Chromatographic Separation of Preconcentrated Vitamin E from Palm Fatty Acid Distillate

The importance of vitamin E as a lipid-soluble antioxidant that protects unsaturated fatty acids against oxidative deterioration is widely known. An important source of vitamin E is the fatty acid distillate from deodorization during the refining of vegetable oils. The purpose of this study was to develop a method for separating vitamin E from palm fatty acid distillate (PFAD) obtained from palm oil refining. Vitamin E in PFAD was first concentrated by enzymatic hydrolysis. The free fatty acids liberated by the hydrolysis, together with those already present in PFAD, were neutralized by sodium hydroxide. A vitamin E-rich fraction was then extracted from the hydrolyzed and neutralized PFAD using hexane. The fraction obtained was finally subjected to normal phase adsorption chromatography with packed silica gel. Two elutions were done in the chromatography: the vitamin E in hexane was adsorbed on silica gel and the extraneous matter eluted out in the first elution, while the second elution released the vitamin E from the silica gel.Reaction variables such as temperature, lipase concentration and water content in the reaction mixture affected the enzymatic hydrolysis of PFAD. Regression models generated from Response Surface Methodology adequately explained the data variation and significantly represented the actual relationships between the reaction parameters and responses. It was suggested from this study that for the maximum vitamin E concentration, the hydrolysis should be carried out with 2.5% w/w lipase and 45.2−47.3% v/w water for 5.5−5.7 h. Screening tests concluded that silica gel was suitable for adsorption of vitamin E. Batch mode adsorption and desorption experiments were employed to study the equilibrium and kinetics of the adsorption and desorption processes. Vitamin E uptake by silica gel was rapid with adsorption equilibrium achieved in about 5 min. The adsorption isothermic data were in good agreement with the Langmuir model, suggesting that the vitamin E adsorbed on silica gel was monolayer. Kinetics study of batch adsorption revealed that the rate of vitamin E uptake by silica gel followed a pseudo-second order reaction and involved both external mass transfer and intraparticle diffusion. Intraparticle diffusion was the rate-limiting step during the adsorption as it needed a higher magnitude of activation energy (–25.45 and –54.13 kJ mol-1 for external mass transfer and intraparticle diffusion, respectively). The adsorption of vitamin E on silica gel was exothermic, while the reverse was true for desorption. Desorption exhibited a bi-phasic characteristic with an initial fast release of the vitamin followed by a much slower phase. The two distinct rates were probably due to heterogeneities in the adsorbing surfaces. Since the silica gel surface is composed of both high-energy silanol groups and low-energy siloxane groups, it was postulated that the releases of vitamin E molecules from these two groups were responsible for the slow and rapid desorption processes, respectively. Entrapment of the vitamin E molecules in the micropores of the silica gel may also have contributed to the slow desorption in the second phase. The desorption isotherm could be fitted in the Freundlich model. Adsorption of vitamin E on silica gel was also tested in a fixed-bed column. The breakthrough curve of vitamin E adsorption in the column showed a typical S-shaped profile. The service time of the column increased with the column bed height, but decreased with increasing inlet vitamin E concentration, column temperature and flow rate. The column efficiency in terms of adsorbent usage rate could be improved by decreasing the inlet vitamin concentration and flow rate. Since adsorbing vitamin E on silica gel was exothermic, increasing the column temperature decreased the column capacity. The desorption of vitamin E in a column system reflected the “two-distinct-rate” desorption behavior found in batch desorption systems. This slow desorption was the rate-controlling step in the recovery of vitamin E. The desorption rate increased with column temperature but decreased with column bed height and flow rate. The recovery of vitamin E was high for all systems - 94.8 to 98.8% - with a vitamin E concentration in the extract of 18.5−21.5%. The results from this work demonstrate the potential applicability of the current separation method for recovering vitamin E from PFAD. Detailed descriptions of the enzymatic hydrolysis and adsorption/desorption of vitamin E in adsorption chromatography have provided useful information for a better understanding of the current separation process. The method described offers an alternative to the existing vitamin E separation methods. It can be applied as one of a series of steps in producing a high-purity vitamin E concentrate from PFAD

Binding of <i>Clitoria ternatea</i> L. flower extract with α-amylase simultaneously monitored at two wavelengths using a photon streaming time-resolved fluorescence approach

The binding of an extract from the flowers of Clitoria ternatea L. to the digestive enzyme α-amylase was investigated. This extract is a mixture of flavonoids, including anthocyanins, and has been previously shown to inhibit the activity this enzyme. This has implications for modulating starch digestion. Since the extract contains a mixture of flavonoids, including anthocyanins, in order to investigate the kinetics, we made use of time-resolved fluorescence to simultaneously monitor two different emission bands emanating from the extract. This measurement was enabled by the use of a “photon streaming” approach and changes in fluorescence lifetime and intensity were used to follow the interaction. A longer wavelength band (655nm) was ascribed to anthocyanins in the mixture and these were observed to bind at a rate an order of magnitude slower than other flavonoids present in the extract, monitored at a shorter wavelength (485 nm). Changes in the fluorescence emission of the extract upon binding were further assessed by the use of decay associated spectra

