J Am Oil Chem Soc
DOI 10.1007/s11746-011-1869-x
ORIGINAL PAPER
Interesterification of Lard and Soybean Oil Blends Catalyzed
by Immobilized Lipase in a Continuous Packed Bed Reactor
Roberta Claro da Silva • Fabiana Andreia Schaffer De Martini Soares •
Thaı́s Gonzaga Fernandes • Anna Laura Donadi Castells • Kelly Caroline Guimarães da Silva
Maria Inês Almeida Gonçalves • Chiu Chih Ming • Lireny Aparecida Guaraldo Gonçalves •
Luiz Antonio Gioielli
•
Received: 5 January 2011 / Revised: 1 April 2011 / Accepted: 26 May 2011
Ó AOCS 2011
Abstract Structured lipids (SL), formulated by blends of
lard and soybean oil in different ratios, were subjected to
continuous enzymatic interesterification catalyzed by an
immobilized lipase from Thermomyces lanuginosus (Lipozyme TL IM) in a continuous packed bed reactor. The original
and interesterified blends were examined for fatty acid and
triacylglycerol composition, regiospecific distribution, and
solid fat content. Blends of lard and soybean oil in the proportions 80:20 and 70:30 (w/w), respectively, demonstrated a
fatty acid composition, and proportions of polyunsaturated/
saturated fatty acids (PUFA/SFA) and monounsaturated/
polyunsaturated fatty acids (MUFA/PUFA), that are appropriate for the formulation of pediatric products. These same
blends were suited for this purpose after interesterification
because their sn-2 positions were occupied by saturated fatty
acids (52.5 and 45.4%, respectively), while unsaturated fatty
acids predominantly occupied sn-1,3 positions, akin to human
milk fat. Interesterification caused rearrangement of triacylglycerol species.
R. C. da Silva F. A. S. D. M. Soares T. G. Fernandes
A. L. D. Castells K. C. G. da Silva L. A. Gioielli (&)
Department of Biochemical and Pharmaceutical Technology,
Faculty of Pharmaceutical Sciences, University of São Paulo,
Av. Prof. Lineu Prestes n. 580, B16, São Paulo,
SP CEP 05508-900, Brazil
e-mail: lagio@usp.br
M. I. A. Gonçalves
Department of Pharmacy, Faculty of Pharmaceutical Sciences,
University of São Paulo, Av. Prof. Lineu Prestes n. 580,
B13, São Paulo, SP CEP 05508-900, Brazil
C. C. Ming L. A. G. Gonçalves
Department of Food Technology, Faculty of Food Engineering,
University of Campinas, Caixa Postal 6091, Campinas,
SP CEP 13083-970, Brazil
Keywords Human milk fat substitute Lard
Soybean oil 13C-NMR analyses
Triacylglycerol composition
Introduction
The interesterification of lipids catalyzed by lipases represents an alternative to chemical interesterification. Ecofriendly processes for modifying fats and oils by utilizing
lipases of different microbial origins have been reported by
various researchers [1–3]. Lipase-catalyzed interesterification reactions can be performed in a batch-type reactor or
in a continuous packed bed reactor [4]. The packed bed
reactor is one of the most commonly employed apparatus
for solid–fluid contacting in heterogeneous catalysis
because it: (1) facilitates contact and subsequent separation; (2) and the continuous removal of inhibitory substances; (3) allows reuse of the enzyme without the need
for prior separation; (4) permits the handling of poorlysoluble substrates by using large volumes containing low
concentrations of substrate; (5) leads to more consistent
product quality and improved enzyme stability due to ease
of automation and control [5]; (6) is suitable for long-term
and industrial-scale production, in contrast to stirred-tank
reactors where enzyme granules are susceptible to breaking
down because of mechanical shear stress; and (7) is more
cost effective than batch operations [6–8].
