1. INTRODUCTION
⌅Coconut oil is widely consumed as a food ingredient worldwide, especially in Asia and south India. The oil is also used in cosmetic and indigenous medicinal systems like Ayurveda and Sidha (Joshi et al., 2020Joshi S, Kaushik V, Gode V, Mhaskar S. 2020. Coconut Oil and Immunity: What do we really know about it so far? J. Assoc. Physicians India 68, 67–72. https://doi.org/10.1021/ed085p1550). It is a rich source of the saturated fatty acids lauric and myristic acid (Widianingrum et al., 2019Widianingrum DC, Noviandi CT, Salasia SLO. 2019. Antibacterial and immunomodulator activities of virgin coconut oil (VCO) against Staphylococcus aureus. Heliyon 20, e02612. https://doi.org/10.1016/j.heliyon.2019.e02612). Lauric acid forms the active and digestible monolaurin in the human body, and recently coconut oil and monolaurin have been investigated in managing COVID-19 (Maruyama et al., 2014Maruyama JM, Soares FAD, Agostinho NRD’, Gonçalves MI, Gioielli LA, da Silva RC. 2014. Effects of emulsifier addition on the crystallization and melting behavior of palm olein and coconut oil. J. Agric. Food Chem. 62, 2253–2263. https://doi.org/10.1021/jf405221n; Angeles-Agdeppa et al., 2021Angeles-Agdeppa I, Nacis JS, Capanzana MV, Dayrit FM, Tanda KV. 2021. Virgin coconut oil is effective in lowering C-reactive protein levels among suspect and probable cases of COVID-19. J. Funct. Foods 83, 104557. https://doi.org/10.1016/j.jff.2021.104557). Saturated fatty acids are associated with the risk of cardiovascular disease; however, coconut oil contains medium-chain saturated fatty acids which are readily absorbed by the portal vein and play a greater role as a source of energy than in cholesterol synthesis (Sacks et al., 2017Sacks FM, Lichtenstein AH, Wu JHY, Appel LJ, Creager MA, Kris-Etherton PM, Miller M, Rimm EB, Rudel LL, Robinson JG, Stone NJ, Van Horn LV. 2017. American Heart Association. Dietary fats and cardiovascular disease: a presidential advisory from the American Heart Association. Circulation 136, e1–e23, https://doi.org/10.1161/CIR.0000000000000510). Also, coconut oil is regarded as a super-food as it improves memory, promotes heart health, strengthens immunity, and possesses wound healing, antiviral, antibacterial, and anti-inflammatory properties (Joshi et al., 2020Joshi S, Kaushik V, Gode V, Mhaskar S. 2020. Coconut Oil and Immunity: What do we really know about it so far? J. Assoc. Physicians India 68, 67–72. https://doi.org/10.1021/ed085p1550; Peedikayil et al., 2016Peedikayil FC, Remy V, John S, Chandru TP, Sreenivasan P, Bijapur GA. 2016. Comparison of antibacterial efficacy of coconut oil and chlorhexidine on Streptococcus mutans: An in vivo study. J. Int. Soc. Prev. Commun. Dent. 6, 447–452. https://doi.rg/10.4103/2231-0762.192934; Chen et al., 2022Chen X, Kim DI, Moon HG, Chu M, Lee K. 2022. Coconut oil alleviates the oxidative stress- mediated inflammatory response via regulating the MAPK pathway in particulate matter-stimulated alveolar macrophages. Molecules 27, 2898. https://doi.org/10.3390/molecules27092898). Coconut oil is expensive compared to other common oils and this often leads to adulteration, and palm oil is the most widely used adulterant due to its low price and easy availability (Alkan et al., 2012Alkan D, Tokatli F, Ozen B. 2012. Phenolic characterization and geographical classification of commercial extra virgin olive oils produced in Turkey. J. Am. Oil Chem. Soc. 89, 261–268. https://doi.org/10.1007/s11746-011-1917-6). Physicochemical properties are significant in the purity checking and monitoring of adulteration in oils, and 1H NMR spectroscopy has been used as an efficient tool for the characterization of oils (Siudem et al., 2022Siudem P, Zielinska A, Paradowska K. 2022. Application of 1HNMR in the study of fatty acids composition of vegetable oils. J. Pharm. Biomed. Anal. 212, 14658. https://doi.org/10.1016/j.jpba.2022.114658). A recent work from our group has investigated the application of NMR for the identification of fatty acid constituents and evaluation of physicochemical parameters of Garcinia gummi-gutta seed oil (Priya Rani et al., 2022Priya Rani M, Gokul Raj MR, Rameshkumar KB. 2022. Garcinia gummi-gutta seeds. A novel source of edible oils. J. Sci. Food Agric. 102, 3475–3479. https://doi.org/10.1002/jsfa.11671). The food sector, especially the edible oil sector, demands rapid, precise and reliable alternatives to the existing wet lab methods, and the present study elaborates the application of 1H NMR spectroscopy in the authentication of coconut oil and in the detection of adulteration of coconut oil with palm oil.
