LWT - Food Science and Technology 129 (2020) 109542

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LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

Characterisation of bovine and buffalo anhydrous milk fat fractions along T

with infant formulas fat: Application of differential scanning calorimetry,
Fourier transform infrared spectroscopy, and colour attributes
Abdelmoneim H. Alia,b,c, Wei Weia,b, Xingguo Wanga,b,∗
a
State Key Lab of Food Science and Technology, Jiangnan University, Wuxi, 214122, China
b
International Joint Research Laboratory for Lipid Nutrition and Safety, Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, School of
Food Science and Technology, Jiangnan University, Wuxi, 214122, China
c
Department of Food Science, Faculty of Agriculture, Zagazig University, 44511, Zagazig, Egypt

A R T I C LE I N FO A B S T R A C T

Keywords: In this study, bovine and buffalo milk-derived ghee were fractionated using dry and solvent fractionation
Anhydrous milk fat methods. Three different fractions including olein, stearin 1, and acetone washed-stearin (stearin 2) were ob-
Fractionation tained. The fatty acids composition, thermal profiles, and colour attributes of milk fat fractions were analysed
FTIR spectroscopy along with infant formulas’ fat. Furthermore, Fourier transform infrared spectroscopy was applied in the wa-
Differential scanning calorimetry
venumber region of 4000–500 cm−1 to discriminate the different fractions. The results showed significant
Colour
differences in fatty acids composition among the different types of fat, and stearin 2 fractions presented the
highest saturated fatty acids content (90.38 and 83.30% for bovine stearin 2 and buffalo stearin 2, respectively).
The different fractions of milk fat in addition to infant formulas fat demonstrated wide variations in their thermal
behavior. The chroma and yellowness index values of bovine ghee, olein, and stearin 1 were significantly higher
than those of the other fractions. The potential application of milk fat fractions as human milk fat substitutes
needs further investigation.

1. Introduction Mehta, 2009, pp. 527–559; Sharma, 1981).

Ghee is classified as an anhydrous milk fat contributing a prominent
proportion in the Egyptian diet. In Egypt, ghee is frequently prepared
from the butter of domestic water buffalos or dairy cows. According to
the Codex Alimentarius (2018), ghee is defined as the product entirely
manufactured from milk, cream, or butter through different processes,
which nearly cause a total removal of the non-fat solids and water, with
particularly developed physical structure and flavour. It can be pro-
duced through the heat treatment of cream or the butter obtained from
fresh or sour cream or dahi produced by milk fermentation (Srinivasan,
1976). From the chemical viewpoint, ghee is considered a complex
combination of glycerides, phospholipids, free fatty acids, sterols, vi-
tamins, carbonyls, hydrocarbons, tocopherol, carotenoids (in bovine
milk-derived ghee only), minor quantities of casein and few amounts of
phosphorus, iron, calcium, etc. Glycerides represent approximately
98% of the total contents, and the moisture content in ghee does not
exceed 0.3%. In the ~2% remaining ghee components, sterols (mainly
cholesterol) contribute 0.5% (Deosarkar, Khedkar, & Kalyankar, 2016;
The complicated triacylglycerols composition of milk fat makes it
inappropriate for several applications because it causes a wide melting
range, typically between −40 °C and 40 °C, though there are discrete
triacylglycerols with melting points beyond that range. Generally, the
high-melting components exhibit melting points more than 38 °C, and
they are usually used in the bakery industry (Munro & Illingworth,
1986; Rodenburg, 1973). While, the melting point of low-melting
components usually ranges between 21 and 28 °C. In this context, the
fractionation of milk fat has gained a rapid attention since the middle of
the 20th century. In the 21st century, the fractionated forms of milk fat
have been extensively used in diverse numerous applications (Hartel &
Kaylegian, 2001; Illingworth, Patil, & Tamime, 2009). Milk fat frac-
tionation could be attained by using different methods, for instance the
dry fractionation (Breitschuh & Windhab, 1998; Vanhoutte,
Dewettinck, Vanlerberghe, & Huyghebaert, 2003), solvent fractionation
(Cisneros, Mazzanti, Campos, & Marangoni, 2006), and the super-
critical carbon dioxide fractionation (Buyukbese, Rousseau, & Kaya,
2017). There are three sources of fat indicated on commercial infant

∗
Corresponding author. State Key Lab of Food Science and Technology, Jiangnan University, Wuxi, 214122, China.
E-mail address: xingguow@jiangnan.edu.cn (X. Wang).

