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Article

Influence of Casein and Milk Phospholipid Emulsifiers on the Digestion and Self-Assembled Structures of Milk Lipids

by
Malinda Salim
1,
Alastair K. H. MacGibbon
2,
Cameron J. Nowell
3,
Andrew J. Clulow
1,4 and
Ben J. Boyd
1,5,*
1
Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville, VIC 3052, Australia
2
Fonterra Research Centre, Private Bag 11 029, Palmerston North 4442, New Zealand
3
Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville, VIC 3052, Australia
4
Australian Synchrotron, ANSTO, 800 Blackburn Road, Clayton, VIC 3168, Australia
5
Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark
*
Author to whom correspondence should be addressed.
Colloids Interfaces 2023, 7(3), 56; https://doi.org/10.3390/colloids7030056
Submission received: 23 May 2023 / Revised: 8 August 2023 / Accepted: 16 August 2023 / Published: 28 August 2023
Browse Figures
Versions Notes

Abstract

:
Interfacial compositions of fat globules modulate the digestion behaviour of milk triglycerides in the gastrointestinal tract, thereby affecting lipid metabolism and delivery of nutrients. In this study, we aim to understand the impact of emulsifiers on lipid digestibility and the self-assembled liquid crystal structures formed by anhydrous milk fat (AMF) during digestion. AMF was emulsified with casein and milk phospholipids, and digestion was performed in both gastric and small intestinal conditions to account for changes at the oil/water interface following enzymatic digestion in the gastric phase. Small angle X-ray scattering was used to characterise the self-assembled structures of the digestion products, while coherent anti-Stokes Raman scattering microscopy was utilised to probe changes in lipid distribution at the single droplet level during digestion. Our findings confirmed that emulsifiers play a key role in the digestion of AMF. Milk phospholipids exhibited a protective effect on milk triglycerides against pancreatic lipase digestion by slowing digestion, but this effect was slightly negated in emulsions pre-digested under gastric conditions. The overall types of liquid crystal structures formed after digestion of casein- and milk phospholipids-emulsified AMF were comparable to commercial bovine milk irrespective of gastric pre-treatment. However, emulsification of AMF with milk phospholipids resulted in changes in the microstructures of the liquid crystal phases, suggesting potential interactions between the digested products of the fat globules and milk phospholipids. This study highlights the importance of emulsifiers in regulating lipid digestion behaviour and lipid self-assembly during digestion.

1. Introduction

Milk or nutritional substitutes provide the sole source of nutrients that play critical roles in the growth and development of children in the early stages of life. Between 1993 and 2013, less than about 40% of infants under 6 months of age worldwide were exclusively breastfed [1,2], and although the World Health Organization (WHO) and a number of countries have imposed strategies and established platforms to improve breastfeeding practices [3,4], infant formulas remained the only supplement for mothers who are not able to breastfeed due to medical, work, and lifestyle issues. Designing infant formulas that can deliver adequate nutrients to children is therefore critical, and continuous efforts to match the compositions of infant formulas to human milk have been made in the past decades. This has included efforts to synthesise structured lipids [5,6] and, more recently, supplementation of infant formula with milk fat globule membrane (MFGM; an enveloping membrane material from natural milk globules) [7,8,9], prebiotics [10,11], and probiotics [12,13]. However, complete replication is challenging given the inherent complexity of human milk and an incomplete understanding of the physiology of the gastrointestinal tract in children as well as the implications of milk compositions on the bioavailability of nutrients delivered orally.
Our group has recently shown that not all infant formulas behave similarly to human milk in the context of digestion and lipid self-assembly under small intestinal conditions [14,15]. Differences in the formation of liquid crystalline structures were mainly attributable to variations in the lipid compositions of the formulas, which could potentially affect the delivery of nutrients since digestion of lipids is known to play a critical role in determining the solubilisation and bioavailability of poorly water-soluble compounds, such as small molecule drugs and poorly soluble vitamins, when delivered orally [16,17,18,19,20,21]. While various structures were formed from the self-assembly of lipid digestion products in infant formulas [14,15], digestion of human milk in the small intestinal condition produced a mixture of calcium soaps with a discontinuous micellar cubic phase that had an Fd3m space group consisting of inverse micelles arranged in a cubic lattice [22]. Meanwhile, the digestion products of bovine milk self-assemble into an inverse bicontinuous cubic phase (lipid bilayers separated by intertwining and interconnected water network) with Im3m space group and an inverse hexagonal structure H2 (cylindrical inverse micelles ordered in a hexagonal lattice) [23,24]. Understanding the factors that influence digestion behaviour and colloidal structure formation in milk and infant formulas would therefore enable rational design of both vegetable oil-based and bovine milk fat-based infant formulas that replicate the lipid self-assembly of human milk. While recent efforts are making in-roads into understanding the impact of the core triglyceride composition on structure formation, the effect of interfacial lipids, emulsifiers, and proteins has not been addressed to date.
In this study, we investigate the impact of milk-based emulsifiers on the digestibility and self-assembled liquid crystal structures of lipid emulsions using anhydrous bovine milk fat (AMF) as the nominal triglyceride core. It is worth noting that, whilst in vitro digestion of lipids and the impact of emulsifiers on digestibility of the lipid droplets in gastric and small intestinal conditions have been widely investigated [25,26,27], studies relating the interfacial composition to liquid crystalline structure formation mentioned in the previous paragraph are absent, even though such findings are important when considering a selection of emulsifiers during the development of milk lipid-based formulations. Casein and milk phospholipids are used as the emulsifiers in this study because casein is a milk serum protein [28] and milk phospholipids are important components in MFGM [29,30,31]. The complex mixture of components in MFGM has been shown to pose various health benefits, including modulating gut microbiome and enhancing the intestinal epithelial barrier function [32,33], and has thus been increasingly supplemented in infant formulas and other functional foods [34]. Small angle X-ray scattering (SAXS) was the technique used to probe the evolution of liquid crystal structures during digestion. Effects of gastric digestion on the structure formation by the digesting lipids under small intestinal conditions were also studied to understand potential changes that occurred at the oil/water interface following gastric pre-treatment. Schematic representation of the concepts of this study are summarised in Figure 1.

