Effect of Chemical and Enzymatic Modifications on the Identity Characteristics of Lard: A Review

Abstract

Lard is a fat substance extracted from the adipose tissues of swine. Due to its specific physical and functional characteristics, lard has found numerous uses in the food and feed industry. Lard has often been subjected to modification through techniques, such as fractionation, partial hydrogenation, and interesterification. Although the application range of lard in food would have widened, these modification techniques could also change the original identity characteristic properties of lard, which enable its detection in food by various analytical methods. The purpose of this review is to present an update of the current literature in this topic and provide some directions for future research.

Keywords:

• Animal fat
• DSC
• Interesterified lard
• Lard detection
• Lard stearin
• Lard olein

INTRODUCTION

Lard is a fatty substance extracted from the adipose tissues of swine by a rendering process. Although it is a taboo for certain communities, it has found many uses in food preparations, mainly for its flavors and functional properties.[1] Particularly, it has been reported to contribute a unique flavor and function to Chinese-style bakery products. Lard has also been used in the manufacture of bread, biscuits, crackers, cakes, cookies, and so on. The back fat of swine, which is extracted from the dorso-lumber region of the animal, is preferred in processed meat products because of its effect on the taste and texture.[2] In the commercial world, lard of many different kinds may exist depending on the needs of the industry. For instance, lard may be used after modification by methods, such as hydrogenation, interesterification, and fractionation.[3,4] Whenever oxidative stability and firmness have become the key concern, lard was subjected to hardening through hydrogenation. Partially hydrogenated lards were used as a general purpose shortening as well as a medium for commercial frying.[5] In the preparation of certain types of commercial shortenings, lard was transformed by an interesterfication process, which would alter the triacylglycerol molecular structure of lard by reshuffling of the component fatty acids.[6] If lard was subjected to fractional crystallization, two modified forms of lard known as lard stearin and lard olein would be obtained. Generally, the aim of fractionation of lard is to recover a high-melting solid fat and liquid oil, which may have different physical and functional properties. As lard in different forms has been used in food systems, it has become a concern for consumers whose religious restriction prevents the use of lard in food.[7,8] However, the true identity of lard might change to a varying degree due to the above mentioned modification processes, which would make its detection in food systems really challenging. The objective of this article is to highlight how the identity characteristics of native lard (NLD) would be affected under different methods of modification.

IDENTITY CHARACTERISTICS OF LARD BY FATTY ACID COMPOSITION

Assignment of characteristic marker fatty acids for the identification of NLD based solely on the overall fatty acid composition may be difficult. It is because of the fact that the variation in the overall fatty acid compositions is wider as reported from different sources[8 17] (Table 1). NLD is generally found to have more unsaturated fatty acids (51.3 to 65.9%) than saturated fatty acids (34.1 to 48.7%). In the majority of the cases, oleic acid (24.0 to 51%) has emerged as the most dominant fatty acid, followed by palmitic (20 to 29%) acid (Table 1). Although stearic acid is the third most abundant fatty acid of NLD, at times linoleic acid (17.1 to 19.3%) has been found to take over the position of stearic acid. In fact the causes for the variations in the distribution of fatty acids of NLD could be multi-factorial. According to Kincs,[1] the type of diet fed to the animal may have a strong influence on the fatty acid composition of NLD. For instance, feeding the animals with diets containing polyunsaturated oils could make NLD become more unsaturated.[18] At times, fatty acid composition of NLD might differ based on the part of the animal from where it has been extracted.[19,20] For instance, the back fat of swine is often found to be more saturated than leaf lard, which is extracted from fat around the kidneys of the animal. Further, even natural factors, such as genetics and sex of the animal could have some influence on the fatty acid composition of NLD.[21 24]

Table 1 Fatty acid compositional variations of lard (NLD) from different sources. ¹