<i>Clitoria ternatea</i> L. flower extract inhibits α-amylase during <i>in vitro </i>starch digestion

This study aimed to investigate the inhibitory effect of Clitoria ternatea flower against α-amylase during simulated in vitro wheat starch digestion. The dark-blue tropical flower is used as a food colorant but its ability to modulate starch digestion has not been tested before. The aqueous extract of the flower containing anthocyanins was a competitive inhibitor against α-amylase with an IC50 value (concentration of inhibitor required to reduce the enzyme activity by half) and inhibition constant, Ki, of 0.91 mg/mL and 0.75 mg/mL,respectively. Subjecting the extract to pasteurisation (72oC for 15 s) and boiling (for 30 min) it significantly (P&lt;0.05) decreased the anthocyanin content as determined by a pH-shift method, although the light absorbance profile of the extract remained virtually unchanged, suggesting that the equilibrium mixture of anthocyanin species was unaffected. The thermal degradation of the anthocyanins explained the partial loss of inhibition activity of the extract, as indicated by the decrease in Michaelis-Menten constant, Km, from 14.8 mg/mL in thesystems with unheated extract to 11.3 and 6.1 mg/mL in pasteurised and boiled extracts, respectively. The thermal treatments, however, did not change the type (competitive) of inhibition. The results from this work demonstrated the potential of C. ternatea flower extract in inhibiting α-amylase during starch digestion, which might lead to development of functional food/drink for controlling postprandial blood glucose level

The effect of different frequencies of ultrasound on the activity of horseradish peroxidase

Ultrasound technology has been studied by food researchers as an alternative method for thermal processing. The use of ultrasound as a way to inactivate and/or activate enzymes has been widely studied at low frequencies (20–40 kHz), however, little research on the effect of high frequencies has been reported. Thus, the effect of high and low frequency ultrasound on commercial horseradish peroxidase with a concentration of 0.005 mg mL−1 is described. Experiments were performed for 60 min using 20, 378, 583, 862, 995, 1144 and 1175 kHz ultrasound at power levels (acoustic energy) between 2.1 and 64 W. Residual activity was monitored using a spectrophotometric method and data analysis was performed using ANOVA. A significant enhancement of enzyme inactivation (p &lt; 0.05) was observed at each frequency with an increase of sonication time and power. Inactivation of peroxidase by ultrasound followed first order kinetics and an increase of the rate constant with the power applied was observed for all the frequencies studied. Overall, low frequency (20 kHz) and low power are not effective on the enzyme inactivation and the level of residual activity remained high. The use of 378 and 583 kHz (48 W) is particularly effective for complete enzyme inactivation

Performance of Lipase-Transesterified Frying Shortening for Frying of Banana Chips

Palm stearin (POs), the solid fraction of palm oil is a useful hard stock that can substitute the hydrogenated oils, which contain trans fatty acids, in the production of plastic frying shortening. This study was initiated to produce a zero-trans plastic frying shortening through enzymatic transesterification of POs and palm kernel olein (PKOo) blend. In the first part of this study, commercial plastic frying shortenings (domestic and imported) in Malaysia were characterised in order to obtain their physical properties which are related to their functionality. The domestic samples were found to have their plastic range at higher temperatures (21°C to 27°C) than the imported ones (15°C to 27°C). They were also more β-tending and generally had a higher slip melting point (SMP, ranging from 44.0°C to 49.7°C) than the imported samples (42.3°C to 43.0°C). Based on the physical properties of the commercial samples, an experimental plastic frying shortening was formulated from POs and PKOo at a ratio of 1:1 (by weight) through enzymatic transesterification, using Lipozyme IM60 lipase (Rhizomucor miehei). Transesterification had successfully changed the triacylglycerol composition of the blend, and consequently altered its physical properties. The SMP of the transesterified POs/PKOo blend was reduced and had a lower solid fat content at all temperatures investigated compared to the control. The transesterified blend was also found to have similar or better product characteristics compared to the domestic samples. The SMP of the transesterified blend fell within the range of the commercial samples' and it had a wider plastic range or better workability compared to the commercial shortenings investigated However, transesterification had reduced the antioxidant activity that was naturally present in POS and PKOo. The transesterified blend was more susceptible to oxidation during deep fat flying and storage compared to its control. The banana chips fried with the transesterified blend also showed similar results. Nevertheless, the transesterified blend was still quite stable compared to a selected commercial frying shortening (Sample D) since it had a significantly (P < 0.05) lower iodine value (IV = 31.6 g of I2/100 g of oil) compared to the commercial shortening (IV = 89.4 g of I2/100g of oil). The results in this study had provided a better understanding on the functionality of the plastic frying shortening and also serves as a guideline in producing a similar product in the industries

Time-resolved fluorescence observation of di-tyrosine formation in horseradish peroxidase upon ultrasound treatment leading to enzyme inactivation

