Use of GC and PDSC methods to characterize human milk fat substitutes obtained from lard and milk thistle oil mixtures
Use of GC and PDSC methods to characterize human milk fat substitutes obtained from lard and milk thistle oil mixtures
Joanna Brys´ 0 1 2
lneˆs Filipa Vaz Flores 0 1 2
Agata Go´ rska 0 1 2
Magdalena Wirkowska-Wojdyła 0 1 2
Ewa Ostrowska-Lige˛za 0 1 2
Andrzej Brys´ 0 1 2
0 Faculty of Biotechnology, Catholic University of Portugal , Dr. Anto ́nio Bernardino de Almeida st., 4200-072 Porto , Portugal
1 Faculty of Food Sciences, Warsaw University of Life Sciences (WULS - SGGW) , Nowoursynowska st. 159c, 02-787 Warsaw , Poland
2 Faculty of Production Engineering, Warsaw University of Life Sciences (WULS - SGGW) , Nowoursynowska st. 164, 02-787 Warsaw , Poland
The aim of this study was the analytical evaluations of human milk fat substitutes (HMFS) synthesized via enzymatic interesterification of lard and milk thistle oil mixtures by a immobilized commercial sn-1,3-specific lipase, using calorimetric and chromatographic methods. The mixtures of lard and milk thistle oil at mass ratio 6:4 and 8:2 were interesterified for 2, 4 and 6 h at the temperature of 60 C. The determination of fatty acid composition was carried out by gas chromatographic analysis of fatty acid methyl esters. The positional distribution of fatty acids in the sn-2 and sn-1,3 positions of triacylglycerols was based on the ability of the pancreatic lipase to selectively hydrolyze ester bonds in the sn-1,3 positions. Pressure differential scanning calorimetry (PDSC) method was used for the determination of the oxidative stability of HMFS. The oxidative induction time was obtained from the PDSC curves. Due to enzymatic interesterification of mixtures of lard and milk thistle oil, new HMFS that have a similar regiospecific structure of triacylglycerols to human milk fat can be produced. The induction time obtained The results of this research were presented at the 11th Seminar to the memory of prof. Stanislaw Bretsznajder.
Lard; Interesterification substitutes; Milk thistle oil; PDSC; Oxidative stability; Human milk fat
There is the commonly accepted opinion that breastfeeding
is recognized to enable the best development and growth of
newborns; however, infant formulas became an alternative
among mothers who cannot breastfeed their baby. The
development of infant milk fat similar to human milk fat
(HMF) catches the attention and became a challenge for
food industry . Modified lipids resembling the
triacylglycerols (TAG) of human milk can be produced by
chemical or enzymatic modification of TAG, but in the past
few years enzymatic modification of TAG has prevailed.
According to previous reports [1–3], enzymatic acidolysis
and interesterification using sn-1,3-specific lipases are
strategies that have received considerable attention in the
literature. In contrast to chemical methods, enzymatic
synthesis as a tool for fat modification has many
advantages. Catalyzed with lipases, interesterification reactions
can be directed in controlled ways, and specific or totally
new products can be produced . Microbial lipases have a
great potential for commercial application due to their
stability, selectivity and broad substrate specificity.
Lipasecatalyzed reactions are carried out under milder conditions
and with a higher selectivity than chemically catalyzed
reactions. The use of 1,3-selective lipases allows to
maintain the fatty acids in the sn-2 position of the acylglycerols.
This is nutritionally desirable and not possible to attain by
chemical catalysis [3, 5].
Breast milk is considered the main source of nutrients
and energy for many infants in their early life because of
nutritional, immunological and food safety points of view.
Lipids in human milk provide a major source of energy
and essential structural components for the cell
membranes of the newborn [2, 6]. Human milk consists of
3–5% of lipids and approximately 99% of them are TAG.