Fatty acid	NLD-A	NLD-B	NLD-C	NLD-D	NLD-E	NLD-F	NLD-G	NLD-H	NLD-I	LS	LO
C10:0	—	—	—	—	—	0.1	—	—	—	—	—
C12:0	—	—	—	—	—	0.2	—	0.1	0.07	—	—
C14:0	1.60	1.5	1.8	1.2	—	1.6	—	1.0	1.1	1.23	1.46
C15:0	—	—	—	—	—	—	—	—	0.08	—	—
C15:1	—	—	—	—	—	—	—	—	—	—	—
C16:0	23.67	25.4	29.0	24.0	24.0	25.7	24.6	23.9	20.30	31.68	21.76
C16:1	4.0	2.4	2.6	2.79	—	1.6	—	—	1.48	0.72	2.30
C17:0	—	—	—	—	—	0.4	—	—	0.40	—	—
C17:1	—	—	—	—	—	—	—	—	0.24	—	—
C18:0	15.05	13.3	17.9	12.69	16.0	14.7	15.8	15.4	11.21	25.15	6.38
C18:1	34.67	44.0	41.9	51.24	47.0	42.7	42.9	41.6	23.92	24.97	42.76
C18:2	17.13	3.4	6.8	5.70	9.0	9.7	9.9	13.2	19.29	14.04	23.62
C18:3	2.05	—	—	0.74	0.3	1.0	—	0.4	1.31	—	—
C20:0	0.55	—	—	1.63	—	0.6	—	—	0.98	—	—
C20:1	—	—	—	—	—	1.1	—	—	0.08	—	—
C22:1	—	—	—	—	—	0.7	—	—	—	—	—
SFA	40.87	39.9	48.7	39.52	40.0	43.2	40.4	40.4	34.14	58.06	29.6
USFA	59.13	60.1	51.3	60.48	60.0	56.8	59.6	59.6	65.86	39.73	68.68
Ref.	[8]	[9]	[10]	[11]	[12]	[13]	[14]	[15]	[16]	[17]	[17]
^1LS: lard stearin; LO: lard olein; SFA: saturated fatty acid; USFA: unsaturated fatty acid.

If NLD is fractionated into component fat derivatives, such as lard stearin (LS) and lard olein (LO), there could be drastic changes in the fatty acid compositions. Fractionation of NLD could be accomplished through techniques, such as short-path distillation,[25] supercritical carbon dioxide extraction,[26] or dry and solvent crystallization.[27,28] As triacylglycerol (TAG) molecules with more saturated fatty acids would undergo crystallization easily, there could be a considerable amount of saturated fatty acids migrating into the solid phase, leaving more unsaturated fatty acids in the liquid phase. As a consequence, the palmitic and stearic acids contents of the solid-phase would normally be higher than those of NLD.[26] As reported by Yanty et al.,[17] palmitic acid would have become the most dominant fatty acid (31.68%) in LS followed by stearic (25.15%). Meanwhile, in LO, there would be some increase in the proportions of the unsaturated fatty acids, such as oleic and linoleic. According to some other reports, unsaturated to saturated fatty acid ratio of the lower melting components isolated from the multi-step crystallization of NLD was found to increase progressively.[25,26] As a result of the compositional changes, the iodine values of the fat derivatives would differ very much from that of NLD. For instance, a remarkable reduction in the iodine value of LS was observed as the saturated fatty acid content has gone down. Similarly, the iodine value of LO was increased tremendously while the unsaturated fatty acid content has gone up.[28]

Unlike most plant-based lipids, lard having a higher proportion of palmitic and lower contents of oleic and linoleic acids at the sn-2 position of the glycerol backbone is a unique feature, which has been established in several previous studies.[8,17] Hence, the examination of the fatty acid distribution of the sn-2 position of the glycerol backbone has been continually used as an identity characteristic of NLD for its detection in food.[29] Interestingly, this characteristic feature still remains intact even after fractionation of NLD into components, namely, LS and LO.[17] The proportion of C16:0 at the sn-2 position of LS is found to be 76.57%, while the proportion of the same fatty acid at the sn-2 position of LO is 79.18%.