2. Materials and Methods

2.1. Materials

Pasteurised anhydrous milk fat and milk phospholipids (PC700) [35] were provided by Fonterra Co-operative Group Ltd. (Palmerston North, New Zealand). The PC700 contained about 84 wt% total fat, 24 wt% neutral lipid, 60 wt% total phospholipids (of which phosphatidylcholine = 19.1 wt%, phosphatidylethanolamine = 17.0 wt%, sphingomyelin = 16.6 wt%, phosphatidylserine = 2.4 wt%, phosphatidylinositol = 2.0 wt%), 6.2 wt% lactose, 7.4 wt% ash, and 2 wt% moisture. Casein from bovine milk (technical grade), Trizma maleate reagent grade, pepsin from porcine gastric mucosa (3200–4500 U/mg), and sodium azide (≥99%, Fluka) were purchased from Sigma Aldrich (Saint Louis, MO, USA). Fungal gastric lipase (activity: 80,000 TBU/g powder) was a gift from Connell Bros (Croydon South, VIC, Australia). Pancreatic lipase (USP grade pancreatin extract) was purchased from Southern Biologicals (Alphington, VIC, Australia). Sodium chloride (>99%) was purchased from Chem Supply (Gillman, SA, Australia). Calcium chloride dihydrate (>99%) and sodium hydroxide pellets (min. 97%) were purchased from Ajax Finechem (Seven Hills, NSW, Australia). Hydrochloric acid (36%) was purchased from LabServ (Longford, Ireland). Bacterial microfibrous cellulose (0.14 w/v% in water) was a gift from Associate Professor Patrick Spicer (UNSW, Sydney, NSW, Australia) [36]. Water was distilled and deionised from a Milli-Q Millipore purification system (Merck Millipore, Bayswater, VIC, Australia). Unless otherwise stated, all chemicals were used without further purification.

2.2. Methods

2.2.1. Preparation of Milk Fat Emulsions

Emulsions containing 3.8 w/v% anhydrous milk fat (AMF) were prepared in tris buffer (50 mM Trizma maleate, 5 mM calcium chloride dihydrate, 150 mM sodium chloride and 6 mM sodium azide; pH 6.5) with 2 w/v% casein or milk phospholipids as the emulsifier. A concentration of 2 w/v% emulsifier was selected to provide sufficient surface coverage of the fat globules for particle stabilisation [37], while a 3.8 w/v% fat was chosen on the basis of fat content in milk [29]. Briefly, AMF (0.76 g) was added to 20 mL of casein solution in tris buffer (2 w/v%) or 20 mL of tris buffer containing 0.40 g dispersed PC700 powder. Samples were incubated for about 5–10 min in a 60 °C oven until the AMF was molten, followed by sonication at 25 amplitude, 2 s on/off for 3 min (Misonix S-4000-040, 3 mm diameter probe tip) whilst still hot. Emulsions of AMF containing mixed emulsifiers of casein and PC700 with a 1:1 weight ratio (while maintaining total emulsifier concentrations of 2 w/v%) were also prepared using the same processing conditions.

2.2.2. In Vitro Digestion of Milk Fat Emulsions

Casein and PC700-emulsified fat globules were prepared based on methods described in Section 2.2.1. Digestion of the milk fat emulsions by pancreatic lipase was performed as follows: 18 mL of emulsion was transferred to a thermostatted glass digestion vessel (37 °C), and the pH of the sample was adjusted to 6.500 ± 0.003 prior to the injection of 2 mL reconstituted freeze-dried pancreatic lipase [38] dispersed in tris buffer to initiate digestion. The lipolytic activity of the pancreatic lipase was ~700 TBU, measured by digesting tributyrin (5.8 mL) in tris buffer at pH 7.5 (18 mL) with the reconstituted freeze-dried pancreatic lipase (2 mL) at 0.217 g/mL. NaOH (1.0 M) was used to maintain the pH of the sample at 6.5 during digestion by the automatic dosing unit of the pH-stat apparatus (902 STAT titration system, Metrohm AG, Herisau, Switzerland). In some cases, the milk fat emulsions were also exposed to gastric conditions prior to intestinal digestion (Figure 2). Herein, 18 mL of the milk fat emulsions were transferred to a digestion vessel maintained at 37 °C, and the pH of the sample was adjusted to 3.0 using 5 M HCl. Pepsin (~2000–2810 U/mL) and/or fungal gastric lipase (~60 TBU measured at pH 5.5) [39] powders were added, and the sample was allowed to digest at 37 °C for 1 h under constant magnetic stirring. After gastric digestion, the pH of the sample was re-adjusted to 6.5 using 5 M NaOH prior to the injection of pancreatic lipase.
Digestibility of the milk fat emulsions, presented as the extent of digestion, was calculated based on the amount of total fatty acids released in forward titration (digestion at pH 6.5) and back-titration (pH increased to 9.0 after 1 h intestinal digestion at pH 6.5 to ionise all fatty acids) to account for both ionised and non-ionised fatty acids released during digestion, respectively. The total amount of fatty acids released at individual digestion time points (in mmol) were determined by multiplying the titrated fatty acids (or ionised fatty acids) in forward titration with the correction factor [40] calculated from Equation (1):
Correction factor = Ionised fatty acids released/Total fatty acids released
where total fatty acids were the summation of ionised and non-ionised fatty acids. The extent of digestion (%) was subsequently calculated using Equation (2):
Extent of digestion (%) = (Total fatty acids released/Theoretical fatty acids released) × 100
Theoretical number of fatty acids was determined based on the assumption that 1 mol of triglycerides released 2 mol of fatty acids. The average molecular weight of milk triglycerides was taken to be 737 g/mol [41].