Palmitic acid (16:0), oleic acid (18:1) and linoleic acid
(18:2n-6) are the three major fatty acids of TAG, which
constitutes 25, 30 and 15% of total fatty acids,
respectively . HMF similarly to other milk fats contains high
content of the saturated palmitic acid, but its unique
feature is also a high content of the polyunsaturated fatty
acids (PUFA) [8, 9]. The polyunsaturated fatty acid
components of human milk are complex, including both
the C18 precursors, linoleic acid (18:2 n-6) and
a-linolenic acid (18:3 n-3), and the bioactive very long-chain
polyunsaturated fatty acids (LCPUFA) of both the n-6 and
the n-3 families . The major LCPUFA in HMF are
eicosapentaenoic acid (C20:5n-3, EPA), docosahexaenoic
acid (C22:6n-3, DHA) and arachidonic acid (C20:4n-6,
ARA). ARA and DHA are membrane fatty acids required
for growth and development of the central nervous system
of infants. Recent studies also show the importance of
ARA and DHA for bone mineralization and bone mass,
probably an effect caused by the influence of the fatty
acids on biosynthesis of prostaglandins. Therefore, infant
formula should also contain these LCPUFA in appropriate
amounts . The structure of human milk TAG is also
unique, as 60–70% of palmitic acid is located at the sn-2
position and the sn-1 and sn-3 positions are taken by 18:0,
18:1 and 18:2 fatty acids . This intramolecular
structure is an advantageous aspect for the efficiency of
calcium absorption .
Therefore, HMF is the component that supplies not only
the highest fraction of the infant’s required dietary energy
. The specific fatty acid compositions and structure of
HMF provide also an efficient fat absorption by infants.
The main goals of production of the HMFS were to obtain
fatty acid composition and positional acyl distribution as
close as possible to those found in HMF .
In order to produce HMFS that mimic HMF, several
studies have been carried out using immobilized lipases as
catalysts. In the majority of these studies, HMFS
resembling HMF were obtained by enzymatic interesterification
or acidolysis reactions of tripalmitin or lard with free fatty
acids from different sources. Lard has been recommended
as a potential alternative because it is the only fat
resembling HMF in terms of fatty acid composition .
Compared with HMF, lard has a similar palmitic and oleic acids
content but is characterized by lower content of essential
fatty acids. Vegetable oils like milk thistle oil are a source
of PUFA. A combination of tripalmitin or lard with
vegetable oils has been chosen to obtain a product possessing
similar fatty acids composition and their distribution in
TAG to HMF [2, 5, 15].
As Nielsen et al.  supposed, for new HMFS products
which are in development, it is necessary to optimize the
nutritional and physical properties as well as ensure that
they possess acceptable oxidation stability. Lipids are one
of the mostly chemically unstable food components, and
when lipid oxidation occurs, it gives rise to the formation
of lipid hydroperoxides. Free fatty acids (FFA) and
formation of lipid hydroperoxides increase the formation of
secondary volatile products of oxidation that influence
unpleasant odor. In order to maintain a healthy product, it
is important that lipids are oxidatively stable .
Differential scanning calorimetry (DSC) is one of the most
popular methods for the appraisal of the oxidative stability
of oils and fats . It is based on measuring of the
induction times of oils oxidation. The peroxide value (PV)
is widely used for measuring primary products of
The aim of this study was the analytical evaluations of
HMFS synthesized via enzymatic interesterification of lard
and milk thistle oil mixtures by a immobilized commercial
sn-1,3-specific lipase, using calorimetric and
Materials and methods
Chemicals and materials
All the solvents and reagents were purchased from Avantor
Performance Materials Poland S.A. (Gliwice, Poland)
except for porcine pancreatic lipase (Type II) which was
supplied together with the standard compounds by
SigmaAldrich (Saint Louis, MO, USA). Immobilized Lipozyme
RM IM, used in this investigation as a catalyst of
interesterification, was also procured from Sigma-Aldrich.
Lipozyme RM IM is a food-grade granulated silica
preparation of a microbial 1,3-specific lipase from
Rhizomucor miehei (activity 150 IUN g-1). The silica gel
TLC plate was purchased from Merck (Darmstadt,
Germany). All the solvents and reagents used in analyses were
of chromatographic or analytical grade. Lard and milk
thistle oil were provided by commercial company. The
mixtures of L and MTO in mass proportions 6:4 or 8:2
were used in this investigation.