If NLD is modified through either chemical or enzymatic interesterification process, the overall fatty acid compositions of the fat derivatives remain almost the same.[9,30] However, significant changes would be expected in the fatty acid distribution at the sn-2 position of the glycerol backbone after chemical interesterification. According to Rashood et al.,[8] total C_16:0 content at the sn-2 position was reduced from 67.67 to 25.85% in chemically interesterified lard (CLD). Subsequent investigation on the chemical interesterification of NLD has also shown a similar pattern of changes.[31] The main reason for this would be the fact that chemical interesterification causes relocation of the fatty acids attached to the glycerol backbone. On the other hand, enzymatic interesterification of NLD by using pseudomonas sp. lipase was able to replace the palmitic acid occupation in the sn-2 position of glycerol only partly. As such, palmitic acid content of sn-2 position was decreased from 68.2 to 58% while the oleic acid content was increased from 16.1 to 23.60%.[32]

IDENTITY CHARACTERISTICS OF LARD BY TRIACYLGLYCEROL COMPOSITION

The TAG molecular distributional pattern of NLD from different sources has been reported in the literature (Table 2). According to most studies, PLL, OOL, LPO, OPO, PPO, and SPO (O = oleic acid; P = palmitic acid; S = stearic acid; L = linoleic acid) are the TAG species occurring in higher amounts in NLD. In majority of the cases, OPO has emerged as the most dominant TAG molecular species, although considerable variations could be observed in the proportional distribution of other TAG molecules. According to Marikkar et al.,[30] LPO, and SPO are the second and third most abundant TAG molecules of NLD. This is in agreement with the previous findings reported by Rashood et al.[8] and Yanty et al.[17] (Table 2). However, the results reported by Liu et al.[33] and Silva et al.[9] were somewhat dissimilar with regard to the distribution of other TAG molecules. According to Liu et al.[33] POS (31.9%) and POP (19.0%) were the second and third most abundant TAG molecular species in NLD. Likewise, TAG profile of NLD reported by Silva et al.[9] showed that PSO (18.3%), and PLL (12.2%) were the second and third most abundant TAG molecular species in NLD. These differences in TAG composition could be attributed to the factors that have been mentioned earlier.

Table 2 Triacylglycerol (TAG) compositional variations of lard and its fat derivatives. ¹

TAG	NLD-A	NLD-B	LS	LO	NLD-C	ELD	CLD
LLLn	0.11	1.54	0.22	2.29	1.07	0.86	1.00
LLL	1.03	0.68	0.23	1.43	0.81	0.70	0.15
OLL	3.72	4.68	2.11	6.01	4.07	4.05	4.70
PLL	6.92	7.05	3.26	9.33	6.38	5.20	5.00
OOL	5.49	6.93	3.40	8.48	6.70	8.10	10.30
LPO	19.99	20.0	9.32	24.52	22.55	19.23	14.90
PPL	—	2.62	3.96	2.63	2.47	4.00	4.90
OOO	8.08	4.33	2.46	5.61	5.58	7.10	6.90
OPO	19.31	20.67	9.48	26.11	26.58	24.20	19.90
PPO	13.46	10.63	22.87	3.05	8.86	10.95	11.60
OOS	4.19	3.62	1.79	4.30	3.55	5.05	8.15
SPO	13.17	12.52	30.19	2.16	10.90	9.00	9.85
PPS	1.00	0.81	2.53	ND	0.37	1.00	2.40
SSO	0.99	0.83	2.29	ND	—	—	—
SSS	—	1.31	4.14	ND	—	—	—
Unknown	—	1.84	1.78	4.12	—	—	—
Ref.	[8]	[17]	[17]	[17]	[30,31]	[31]	[31]
^1NLD: natural lard; LS: lard stearin; LO: lard olein; ELD: enzymatically interesterified lard; CLD: chemically interesterified lard.