Lipids are the major source of energy in human milk or
infant formulas [9]. Hence, modification of fats and oils for
infant formulas in order to obtain not only the correct fatty
acid (FA) composition but also the same positional distribution as in human milk fat (HMF) via interesterification is
a focus of investigation. The major saturated fatty acid
(SFA) is palmitic acid (16:0), which represents about a
123
J Am Oil Chem Soc
fourth of the fatty acids present in breast milk and is
esterified mainly at the sn-2 position of triacylglycerols
(TAG). Stearic, oleic and linoleic acids are generally
esterified at the sn-1,3 positions of TAG [10, 11]. This
feature lends HMF a unique structure, and lard is the only
animal fat that has a similar structure [12]. The palmitic acid
residue at the sn-2 position is not hydrolyzed by pancreatic
lipase and, as 2-monopalmitin, forms a mixed micelle with
bile salt which is efficiently absorbed [13]. The palmitic
acid residues esterified at the sn-1,3 positions are hydrolyzed by pancreatic lipase, producing free palmitic acid
residues which form poorly absorbed calcium soaps in the
intestinal tract, resulting in reduced absorption of both
calcium and fat [14]. Formation of such calcium soaps can
lead to stool hardness, constipation and in some cases,
bowel obstruction. Thus, the presence of palmitic acid acyl
residues at the sn-2 position of HMF increases absorption of
16:0 in infants and reduces calcium losses in the feces [15].
The main aim of the present study was to produce SL via
EIE of lard and soybean oil blends catalyzed by an immobilized commercial sn-1,3-specific lipase from Thermomyces lanuginosus in a continuous packed bed reactor, and to
characterize the chemical properties of the structured lipids
thus produced. Lard and soybean oil were chosen due to their
desirable nutritional qualities for the development of a fat
substitute for human milk fat, and also because they are low
cost bio-resources produced on a large scale in Brazil.
respectively. The blends were prepared after complete
melting of the fats at 70–80 °C for 30 min under magnetic
stirring. The blends thus obtained were stored at -18 °C
until analysis.
Performance of Interesterification Reactions
The EIE was carried out in a continuous tubular glass bioreactor (height 34 cm, internal diameter 2 cm) equipped
with an external jacket to maintain constant temperature and
fixed bed to support the enzyme (70 g), equipped with a
peristaltic pump (VC 360II Ismatec, Switzerland). Soybean
oil was initially introduced into the reactor at a flow rate of
1 mL/min to remove air and water from the enzyme until the
free fatty acids of the interesterified oil presented a stable
value. The residence time was experimentally determined
and was defined as the time needed to fill the empty spaces of
the reactor packed with immobilized enzyme, using a flow
rate of 1 mL/min. The jacketed column was heated by water
bath (RE 112, Lauda, Germany) to maintain the bed enzyme
at 60 °C (according to the manufacturer’s recommendations). After conditioning of the enzyme, the blends were
pumped into the reactor at the flow rate of 1 mL/min (residence time of 60 min). Each different interesterified sample
was collected (200 mL) after discarding the first 200 mL to
avoid cross-contamination with the previous blend, and
stored at -18 °C until analysis.
Determination of Fatty Acid Composition
Materials and Methods
Materials
Lard and soybean oil were obtained from local commerce
(São Paulo, Brazil). Commercial immobilized lipase from
Thermomyces lanuginosus (Lipozyme TL IM) was kindly
supplied by Novozymes Latin America Ltd. (Araucária,
Brazil). The enzyme activity of the lipase was 250 IUN/g.
All other reagents and solvents were analytical or chromatographic grade.
Betapol was kindly provided by Loders Croklaan, Lipid
Nutrition (Wormerveer, Netherlands).
The human milk fat was obtained from samples of
human milk donated by the Human Milk Bank of the
University of São Paulo.
