
==== Front
Ultrason Sonochem
Ultrason Sonochem
Ultrasonics Sonochemistry
1350-4177
1873-2828
Elsevier

S1350-4177(24)00306-7
10.1016/j.ultsonch.2024.107058
107058
Original Research Article
Modifying physicochemical properties, rheology, and creaming stability of milk fat globule and membrane through ultrasound treatment
Sun Yanjun ab
Roos Yrjö H. b
Miao Song Song.Miao@teagasc.ie
ac⁎
a Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland
b School of Food and Nutritional Sciences, University College Cork, Cork, Ireland
c China-Ireland International Cooperation Centre for Food Material Sciences and Structure Design, Fujian Agriculture and Forestry University, China
⁎ Corresponding author. Song.Miao@teagasc.ie
04 9 2024
12 2024
04 9 2024
111 1070581 7 2024
14 8 2024
2 9 2024
© 2024 Published by Elsevier B.V.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The healthy benefits of milk fat globules and membrane (MFGs/MFGM) ingredients are increasingly recognized in the dairy industry. In this research, we examined the effects of ultrasonic treatment on the physicochemical and rheological properties, as well as the emulsions stability of MFGs/MFGM derived from bovine raw milk. Fresh milk was subjected to sonication at frequencies of 20 kHz and 40 kHz, either individually or simultaneously, for durations of 5 min or 15 min, using work/rest cycles of 5 s on and 3 s off. Bovine milk, without any treatment, served as the control. Regardless of the intensity difference, ultrasonic treatment for 5 min resulted in more pronounced changes in the regions of Amide Ⅱ (1600–1500 cm−1), Amide Ⅲ (1500–1200 cm−1), and fingerprint region (1200–1900 cm−1) compared to both the 15 min treatments and control MFGs/MFGM. Principal component analysis (PCA) conducted on the entire spectra, as well as in the regions of Amide Ⅰ, Amide Ⅱ, and the fingerprint spectra, clustered the 5 min treatment distinctly from the control and MFGs/MFGM ultrasonically treated for 15 min. MFGs/MFGM samples following 20 kHz and 40 kHz synchronous treatment for 15 min exhibited lower absorbance bands at 1727–1726 cm−1, whereas a higher content at 1740 cm−1 was observed compared to control MFGs/MFGM. Additionally, a more significant reduction in the intramolecular β-sheet content in 20 + 40 kHz/ 15 min treatment was observed. According to the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) patterns, a diminished intensity of Periodic Acid Schiff 6/7 (PAS 6/7) bands was observed across all the MFGs/MFGM. Ultrasonic treatment retained more caseins while reducing the β –LG levels compared to the controls, enhancing the stability of MFGs/MFGM, except in MFGs/ MFGM subjected to 20 and 40 kHz simultaneously treated for 15 min. The irregular sphericity of fat globules was noted particularly in MFGs/MFGM treated at 20 kHz independently or in combination with 40 kHz for 15 min. According to the confocal laser scanning microscopy (CLSM), ultrasonic treatment facilitated the binding of caseins or whey proteins to the MFGs surface and induced flocculation of membrane proteins. Hierarchical cluster analysis (HCA) heat map further underscored the impact of ultrasonic treatments on the structural and compositional changes, as well as rheology and emulsions stability, of MFGs/MFGM.

Keywords

Milk fat globules
Ultrasonic treatment
FTIR
Rheological property
Physical instability
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pmc1 Introduction

Milk fat primarily exists in the form of globules (MFGs) characterized by a core of triacylglycerol enveloped by a tri-layer structure known as milk fat globule membrane (MFGM). The MFGM is rich in phospholipids and glycoproteins, which are crucial for the neurocognitive function and confer brain-immune-gut benefits [1], [2]. Dairy producers, such as Fonterra, Arla, and FrieslandCampina, have developed whey protein concentrate (WPC) ingredients enriched with MFGM compositions derived from cheese whey or butter milk [3]. These commercial ingredients are used to fortify infant formula (IF) to mimic the natural nutritional profile of human milk, addressing the deficiency of functional lipids and membrane-bound proteins in existing IF products. Despite these advancements, the industry lacks standardized quality regulations to oversee the production of MFGM ingredients, and analytical methods for MFGM proteins remain undeveloped [4]. Consequently, the compositions of the MFGM ingredients vary significantly between different manufacturers, leading to heterogeneous outcomes. Whey proteins predominate in these formulations, with phospholipid content typically ranging from 6–10 %; however, membrane proteins are often not specified on product labels [3].

In addition to nutritional enhancement, the role of MFGM fragments in improving the technical functionalities of IFs and emulsions has been extensively studied. This includes investigations into the interfacial composition and structure of MFGs within IF products [5], as well as the stability of emulsion and colloidal phases in homogenized milk [6]. MFGM fragments have been shown to alter the composition and conformational structure at the oil–water interface, thereby influencing the stability of dairy emulsions [7]. MFGM ingredients are employed as a natural emulsifier in dairy industry due to the amphipathic nature of polar lipids and glycoproteins.

Moreover, the rheological properties of milk system are critical for processing efficiency in the dairy industry. An in-depth understanding of the rheological behaviours of milk liquids is essential for the design and evaluation of process units and operational equipment, such as the heat exchangers, heating and cooling rates, and the pressure losses in pipelines [8]. The rheological behaviour of milk products is complex and significantly affected by temperature and the concentration of dispersed phases [9]. For instance, the viscosity of the concentred skim milk must be strictly controlled to avoid detrimental effects on the properties of milk powder [10].

The structure, composition, and techno-functionalities of MFGs or MFGM have been documented in previous reviews [11]. However, there is a compelling need to explore the impact of various preparation steps on MFGM functionalities, including heating, membrane filtration, mechanical shearing, and non-thermal treatments like ultrasonic processing. Ultrasound induces physical and chemical effects in foods due to acoustic cavitation, characterized by the continuous formation and implosion of microbubbles [12]. Research has shown that ultrasonic treatment can enhance the solubility of MFGM (rich in milk phospholipids, MPL), improve the emulsifying stability of MPL-based emulsions [13], and retain more MFGM proteins compared to shear homogenisation [14]. These studies predominantly utilized commercial MFGM materials, where the native structure of fat globules was disrupted and MFGM existed in fragmented form. However, studies examining the impact of dairy processing units on MFGM derived from raw milk are limited. The gap underscores the importance of further investigations to better understand and optimize the processing of MFGs or MFGM ingredients in their most natural state.