If NLD is subjected to fractional crystallization, the majority of the tri- and di-saturated TAG molecules would tend to go into the solid phase, leaving mono-saturated and tri-unsaturated TAG molecules in the solvent phase.[27] Hence, the solid and liquid fractions would display TAG compositions, which could differ considerably from that of NLD. For instance, in the multi-step crystallization of NLD as reported by Wang and Lin,[26] the most high-melting component has become enriched with more saturated TAG molecules. As a consequence, LS would tend to have increased amounts of PPO and SPO and decreased amounts of LPO and OPO. In the same study, the components isolated from the third and fourth steps of crystallization were tended to become enriched with TAG molecules, which are mainly esterified with unsaturated fatty acids. These results are largely in agreement with the findings obtained for fractionation of NLD by Yanty et al.[17] In this study, SPO (30.19%) was the most dominant TAG molecular species in LS followed by PPO (22.87%) and LPO (9.32%). On the other hand, LO has been found to possess OPO as the most dominant TAG molecule followed by LPO. In fact, the proportions of LPO and OPO in LO were remarkably higher than those of the same molecules in either NLD or LS (Table 2).

The TAG distributional pattern of NLD could also change significantly, if it were subjected to interesterification, which would cause exchange of fatty acids within a TAG molecule and in between TAG molecules. Depending on the type of interesterification, the interesterified product might have TAG molecular composition, which would be different from the starting material. Some amount of hydrolysis of fatty acid might also happen leading to the formation of partial acylglycerols in the end product.[31,32] According to past studies, both chemical and enzymatic interesterification of NLD were found to cause increases in OOL, PPL, PPO, OOS, and PPS with the concurrent decreases in the proportions of PLL, LPO, and OPO (Table 2). However, POO has still been remained as the most dominant TAG molecular species of both CLD and enzymatically interesterified lard (ELD). These results were in accordance with the previous findings reported by Rashood et al.,[8] who also found that the most dominant TAG molecular species of CLD was OPO followed by LPO. Although ELD has been found to possess OPO as the most dominant TAG molecule, its second most abundant TAG molecule was LPO.

IDENTITY CHARACTERISTICS OF LARD BY DIFFERENTIAL SCANNING CALORIMETRY (DSC)

Thermal analysis by DSC has been found to be a potentially useful approach in food authentication. Unlike chromatographic techniques, which involve sample derivatization and use of solvents, the DSC approach is more direct and easy to handle. Generally, thermal events associated with lipids are represented by both cooling and melting thermograms obtained by DSC. According to the current literature, the thermal profiles of plant lipids and animal lipids including NLD have been documented by several research groups.[7,8,11,17,30,32] In most of the instances, cooling curves of NLD were comparably similar and found to display exothermic transitions in both high (>0°C) and low (<0°C) temperature regions (Fig. 1). In an earlier study, Huyghebaert et al.[34] found that the cooling profile of NLD had three thermal transitions, of which the one appearing at the onset of crystallization was so small that it might not even be visible, while the other two were relatively larger. In the majority of the cases, the thermal transitions displayed by NLD were sharp in appearance and positioned wide apart in the cooling curves.[17,30] These results were largely in good agreement with the description DSC cooling curves reported by other investigators,[8,35] although some minor differences could be noticeable in the positions of the thermal events, due to sample to sample variations in TAG and fatty acid compositions (Tables 1 and 2). While the transition appearing in the high temperature region (>0°) could have been caused by the high melting TAG groups (HMG), the one appearing in the high temperature region (<0°) could be due to the low melting TAG groups (LMG). These two thermal transitions of NLD might play a crucial role in the detection of NLD in commonly used vegetable oils. When palm oil was adulterated with NLD in the range of 1–20% (w/w), an exothermic peak in the lower temperature region of the cooling curve appeared at –37°C indicating NLD contamination.[30] This phenomenon could be due to the influence of LMG transition on the overall thermal properties of palm oil. Likewise, the HMG thermal transition has been found to be useful for detection of NLD in canola and sunflower oils as the gradual increase of NLD in canola and sunflower oils has given rise to an extra sharp peak in the high-melting region of the DSC heating curve.[7]