Methods
Reactant Blend Preparation
Lard and soybean oil were blended at 80:20, 70:30, 50:50,
60:40, 40:60, 30:70 and 20:80 (w/w) proportions,
123
Fatty acids in the triacylglycerols of the interesterified
blends were converted into fatty acid methyl esters
(FAME). Rapid preparation of methyl esters for gas chromatographic analysis was accomplished by saponifying
fats with 0.5 mol equiv./L methanolic potassium hydroxide, followed by refluxing with a solution of ammonium
chloride and sulphuric acid in methanol. Rigorous conditions during the saponification and conversion of soaps into
methyl esters, and the precipitation of alkali sulfates during
the reaction, were avoided, and the degree of esterification
was approximately 99.5 g/100 g [16, 17]. Analyses of
FAMEs were carried out on a Varian GC gas chromatograph (model 430 GC, Varian Chromatograph Systems,
Walnut Creek, CA, USA), equipped with a CP 8412 auto
injector. The Galaxie software was used for quantification
and identification of peaks. Injections were performed on a
100-m fused silica capillary column (ID = 0.25 mm)
coated with 0.2 lm of polyethylene glycol (SP-2560, Supelco, USA) using helium as the carrier gas at an isobaric
pressure of 37 psi; linear velocity of 20 cm/s; with makeup gas: helium at 29 mL/min at a split ratio of 1:50; volume injected: 1.0 lL. The injector temperature was set at
250 °C and the detector temperature was set at 280 °C. The
60.5
24.4
15.1
0.0 ± 0.0
0.0 ± 0.0
6.3 ± 0.1
54.2 ± 0.3
1.5 ± 0.0
22.9 ± 0.1
3.4 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
Soybean oil
(after)
11.6 ± 0.5
60.7
52.8
27.6
24.5
14.9
19.6
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
5.1 ± 0.2
6.3 ± 0.0
54.4 ± 0.0
47.7 ± 1.2
1.6 ± 0.1
1.6 ± 0.1
22.8 ± 0.1
25.5 ± 1.0
4.9 ± 0.3
3.4 ± 0.1
0.0 ± 0.0
0.5 ± 0.0
14.2 ± 0.3
11.4 ± 0.0
0.4 ± 0.0
0.0 ± 0.0
20:80 (after)
Soybean oil
(before)
51.4
48.0
30.6
28.4
19.9
21.4
0.0 ± 0.0
0.0 ± 0.0
0.3 ± 0.0
0.4 ± 0.0
4.7 ± 0.0
5.1 ± 0.1
46.3 ± 0.5
43.4 ± 0.0
1.7 ± 0.01
1.7 ± 0.0
25.9 ± 0.2
27.7 ± 0.0
6.0 ± 0.01
3.1 ± 0.0
0.5 ± 0.0
0.7 ± 0.0
15.0 ± 0.0
0.4 ± 0.0
0.4 ± 0.0
30:70 (after)
20:80 (before)
14.5 ± 0.5
44.6
47.6
30.7
32.1
23.5
21.6
0.0 ± 0.0
0.0 ± 0.0
0.5 ± 0.1
0.4 ± 0.1
4.6 ± 0.0
4.2 ± 0.0
40.1 ± 0.1
43.0 ± 0.1
1.8 ± 0.0
1.9 ± 0.0
28.8 ± 0.0
27.9 ± 0.0
6.1 ± 0.0
6.6 ± 0.0
0.9 ± 0.0
16.3 ± 0.0
15.1 ± 0.0
0.6 ± 0.0
0.5 ± 0.0
40:60 (after)
30:70 (before)
0.7 ± 0.0
29.9
47.0
30.7
38.1
31.9
22.1
0.0 ± 0.0
0.4 ± 0.0
0.6 ± 0.0
0.0 ± 0.0
4.5 ± 0.1
2.3 ± 0.0
27.2 ± 0.1
42.5 ± 0.6
1.8 ± 0.0
2.2 ± 0.0
33.7 ± 0.1
28.2 ± 0.5
6.3 ± 0.2
9.4 ± 0.1
1.6 ± 0.0
21.4 ± 0.2
15.4 ± 0.3
1.2 ± 0.0
0.5 ± 0.0
70:30 (after)
40:60 (before)
0.7 ± 0.0
25.8
26.0
28.5
40.5
37.7
40.8
33.4
33.5
32.9
0.5 ± 0.0
0.4 ± 0.0
0.5 ± 0.0
0.6 ± 0.0
0.6 ± 0.0
0.5 ± 0.1
1.7 ± 0.0
2.2 ± 0.0
1.7 ± 0.0
23.5 ± 0.1
23.8 ± 0.3
26.9 ± 0.5
2.3 ± 0.0
2.1 ± 0.0
2.3 ± 0.0
36.2 ± 0.0
35.8 ± 0.5
33.3 ± 0.8
10.3 ± 0.2
9.2 ± 0.4
10.6 ± 0.1
1.7 ± 0.0
21.7 ± 0.0
21.9 ± 0.9