In our previous study, we conducted a comparative analysis of the changes of interfacial and thermal-dynamic properties of MFGs/MFGM isolated from fresh milk exposed to ultrasound at varying intensities [15]. For this study, a follow-up research on the physicochemical and rheological properties, as well as the emulsions stability of MFGs/MFGM was investigated. To deepen our understanding of the technical functionalities of natural MFGs and MFGM, bovine raw milk was used as the raw material. Prior to the isolation of MFGs/MFGM materials, ultrasound at different intensities was applied to the raw milk. The changes in MFGM proteins and their interactions with non-membrane proteins were assessed using a combination of analytical techniques, including sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Fourier transform infrared spectroscopy (FTIR), and confocal laser scanning microscopy (CLSM). Furthermore, shearing scanning was performed on all the MFGs/MFGM to characterize their rheological properties, while an accelerated centrifugation method was used to evaluate the stability of emulsion stabilized by MFGs/MFGM. The findings from this research may provide valuable insights into the innovative development of MFGs/MFGM products and their prospective uses in the dairy field.

2 Materials and methods

2.1 Materials and reagents

Fresh bovine milk samples were obtained from Teagasc animal & grassland research and innovation centre (Moorepark, Fermoy, Co. Cork, Ireland). Immediately after milking, the fresh milk samples were cooled down to 4 °C by plate heat exchanger and maintained at this temperature until further analysis. The compositions of the collected milk was analysed using the DairySpec FT–15 (Bentley Instruments Inc., Chaska, MN, USA), revealing the mean values of fat at 4.09 ± 0.01 %, protein at 3.36 ± 0.03 %, dry matter at 12.94 ± 0.03 %, pH at 6.68 ± 0.02.

Nile Red, Fast Green FCF, wheat germ agglutinin (WGA), Polyethylene glycol 200, and phosphate buffer saline (PBS) were purchased from Sigma–Aldrich® (Merck Life Science Ltd., Cork, Ireland). Thermo Scientific Inc. (Thermo Fisher Scientific Inc., Cork, Ireland) was the supplier of all the chemicals for gel electrophoresis.

2.2 Ultrasonic processing

Bovine raw milk was subjected to ultrasonic treatments of different intensities referring to the methodologies detailed in our previous research [15]. The procedure involved preheating the raw milk to 30–35 °C before subjecting it to sonication at frequencies of 20 kHz and 40 kHz, using both independent and synchronous operating models. An ultrasonic processor (WKS1200/2J, Jiangsu Jiangda Wukesong Biotechnology Co., LT, Zhenjiang, China) was employed, featuring a 2-cm diameter titanium probe. The durations of the ultrasonic treatments were 5 and 15 min using the 5 s: 3 s work/rest working model. Raw milk that did not undergo ultrasonic treatment served as the control groups.

2.3 Separation of MFGs/MFGM ingredients

The separation of MFGs/MFGM fractions was carried out using ultra-centrifugation (10000 × g, 15 min at 4 °C) (Kendro Laboratory Products, Asheville, NC) following the method of Ren, et al. [16] with some modifications. The upper cream layer was collected and washed with 3 volumes of phosphate buffered saline (PBS, 0.1 mol/L, pH 6.80). This rinsing procedure was repeated 3 times. Subsequently, the upper layer obtained from the final centrifugation was freeze-dried for 72 h. The MFGs/MFGM were individually labelled according to the ultrasonic conditions: 20 kHz/ 5 min, 20 kHz/ 15 min, 40 kHz/ 5 min, 40 kHz/ 15 min, 20 + 40 kHz/ 5 min, and 20 + 40 kHz/ 15 min. The resulted total protein and fat content were noted in our previous study [15].

2.4 Fourier transform infrared spectroscopy (FTIR)

FTIR spectra was acquired using a Bruker Tensor 27 (Bruker Optik GmbH, Ettlingen, Germany) equipped with an Optics Platinum Attenuated Total Reflectance (ATR) cell (Pike Technology Inc., Madison, WI, USA). For the analysis, 20 mg of each sample was placed on the zinc ATR crystal aperture. Spectra were captured within the spectral range of 4000 to 900 cm−1, averaging 120 scans at a resolution of 4 cm−1. Each sample was analysed in triplicate. Prior to each determination, a background measurement was conducted under identical conditions. The collected spectral data was initially pre-processed using OPUS 7.5 software (Bruker Optik GmbH, Ettlingen, Germany) to eliminateatmospheric vapour and carbon dioxide interference, and vector normalization was applied to mitigate scaling effects.

Noise reduction, baseline subtraction, second derivation, and peaks fitting and deconvolution were performed using OriginPro 2023 (OriginLab, Northampton, MA, USA) to reveal hidden and overlapping bands. The Savityzky–Golay function was utilized for smoothing, and the baseline points were anchored using the 2nd derivative method. The fitting of peaks was carried out using Gaussian, Voigt, or Lorentz functions across various spectral regions to achieve the optimal fit. Parameters such as peaks widths and centres, along with baseline parameter, were adjusted until achieving a fit with R2 > 0.990. Finally, unsupervised chemometric analysis, principal component analysis (PCA), was used to visually depict the variation within and between the spectra of all MFGs/MFGM and to classify samples according to treatments. Namely, loading plots highlighted differentiated wavenumbers, and PCA plots illustrated distinct groups based on their spectral profiles.