https://doi.org/10.1016/j.lwt.2020.109542
Received 9 December 2019; Received in revised form 1 May 2020; Accepted 3 May 2020
Available online 06 May 2020
0023-6438/ © 2020 Elsevier Ltd. All rights reserved.
A.H. Ali, et al.
formulas labels, including vegetable oils formula, bovine milk formula,
and goat milk formula, which by the way could cause fatty acids
composition variations. It is particularly worth observing that each
group has a variety of fatty acids composition, and bovine milk- and
goat milk-based formulas are not entirely dairy fats. In order to mimic
the human milk fatty acids composition, bovine milk formulas and goat
milk formulas are usually supplemented by small quantities of vege-
table oils (Sun et al., 2016).
Fourier transform infrared (FTIR) spectroscopy coupled with che-
mometrics, such as soft independent modeling of class analogy
(SIMCA), partial least square (PLS), and principal component analysis
(PCA) models has been applied for the detection of several food raw
materials adulteration (Jaiswal et al., 2015; Jha, Jaiswal, Borah,
Gautam, & Srivastava, 2015), edible oils in extra virgin olive oil
(Rohman & Man, 2010), lard in chocolate and related-products (Man,
Syahariza, Mirghani, Jinap, & Bakar, 2005), detection of virgin coconut
oil adulteration with paraffin oil (Jamwal, Kumari, Dhaulaniya, Balan,
& Singh, 2020), and in the characterisation of ghee (Antony, Sharma,
Mehta, Ratnam, & Aparnathi, 2018; Upadhyay, Jaiswal, & Jha, 2016).
Colour is considered as an important parameter for consumer's ac-
ceptability. It was reported that consumers were less sensitive to the
changes in fat content when the associated appearance variances were
ignored, approving the influence of colour on the organoleptic prop-
erties of dairy products (Cheng, Barbano, & Drake, 2018). The colour of
ghee depends on the type of milk used for production, since bovine
ghee looks yellow owing to β-carotene presence (Deosarkar et al.,
2016). Moreover, milk fat fractionation resulted in β-carotene separa-
tion with the olein fraction which by the way attains a golden-yellow
colour, while the hardest stearin fractions are almost white. The colour
of fractionated milk fat plays a functional role in several food appli-
cations, since the soft fractions are mainly used in confectionery, while
the hard fractions are used in laminated and puff pastries (Early, 2012,
pp. 417–445).
The main objective of the present study was to prepare different
fractions of bovine and buffalo milk fat by using the dry and solvent
fractionation methods. The obtained fractions were compared with the
fat extracted from commercially available 0–6 months' infant formulas
and 6–12 months’ infant formulas regarding their fatty acids composi-
tion, thermal profiles, and colour attributes. In addition, FTIR spec-
troscopy was applied in a wavenumber range of 4000–500 cm−1 to
characterise the different fractions of milk fat.
2. Material and methods
2.1. Materials
Fresh raw bovine and buffalo milk were obtained from a local farm
(Zagazig, Egypt). Six different brands of infant formulas (three 0–6
months' infant formulas and three 6–12 months’ follow-on formulas)
available in the Egyptian market were collected. Starter cultures in-
cluding Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. lactis
biovar diacetylactis, and Leuconostoc mesenteroides subsp. cremoris were
purchased from Christian Hansen Laboratory Copenhagen, Denmark.
All chemicals and solvents used in extraction and analysis were of high
purity and analytical grade.
2.2. Fractionation of milk fat
Bovine and buffalo milk were immediately pasteurised at 70 °C for
5 min and cooled to 42 °C. Cream was separated from milk by using an
Alfa Laval separator (2010.1.21A, Alfa Laval Separator Co., Ltd.
Hamilton, New Zealand), and the obtained cream was pasteurised at
90–95 °C for 15 s, cooled to 20 °C, inoculated with the starter cultures
(2 g/100 g), and incubated for 10–14 h until a pH value of 4.4–4.6 was
reached. Then, the cream was churned in a butter churner at 8–10 °C,
and buttermilk was separated after butter formation. The produced
2
LWT - Food Science and Technology 129 (2020) 109542
butter was heated at 60 °C in a thermostatically controlled water bath
(SHA-B, Shanghai Jiulian Precision Instrument Co., Ltd. China). Butter
was then clarified at temperatures ranging from 110 to 120 °C with
continuous stirring, and the foam formed above the oil phase was fre-
quently collected with a spoon. The oil phase above the aqueous phase
was allowed to separate for 2 h, and then filtered through several layers
of cheese cloth into amber coloured glass bottles and stored at 4 °C until
used.
The fractionation of milk fat was performed according to the
method of Sebben, Gao, Gillies, Beattie, and Krasowska (2019) with
slight modifications. Three hundred g of the obtained bovine and buf-
falo ghee were weighed in two large beakers and transferred to a
temperature-controlled chiller bath. In the beginning, the bath was
heated to 70 °C for 40 min in order to melt the ghee and erase crystal
nuclei memory. Bovine- and buffalo-ghee samples were then cooled at a
constant rate to 28 °C over a period of 10 h, and kept for 4 h at 28 °C.
The resulting mixture of solid and liquid fat was subsequently filtered
under vacuum through a 0.45 μm Whatman filter paper for the initial
separation of liquid fat (olein). The obtained product after the initial
filtration was denoted as hard milk fat (stearin 1). Half of the stearin 1
fraction was transferred into a clean bottle, and the remaining half was
subjected to solvent washing. Briefly, the remaining stearin 1 was wa-
shed 3 times by using cold acetone (~4 °C), sonicated for 2 min, and
then under vacuum-filtered. The acetone-washed stearin (stearin 2) was
transferred to a clean beaker and further dried in a vacuum desiccator
in order to remove any remaining solvent. The different fractions of
milk fat were stored in amber glass tubes under N2 to avoid oxidation.
2.3. Analysis of the fatty acids composition
The extraction of lipids from infant formula samples was carried out
according to the procedure reported by Folch, Lees, and Stanley (1957).
Briefly, the samples were dissolved in a mixture of chloroform/me-
thanol (200 mL; 2:1), and the mixture was thoroughly mixed for 15 min
and centrifuged at 2054 xg for 15 min (Centrifuge 5810R, Eppendorf
AG, Germany). The organic phase-containing fat was collected and
equilibrated with fourth volume of a saline sodium chloride solution
(0.86 g/100 g). The lower chloroform layer was then filtered, and the
solvent was evaporated in a rotary evaporator under vacuum at 40 °C.
Fatty acids methyl esters of milk fat fractions and infant formulas fat
were prepared and analysed by using the gas chromatography as re-
ported by Ali et al. (2018). Briefly, 50 mg of the obtained fractions were
weighed in sealable tubes, and then 2 mL of hexane and 0.5 mL of
KOH–CH3OH (2 mol/L) were added. The mixture was vigorously mixed
for 5 min using a vortex, and then 3 mL of a saturated NaCl solution was
added for phase separation. The supernatant was collected, dried over
sodium sulfate anhydrous, and then injected to an Agilent 7820A gas
chromatography (Agilent Corp., USA) equipped with an autosampler,
an ionic liquid capillary column (TRACE TR-FAME,
60 m × 0.25 mm × 0.25 μm, Thermo Fisher, USA), and a flame io-
nisation detector. The oven temperature was adjusted at 60 °C, and the
running time of each sample was set at 40 min. The injector and de-
tector temperatures were adjusted at 250 °C. The analysis of fatty acids
was accomplished using a temperature gradient program from an initial
temperature of 60 °C (held for 3 min), elevated by 5 °C/min to 175 °C
(held for 15 min), and then elevated by 2 °C/min to a final temperature
of 220 °C for 10 min. Nitrogen was used as a carrier gas with a flow rate
of 1.2 mL/min; split ratio 1:100; detector gas 30 mL/min hydrogen;
400 mL/min air and 25 mL/min nitrogen. Fatty acids identification was
achieved by comparing the retention times of gas chromatography
peaks with those of corresponding known standards.
2.4. Crystallisation and melting profiles
The thermal profile analysis of milk fat fractions and infant formulas
fat was conducted by using a differential scanning calorimetry (DSC
 A.H. Ali, et al.