Flasks containing of the initial mixtures were prepared and
positioned in a thermostated mineral-oil bath shaker. After
thermal equilibration of sample at 60 C, the enzymatic
catalyst (8% w/w) was added. The interesterifications were
performed with continuous shaking and stopped by filtering
off the biocatalyst after a predetermined time (2, 4 or 6 h).
Acid values and free fatty acids content
Acid value (AV) was determined by titration of fat samples
with 0,1 M ethanolic potassium hydroxide solution. Fatty
acid concentration was calculated based on acid values and
the value of molar mass of oleic acid. Acids values were
determined according to ISO method 660:2000 .
The peroxide value (PV) of mixture was determined by
iodometric technique with visual endpoint detection in
accordance with Standards ISO 3960:2007 .
A differential scanning calorimeter (DSC Q20 TA) coupled
with a high-pressure cell (PDSC—pressure differential
scanning calorimetry) was used. The instrument was
calibrated using high-purity indium as a standard. Fat samples
of 3–4 mg were weighted into an aluminum open pan and
placed in the sample chamber under oxygen atmosphere
with an initial pressure of 1 400 kPa and with the
100 mL min-1 oxygen flow rate. The isothermal
temperature for each sample was 120 C. Obtained diagrams were
analyzed using TA Universal Analysis 2000 software. For
each sample, the output was automatically recalculated and
presented as amount of energy per one gram. The
maximum PDSC oxidation time (induction time) was
determined based on the maximum rate of oxidation
(maximum rate of heat flow).
Fatty acid composition / GC measurements
The determination of fatty acid composition was carried
out by gas chromatographic (GC) analysis of fatty acid
methyl esters. Methyl esters of fatty acids were prepared
through transesterification with sodium methoxide
according to ISO 5509:2001. An YL6100 GC
chromatograph equipped with a flame ionization detector and
BPX-70 capillary column of 0.20 mm i.d. 9 60 m length
and 0.25 lm film thickness was used. The oven
temperature was programmed as follows: 60 C for 5 min
and then it was increased by 10 C min-1 to 180 C;
from 180 to 230 C, it was increased by 3 C min-1 and
then kept at 230 C for 15 min. The temperature of the
injector was 225 C, with a split ratio of 1:100, and the
detector temperature was 250 C. Nitrogen flowing with
the rate of 1 mL min-1 was used as the carrier gas. The
results were expressed as relative percentages of each
fatty acid (% of the fatty acids peaks area was
calculated). Fatty acids were identified by comparing the
relative retention times of FAME peaks with FAME
Positional distribution of fatty acids in the sn-2
and sn-1,3 positions of TAG
The positional distribution of fatty acids in the sn-2 and
sn1,3 positions of TAG is based on the ability of the
pancreatic lipase to selectively hydrolyze ester bonds in the
sn1,3 positions. Briefly, 20 mg of purified pancreatic lipase
(porcine pancreatic lipase, crude type II), 1 mL of Tris
buffer (pH 8.0), 0.25 mL of bile salts (0.05%) and 0.1 mL
of calcium chloride (2.2%) were added to 50 mL centrifuge
tubes and vortexed with 0.1 g of fat sample. The mixture
was incubated at 40 C in a water bath for 5 min and then
1 mL of 6 mol L-1 HCl and 1 mL of diethyl ether were
added and the mixture was centrifuged. Diethyl ether layer
was collected to test tubes and evaporated under nitrogen
gas to obtain 200 uL volume. A 200 uL aliquot was loaded
onto a silica gel TLC plate with fluorescent indicator
254 nm and developed with hexane/diethyl ether/acetic
acid (50:50:1, v:v:v). The 2-monoacylglycerol (2-MAG)
band was visualized under UV light. The 2-MAG band was
scraped off into a screw-capped test tube, extracted twice
with 1 mL of diethyl ether and centrifuged. The ether layer
was collected and entirely evaporated under nitrogen, and
then the sample was dissolved in n-hexane and methylated
as described above.
Relative standard deviation was calculated, where
appropriate, for all data collected using Microsoft Excel 2012
software. One-way analysis of variance ANOVA was
performed using the Statgraphics Plus, version 5.1.
Differences were considered to be significant at a p-value of
0.05, according to Tukey’s multiple range test.
