min were observed
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between buffalo milk and the results reported by Devle et al. (2014) on cows milk. The S-CNs
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appeared more resistant to digestion than the - and -CN in buffalo milk, and these results are
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more consistent with the results of Gallier, Ye, and Singh (2012). Almaas et al. (2006) reported
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that the protein composition in different species may have an impact on protein digestibility, as
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higher degradation of goat milk CN compared with bovine milk CN was observed. These results
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were confirmed by Inglingstad et al. (2010) who reported high CN degradation variability
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amongst the milk from cow, goat, horse, and human. Buffalo milk CN degradability seems to
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differ from other species in respect to differences in the relative ratios of S1-, S2-, - and -CN
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(Islam et al., 2014; Miranda et al., 2004). The differences in the amino acids may also play an
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important role in the differences in milk protein digestion from these species.
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Devle et al. (2014) reported complete resistance of -Lg in full fat cows milk after 120
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min of duodenal digestion. In whole buffalo milk, -Lg was partially digested after 5 min of
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duodenal digestion and fully digested after 120 min (Fig 1A, D05 and D120). The fat in buffalo
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milk did not affect the digestion of -Lg as it did in cows milk reported by Devle et al. (2014).
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Islam, Ekeberg, Rukke, and Vegarud (2015) also found intact -Lg after 120 min of ex vivo
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duodenal digestion of full-fat Red Chittagong Cattle (RCC) milk from Bangladesh. The digestion
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conditions were similar to the present study; however, they did not report the skimmed RCC
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milk digestion. In the present study, the concentration of bile acid was 2.4 mM in the HDJ
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aspirate, whereas in the study reported by Devle et al. (2014), the concentration of bile acid was
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1.0 mM. According to Gass, Vora, Hofmann, Gray, and Khosla (2007), a concentration of 2 mM
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bile acid may accelerate -Lg digestion. The variations in the bile acid concentration may
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explain some of the variation between the present study and the results obtained by Devle et al.
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(2014). Binding of certain bile acids to the hydrophobic pockets of the protein may destabilise
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protein structure and make available some additional interior domains of the protein for protease
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action (Gass, Vora, Hofmann, Gray, & Khosla, 2007). Devle et al. (2014) proposed two possible
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mechanisms for the reduced digestion of -Lg in whole cows milk compared with skimmed cow
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milk. These were: (i) binding of released fatty acids to -LG during the digestion of the whole
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milk may interrupt the binding of bile acids to the -LG and hence the destabilisation of the -
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LG before being digested, and (ii) the availability of the bile acids to bind with the protein may
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be reduced due to the assimilation of some of the bile acids with the lipolytic products of the
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milk fat into the mixed micelles. Gass, Vora, Hofmann, Gray, and Khosla (2007) also reported
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that the presence of mixed micelles alters the effect of bile acids on protein degradation. Hence, a
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higher concentration of bile acids in the digestive juices will result in more available to bind with
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the -LG to destabilise the protein and make it more susceptible to protease action. The genetic
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variant may also be a factor in -Lg degradation variability as reported by Tidona et al. (2014).
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These authors reported that in donkeys milk, -Lg showed rapid degradation when it consists of
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only -Lg I, more specifically in the gastric digestion. El-Zahar et al. (2005) also reported more
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rapid degradation of -Lg variant B compared with that of the variant A in ovine milk. The
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replacement of the amino acid(s) could change the tertiary structure and surface hydrophobicity