Table 1
Fatty acids composition (%) of bovine milk fat fractions, buffalo milk fat fractions, and infant formulas fat.
Fatty acids Types of fat

Bovine ghee Bovine olein Bovine stearin 1 Bovine stearin 2 Buffalo ghee Buffalo olein Buffalo stearin 1 Buffalo stearin 2 IF 1 IF 2

c c d c b c c d e
4:0 4.28 ± 0.01 4.59 ± 0.04 3.80 ± 0.01 4.29 ± 0.03 5.62 ± 0.05 4.70 ± 0.01 4.80 ± 0.03 3.99 ± 0.02 1.75 ± 0.03 6.70 ± 0.05a
6:0 1.01 ± 0.03a 1.13 ± 0.01a 0.90 ± 0.04a 0.12 ± 0.01b 1.02 ± 0.01a 1.18 ± 0.02a 1.00 ± 0.02a 0.45 ± 0.01b 0.01 ± 0.01c –
8:0 0.61 ± 0.01a 0.23 ± 0.02a 0.56 ± 0.01a 0.07 ± 0.02b 0.47 ± 0.03a 0.53 ± 0.02a 0.45 ± 0.01a 0.22 ± 0.02a 0.72 ± 0.04a 0.85 ± 0.01a
10:0 1.37 ± 0.03a 1.47 ± 0.01a 1.29 ± 0.03a 0.29 ± 0.01a 0.97 ± 0.01a 1.08 ± 0.01a 0.96 ± 0.03a 0.61 ± 0.04b – –
12:0 1.79 ± 0.02b 1.86 ± 0.04b 1.75 ± 0.01b 0.80 ± 0.01c 1.55 ± 0.04b 1.65 ± 0.03b 1.55 ± 0.01b 1.34 ± 0.01b 12.27 ± 0.01a 12.11 ± 0.03a
14:0 7.89 ± 0.04b 7.85 ± 0.10b 7.98 ± 0.05b 7.43 ± 0.04b 9.64 ± 0.11a 9.67 ± 0.07a 9.80 ± 0.10a 10.73 ± 0.20a 4.71 ± 0.02c 4.70 ± 0.01c
14:1(n-5) 1.01 ± 0.01a 1.09 ± 0.02a 0.95 ± 0.01a 0.38 ± 0.01b 1.18 ± 0.03a 1.29 ± 0.02a 1.16 ± 0.02a 0.82 ± 0.01a 0.03 ± 0.00c 0.04 ± 0.02c
15:0 1.94 ± 0.01a 1.96 ± 0.04a 1.93 ± 0.03a 1.74 ± 0.04ab 2.46 ± 0.02a 2.50 ± 0.01a 2.47 ± 0.03a 2.45 ± 0.00a – –
15:1(n-7) 0.50 ± 0.02a 0.49 ± 0.01a 0.49 ± 0.01a 0.47 ± 0.01a 0.61 ± 0.01a 0.61 ± 0.02a 0.61 ± 0.01a 0.62 ± 0.03a – –
16:0 29.76 ± 0.31de 27.89 ± 0.11e 31.08 ± 0.22d 46.25 ± 0.26a 33.13 ± 0.20c 32.06 ± 0.30cd 34.02 ± 0.22c 40.58 ± 0.30b 27.45 ± 0.11e 26.64 ± 0.20e
16:1(n-7) 1.24 ± 0.03b 1.42 ± 0.02b 1.14 ± 0.01b 0.03 ± 0.02c 2.14 ± 0.02a 2.41 ± 0.03a 2.05 ± 0.04a 0.13 ± 0.02c 0.12 ± 0.02c 0.13 ± 0.02c
17:0 0.93 ± 0.01a 0.86 ± 0.03a 0.96 ± 0.02a 1.50 ± 0.01a 1.15 ± 0.05a 1.07 ± 0.01a 1.18 ± 0.03a 1.53 ± 0.01a 0.08 ± 0.01b 0.11 ± 0.04b