2.5 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)

The protein profiles of control and ultrasonically-treated MFGs/MFGM isolates were characterized by SDS-PAGE with a modified method described by Sun, et al. [17]. The non-reducing SDS-PAGE was performed at 120 V for a duration of 45 min, after which the proteins were stained with Coomassie Blue. Subsequently, the gels were detained using a blend of 45 % ethanol and 5 % acetic acid until a clear background was achieved. Relative quantification of caseins, whey proteins, and specific MFGM proteins was performed to assess the impact of ultrasonic treatments. The densities of protein bands were analysed by ImageJ software 1.36b (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA), and the results were normalized to a total of 100 %.

2.6 Rheological analysis

The rheological properties of both control and ultrasound-treated MFGs/MFGM were evaluated using an ARG 2 Rheometer (TA Instruments, Crawley, UK). Before analysis, the freeze-dried MFGs/MFGM was kept at 35–40 °C until melting into the liquid state. The measuring geometry consists a 60 mm diameter aluminium parallel plate. The gaps size was set to 300 μm during the analysis. The shear rate was increased from 0.1 s−1 to 1000 s−1, during which both shear (apparent) viscosity and normal stress were recorded as a function of shear rate. All measurements were conducted at a constant temperature of 25 °C. A lid was used to cover the MFGs/MFGM during the rheological analysis to reduce the moisture evaporation. Each analysis was replicated three times. Given that raw milk and cream predominantly exhibit non-Newtonian behaviour under conditions with temperature < 40 °C and at low shear rate, the experimental data were fitted using the power law model and Herschel-Bulkley to characterize the nonlinear behaviour of the MFGs/MFGM samples [18].

Power law,(1) σ=KY¨n

and Herschel-Bulkley,(2) σ=σ0+KY¨n

where σ is the shear stress (Pa), while Y¨ denotes the shear rate (1/s). The parameter K (Pa sn) serves as the consistency index, and n is the flow behavior index. Additionally, σ0 refers to the yield stress (Pa). The power-law model is utilized to predict the rheological behaviour of fluids, characterizing them as shear-thinning for 0 < n < 1, Newtonian for n = 1, and shear-thickening for n > 1.

2.7 Accelerated emulsion stability

Lumisizer was employed (LUM GmbH, Berlin, Germany) to investigate the impact of ultrasonic treatments on the physical stability of MFGs/MFGM. Freeze-dried MFGs/MFGM materials were initially pre-heated to a liquid phase at temperatures ranging from 35 °C to 40 °C. Subsequently, 400 μL of these liquid samples were loaded into a polyamide cell (2 mm light path) and subjected to a centrifugal force of 3000 rpm for 45 min at a controlled temperature of 37 ± 2 °C. The intensity of near-infrared (NIR) light (wavelength at 865 nm) transmitted through the length of samples was recorded every 10 s intervals during centrifugation. The SEP View software (SepView 4.1, LUM GmbH) was used for the data recording and instability calculation. To calculate the instability index, the clarity of the emulsion caused by phase separation was measured and then divided by the maximum possible transmission at the given centrifugal force. This dimensionless index spans from 0 to 1, where 0 means no alteration in NIR transmission and 1 means complete phase separation [19].

2.8 Confocal laser scanning microscopy (CLSM)

CLSM, as reported in the literature, provides reliable qualitative identification of protein adsorption on the surfaces of MFGs and has demonstrated the aggregation of protein and fat globules [20]. In this study, CLSM (Leica TCS SP5, Leica Microsystems, Germany) was used to examine the ultrasound-induced structural and surface compositional changes of the fat globules. Samples preparation and the instrument settings were based on our published report with modifications [17]. Generally, non-polar lipids, predominantly triglycerides, were stained with Nile Red, while non-membrane proteins and glycoproteins were labelled using Fast Green FCF and wheat germ agglutinin (WGA), respectively. All the MFGs/MFGM underwent dual staining by 10 µL mixture comprising 0.1 % (w/v) Nile Red in polyethylene glycol-200 and 0.1 % (w/v) Fast Green FCF in distilled water, at a ratio of 3:1 (v/v) (FCF: Nile Red). Additionally, 50 µL of 1 mg/mL WGA in PBS was combined with Nile Red at a 1:1 (v/v) ratio. All MFGs/MFGM were subsequently incubated in darkness for half an hour to ensure thorough staining. The excitation of Nile Red, Fast Green FCF, and WGA were performed with a helium–neon (He-Ne) laser at 565–625 nm, 632–719 nm, and 493–550 nm, respectively. For each sample, a minimum of 10 images were recorded, from which one representative image was selected for each experiment.

2.9 Statistical analysis

All analyses were conducted in triplicate, unless otherwise indicated. The tested results were expressed as mean value ± standard deviation (SD). The variant analysis was performed using Analysis of Varian (ANOVA), and the significance of mean differences was assessed using Fisher’s test (p < 0.05). Additionally, Multivariate Statistical Analysis was executed in OriginPro, the results of which were visually represented through a heat map, derived from the Hierarchical Cluster analysis (HCA). All these statistical evaluation were conducted using the OriginPro 2023 software system (OriginPro 2023, OriginLab Corporation, Northampton, USA).