Results and Discussion
Fatty acids composition and their distribution
in TAG in studied fats
HMF contains a similar amount of monounsaturated fatty
acids (MUFA) and saturated fatty acids (SFA), 39.1 and
40.6% respectively, and about 19.5% of PUFA . Lard,
which was the subject of research, is a rich source of SFA,
among palmitic acid (23.0%) and stearic acid (14.0%). It
also contains oleic acid (46.1%) belonging to MUFA and
linoleic acid (9.7%) belonging to PUFA in significant
amounts. These 4 major fatty acids match approximately
92.8% of the total fatty acids. The fatty acid compositions
of lard are in agreement with results published by
LopezLopez . Comparing to HMF, lard is characterized by a
similar SFA content (about 39.5%), but higher MUFA and
less PUFA content, 49.7 and 10.7%, respectively (Fig. 1).
The fatty acids composition of milk thistle oil was different
compared to lard. Linoleic acid in milk thistle oil accounts
for 52.0% of the total fatty acids, while oleic acids content
reached 26.6%. The other fatty acid was palmitic acid
which constitutes only 7.6% of the total fatty acids.
Analyzing the fatty acids profile of the milk thistle oil, PUFA
constitute about 55.1% of the total fatty acids, which is a
higher amount compared to HMF that contains only 19.5%
of PUFA. On the other hand, the SFA and MUFA content
were lower compared to HMF, 17.1 and 27.8%
In order to enrich TAG derived from L in PUFA, the
enzymatic interesterification was applied. As a source of
PUFA, milk thistle oil was used. As a result of
interesterification, the essential fatty acids from milk thistle oil were
incorporated into TAG structures of lard. The
interesterified fats contained from 19.5 to 31.3% of PUFA (Table 1;
Fig. 1). Mixtures after interesterification showed from 17.9
to 29.0% of linoleic acid belonging to omega-6 essential
fatty acid, while the original lard only 9.7% of this acid was
observed. TAG of interesterified fats contained also from
0.5 to 0.9% of a-linolenic acid belonging to omega-3
essential fatty acid. The enzymatic interesterification of
lard and thistle oil blends were performed at various
proportions and times to obtain a fatty acid profile matching
that of HMF. The SFA content in the blends after
interesterification ranged from 26.9 to 36.6%, the MUFA from
39.9 to 50.7% and PUFA from 19.4 to 28.9%. In general,
percentage of SFA in all mixtures after interesterification
was lower in comparison with that of HMF (40.6%). The
MUFA contents in the blends of lard and oil mixed in
proportions 6:4 after interesterification were about 39%,
and these values were similar to those of to HMF (39.1%).
In the interesterified blend of lard and oil mixed in
proportions 8:2, similar amount of PUFA (19.5–22.3%) was
detected compared to the reference HMF (19.5%). The
blends of lard and oil mixed in proportions 8:2 after 2 and
6 h of reaction were characterized by higher amount of
MUFA, about 50.7%, compared to the reference HMF
(39.1%). In the blends of lard and vegetable oil mixed in
proportions 6:4, the PUFA content was higher than in the
mixture 8:2 after interesterification. The results obtained in
this study suggest that in blends after interesterification no
significant differences in the fatty acid profile were
Fig. 1 Content of fatty acids
(SFA saturated fatty acids,
MUFA monounsaturated fatty
acids, PUFA polyunsaturated
fatty acids) for mixtures after
interesterification and for
human milk fat. *Human milk
fat. Values are derived from
Lopez-Lopez et al. 
Table 1 Fatty acids composition (%) of mixtures after interesterification
observed independently of the processes conditions used
(time of interesterification). HMF contains also long-chain
polyunsaturated fatty acids (LCPUFA) 20:4n-6 and
3. Nevertheless, these fatty acids were not found in blends
after interesterification. However, these mixtures contain
other essential fatty acids like linoleic acid and a-linoleic
acid, which can be converted in our organism to LCPUFA.
