Estimating the content of free fatty acids in high‐oleic sunflower seeds by near‐infrared spectroscopy

The content of free fatty acids (FFA) in vegetable oils represents an important quality factor in oil crops. The objective of the investigation was to develop a near‐infrared (NIR) calibration for estimating the FFA content in high‐oleic sunflower seeds. A sample set of different varieties from the harvest of 2004 as well as of 2005 from two locations in Germany was used; additionally seeds from 2003 were stored under unsuitable conditions to obtain samples utilised for calibration with an extended FFA range. A direct titration method for FFA determination was developed and adjusted to the official AOCS method. The modified method is sufficiently reliable, much faster than the AOCS method and therefore suitable for use in the calibration of NIR spectrometers. The developed NIR spectroscopy (NIRS) calibration was calculated with a modified partial least square algorithm, standard normal variate and detrend scatter correction and the 2^nd derivative of the spectra of ground sunflower seeds. The standard error of prediction of the validated calibration was 0.20, and the multiple coefficient of determination (RSQ_val) reached 0.94. The obtained results demonstrated clearly the efficiency and how cost effective the NIRS method is for the estimation of FFA content in sunflower seeds.     

Comparative study of the extraction and measurement of cottonseed free fatty acids

Crude oil was extracted from cottonseed by three different methods to study the influence of extraction technique on the free fatty acid (FFA) concentration. Extraction procedures that recovered more oil had higher levels of FFA. In addition, the highest concentration of FFA was found in oil recovered by Soxhlet reextraction of a meal initially defatted by a room-temperature extraction process. The FFA concentrations of oils recovered by Soxhlet extraction were highly correlated with the FFA concentration of oils recovered by the other extraction methods studied (R ²>0.96). Titration of oil and gas chromatography of silylated oil were compared as methods to determine FFA concentration. The methods compared well (R ²=0.998) with the titration method, giving ∼5% higher values for FFA than the chromatography method. Half of this difference appeared to be due to the oleic acid approximation used in the titration approach. The other half of the difference is likely due to the detection of other acidic components in crude oil.     

Determination of free fatty acids in palm oil by near-infrared reflectance spectroscopy

A near-infrared (NIR) spectroscopy calibration was developed for the determination of free fatty acids (FFA) in crude palm oil and its fractions based on the NIR reflectance approach. A range of FFA concentrations was prepared by hydrolyzing oil with 0.15% (w/w) lipase in an incubator at 60°C (200 rpm). Sample preparation was performed in Dutch cup, and the spectra were measured in duplicate for each sample. The optimized calibration models were constructed with multiple linear regression analysis based on C=O overtone regions from 1850–2050 nm. The best wavelength combinations were 1882, 2010, and 2040 nm. Multiple correlation coefficients squared (R ²) were: 0.994 for crude palm oil, 0.961 for refined-bleached-deodorized (RBD) palm olein, and 0.971 for RBD palm oil. Calibrations were validated with an independent set of 8–10 samples. R ² of validation were 0.997, 0.943, and 0.945, respectively. The developed method was rapid, with a total analysis time of 5 min, and environmentally friendly, and its accuracy was generally good for raw-material quality control.     

Phase equilibria of free fatty acids enriched vegetable oils and carbon dioxide: Experimental data,distribution coefficients and separation factors

In present work, the oils were treated by saponification procedure to release fatty acids from triglycerides to obtain free fatty acids (FFA) enriched oils and glycerol. Phase equilibria data for systems FFA enriched rapeseed oil–CO₂, FFA enriched corn germ oil–CO₂ and FFA enriched borage oil–CO₂ were determined at temperatures 35, 45, 65 and 85 °C, and in pressure range from 100 to 550 Bar. Experiments using FFA enriched oils were performed using high-pressure variable–volume view cell, where phase inversions were also observed at temperatures 35 and 45 °C. Samples from liquid and vapor phases were analyzed by gas chromatography for the content of free fatty acids, and based on obtained data; the distribution coefficients and the separation factors between FFA and glycerol were calculated.     