2.2.3. Self-Assembled Structures Formed during Digestion of Fat Globules: Synchrotron SAXS Measurements

Samples from the digestion vessel (18 mL of the milk fat emulsions, either freshly prepared or treated with pepsin and/or fungal gastric lipase) were aspirated to a fixed quartz capillary mounted in the X-ray beam (wavelength/photon energy = 1.033 Å/12.0 keV; or 0.954 Å/13.0 keV) using a peristaltic pump operating at ~10 mL/min. Pancreatic lipase (2 mL) was remotely injected into the vessel to initiate digestion. Two-dimensional SAXS images were recorded using a Pilatus 2M detector with a 5 s acquisition time and 15 s delay between measurements. Sample-to-detector distances were 1397 mm (12 keV) or 1615 mm (13 keV) to cover a q range of 0.01< q < 1.12–1.22 where q = (4π⁄(λ))sinθ: q = scattering vector, 2θ = scattering angle, and λ = X-ray wavelength. The raw data were reduced to scattering functions I(q) vs. q using the in-house developed software package Scatterbrain version 2.71.
The identities of the liquid crystal structures that formed during digestion were determined by the relative positions of the diffraction peaks observed [42,43]. For a lamellar phase, the relative peak positions are 1, 2, 3, etc. The relative peak positions for a bicontinuous cubic phase with Im3m space group are √2, √4, √6, √8, √10. For the H2 phase, the relative peak positions are 1, √3, √4, √7, √9. Lattice parameters for lamellar phases, i.e., the distance between the lipid bilayers, are (2π/q). For Im3m, the lattice parameters (distance between the cubic cell edge in a cubic phase containing a lipid bilayer arranged as networks of connected rods cubically joined 6 by 6) are {[√(h2 + k2 + l2)]2π/q}, where h, k, l are the Miller indices (110, 200, 211, 220, 310, 222, etc.) and 2π/q is the repeat distance of the lattice. For H2, the lattice parameters (distance between centres of adjacent, hexagonally packed cylindrical water channels) are [4π/(√3 q)].

2.2.4. Particle Size Distributions of Fat Globules: Laser Light Scattering

Particle size distributions of the emulsified fat globules were determined using a laser light scattering apparatus (Mastersizer 2000, Malvern Panalytical, Malvern, UK) equipped with a He-Ne laser (λ = 633 nm; red light) and a light-emitting diode light source (λ = 466 nm; blue light). Samples were briefly vortexed and added to circulating water in the liquid dispersion unit to achieve obscuration between 5 and 10% for the red light. The volume size distributions were generated by the onboard analysis software, and, where referred to, particle sizes will be given as the volume-weighted mean diameter (D4,3). The refractive indices of the particles and dispersant were 1.46 and 1.33, respectively, and the absorbance of the fat droplets was taken to be 0.001 for analysis.

2.2.5. Imaging of Fat Globules: CARS Microscopy

Coherent anti-Stokes Raman scattering (CARS) microscopy was used as a label-free technique to image individual casein- and PC700- emulsified fat globules during digestion to investigate changes relating to the morphology of the fat globules and distributions of the digestion products by the action of pancreatic lipase. CARS microscopy was performed on a Leica SP8 CARS system using a 40x IR PlanApo NA1.2 objective. Excitation was provided by an Ape PicoEmerald S OPO laser with a tunable pump line and a fixed 1032 nm stokes beam. Emission was captured to an external non-descanned detector through a 670/125 band pass filter. About 12 µL of lipid emulsions (prepared as in Section 2.2.1 but with 10 s sonication time to obtain large-sized globules) and microfibrous cellulose mixtures (1:1 volume ratio, respectively) were loaded onto a microscope glass slide covered with a glass coverslip. The temperature of the slide was maintained at 37 °C, but digestion of the fat globules was not pH controlled. The CARS intensity was measured at 2845 cm−1 to determine the total lipid signals (triglycerides and the digested products) and 2940 cm−1 for the undigested triglycerides since their spectral intensities at this wavenumber were higher compared to the digested products [44].

3. Results

3.1. Digestion and Structure Formation of Casein- and PC700-Emulsified Fat Globules under Small Intestinal Conditions

Emulsions of anhydrous milk fat (AMF) emulsified with either 2 w/v% casein or PC700 exhibited similar size distributions (average D4,3 ~1.5–1.8 µm, which is the range typical in raw milk [45]; Table S1), but their rates of digestion by pancreatic lipase under simulated small intestinal conditions were significantly different (Figure 3). Digestion of milk fat emulsions prepared from casein + AMF was significantly faster compared to PC700 + AMF during the initial course of digestion, with no significant differences observed in the final digestibility of the lipids at 60 min. Particle size distributions of the emulsions during and after digestion are shown in Figure S1. As was expected, rapid increase in particle size was observed for emulsion that underwent faster digestion (casein + AMF), and a comparable size was observed after digestion completion at 60 min. Faster digestion of lipids from casein-emulsified oil/water interfaces was also evident from the small angle X-ray scattering (SAXS) profiles in Figure 4. Diffraction peaks attributed to lamellar fatty acid calcium soaps (q ~ 0.13, 0.26, 0.39 Å−1), the intensity of which typically correlates with the extent of lipid digestion [23], were seen 2 min after injection of pancreatic lipase. Meanwhile, in PC700 + AMF emulsions, calcium soaps were formed between 10 and 15 min after commencing digestion.
The SAXS profiles in Figure 4 also show that, in addition to the lamellar phases associated with calcium soaps, digestion of casein- and PC700-emulsified fat globules resulted in the formation of an inverse hexagonal H2 phase and an inverse bicontinuous cubic phase with an Im3m space group, both of which are characteristic of the lipid self-assembly that occurs in the digestion of bovine milk [23,24]. These structures were formed relatively early during digestion in casein + AMF samples due to the more rapid release of fatty acids and monoglycerides from the milk fat droplets. Comparisons between the liquid crystal structures formed from casein + AMF and PC700 + AMF at the end of digestion (60 min for casein and 80 min for PC700) showed that, although the overall types of structures remained identical, the lattice parameters for the hexagonal phase were slightly different from each other: about 66 Å for casein + AMF (which was similar to commercial homogenised bovine milk, 65 Å [23]) and 71 Å for PC700 + AMF.
Figure 5 shows visualisation of the lipid droplets in PC700-emulsified fat globules during digestion. Images of the time-resolved CARS microscopy for a selected representative globule showed a clear progressive decrease in intensity of the triglycerides (2940 cm−1) with digestion and localisation of the digestion products on the PC700 + AMF fat droplets [44]. Similar behaviour was observed for casein + AMF (Figure S3). A closer look at the fat globules during digestion (Figure 6) also revealed an ingress of water (-OH signal at 3200 cm−1) into the droplet as the triglycerides were digested resulting in a less pronounced oil/water interface. The ingress of water and generation of lipolytic products could therefore result in the formation of localised liquid crystal structures that are constituted of intricate, geometrically arranged water channels. Probing these structures in a digesting droplet in situ is challenging and may require the use of a microfocus SAXS. Interestingly, the plots of intensity of the -OH signal in the horizontal and vertical directions across the fat droplet showed a higher signal accumulation at the boundaries (Figure 6).