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of the -Lg and thus may affect its degradation (Ulleberg, 2011; El-Zahar et al., 2005).
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3.2.
Site of proteolysis and peptides
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The peptides identified by UPLC/Q-TOF MS were matched with the whole sequence of
316
the protein to localise the proteolytic cutting sites (Fig. 2). Among the proteins in both types of
317
milk, S1-CN and -CN showed extensive proteolysis during both phases of digestion. The whole
318
and skimmed buffalo milk showed minimum variability in the peptide pattern, and only by a few
319
residues at the same region of proteolysis or by very few (one to three) new regions of
320
proteolysis (Fig. 2). In whole and skimmed buffalo milk, almost all the -CN peptides observed
321
were formed during gastric digestion, whereas most peptides from -Lg were observed during
322
duodenal digestion. These results are consistent with the results of SDS-PAGE (Fig. 1). The
323
variability of peptide regions among different phases and steps of digestion in Fig. 2 reveals the
324
subsequent hydrolysis of peptides and release of amino acids, or hydrolysis at a new site due to
325
different specificity of the GI enzymes. A relatively lesser number of peptides were observed
326
from UPLC/Q-TOF MS analysis during the digestion (Table 1) compared with the degradation
327
of the proteins on SDS-PAGE (Fig. 1A,B). This is may be due to the limitations of the UPLC/Q-
328
TOF MS with an identification range of peptides of 8004500 Da. Moreover, the free amino
329
acids were not analysed, so it is not possible to predict the extent of degradation of proteins to its
330
component residues.
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A maximum number of peptides were identified from -CN and S1-CN, followed by -
332
CN, S2-CN and -Lg (Table 1). Some peptides were identified both in the gastric and duodenal
333
phases. The multiple sequence alignments of the peptides generated from the gastric and
334
duodenal digestion (identified by UPLC/Q-TOF MS analysis) showed very little variation
335
between whole and skimmed buffalo milk (Fig. 3). Most of the peptides contained proline
15
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neighbouring hydrophobic residue(s), i.e., leucine, isoleucine, valine, phenylalanine, alanine, or
337
glycine, that could provide a motif for preferred uncleaved peptide bonds. This is reflected by the
338
consensus sequence obtained from Jalview 2.8 and 2.8.2. This is in agreement with results
339
discussed by Almaas et al. (2011) on peptides generated from -CN, -CN, -Lg and
340
glycomacropeptide by the digestion of goats milk. Jornvall and Persson (1983) reported that
341
proline restricts further proteolytic processing, especially toward protease with trypsin-type
342
specificity.
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343
3.3.
Lipid analysis
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345
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The pattern of lipolysis of neutral lipids during gastric and duodenal digestion of whole
buffalo milk is shown in Fig. 4. No lipolysis was observed after 40 min of gastric digestion
348
(G40). A greater extent of lipolysis was shown during the first 30 min of duodenal digestion.
349
Thereafter, only very little extra lipolysis was observed. These results are in agreement with
350
Devle et al. (2014) on cows milk. According to Pafumi et al. (2002), 1030% of triacylglycerols
351
can be hydrolysed by the gastric lipases in the stomach. The lack of lipolysis in our study may be
352
due to insufficient secretion during aspiration of the fasted volunteers or the enzymes were
353
inactive in the simulated conditions for the ex vivo gastric digestion (Devle et al., 2014). In the
354
present study, and also in Devle et al. (2014), the pH of the milk was first adjusted to 5.0 before
355
HGJ was added (milk:HGJ = 1:0.8). The natural pH of the HGJ varies from 1.5 to 2.5, and this
356
lowers the pH of the milk samples well below 5.0. However, the optimum pH for gastric lipase
357
activity is around 5.0 to 6.0 (Carriere, Barrowman, Verger, & Laugier, 1993), which may explain
358
why gastric lipases were not properly activated during G20, and as the pH was further lowered to
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2.5, there was almost no chance of gastric lipase activity during G40. The gastric lipase activity
360
in the aspirated digestive juice should have been measured, but we were unable to do this
361
because of a lack of appropriate kit and/or method to measure the activity at such a low pH of
362
1.5-2.5. However, gastric digestion is reported to be important for further duodenal lipolysis
363
(Gallier et al., 2012; Ye, Cui, & Singh, 2011). The accumulation of lipid digestion products is the
364
possible cause of very little lipolysis during 60 and 120 min of duodenal digestion. During
365
lipolysis, in addition to FFA, diglycerides and monoglycerides will be produced. But in the
366
present study, diglycerides and monoglycerides were included with the triglycerides in neutral
367
lipids and FFA was considered as the only lipolytic product.
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The concentration of FFAs during the digestion are summarised in Table 2, including the
extent of lipolysis. The amount of each fatty acid in the FFA increased sharply after 30 min of
370
duodenal digestion, and thereafter, showed very little change, as shown in Fig. 4. The average
371