Influence of triacylglycerol characterics on the determination of free fatty acids in vegetable oils by fourier transform infrared spectroscopy

A rapid and direct Fourier transform infrared (FTIR) spectroscopic method using a 25-μm NaCl transmission cell was developed for the determination of free fatty acids (FFA) in six important vegetable oils (corn, soybean, sunflower, palm, palm kernel, and coconut oils) that differ in fatty acid profile. The calibrations were established by adding either standard FFA (oleic, lauric acids) or a representative mixture of FFA obtained after saponification of the refined oils. For all oils, up to a FFA level of 6.5% for coconut oil, the best correlation coefficient was obtained by linear regression of the free carboxyl absorption at 1711 cm⁻¹. All correlation coefficients were greater than 0.993, and no significant difference between the calibration methods could be detected. Upon validation of the calibration, no significant difference (α=0.05) between the “actual” and the “FTIR predicted” FFA values could be observed. The calibration models developed for the six oils differed significantly and indicate the need to develop a calibration that is specific for each oil. In terms of repeatability and accuracy, the FTIR method developed was excellent. Because of its simplicity, quick analysis time of less than 2 min, and minimal use of solvents and labor, the introduction of FTIR spectroscopy into laboratory routine for FFA determination should be considered.     

Rapid Characterization of Fatty Acids in Oleaginous Microalgae by Near-Infrared Spectroscopy

The key properties of microalgal biodiesel are largely determined by the composition of its fatty acid methyl esters (FAMEs). The gas chromatography (GC) based techniques for fatty acid analysis involve energy-intensive and time-consuming procedures and thus are less suitable for high-throughput screening applications. In the present study, a novel quantification method for microalgal fatty acids was established based on the near-infrared spectroscopy (NIRS) technique. The lyophilized cells of oleaginous Chlorella containing different contents of lipids were scanned by NIRS and their fatty acid profiles were determined by GC-MS. NIRS models were developed based on the chemometric correlation of the near-infrared spectra with fatty acid profiles in algal biomass. The optimized NIRS models showed excellent performances for predicting the contents of total fatty acids, C16:0, C18:0, C18:1 and C18:3, with the coefficient of determination (R²) being 0.998, 0.997, 0.989, 0.991 and 0.997, respectively. Taken together, the NIRS method established here bypasses the procedures of cell disruption, oil extraction and transesterification, is rapid, reliable, and of great potential for high-throughput applications, and will facilitate the screening of microalgal mutants and optimization of their growth conditions for biodiesel production.     

Reduction of Free Fatty Acids in Acidic Nonedible Oils by Modified K10 Clay

Biodiesel is a biofuel obtained from vegetable oils. The oils used as raw materials are usually refined edible vegetable oils. Nonedible acidic oils are unsuitable for biodiesel production unless reduction of the high content in free fatty acids (FFA) of these materials had been achieved. Obtaining a good raw material from unprofitable oils becomes an important research field. Additionally clays have a long history in industrial sorption and catalysis, some being commercially available and with properties that can be modified. In this work we present the results of the use of the montmorillonite clay K10 and two acid modified clays K10(I) and K10(II), in the esterification of stearic acid with methanol and 95 % of methyl stearate was obtained with K10(II). These clays were then used for the first time to reduce the acidity of enhanced FFA sunflower oil and they show to be very effective. Reduction of FFA from 11 to 4 % was obtained with K10(II) mainly due to 94 % conversion of FFA into fatty acid methyl esters (FAME). These clays were also tested with two waste oils, one from domestic use and the other gathered from different restaurants, and showed their ability to lower the acidity of these oils. Reactions were followed by ¹H NMR as well as quantitative determination of FFA and FAME. Clays were characterized by FTIR and XRD.     