3.2. Effects of Gastric pre-Digestion on the Digestion of Casein- and PC700-Emulsified Fat Globules under Small Intestinal Conditions

3.2.1. Individual Emulsifiers

As digestion of lipids in the small intestine requires passage of food through the stomach environment, we aimed to understand how pre-treatment of the casein- and PC700-emulsified fat globules with gastric pepsin and lipase influences the accessibility of the fat droplet interfaces towards pancreatic lipase. Figure 7a showed that pre-treatment of casein + AMF with pepsin (at pH 3.0 for 1 h) did not alter the overall extent of digestion of the emulsion by pancreatic lipase, although a slight increase in the initial rate of lipolysis could be seen. Similarly, digestion of AMF by pancreatic lipase was accelerated after the PC700-emulsified fat globules were exposed to pepsin (Figure 7b). This resulted in the formation of an inverse bicontinuous cubic phase with an Im3m space group at earlier digestion times (between 40 and 50 min, Figure 7e) compared to non-treated fat globules (between 50 and 60 min, Figure 5b). Analysis of the particle size distributions after pepsin pre-treatment and prior to pancreatic lipase injection at pH 6.5 revealed that rapid digestion of the fat globules by pancreatic lipase in PC700 + AMF was not caused by particle size reduction because only a slight increase in D4,3 was observed (Table S1 and Figure S4 in the Supplementary Materials).
Following the addition of fungal gastric lipase to model an in vitro gastric lipolysis, we showed that the overall extent of digestion of casein + AMF under simulated intestinal conditions was lowered by 10%, which was deemed to be due to pre-digestion by FGL that was not accounted for by the pH-stat measurement under intestinal conditions (Figure 7a). Release of fatty acids by fungal gastric lipase (in the presence of pepsin) was evident from the SAXS profile in Figure 7d, where lamellar peak at q = 0.13 Å−1 associated with calcium soaps of liberated fatty acids was seen at 0 min prior to injection of pancreatic lipase. Emulsions that were pre-treated with pepsin and fungal gastric lipase also showed average particle sizes greater than that without the gastric lipase (Table S1 and Figure S4 in the Supplementary Materials) due to the fatty acid-derived calcium soaps [23]. Interestingly, the intrinsic lamellar peak at q = 0.13 Å−1 was not apparent after PC700 + AMF were digested with fungal gastric lipase (Figure 7f), and no significant increase in particle sizes were observed (Table S1). Importantly, pre-treatment of casein and PC700-emulsified fat globules with simulated gastric conditions did not alter the overall types of liquid crystal structures.

3.2.2. Mixed Emulsifiers

The role of mixed casein and milk phospholipids emulsifiers on digestibility and structure formation of milk fat emulsions was subsequently investigated to gain further insights into the underlying emulsification properties of these systems under simulated gastric and small intestinal conditions. Figure 8a shows that injection of pancreatic lipase to mixtures of casein + PC700 + AMF emulsion induced rapid lipolysis of the emulsion, correlating with the SAXS data in Figure 8c, which illustrates the formation of a lamellar phase at q = 0.13 Å−1 less than 2 min after lipase injection and an Im3m cubic phase between 10 and 15 min after commencing digestion. However, contrasting behaviour was observed for the digestion of the casein + PC700 + AMF emulsion that was pre-treated with fungal gastric lipase and pepsin. Figure 8a,b showed that digestion of fat globules with mixed emulsifiers under small intestinal conditions was significantly slower after gastric pre-digestion compared to digestion under intestinal conditions alone, and that larger size distributions were observed. It is worth noting that no pronounced increase in particle sizes were observed in casein-only and PC700-only emulsified fat globules (Table S1). In addition, we found that changes to the PC700 structure were rapid after pancreatic lipolysis (boxed region, Figure 8c) and the characteristic lamellar peak at q = 0.09 Å−1 shifted to a greater degree compared to PC700 + AMF (Figure 4b, 15 min) at the similar extent of AMF digestion (Figure S5). The effects of casein on structural changes of PC700 were further accentuated after gastric pre-treatment of the mixed emulsions (t = 0 min in Figure 8d).
Figure 9 compares the SAXS profiles of PC700 in tris buffer (no AMF) and in milk fat emulsions with and without gastric digestion after pH adjustments to 6.5. Amphiphilic phospholipid components in the PC700 self-assembled into a lamellar/vesicular structure (q = 0.09 and 0.18 Å−1) in tris buffer with lamellar repeat distances of about 69 Å. The lamellar structure was still observed after emulsification with fat globules, but interactions between the PC700 components and the non-polar milk triglycerides resulted in broadening of the lamellar peaks and a slight increase in the q values. The self-assembled structure of PC700 remained similar after incubation of fat globules with fungal gastric lipase and pepsin, provided that no gastric lipolysis occurred, as was the case in the mixed emulsifiers system.