3 Results and discussion

3.1 General profiling of composition changes induced by ultrasonic treatment

The averaged FTIR spectra of MFGs/MFGM fractions extracted from bovine raw milk treated with ultrasound at varying intensities are depicted in Fig. 1A. Additionally, a second derivative analysis was conducted to search for the hidden peaks within each spectrum (Fig. 1B). The entire spectral range between 4000 and 900 cm−1 has been classified into six regions based on different milk components referring to published articles. In particular, the region from 3000–2800 cm−1 is attributed to the stretching modes of CH2 groups of acyl chains in triglycerides and phospholipids [21] (Region 1); 1800–1700 cm−1 represents the C <svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="20.666667pt" height="16.000000pt" viewBox="0 0 20.666667 16.000000" preserveAspectRatio="xMidYMid meet"><metadata> Created by potrace 1.16, written by Peter Selinger 2001-2019 </metadata><g transform="translate(1.000000,15.000000) scale(0.019444,-0.019444)" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z M0 280 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z"/></g></svg> O stretching vibrations of ester, aldehydes and ketones [22] (Region 2); Protein secondary structure is commonly described by the Amide Ι (1700–1600 cm−1) (Region 3), Amide Ⅱ (1600–1500 cm−1) (Region 4) and Amide Ⅲ (1500–1200 cm−1) [23] (Region 5); and the1200–900 cm−1, known as the fingerprint region, corresponds to the lipid components of membranes, protein-lipid complexes, and lactose groups [24] (Region 6).Fig. 1 FTIR spectra for MFGs/MFGM materials: original (A) and 2nd derivative (B). (Spectra between 2800 and 1800 cm−1 are not shown as no peak was detected for any of the MFGs/MFGM, with all spectra displaying similar flat profiles). Numbers 1–6 above the figure indicate different wavenumber ranges: 1: 3000–2800 cm−1; 2: 1800–1700 cm−1; 3: Amide Ⅰ, 1700–1600 cm−1; 4: Amide Ⅱ, 1600–1500 cm−1; 5: Amide Ⅲ, 1500–1200 cm−1; 6: Finger print region, 1200–1900 cm−1.

The original and 2nd deprivation spectra in regions numbered 1–3 show no noticeable differences, whereas more pronounced changes are observed in regions 4–6 for MFGs/MFGM treated for 5 min compared to those treated for 15 min and the control (Fig. 1A and B).

PCA conducted on the full spectra range from 4000–900 cm−1 grouped the MFGs/MFGM into two distinct categories along the axis of Principle Component 1 (PC1), which accounts for 44.30 % of the variance (Fig. S1A). MFGs/MFGM treated for 5 min were differentiated from both control and 15 min ultrasonic treatment samples. This division is mainly due to different loadings for the wavenumber ranges of 3000–2800 cm−1 and 1700–900 cm−1 (Fig. S1B). Further PCA was performed on these specific wavenumber regions to explore the effects of ultrasonic treatments on the lipids, proteins, and the interactions of membrane proteins in the MFGs/MFGM materials. For the first region between 3000 cm−1 and 2800 cm−1, the control, 20 kHz/ 15 min, and 40 kHz/ 15 min MFGs/MFGM were separated from the 20 + 40 kHz/ 15 min and the 40 kHz/ 5 min ultra-sonicated MFGs/MFGM along the PC2 (Fig. S2A) with a variation of 9.2 %. This separation is due to peaks at 2916/2850 cm−1 and 2900 cm−1, which are ascribed to the methylene groups and C—H stretching of lipids, respectively (Fig. S2B) [21].

In the region of 1800–1700 cm−1, the 20 + 40 kHz/ 15 min MFGs/MFGM were distinctly separated from other treatments along the direction of PC2 (9.1 %) (Fig. S3A). This division is primarily due to differences in the wavenumber at 1742 cm−1, which is assigned to the ester functional group of lipids and their interactions with proteins via CO stretching vibrations (Fig. S3B) [22]. In addition, hidden peaks at the wavenumber of 1726 cm−1 in each sample were identified using a peak fitting function (Fig. S4A–G). The band at 1726 cm−1 is typically indicative of the presence of hydrogen bonded carbonyl groups in esters [25]. Quantitative assessments of the respective corresponding groups at band of 1740 cm−1 and 1727 cm−1 in each MFGs/MFGM were conducted to elaborate on changes in lipids groups (Table 1). Bands between 1727–1726 cm−1 in control MFGs/MFGM were significantly higher than those in MFGs/MFGM fractions treated by 20 and 40 kHz for 15 min (p < 0.05), whereas the opposite trend was observed at 1742–1739 cm−1, where a stronger spectrum was noted in the MFGs/MFGM sourced from 20 and 40 kHz ultra-sonicated raw milk for 15 min MFGs/MFGM compared to the control.Table 1 Quantitative estimation of the functional ester group of lipids at the band of 1727–1726 cm−1 and 1742–1739 cm−1 in all MFGs/MFGM ingredients obtained from ultrasound-treated bovine raw milk (1800–1700 cm−1).

	1727–1726 cm−1	1742–1739 cm−1	
Control	94.29 ± 0.22a	5.71 ± 0.22b	
20 kHz/ 5 min	91.37 ± 0.52ab	8.63 ± 0.52ab	
20 kHz/ 15 min	87.41 ± 0.10ab	12.59 ± 0.10ab	
40 kHz/ 5 min	86.22 ± 4.25ab	13.78 ± 4.25ab	
40 kHz/ 15 min	91.94 ± 0.92ab	8.06 ± 0.92ab	
20 + 40 kHz/ 5 min	91.92 ± 1.45ab	8.08 ± 1.45ab	
20 + 40 kHz/ 15 min	81.38 ± 17.19b	18.62 ± 17.19a	
Superscript letters that differ in the same column denote significant differences between MFGs/MFGM (p < 0.05). ‘ns’ stands for no significant difference (p > 0.05).

For the Amide I and Amide II regions, PC2 classified all of the MFGs/MFGM into two separate groups, respectively (Fig. S5B and S6B). MFGs/MFGM treated at 20 kHz/ 5 min and 40 kHz/ 5 min exhibited similar distributions, while the control and those treated for 15 min clustered into another group. Notably, MFGs/MFGM from the 20 + 40 kHz/ 5 min treatments demonstrated poor repeatability, as indicated by their dispersed distribution in both Fig. S5A and S6A. The separation observed in the Amide I region is due to the loading for the wavenumber at 1654 cm−1, which can be associated with changes in the α-helix content of proteins. The loading for 1574 cm−1 and 1540 cm−1 is responsible for the group separation in the Amide II band, which has been reported to associate with the –COO-asymmetric stretching vibration of Asp and Glu residues and C—N and N—H stretching, respectively [26].