Identification of oil palm breeding lines producing oils with low acid values

In the oil palm (Elaeis guineensis Jacq.), an active endogenous lipase is responsible for the massive release of free fatty acids (FFA) in the mesocarp of fruits at maturity. This can lead to the production of oils with unacceptable acid values. We have investigated the lipase activities in 39 genotypes of oil palm presently used in breeding programs. While most E. guineensis genotypes exhibited high lipase activity values, four of them with negligible activities were identified. We analyzed in situ lipolysis in the mesocarp upon severe wounding of fruits. The FFA content of high‐lipase fruits ranged from 17 to 55%, while all low‐lipase fruits contained less than 7.5% FFA. The analysis of oil from fallen overripe fruits indicated that all low‐lipase genotypes contained less than 1.2% FFA (oil acidity <5% FFA, FAO‐WHO international norms), while all but one high‐lipase genotypes had FFA contents much higher than the acceptable standards. Because the identified low‐lipase lines are of high agronomical value and at least one of them is an elite genotype, it will soon be possible to provide farmers with new low‐lipase oil palm lines allowing significant savings on labor costs, without any need for further breeding.     

Rapid quantitative determination of free fatty acids in fats and oils by fourier transform infrared spectroscopy

Rapid direct and indirect Fourier transform infrared (FTIR) spectroscopic methods were developed for the determination of free fatty acids (FFA) in fats and oils based on both transmission and attenuated total reflectance approaches, covering an analytical range of 0.2–8% FFA. Calibration curves were prepared by adding oleic acid to the oil chosen for analysis and measuring the C=O band @ 1711 cm^–1 after ratioing the sample spectrum against that of the same oil free of fatty acids. For fats and oils that may have undergone significant thermal stress or extensive oxidation, an indirect method was developed in which 1% KOH/methanol is used to extract the FFAs and convert them to their potassium salts. The carboxylate anion absorbs @ 1570 cm^–1, well away from interfering absorptions of carbonyl-containing oxidation end products that are commonly present in oxidized oils. Both approaches gave results comparable in precision and accuracy to that of the American Oil Chemists’ Society reference titration method. Through macroprogramming, the FFA analysis procedure was completely automated, making it suitable for routine quality control applications. As such, the method requires no knowledge of FTIR spectroscopy on the part of the operator, and an analysis takes less than 2 min.     

Development of calibration equations to predict oil content and fatty acid composition in brassicaceae germplasm by near-infrared reflectance spectroscopy

The use of nondestructive analytical methods is critical for the evaluation of very small seed samples such as those from germplasm collections. The objective of this study was to evaluate the potential of near-infrared reflectance spectroscopy (NIRS) for the simultaneous analysis of seed oil content and concentration of major fatty acids in intact-seed samples of the family Brassicaceae. A total of 495 samples from 56 genera and 128 species were analyzed by NIRS. The fatty acid composition of the seed oil was determined in all the samples by gas-liquid chromatography (GLC). The total seed oil content was determined by solvent extraction in 129 samples from 22 genera. Calibration equations for oil content (n=97) and individual fatty acids (n=410) were developed and tested through external validation with the samples not included in the calibration sets. The calibration equations for oil content (r ²=0.97 in validation) and concentrations of C_18:1 (r ²=0.93), C_18:3 (r ²=0.95), and C_22:1 (r ²=0.94) showed very good performance and provided reliable estimations of these traits in the samples of the validation set. The calibration equations for C_16:0, C_18:0, and C_18:2 content were less reliable, with r ² from 0.67 to 0.73. There was practically no response of NIRS to differences in C_20:1 (r ²=0.31). These results demonstrated that the oil content and concentrations of C_18:1, C_18:3, and C_22:1 can be estimated reliably within the family Brassicaceae by using NIRS calibration equations integrating broad taxonomic variability.     