3
17:1(n-7) 0.36 ± 0.03a 0.41 ± 0.03a 0.34 ± 0.04a 0.05 ± 0.02b 0.45 ± 0.01a 0.51 ± 0.04a 0.44 ± 0.03a 0.21 ± 0.02a 0.03 ± 0.03b 0.06 ± 0.01b
18:0 13.18 ± 0.11c 11.58 ± 0.15d 14.16 ± 0.10c 26.97 ± 0.20a 13.07 ± 0.11c 11.53 ± 0.09d 13.75 ± 0.21c 20.52 ± 0.15b 4.54 ± 0.04e 4.58 ± 0.05e
18:1(n-9) 28.76 ± 0.20c 31.50 ± 0.19a 27.46 ± 0.15c 7.55 ± 0.10g 22.49 ± 0.20e 24.95 ± 0.12d 21.84 ± 0.16e 13.30 ± 0.21f 32.70 ± 0.13a 30.34 ± 0.24b
18:2t 0.62 ± 0.03a 0.53 ± 0.04a 0.61 ± 0.04a 0.60 ± 0.02a 0.67 ± 0.02a 0.63 ± 0.03a 0.63 ± 0.01a 0.59 ± 0.03a 0.01 ± 0.00b 0.05 ± 0.01b
18:2(n-6) 2.91 ± 0.07b 3.16 ± 0.01b 2.79 ± 0.01b 0.42 ± 0.01d 1.54 ± 0.01c 1.66 ± 0.04c 1.50 ± 0.01c 0.64 ± 0.02d 13.99 ± 0.06a 12.29 ± 0.10a
18:3(n-6) 0.26 ± 0.01a 0.29 ± 0.03a 0.25 ± 0.02a 0.02 ± 0.04b 0.32 ± 0.00a 0.36 ± 0.01a 0.31 ± 0.03a 0.10 ± 0.04b – –
18:3(n-3) 0.30 ± 0.02b 0.34 ± 0.01b 0.28 ± 0.01b 0.04 ± 0.01c 0.28 ± 0.02b 0.32 ± 0.03b 0.27 ± 0.02b 0.12 ± 0.01bc 1.16 ± 0.01a 0.96 ± 0.05a
20:0 1.06 ± 0.01a 1.10 ± 0.03a 1.05 ± 0.04a 0.92 ± 0.02a 0.97 ± 0.01a 1.00 ± 0.04a 0.97 ± 0.03a 0.88 ± 0.02a 0.33 ± 0.02b 0.33 ± 0.02b
20:1(n-9) 0.23 ± 0.04a 0.26 ± 0.00a 0.22 ± 0.00a 0.07 ± 0.03b 0.27 ± 0.03a 0.29 ± 0.02a 0.26 ± 0.01a 0.17 ± 0.03ab 0.11 ± 0.01ab 0.10 ± 0.01ab
SFA 63.82 ± 0.25e 60.51 ± 0.39f 65.46 ± 0.22d 90.38 ± 0.32a 70.05 ± 0.16c 66.97 ± 0.32d 70.95 ± 0.21c 83.30 ± 0.13b 51.85 ± 0.24h 56.02 ± 0.18g
MUFA 32.09 ± 0.20b 35.17 ± 0.16a 30.60 ± 0.10c 8.54 ± 0.12f 27.14 ± 0.05d 30.06 ± 0.20c 26.35 ± 0.09d 15.24 ± 0.07e 32.99 ± 0.12b 30.68 ± 0.05c
PUFA 4.09 ± 0.10c 4.31 ± 0.08c 3.93 ± 0.04c 1.08 ± 0.02e 2.81 ± 0.01d 2.97 ± 0.02cd 2.70 ± 0.04d 1.45 ± 0.01e 15.16 ± 0.06a 13.30 ± 0.11b
LA/ALA 9.62 ± 0.20c 9.31 ± 0.15c 9.83 ± 0.25c 11.76 ± 0.20b 5.57 ± 0.06d 5.21 ± 0.12d 5.57 ± 0.05d 5.32 ± 0.10d 12.03 ± 0.25a 12.74 ± 0.15a