2. MATERIALS AND METHODS
⌅2.1. Sample preparation
⌅Authentic samples of coconut oil and palm oil were procured from oil mills in Thiruvananthapuram, Kerala. For the preparation of blends (5-50% v/v), varying concentrations of coconut and palm oils were mixed and the resulting blends were homogenized at 80 ºC.
2.2. 1H NMR analysis
⌅For 1H NMR analysis, CDCl3 was used as the solvent along with TMS (Tetramethyl silane) as the internal standard. The spectra were taken using Bruker Avance III HD, 400 MHz spectrometer, at 30° pulse. The integral values for the peaks obtained from 1H NMR analysis were used for the evaluation of various physicochemical parameters. For the authentication of coconut oil, various physico-chemical parameters such as average chain length, saponification value, molecular weight, iodine value, peroxide value and percentage unsaturation were calculated in triplicate (n = 3) from 1H NMR as described previously (Skiera et al., 2012Skiera C, Steliopoulos P, Kuballa T, Holzgrabe U, Diehl B. 2012. 1H-NMR spectroscopy as a new tool in the assessment of the oxidative state in edible oils. J. Am. Oil Chem. Soc. 89, 1383–1391. https://doi.org/10.1007/s11746-012-2051-9; Siudem et al., 2022Siudem P, Zielinska A, Paradowska K. 2022. Application of 1HNMR in the study of fatty acids composition of vegetable oils. J. Pharm. Biomed. Anal. 212, 14658. https://doi.org/10.1016/j.jpba.2022.114658).
2.3. Determination of physico-chemical parameters using the 1H NMR spectroscopic method
⌅1H NMR values for oils and fats depict specific resonances for the protons present in the structural patterns of triglycerides, especially in the methylene groups (-CH2), double bonds (-CH=CH-) and in the terminal methyl groups (-CH3) (Ivanova et al., 2022Ivanova M, Hanganu A, Dumitriu R, Tociu M, Ivanov G, Stavarache C, Popescu L, Ghendov-Mosanu A, Sturza R, Deleanu C, Chira NA. 2022. Saponification value of fats and oils as determined from 1H-NMR data: The case of dairy fats. Foods 11, 1466. https://doi.org/10.3390/foods11101466). Average chain length, which is determined by adding ‘one’ to the total integral values corresponds to the characteristic peaks (δ 0.89, 1.30, 1.60, 2.00, 2.30, 2.80, 4.30, 5.26 and 5.36) in the 1H NMR spectrum. These chemical shift values correspond to unsatuarated protons as well as protons of methylene groups of the fatty acyl chains. Saponification value (SV) is the number of milligrams of potassium hydroxide required to saponify 1 g of the sample completely (Reda et al., 2007Reda SY, Costa B, Freitas RJS. 2007. Determination of iodine value in ethylic biodiesel samples by 1H NMR. Ann Magn Reson 6, 69–75.). Saponification value can also be calculated from 1H NMR values using the equation (Carvalho Dos et al., 2018Carvalho Dos SR, Alves Chagas E, Melo Filho A, Takahashi J, Montero Fernandez I, Dos Santos FG, Cardoso Chagas P, Goncalves Reis De Melo A. 2018. Chemical characterization of oils and fats from Amazonian fruits by 1H NMR. Chem. Eng. Trans. 64, 235–240. https://doi.org/10.3303/CET1864040):
where the value 398.42 is the average mass of the fatty acids.