Figure 1 DSC cooling curves of natural lard (NLD), lard stearin (LS), lard olein (LO), chemically-interesterified lard (CLD), and enzymatically-interesterified lard (ELD).^[17,31]

Fat derivatives isolated from NLD through fractionation would have their own characteristic thermal curves since differing compositions may influence the thermal properties. As reported by Yanty et al.,[17] high-melting component isolated from fractionation of NLD would become enriched with more saturated TAG molecules, while the liquid component would become concentrated with more unsaturated TAG molecules. Since solid component is found to lose a greater proportion of the unsaturated TAG molecules, the heat of transition (ΔH) of the high-melting peak would become stronger as well as its position (T _peak) could be shifted towards the higher temperature region (Fig. 1, Curve-LS). On the other hand, in the liquid phase, the increase in unsaturated TAG molecules would make the heat of transition (ΔH) of the low-melting peaks stronger as well as their positions (T _peak) be shifted towards the lower temperature region (Fig. 1, Curve-LO). These changes in the thermal characteristics of solid stearin and liquid olein would make them distinctly different in identifying from that of NLD (Fig. 1). The actual effect of the observed shift in the positions of the LMG and HMG thermal transitions of derivatives of NLD on their detection in vegetable oils, such as palm, sunflower, and canola oils, is yet to be determined.

DSC thermal profile might also change due to interesterification of NLD either by chemical or enzymatic method. As pointed out earlier, this could have been attributed to the changes taking place in TAG composition due to the reshuffling of fatty acids in the original sample.[8,32] According to Marikkar,[31] CLD exhibited two sharp peaks at 17.75 and −43.7°C, and a strong broader peak at 2.8°C. Apart from these, a minor peak was also found to appear at 7.8°C. Overall, the number of thermal transitions increased from two to three with the changing TAG composition and in addition, the high-melting peak was reduced in size while the low-melting peak was transformed to two peaks with substantial broadening (Fig. 1). Likewise, after NLD being modified by enzymatic transesterification (ELD), thermal transitions occurred in four major steps with peak maxima at 16.20, 6.75, −27.65 and −41.40°C.[32] These four thermal transitions were also found to overlap with each other to exhibit peak broadening (Fig. 1). As noticed earlier, these changes could be attributed to the TAG compositional changes as presented in Table 2. The possibility of detecting CLD and ELD in vegetable oils has also been explored. When palm oil was adulterated with either by CLD or ELD in the range of 1–20% (w/w), an exothermic peak again appeared at –37°C.[30,32] This observation suggested that the adulteration peak appearing at –37°C of the DSC cooling curve of palm oil could be a common indicator for detection of NLD or its modified forms, such as CLD and ELD in palm oil.

CONCLUDING REMARKS

Determining the common characteristic features between NLD and its modified forms is important for their detection in food systems. NLD having an extremely higher proportion of C16:0 at the sn-2 position is a unique feature, which has been used frequently for identification purposes. This feature is found to remain intact even after fractionation of NLD into LS and LO. When NLD was transformed into interesterified lard by using a chemical catalyst, this characteristic feature is entirely lost. Based on TAG composition, the major TAG molecular species occurring in higher amounts in NLD are PLL, OOL, LPO, OPO, PPO, and SPO. Although proportional variations in the distribution of the TAG molecules have been noticed for samples from different sources, OPO has emerged as the most dominant TAG molecular species in the majority of the cases. However, OPO has still been the most dominant TAG molecular species even after modification through interesterification using either chemical or biological catalysts. After fractionation of NLD into solid and liquid components, OPO remains as the most dominant TAG species only in LO, while SPO becomes the most dominant TAG species of LS. As DSC thermal profiles of lipids are highly sensitive for subtle TAG compositional changes, lard in other forms, such as LS, LO, CLD, and ELD, display thermal profiles, which are significantly different from that of NLD.