22.4 ± 1.6
1.1 ± 0.0
1.2 ± 0.1
1.3 ± 0.2
80:20 (before)
80:20 (after)
70:30 (before)
1.8 ± 0.1
1.7 ± 0.2
17.0
16.9
44.5
43.3
39.7
38.7
0.6 ± 0.1
0.5 ± 0.2
0.6 ± 0.2
0.7 ± 0.1
0.6 ± 0.0
0.6 ± 0.0
15.9 ± 0.7
15.7 ± 0.2
2.5 ± 0.0
2.4 ± 0.3
37.5 ± 2.7
39.0 ± 0.7
12.2 ± 0.2
Linolenic
(18:3)
Linoleic
(18:2)
Elaidic
(18:1t)
Oleic
(18:1)
Stearic
(18:0)
11.1 ± 1.2
2.7 ± 0.7
2.2 ± 0.11
24.9 ± 1.1
26.6 ± 3.8
As expected, EIE does not affect the degree of saturation
nor cause isomerization [23]. Our results confirmed that
1.5 ± 0.2
Fatty Acids Composition
2.0 ± 0.7
Results and Discussion
Palmitoleic
(16:1)
A proton-decoupled 13C NMR was used to analyze the
positional distribution of fatty acids on the triacylglycerol
backbone [20–22]. Lipid samples (250 mg) were dissolved
in 0.5 mL of deuterated chloroform (CDCL3) in 5-mm
NMR tubes, and NMR spectra were recorded on a Bruker
Advance DPX spectrometer operating at 300 MHz. The
13
C spectra of the lipid samples were acquired with a
spectral width of 2,332.090 Hz, pulse of 10.2 ls, and a
relaxation delay of 30 s. Determination of 13C was performed at a frequency of 75.8 MHz with a 5 mm multinuclear probe operating at 30 °C, using the method
described by Vlahov [20]. The results showed the compositions of saturated fatty acids, oleic acid and linoleic ? linolenic acids in sn-2 and sn-1,3 positions.
Palmitic
(16:0)
C-NMR Analyses
Myristic
(14:0)
13
Table 1 Fatty acid composition (g/100 g) of lard, soybean oil and blends, before and after continuous enzymatic interesterification
Analyses of triacylglycerols were carried out on a gas
chromatograph (model CGC, Agilent 6850 Series CG
System, Santa Clara, USA). A capillary column (50%
phenylmethylpolysiloxane, 15 m length 9 0.25 mm internal diameter and 0.15 lm film) DB-17HT from Agilent
(Santa Clara, CA, USA) was used. Conditions were as
follows: split injection, ratio of 1:100; column temperature:
250 °C, programmed up to 350 °C at 5 °C/min; carrier gas:
helium, flow rate of 1.0 mL/min; injector temperature:
360 °C; detector temperature: 375 °C; volume injected:
1.0 lL; sample concentration: 20 mg/mL of tetrahydrofuran. Identification of triacylglycerol groups was performed
by comparing retention times, according to Antoniosi Filho, Mendes and Lanças [19].
Eicosenoic
(20:1)
Triacylglycerol Composition
Lard (before)
Eicosadienoic
(20:2)
SFA
MUFA
oven temperature was initially held at 140 °C for 5 min,
programmed to increase to 240 °C at a rate of 4 °C/min,
and then held isothermally for 30 min. Qualitative FA
composition of the samples was determined by comparing
the retention times of the peaks produced after injecting the
methylated samples with those of the respective standards
of fatty acids. The quantitative composition was obtained
by area normalization and expressed as mass percentage,
according to the AOCS Official Method Ce 1-62 [18]. All
samples were analyzed in triplicate and the reported values
are the average of the three runs.
