1. Introduction
Mother’s milk is the major source of nutrients for infants. The ingredient that provides the largest part of the infant’s required nutritional energy is human milk fat (HMF) [
1]. Therefore, fat in breast milk is the main source of energy for infants, especially in the first period of their life. Additionally, HMF delivers important structural components for neonatal cell membranes [
2]. The structure of HMF and its fatty acid (FA) composition are unique. It is characterized by a high content of palmitic acid (20–25%) belonging to saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA) [
3]. Among the PUFA in human milk, there are both essential FAs precursors, linoleic acid (18:2 n-6) and α-linolenic acid (18:3 n-3), and a very long chain of bioactive PUFA of both the n-6 and the n-3 families [
4,
5]. Triacylglycerols (TAG) are the main component of HMF (98% of HMF). The structure of human milk TAG is special, as 60–70% of palmitic acid (16:0) is found in the internal position of TAG, and unsaturated FAs are positioned externally. The position of palmitic acid in TAG affects digestion and absorption of fats and minerals from the infant’s intestines [
1,
6].
The World Health Organization and other important health organizations recommend breastfeeding as the best method that provides distinctive benefits for the child and mother. In order to assess the suitability of a given infant formula for feeding, its ability to promote growth and development comparable to that of human milk was determined. The biochemical, functional, and metabolic responses of modified formula-fed infants are compared to those of breast-fed infants [
7]. Vegetable oils, which are typically used in infant formulas, contain palmitic acid in the external positions of TAG. The use of vegetable oils as a substitute of HMF in infant formulas may cause calcium deficiency due to the formation of insoluble calcium soaps with saturated FA released by the action of the specific pancreatic lipase [
6,
8,
9]. Recently, many scientific studies have been carried out and attempts have been made to obtain structured lipids (SL) with FA content and TAG structure similar to HMF. SL are often called nutraceuticals or next generation fats. Nutraceuticals are foods or parts of foods that not only provide basic nutrition but also medical and health benefits and prevent and even treat certain diseases. SL are produced by enzymatic or chemical modification of the TAG [
10,
11]. Due to the numerous advantages of using enzyme catalysts in order to obtain SL with specific functional properties, the number of studies on this type of modification is constantly increasing [
6,
8]. Enzymatic modification has many advantages compared to chemical methods used during fat modification. Lipases (triacylglycerol acylhydrolases, EC. 3.1.1.3) hydrolyze esters in an aqueous medium. However, if access to water is restricted, interesterification reactions can also be catalyzed with lipases. Lipase catalyzed reactions are performed under milder circumstances and with greater selectivity than chemically catalyzed reactions. Additionally, the use of regioselective lipases allows the FA to be kept in the internal position of the acylglycerols. This is desirable for nutritional reasons and impossible to achieve via chemical catalysis [
12].
Several studies were carried out by means of immobilized lipases as catalysts to produce HMFS which mimics HMF. In most of them, SL reminiscent of HMF were obtained via enzymatic acidolysis or interesterification of lard or tripalmitin with free FA from various sources. Lard is the only fat resembling HMF in terms of FA composition and their distribution in glycerol backbone. Compared to HMF, lard is characterized by a similar palmitic and oleic acids content, but it is a worse source of essential FAs. Vegetable oils are rich in polyunsaturated fatty acid. Taking the above-mentioned into account, a mixture of tripalmitin or lard and vegetable oils were selected to obtain a product of similar FAs composition and their distribution in triacylglycerols (TAG) to HMF [
13,
14].
