Comparative health-related fatty acid profiles, atherogenicity and desaturase indices of marula seed cake products from South Africa and Eswatini Doctor

Marula seed cake (MSC) is a nutritionally-rich natural feed resource that can enhance the healthiness of animal-derived foods (ADFs) for human consumption. This study compared the health-related fatty acid (FA) profiles of MSC products from South Africa and Eswatini. Composite samples monthly collected from both countries were analysed for FAs. MSC products from both countries were found to be dominated by oleic acid (>70%), followed by palmitic, linoleic and stearic acids. Consequently, both products had their FA totals dominated by ƩMUFA followed by ƩSFA, ƩPUFA, Ʃn-6 PUFA and Ʃn-3 PUFA. Both oleic and stearic Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 12 November 2019 doi:10.20944/preprints201911.0132.v1 © 2019 by the author(s). Distributed under a Creative Commons CC BY license. 2 acids were higher (P < 0.01) whilst linoleic (P < 0.001), α-linolenic (P < 0.05), margaric (P < 0.05), palmitoleic (P < 0.05) and eicosatrienoic (P < 0.05) acids were lower in South African in comparison to Eswatini MSC. Consequently, South African MSC had higher ƩMUFA (P < 0.01) but lower ƩPUFA (P < 0.001), Ʃn-6 PUFA (P < 0.001) and Ʃn-3 PUFA (P < 0.05). Also, Eswatini MSC had higher n-6 : n-3 PUFA, PUFA : SFA (P = 0.001) and PUFA : MUFA (P < 0.05) ratios. Further, MSC products from both countries had similarly (P > 0.05) low atherogenicity and high desaturase indices. In conclusion, both country products are rich particularly in oleic acid and their incorporation into farm animal diets would increase content of the MUFA in ADFs and, consequently, improve health benefits to human consumers.


Introduction
One major challenge facing the world particularly in Africa is massive human population growth at a rate that surpasses the capacity to produce sufficient food to nourish the growing masses. The global human population is forecast to surpass 9 billion by 2050, necessitating > 50% increase in food productivity [1]. Apart from plants (crops), animal-derived foods (ADFs) represent the major source of nutrition to the ever growing human populations and constitute a significant portion of current national diets in Africa and globally [2,3]. For example, from 2003 to 2015 the South African per capita consumption of total meats (including beef, pork, veal, lamb, poultry, fish, and shellfish) and milk increased by 54.4% to 66.83 kg/year [4].
However, the healthiness and nutritional quality of ADFs is compromised by the feedstuffs that are fed to animals on farms. In an attempt to enhance animal productivity, reduce feed costs and increase profits, industrialized livestock and poultry producers incorporate in their animal diets a myriad of unnatural and sometimes ethically questionable feedstuffs that raise concerns for public health. These include rendered animal products, animal wastes such as chicken (broiler) litter and manure, rumen contents, hormones, antibiotics, organo-arsenicals and others [5 -7]. Incorporation of these ingredients into animal feeds can result in the presence of a range of biological, chemical, and other etiologic agents in feed that can affect the quality and safety of ADFs and pose potential risks to human health [8,9]. Indeed, a growing body of evidence shows that the use of such unnatural feed resources as animal feed is detrimental to the health and wellbeing of human consumers [6,10,11] and may underlie the rapid proliferation of the so-called lifestyle diseases of Western civilization such as cardiovascular disease (CVD), diabetes, cancer, stroke and others [12,13]. This may explain the significant decline in the demand for certain traditional types of meat such as beef and mutton over the last few decades, as perceived health concerns surrounding red meat consumption increase [14,15].