Lard (after)
PUFA
J Am Oil Chem Soc
123
J Am Oil Chem Soc
Fig. 1 Ratios of 18:2/18:3,
PUFA/SFA and MUFA/PUFA
of lard, soybean oil and binary
blends, before and after
continuous enzymatic
interesterification
interesterification did not cause a significant alteration in
the fatty acid profile of the initial blends (Table 1). The
content of linoleic acid in human milk is dependent on diet
and varies according to the feeding habits and geographic
region of the population. An adequate intake of essential
fatty acids (linoleic and linolenic acids) is crucial for
newborn babies. Hence, infant formulas must have a ratio
of linoleic/linolenic acids between 5 and 15. In human milk
in general, this ratio is ca. 14.4 [10] whereas in human milk
from Brazilian mothers, this proportion was found to be
15.2 [11]. In the present study, the ratios of all blends of
lard and soybean oil were in the 5–15 range (Fig. 1). Both
Food and Agriculture Organization/World Health Organization (FAO/WHO) and the European Union Committee
recommend a minimum polyunsaturated/saturated fatty
acids ratio (PUFA/SFA) of 1.0 in infant formulas. The
PUFA/SFA ratio of lard and soybean oil blends ranged
from 0.8 to 2.7. Furthermore, the literature reports values in
human milk ranging from 0.23 [10] to 0.49 [11], levels that
fall below those recommended. The commercial structured
lipid Betapol has a proportion of 0.90 [10]. According to
Jensen [9], the amount of oleic acid in breast milk is
important to reduce the melting point of the triacylglycerols, thus providing the liquidity required for the formation,
transport and metabolism of fat globules in breast milk.
Human milk fat provides an MUFA/PUFA ratio of 1.62
[11], while the 80:20 blend had a similar proportion
(Fig. 1).
Triacylglycerol Composition
In this study, the most abundant TAG in lard were: POO
(29.6 g/100 g), POL (15.1 g/100 g), PStO (14.2 g/100 g),
PPO (5.6 g/100 g), OOL (5.6 g/100 g) and PPL (5.5 g/100 g).
123
These results for TAG composition of lard are similar to
those previously described in the literature [11].
The most abundant TAG in soybean oil were: LLL
(21.0 g/100 g), OLL (18.3 g/100 g), PLL (14.0 g/100 g),
OOL (12.9 g/100 g), POL (9.5 g/100 g) and LLLn (6.5 g/
100 g). Ribeiro et al. [24] described a similar composition
of triacylglycerols in soybean oil. The TAG composition of
the binary fat blends represents a linear combination of the
fats constituting the blends (Table 2).
The triacylglycerols compositions of the samples, before
and after interesterification, were divided into classes based
on total number of carbons (excluding the carbons of
glycerol) and saturation and unsaturation [23, 25]. Based
on the number of carbons of triacylglycerols in lard and
80:20, 70:30 and 60:40 blends before interesterification,
there was a predominance of C52, while soybean oil and
40:60, 30:70 and 20:80 blends showed a predominance of
C54. Continuous EIE promoted an increase in the C48
group for lard and 80:20, 70:30 and 60:40 blends, in the
C50 group (except for 40:60 blend) and in the C54 group
(except for 40:60 blend). On the other hand, this caused a
reduction in the C52 group across all samples, except for
the 40:60 blend (Table 2).
For food product formulation, the physical properties of
a fat are more easily interpreted when triacylglycerols are
designated by their degree of saturation and unsaturation:
SSS (trisaturated), SSU (disaturated–monounsaturated),
UUS (monosaturated–diunsaturated) and UUU (triunsaturated), instead of by the individual triacylglycerol species
[24]. The SSS, SSU, SUU and UUU contents of TAG
before and after interesterification are shown in Fig. 2.