lipolysis of saturated short (C4:0 to C8:0), medium (C10:0 to C16:0) and long ( C17) chain FA
372
were 41%, 33% and 43%, respectively. Among the individual SFAs, C4:0 showed the highest
373
extent of lipolysis which was 48%, and C6:0 was only 1.5% less than this. However, complete
374
measurement of C4:0-C8:0 is challenging because of the volatility, and concentration of free
375
C4:0 in the Table 2 is absent. Amongst the unsaturated fatty acids (USFAs), only C18:1 others
376
and C16:1 n-7 cis showed more than 30% lipolysis. The lowest lipolysis was observed in C12:0
377
(27%) and C8:0 (28%) among the SFAs, and also for C18:2 n-6 cis (14%) and C18:2 n-7 trans
378
(17%) among the USFAs. The extent of lipolysis of total SFAs was 9% higher than the total
379
USFAs.
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380
The pancreatic lipase has a preference to attack the sn-1 and sn-3 positions of the
381
triglyceride (Armand, 2007; Rogalska, Ransac, & Verger, 1990). Compared with medium chain
17
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FAs, a prevalence of short and long chain fatty acids in the sn-1 and 3 positions is reported
383
(Angers, Tousignant, Boudreau, & Arul, 1998; Blasi et al., 2008). The presence of short chain
384
fatty acids at sn-1 and sn-3 ranges from 76100%; which is 66100% and 3388% for the long
385
chain fatty acids and medium chain fatty acids, respectively. This explains why the average
386
lipolysis of medium chain FA was less than that of the long and short chain FA. However,
387
factors such as the molecular size of the triacylglycerol, the type of fatty acid (saturated or
388
unsaturated) and the species of mammal will influence the stereospecific distribution of fatty
389
acids.
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The overall results show some variability with the cows milk results reported by Devle
391
et al. (2014). The possible reasons are, firstly, the average buffalo milk fat globule (12.3 m) is
392
larger than that in cows milk (4.2 m, Islam et al., 2014), and moreover, in the cows milk study
393
homogenised whole milk was used. Smaller globules provide more total surface area for lipase
394
action compared with larger globules. Secondly, the reported variability in the FA distribution in
395
the triacylglycerols of cows and buffalo milk (Angers et al., 1998; Blasi et al., 2008; Lindmark-
396
Mnsson, 2008) may also contribute to differences in the lipolysis of the fatty acids.
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399
400
4.
Conclusions
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Ex vivo digestion of whole and skimmed buffalo milk was carried out to study protein
401
and lipid degradation and the effect of milk lipids on protein digestion, specifically the
402
immunogenic S1-casein and -lactoglobulin. Generation of peptides and free fatty acids were
403
also determined. In whole and skimmed buffalo milk, except for S-caseins, all the caseins were
404
rapidly digested during the gastric phase, and completely digested after 40 min. During the
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duodenal phase, S-caseins were digested completely after 5 min. In addition, -lactoglobulin
406
was readily digested after 5 min of duodenal digestion in both skimmed and whole buffalo milk.
407
Minimal variations in the peptide patterns were observed between the whole and skimmed milk.
408
In buffalo milk, milk fat had almost no effect on the milk protein digestion and in peptide
409
generation.
410
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A rapid lipolysis of neutral lipids was observed during the first 30 min of duodenal
digestion and the extent of lipolysis was 30%. The short (C4:0C8:0) and long ( C17:0) chain
412
fatty acids showed 810% more lipolysis than the medium (C10:0C16:0) chain fatty acids. The
413
lipolysis of total unsaturated fatty acids was 9% less than the total saturated fatty acids. A fast
414
digestion of the neutral lipids and the immunogenic proteins, S1-casein and -Lg, in buffalo
415
milk may be of nutritional importance for consumers.
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416
Acknowledgements
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The authors extend their thanks to Irene Comi for aspiration of human digestive juices
420
with the cooperation of Lovisenberg Diakonale Hospital, Oslo, Norway and making the juices
421
ready to use. Our thanks also to Professor Dr. Abdul Wadud, Department of Dairy Science, and
422
Professor Dr. Mohammad Al-Mamun, Department of Animal Nutrition, Bangladesh Agricultural
423
University, for checking the language of the manuscript.
425
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Department of Chemistry, Biotechnology and Food Science (IKBM), Norwegian
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University of Life Sciences (UMB), s, Norway.
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Ulleberg, E. K., Comi, I., Holm, H., Herud, E. B., Jacobsen, M., & Vegarud, G. E. (2011).
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vitro gastric digestion of milk. Journal of Dairy Science, 94, 2762-2770.
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Ye, A., Cui, J., & Singh, H. (2011). Proteolysis of milk fat globule membrane proteins during in
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Chapt. 2). London, UK: CRC Press. Taylor & Frances Group.
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Table 1
Number of peptides identified from whole (W) and skimmed (S) buffalo milk after gastric and
duodenal digestion. a
G20
G40
D05
S1-CN
18
18
17
19
11
S2-CN
-CN
-CN
-Lg
28
09
-
03
29
11
-
03
25
05
01
04
26
08
-