Molecular weight (Mw) represents the weight corresponding to the fatty acid-derived triglycerides. While calculating the molecular weight of TAG (Triacyl glycerol), the terminal -CH3 and glycerol entity along with the carbonyl group of the fatty chain are given a fixed molecular weight, while the number of -CH2- and -CH=CH- may vary. Molecular weight can be calculated using the equation:
where, T represents the total number of hydrogen and V represents the peaks of 1H NMR spectrum assigned to unsaturated fatty acids. T value was obtained by the sum of integral values of hydrogen in the 1H NMR spectrum (A-I) divided by an area of a single proton (PA).
where, G = the sum of two hydrogen peaks attached to methylene glycerol carbons at δ 4.3 and 4.1 ppm.
The peak area of olefinic hydrogen at δ 5.4 ppm (I) and 5.3 ppm (H) were used to obtain V by the equation:
Iodine value gives the number of grams of iodine absorbed per 100 g of sample, and gives the average amount of unsaturation (Reda et al., 2007Reda SY, Costa B, Freitas RJS. 2007. Determination of iodine value in ethylic biodiesel samples by 1H NMR. Ann Magn Reson 6, 69–75.),
As a milliequivalent of iodine per 1 kg of sample, peroxide value is determined by adding potassium iodide under specific conditions (Skiera et al., 2012Skiera C, Steliopoulos P, Kuballa T, Holzgrabe U, Diehl B. 2012. 1H-NMR spectroscopy as a new tool in the assessment of the oxidative state in edible oils. J. Am. Oil Chem. Soc. 89, 1383–1391. https://doi.org/10.1007/s11746-012-2051-9). Peroxide value gives the extent of the oxidative deterioration of the oil sample. It corresponds to the sum of the integral values at δ 2.6 to 2.9 and δ 5.1 to 5.6 divided by the integral values at δ 0.6 to 2.5. Percentage unsaturation corresponds to the signals at δ 2.0, 2.8 and 5.4 ppm which are due to the -CH2CH=CHCH2=CHCH2CH= and -CH=CH- respectively of the fatty chain group of the triglyceride.
3. RESULTS AND DISCUSSION
⌅The physicochemical parameters play an important role in assessing the quality and authenticity of oils and oil-derived value-added products. The conventional wet lab methods for physico-chemical evaluations are time consuming, destructive and require a large sample size, while 1H NMR has emerged as a standardized and validated method for the determination of physico-chemical parameters (Skiera et al., 2012Skiera C, Steliopoulos P, Kuballa T, Holzgrabe U, Diehl B. 2012. 1H-NMR spectroscopy as a new tool in the assessment of the oxidative state in edible oils. J. Am. Oil Chem. Soc. 89, 1383–1391. https://doi.org/10.1007/s11746-012-2051-9; Siudem et al., 2022Siudem P, Zielinska A, Paradowska K. 2022. Application of 1HNMR in the study of fatty acids composition of vegetable oils. J. Pharm. Biomed. Anal. 212, 14658. https://doi.org/10.1016/j.jpba.2022.114658). The 1H NMR- derived (Figure 1) physicochemical parameters of coconut oil, and the mixture with palm oil are given in Table 1.
| Parameters | Blend sample ratio (%) | ||||||
|---|---|---|---|---|---|---|---|
| Coconut oil | CO:PO (95:5) | CO:PO (90:10) | CO:PO (75:25) | CO:PO (50:50) | Palm oil | Wet lab results for CO | |
| Average chain length | 14.25±0.59 | 15.09±0.23 | 15.46±0.46 | 15.86± 0.40 | 16.77±0.28 | 20.05±0.41 | 12.3-13.3 (Crowther, 2008Crowther MW. 2008. NMR and IR spectroscopy for the structural characterization of edible fats and oils. An instrumental analysis laboratory. J. Chem. Educ. 85, 1550–1554.) |
| Saponification index (g KOH/kg oil) | 244.66±0.67 | 240.50±0.51 | 236.44±0.40 | 229.53±0.48 | 220.47±0.39 | 191.91±0.37 | 250-260 (APCC, 2009Asian Pacific Coconut Community (APCC). 2009. APCC standards for virgin coconut oil. Asian and Pacific Coconut Community, Jakarta, Indonesia.) |
| Molecular weight | 652.12±0.49 | 671.05±0.39 | 686.91±0.42 | 716.24±0.61 | 754.67±0.59 | 875.78±0.49 | 680 (Alander, 2004Alander J. 2004. Process for the preparation of fat composition containing sterol esters a product obtained by said process and the use thereof, US10/451 (Patent).) |
| Iodine value (g/100 kg) | 8.27±0.28 | 12.18±0.49 | 13.16±0.36 | 19.49±0.27 | 32.37±0.51 | 57.82±0.41 | 4-11 (APCC, 2009Asian Pacific Coconut Community (APCC). 2009. APCC standards for virgin coconut oil. Asian and Pacific Coconut Community, Jakarta, Indonesia.) |