Means with different superscript letters in the same row are significantly different (P < 0.05).
Bovine stearin 1, bovine stearin obtained after filtration; Bovine stearin 2, acetone washed-bovine stearin; Buffalo stearin 1, buffalo stearin obtained after filtration; Buffalo stearin 2, acetone washed-buffalo stearin; IF 1,
0–6 months formulas; IF 2, 6–12 months formulas.
SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; LA/ALA, linoleic acid/α-linolenic acid.
LWT - Food Science and Technology 129 (2020) 109542
A.H. Ali, et al.
Fig. 1. Crystallisation (A), and melting (B) profiles of bovine milk fat fractions. Bovine ghee (black curve), bovine olein (red curve), bovine stearin 1 (blue curve),
bovine stearin 2 (green curve). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Q2000 V4.7A Build 121, TA Instruments, New Castle, Delaware, USA).
During the analysis of each sample, the system was purged by N2 with a
flow rate of 20 mL/min, simultaneously the gas was used to decrease
the system temperature owing to its ability to act as a refrigerant. The
system was calibrated using indium (melting point = 156.6 °C,
ΔHf = 28.45 J/g). The melted fat samples (5–10 mg) were weighed and
hermetically sealed in aluminum measuring pans, and in the same time
an empty sealed pan was used as a reference. The DSC technique was
applied to acquire heat flow curves contrasted with temperature curves
(Ali et al., 2018). The applied temperature/time program for the non-
isothermal profile of the different samples was in this manner; the
samples were initially heated to 60 °C and kept for 15 min in order to
guarantee an entirely liquid state and eradicate all the crystal nuclei.
Then, the temperature was decreased by cooling to −40 °C at a rate of
10 °C/min with holding for 5 min, and finally the samples were heated
back to 60 °C at a rate of 10 °C/min. The obtained thermographs were
analysed using the thermal solutions software (TA Instruments, V 4.7A).
4
LWT - Food Science and Technology 129 (2020) 109542
2.5. FTIR spectra acquisition
The neat forms of milk fat fractions and infant formulas fat were
applied on the Diamond crystal cell attenuated total reflectance (ATR)
crystal having a path length of 1.66 mm. The absorption spectra of the
different samples were recorded by using a Nicolet FTIR spectroscope
(Nexus 670, Waltham, MA, USA) at a scan speed of 0.2 cm/s and a
resolution of 4 cm−1. Firstly, the samples were heated at 60 °C in order
to keep them at entirely molten phase during the spectra acquiring.
Sixteen scans were recorded for each sample in the wavenumber range
of 4000−500 cm−1. The obtained spectra were subtracted against a
background air spectrum. For spectra analysis, OMNIC Nicolet Software
(version 8.1) was used. The ATR crystal was in situ cautiously cleaned
by wiping it using a soft tissue soaked with isopropanol, after that it was
completely dried before acquiring the subsequent sample spectra.
A.H. Ali, et al.
Fig. 2. Crystallisation (A), and melting (B) profiles of buffalo milk fat fractions. Buffalo ghee (black curve), buffalo olein (red curve), buffalo stearin 1 (blue curve),
buffalo stearin 2 (green curve). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
2.6. Colour measurements
The colour values of the different milk fat fractions and infant for-
mulas fat were determined by using a high accuracy colorimeter
(UltraScan PRO, HunterLab; Shanghai Beifu Chem. Technol. Ltd. Co.),
including L, a* and b* values. The instrument was firstly calibrated by
using black and white references. The results were described as L
(lightness), a* (redness to greenness), and b* (yellowness to blueness).
Three reads were taken for each sample during the analysis. a* and b*
values were used to calculate hue angle (h*) and chroma (C*). The
mathematic h* and C* were calculated from the formulas;
h* = arctan [b*/a*] and C* = [a*2 + b*2]0.5 as reported by Ahmed,
Akter, Lee, and Eun (2010).
The colour of milk fat fractions and infant formulas fat was ex-
pressed as the whiteness index (WI) using the formula;
WI = 100 – [(100˗L)2 + a*2 + b*2]0.5
In addition, the yellowness index (YI) was calculated from the
5
LWT - Food Science and Technology 129 (2020) 109542
formula;
YI = 142.86 x (b*/L) (Francis & Clydesdale, 1975).
2.7. Statistical analysis
The different analyses were repeated three times, and the obtained
results were described as mean values ± standard deviations. One-way
analysis of variance (ANOVA) test was performed to analyse the ex-
perimental data, followed by Duncan's multiple range test by using the
CoStat statistical analysis software (version 6.4, Monterey, CA, USA).
The results were considered to be statistically significant for P values of
less than 0.05. In addition, SIMCA software (version 14.1.0.2047, MKS
Umetrics AB, Malmö, Sweden) was applied to perform the PCA of FTIR
spectral data.
A.H. Ali, et al.
Fig. 3. Crystallisation and melting profiles of infant formulas fat. 0–6 months'
formulas (black curve), 6–12 months' formulas (red curve). (For interpretation
of the references to colour in this figure legend, the reader is referred to the
Web version of this article.)
3. Results and discussion
3.1. Analysis of fatty acids composition
The fatty acids composition of bovine and buffalo milk fat fractions
along with infant formulas fat is presented in Table 1. The obtained
results show that there were significant variations in the fatty acids
composition between the different samples. The major fatty acids in
both bovine and buffalo milk fat fractions were palmitic, oleic, stearic,
myristic, and butyric. On the other hand, oleic, palmitic, linoleic, and
lauric were the major fatty acids in infant formulas fat. The stearin 2
fractions possessed significantly (P < 0.05) higher amounts of satu-
rated fatty acids (making up 90.38% in bovine stearin 2 and 83.30% in
buffalo stearin 2) and significantly (P < 0.05) lower polyunsaturated
fatty acids contents (making up 1.08% in bovine stearin 2 and 1.45% in
buffalo stearin 2). The higher concentration of saturated fatty acids
confers better oxidation stability to the stearin fractions when com-
pared to the regular natural anhydrous milk fat, which considers an
advantage from the technological viewpoint. It was also shown that