03
37
02
02
10
02
03
04
39
05
04
37
04
21
02
SC
D120
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Protein
AC
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EP
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D
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Abbreviations are: G20, gastric digestion at pH 5.0 for 20 min; G40, gastric digestion at pH 2.5
for 20 min after 20 min gastric digestion at pH 5.0; D05, duodenal digestion for 5 min, pH 7.0;
D120: duodenal digestion for 120 min, pH 7.0; CN: casein; -Lg, -lactoglobulin.
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Table 2
Fatty acid (FA) concentrations in the free fatty acid fraction of whole buffalo milk during
digestion with human gastric and duodenal juices, and lipolysis (%) of individual FA in the
neutral lipids (NL) fraction after 120 min duodenal digestion.
Lipolysis (%)
48.0
46.5
28.4
34.1
27.5
33.6
33.0
36.5
43.3
42.8
42.2
37.6
24.8
31.9
27.9
38.3
14.1
17.0
28.3
RI
PT
D120
nd
225
309
1144
1866
123050
3368
4660170
162.32.7
290080
35.02.0
9670280
78.42.6
3829
4040230
3595
2167
49.82.0
153
16.41.6
5150240
SC
D60
nd
20.71.3
354
1185
1916
125830
3336
4710140
1524
2805120
34.02.4
9700300
82.50.9
3936
4270190
3526
225.21.2
54.62.0
153
15.92.1
5410200
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D30
nd
333
456
11010
17717
111070
25911
4000270
1105
2013130
234
7900500
664
31213
3170290
23719
18612
394
11.31.4
163
4000300
EP
Values are given as g mL-1 milk standard deviation. Abbreviations are: G40, gastric
digestion at pH 2.5 for 20 min after 20 min gastric digestion at pH 5.0; D30, D60, D120:
duodenal digestion for 30, 60 and 120 min, respectively, at pH 7.0; SFA, total saturated fatty
acids; USFA, total unsaturated fatty acids; nd, not detected. Lipolysis calculated as [(FA in
undigested NL FA in D120 NL)/FA in undigested NL] 100.
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G40
nd
1.70.0
1.40.5
2.40.4
1711
28.82.5
3.60.7
159.22.8
3.30.7
1634
nd
3817
2.20.4
4.30.9
7911
nd
7.62.1
nd
nd
nd
9311
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FA
C4:0
C6:0
C8:0
C10:0
C12:0
C14:0
C15:0
C16:0
C17:0
C18:0
C20:0
SFA
C14:1 n-5 cis
C16:1 n-7 cis
C18:1 n-9 cis
C18:1 others
C18:2 n-6 cis
C18:2 n-7 trans
C18:3 n-3 cis
C20:4 n-6 cis
USFA
ACCEPTED MANUSCRIPT
Figure legends
Fig. 1. Protein degradation profile in (A) whole buffalo milk and (B) skimmed buffalo milk, after
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human gastric and duodenal digestion. The pH at G20 and G40 was 5.0 and 2.5, respectively,
and at D05, D30, D60 and D120 was 7.0. Abbreviations: SA, serum albumin; CN, casein; -Lg,
-lactoglobulin; -LA, -lactalbumin; a-d, digestive enzymes in the human duodenal juices
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(Devle et al., 2014). Lanes for both panels are: lane 1, low molecular mass marker; lane 2,
undigested sample; lanes 3 and 4, gastric digestion for 20 and 40 min, respectively; lanes 5, 6, 7
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and 8, duodenal digestion for 05, 30, 60 and 120 min, respectively. In panel A, the arrows
marked 1 and 2 indicate -lactoglobulin and -lactalbumin (?) with -lactoglobulin, respectively,
while in panel B the arrows marked 1 and 2 both indicate -lactalbumin with -lactoglobulin;
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note that the database search suggests -lactalbumin is of bovine origin.
Fig. 2. Comparative peptide regions derived from the different milk proteins of buffalo whole
(grey) and skimmed milk (underlined) after ex vivo gastric and duodenal digestion. Single letter
EP
amino acid code used. Bold and italic residues, signal peptide. The pH at G20 and G40 was 5.0
and 2.5, respectively, and at D05 and D120 was 7.0. Abbreviations: G20 and G40, gastric
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digestion for 20 and 40 min, respectively; D05 and D120, duodenal digestion for 5 and 120 min,
respectively.
Fig. 3. Multiple sequence alignment [software: CLUSTAL O (1.2.0 and 1.2.1)] of peptides
generated from different proteins of whole (BW) and skimmed (BS) buffalo milk by gastric (G)
and duodenal (D) digestion. Numbers on the left are the serial numbers of the minimal
ACCEPTED MANUSCRIPT
overlapped peptides. Numbers on the right indicate the number of amino acid residues in that
peptide. Numbers on the right in the parenthesis are experimental molecular weights and scores.
Consensus obtained from Jalview 2.8 and 2.8.2: (.), residues with weakly similar properties and
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conserved; (:), residues with strongly similar properties and conserved; (*), residue which is fully
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conserved.
Fig. 4. Proportion (%) of lipolysis of neutral lipids () of whole buffalo milk and subsequent
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formation of free fatty acids () during digestion with human gastric and duodenal juices.
Abbreviations: G40, gastric digestion at pH 2.5 for 20 min after 20 min gastric digestion at pH
AC
C
EP
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D
5.0; D30, D60 and D120, duodenal digestion for 30, 60 and 120 min, respectively, at pH 7.0.
ACCEPTED MANUSCRIPT
Figure 1
RI
PT
A)
kDa
SA
97.0
-CN
45.0
30.0
-CN
20.1
-Lg
-LA
b
c
d
1
2
14.4
G20
G40
D05
D30
D60
D120
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D
STD
B)
kDa
97.0
EP
SA
M
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S-CN
SC
66.0
66.0
45.0
b
c
d
AC
C
S-CN
-CN
30.0
-CN
-Lg
-LA
20.1
14.4
1
2
STD
G20
G40
D05
D30
D60 D120
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Figure 2
S1-Casein (Accession number: O62823)

