Jale Acar

Enzymatically validated liquid chromatographic method for the determination of ascorbic and dehydroascorbic acids in fruit and vegetables.

A liquid chromatographic method has been described for the determination of total vitamin C, ascorbic acid (AA) and dehydroascorbic acid (DHAA) in fruits and vegetables. The complete separation of AA and DHAA could be achieved on a C18 column using 0.2 M KH2PO4 (pH adjusted to 2.4 with H3PO4) as the mobile phase at a flow-rate of 0.5 ml/min. Since the detection sensitivity was poor for DHAA even at 210 nm, it was estimated as the difference between the total AA after DHAA reduction and AA content of the original sample, using dithiothreitol (DTT) as the precolumn reductant. The reaction times for the complete conversion of DHAA to AA at room temperatures were 150, 120, 90 and 75 min for 1, 2, 4 and 8 mmol DTT per mmol of DHAA, respectively. The percentage recovery ranged from 81.7 to 105.9. AA contents of some selected fruits and vegetables were analyzed comparatively by liquid chromatography and enzymatic assay to validate the method.

The use of commercial pectinase in fruit juice industry. Part 3: Immobilized pectinase for mash treatment

Abstract Enzymatic mash treatment is a well-known modern process for gaining more juice from fruits and vegetables. According to the technique, cell wall and middle-lamina pectin of the fruit are degraded by pectinase activities. Besides increasing press capacity and the yield of juice up to 20%, it has also a positive effect to achieve high carotene and dry matter content of the product. The aim of the research was to investigate the activity and reusability of immobilized commercial pectinase named as “Pectinex Ultra SP-L” on carrot puree. Immobilization process was carried out by using ion exchange resin particles washed with 0.05 M phosphate buffer at pH 4.5. Pectinase activity of immobilized enzyme was determined by the measurement of viscosity reduction of pectin solution model system at pH 4.5 and 35°C and found to be 1.252-pectin (w/v, %)/s ml. Activity loss was only 20% after nine batches run model system studies. The optimum initial enzyme concentration was detected by measuring the highest pectinase activity in pectin solution and found to be 6% (v/v). Enzyme immobilized particles were added to the carrot puree with an amount of 1.5 g particle/100 g puree at pH 4.5 and 35°C to degrade soluble and insoluble pectin and haze-provoking polysaccharides. The activity of immobilized enzyme was determined by measuring pH, dry matter content and viscosity of the puree. Immobilized enzyme preparation reduced the viscosity of the carrot puree from 90 to 6.5 Poise, after 60 min of incubation. While the viscosity and pH of the puree were decreased, dry matter content and total yield were found to be increased because of the polysaccharide degradation. An average yield increment was 30.23% with respect to the yield obtained from non-enzymic processed carrot juice. Immobilized enzyme was used 5 times in carrot puree medium at the above described conditions and the activity loss was found to be only 6.5%. Activity of the immobilized enzyme was quite stable.

Simultaneous determination of 5-hydroxymethylfurfural and patulin in apple juice by reversed-phase liquid chromatography.

A rapid, simple and economical method was described for the simultaneous determination of 5-hydroxymethylfurfural (HMF) and patulin in apple juice. The sample was extracted with ethyl acetate and the extract was then cleaned up by extraction with a sodium carbonate solution. Then HMF and patulin were determined by reversed-phase liquid chromatography using a C18 column and a photodiode array detector. HMF and patulin could be completely resolved by using the mixture water-acetonitrile (99:1, v/v) as the mobile phase with a flow rate of 1.0 ml/min. Mean recoveries of HMF ranged from 86% to 100% with an overall mean of 94%, that of patulin ranged from 94% to 125% with an overall mean of 103%, for different spiking levels. The limits of detection for HMF and patulin in apple juice were found to be < 0.01 mg/l and < 5 micrograms/l, respectively.

Incidence of patulin in apple juice concentrates produced in Turkey.

A total of 215 apple juice concentrate samples from three different producers in Turkey were analyzed for patulin using reversed-phase high-performance liquid chromatography. The detection limit of patulin in single strength apple juice at a sugar content of 11.2 degrees Brix was lower than 5 micrograms/l. Patulin was detected in all of the samples analyzed, at concentrations ranging from 7 to 375 micrograms/l. Of the samples, 43.5% were found to exceed a patulin contamination level of 50 microgram/l.

The use of commercial pectinase in the fruit juice industry, part 2: Determination of the kinetic behaviour of immobilized commercial pectinase

Abstract In this study, commercial pectinase was immobilized onto anion exchange resin particle with electrostatic adsorption. The kinetics of immobilized pectinase was studied in a batch reactor. The pectolytic activities of free and immobilized Pectinex Ultra SP-L were measured from the viscosity reduction of pectin solution at pH 4.5°C and 35°C. Kinetic constant were found as Vmax 0.0046% (w/v)/s and Km 1.137% (w/v) pectin for free enzyme and Vmax 0.0091% (w/v)/s and Km 2.172% (w/v) pectin for immobilized pectinase. The temperature dependence of the reaction rate obeyed the Arrhenius law. The activation energy of biochemical reaction catalyzed by free and immobilized commercial pectinase was calculated as 9.424 and 11.98 kcal mol−1, respectively.