During the development of new products such as HMFS, it is significant to optimize the physical and nutritional properties, but the oxidative stability of these products should also be guaranteed [
2]. Oxidation of lipids is a very important process occurring in food systems. Primary and secondary oxidation products can adversely affect the nutritional value and food safety [
15,
16]. The production process for SL increases free fatty acids (FFA) concentration, which are responsible for off-flavor and odor characteristic of oxidation. A high concentration of FFA in SL induces an unacceptable rancid and bitter taste. Moreover, structured lipids produced with lipids that contain unsaturated FAs can deteriorate during storage. Unsaturated FAs in SL may be rapidly oxidized to hydroperoxides. They can then be quickly broken down into secondary oxidation products, such as aldehydes, alkenes, and ketones responsible for their unwanted flavor. Numerous products of oxidative degradation of edible oils and fats are detrimental to human healthiness. These products damage vitamins and enzymes and may cause mutations or gastrointestinal problems [
17,
18,
19,
20,
21].
Many methods are used to monitor the autooxidation of fats and oils. Currently, instrumental methods that are faster, more accurate, more objective, and have a wider detection range are used. Differential scanning calorimetry (DSC) is a nonchemical method that can be used to determine fat quality parameters [
22,
23,
24,
25].
The purpose of this work was to study the properties of HMFS obtained by enzymatic modification of a mixture of lard and rapeseed oil using DSC as well as chromatography (GC) and thin layer chromatography (TLC).
4. Discussion
HMF provides approximately 50% of infants’ total energy intake and consists mainly C18:1, C16:0, and C18:2 FAs [
34]. The long-chain polyunsaturated FAs (LCPUFA) are also present in HMF at levels tied to the mother’s eating habits. The main LCPUFAs in HMF are arachidonic acid (ARA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA). DHA and ARA are membrane FAs necessary for development and growing of the infants’ central nervous system. FAs such as DHA and ARA also influence bone mineralization, bone mass growth, and the synthesis of eicosanoids, i.e., tissue hormones. Therefore, the human milk fat substitutes should also contain these LCPUFAs in appropriate amounts [
35]. In comparison with HMF, lard is a source of palmitic and oleic acids at a similar level, but it is characterized by less content of linoleic and higher amounts of stearic acid [
12]. Rapeseed oil (RO) contains twice as many of MUFA and PUFA compared to HMF. The addition of the rapeseed oil to lard increased the unsaturated FAs contents.
Brest milk fat contains about 40.6% of the SFA 39.1% of the MUFA and 19.5% of the PUFA [
30], while in the case of obtained SL, SFA amount was determined at the level of from 40.2 to –41.9%, MUFA-from 44.7–46.2%, and PUFA-13.4–13.6%, including significant essential FAs from n-3 group and from n-6 group, like α-linolenic acid and linoleic acid. Content of oleic and palmitic acid for obtained SL is similar to HMF. Obtained SL does not contain valuable DHA, EPA, ARA, but it is a source of precursors of these FAs. FAs such as DHA, EPA, and ARA can be formed in the human body by metabolism of FAs, i.e., α-linolenic acid and linoleic acid. Therefore, the enzymatic modification of a mixture of lard and rapeseed oil allows for an accumulation of new fats with oleic and palmitic acids at quantities comparable to HMF, as well as containing crucial essential FA from the omega-3 group.
The types of FAs and its distribution in the molecule of TAG affect the physical properties of fats as well as the behavior of fats during digestion and absorption [
36,
37]. Breast milk contains approximately 70% palmitic acid in the middle position of TAG, while in most vegetable oils this FA is located in the outer positions of the TAG. Taking into account the obtained results, it can be concluded that lard, like HMF, contains about 85.3% palmitic acid in the internal position of TAG, while the percentage of this FA in the internal TAG position in RO was only 23.3%.