The triacylglycerol composition of lard was dominated
by the SUU and SSU groups, while soybean oil and the
80:20, 70:30, 60:40, 40:60, 30:70 and 20:80 blends showed
TAG
Lard
Before
Lard
After
80-20
Before
80-20
After
70-30
Before
70-30
After
60-40
Before
60-40
After
40-60
Before
40-60
After
30-70
Before
30-70
After
20-80
Before
20-80
After
Soybean oil
Before
Soybean oil
After
PPP
0.2
1.0
0.2
0.9
0.4
0.9
0.2
0.8
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
MPO
0.8
1.5
0.6
1.1
0.8
1.0
0.5
0.7
0.0
0.0
0.0
0.5
0.0
0.0
0.0
0.0
PPSt
1.2
1.8
1.0
1.3
0.7
1.2
0.4
0.8
0.1
0.3
0.1
0.5
0.0
0.0
0.0
0.0
PPO
5.6
7.8
4.8
6.4
4.5
5.8
3.7
4.6
1.9
2.1
1.9
2.8
1.3
0.3
1.1
1.3
PPL
5.5
5.2
4.6
5.2
4.0
4.7
3.7
4.5
2.7
3.1
2.9
4.2
2.5
6.2
2.7
2.8
MOL
1.7
1.2
1.2
1.2
0.9
1.2
0.7
0.8
0.0
0.0
0.2
0.0
0.0
0.0
0.0
0.0
PStSt
1.5
1.1
0.8
0.5
0.8
0.5
0.1
0.5
0.3
0.9
0.0
0.0
0.0
0.0
0.0
0.0
PStO
14.2
10.8
10.9
7.1
9.7
5.5
8.2
4.9
4.8
4.6
4.0
1.6
3.2
1.1
0.5
0.3
POO
29.6
23.4
24.3
18.3
23.1
15.6
19.7
13.1
12.9
12.2
11.6
7.1
9.2
5.7
4.4
3.2
POL
15.1
14.1
14.7
15.0
13.1
15.5
12.8
15.2
12.0
11.6
11.4
13.6
11.4
12.5
9.5
9.7
PLL
PLLn
3.8
0.0
2.6
0.0
5.1
0.0
5.2
0.0
6.3
0.0
6.7
0.0
7.2
0.0
8.5
0.0
11.0
0.0
10.9
0.0
10.7
0.4
11.6
0.3
12.3
0.0
12.0
0.0
14.0
3.0
12.2
2.7
StStO
1.0
1.7
1.0
1.2
0.7
0.7
0.8
0.9
0.3
2.3
0.0
0.0
0.0
0.0
0.0
0.0
StOO
3.9
5.6
3.3
4.0
2.4
3.0
2.7
2.8
1.6
0.5
1.6
1.3
1.2
1.3
0.7
0.5
OOO
3.6
5.6
4.6
5.3
4.5
5.4
4.3
4.9
3.8
5.2
3.5
2.2
3.2
2.3
2.8
1.5
StOL
4.5
5.6
3.7
6.4
3.4
5.8
4.5
5.5
3.5
3.3
4.0
5.4
3.4
4.2
2.7
2.4
OOL
5.6
8.2
7.7
11.4
8.4
12.6
9.0
13.6
7.6
10.8
11.1
14.9
11.8
14.4
12.9
12.2
OLL
2.4
2.7
6.1
7.4
7.8
9.1
9.5
11.1
17.9
13.4
15.0
18.6
16.8
21.1
18.3
23.3
LLL
0.0
0.0
4.6
2.3
6.7
3.8
9.4
5.5
15.3
14.4
16.3
12.5
18.7
14.8
21.0
21.5
LLln
0.0
0.0
0.0
0.0
0.0
0.0
2.7
0.6
4.3
4.4
5.2
2.9
5.1
4.1
6.5
6.5
LnLnL
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
48
1.1
2.5
0.8
2.0
1.2
1.9
0.7
1.6
0.0
0.0
0.0
0.5
0.0
0.0
0.0
0.0
50
14.0
16.0
11.6
14.1
10.2
12.9
8.5
10.8
5.9
5.5
6.2
7.5
5.0
6.6
3.8
4.1
52
64.1
52.0
55.7
46.0
53.1
44.2
48.0
42.4
41.9
42.4
40.0
34.2
37.8
31.3
31.3
28.1
54
20.9
29.5
31.9
37.9
35.6
41.0
42.8
45.2
52.1
52.1
53.8
57.7
57.2
62.1
64.9
67.8
CN
J Am Oil Chem Soc
Table 2 Triacylglycerol composition of lard, soybean oil and binary blends before and after enzymatic interesterification
123
J Am Oil Chem Soc
Fig. 2 Distribution of
triacylglycerols in lard, soybean
oil and binary blends, before
and after continuous enzymatic
interesterification (UUU
triunsaturated, UUS/USU
diunsaturated–monosaturated,
SSU/SUS disaturated–
monounsaturated, SSS
trisaturated)
a predominance of UUU and SUU groups. Continuous EIE
increased the amount of UUU triacylglycerols (all samples)
and SSS (with the exception of soybean oil plus 30:70 and
20:80 blends), while the levels of UUS (all samples) and
SSU (with the exception of lard and soybean oil)
decreased, indicating that there were exchanges of fatty