A modified method for determining free fatty acids from small soybean oil sample sizes

A modification of the AOCS Official Method Ca 5a-40 for determination of free fatty acids (FFA) in 0.3 to 6.0-g samples of refined and crude soybean oil is described. The modified method uses only about 10% of the weight of oil sample, alcohol volume, and alkali strength recommended in the Official Method. Standard solutions of refined and crude soybean oil with FFA concentrations between 0.01 and 75% were prepared by adding known weights of oleic acid. The FFA concentrations, determined from small sample sizes with the modified method, were compared with FFA percentages determined from larger sample sizes with the Official Method. Relationships among determinations obtained by the modified and official methods, for both refined and crude oils, were described by linear functions. The relationship for refined soybean oil had an R ² value of 0.997 and a slope of 0.99±0.031. The values for crude soybean oil are defined by a line with R ²=0.9996 and a slope of 1.01±0.013.     

Extrusion deactivation of rice bran enzymes by pH modification

Rice bran is considered in Mexico as “waste”, useful only for feeds. As considerable amounts of oil are available in rice bran, it might be worthwhile to stabilize it and extract the edible oil before using it for feedstuffs. Precisely these oils are responsible for rice bran rapid deterioration, particularly in climatic conditions such as those prevalent in Mexico's tropical areas (high humidity and high temperature). This paper deals with the study of the effect of pH during extrusion of fresh rice bran in order to inactivate lipid‐breaking enzymes. Hydrochloric acid or calcium hydroxide, Ca(OH)₂, were added at 0, 1, 5, 10% (dry basis), and moisture content of the bran samples was varied (20, 30, 40%, dry basis) in a 3² factorial design to corroborate its effect at acid and alkaline pH range. Free fatty acids (FFA) increase was the control variable. Extruded samples were stored at room temperature (between 20 and 28 °C) using a non‐extruded sample as control to assess the shelf life effects. Results indicate that in acid‐extruded samples, the increase in FFA concentration after 98 days was much less than in the unmodified‐pH or alkaline samples. The lowest FFA increase after 3 months of storage time was <10 mg FFA/g rice bran using extrusion with no water or chemicals added or using extrusion adding HCl, irrespective of the moisture content of rice bran.     

Multidimensional Proton Nuclear Magnetic Resonance Relaxation Morphological and Chemical Spectrum Graphics for Monitoring and Characterization of Polyunsaturated Fatty-Acid Oxidation

Polyunsaturated fatty acids (PUFA) are components of many commercial products such as edible oils, foods, cosmetics, medication, and in biological systems such as phospholipids of cellular membranes. Although PUFA aggregates are important functional components, they are also related to system degradation, because PUFA are susceptible to oxidation via their multiple double bonds and allylic carbons. Current technologies are not effective in characterizing the morphological and chemical structural domains of saturated, monounsaturated fatty acids (MUFA) and PUFA materials, or how the morphological structures of fatty acids, at the mesomolecular, nanomolecular, and molecular levels, affect their oxidation mechanisms. In this article, the ¹H low-field (LF) NMR energy relaxation time technology is proposed as a tool to analyze PUFA oils undergoing thermal oxidation. This technology generates two-dimensional (2D) chemical and morphological spectra using a primal-dual interior method for the convex objectives (PDCO) optimization solver for computational processing of the energy relaxation time signals T₁ (spin–lattice) and T₂ (spin–spin). The 2D graphical maps of T₁ vs. T₂ generated for butter, rapeseed oil, soybean oil, and linseed oil show that the different degrees of unsaturation of fatty-acid oils affect their chemical and morphological domains, which influences their oxidative propensity. The technology of the ¹H LF-NMR energy relaxation time proved to be an effective tool to characterize and monitor PUFA oxidation.     