In contrast, use of natural plant-based feedstuffs and oils results in production of ADFs that are healthy for human consumption. Many studies have demonstrated that use of such natural feed resources enhances the healthiness of ADFs by altering their fatty acid (FA) composition away from the saturated (SFA) towards the unsaturated -MUFA and PUFAprofile and lower n-6 : n-3 PUFA ratios [16 -19]. Also, supplementation of beef cattle diets with the leaf meal of Melia azedarach in replacement of broiler litter, inter alia, increased beef kidney fat α-linoleic acid (n-3), conjugated linoleic acid (CLA) and Ʃn-3 PUFA whilst it decreased the n-6 : n-3 PUFA ratio [20]. Meat with high levels of n-3 PUFAs and CLA, as well as a low n-6 : n-3 PUFA ratio (optimal values ≤ 4), is said to decrease the risk of cardiovascular disease and other 4 chronic disorders [21]. There is therefore a need for a paradigm shift in the way farm animals are fed towards use of natural feedstuffs that are not only healthy to them but to human consumers as well [22].
The MSC is a by-product of oil extraction from the kernels (nuts) of the ripe fruit seeds of marula  [20,29]. Of particular interest, MSC is naturally endowed with a highly desirable FA composition that can enormously enhance the oxidative stability and healthiness of ADFs for human consumption.
In this regard, its abundant residual oil (EE: 343.5 -411.32 g/kg DM) has an extremely high content of oleic acid (74% -85%) [20,30], a hypo-cholesterolemic [31,32], antiatherosclerotic and anti-diabetic [33 -36] MUFA with an extremely high oxidative stability that is 10x more than that of olive oil [37 -39]. The oil's remarkable oxidative stability has long been exploited in Africa, especially for meat preservation [40]. Also, the oil in MSC contains some essential FAs (EFAs) linoleic and α-linolenic acids whose dietary incorporation into ADFs would help in the fight against human illnesses like rheumatoid arthritis [41] and diabetics [42]. Therefore, incorporation of MSC into animal diets in Southern African where this product is produced would not only supply much needed protein, energy and other nutrients, but would also enhance the shelf-life and health status of meat and other ADFs that would, consequently, improve health benefits to human consumers. However, in light of the high phenotypic variation in nut and kernel traits [43] as well as intra-population genetic diversity 5 among S. birrea populations [44], we hypothesized that there would also be significant differences in the FA composition and healthiness of MSC from different regions in Southern Africa. The objective of this study was therefore to compare the FA composition of MSC products produced in South Africa and Eswatini. A lipid aliquot (±30 mg) of sausage batter lipid were converted to methyl esters by basecatalysed trans-esterification in order to avoid CLA isomerisation, with sodium methoxide (0.5 6 M solution in anhydrous methanol) during 2 h at 30 °C, as proposed by Park et al. [46], Kramer et al. [47] and Alfaia et al. [48]. Fatty acid methyl esters (FAMEs) from sausage batter lipid were quantified using a Varian 430 flame ionization GC, with a fused silica capillary column, Chrompack CPSIL 88 (100 m length, 0.25 mm ID, 0.2 μm film thicknesses). Analysis was performed using an initial isothermic period (40 °C for 2 minutes). Thereafter, temperature was increased at a rate of 4 °C / minute to 230 °C. Finally an isothermic period of 230 °C for 10 minutes followed. FAMEs n-hexane (1μl) was injected into the column using a Varian CP 8400

Samples
Autosampler. The injection port and detector were both maintained at 250 °C. Hydrogen, at 45 psi, functioned as the carrier gas, while nitrogen was employed as the makeup gas. Galaxy Chromatography Data System Software recorded the chromatograms.  Results are reported as means ± standard deviation (n = 2). Non-detected FAs were considered as 0 value for statistical analysis. Normal distribution was checked for all data with the onesample Kolmogorov-Smirnoff test and homogeneity of variances with the Levene test.
Differences between pairs of means were tested using Student's t-test. In all tests used, statistical significance was accepted at P < 0.05 level of probability. All statistical analysis were carried out using the Minitab [50] package.