4. Discussion

The role of lipids in providing sufficient energy and facilitating the delivery of essential fatty acids and poorly water-soluble nutrients to infants has been well-documented. Considerable efforts have therefore been made to design milk formulas that more closely resemble human milk by matching their fatty acids compositions. These milk formulas should, ideally, form digestion-driven liquid crystal structures that are characteristic of human milk, and our group has recently identified key fatty acids/monoglycerides pertaining to these liquid crystal structures [14] as well as homotriglyceride mixtures capable of mimicking human milk structuring [38]. However, whilst the role of lipid composition in self-assembly during digestion is becoming clearer, much remains unknown about how the inherent emulsifiers of fat globules in mammalian milk and infant formulas affect the self-assembly of the lipolytic products.
Our studies showed that the types of liquid crystal structures formed during digestion were primarily driven by the self-assembly of lipid digestion products, as both casein-emulsified and milk phospholipids (PC700)-emulsified fat globules self-assembled into the three primary structures characteristic of bovine milk (i.e., lamellar, H2, and Im3m cubic phases) after digestion. The types of emulsifiers (at least in the case of casein and PC700 at 2 w/v% of emulsions containing 3.8% fat) and/or their digestion counterparts could, however, interact and self-assemble with the lipid digestion products, which was evident from differences observed in the lattice parameters of the liquid crystal phases (Table S2). In PC700 + AMF emulsions, digestion of the fat under small intestinal conditions caused a greater degree of swelling of the H2 phase (lattice parameter 71 Å) compared to casein + AMF (lattice parameter 66 Å, which is similar to bovine milk). This may be due to enlargement of the water channels caused by mixing and self-assembly between lipolytic products from the milk fat and constituents in PC700 that contained large, potentially charged phospholipid headgroups. Meanwhile, the hexagonal structures formed in casein + AMF were similar to those reported previously for homogenised and pasteurised bovine milk; and smaller differences were seen between the lattice parameters of the Im3m structure in casein + AMF and bovine milk compared to PC700 + AMF (Table S2). The larger lattice parameter for the bicontinuous cubic Im3m structure in PC700 + AMF compared to milk and casein + AMF signified enlargement of the water channels caused by an increase in the effective headgroup areas [42,46]. Previous studies have shown that fat globules in processed bovine milk are primarily emulsified by casein (and/or other milk serum proteins) after the MFGM is disrupted during homogenisation [47,48]. This is consistent with the finding of this work that the digestion behaviour of processed milk was closer to that of the casein + AMF emulsions, even though MFGM fragments would still be present in the processed milk. However, it is worth noting that the ratio of phospholipids to milk fat in PC700 + AMF emulsions used in this study (about 22 wt% phospholipids in total fat) was significantly higher than that in bovine milk (about 0.5–1.0 wt%) [49], which may explain the absence of an observable lamellar peak that is characteristic of milk phospholipids vesicles at q = 0.09 Å−1 in the latter and the slower extent of milk fat digestion in PC700-rich emulsions. A closer examination of the progression of liquid crystalline structure formation during digestion under small intestinal conditions also revealed slower release of lipolytic products from AMF emulsified with PC700 when compared with the AMF-casein emulsions (Figure 4b). Milk phospholipids could therefore exhibit greater resistance towards hydrolysis by pancreatin (which contains mixtures of lipases, proteases, and amylases) compared to casein, which may contribute to the slower digestion of raw milk compared to processed milk in addition to the effects of larger particle size populations of the former [23,27,50]. The slower hydrolysis of PC700 and the presence of interfacial-rich phospholipids could also serve as a barrier to limit access of pancreatic lipase to the triglyceride core, thereby resulting in a decreased rate of lipid digestion in PC700 + AMF compared to casein + AMF emulsions (Figure 3a). Although there is evidence to suggest that the digestion of phospholipids is largely dependent on their chain lengths and head groups, digestibility of the individual phospholipid components in PC700 was not investigated in this study.
The discussions thus far have focused on the effects of casein and PC700 emulsifiers on structure formation and digestibility of milk fat emulsions under small intestinal conditions. Whilst it has been recognised that the small intestine is the primary site for lipid digestion and absorption, with gastric lipolysis accounting for typically less than 25–30% of overall digestion [25,51], structural changes in fat globules may occur during gastric digestion due to particle aggregation and/or partial hydrolysis of the emulsifiers and lipids [25]. Our findings revealed that milk fat was digested by fungal gastric lipase when emulsified by casein, but that digestion of the fat globules in PC700 + AMF emulsions was not significant. Release of fatty acids in the gastric phase was characterised by the formation of fatty acid-derived calcium soaps (observed in SAXS as a lamellar phase with the first diffraction peak around q = 0.13 Å−1) when the pH of the emulsions was increased to 6.5 after gastric digestion due to the deprotonation of the free fatty acids released. The susceptibility of milk fat emulsions to fungal lipase digestion could be related to casein aggregation at low pH and proteolysis by pepsin in the gastric phase [52], enabling increased access to the oil/water interface (inset in Figure 7a) which is less important for the PC700-stabilised emulsions. Pancreatic lipolysis of the gastric-treated casein-emulsified fat globules subsequently resulted in an apparently lower overall extent of digestion than the emulsions that were only subjected to intestinal digestion because some lipolysis had already occurred in the gastric digestion that was not accounted for in the titration of fatty acids released during intestinal lipolysis (Figure 7a).
In contrast to casein, addition of fungal gastric lipase and pepsin to PC700-emulsified milk fat globules did not seem to result in significant lipolysis of the fat by gastric lipase. The lack of lipid digestion can also be inferred from the absence of a diffraction peak associated with lamellar phase at q = 0.13 Å−1 after pH adjustment from gastric to the small intestinal condition and the similar extents of intestinal lipolysis observed in PC700 + AMF emulsions, whether or not they were subjected to gastric digestion. This suggests higher resistance of milk phospholipids to the gastric enzymes (pepsin + fungal gastric lipase) compared to casein due to the absence of phospholipases [53]. However, exposure of PC700 + AMF emulsions to gastric enzymes may lead to adsorption of the enzyme to the phospholipid interface, thereby altering the surface onto which pancreatic lipase would adsorb. These changes were evident from the significantly faster digestion of milk fat by pancreatic lipase after exposure to pepsin, irrespective of the absence or presence of fungal gastric lipase (Figure 7b) [54]. Interaction between pepsin and phospholipids and the potential adsorption of pepsin to the oil/water interface via surface defects are therefore likely to provide greater accessibility to pancreatic lipase.
When considering gastrointestinal digestion of lipids, one of the key considerations (other than the rate and extent of lipolysis) was to evaluate how pre-digestion of lipids under gastric conditions affected the overall structures that were formed from the self-assembly of lipids and other digestible products. Whilst pre-treatment of PC700 + AMF emulsions with pepsin only and pepsin + fungal gastric lipase did not change the liquid crystal structure formation during the subsequent pancreatic lipolysis (Table S2), subtle differences in the lattice parameters for the cubic phases could be seen for casein + AMF emulsions that were exposed to fungal gastric lipase. This was not unexpected since part of the fatty acids were released by the action of fungal gastric lipase (which formed calcium soaps at intestinal pH), thereby changing the overall distribution of fatty acid species available for pancreatic digestion. However, the progression of liquid crystalline structures from lamellar to hexagonal to bicontinuous cubic phases remained unchanged. This was consistent with previous observations in milk-mimicking emulsions replicating bovine milk, which showed that for typical levels of gastric digestion (≤25% lipid digestion [55,56]) only calcium soaps were present at pH 6.5, with other liquid crystalline phases only observed at greater extents of digestion after more extensive intestinal lipolysis [38]. It should be noted that the range of gastric lipase activity is wide, and further studies are needed to elucidate the impact of higher levels of gastric lipase activity on liquid crystal structures, particularly the use of recombinant human gastric lipase. The use of a higher level of gastric lipase and/or digestion at a higher pH may also reflect more closely the state of lipid digestion in the stomach of infants, since lipid digestion of up to about 40% has been previously reported [57].
Finally, in an attempt to extrapolate the understanding gained from these assembled emulsions to lipid digestion in MFGM-supplemented milk containing endogenous milk serum proteins, AMF emulsions were prepared with mixtures of casein and PC700. The total amount of emulsifiers was kept constant at 2 w/v%, and the ratio of casein:PC700 tested was 1:1 w/w. Herein, we observed that the digestion of casein + PC700 + AMF did not exhibit mixed behaviour between those of the casein + AMF and PC700 + AMF only emulsions; instead, emulsification of fat globules with casein + PC700 + AMF produced a more favourable environment for initial access to pancreatic lipase, where 72 ± 1% of fat was digested after 15 min in the mixed emulsifiers compared to 65 ± 2% in casein + AMF and 25 ± 3% in PC700 + AMF. Interestingly, the response of casein + PC700 + AMF to exposure to fungal gastric lipase and pepsin had the opposite effect on the rate of intestinal digestion to that of PC700 + AMF (Figure 7f). Exposure of casein + PC700 + AMF to fungal gastric lipase and pepsin did not accelerate the release of fatty acids by pancreatic lipase. This antagonistic effect may be caused by the formation of large aggregates due to destabilisation of fat globules following gastric lipolysis (diffraction peak associated with lamellar calcium soaps at q = 0.13 Å−1 was present at t = 0 min) (Figure 8b). In such cases, release of fatty acids in the small intestinal condition might be the rate limiting step for dissolution of poorly water-soluble nutrients, leading to delayed absorption in vivo. Moreover, analysis of the SAXS profiles (Figure 8d and Figure 9) revealed that the PC700 underwent significant changes in its self-assembled structure (lamellar phase) after gastric digestion in the presence of casein, and this partial loss of structure may be caused by interactions between milk phospholipids and the digested products of milk lipids and casein in the gastric phase. Future research requires investigations into the effects of different casein to PC700 ratios and the addition of whey (another major milk serum protein) on lipid digestion, which is crucial to the design of infant formulas.