The selective pancreatic lipase plays an significant role in the digestion of fat; it hydrolyzes TAG molecules in the external positions to free FAs and 2-monoglyceride. Therefore, the products of human fat digestion will be 2-monopalmitin that is effectively absorbed and unsaturated free FAs. However, if free palmitic acid is present after hydrolysis, insoluble calcium soaps may form in the intestine. This reduces the absorption of both calcium and fat. Therefore, the position of palmitic acid in TAG is important for the absorption of minerals and fat in infants [
2,
5]. Given the results on the distribution of FAs in the TAG molecules of investigated SL, it can be stated that this distribution is very similar to that of HMF. The percentage of palmitic acids in internal position of TAG in all SL exceeded 33%, which means that it is mostly in the sn-2 TAG position. Considering the percentage of unsaturated FAs in the internal TAG position in SL, it can be deduced that they are mainly in the external TAG positions. These are especially linoleic acid and oleic acid. Similar results were obtained in the work related to enzymatic modification of lard with milk thistle oil [
4]. Taking into account the obtained results, it can be concluded that the time of interesterification affects the distribution of FAs in the TAG both in the middle and in the outer position. The modification of fats with the use of Lipozyme RM IM occurred predominantly in the outer positions of the TAG. Because interesterification in the presence of regiospecific enzymes only occurs at the external positions, the sn-2 position of the TAG remains unchanged [
33]. Changes in internal position of TAG may be caused by possible acyl migrations within and between TAG molecules during a longer interesterification period, as evidenced by Xu et al. [
38].
Literature data indicate that fats after interesterification are characterized by a reduced oxidative stability compared to the starting blends [
19,
20,
23]. The obtained results confirm that the enzymatic modification of fats reduces their oxidative stability. The induction time for samples interesterified for a long time (24 h) is higher than for those interesterified for short time (4 h). The results obtained by DSC are consistent with tests of peroxide value (PV) determination. The high PV is a measure of the content of primary oxidation products from which secondary oxidation products can be formed very quickly. Bryś et al. [
23] report that there is an inverse relationship between PV and induction time. An increased index of PV in the blends after interesterification may reduce its oxidative stability. This parameter is a very important factor that affects the quality of oils, fats, and foods. Usually, foods containing high amounts of unsaturated FAs are characterized by a low oxidative stability. Within the FA family, PUFA are highly unstable molecules and are susceptible to oxidation processes, which results in the formation of free radicals, polymers and hydroperoxides which may lead to quality loss, both in terms of technology and health [
20]. The presence of free FAs in the oils can induce oxidation due to the catalytic effect of FAs carboxyl groups on the formation of free radicals. Frega et al. [
39] investigated the effect of free FA content on the oxidative stability of vegetable oils. The scientists observed the pro-oxidative effect of free FAs with all filtered oils. A higher reduction in oxidative stability was also observed in the case of modified oils and fats, when the product after interesterification contained a higher level of free FAs, monoacylglycerols and diacylglycerols [
20]. Triacylglycerols are the core components of fats. Fats also include certain quantities of incomplete acylglycerols and free FAs. As a result of the hydrolysis process in the presence of lipases free FAs, partial acylglycerols and glycerol are obtained. The hydrolysis reaction is reversible and, if the water level is lowered, the formation of new acylglycerols will dominate the hydrolysis [
40]. Oxidative stability does not only depend on the FA composition and free FA content. Stability may also be affected by the content of antioxidants. Vegetable oils like rapeseed oil contain natural antioxidants such as tocopherols, tocotrienols, phenolic compounds, or phytosterols. The inferior stability of mixtures of lard and rapeseed oil after interesterification, as compared to the starting materials, may be related to the loss of these natural antioxidants during the process [
20,
41]. The oxidative stability of fats and oils is also related to the distribution of FAs in the TAG molecules. During interesterification the distribution of FAs changes and it may affect the oxidative stability [
42].
The activation energies for the thermal oxidative decomposition of the fats obtained in this work correlate with the data mentioned in the literature [
24]. The results indicate that E
a should not be the only parameter of comparison for analyzed fats and oils. Interesterified lard with rapeseed oil is a very complex blend, containing mainly triacylglycerols. These compounds are highly diverse in terms of chain length and degree of unsaturation of FAs and their position. Such variety influences the oxidative stability. During the measurement at the same time there are some reactions that are characterized by a different constant rates and DSC detects only the reactions with the highest exothermic effects. This can be an explanation of why changes in the effective activation energy, according to the compensation theory, have no physical significance [
24].