Consequently, the South African MSC oil had higher ƩMUFA (P < 0.01) but lower ƩPUFA (P < 0.001), Ʃn-6 PUFA (P < 0.001) and Ʃn-3 PUFA (P < 0.05) (  [20,30,58]. The variation in the MSC oil FA composition and FA totals between the two countries might be due to marula tree genetic differences or variations in soil type, soil fertility status, rainfall patterns and harvesting time. Indeed, the FA composition and oil content of marula kernels can be affected by harvesting time, with an increase (up to 63% of dry weight) in the oil content obtained at the end (June) in comparison to the start (March) of the harvesting period [39]. Also, marula trees exhibit wide genetic variations [60], with their growth rate and fruit production also marginally linked to rainfall amount [61].
Our data also showed the oil from both South Africa and Eswatini MSC products to have remarkably high n-6 : n-3 PUFA ratios (Table 2). Interestingly, this ratio was higher in the oil from Eswatini MSC than in that from the South African product (P = 0.001). Notwithstanding, both the South African and Eswatini MSC products had oil with exceedingly high n-6: n-3 PUFA ratios in comparison to those of modern (10:1 -15:1) and primitive man (1:1) diets [62,63]. This arises from the high content of linoleic acid in comparison to the Ʃn-3 PUFAs in the oil from both country MSC samples. A high n-6 : n-3 PUFA ratio is said to promote many noncommunicable diseases such as CVD, atherogenesis, arthritis, cancer, osteoporosis and inflammatory and autoimmune diseases whereas lower ratios have suppressive effects [63 -65]. However, despite the fact that the Eswatini MSC had a higher n-6 : n-3 PUFA ratio in comparison to the South African product, it is unlikely that consumption of either product would cause any ill health to humans or animals as both marula by-products are extremely rich in oleic acid, a n-9 MUFA with hypo-cholesterolemic [31,66,67], anti-diabetic [68], antistroke [69], anti-cancer [70,71], anti-obesity and anti-hypertension [72,73] properties. If anything, the Eswatini MSC would appear to be even more healthier as its oil had higher PUFA : SFA (P = 0.001) and PUFA : MUFA (P < 0.05) ratios, as well as higher Ʃn-3 PUFA (P < 0.05), in comparison to the South African product (Table 2). Increased intake of PUFAs relative to SFAs and n-3 PUFAs relative to n-6 PUFAs is highly recommended in the modern era of chronic diseases [62,63,74].
Further, both the South African and Eswatini MSC products had similarly (P > 0.05) low AI and high DI ( Table 2). This is the first time that these indices have been determined in MSC.
Nonetheless, the observed AI values are lower than those previously found in mucuna bean (0.26; Mthiyane et al., unpublished) and meat fats (0.5 -1.0) [75,76]. High AI values indicate greater atherogenicity risk [77]. With low AI values, both the South African and Eswatini MSC products used in this study would therefore appear to be healthy for both human and animal consumption. On the other hand, the DI values of MSC products used in this study are noticeably higher than that (1.25) found in mucuna bean. Whilst the literature reports that a high DI is associated with obesity [78 -80], hypertriacylglycerolaemia [81] and the metabolic syndrome [82], as well as with an increased risk to develop insulin resistance [83], cardiovascular death and total death [84], there are currently no recommended or normal values that should be expected in food/feed products.

Conclusion
Our data showed the FA composition of oil in both MSC products from South Africa and Eswatini to be dominated by oleic acid, followed by palmitic, linoleic and stearic acids, with smaller amounts of arachidic, α-linolenic acid, margaric, lignoceric, palmitoleic, eicosatrienoic, and other acids. Consequently, both products had their FA totals dominated by the ƩMUFA followed by the ƩSFA and low amounts of ƩPUFA, Ʃn-6 PUFA and Ʃn-3 PUFA. Both oleic and stearic acids were higher whilst linoleic, α-linolenic, margaric, palmitoleic and eicosatrienoic acids were lower in oil from South African MSC in comparison to the Eswatini product. Consequently, the South African product's oil had higher ƩMUFA but lower ƩPUFA,