Peak fitting was applied to the Amide I region (Fig. S7A–G), the results of which demonstrated that the ultrasonic treatment had no significant effect on the structure of β-turn and side chain in all MFGs/MFGM (p > 0.05) (Table 2). Comparing with control, MFGs/MFGM exposed to 20 + 40 kHz for a duration of 15 min significantly reduced the intramolecular β-sheet content (p < 0.05). Additionally, more random coil were identified in MFGs/MFGM ingredients subjected to 40 kHz for 15 min than that in control. Under the same ultrasonic conditions, 15 min treatments significantly enhanced the random coil structure compared to 5 min treatments, except for the 20 kHz and 40 kHz concurrent treatment (p < 0.05). Conversely, α-helix structures were more prevalent in MFGs/MFGM treated for 5 min compared to those treated for 15 min. This finding is inconsistent with the PCA results for the band of Amide I, where differences in α-helix were a primary factor in grouping 5 min treated MFGs/MFGM separately from control and 15 treatments. Peak fitting in the spectrum of Amide II indicated an apparent band migration from 1582–1584 cm−1 to 1572–1573 cm−1 as the duration of ultrasonic treatment prolonged to 15 min (Fig. S8A–G). This supports the previous PCA results that distinguished 5 min MFGs/MFGM from those treated for 15 min and the control along the PC2 at the band at 1573 cm−1.Table 2 Relative content of secondary structures in Amide I region (1700–1600 cm−1) in all MFGs/MFGM compounds sourced from bovine raw milk subjected to ultrasound treatment.

	Intramolecular β-sheet	Random coil	α-helix	β-turn	Side chain	
Control	11.08 ± 0.35a	36.26 ± 6.00bcd	26.96 ± 3.48b	17.78 ± 7.70ns	7.92 ± 1.82 ns	
20 kHz/ 5 min	9.99 ± 1.18ab	28.60 ± 7.10d	43.13 ± 10.46a	12.32 ± 3.52	5.96 ± 1.07	
20 kHz/ 15 min	8.69 ± 4.53ab	41.18 ± 6.77abc	26.90 ± 3.37b	14.68 ± 2.32	8.54 ± 1.86	
40 kHz/ 5 min	7.39 ± 2.87ab	31.67 ± 5.46cd	40.00 ± 6.32a	12.05 ± 3.72	8.88 ± 2.56	
40 kHz/ 15 min	6.24 ± 2.85b	49.95 ± 6.54a	20.18 ± 5.88b	15.60 ± 0.62	8.03 ± 3.64	
20 + 40 kHz/ 5 min	8.53 ± 3.09ab	35.62 ± 10.68bcd	28.26 ± 0.22b	17.19 ± 2.69	8.81 ± 2.34	
20 + 40 kHz/ 15 min	6.34 ± 1.62b	45.70 ± 2.59ab	23.72 ± 2.52b	16.13 ± 1.83	8.11 ± 1.20	
Superscript letters that differ in the same column denote significant differences between MFGs/MFGM (p < 0.05). ‘ns’ stands for no significant difference (p > 0.05).

Similar group distinctions observed in the Amide I and Amide II regions were also noted when analysing the fingerprint region of 1200–900 cm−1 via PCA. The analysis demonstrated that MFGs/MFGM treated for 5 min were differentiated from control and 15 min treatment MFGs/MFGM (Fig. S9A). This differentiation was largely attributed to absorbance peaks at 987 cm−1, 1035 cm−1, and 1153 cm−1 along the PC2 axis, which represented 80.40 % of the observed variance.

The band at 987 cm−1 has been reported to be related to the release of colloidal calcium phosphate (CCP) from the phosphate residues due to pressure effects [27]. The bands around 1035 cm−1 ascribes to –CH2 bending, and peak around 1159 cm−1 represents the C—O group (esters) [24]. Further insights were gained through peak fitting analysis of the region between 1200–900 cm−1 (Fig. S10A–G), where peaks at bands 1003 cm−1 and 1058 cm−1 were absent in 5 min treatment MFGs/MFGM. Meanwhile, absorbance peaks positioned at 1035 cm−1 and 1141 cm−1 in both control and 15 min treatment MFGs/MFGM shifted to 1043 cm−1 and 1153 cm−1, respectively, in 5 min treatment MFGs/MFGM.

In summary, PCA analysis of the spectral bands from Amide I (1700–1600 cm−1), Amide II (1600–1500 cm−1), and 1200–900 cm−1 revealed that all treatments could be categorized into two distinct groups: one compromising the 5 min treatment, and another encompassing the control and 15 min treatment, a finding further substantiated by the peak fitting results.

3.2 Effect of ultrasonic treatments on the distribution of milk fat globule membrane proteins

Non-reducing SDS-PAGE was conducted to illustrate the impact of varying ultrasound intensities on the MFGs/MFGM proteins, with the resulting SDS-PAGE patterns depicted in Fig. 2A. Notable non-membrane proteins identified included casein, β-lactoglobulin (β-LG), α-lactalbumin (α-LA), and six key membrane proteins: MUC1, the redox enzyme xanthin oxidase (XO), periodic acid Shciff 3 (PAS Ⅲ), CD 36, butyrophilin (BTN), and periodic acid Schiff 6/7 (PAS 6/7). Generally, the intensity of PAS 6/7 bands was observed to be lower compared to other proteins, likely due to its classification as a peripheral protein, which is more susceptible to removal through mechanical shearing during sample preparation [17]. In contrast, casein, β-LG, and α-LA were distinctly present in the control, persisting through the centrifugation and rinsing processes (Fig. 2A), contrary to findings by Zhao, et al. [28] where more intensive centrifugation and rinsing were applied, resulting in the removal of most caseins and whey proteins during centrifugation. The concentration of MFGM proteins in MFGs/MFGM can further be improved by washing with SDS, followed by sonication [29].Fig. 2 SDS-PAGE patterns obtained under non-reducing conditions of fat globule materials from control and ultra-sonicated raw milk. (A) Lane 1–7, control, processed at 20 kHz for 5 min and 15 min, 40 kHz for 5 min and 15 min, 20 + 40 kHz for 5 min and 15 min.