bovine and buffalo milk-derived olein contained significantly
(P < 0.05) higher amounts of the monounsaturated fatty acids (35.17
and 30.06%, respectively) as compared to the other fractions of milk
fat.
As compared to the different fractions of bovine and buffalo milk
fat, infant formulas fat demonstrated significantly (P < 0.05) higher
polyunsaturated fatty acids content (making up 15.16% in 0–6 months'
formulas and 13.30% in 6–12 months' formulas). This was attributed to
their higher content of linoleic acid, since 0–6 months' formulas con-
tained 13.99% and 6–12 months' formulas contained 12.29%. Sun et al.
(2016) reported that the content of linoleic acid ranged between 12.73
and 25.65% for 0–6 months' infant formulas and from 10.18 to 22.64%
for 6–12 months' formulas. In human milk fat substitutes, it is re-
commended that the ratio between linoleic acid and α-linolenic acid
should be within the range of 5:1 and 15:1 owing to the competition
between these two fatty acids for the same elongation and desaturation
enzymes (Koletzko et al., 2005). It is shown from the obtained results
that the ratio between linoleic acid and α-linolenic acid was in the
recommended range, and infant formulas fat displayed the highest ratio
(12.03% in 0–6 months' formulas and 12.74% in 6–12 months' for-
mulas). It was also noted that 6–12 months' formulas had a significantly
higher content of butyric acid (6.70%) in comparison with 0–6 months'
formulas (1.75%) and the different bovine and buffalo milk fat frac-
tions. While, the content of butyric acid in vegetable oils-based 6–12
months’ formulas ranged between 0 and 3.66% (Sun et al., 2016). The
differences in the fatty acids composition in the present study and those
reported in other literature may attributed to several reasons, such as
6
LWT - Food Science and Technology 129 (2020) 109542
the animal species, animal diet, season and period of lactation, and the
analysis methods for milk fat fractions, and the source and composition
of the oils used in infant formulas preparation.
3.2. Crystallisation and melting profiles
The anhydrous milk fat has a wide range of melting points from
−40 °C to 40 °C, different from the pure molecules that demonstrate a
sharp melting temperature. Anhydrous milk fat becomes entirely solid
until the temperature reaches −40 °C or below; and in order to guar-
antee the complete melting of triacylglycerols, it should be heated at
least to 40 °C. The melting and crystallisation profile of milk fat has
been studied by using the DSC (Lopez, Bourgaux, Lesieur, & Ollivon,
2007; Ten Grotenhuis, Van Aken, Van Malssen, & Schenk, 1999). A
characteristic crystallisation curve of anhydrous milk fat displays two
major exotherms through cooling, referring to a group of triacylgly-
cerols with high crystallisation temperatures and another group with
low crystallisation temperatures. While, a distinctive melting curve of
milk fat displays three patterns of overlapped endotherms that can be
classified based on their melting points into low melting point-fractions,
medium melting point-fractions, and high melting point-fractions
(Lopez, 2018). These curves correspond to a large group of triacylgly-
cerols that separately melt and act as solid solutions. The low melting
point endotherm refers to the melting of triacylglycerols with a high
content of long-chain unsaturated fatty acids, such as linoleic and li-
nolenic and short-chain saturated fatty acids, such as butyric and ca-
proic. The low melting point-fractions are existed in liquid state at room
temperature. The main triacylglycerols that melt in the medium melting
point fraction comprise one short-chain fatty acid or one unsaturated
fatty acid. The high melting point-fractions are distinguished by the
presence of higher quantities of the long-chain saturated fatty acids.
The melting of triacylglycerols in the medium melting point fraction
plays a significant role in the mouth feeling properties of milk fat (Sato,
2018).
Figs. 1–3 show the crystallisation and melting curves for bovine
milk fat fractions, buffalo milk fat fractions, and infant formulas fat,
respectively. The cooling and heating peaks of the natural anhydrous
milk fat or ghee (black curves) follow the typical profile of milk fat
thermal behavior. It is obvious that the thermal profiles of milk fat have
been influenced by the fractionation, and the impact of solvent washing
procedure was certainly identified. The first liquid fraction (olein, red
curve) of both bovine and buffalo milk fat demonstrated one peak at
11.45 °C and 13.68 °C, respectively, corresponding to the low crystal-
lisation temperature triacylglycerols. While, the hard milk fat fraction
(stearin 1, blue curve) possessed two crystallisation curves at 21.58,
11.49 °C and 21.57, 13.55 °C for bovine and buffalo milk fat, respec-
tively. The acetone washed-stearin (stearin 2, green curve) exhibited
one major strong crystallisation curve at 36.49 °C and 31.38 °C, re-
spectively corresponding to the high crystallisation temperature tria-
cylglycerols. Regarding the melting profiles, it was shown that the
melting curves of the stearin 1 fractions (blue curve) were relatively
similar to that of the raw ghee (black curve). On the other hand, pre-
dominant endothermic curves for the acetone washed-stearin (green
curve) were observed at 49.92 °C and 46.51 °C, respectively. Further-
more, the stearin 2 fractions exhibit low intensity peaks associated with
the low melting points-triacylglycerols. Similar results were reported by
Sebben et al. (2019). Olein fractions were enriched in short-chain and
unsaturated fatty acids, while stearin fractions were enriched in long-
chain fatty acids. Lopez, Bourgaux, Lesieur, Riaublanc, and Ollivon
(2006) reported that compared to the whole milk fat, olein fraction
comprised higher amounts of triacylglycerols containing (a) two
monounsaturated long-chain fatty acids, (b) one monounsaturated and
two medium-chain fatty acids, and (c) a short chain fatty acid and two
saturated long-chain fatty acids or one monounsaturated fatty acid or
two monounsaturated long-chain fatty acids. Stearin fraction comprised
higher amounts of triacylglycerols containing (a) three saturated long-
A.H. Ali, et al. LWT - Food Science and Technology 129 (2020) 109542