| Peroxide value (meqO2/kg) | 0.02±0.01 | 0.02±0.01 | 0.02±0.01 | 0.03±0.02 | 0.04±0.01 | 0.06±0.01 | <3 (APCC, 2009Asian Pacific Coconut Community (APCC). 2009. APCC standards for virgin coconut oil. Asian and Pacific Coconut Community, Jakarta, Indonesia.) |
| Percentage unsaturation | 7.81±0.52 | 15.23±0.47 | 17.10 ±0.52 | 22.50 ±0.48 | 35.52 ±0.39 | 66.51±0.43 | 9 (APCC, 2009Asian Pacific Coconut Community (APCC). 2009. APCC standards for virgin coconut oil. Asian and Pacific Coconut Community, Jakarta, Indonesia.) |
aCO: Coconut Oil; PO: Palm Oil
In the present study, the chain length value and percentage unsaturation of coconut oil increases upon adding palm oil. Coconut oil possessed an exceptional saponification value of 244.66 g KOH/kg, due to the presence of lauric (12:0) and myristic (14:0) acids, and the addition of palm oil led to significant decrease in saponification value. Therefore, the saponification value could be used as a reliable parameter for detecting adulteration in coconut oil with palm oil. Iodine value was determined to identify the extent of fat unsaturation, which varies with the type and proportion of unsaturated fatty acid present in the oil. The iodine value for pure coconut oil was 8.27 g/kg, and the addition of palm oil to coconut oil caused a gradual and significant increase in iodine value, and a higher iodine value can be taken as an index of adulteration of coconut oil with palm oil. The percentage value of unsaturation also increases with the increase in blending of palm oil with coconut oil (7.81% - 66.51%). Pure coconut oil had lower peroxide value (0.02 meqO2/kg) than that of pure palm oil (0.06 meqO2/kg), indicating the oxidative stability of coconut oil. The adulteration of pure coconut oil with palm oil caused a significant increase in peroxide value of the mixture, due to the high content of unsaturated fatty acids in palm oil (Table 1). The physico-chemical parameters of coconut oil were found to be well in agreement with the values provided by APCC (Alander, 2004Alander J. 2004. Process for the preparation of fat composition containing sterol esters a product obtained by said process and the use thereof, US10/451 (Patent).).
Further, the adulteration of coconut oil using palm oil was detected through variation in peak area of characteristic 1H NMR signals that arise due to blending with palm oil. The 1H NMR peaks E, F and I of the triglyceride (Figure 1) are characteristic of unsaturated triglycerides. The extent of unsaturation in palm oil is about 9 times higher than that of coconut oil and this feature can be explored for detecting the presence of palm oil in coconut oil (Figure 2). The peak areas at δ 5.4, 2.8 and 2.0 ppm in the 1H NMR data represent unsaturation in the TAG moiety in palm oil (-CH=CH-, =CHCH2CH= and -CH2CH=CHCH2- respectively). The non-destructive NMR technique offers advantages over traditional wet lab experiments by providing rapid and accurate analysis of key parameters such as average chain length, saponification index, molecular weight, iodine value, peroxide value, and percentage of unsaturation. Furthermore, the ability to discern the chemical shift values of olefinic protons enables precise determination of the extent of palm oil adulteration.
4. CONCLUSIONS
⌅The authentication of oil represents one of the most challenging analytical problems in the food sector due to the complexity of lipid components in the oil. Though coconut oil is widely consumed, literature reports on authentication of coconut oil are scarce, except those based on conventional wet lab methods. The present work highlights 1H NMR as a rapid and reliable analytical tool for the authentication of coconut oil as well as for the detection of adulteration of coconut oil using palm oil. The findings are of relevance to the food sector, especially in the context of the emerging international market for coconut oil. The results highlight the potential of NMR spectroscopy as an efficient tool for safeguarding the authenticity and purity of coconut oil across various domains, including food and traditional medicinal applications.