1
1
1
1
MKLLILTCLVAVALARPKQPIKHQGLPQGVLNENLLRFFVAPFPEVFGKEKVNELSTDIG
G20
G40
D05
D120
61
61
61
61
G20
G40
D05
D120
121
121
121
121
VPQLEIVPNLAEEQLHSMKEGIHAQQKEPMIGVNQELAYFYPQLFRQFYQLDAYPSGAWY
G20
G40
D05
D120
181
181
181
181
YVPLGTQYPDAPSFSDIPNPIGSENSGKTTMPLW
M
AN
U
SC
SESTEDQAMEDIKQMEAESISSSEEIVPISVEQKHIQKEDVPSERYLGYLEQLLRLKKYN
S2-Casein (Accession number: Q3Y443)
MKFFIFTCLLAVALAKHTMEHVSSSEESIISQETYKQEKNMAIHPSKENLCSTFCKEVIR
G20
G40
D05
D120
61
61
61
61
G20
G40
D05
D120
121
121
121
121
LNPWDQVKRNAVPITPTLNREQLSTSEENSKKTVDMESTEVITKKTKLTEEDKNRLNFLK
181
181
181
181
KISQHYQKFTWPQYLKTVYQYQKAMKPWTQPKTKVIPYVRYL
TE
D
1
1
1
1
EP
NANEEEYSIGSSSEESAEVATEEVKITVDDKHYQKALNEINQFYQKFPQYLQYLYQGPIV
AC
C
120
120
120
120
180
180
180
180
214
214
214
214
G20
G40
D05
D120
G20
G40
G05
D120
60
60
60
60
RI
PT
G20
G40
D05
D120
222
222
222
222
60
60
60
60
120
120
120
120
180
180
180
180
ACCEPTED MANUSCRIPT
-Casein (Accession number: Q9TSI0)