Fig. 4. Total ATR-FTIR spectra of bovine milk fat fractions, buffalo milk fat fractions and infant formulas fat in the wavenumber range 4000−500 cm−1 (A), bovine
milk fat fractions spectra (B), buffalo milk fat fractions spectra (C), and infant formulas fat spectra (D).

7
A.H. Ali, et al.
Fig. 5. Principal component scores plot of bovine milk fat fractions, buffalo milk fat fractions, and infant formulas fat in the wavenumber range of 2000−1500 cm−1.
chain fatty acids, (b) one or two saturated medium-chain fatty acids and
a saturated long-chain fatty acid, and (c) one monounsaturated long-
chain fatty acid and two saturated long-chain fatty acids.
The crystallisation and melting profiles of infant formulas fat are
shown in Fig. 3. Both the two types of infant formulas exhibited dif-
ferent thermal patterns. The crystallisation peaks of 0–6 months for-
mulas fat were observed at 8.38 and −14.40 °C, while they were de-
tected at −0.03 and −19.40 °C for the 6–12 months formulas fat.
Regarding the melting profile, low melting fractions, medium melting
fractions, and high melting fractions were observed at 0.09, 13.87, and
30.42 °C, respectively for 0–6 months' formulas fat. On the other hand,
the 6–12 months’ formulas fat showed its melting curves at −20.35,
1.06, and 21.46 °C, respectively. Both the two types of infant formulas
fat melted at the normal body temperature (about 37 °C). Human milk
fat substitutes with high melting temperatures are not easy to be han-
dled, transported and mixed with other non-lipid constituents during
the preparation of the formula (Jensen & Patton, 2000; Tomarelli,
Meyer, Weaber, & Bernhart, 1968).
3.3. FTIR spectra of milk fat fractions and infant formulas fat
FTIR spectroscopy is one of the non-destructive procedures ex-
tensively applied to acquire molecular information of constituents with
slight sample preparation requirements. Progressive FTIR method in
microscopic pattern further allows in-depth identification of chemical
compositions, mainly proteins and lipids distribution, and their con-
firmation in diverse layers of the micro particles (Leroy, Lafleur, Auger,
Laroche, & Pouliot, 2013). In the same way, FTIR is distinguished by its
effectiveness in offering comprehensive information about the nature
and content of lipids existing in a material, since the absorption peak
corresponding to the carbonyl bonds appears in the range between
1800 and 1700 cm−1 considers a distinctive pattern for the total lipids.
While, the olefinic double bonds (−HC]CH–) of the unsaturated fatty
acids show their absorption peaks in the range between 3050 and
3000 cm−1. Furthermore, FTIR spectra can be applied to obtain com-
prehensive data about the molecular structure and chemical bonding of
materials, which are not easy to be accomplished by using the regular
microscopic techniques (Araki et al., 2015). Additionally, FTIR proce-
dure can be applied to perform multi-component investigates (Rohman
& Man, 2010).
The illustrative peaks for milk fat fractions and infant formulas fat in
the mid-infrared region specifically 4000–500 cm−1 attained by ATR-
8
LWT - Food Science and Technology 129 (2020) 109542
FTIR are presented in Fig. 4. The spectra patterns of the different types
of fat were similar and exhibited distinguishing absorption bands of
edible oils (Upadhyay et al., 2016). Nevertheless, the obtained peak
intensities within the wavenumber range were shown to be non-over-
lapping peaks demonstrating that no two samples exhibited precisely
comparable absorbance in the examined wavenumber range. The two
types of infant formulas fat showed a major peak in the wavenumber
range of 753–756 cm−1 in comparison with the other fractions of milk
fat (Fig. 4D). The obtained distinctive spectra in the mid-infrared FTIR
spectroscopy are corresponding to particular wavenumbers absorbed by
particular functional groups. The robust absorptions for oils and fats are
detected in the region between 3000 and 2800 cm−1 arising from the
stretching vibrations of C–H groups. In addition, other functional
groups display an absorption at 2959 cm−1 correspond to C–H(CH3),
and the absorption peaks observed at 2915 and 2848 cm−1 refer to
–C–H(CH2) (Nurrulhidayah et al., 2013). Furthermore, the peaks de-
tected at 1744 and 3004 cm−1 are described to be related to the C]O
stretching vibrations of aliphatic esters and C–H stretching vibrations of
the cis-CH out-of-plane vibration, respectively (Koca, Kocaoglu-Vurma,
Harper, & Rodriguez-Saona, 2010). The absorption of two asymmetric
vibrations comprising of C–C(=O)–O and O–C–C of the C–O bond of
esters; and some bending vibrations of methylene groups take place in
the absorption wavenumber between 1320 and 1145 cm−1 respec-
tively, as previously stated by Guillen and Cabo (1997). The absorption
peaks observed at 1467, 1382, 1150, 1117, 1097, and 1057 cm−1 are
corresponding to C–H–CH2, C–H–CH3, =C–H– (cis), –C–H-bending,
–C–H–, and –C–O–CH2– functional groups, respectively. Nurrulhidayah
et al. (2013) suggested that the wider range of 1500–1000 cm−1 can be
used as the fingerprint areas to detect butter adulteration with beef fat.
Conversely, in case of infant formulas fat, 753–756 cm−1 could be
considered a significant region for the differentiation between infant
formulas fat with bovine and buffalo milk fat fractions because of the
existence of the peak in this wavenumber range as discussed above. The
observed variations in the spectral regions of milk fat fractions and
infant formulas fat could be attributed to the differences in the com-
position of these fat fractions regarding chain length and unsaturation
degree. Antony et al. (2018) observed the FTIR spectra for two different
kinds of ghee (buffalo and bovine) to analyse the peaks and their ab-
sorbance strengths. It was indicated that the total peaks number and
their locations were nearly similar, and no variances in the spectra of
the two types of ghee samples were detected.
PCA is a statistical technique comprising factor analysis, which has
A.H. Ali, et al. LWT - Food Science and Technology 129 (2020) 109542

been acknowledged as an investigative tool in multivariate statistical

−4.74 ± 0.00c

77.43 ± 0.08d
31.22 ± 0.13d
90.00 ± 0.02b

19.67 ± 0.08c
76.44 ± 0.04c
20.23 ± 0.08c
analysis and considered a mathematical accurate method displaying
differences in the dataset using few factors (Granato, Santos, Escher,
Ferreira, & Maggio, 2018). PCA of the spectral data was performed in
IF 2

the wavenumber range of 2000−1500 cm−1 in order to observe sam-

ples clustering (Fig. 5). The figure shows the scores plot for the first two
principal components (PCs) of milk fat fractions and infant formulas fat
−5.87 ± 0.02c

35.61 ± 0.28d
b

74.48 ± 0.17e
22.10 ± 0.16c
75.12 ± 0.05c
22.87 ± 0.17c
88.67 ± 0.05

with 78% of the data described by PC1 and 12% by PC2. The observed
variances among the different fractions indicate the importance of FTIR
in the characterisation of milk fat fractions and infant formulas fat. The
IF 1

variances in FTIR spectra which are responsible for the clusters ob-
served in PCA score plots could be identified by the analysis of PC plots
together with the FTIR spectra. It was shown that stearin 2 fractions
Buffalo stearin 2

89.22 ± 0.00a

90.01 ± 0.01a
12.26 ± 0.03f

were clustered in the lower left-hand corner, which reveals the corre-
94.12 ± 0.01
0.11 ± 0.02b
8.08 ± 0.02e