5. Conclusions

Digestion and lipid self-assembly in milk are important, unexplored aspects to the delivery of nutrients in children. In this study, we sought to understand the impact of milk-derived emulsifiers, casein and milk phospholipids (PC700), on the self-assembled liquid crystal structures that form in anhydrous milk fat (AMF) during digestion. Our studies conclude that the identity and sequence of self-assembled structures of milk lipids during digestion were not affected by the emulsifiers present, but they were primarily dictated by the triglyceride lipid species in milk. However, subtle differences in the microstructures of the liquid crystal phases occurred when the fat globules were emulsified with the different emulsifiers. Digestion of milk phospholipids-emulsified fat globules (PC700 + AMF) in the small intestinal condition was slower compared to casein-emulsified fat globules, but the overall digestibility of the milk lipids over 1 h was not affected by the types of emulsifiers. When exposed to gastric enzymes (pepsin and fungal gastric lipase), PC700 + AMF showed greater resistance to gastric lipolysis than casein + AMF, presumably due to proteolytic activity of pepsin against casein exposing the emulsified lipids. However, pre-exposure of PC700 + AMF to the gastric enzymes slightly accelerated the lipid digestion in the small intestinal condition, and this slight change in the kinetics of lipid digestion could be caused by changes to the interfacial properties at the oil/water interface. Tailoring the compositions of lipids and emulsifiers in lipid-based formulations could therefore allow fine tuning of the digestibility and self-assembly of the lipids to enhance the bioavailability of nutrients delivered orally, and emerging techniques, including CARS microscopy, will increasingly enlighten the effects of digestion at the individual droplet scale.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/colloids7030056/s1, Figure S1: Particle size distributions of casein- and PC700-emulsified fat globules during digestion by pancreatic lipase, Figure S2: Polarised light microscopy image of hydrated bulk milk phospholipids (PC700) in water at 37 °C indicating birefringence lamellar textures and cryogenic electron microscopy image of dispersed PC700, Table S1: Average volume weighted mean particle size (D4,3) of anhydrous milk fat (AMF) emulsified with casein and milk phospholipids (PC700) under different conditions, Figure S3: CARS microscopy images of casein-emulsified fat globules during digestion by pancreatic lipase, Figure S4: Particle size distributions of casein- and PC700-emulsified fat globules before and after gastric digestion, Table S2: Lists of main liquid crystal structures formed (and their associated lattice parameters) after 60 min or 80 min digestion of casein-emulsified fat globules (casein + AMF) and PC700-emulsified fat globules (PC700 + AMF) by pancreatic lipase at pH 6.5, Figure S5: SAXS profiles of PC700 and casein + PC700-emulsified fat globules before and after digestion by pancreatic lipase at pH 6.5.

Author Contributions

Conceptualization, M.S., A.K.H.M. and B.J.B.; methodology, M.S., A.K.H.M., C.J.N., A.J.C. and B.J.B.; validation, M.S., A.K.H.M., A.J.C. and B.J.B.; formal analysis, M.S.; investigation, M.S. and A.J.C.; resources, A.K.H.M. and B.J.B.; data curation, M.S. and A.J.C.; writing—original draft preparation, M.S.; writing—review and editing, M.S., A.K.H.M., A.J.C. and B.J.B.; visualization, M.S., A.K.H.M. and B.J.B.; supervision, B.J.B.; project administration, B.J.B.; funding acquisition, A.K.H.M. and B.J.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by ARC Linkage (project ID LP180101147). Funding is also acknowledged from the Australian Research Council under the Discovery Projects scheme (DP160102906) and A.J.C. was the recipient of a discovery early career research award (DE190100531) at the time of this work.

Data Availability Statement

Data is contained within the article or supplementary material.