The type of fatty acid and its stereospecific position in
TAG determine both, the physical behavior of dietary fats
and their fate during digestion and absorption 
Considering the results obtained, it can be concluded that
interesterification affects the distribution of fatty acids in
TAG in both, internal and external positions. HMFS are
characterized by a similar fatty acid composition in sn-2
position of TAG, regardless of time of process. Palmitic
acid was the most abundant fatty acid located in sn-2
position of interesterified mixtures. The reaction time
influenced to a small extent to reduce the palmitic acid
content and increase in the stearic and oleic acid content in
sn-2 position of TAG. Due to the positional sn-1,3 specifity
of the lipase, the interesterification catalyzed by Lipozyme
RM IM occurred mainly in external positions of TAG. As
the enzyme operated on the external positions, the
percentages of given fatty acids in sn-2 positions of
interesterified TAG in comparison with their counterparts for
initial blends remain nearly unchanged . The changes
in sn-2 percentages can be caused by possible acyl
migrations in TAG molecules during prolonged time of
interesterification, as it was proved by Xu et al. .
Increasing the amount of oil in the sample from 20 to 40%
caused the reduction in the palmitic acid content in sn-2
position of TAG from 44.8–50.9 to 34.7–36.4%.
However, in order to obtain the confirmation of fatty
acid distribution between regiospecific TAG positions, it
was essential to recalculate the fatty acid composition of
sn-2 position into relative fatty acid in sn-2 position .
As reported by Lopez-Lopez et al. , fatty acid
composition of sn-1,3 and sn-2 in TAG positions is not the
most suitable way of presenting data of sn-2 fatty acid
composition without taking into account the total
percentage of fatty acids in whole TAG molecules. Hence,
Fig. 2 presents the percentage of most abundant fatty
acids in sn-2 position of TAG, expressed as relative fatty
acid in analyzed fats. Human milk contains approximately
70% of palmitic acid in the sn-2 position, whereas most
animal and vegetable fats contain this fatty acid primarily
in the sn-1 and sn-3 positions . Lard, like HMF,
contains approximately 85.7% of palmitic acid in the sn-2
position of TAG, whereas percentage of this fatty acid in
internal position of TAG in oil reached only 14.6%.
Analyzing the results regarding the percentage of the fatty
acids esterified at sn-2 position of TAG in HMFS, it can
be concluded that the structure of TAG is very similar to
the HMF. Percentage of the palmitic acids at sn-2 of TAG
in all HMFS exceeded 33%, which means that it is located
mainly in the internal position of TAG. This location
increases the efficiency of calcium absorption by infants.
Taking into account the percentage of unsaturated fatty
acids in the sn-2 position of TAG in HMFS, it can be
stated that they are located mostly in external positions of
TAG. This is mainly oleic acid. Percentage of oleic acid at
the sn-2 of TAG in HMFS ranged from 14.1 to 22.8%,
which confirms that it is located mainly in sn-1,3 positions
Fig. 2 Percentage of a given fatty acid in sn-2 position of TAG of interesterified fats. *Human milk fat. Values are derived from Lien et al. 
Table 2 Acid value, peroxide value and oxidation induction time of
raw materials and mixtures after interesterification
Data expressed as mean ± standard deviation
The different lowercase letters (a–e) in the same column indicate
significantly different values (p \ 0.05)
The quality assessment of studied fats
All fats and oils are susceptible to oxidation processes,
giving rise to free radicals, hydroperoxides and polymers
. The peroxide value is a method used for determining
the amount of oxygen chemically bound to an oil or fat as
peroxides, particularly hydroperoxides which are primary
oxidation products. The PV of the raw materials and HMFS
are listed in Table 2. The blends after interesterification
were characterized by lower PV, in contrary to milk thistle
oil, for which the parameter reached the values greater than
5.8 meq O2 kg-1 fat. High PV means a high production of
hydroperoxides, which can alter or even lose the nutritional
quality of fats. The products obtained by enzymatic
reactions which were carried out for 2 h were characterized by
the higher PV which means a faster progression of
oxidation. Comparing the different proportions of substrates but
maintaining the same time, HMFS obtained from
interesterified mixture of lard and oil in ratio of 8:2 showed
lower PV than the samples with a ratio of 6:4. This is the
consequence of the fact that the samples with ratio of 6:4
contained bigger amount of oil which oxidizes quickly.