8.08 ± 0.02e

lation with their higher content of saturated fatty acids as compared to

the other fractions of milk fat and infant formulas fat. Furthermore,
bovine stearin 1, buffalo ghee, buffalo olein, and buffalo stearin 1
fractions were clustered near to the center, reflecting their comparable
saturated fatty acids content. On the other hand, the two categories of
Buffalo stearin 1

−2.17 ± 0.03c

infant formulas fat were located in the lower right-hand corner, which
13.25 ± 0.08d

13.43 ± 0.08d
b

80.71 ± 0.20b

83.24 ± 0.00b
21.04 ± 0.11e
89.96 ± 0.10

L, lightness; a*, redness to greenness; b*, yellowness to blueness; h*, hue angle; C*, chroma or saturation; WI, whiteness index; YI, yellowness index.

might attributed to their significantly (P < 0.05) higher content of

polyunsaturated fatty acids than bovine and buffalo milk fat fractions.

3.4. Colour measurements of milk fat fractions and infant formulas fat

The different colour measurement parameters (L, a*, b*, h*, C*, WI,
−5.10 ± 0.06c
d

59.20 ± 0.21d

72.15 ± 0.11f
16.51 ± 0.02f
74.00 ± 0.13

8.55 ± 0.02e

9.96 ± 0.05e

and YI) of milk fat fractions and infant formulas fat are shown in
Buffalo olein

Table 2. The highest L values were observed for the buffalo stearin 2,
bovine stearin 2, and 6–12 months formula making up 94.12, 93.08,
and 90.00, respectively. While, olein possessed the lowest values (68.10
and 74.00 for bovine and buffalo milk fat, respectively) as compared to
the other fractions of milk fat. Regarding the a* value, bovine milk fat
−3.44 ± 0.01c
13.27 ± 0.02d

13.71 ± 0.02d
b

21.84 ± 0.01e
75.48 ± 0.06c

80.99 ± 0.02c
86.83 ± 0.05

fractions had the highest values with positive numbers, while buffalo
Buffalo ghee

milk fat fractions and infant formulas fat showed lower a* values with
negative numbers. Büyükbeşe, Emre, and Kaya (2014) reported that the
a* value of bovine anhydrous milk fat was −3.06. In addition, bovine
ghee, olein, and stearin 1 demonstrated significantly higher b* values
Means with different superscript letters in the same row are significantly different (P < 0.05).

(44.55, 47.43, and 34.25, respectively) than the other fractions. Infant
Bovine stearin 2

86.52 ± 0.04a

89.33 ± 0.00a
12.44 ± 0.01f

formulas fat showed significantly lower h* values (75.12 and 76.44,

93.08 ± 0.01
0.49 ± 0.00b
8.10 ± 0.01e

8.12 ± 0.01e

respectively) as compared to bovine and buffalo milk fat fractions. The

C* and YI values of bovine ghee, olein, and stearin 1 fractions were
significantly higher than bovine stearin 2, buffalo milk fat fractions, and
infant formulas fat, meaning that their colour looked dark. The yellow
colour of bovine milk fat fractions was attributed to the coloured
Bovine stearin 1

34.25 ± 0.01b

34.36 ± 0.02b
62.65 ± 0.00g
85.36 ± 0.02a

compounds that predominantly consist of carotenoids and comparable

57.33 ± 0.01c
85.35 ± 0.02
2.78 ± 0.01a

compounds, known as carotenes (Evans, 1976).

Colour measurements of milk fat fractions and infant formulas fat.

4. Conclusion

The fractionation of bovine and buffalo milk fat was performed by

47.43 ± 0.03a
87.25 ± 0.05a
47.48 ± 0.03a

99.50 ± 0.06a
e

the dry and solvent fractionation methods, and three different fractions
42.79 ± 0.03i
68.10 ± 0.00
2.28 ± 0.04a
Bovine olein

were obtained. The process of fractionation resulted in significant dif-

ferences in the fatty acids compositions, thermal behaviors, and colour
For treatment abbreviations, see Table 1.

attributes of the different fractions of milk fat as compared to infant

formulas fat. Olein fractions were enriched in monounsaturated fatty
acids, while stearin fractions were enriched in saturated fatty acids. In
51.95 ± 0.03h
77.46 ± 0.05b
44.55 ± 0.03a
86.84 ± 0.00a
44.62 ± 0.03a

addition, infant formulas fat presented the highest polyunsaturated

c
82.17 ± 0.01
2.46 ± 0.00a
Bovine ghee
Types of fat

fatty acids content. The variations in fatty acids composition among the
different types of fat reflected diverse crystallisation and melting pro-
files. The FTIR spectroscopy revealed that the absorption values of the
tested samples in the spectral wavenumbers of 4000–500 cm−1 were
different, which may be attributed to the differences in the motion type
Parameters

of functional chemical bonds causing diverse intensities and vibrations.

Moreover, the colour of milk fat fractions was influenced by fractio-
Table 2

nation, since bovine ghee, bovine olein, and bovine stearin 1 displayed
WI
C*
a*

h*
b*

YI
L

higher C* and YI values. The finding of this study could pave the way

9
A.H. Ali, et al.
towards the synthesis of novel structured lipids composed of anhydrous
milk fat fractions and polyunsaturated fatty acids for potential use as
the fat source in infant formulas preparation. Furthermore, we re-
commend further studies to evaluate the incorporation of milk fat
fractions in innovative food applications.
CRediT authorship contribution statement
Abdelmoneim H. Ali: Conceptualization, Formal analysis,
Investigation, Writing - original draft. Wei Wei: Visualization, Data
curation, Writing - review & editing. Xingguo Wang: Supervision,
Project administration.
Declaration of competing interest
No conflict of interest to be declared.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (31701558) and the Young Elite Scientists
Sponsorship Program by CAST (2017QNRC001).
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