Shimada et al. (2000) converted free sterols in SODD to sterol esters and completely hydrolyzed acylglycerols by applying an enzymatic reaction to the purification of tocopherols and sterols. Fractionation of these two compounds of interest was carried out by short-path distillation. It was found that C. rugosa lipase recognized sterols as substrates but not tocopherols, and that esterification of sterols with FFA could be effected with negligible influence on the water content. High boiling point substances, including steryl esters, were removed from the SODD by distillation, and the resulting distillate (SODDTC) was used as a starting material for tocopherol purification. Several factors affecting esterification of sterols were investigated. It was observed that approximately 80% of sterols were esterified when tocopherols were unmodified. After the reaction, tocopherols and FFA were recovered as a distillate by molecular distillation of the oil layer. To enhance further removal of the remaining sterols, the lipase-catalyzed reaction was repeated on the distillate. As a result, more than 95% of the sterols were esterified in total. The resulting reaction mixture was fractionated to four distillates and one residue. The main distillate fraction contained 65% tocopherols with low contents of FFA and sterols, and the residue fraction contained high-purity steryl esters. It was suggested that due to the fact that the process presented in this study included only an organic solvent-free enzymatic reaction and a molecular distillation, it could be feasible as an industrial purification method for tocopherols. However, the process had the drawback that FFA and tocopherols were not efficiently fractionated since the boiling points of the two compounds were close.

Watanabe et al. (2004) introduced the chemical modification of the DD to convert FFA to their methyl or ethyl esters, followed by short-path distillation of the reaction mixture for the elimination of fatty acid esters and thus the purification of tocopherols and sterols esters.

Tocopherols and sterols in the SODD were first recovered by short-path distillation, which was named SODD tocopherol/sterol concentrate (SODDTSC). The SODDTSC which contained MAG, DAG, FFA and unidentified hydrocarbons in addition to the two compounds of interest was then treated with a lipase to convert the free sterols to fatty acid steryl esters (FASEs), acylglycerols to FFA and FFA to FAME. It was observed that methanol inhibited the esterification of the sterols. Hence, a two-step in situ reaction was conducted and a conversion of 80% of the initial sterols to FASEs, complete hydrolysis of the acylglycerols and a 78% decrease in the initial FFA content by methyl esterification, was achieved. To enhance the degree of steryl and methyl esterification, the reaction products (FASEs and FAME) were removed by short-path distillation, and the resulting fraction containing tocopherol, sterols and FFA was again treated with the lipase. Distillation of the reaction mixture purified the tocopherols to 76% (recovery, 89%) and sterols to 97% as FASEs (recovery, 86%).

Nagao et al. (2005) described a process where SODD was first distilled and the sterols and tocopherols were enriched. The obtained fraction was SODDTSC. In this study, esterification of sterols was improved by removing water with a degree of esterification of 95%. The second-step reaction was then conducted in which

95% FFAs were converted into FAME. Finally, tocopherols and steryl esters were purified from the reaction mixture by short-path distillation. Tocopherols were purified to 72% (88% yield) and steryl esters were purified to 97% (97% yield).

Purification on a larger scale was performed with 1.5 kg SODDTSC and the procedure is shown in Fig. 22.5.

Albiez et al. (2004) described a process for concentrating and isolating sterols and/or tocopherols from physically refined DD that consisted in hydrolysis to split the glycerides present into FFA and glycerol, followed by the glycerol- containing hydrolysis water removal and distillation of the FFA and readily volatile unsaponifiable components. The distillation residue was additionally hydrolyzed to split the sterol esters into FFA and free sterols, followed by the distillation of the later one.

Accordingly, the problem addressed by the present patented invention was to provide a process for the simultaneous production of tocopherol and sterol which would be applicable to many different starting mixtures, which would not involve the use of toxicologically and ecologically unsafe solvents, which would use even low-concentration starting materials sparingly and which would still give high yields without the use of metal-containing catalysts. In addition, the process would be economically workable on an industrial scale.

Top et al. (1993) described a process for the production of tocopherols and tocotrienols where the PFAD was first modified and the resulting fractions were further purified applying different steps. The process includes the conversion of FFA and glycerides in PFAD into alkyl esters by esterification and transesterification, followed by distillation of the resulting product under reduced pressure to remove a major part of the alkyl esters and leave the tocopherols and tocotrienols and other higher boiling point substance in the residue. The residue was cooled to induce crystallization of higher melting substances and other impurities and the crystalline material was filtered off to leave the tocopherols and tocotrienols in the filtrate. The filtrate was further treated by an ion-exchange procedure with a high selectivity anionic resin to produce a concentrated fraction of tocopherols and tocotrienols, the solvent was removed by evaporation. The tocopherols and tocotrienols fraction was washed dried and then subjected to molecular distillation and deodorization to produce a further concentrated product of tocopherols and tocotrienols. After evaporation step, the concentrations of tocopherols and tocotrienols were 83% and 87%, respectively.

In the same invention, an alternative process was described, where the PFAD was pretreated to remove the majority of the FFA by distillation before sending it to the process described above.

Martins et al. (2005) described a process where the SODD was chemically modified, submitted to molecular distillation for fatty acids elimination and the product obtained was submitted to an ethanolic extraction for tocopherols and concentrations of phytosterols. Chemical modification of SODD was conducted by a saponification at 65°C, followed by an acidulation step. With this procedure it was possible to release conjugated fatty acids of acylglycerols molecules. Therefore, not only FFA can be removed from the mixture by molecular distillation, but conjugated fatty acids of acylglycerols also lead to a higher tocopherol concentration. The applied molecular distillation was characterized by using high vacuum, reduced temperature and low residence time. SODD, containing about 75% of FFA, was submitted to four steps of molecular distillation to remove the FFA from the mixture. The separation of tocopherols from sterols was difficult because they have similar molecular weights, boiling points and vapor pressure, and consequently, they are distilling together. Therefore, the resulting product of molecular distillation was submitted to an ethanolic extraction at 0°C to separate the tocopherols from the sterols. As tocopherols are soluble in ethanol, it was possible to separate and to concentrate phytosterols and tocopherols. This process obtained a purity of 26% of tocopherols and 52% of sterols.

A recent patent (Zima et al., 2009) describes a process for preparing a phytolipid composition containing squalene, phytosterols, tocopherols and vegetable wax that consist two steps of distillation at different temperatures and pressure, extraction and precipitation (Fig. 22.6). The final phytolipid product may contain ca. 10% to about 40% squalene, 2% to about 20% phytosterols, 1% to about 10% of mixed tocols and 40% to about 80% vegetable wax with possible applications in the cosmetic, nutraceutical or food industry.