acids between triacylglycerols. The increase in SSS triacylglycerols of blends of lard and soybean oil after continuous EIE can be explained by the presence of palmitic
acid at the sn-2 position in lard and the presence of saturated fatty acids in sn-1,3 positions in soybean oil. Soybean
oil (before and after EIE) showed more than 60 g/100 g of
triacylglycerols of the UUU group and therefore did not
present solid fat content even at refrigeration temperature
(5 °C), because the melting point of this group is in the
range -13 to 1 °C, remaining liquid at 5 °C.
13
C-NMR Analyses
Analysis of the regiospecific distribution of fatty acids in
triacylglycerols by NMR is desirable, as the method does
not require hydrolysis by pancreatic lipase, with further
separation of partial acylglycerols performed by thin layer
chromatography and finally, analysis of fatty acids by gas
chromatography [26]. However, the technique cannot discriminate between saturated fatty acids, and cannot discriminate between linoleic and linolenic acids, which are
assessed together. The NMR analysis technique also allows
the determination of fatty acid composition, albeit with the
limitations described above for the distinction of saturated
123
fatty acids and linoleic acid/linolenic acid. The results for
the fatty acid composition (saturated, 18:1 cis ? 18:1 trans
and 18:2 ? 18:3) by NMR were similar to those obtained
by gas chromatography for all samples of this study.
Another point that shows the feasibility of the NMR
technique is that the signal of the spectrum corresponding
to the sn-2 position is always equivalent to around 33.3 g/
100 g, while the signal corresponding to sn-1,3 positions is
always equivalent to around 66.6 g/100 g.
In this study, HMF showed 78.3 g/100 g saturated fatty
acids, 12.8 g/100 g of oleic acid and 9.0 g/100 g linoleic
acid ? linolenic acid in the sn-2 position of triacylglycerols (Fig. 3), while values for sn-1,3 positions were 46.0 g/
100 g saturated fatty acids, 33.1 g/100 g of oleic acid and
20.8 g/100 g linoleic acid ? linolenic acid (Fig. 4).
The structured lipid called Betapol showed a different
distribution of fatty acids at the sn-2 position in relation to
HMF, containing 51.4 g/100 g of saturated fatty acids,
28.4 g/100 g of oleic acid and 20.3 g/100 g of linoleic
acid ? linolenic acid. This lipid contained 50.4 g/100 g of
saturated fatty acids, 37.0 g/100 g of oleic acid and
12.6 g/100 g of linoleic acid ? linolenic acid at sn-1,3
positions.
Lard has a similar regiospecific distribution to that of
human milk fat in the sn-2 position, with 73.0 g/100 g of
saturated fatty acids, 18.4 g/100 g of oleic acid and 8.6 g/
100 g linoleic acid ? linolenic acid (Fig. 3). However, the
distribution at sn-1,3 positions differs to that of HMF, with
24.4 g/100 g of saturated fatty acids, 54.1 g/100 g of oleic
acid and 21.5 g/100 g linoleic acid ? linolenic acid.