Acknowledgments

We thank the SAXS/WAXS beamline scientists at the Australian Synchrotron, part of ANSTO, for their technical support. The SAXS experiments for this work were conducted on the SAXS/WAXS beamline of the Australian Synchrotron, part of ANSTO. We thank Simon Crawford for his technical support with cryogenic electron microscopy, and the authors acknowledge the use of instruments and assistance at the Monash Ramaciotti Centre for Cryo-Electron Microscopy, a Node of Microscopy Australia. We also thank James Dekker and Sophia Stathopoulos for discussions and providing insights to the project.

Conflicts of Interest

Alastair MacGibbon is an employee of Fonterra Co-operative Group Ltd. (Palmerston North, New Zealand), supplier of AMF and PC700 materials used in this report.

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Figure 1. Schematic representation of the concepts of this study. Anhydrous milk fat (AMF) was emulsified with casein, milk phospholipids (PC700), or mixtures of casein and PC700. Effects of emulsifiers on the digestibility of AMF and the liquid crystalline structures formed during digestion were probed. La (lamellar) → H2 (inverse hexagonal) → V2 (Im3m) (bicontinuous cubic with Im3m space group) were the typical liquid crystalline structures formed by bovine milk fat during digestion by pancreatic lipase.
Figure 1. Schematic representation of the concepts of this study. Anhydrous milk fat (AMF) was emulsified with casein, milk phospholipids (PC700), or mixtures of casein and PC700. Effects of emulsifiers on the digestibility of AMF and the liquid crystalline structures formed during digestion were probed. La (lamellar) → H2 (inverse hexagonal) → V2 (Im3m) (bicontinuous cubic with Im3m space group) were the typical liquid crystalline structures formed by bovine milk fat during digestion by pancreatic lipase.
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Figure 2. A simplified view of the experimental workflow.
Figure 2. A simplified view of the experimental workflow.
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Figure 3. (a) Extent of digestion of anhydrous milk fat (AMF) emulsified with 2 w/v% casein and 2 w/v% milk phospholipids (PC700) under simulated small intestinal conditions (no gastric digestion), pH 6.5 with pancreatic lipase. Digestions were performed in triplicate (n = 3 ± standard error of mean). (b) The corresponding size distributions of the fat globules prior to pancreatic lipase injection. Particles > 10 µm were not observed.
Figure 3. (a) Extent of digestion of anhydrous milk fat (AMF) emulsified with 2 w/v% casein and 2 w/v% milk phospholipids (PC700) under simulated small intestinal conditions (no gastric digestion), pH 6.5 with pancreatic lipase. Digestions were performed in triplicate (n = 3 ± standard error of mean). (b) The corresponding size distributions of the fat globules prior to pancreatic lipase injection. Particles > 10 µm were not observed.
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Figure 4. SAXS profiles of anhydrous milk fat (AMF) emulsified by (a) casein and (b) milk phospholipids (PC700) at 2 w/v% as a function of digestion time in min, and brackets contain the average extent of digestion at that time point in mol% (ext. dig) expressed as a % of triglycerides digested under small intestinal conditions at pH 6.5. NA = ext.dig was not measured beyond 1 h digestion time. Pancreatic lipase was injected at time 0 min. Diffraction peaks characteristic of lamellar phases were labelled as *, hexagonal phases as +, and bicontinuous cubic Im3m phases as #. (c) SAXS profiles of casein + AMF and PC700 + AMF at the conclusion of digestion (60 min for casein and 80 min for PC700) were shown for comparison purposes. Dashed arrows point to an additional lamellar phase that was formed after digestion. (d) Characteristic SAXS pattern of multilamellar vesicles formed by PC700 in buffer (no AMF) at 2 w/v% concentration. Polarised microscopy image of hydrated PC700 and the cryogenic electron microscopy image of dispersed PC700 supporting the SAXS profiles are presented in Figure S2 in the Supplementary Materials section.
Figure 4. SAXS profiles of anhydrous milk fat (AMF) emulsified by (a) casein and (b) milk phospholipids (PC700) at 2 w/v% as a function of digestion time in min, and brackets contain the average extent of digestion at that time point in mol% (ext. dig) expressed as a % of triglycerides digested under small intestinal conditions at pH 6.5. NA = ext.dig was not measured beyond 1 h digestion time. Pancreatic lipase was injected at time 0 min. Diffraction peaks characteristic of lamellar phases were labelled as *, hexagonal phases as +, and bicontinuous cubic Im3m phases as #. (c) SAXS profiles of casein + AMF and PC700 + AMF at the conclusion of digestion (60 min for casein and 80 min for PC700) were shown for comparison purposes. Dashed arrows point to an additional lamellar phase that was formed after digestion. (d) Characteristic SAXS pattern of multilamellar vesicles formed by PC700 in buffer (no AMF) at 2 w/v% concentration. Polarised microscopy image of hydrated PC700 and the cryogenic electron microscopy image of dispersed PC700 supporting the SAXS profiles are presented in Figure S2 in the Supplementary Materials section.
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Figure 5. Images from CARS microscopy of PC700-emulsified fat globules during digestion (from top to bottom) by pancreatic lipase, showing distributions of triglycerides (TG, middle column) and all lipids, including the digestion products (left column), and the ratio between the two signals (right column). Time between each acquisition was about 5 min, and the first image was acquired about 5–10 min after lipase injection. The progression of digestion did not represent the actual digestion time scale, and it is worth noting that the extent of lipolysis for individual droplets was not identical, and the pH of the samples were not controlled. White arrows point to the accumulation of digestion products.
Figure 5. Images from CARS microscopy of PC700-emulsified fat globules during digestion (from top to bottom) by pancreatic lipase, showing distributions of triglycerides (TG, middle column) and all lipids, including the digestion products (left column), and the ratio between the two signals (right column). Time between each acquisition was about 5 min, and the first image was acquired about 5–10 min after lipase injection. The progression of digestion did not represent the actual digestion time scale, and it is worth noting that the extent of lipolysis for individual droplets was not identical, and the pH of the samples were not controlled. White arrows point to the accumulation of digestion products.