Rice bran FFA determination by diffuse reflectance IR spectroscopy

Rice bran with FFA levels above 0.1% cannot be used as a food ingredient due to oxidative off-flavor formation. However, extracting high FFA oil from bran by in situ methanolic esterification of rice bran oil to produce methyl ester biodiesel produces greater yields relative to low-FFA rice bran oil. Therefore, high-FFA bran could be exploited for biodiesel production. This study describes an FTIR spectroscopic method to measure rice bran FFA rapidly. Commercial rice bran was incubated at 37°C and 70% humidity for a 13-d incubation period. Diffuse reflectance IR Fourier transform spectra of the bran were obtained and the percentage of FFA was determined by extraction and acid/base titration throughout this period. Partial least squares (PLS) regression and a calibration/validation analysis were done using the IR spectral regions 4000-400 cm⁻¹ and 1731-1631 cm⁻¹. The diffuse reflectance IR Fourier transform spectra indicated an increasing FFA carbonyl response at the expense of the ester peak during incubation, and the regression coefficients obtained by PLS analysis also demonstrated that these functional groups and the carboxyl ion were important in predicting FFA levels. FFA rice bran changes also could be observed qualitatively by visual examination of the spectra. Calibration models obtained using the spectral regions 4000-400 cm⁻¹ and 1731-1631 cm⁻¹ produced correlation coefficients R and root mean square error (RMSE) of cross-validation of R=0.99, RMSE=1.78, and R=0.92, RMSE=4.67, respectively. Validation model statistics using the 4000-400 cm⁻¹ and 1731-1631 cm⁻¹ ranges were R=0.96, RMSE=3.64, and R=0.88, RMSE=5.80, respectively.     

Determination of free fatty acids in crude palm oil and refined-bleached-deodorized palm olein using fourier transform infrared spectroscopy

A rapid direct Fourier transform infrared (FTIR) spectroscopic method using a 100 μ BaF₂ transmission cell was developed for the determination of free fatty acid (FFA) in crude palm oil (CPO) and refined-bleached-deodorized (RBD) palm olein, covering an analytical range of 3.0–6.5% and 0.07–0.6% FFA, respectively. The samples were prepared by hydrolyzing oil with enzyme in an incubator. The optimal calibration models were constructed based on partial least squares (PLS) analysis using the FTIR carboxyl region (C=O) from 1722 to 1690 cm⁻¹. The resulting PLS calibrations were linear over the range tested. The standard errors of calibration (SEC) obtained were 0.08% FFA for CPO with correlation coefficient (R ²) of 0.992 and 0.01% FFA for RBD palm olein with R ² of 0.994. The standard errors of performance (SEP) were 0.04% FFA for CPO with R ² of 0.998 and 0.006% FFA for RBD palm olein with R ² of 0.998, respectively. In terms of reproducibility (r) and accuracy (a), both FTIR and chemical methods showed comparable results. Because of its simpler and more rapid analysis, which is less than 2 min per sample, as well as the minimum use of solvents and labor, FTIR has an advantage over the wet chemical method.     

Determination of seed oil content and fatty acid composition in sunflower through the analysis of intact seeds, husked seeds, meal and oil by near-infrared reflectance spectroscopy

A methodological study was conducted to test the potential of near-infrared reflectance spectroscopy (NIRS) to estimate the oil content and fatty acid composition of sunflower seeds. A set of 387 intact-seed samples, each from a single plant, were scanned by NIRS, and 120 of them were selected and further scanned as husked seed, meal, and oil. All samples were analyzed for oil content (nuclear magnetic resonance) and fatty acid composition (gas chromatography), and calibration equations for oil content and individual fatty acids (C_16:0, C_16:1, C_18:0, C_18:1, and C_18:2) were developed for intact seed, husked seed, meal, and oil. For intact seed, the performance of the calibration equations was evaluated through both cross- and external validation, while cross-validation was used in the rest. The results showed that NIRS is a reliable and accurate technique to estimate these traits in sunflower oil (validation r ² ranged from 0.97 to 0.99), meal (r ² from 0.92 to 0.98), and husked seeds (r ² from 0.90 to 0.97). According to these results, there is no need to grind the seeds to scan the meal; similarly accurate results are obtained by analyzing husked seeds. The analysis of intact seeds was less accurate (r ² from 0.76 to 0.85), although it is reliable enough to use for pre-screening purposes to identify variants with significantly different fatty acid compositions from standard phenotypes. Screening of intact sunflower seeds by NIRS represents a rapid, simple, and cost-effective alternative that may be of great utility for users who need to analyze a large number of samples.     