1
1
1
1
MKVLILACLVALALARELEELNVPGEIVESLSSSEESITHINKKIEKFQSEEQQQMEDEL
G20
G40
D05
D120
61
61
61
61
QDKIHPFAQTQSLVYPFPGPIPKSLPQNIPPLTQTPVVVPPFLQPEIMGVSKVKEAMAPK
120
120
120
120
G20
G40
D05
D120
121
121
121
121
HKEMPFPKYPVEPFTESQSLTLTDVENLHLPLPLLQSWMHQPPQPLPPTVMFPPQSVLSL
180
180
180
180
G20
G40
D05
D120
181
181
181
181
SQSKVLPVPQKAVPYPQRDMPIQAFLLYQEPVLGPVRGPFPIIV
M
AN
U
SC
RI
PT
G20
G40
D05
D120
60
60
60
60
224
224
224
224
-Casein (Accession number: A8KRP5)

1
1
1
1
MMKSFFLVVTILALTLPFLGAQEQNQEQPIRCEKEERFFNDKIAKYIPIQYVLSRYPSYG
G20
G40
D05
D120
61
61
61
61
G20
G40
D05
D120
121
121
121
121
HPHLSFMAIPPKKNQDKTEIPTINTIVSVEPTSTPITEAIENTVATLEASSEVIESVPET
G20
G40
D05
D120
181
181
181
181
NTAQVTSTVV
NTAQVTSTVV
NTAQVTSTVV
NTAQVTSTVV
TE
D
G20
G40
D05
D120
AC
C
EP
LNYYQQKPVALINNQFLPYPYYAKPAAVRSPAQILQWQVLPNTVPAKSCQAQPTTMTRHP
190
190
190
190
60
60
60
60
120
120
120
120
180
180
180
180
ACCEPTED MANUSCRIPT
-Lactoglobulin (Accession number: C3W955)
G20
G40
D05
D120
61
61
61
61
G20
G40
D05
D120
121
121
121
121
RI
PT
MKCLLLALGLALACGAQAIIVTQTMKGLDIQKVAGTWYSLAMAASDISLLDAQSAPLRVY
VEELKPTPEGDLEILLQKWENGECAQKKIIAEKTKIPAVFKIDALNENKVLVLDTDYKKY
LLFCMENSAEPEQSLACQCLVRTPEVDDEALEKFDKALKALPMHIRLSFNPTQLEEQCHV
SC
1
1
1
1
AC
C
EP
TE
D
M
AN
U
G20
G40
D05
D120
60
60
60
60
120
120
120
120
180
180
180
180
ACCEPTED MANUSCRIPT
Figure 3
A) Peptides from S1-casein (gastric phase)
SC
TE
D
B) Peptides from S1-casein (duodenal phase)
EP
----SDIPNPIGSENSGK---------SDIPNPIGSENSGK-----------------EIVPNLAEEQLH
------------EIVPNLAEEQLH
--------------YLGYLEQLL-HIQKED-VPSE-----------EGIHAQQKEPMIGVNQEL-----EGIHAQQKEPMIGVNQEL-----QPIK-HQGLPQGVLNENL-----QPIK-HQGLPQGVLNENL-----------YYVPLGTQYPDAPL---------YFYPQ------------Y-EPIHAQQKEIPPGVLEENLGK
AC
C
BWD-03
BSD-01
BWD-07
BSD-04
BWD-01
BWD-06
BWD-04
BSD-05
BWD-02
BSD-03
BWD-05
BSD-02
Consensus
20
20
18
18
27
27
16
16
11
15
7
7
17
17
11
17
15
15
15
15
8
(2265.1063;
(2265.0667;
(2036.0692;
(2036.0191;
(3073.5257;
(3073.3849;
(1865.9167;
(1865.9569;
(1370.7070;
(1854.1204;
(1000.4728;
(1000.4876;
(1746.7914;
(1746.7280;
(1362.6406;
(1843.9624;
(1742.8690;
(1742.8242;
(1716.7032;
(1716.7530;
( 865.2948;
RI
PT
----------------------------RPKQPI-KHQGLPQGVLNENL
----------------------------RPKQPI-KHQGLPQGVLNENL
-------------FVAPFPEVFGKEKVNELS------------------------------FVAPFPEVFGKEKVNELS---------------------------------------AEEQLHSMKEGIHAQQKEPMIGVNQEL
----------------------AEEQLHSMKEGIHAQQKEPMIGVNQEL
---------------EIVP-NLAEEQLHSMKE-------------------------------EIVP-NLAEEQLHSMKE-------------------------------------------LRLKKYNV-----PQL----------------------------LEQLLRLKKYNV-----PQL----------------------------FRQFYQL-----------------------------------------FRQFYQL---------------------------------SDIPNPIGSENSGKTTM-------------------------------SDIPNPIGSENSGKTTM----------------------------IQKEDVPSERY----------------------------PLGTQYPDAPLFSDIPN----------------------------YYVPLGTQYPDAPLF---------------------------------YYVPLGTQYPDAPLF---------------------------------YYVPLGTQYPDAPSF---------------------------------YYVPLGTQYPDAPSF---------------------------------------DAYPSGAW----------------------------------YYVPLGTQYPDAPLFEDIPNNFAEEQLHRLKEGIHAHQGEPQGGLNEEL
M
AN
U
BWG-05
BSG-01
BWG-02
BSG-02
BWG-08
BSG-05
BWG-09
BSG-06
BWG-06
BSG-03
BWG-07
BSG-04
BWG-01
BSG-07
BSG-08
BSG-09
BWG-03
BSG-11
BWG-04
BSG-10
BSG-12
Consensus
14
14
12
12
9
10
18
18
17
17
14
5
(1413.6538;
(1413.6358;
(1390.5764;
(1390.6456;
(1110.5436;
(1180.5698;
(2019.9276;
(2019.9830;
(1883.9524;
(1883.9592;
(1595.6493;
( 716.2650;
79)
83)
30)
35)
32)
28)
47)
39)
31)
23)
17)
19)
90)
90)
30)
40)
66)
88)
90)
65)
58)
28)
24)
25)
66)
88)
34)
13)
24)
17)
24)
24)
27)
ACCEPTED MANUSCRIPT
Figure 3 continued
Consensus
VYQYQKAMKPWTQPKTNVIPYVRYL 25 (3113.7213; 36)