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Figure 6. Images from CARS microscopy of casein-emulsified fat globules during digestion (from A–G; time between each acquisition about 5 min) by pancreatic lipase, showing distributions of all lipids, including the digestion products (top row); the hydroxyl (-OH) group contribution (middle row); and an overlay between the two (bottom row). The stack plots show the intensity of -OH groups across the horizontal (left to right) and vertical (top to bottom) directions of the droplet, the lines of which are shown by the dashed lines in A-middle row.
Figure 6. Images from CARS microscopy of casein-emulsified fat globules during digestion (from A–G; time between each acquisition about 5 min) by pancreatic lipase, showing distributions of all lipids, including the digestion products (top row); the hydroxyl (-OH) group contribution (middle row); and an overlay between the two (bottom row). The stack plots show the intensity of -OH groups across the horizontal (left to right) and vertical (top to bottom) directions of the droplet, the lines of which are shown by the dashed lines in A-middle row.
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Figure 7. Extent of digestion of anhydrous milk fat (AMF) globules emulsified with (a) 2 w/v% casein and (b) 2 w/v% milk phospholipids (PC700) under simulated small intestinal conditions at pH 6.5 with no gastric pre-treatment, post-pepsin treatment, and post-fungal gastric lipase (FGL) + pepsin treatment. Inset in panel (a) shows the expanded digestion profile for casein + AMF from 0–3 min where pre-digestion of casein + AMF with pepsin reduced the duration of the lag time for the onset of digestion. Digestions were performed in triplicate (n = 3 ± standard error of mean). SAXS profiles of (c,d) the casein-emulsified fat globules and (e,f) the PC700-emulsified fat globules as a function of digestion time (min) and the corresponding average extent of digestion in mol% (ext. dig) after exposure to pepsin and FGL + pepsin. Lipase was injected at time = 0. Peaks characteristic of lamellar phases were labelled as *, hexagonal phases as +, and bicontinuous cubic Im3m phases as #.
Figure 7. Extent of digestion of anhydrous milk fat (AMF) globules emulsified with (a) 2 w/v% casein and (b) 2 w/v% milk phospholipids (PC700) under simulated small intestinal conditions at pH 6.5 with no gastric pre-treatment, post-pepsin treatment, and post-fungal gastric lipase (FGL) + pepsin treatment. Inset in panel (a) shows the expanded digestion profile for casein + AMF from 0–3 min where pre-digestion of casein + AMF with pepsin reduced the duration of the lag time for the onset of digestion. Digestions were performed in triplicate (n = 3 ± standard error of mean). SAXS profiles of (c,d) the casein-emulsified fat globules and (e,f) the PC700-emulsified fat globules as a function of digestion time (min) and the corresponding average extent of digestion in mol% (ext. dig) after exposure to pepsin and FGL + pepsin. Lipase was injected at time = 0. Peaks characteristic of lamellar phases were labelled as *, hexagonal phases as +, and bicontinuous cubic Im3m phases as #.
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Figure 8. (a) Extent of digestion of anhydrous milk fat (AMF) globules emulsified with 1 w/v% casein and 1 w/v% milk phospholipids (PC700) under simulated small intestinal condition at pH 6.5 with no gastric pre-treatment or treatment with fungal gastric lipase (FGL) + pepsin. Digestions were performed in triplicate (n = 3 ± standard error of mean). (b) Particle size distributions of the samples at pH 6.5 and (c,d) the SAXS profiles of the digesting media as a function of digestion time (in min) and the average extent of digestion in mol% (ext. dig). Lipase was injected at time = 0. Peaks characteristic of lamellar phases were labelled as *, hexagonal phases as +, and bicontinuous cubic Im3m phases as #.
Figure 8. (a) Extent of digestion of anhydrous milk fat (AMF) globules emulsified with 1 w/v% casein and 1 w/v% milk phospholipids (PC700) under simulated small intestinal condition at pH 6.5 with no gastric pre-treatment or treatment with fungal gastric lipase (FGL) + pepsin. Digestions were performed in triplicate (n = 3 ± standard error of mean). (b) Particle size distributions of the samples at pH 6.5 and (c,d) the SAXS profiles of the digesting media as a function of digestion time (in min) and the average extent of digestion in mol% (ext. dig). Lipase was injected at time = 0. Peaks characteristic of lamellar phases were labelled as *, hexagonal phases as +, and bicontinuous cubic Im3m phases as #.
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Figure 9. SAXS profiles of PC700 (2 w/v%) emulsified in tris buffer and PC700-emulsified fat globules in presence or absence of casein (total emulsifier 2 w/v% and ratio of PC700 to casein was 1:1 w/w), fungal gastric lipase (FGL), and pepsin. Peaks characteristic of lamellar phases were labelled as *, arrows in the figure pointed to an additional peak from an unknown phase (?), and *’ was the broad lamellar peak from fatty acids-calcium soaps.
Figure 9. SAXS profiles of PC700 (2 w/v%) emulsified in tris buffer and PC700-emulsified fat globules in presence or absence of casein (total emulsifier 2 w/v% and ratio of PC700 to casein was 1:1 w/w), fungal gastric lipase (FGL), and pepsin. Peaks characteristic of lamellar phases were labelled as *, arrows in the figure pointed to an additional peak from an unknown phase (?), and *’ was the broad lamellar peak from fatty acids-calcium soaps.
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MDPI and ACS Style

Salim, M.; MacGibbon, A.K.H.; Nowell, C.J.; Clulow, A.J.; Boyd, B.J. Influence of Casein and Milk Phospholipid Emulsifiers on the Digestion and Self-Assembled Structures of Milk Lipids. Colloids Interfaces 2023, 7, 56. https://doi.org/10.3390/colloids7030056

AMA Style

Salim M, MacGibbon AKH, Nowell CJ, Clulow AJ, Boyd BJ. Influence of Casein and Milk Phospholipid Emulsifiers on the Digestion and Self-Assembled Structures of Milk Lipids. Colloids and Interfaces. 2023; 7(3):56. https://doi.org/10.3390/colloids7030056

Chicago/Turabian Style

Salim, Malinda, Alastair K. H. MacGibbon, Cameron J. Nowell, Andrew J. Clulow, and Ben J. Boyd. 2023. "Influence of Casein and Milk Phospholipid Emulsifiers on the Digestion and Self-Assembled Structures of Milk Lipids" Colloids and Interfaces 7, no. 3: 56. https://doi.org/10.3390/colloids7030056

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