Therefore, all blends after interesterification showed a
PV quite similar and lower than 2 meq O2 kg-1 fat. The
HMFS were characterized by lower PV than oil and that
made them more stable to oxidation. However, many
studies have reported a decrease in oxidative stability of
HMFS compared to initial fats [1, 23]. The evidence about
oxidative stability of HMFS is diverse; the main factors
that determine the oxidative stability of HMFS are the
different analytical methods of purification and production,
the different nature of oil and lard sources and the presence
of antioxidants during the production .
The main components of fats are TAG. Fats also contain
certain quantities of monoacylglycerols, diacylglycerols
and fatty acids contents. The determinant of free fatty acids
contents in fat is acid value (AV). The AV and free fatty
acids content (FFA) of lard, milk thistle oil and blends after
interesterification are shown in Table 2. Analyzing the
results, it can be observed that AV of mixtures after
interesterification was higher compared to lard and oil,
which indicate a bigger presence of FFA. Lard was
characterized by the lowest FFA content (\1%), similarly to
studied vegetable oil with an amount of FFA at the level of
\2%. Analyzing and comparing the results, the FFA
content in mixtures increased after enzymatic
interesterification reaction. These results are in agreement with
Brys et al. , who indicated that enzymatic
interesterification process caused the increase in FFA amount. The
AV and FFA contents depend significantly on the time of
the reaction. In the case of interesterified mixtures, the AV
was lower when reaction time was shorter (*2 h). General
blends of lard and milk thistle oil after interesterification at
the presence of Lipozyme RM IM contained 5.1–8.2% of
FFA. Therefore, it was demonstrated that increase in AV is
related to increase in FFA content in the sample. Hamam
and Shahidi  suggested that the presence of FFA in the
reaction mixture may induce oxidation due to a catalytic
effect of the carboxylic groups of the fatty acids contents
on the formation of free radicals. In general, the higher the
level of fatty acids contents, monoacylglycerols and
diacylglycerols in the obtained product after interesterification
with respect to the level of TAG is, the higher is the
reduced oxidative stability . It should be concluded that
before using HMFS for potential applications in functional
foods, the FFA should be removed.
Numerous methods have been developed for
monitoring fat and oil autoxidation [25–28]. DSC is a very popular
method for the assessment of the oxidative stability of oils
and fats. The DSC experiments are performed with a linear
increase in temperature (dynamic conditions) or at
constant temperature (isothermal conditions). The oxidation
medium (oxygen or air) can be maintained at normal
(atmospheric) pressure or at increased pressure (PDSC) .
The results of PDSC measurements expressed as the
oxidation induction times are shown in Fig. 3. The induction
times obtained for analyzed fats can be used as primary
parameters for the assessment of the resistance of tested
fats to their oxidative decomposition. Mostly, samples
with longer induction time are more stable than those for
which the induction time obtained is shorter . The
PDSC tests for HMFS performed at isothermal
temperature of 120 C showed that their induction times
(28.9–34.8 min) were reduced compared with the lard
(46.8 min). Comparing raw materials, milk thistle oil was
characterized by lower induction time (32.7 min) than
lard. In general, oils contain a high concentration of
unsaturated fatty acids and they have worse oxidative
stability, consequently a short induction time. Comparing
the proportions between mixtures of lard and milk thistle
oil, the induction time should be lower in samples with
ratios of 6:4. This is a result of higher concentration of oil
unsaturated fatty acids. Most studies have reported a
decrease in oxidative stability of interesterified fats,
especially fats rich in unsaturated fatty acids, compared to
the raw materials [1, 23, 29]. Same studies have suggested
also the loss of antioxidants during the production of
Fig. 3 Oxidation induction
time of interesterified fats and
raw materials. Different letters
indicate that the samples are
significantly different at
p \ 0.05
interesterified fats as the main reason for explaining a
worse oxidative stability found for fats after modification
even regardless of the production method used or of the
polyunsaturated nature of fatty acids. This loss of
endogenous antioxidants is especially important when
vegetable oils are used as a source of fatty acids for the
production of interesterified fats, due to their natural
content in antioxidants, such as tocols, both tocopherols and
tocotrienols, phytosterols, or phenolic compounds .