LYQGPIVLNPWDQVKRNAVPITPTL 25 (2831.6224; 46)
LYQGPIVLNPWDQVKRNAVPITPTL 25 (2831.5792; 63)
:**
.::** * * *.:* . *
LYQGPIVLNPWDQVKRNAVPITPTL
D) Peptides from S2-casein (duodenal phase)

14
17
10
14
9
AC
C
EP
TE
D
Consensus:
------ALNEINQFYQKFPQ
LYQGPIVLNPWDQVKRN---------LTEEDKNRLN-------TKLTEEDKNRLNFL-------ITVDDKHYQ---:. ::
-------LTEEDKNRLNF-
(1738.8258;
(2039.0798;
(1230.5986;
(1719.8695;
(1117.4660;
52)
44)
41)
56)
42)
M
AN
U
BWD-02
BWD-03
BWD-01
BSD-02
BSD-01
SC
BSG-01
BSG-02
BWG-01
RI
PT
C) Peptides from S2-casein (gastric phase)
ACCEPTED MANUSCRIPT
Figure 3 continued
E) Peptides from -casein (gastric phase)

30
34
26
40
16
18
19
16
16
20
20
20
18
13
13
26
28
20
20
36
23
13
(3830.0265;
(3829.9185;
(2901.5692;
(4557.2921;
(1873.9822;
(2015.1388;
(2201.1678;
(1872.9044;
(1872.9274;
(2240.1566;
(2240.0686;
(2253.3044;
(1993.0804;
(1511.7666;
(1511.7850;
(2876.4364;
(3136.7745;
(2192.2285;
(2192.1733;
(3949.1213;
(2502.3204;
(1441.7760;
SC
RI
PT
-------GVSKVKE--AMAPKHKEMPFPK--YPVEPFTESQ--------------------------GVSKVKE--AMAPKHKEMPFPK--YPVEPFTESQSLTL-----------------------------------------------PVEPFTESQSLTLTDVENLHLPLPLL------------------AMAPKHKEMPFPK--YPVEPFTESQSLTLTDVENLHLPLPLL-----------------------------------------------TDVENLHLPLPLLQSW
------------------------------------------LTLTDVENLHLPLPLLQS------------------------------------------LTLTDVENLHLPLPLLQSW
-----QSWMHQPPQ-----------PLPPTVM---------------------------------QSWMHQPPQ-----------PLPPTVM------------------------------------MHQPPQ-----------PLPPTVMFPPQ----SVL-------------------------MHQPPQ-----------PLPPTVMFPPQ----SVL-----------------FLLYQEPVLGPVRGPFPIIV------------------------------------------LYQEPVLGPVRGPFPIIV-----------------------------------------------------------LQDKIHPFAQTQS-----------------------------------------------LQDKIHPFAQTQS------------------------------SLSQSKVLPVP--------QKAVPYPQRDMPIQA--------------------------SLSQSKVLPVP--------QKAVPYPQRDMPIQAFL----------------------------------------------------PVVVPPFLQPEIMGVSKVKE----------------------------------------PVVVPPFLQPEIMGVSKVKE-----------------LVYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPEIM------------------------LVYPFPGPIPNSLPQNIPPLTQT-----------------------------------QSLVYPFPGPIPK--------------------------------------------LLQQSLVHPVPGPIPMALKQKIMPLPQTVMPVPPFLESQILTLTDVENLHLPLPLLQSW
M
AN
U
BWG-08
BSG-01
BWG-04
BSG-02
BWG-06
BWG-05
BSG-03
BWG-02
BSG-06
BWG-03
BSG-05
BWG-07
BSG-04
BWG-09
BSG-08
BWG-01
BSG-09
BWG-10
BSG-07
BWG-11
BSG-11
BSG-10
Consensus:
EP
--RELEELNVPGEIVE---------------------------SWM----HQPPQPLPPTV--------------------------WM----HQPPQPLPPTVM--FPPQS-------------V----WM----HQPPQPLPPTVM--FPPQS-------------V-----M----HQPPQPLPPTVM--FPPQS-------------VL----M----HQPPQPLPPTVM--FPPQS-------------VLS-SLTLTDVENLHLPLPL---------------------------SLTLTDVENLHLPLPL----------------------------LTLTDVENLHLPLPLL---------------------------LTLTDVENLHLPLPLL--------------------------AQTQSLVYPFPGPIPK---------------------------AQTQSLVYPFPGPIPK--------------------------------LVYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPEIM-------LVYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPEIM------------------------------PVVVPPFLQPEIMG-----------------SLPQNIPPLTQTPVVVPPFLQPEIMGVS
LYQEPVLGPVRGPFPIIV-------------------------LYQEPVLGPVRGPFPIIV--------------------------QMEDELQDKIHPF------------------------------QMEDELQDKIHPF-------------------------------------HKEMPFPK-----------------------------------HKEMPFPK---------------------------SLMETDVHPPPGPLPPTVMQNFPPQSQTPVVVPPFLQPEIMG-
AC
C
BWD-01
BWD-03
BWD-04
BSD-04
BWD-05
BSD-05
BWD-06
BSD-01
BWD-07
BSD-02
BWD-10
BSD-06
BWD-11
BSD-08
BWD-12
BSD-07
BWD-08
BSD-09
BWD-02
BSD-03
BWD-09
BSD-10
Consensus
TE
D
F) Peptides from -casein (duodenal phase)
14
14
20
20
20
21
16
16
16
16
16
16
36
36
14
28
18
18
13
13
8
8
(1624.7688;
(1613.7868;
(2313.1102;
(2313.0692;
(2239.9734;
(2327.1224;
(1773.9524;
(1773.8804;
(1800.0126;
(1800.0397;
(1741.8370;
(1741.8296;
(3949.1341;
(3949.2249;
(1521.8566;
(2997.6566;
(1993.0918;
(1993.0100;
(1628.6728;
(1628.7144;
(1012.5234;
(1012.5164;
52)
16)
16)
23)
40)
20)
53)
53)
65)
62)
84)
84)
26)
28)
52)
10)
37)
30)
71)
36)
36)
28)
21)
21)
91)
44)
24)
50)
22)
25)
19)
22)
52)
11)
31)
85)
84)
98)
16)
42)
50)
23)
09)
94)
ACCEPTED MANUSCRIPT
Figure 3 continued
G) Peptides from -casein (gastric phase)

19
19
12
9
25
26
13
15
10
10
9
9
11
12
SC
H) Peptides from -lactoglobulin (duodenal phase)
AC
C
EP
Consensus:
----KIDALNEN--KVL---------KIDALNEN--KVLV----VYVEELKPTPEGDLEILLQ---VYVEELKPTPEGDLEILLQ-----------TPEVDDEALEKFDK--------TPEVDDEALEKFDKA
: : *
VYVEEIDATPEGDDEALEKFDK-
TE
D
BWD-01
BSD-01
BWD-02
BSD-03
BWD-03
BSD-02
(2138.1514;
(2138.0440;
(1392.6686;
(1108.5288;
(2861.4964;
(2974.6690;
(1611.8368;
(1824.0558;
(1227.6418;
(1227.5218;
(1146.5758;
(1146.5458;
(1267.6392;
(1430.7524;
28)
35)
14)
22)
57)
66)
13)
25)
39)
52)
43)
44)
35)
19)
RI
PT
--------------------MAIPPKKNQDKTEIPTINT
--------------------MAIPPKKNQDKTEIPTINT
----------------GLNYYQQKPVAL-----------------------------YYQQKPVAL--------------------INNQFLPYPYYA-KPAAVRSPAQILQ-----------LINNQFLPYPYYA-KPAAVRSPAQILQ--FNDKIAKYIPIQY-------------------------FNDKIAKYIPIQYVL---------------------------------TRHPHPHLSF----------------------------TRHPHPHLSF-------------------------YVLSRYPSY-----------------------------YVLSRYPSY------------------------------VLSRYPSYGLN---------------------------VLSRYPSYGLNY------------------FNDKIAKYVLIRYPSYGLNYYAIKPAALQDKAEILQINT
M
AN
U
BWG-01
BSG-02
BWG-06
BSG-07
BWG-07
BSG-06
BWG-02
BSG-01
BWG-03
BSG-03
BWG-04
BSG-05
BWG-05
BSG-04
Consensus:
11
12
19
19
14
15
(1255.7322;
(1354.7070;
(2184.0770;
(2184.1794;
(1634.7518;
(1705.7671;
34)
20)
38)
45)
75)
31)
ACCEPTED MANUSCRIPT
Figure 4
RI
PT
100%100
90%
80% 80
SC
70%
Lipids (%)
60% 60
M
AN
U
50%
40% 40
30%
20% 20
0%
G40
AC
C
EP
Undigested
TE
D
10%
D30
D60
D120