The intensity of caseins in ultrasonically treated MFGs/MFGM increased dramatically, whereas a reduced intensity of β –LG bands was identified compared to control. From the quantitative analysis results in Fig. 2B, 20 kHz and 40 kHz treatments retained 55.44–63.28 % of caseins, compared to 36.53 % in the control. With prolonged ultrasonic durations, a higher retention of caseins was observed, except in the 40 kHz treatments, consistent with the study by Lu, et al. [29] which reported that homogenization damages MFGM while aqueous phase proteins like caseins and whey proteins adsorb onto the newly formed MFGM. Conversely, the reduction of β –LG intensity in ultrasonic MFGs/MFGM may be attributed to the formation of aggregates between β –LG and MFGM proteins via disulphide bands [30]. Subsequent centrifugation aided in the removal of these aggregates, concurrently removing the PAS Ⅲ and XO. This accounts for the observed reductions in the intensities of PAS Ⅲ and XO bands. Notably, treating at 20 + 40 kHz ultrasound for 15 min most efficiently facilitated the removal of β –LG from MFGs/MFGM ingredients, followed by 5-min treatments at 20 + 40 kHz, then 40 kHz, and lastly 20 kHz.

Additionally, the increased intensity of CD 36 in ultrasonic MFGs/MFGM, particularly in the 20 + 40 kHz treatments, suggests that ultrasonic treatment enhances the transfer of CD 36 into the rinsing phase from the MFGM structure. In contrast, lower amounts of BTN and MUC1 were detected in MFGs/MFGM after ultrasonic treatment compared to control. This variability in the effects of ultrasonic treatments on CD36, BTN, and MUC1 levels may be due to their distinct localizations within the three layers of membrane; CD 36 primarily localizes in the apical membrane [31], MUC1, characterized by its extended rod-like structure, forms part of the glycocalyx on various epithelial cell surfaces and milk membranes [32], and BTN is mainly found in a 'peripheral' cytoskeletal-like component [33]. Comparatively, the 20 kHz/ 15 min MFGs/MFGM contained fewer MFGM proteins but higher levels of non-membrane proteins than other MFGs/MFGM, indicating that more intensive ultrasonic treatment promotes greater loss of MFGM proteins.

3.3 Rheological properties

As shown in Fig. 3, the apparent viscosity of MFGs/MFGM consistently decreased with an increase in shear rate (0.1 s−1 to 1000 s−1) across all MFGs/MFGM, demonstrating shear-thinning behaviour (non-Newtonian), regardless of the ultrasonic treating intensities. There was a dramatic decrease in viscosity observed in the control, 20 kHz, and 20 + 40 kHz MFGs/MFGM within the shear rate range of 0.1 to 2 s−1, with the exception of those treated at 40 kHz, suggesting a more pronounced degree of Newtonian behavior at higher shear rates. This observation of shear-thinning behaviour in the control is in disagreement with findings from Ayyash, et al. [34]. They demonstrated that non-fermented raw milk exhibited little or no shear-thinning behavior. This discrepancy in the magnitudes of viscosity could be due to differences in experimental conditions, particularly the higher shear rate range (10–1000 s−1) used in their study.Fig. 3 Apparent viscosity of MFGs/MFGM components derived from bovine raw milk exposed to ultrasound with different intensities.

The initial viscosity of the control was significantly lower compared to those treated at 20 kHz and 20 + 40 kHz, while the 40 kHz MFGs/MFGM exhibited the lowest viscosity. Additionally, a slight variation in the viscosity of 40 kHz MFGs/MFGM was observed throughout the analysis. This trend is in accordance with the regression results in Table 3. The flow behavior indexes (n) for the 40 kHz MFGs/MFGM, derived from Herschel-Bulkley models, are 1 or close to 1, indicating Newtonian or shear thickening behaviour. This characteristic may be attributed to the increase in volume density (%) of larger particles observed at 40 kHz, as noted in the prior research [15]. A heterogeneous distribution of particles, induced by higher stresses, may lead to the shear thickening behavior observed in skim milk and 3.5 % fat milk at temperature of 25 and 40 °C [9]. Furthermore, the flow behavior indexes (n) of the control, fitted from the power law model, were higher than those exposed to 20 kHz or 20 kHz and 40 kHz combination treatment, indicating that ultrasonic treatment may enhance the shear thickening behaviour of MFGs/MFGM materials. Notably, the ultra-sonicated treatment at 20 kHz for 15 min showed the highest consistency indexes (K, Pa sn) among all ultrasonic treatments. As reported in our previous research, MFGs/MFGM isolates sourced from the bovine milk treated at 20 kHz with a duration of 15 min samples contained more protein content than other treatments [15]. This observation is opposite to findings by Kieferle, et al. [35], who reported the consistency indexes in fresh or reconstituted milk protein concentrates increased with rising protein content.Table 3 Regressed parameters of the Herschel-Bulkley models used to describe the flow curve of MFGs/MFGM components derived from bovine raw milk exposed to ultrasound at different intensities.

Samples	Herschel-Bulkley	
	σ0 (Pa)	K(Pa sn)	n	R2	
Control	0.013	0.002	0.950	0.999	
20 kHz/ 5 min	0.016	0.054	0.448	0.997	
20 kHz/ 15 min	0.070	0.032	0.529	0.990	
40 kHz/ 5 min	0.007	0.001	1.022	0.999	
40 kHz/ 15 min	0.008	0.000	1.096	0.999	
20 + 40 kHz/ 5 min	0.053	0.023	0.571	0.995	
20 + 40 kHz/ 15 min	0.064	0.021	0.608	0.988	

The flow curves of the control and MFGs/MFGM treated at 40 kHz exhibited the best fits, with R2 = 0.999, followed by the 20 kHz/ 5 min MFGs/MFGM with an R2 of 0.997 (Table 3). Our preliminary results showed that the Herschel-Bulkley model provided a better fit than the power law model across all MFGs/MFGM, with higher R2 values ranging from 0.988 to 0.999. The predicted shear stress values from both the power law and Herschel-Bulkey models were higher than the expreimental values across the shear rate of 0–600 s−1. Notably, the shear stress predicted by power law was higher than that predicted by the Herschel-Bulkey model in the shear rate of 0–600 s−1, but was lower when the shear rate > 600 s−1 (Fig. S11). The differentiation in predicted shear stress between these models can be explained by the absence of yield stress (σ0) in the power law model [36].