J Am Oil Chem Soc
Fig. 3 Distribution of fatty
acids in the sn-2 position of
triacylglycerols in human milk
fat, Betapol, lard, soybean oil
and their blends, before and
after continuous enzymatic
interesterification
Fig. 4 Distribution of fatty
acids in the sn-1,3 positions of
triacylglycerols in human milk
fat, Betapol, lard, soybean oil
and their blends, before and
after continuous enzymatic
interesterification
Therefore, the main differences in these positions involve
saturated fatty acids and oleic acid (Fig. 4). A low intensity
signal in the sn-1,3 positions corresponding to palmitoleic
acid (16:1) was also observed, but was not quantified.
Soybean oil is rich in polyunsaturated fatty acids and
contains 23.6 g/100 g of oleic acid and 73.7 g/100 g of
linoleic acid ? linolenic acid at the sn-2 position. Therefore, soybean oil is practically free of saturated fatty acids
in this position, a typical feature of vegetable oils. However, the literature describes about 3–4 g/100 g of saturated
fatty acids, mainly palmitic acid, in the sn-2 position when
determined by other methods such as enzymatic hydrolysis
[26]. In sn-1,3 positions, soybean oil showed 24.8 g/100 g
of saturated fatty acids, 25.1 g/100 g of oleic acid and
50.1 g/100 g of linoleic acid ? linolenic acid, similar
values to those found in the literature [26].
123
J Am Oil Chem Soc
According to Quinlan, Lockton, Irwin and Lucas [27],
unsaturated fatty acids initially located at the sn-2 position
should largely remain in this position after using sn-1,3
specific lipase, although some degree of acyl migration to
the sn-1,3 positions can occur [28]. The blends of lard and
soybean oil showed lower values of saturated fatty acids at
the sn-2 position compared to HMF (Fig. 3). However,
these values were higher than those found in most infant
formulas [29].
In lard and soybean oil blends, the sn-1,3 positions are
predominantly occupied by unsaturated fatty acids, mainly
oleic acid, when there was a predominance of lard, and linoleic ? linolenic acids, when there was a predominance of
soybean oil. Diets rich in unsaturated fatty acids are beneficial
for infant nutrition, because these fatty acids are more easily
absorbed than saturated fatty acids in sn-1,3 positions [30].
Lard and the 80:20, 70:30 and 60:40 blends showed a
decrease in saturated fatty acids at the sn-2 position after
EIE, while 40:60, 30:70 and 20:80 blends had increased
levels of saturated fatty acids. However, the opposite
occurred for saturated fatty acids in sn-1,3 positions, as the
EIE promoted an increase in lard and 80:20, 70:30 and
60:40 blends, and led to a decrease in the saturated fatty
acids in 40:60, 30:70 and 20:80 blends.
From a nutritional standpoint, blends containing predominantly lard (80:20 and 70:30) after EIE, proved the
most interesting distributions as a HMF substitute, since
their sn-2 positions are occupied mainly by saturated fatty
acids (52.5 g/100 g and 45.4 g/100 g, respectively), while
the unsaturated fatty acids predominantly occupy the sn-1,3
positions.
Conclusions
The development process presented is an eco-friendly
approach for the use of relatively low cost bioresources
such as lard and soybean oil, exploiting their intrinsic
nutritional and physical properties. Blends of lard with
soybean oil in the proportions 80:20 and 70:30, respectively, demonstrated a fatty acid composition and proportions of fatty acids, which are appropriate for the
formulation of pediatric products. These same blends were
more suited for this purpose after continuous EIE because
their sn-2 positions were predominantly occupied by saturated fatty acids while unsaturated fatty acids tended to
occupy sn-1,3 positions, akin to HMF.
Acknowledgments The authors gratefully acknowledge the generous support from the Brazilian research funding agencies, Conselho
Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq),
Coordenação de Aperfeiçoamento de Pessoal de Nı́vel Superior
(CAPES), and Fundação de Amparo à Pesquisa do Estado de São
Paulo (FAPESP).
123
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