The interesterified mixtures of L and MTO were
satisfactory to produce HMFS rich in PUFA. The essential fatty
acids from MTO were incorporated into TAG structures of
lard after modification. Major fatty acids determined in the
studied fats after interesterification were palmitic, oleic and
linoleic acids, similarly to HMF. The interesterified blends
were characterized also by a similar ratio of SFA, MUFA
and PUFA like HMF, especially blend of lard and oil
mixed in proportions 8:2 after 4 h of reaction.
The oxidative stability of the HMFS showed that
products based on blends of lard and milk thistle oil after
interesterification were more stable to oxidation than oil.
The interesterified fats showed a short induction time
compared to lard, which means their worse oxidative
stability. PDSC is a fast and reliable method that can be used
to assess the oxidation parameters of interesterified
mixtures of lard and milk thistle oil. The induction times
obtained for analyzed fats can be used as primary
parameters for the assessment of the resistance of tested fats to
their oxidative decomposition. The products of enzymatic
reactions which were carried out for 2 h underwent faster
progression of oxidation. This means a worse oxidative
stability for blends which were carried out during short
time. The mass ratio of substrates in blends also influenced
the oxidative stability. In blends which contained a higher
amount of vegetable oil, the progression of oxidation was
In general, FFA content of mixture increased after
interesterification reaction. The presence of free fatty acids
after the reaction can decrease the oxidative stability of
interesterified fats. It should be concluded that before using
HMFS for potential applications in functional foods, the
FFA should be removed.
Positional distribution of fatty acids in the TAG of
interesterified blends was similar to that of HMF. The
sn1,3 positions predominantly contain unsaturated FA,
mostly oleic acid and sn-2 position contain mostly palmitic
acid. This position of palmitic acid in TAG is important for
proper absorption of fat and minerals by infants.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://crea
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
1. Maduko C , Park Y , Akoh CC . Characterization and oxidative stability of structured lipids: infant milk fat analog . JAOCS . 2008 ; 85 : 197 - 204 .
2. Yang T , Xu X , He C , Li L. Lipase-catalyzed modification of lard to produce human milk fat substitutes . Food Chem . 2003 ; 80 : 473 - 81 .
3. Wang YH , Qin XL , Zhu QS , Zhou R , Yang B , Li L. Lipasecatalyzed acidolysis of lard for the production of human milk fat substitute . Eur Food Res Technol . 2010 ; 230 : 769 - 77 .
4. Forssell P , Kervinen R , Lappi M , Linko P , Suoptti T , Poutanen K. Effect of enzymatic interesterification on the melting point of tallow-rapeseed oil (LEAR) mixture . JAOCS . 1992 ; 69 : 126 - 9 .
5. Tecelao C , Silva J , Dubreucq E , Ribeiro M , Ferreira-Dias S. Production of human milk fat substitutes enriched in omega-3 polyunsaturated fatty acids using immobilized commercial lipases and Candida parapsilosis lipase/acyltransferase . J Mol Cat B Enzym . 2010 ; 65 : 122 - 7 .
6. Jensen RG . The lipids in human milk . Prog Lipid Res . 1996 ; 35 : 53 - 92 .
7. Mu H. Production and nutritional aspects of human milk fat substitutes . Lipid Technol . 2010 ; 22 : 126 - 9 .
8. Sahin N , Akoh CC , Karaali A. Enzymatic production of human milk fat substitutes containing c-linolenic acid: optimization of reactions by response surface methodology . JAOCS . 2005 ; 82 ( 8 ): 549 - 57 .
9. Xu X. Production of specific-structured triacylglycerols by lipasecatalyzed reactions: a review . Eur J Lip Sci Technol . 2000 ; 102 : 287 - 303 .
10. Lien EL . The role of fatty acid composition and positional distribution in fat absorption in infants . J Pediatr . 1994 ; 125 : 562 - 8 .
11. Huiling M. Production and nutritional aspects of human milk fat substitutes . Lipid Technol . 2010 ; 22 : 126 - 9 .
12. Nielsen NS , Yang T , Xu X , Jacobsen Ch. Production and oxidative stability of a human milk fat substitute produced from lard by enzyme technology in a pilot packed-bed reactor . Food Chem . 2006 ; 94 : 53 - 60 .