3.4 Physical stability

Fig. 4 illustrates the transmission profiles of MFGs/MFGM as a function of time and space during an accelerated emulsion stability test. The red profile at the bottom of the image indicates the initial recorded profile, while the green profile layered on top shows the final transmitted profile. A noticeable shift from red to green profiles suggests the occurrence of creaming, characterized by the migration of the fat phase to the top of the cell, which implies phase separation in all MFGs/MFGM [37]. In the control, the larger and more distinct separation between the initial and final profiles indicates poorer cream stability compared to other MFGs/MFGM [38]. This observation is further confirmed by the visual evidence images in Fig. 5A, where a more pronounced cream layer was observed at the top of the control. Additionally, the final transmission profile of the control tended to be more horizontal, whereas the profiles of the ultrasonically treated MFGs/MFGM gradually inclined (Fig. 4C–H). This inferred that ultrasonic treatments decelerated the phase separation and resulted in a thinner cream layer at the top of the cell [39].Fig. 4 Lumisizer phase separation near infrared (NIR) transmission profiles and instability index of MFGs/MFGM prepared from bovine raw milk treated by different ultrasonic intensities: A, control; B, instability index; C-D, 20 kHz treated for 5 min and 15 min; E-F, 40 kHz treated for 5 min and 15 min; G-H, Combined treatment using 20 kHz and 40 kHz for 5 min and 15 min.

Fig. 5 (A) The visual cream layer of MFGs/MFGM after centrifugation by Lumisizer: A, control; B-C, 20 kHz treated for 5 min and 15 min; D-E, 40 kHz treated for 5 min and 15 min; F-G Combined treatment using 20 kHz and 40 kHz for 5 min and 15 min. (B) Instability indexes of all the MFGs/MFGM at the end of centrifugation (3000 s).

By comparison, the transmission profile area of 40 kHz treated MFGs/MFGM (Fig. 4E and 5F) was smaller than the control and MFGs/MFGM treated with other ultrasonic intensities. This suggested that 40 kHz treatments facilitated phase stability during the centrifugal process. This effect might be associated with decreased volume density (%) of particles sizes between 1 and 10 μm in the 40 kHz treated MFGMs/MFGM, as found in previous research [15]. The 40 kHz treatment likely caused the MFGs/MFGM to fragment into larger aggregates, decelerating their migration towards the top of the cell. Furthermore, the maximum transmission percentage in the control was 60 %, which decreased to 40 % following the 40 kHz treatments, reflecting lower droplet concentrations and improved cream stability. The duration of the ultrasonic treatments did not significantly affect the transmission profile for the 20 kHz and 40 kHz treatments, highlighting the impact of treatment intensity rather than duration.

Except for the overall transmission profile, emulsion stability was quantitatively analysed and expressed as the instability index. The instability index is the slope of the integrated transmission time plots and is a dimensionless number between 0 and 1. A value of 0 signifies no change in NIR transmission, whereas 1 indicates total phase separation [19]. A significant decrease in the instability index for MFGs/MFGM treated at 40 kHz (0.099–0.143) by the end of the measurement period was observed compared to control (Fig. 5B). This result underscored the finding that 40 kHz treatments enhanced the emulsion stability under applied centrifugal forces, as evidenced by the reduced spacing between NIR transmission profile bands for 40 kHz treated MFGs/MFGM. This was in consist with the lower viscosity and Newtonian similar behaviour of the 40 kHz treatments, as discussed in section 3.3. Ultrasonic treatment at lower frequency (20 kHz) shows higher acoustic energy than at higher frequency (40 kHz) [15]. Based on the stability result from this study, we can speculate that ultrasound with moderate acoustic energy may promote the system stability. Contrarily, stringent shearing treatment induced by low frequency at 20 kHz, or 20 + 40 kHz may facilitate the formation of aggregates, which adversely affected the stability of the system.

From the start of the analysis until 700 s, the control exhibited the highest instability index, suggesting the greatest degree of phase separation during this initial period. However, from 700 s onward to the end of the analysis period at 3000 s, the concurrent treatment at 20 kHz and 40 kHz for 15 min displayed a consistently higher instability index compared to other treatments. Meanwhile, at the end of analysis period (3000 s), the instability index for the control equalized to levels similar to those observed in the MFGs/MFGM subjected to dual-frequency treatment at 20 kHz and 40 kHz for 5 min (0.154), 15 min (0.157), and 20 kHz independently for 5 min (0.154) (Fig. 5B). These data suggest that ultrasonic treatments, with the exception of 20 + 40 kHz/ 15 min treatment, generally enhanced the stability of MFGs/MFGM to varying extents compared to control given their wider transmission profiles and higher instability indexes. This observation was in agreement with the FTIR results in section 3.1 which highlighted the poor repeatability of sample distribution for the 20 + 40 kHz/ 15 min treatment. It can be inferred that phase separation may contribute to variations in sampling, thereby influencing the precision of the FTIR results.