The use of commercial pectinase in fruit juice industry. Part I: viscosimetric determination of enzyme activity

The kinetics of commercial pectinase, Pectinex Ultra SP-L, in a batch reactor was studied. The change in viscosity of pectin solution was followed by an enzymatic assay to determine pectolytic activity of the enzyme preparation. The pectolytic activity of Pectinex Ultra SP-L was measured at pH 3.5 and 35°C and found to be 0.025 pectin % (w/v)/sec/enzyme % (v/v). The Michaelis-Menten constant (Km) of enzyme preparation was found to be 1.137 pectin % (w/v). The temperature dependence of the reaction rate was obeying the Arrhenius Law. The activation energy of the biochemical reaction catalysed by commercial pectinase was calculated as 9.316 kcal mol−1.

Rapid reversed-phase liquid chromatographic determination of patulin in apple juice

A rapid, simple and economical method using a limited amount of organic solvent is described for the determination of patulin in apple juice. The sample was extracted with ethyl acetate and the extract was cleaned up by extraction with sodium carbonate solution. Patulin was then determined by reversed-phase liquid chromatography using a MicroPack C18 column and a variable-wavelength UV-Vis detector set at 276 nm. Patulin and 5-hydroxymethylfurfural were completely resolved by using water-acetonitrile (99:1, v/v) as the mobile phase at a flow-rate of 1.0 ml/min. The detection limit was < 5 micrograms/l and the recovery was 98%.

Long-term survey of patulin in apple juice concentrates produced in Turkey

A liquid chromatographi c method described by us elsewhere was evaluated for a long-term survey of patulin in apple juice concentrates. Patulin was separated on a reversed phase C18 LC column with water-acetonitrile (99:1) as the mobile phase and quantitated with a photodiode array (PDA) detector. Relatively low amounts of patulin (< 5 μg/l for single strength juice at 11.2° 8Bx) were detected in apple juice concentrates and confirmed by PDA detector, comparing the corresponding UV spectra with that of patulin standard. Four hundred and eighty two apple juice concentrates produced through 1996-99 were analysed for their patulin contents. Year-to-year variations in patulin levels of apple juice concentrates were found out to be statistically significant. Patulin contamination levels of apple juice concentrates tended to decrease through the years and averaged 63, 43, 19 and 31 μg/l in 1996, 1997, 1998 and 1999, respectively. Percentages of concentrates exceeding the maximum permitted concentration of 50 μg/ l were 52%, 34%, 8% and 8% for 1996, 1997, 1998 and 1999, respectively.

LIQUID CHROMATOGRAPHIC METHOD FOR THE DETERMINATION OF CHLOROPHYLLS, CAROTENOIDS, AND THEIR DERIVATIVES IN FRESH AND PROCESSED VEGETABLES

ABSTRACT A high-performance liquid chromatographic method has been developed to quantify major chlorophylls and carotenoids in some raw and processed vegetables. Vegetable samples were simultaneously homogenized and extracted with methanol in a homogenizer in two steps. A sample to solvent ratio of 1 : 10 (w/v) was sufficient for a complete extraction of pigments from pea. The extraction yields were determined to be 79.1% and 20.9% for the first and the second extraction steps, respectively. A MikroPak C8 reversed-phase column was used for the separation of chlorophylls and carotenoids in an elution time of 20 min using gradient mixture of methanol : water at a flow rate of 0.75 mL/min. A good separation of chlorophyll a (Chl a) and chlorophyll b (Chl b), and of their major derivatives, was achieved. The method has also been shown to be applicable for the separation of yellow (α- and β-carotenes) and orange (xanthophylls) carotenoids in some vegetables. The method was shown to be useful to quantify Chl a and Chl b, and of their Mg-free derivatives of Pheo a and Pheo b, in some fresh and processed peas, as well as to quantify β-carotene in carrots.

Liquid chromatographic determination of β-naphthoxyacetic acid in tomatoes

Abstract An alternative high-performance liquid chromatographic method for the determination of β-naphthoxyacetic acid (BNOA) in tomatoes is described. BNOA was extracted from tomatoes with acetone–dichloromethane (2:1). The extract was cleaned up by Bio-Beads S-X3 gel-permeation chromatography and by partitioning. A reversed-phase C 18 column was used for HPLC analysis. The mobile phase was acetonitrile–2% acetic acid in water (50:50, v/v) pumped at a flow-rate of 1.0 ml/min. Retention time of BNOA was ca. 7 min with a percentage coefficient of variation of 0.71. Resolution of BNOA was good on the column. Percentage recoveries of BNOA were 79.5±6.82, 94.8±2.70 and 86.4±16.43 for the corresponding spiking levels of 0.5, 1.0 and 2.0 μg per g tomato, respectively. Analysis of 10 greenhouse tomato samples from local markets in Ankara showed no BNOA residue.