The application of ultrasound to a solution can induce cavitional phenomena and generate high localised temperatures and pressures. These are dependent of the frequency used and have enabled ultrasound application in areas such as synthetic, green and food chemistry. High frequency (100 kHz to 1 MHz) in particular is promising in food chemistry as a means to inactivate enzymes, replacing the need to use periods of high temperature. A plant enzyme, horseradish peroxidase, was studied using time-resolved fluorescence techniques as a means to assess the effect of high frequency (378 kHz and 583 kHz) ultrasound treatment at equivalent acoustic powers. This uncovered the fluorescence emission from a newly formed species, attributed to the formation of di-tyrosine within the horseradish peroxidase structure caused by auto-oxidation, and linked to enzyme inactivation

Factors affecting droplet size of sodium caseinate-stabilized O/W emulsions containing β-carotene

This work was initiated to prepare an oil-in-water (O/W) emulsion containing -carotene by microfluidization. The -carotene was dissolved in triolein and microfluidized with an aqueous phase containing sodium caseinate (SC) as the emulsifier. Microfluization at 140 MPa resulted in O/W emulsions with a mean droplet diameter of ca. 120 nm, which was further confirmed by transmission electron microscopy analysis. The influences of SC concentration and microfluidization parameters on the droplet size of the emulsions were studied. The results showed that the mean droplet diameter decreased significantly (p <0.05) from 310 to 93 nm with the increase in SC concentration from 0.1 to 2 wt-%. However, a further increase in SC concentration did not much change the droplet diameter, although the polydispersity of the emulsions was slightly improved. The droplet diameter of the emulsions was found to decrease from 200 to 120 nm with increasing microfluidization pressure, with narrower droplet size distribution. The storage study showed that the emulsions were physically stable for about 2 weeks at 4 °C in the dark. The results provide a better understanding of the performance of SC in stabilizing the O/W emulsions

Effect of ultrasonic treatment on enzyme activity and bioactives of strawberry puree

The aim of this study was to evaluate the impact of ultrasound (US) at different frequencies (20, 370, and 583 kHz) and power levels (35 and 48 W) on the residual activity (RA) of peroxidase (POD) and polyphenol oxidase (PPO) in strawberry puree. Total anthocyanin content (TAC), total phenolic content (TPC), ferric ion reducing antioxidant power (FRAP) and trolox equivalent antioxidant capacity (TEAC) were also assessed. Results were compared with untreated, thermally treated at 40 °C (control) and pasteurized (90 °C) strawberry puree. POD and PPO RA were significantly (P &lt; 0.05) reduced, whilst there was a significant (P &lt; 0.05) increase in TAC (5%–19%) in all US-treated samples in comparison with the untreated samples and the controls. US at 20 kHz (35 W) increased significantly (P &lt; 0.05) TPC (9%) and FRAP (6%) in strawberry puree, whereas the effect of 583 kHz (48 W) on these parameters was insignificant (P &gt; 0.05). Pasteurization inactivated POD and PPO, however, decreased dramatically TAC (14%), TPC (17%) and FRAP (9.5%) in strawberry puree. These findings suggest that US is a promising novel non-thermal food technology that can be tailored to improve the quality of strawberry puree by inactivating enzymes responsible for food deterioration whilst maintaining the content of bioactive compounds.</p

Desorption of vitamin E from silica-packed fixed-bed column by isopropanol.

Desorption of vitamin E from silica-packed fixed-bed column was studied as functions of column bed height, column temperature and flow rate of isopropanol. Isopropanol was the desorbing solvent and it was eluted through the columns saturated with vitamin E. The desorption profiles of all systems showed that vitamin E might desorb at two distinct rates simultaneously. The slow desorbing step was the rate-controlling process for recovery of vitamin E. The desorption rate increased with the decrease of column bed height and flow rate, but increased with increasing column temperature. This indicated that the desorption process was an endothermic process. The percentage recovery of vitamin E upon completion of desorption was considered high for all systems, ranging from 94.8 to 98.8%, with vitamin E concentration in the extract of 18.5-21.5%. Although the bed height, column temperature and flow rate were functions of desorption rate, it appeared that percentage recovery and vitamin E concentration in the extract were rather unaffected by the operating conditions tested if the column was eluted by isopropanol for a sufficient time to desorb vitamin E. Nevertheless, the use of isopropanol would be more efficient if desorption was carried out at lower flow rate and higher column temperature

Effect of sucrose on thermal and pH stability of Clitoria ternatea extract

The aim of this work was to investigate the effect of sucrose on the stability of Clitoria ternate extract against thermal and pH degradations. Lyophilised extract of the flower (1 mg/ml) was added into a series of sucrose solutions with concentrations ranging from 0.1% to 20% at pH7. The thermal stability of the extract in the solutions at 60oC was monitored using a UV-VIS spectrophotometer over 24 days. High temperature (60oC) accelerated degradation of the anthocyanin-rich extract but the presence of sucrose appeared to have slowed down the degradation process. However, sucrose asserted no protective effect against pH even at a concentration of 20%. It was thought that sucrose enhanced the thermal stability of anthocyanins by reducing water activity, partially preventing nucleophilic attack at the pyrylium ring of anthocyanins by water molecules. The present work provides some useful information for evaluating the potential of C. ternatea extract on food applications