13. Sahin N , Akoh CC , Karaali A. Enzymatic production of human milk fat substitutes containing c-linolenic acid: optimization of reactions by response surface methodology . JAOCS . 2005 ; 82 ( 8 ): 549 - 57 .
14. Brys ´ J, Wirkowska M , Go´ rska A , Ostrowska-Lige˛za E, Brys´ A. Application of the calorimetric and spectroscopic methods in analytical evaluation of the human milk fat substitutes . J Therm Anal Calorim . 2014 ; 118 : 841 - 8 .
15. Ilyasoglu H. Production of human fat milk analogue containing a-linolenic acid by solvent-free enzymatic interesterification . LWT Food Sci Technol . 2013 ; 54 : 179 - 85 .
16. Ostrowska-Lige ˛za E, Bekas W , Kowalska D , Lobacz M , Wroniak M , Kowalski B. Kinetics of commercial olive oil oxidation: dynamic differential scanning calorimetry and Rancimat studies . Eur J Lipid Sci Technol . 2010 ; 112 : 268 - 74 .
17. ISO 660 : 2009 . Animal and vegetable fats and oils-determination of acid value and acidity . International Organization for Standardization, Geneva, Switzerland.
18. ISO 3960 : 2007 . Animal and vegetable fats and oils-determination of peroxide value-iodometric (visual) endpoint determination . International Organization for Standardization, Geneva, Switzerland.
19. Lopez-Lopez A , Castellote-Bargallo ´ AI, Campoy-Folgoso C , Rivero-Urgel M , Lopez-Sabater MC . Fatty acid and sn-2 fatty acid composition in human milk from Granada (Spain) and infant formulas . Eur J Clin Nutr . 2002 ; 56 : 1242 - 54 .
20. Small D. The effects of glyceride structure on fat adsorption and metabolism . Ann Rev Nutr . 1991 ; 11 : 412 - 34 .
21. Kowalski B , Tarnowska K , Gruczynska E , Bekas W. Chemical and enzymatic interesterification of beef tallow and rapeseed oil blend with low content of tallow . J Oleo Sci . 2004 ; 53 ( 10 ): 479 - 88 .
22. Xu X , Skands ARH , Adler-Nissen J , Høy CE . Production of specific structured lipids by enzymatic interesterification: optimization of the reaction by response surface design . Fett/Lipid . 1998 ; 100 ( 10 ): 463 - 71 .
23. Martin D , Reglero G , Senorans FJ . Oxidative stability of structured lipids . Eur Food Res Technol . 2010 ; 231 : 635 - 53 .
24. Hamam F , Shahidi F. Enzymatic acidolysis of an arachidonic acid single-cell oil with capric acid . JAOCS . 2004 ; 81 : 887 - 92 .
25. Gray JI . Measurement of lipid oxidation: a review . JAOCS . 1978 ; 55 : 539 - 46 .
26. Kowalska D , Gruczynska E , Kowalska M. The effect of enzymatic interesterification on the physico-chemical properties and thermo-oxidative stabilities of beef tallow stearin and rapeseed oil blends . J Therm Anal Calorim . 2015 ; 120 : 507 - 17 .
27. Ratusz K , Popis E , Ciemniewska-Z_ ytkiewicz H , Wroniak M. Oxidative stability of camelina (Camelina sativa L.) oil using pressure differential scanning calorimetry and Rancimat method . J Therm Anal Calorim . 2016 ; 126 : 343 - 51 .
28. Souza AL , Mart´ınez FP , Ferreira SB , Kaiser CR. A complete evaluation of thermal and oxidative stability of chia oil . J Therm Anal Calorim . 2017 ;. doi:10.1007/s10973- 017 - 6106 -x.
29. Brys ´ J, Wirkowska M , Go´ rska A , Ostrowska-Lige˛za E, Brys´ A, Koczon ´ P. The use of DSC and FT-IR spectroscopy for evaluation of oxidative stability of interesterified fats . J Therm Anal Calorim . 2013 ; 113 : 481 - 7 .