3.5 Microstructure

The ultrasound-induced structural changes in MFGs/MFGM are evident in Fig. 6. Most MFGs maintained an intact spherical shape after ultrasonic treatments at different intensities, as seen by the stained triglyceride in green (Fig. 6A–D) or red (Fig. 6a–d) in the core of MFGs. However, 20 kHz independently or combined with 40 kHz ultra-sonicated for 15 min caused the formation of irregular spheroids (Fig. 6B, D, and b). This observation may be associated with the disintegration of fat globules induced by acoustic stress, leading to the compromised MFGs integrity and release of triglycerides. More heterogeneous sizes of MFGs were observed in the ultrasonically treated MFGs/MFGM than that of control, especially noticeable in MFGs/MFGM treated with the combination of 20 kHz and 40 kHz for 15 min (Fig. 6D and d). This further confirms our previous study, which indicated a the multimodal distribution of particle sizes [15]. Lee and Sherbon [40] reported similar findings, noting an increased surface area of MFGs and more pronounced non-uniformity in the MFGM structure in homogenized whole milk. The MFG was wrapped by a tri-layer structure which consisted of phospholipids and membrane proteins. Prior to ultrasonic treatment, most of the membrane proteins were evenly distributed around the fat globules (Fig. 6A). However, these proteins were predominantly found in the whey phase after ultrasonic treatment, especially in samples processed at 20 kHz and 40 kHz for 15 min, with apparent flocculation of MFGM protein clusters evident in the while square. On the contrary, a significant amount of non-membrane proteins attached to the surface of ultrasonically treated fat globules, particularly in the MFGs/MFGM materials exposed to 20 kHz and 40 kHz concurrently ultra-sonicated for 15 min, whereas most of these proteins were randomly distributed in the whey phase of the control. This may be due to an increase in denatured non-membrane proteins adhering to the MFGs. The extent of protein coverage on MFGs, estimated by the surface fraction model (Φm), showed that MFGs/MFGM treated by 20 + 40 kHz/ 15 min exhibited a higher Φm value than other MFGs/MFGM [15]. This observation may be linked to the increased ζ – potential due to the disruption of MFGs and subsequent membrane reorganization, exposing more negatively charged surface [17].Fig. 6 Confocal laser scanning microscopy images of dual-stained MFGs/MFGM ingredients obtained from ultrasound-treated bovine raw milk at different ultrasonic intensities. A-D: control, 20 kHz/ 15 min, 40 kHz/ 15 min, and 20 + 40 kHz/ 15 min. Staining agents Nile Red and wheat germ agglutinin (WGA) were used to color neutral lipids (red) and membrane proteins (amber). White and yellow arrows highlight the membrane proteins present on the surface of MFGs/MFGM and within the whey phase. a-d: control, 20 kHz/ 15 min, 40 kHz/ 15 min, and 20 + 40 kHz/ 15 min. Staining of neutral lipids (green) and non-membrane proteins (red) was performed with Nile Red and fast green FCF. Green and red arrows indicate the non-membrane proteins on MFGs/MFGM surfaces and in the whey phase. The white circle highlights the flocculation of MFGs/MFGM fragments, and the white square marks the aggregated and unattached membrane proteins. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.6 Hierarchical cluster analysis

The HCA results, illustrated in Fig. 7, reveal significant insights into the impact of ultrasonic treatments on the structural and compositional changes, rheological properties and instability indexes of MFGs/MFGM. The dendrogram generated by the HCA distinctly identified two main clusters: one encompassing the control sample and another grouping together the MFGs/MFGM under different ultrasonic intensities. Subsequently, 20 + 40 kHz/ 15 min treatment induced a notably differentiated impact on the analysed functionalities of MFGs/MFGM compared to other ultrasonic treatments. Additionally, the 40 kHz treatments formed a separated cluster, distinct from the 20 kHz and 20 + 40 kHz/ 5 min treatments. The latter group was further divided into two sub-clusters: one consisting solely of 20 kHz/ 5 min MFGs/MFGM, and another comprising MFGs/MFGM treated at both 20 kHz/ 15 min and 20 + 40 kHz/ 5 min.Fig. 7 Hierarchical cluster analysis of MFGs/MFGM ingredients obtained from ultrasound-treated bovine raw milk. The color scale differentiates values as high (red) to low (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

The heat map associated with the HCA results clearly indicated that the ultrasonic treatments applied in this study significantly influenced the structure, composition, rheological properties, and emulsion stability of the MFGs/MFGM. Among all treatments, the 20 + 40 kHz/15 min treatment stood out as having a more pronounced effect compared to the others.

4 Conclusions

Milk fat globules/membrane (MFGs/MFGM) materials derived from bovine raw milk, treated by varying ultrasonic intensities, 20 kHz, 40 kHz, or a combination of 20 kHz and 40 kHz for duration of 5 min and 15 min, exhibited different physicochemical properties, rheological behaviour and emulsion stability compared to the control.

20 kHz and 40 kHz simultaneous processing for 15 min significantly influenced the ester groups of lipids and the interactions of hydrogen-bonded carbonyl groups in esters. The PCA results for Amide I grouped the 15 min with the controls, and the 5 min ultra-sonicated MFGs/MFGM into another group. Nevertheless, qualitative analysis of the protein secondary structures within Amide I revealed inconsistent results; notably, the 15 min treatments induced more significant changes in the intramolecular β-sheet, random coil, and α-helix structure compared to 5 min treatment. This discrepancy underscores the need for the further research to elucidate the underlying causes of these contradictory outcomes.

Ultrasonic treatment promoted the transfer of membrane proteins into the rinsing phase and facilitated the adsorption of non-membrane proteins onto the surfaces of fat globules. Moreover, all the MFGs/MFGM exhibited shear-thinning behaviour, except for the 40 kHz treated MFGs/MFGM, which displayed Newtonian behaviour at higher shear rate, as indicated by flow behaviour indexes (n), of 1 or close to 1, regressed from the Herschel-Bulkley model. Physical stability of MFGs/MFGM emulsions was generally enhanced by ultrasonic treatment with an exception of 20 + 40 kHz/ 15 min treatment, which led to the aggregated membrane proteins, as observed in CLSM images. This differentiation highlights the potential of specific ultrasonic treatment parameters to modify dairy product characteristics in targeted ways, which could be crucial for optimizing processing strategies in the dairy industry.

CRediT authorship contribution statement

Yanjun Sun: Writing – original draft, Visualization, Validation, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Yrjö H. Roos: Writing – review & editing, Supervision. Song Miao: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

The following are the Supplementary data to this article:Supplementary Fig. 1

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Acknowledgements

The project was supported by '10.13039/501100001604 Teagasc -The Irish Agriculture and Food Development Authority (MDDT1392 )', 'The 10.13039/501100004543 China Scholarship Council (No.202008410212 )', and 'The Open Project Program of State Key Laboratory of Dairy Biotechnology (No. SKLDB2022–007)'.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.ultsonch.2024.107058.
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