A method of concentration estimation of trienes,tetraenes, and pentaenes in evening primrose oil

There is no simple method available to estimate the concentration of conjugated fatty acids (CFA) with more than two double bonds in plant oils, because there are no commercially available complete sets of CFAs standards needed for such analysis. This paper presents such a method based on the absorption and fluorescence spectra of available CFAs standards and a computer program calculation. Using this method, the concentrations of the α‐eleostearic acid (conjugated trienoic acid (ELA) 8.8 × 10^?4 mol/L) and cis‐parinaric acid (conjugated tetraenoic acid (PnA) 3.5 × 10^?5 mol/L) in evening primrose oil were estimated. The accuracy of the calculation method is around 10%. Practical applications: This is the first report about the existence of CFAs in evening primrose oil. The described method estimates the total concentration of trienes, tetraenes, and pentaenes in evening primrose oils without using HPLC or other analytical methods. These CFAs are important from a health point of view. They are not synthesized by the human body. Therefore, plant oils containing these fatty acids (FA) are very valuable. Knowledge about the concentrations can be utilized to prepare a mixture of the plant oils with a favourable n‐3 to n‐6 FAs ratio and in addition containing CFAs.     

Effect of Selected Polyhydric Alcohols on Refining Oil Loss in the Neutralization Step

Alkaline neutralization is a classical method for removal of free fatty acids (FFA) in crude oil. It is generally accompanied by neutral oil loss. Thus, reduction of refining losses associated with alkaline neutralization is very desirable. Refined, bleached and deodorized (RBD) palm oils with different FFA contents were used as oil models in this study. FFA in the oil models were neutralized with sodium hydroxide in polyhydric alcohols as neutralization media. Glycerol, propylene glycol and ethylene glycol in water were effective neutralization media. FFA in the oil models were totally removed in one step of neutralization, while percentages of refining losses were different. The losses were increased in the order of water > propylene glycol > ethylene glycol > glycerol used as neutralization media. Also, a higher concentration of polyhydric alcohol in the neutralizing media significantly reduced the percentage of refining loss (p < 0.05). Glycerol (90% in water) was the most effective neutralization media (p < 0.05). When neutralization was carried out on crude palm oil (containing 7.53% FFA), refining loss was reduced from 36.1% (in water) to 20.0% (in 90% glycerol in water).     

Free Fatty Acids Reduction in Waste Cooking Oil by Rhodosporidium toruloides and Simultaneous Carotenoids,Lipids, and PAL Enzyme Production in a Two-Phase Culture System

Waste cooking oil (WCO) is a problematic waste product that contains free fatty acids (FFAs), preventing it from being valorized easily as biodiesel and poses an environmental hazard if incorrectly disposed. The use of WCO as a carbon source for Rhodosporidium toruloides (R. toruloides) using a two-phase culture system is developed. The normal growth of R. toruloides when cultured in WCO (OD₆₀₀ 52) reveals its ability to use a hydrophobic substrate as the carbon source compared to glucose (OD₆₀₀ 51.9). Interestingly, the extracellular lipase activity when R. toruloides is grown on WCO is 14.4 U mL⁻¹ compared to when grown on glucose (2.4 U mL⁻¹). Additionally, FFA levels in WCO are reduced from 2% to 0.2% at end of fermentation, suggesting that R. toruloides can consume FFA. Furthermore, higher yield of beneficial products: β-carotene (4.57 µg mL⁻¹), torularhodin (4.2 µg mL⁻¹), fatty acids (1 mg mL⁻¹), and phenylalanine ammonia-lyase (PAL) enzyme (0.12 µmol mg⁻¹) are produced when WCO is the carbon source, compared to glucose (4.1 µg mL⁻¹ β-carotene, 3.0 µg mL⁻¹ torularhodin, 1 mg mL⁻¹ of fatty acids, and 0.096 µmol mg⁻¹ PAL enzyme). This is a first study that shows R. toruloides can grow on hydrophobic carbon source.