Ex Vivo Digestion of Proteins and Fat in Buffalo Milk

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Ex Vivo Digestion of Proteins and Fat in Buffalo Milk

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Accepted Manuscript

Ex vivo digestion of proteins and fat in buffalo milk

International Dairy Journal

Received Date: 18 February 2014

Accepted Date: 17 August 2015

Ex vivo digestion of proteins and fat in buffalo milk

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

Sciences, P. O. Box 5003, NO-1432 Aas, Norway

*Corresponding author. Tel.: +47 6496 5900

Email address: mohammad.islam0@nmbu.no (M. A. Islam)

Mymensingh-2202, Bangladesh. E-mail address: m.a.islam@bau.edu.bd

Permanent address: Department of Dairy Science, Bangladesh Agricultural University,

lactoglobulin seemed to be resistant during 40 min of gastric digestion. However, by 5 min of

bond(s), were observed.

Gastrointestinal (GI) digestion of dietary lipids is influenced by the physicochemical

and stage of lactation (Abd El-Salam & El-Shibiny, 2011).

70%) or unsaturated (US, 30%) FAs. Considerable variations in the FA composition of

the dietary lipids, including digestion, absorption, and subsequent metabolism.

A complex combination of mechanical, physiochemical and physiological processes are

Materials and methods

Germany), respectively, as previously reported by Islam et al. (2014).

Human gastric and duodenal juices

keeping them overnight on ice, in a cold room at 4-6 C.

proteolytic activity per g milk protein), respectively.

mM bile acids as well.

were homogeneous clear solutions with a yellow colour.

Protein and peptide analysis

2.4.1. Milk protein degradation profiles by SDS-PAGE

2.4.2. In-gel digestion of protein bands and identification by ultra-performance liquid

chromatography and Q-exactive mass spectrometry

liquid chromatography (UPLC; RSLC3000, Dionex/Thermo Fisher Scientific, Bremen,

5 mm, Thermo Fisher Scientific), back flushed onto a 50 cm 75 m analytical column

The Masconvert module of ProteoWizard (http://proteowizard.sourceforge.net/) was used to

oxidised methionine, respectively.

2.4.3. Peptide identification by UPLC and quadrapole-time of flight mass spectrometry

employed NCBInr 20130131 (22749596 sequences; 7819872540 residues).

2.4.4. Multiple sequence alignment of peptides

http://www.ebi.ac.uk/Tools/msa/clustalo/), and for consensus sequence, MS editor Jalview 2.8

chloroform:methanol (2:1, added immediately after digestion) and internal standards in

gas at 37 C. The NL fraction was re-dissolved by adding 2 mL hexane and methylated by

NL and FFA FAMEs were stored at -20 C until analysed by GC-MS.

An Autospec Ultima MS (Micromass Ltd. Manchester, UK) equiped with an electron

(Varian, Middelburgh, The Netherlands).

Results and discussion

(DAmbrosio et al., 2008) reflecting homology between them.

Site of proteolysis and peptides

duodenal digestion (identified by UPLC/Q-TOF MS analysis) showed very little variation

neighbouring hydrophobic residue(s), i.e., leucine, isoleucine, valine, phenylalanine, alanine, or

lipids and FFA was considered as the only lipolytic product.

measurement of C4:0-C8:0 is challenging because of the volatility, and concentration of free

milk may be of nutritional importance for consumers.

Professor Dr. Mohammad Al-Mamun, Department of Animal Nutrition, Bangladesh Agricultural

University, for checking the language of the manuscript.

International Dairy Journal, 16, 961-968.

juice. British Journal of Nutrition, 106, 896-905.

Hydrocolloids, 29, 123-134.

defense against pathogens. Proteomics, 8, 3657-3666.

bovine milk. International Dairy Journal, 36, 6-13.

ovine -lactoglobulin to pepsinolysis. International Dairy Journal, 15, 17-27.

(2013). Identification of lactoferrin peptides generated by digestion with human

gastrointestinal enzymes. Journal of Dairy Science, 96, 75-88.

protein digestion by conjugated bile acids. Gastroenterology, 133, 16-23.

Technology, 90, 549-563.

Chittagong Cattle from Bangladesh. Asian-Australasian Journal of Animal Sciences, 27,