Determination of capsaicin and dihydrocapsaicin in Capsicum anuum and related products by capillary electrophoresis with a mixed surfactant system

An easy, rapid, sensitive, and cheap capillary electrophoresis (CE) method based a mixed surfactant system formed by sodium dodecyl sulphate (SDS) and polyoxyethylene sorbitan monolaurate (Tween 20) as modifier in the buffer was reported. Quantitative analysis of capsaicin and dihydrocapsaicin in Capsicum anuum, pepper sauce and porous capsicum plaster was demonstrated. After conducting a series of optimisations, baseline separation was obtained for the analytes within 5 min under the optimum conditions (15 mM sodium tetraborate–0.05% (v/v) Tween 20–2.2 mM SDS buffer (pH 10.1), 20 kV voltage, 214 nm UV detection). The method resulted in excellent linearity, with r² of regression equation of 0.9994 and 0.9996 for capsaicin and dihydrocapsaicin, respectively. Recoveries were in the range 90–107% and 92–109% for capsaicin and dihydrocapsaicin, respectively.     

Quality assessment of wild European eel (Anguilla anguilla) stored in ice

The quality assessment of wild European eel (Anguilla anguilla) stored in ice and in boxes without ice (3 ± 1 °C) was investigated by the sensory analysis, levels of nucleotide breakdown products and biogenic amines for up to 19 days. Sensory analysis was assessed using the Tasmanian Food Research Unit Scheme. K and related values (K_i, G, P, H and F_r) were used as freshness indicators. Linear regressions (r²) obtained from K, K_i, G, P, H and F_r were 0.95, 0.96, 0.83, 0.96, 0.99 and 0.96, respectively, for eel stored in ice whereas, for eel kept in boxes without ice, the values were 0.86, 0.86, 0.96, 0.91, 0.98 and 0.86, respectively. When eel stored in ice and in boxes without ice were considered at the limit of acceptability by assessors at ∼12–14 days and ∼5–7 days, respectively, the average K, K_i and P values were ∼70–85%, H values were ∼60% and F_r values were ∼10% for both storage conditions. The level of histamine exceeded the legal limit (5 mg/100 g fish) in eel stored without ice after 6–7 days and, in ice, after 13–14 days of storage, at which time eels were rejected by the sensory panel. The concentrations of biogenic amines were higher in eel stored in boxes without ice than in eel kept in ice. The levels of histamine in the muscle of eel kept in boxes without ice and in ice increased to the maximum levels of 17.9 mg/100 g on day 12 and 12.6 mg/100 g on day 19, respectively.     

Nitrosamine formation from different Catha edulis leaves extracts under simulated gastric condition

Nitrosamines are well-known carcinogenic and toxic compounds for humans and animals. The aim of this study was to investigate the possible formation of nitrosamine compounds from aqueous extracts of five different types of Catha edulis leaves using nitrite as a nitrosation agent, either in aqueous solution or under simulated normal fasting stomach conditions (at 37 °C and pH 2) for 1 h. Nitrosoephedrine was used as a reference compound in this study. Nitrosation of aqueous extracts of the different types of Catha edulis leaves with constant concentration of nitrite (14.4 mM) in aqueous solution showed total apparent nitrosamine compounds to be in the range 94–319 mg/100 g DM (dry matter) of CE (Catha edulis) leaves. In contrast, nitrosamines formed in simulated gastric fluid were much lower, in the range 23–79 mg/100 g DM of CE leaves. Based on the moderate formation of nitrosamines with the Sabri Catha edulis leaves, the latter was selected for investigating the effect of varying levels of nitrite in aqueous solution and simulated gastric juice. The nitrosation of aqueous extracts of Sabri Catha edulis with different levels of nitrite in aqueous solution yielded a dose-dependent amount of total apparent nitrosamine compounds, these being undetected at ?0.5 mM sodium nitrite in aqueous solution and ?1 mM in simulated gastric juice. This raises the question of whether the observed high incidence of esophageal and forestomach carcinomas in Yemen could be attributed to the formation of nitrosamines in vivo from the secondary amines present in Catha edulis leaves.     

Effects of binary solvent extraction system,extraction time and extraction temperature on phenolic antioxidants and antioxidant capacity from mengkudu (Morinda citrifolia)

An investigation into the effects of ethanol concentration (0–100%, v/v), extraction time (20–120 min) and extraction temperature (25–65 °C) on the extraction of phenolic antioxidants from mengkudu (Morinda citrifolia) was performed using a single-factor experiment. Total phenolic content (TPC) and total flavonoid content (TFC) assays were used for determination of phenolic compounds. Antioxidant capacity was evaluated by measuring the scavenging effect on 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radicals. Experimental results showed that extraction conditions had significant effect on extraction of phenolic compounds and antioxidant capacities. The optimised conditions were 40% ethanol for 80 min at 65 °C, with values of 919.95 mg GAE/100 g DW for TPC, 472.73 mg CE/100 g DW for TFC, 791.71 μmol TEAC/100 g DW for ABTS and 1928.5 μmol TEAC/100 g DW for DPPH. TPC was significantly correlated with DPPH under the effects of ethanol concentration (r = 0.932) and extraction time (r = −0.938).     

Extraction and on-line concentration of flavonoids in Brassica oleracea by capillary electrophoresis using large volume sample stacking

Flavonoids are bioactive compounds found in plants. Studies indicate consumption of food containing these compounds may reduce the incidences of cancer and cardiovascular diseases. In broccoli, the flavonoids are present at variable concentrations and so far have mainly been determined using high performance liquid chromatography (HPLC). This paper describes a rapid capillary electrophoresis method, involving large volume sample stacking (LVSS), suitable for the analysis of flavonoids in broccoli. Following acid hydrolysis, the two key flavonoids (kaempferol and quercetin) in a broccoli extract were concentrated on-line by LVSS prior to separation by capillary zone electrophoresis (CZE). Using an optimised method, the extract was injected for 50 s into a 50 μm (internal diameter) × 85 cm (total length) capillary followed by stacking/matrix removal at −5 kV for 83 s. The two analytes were then separated in less than 8 min by CZE using a 10 mM sodium borate buffer (pH 8.40) and a separation voltage of +30 kV at 30 °C. A linear relationship in the range 1–20 ppm was observed for the method (r² = 0.9991–0.9995) with detection limits of 0.9 and 0.6 mg/kg of broccoli for kaempferol and quercetin, respectively. This method demonstrated good repeatability for the standard and extract with relative standard deviations of less than 5% for both peak area and migration time measured over five different days (n = 5). The method was successfully applied to quantitatively determine kaempferol and quercetin contents in a commercial broccoli sample as 11.8 and 14.6 mg/kg fresh weight, respectively. This result was validated by HPLC analysis and is within the ranges reported in the literature.     

Qualitative and quantitative analysis of curcuminoids in herbal medicines derived from Curcuma species

A validated and sensitive HPLC–UV–MS method was developed for qualitative and quantitative analysis of curcuminoids in eight herbal medicines derived from four Curcuma species. The samples were separated on a YMC ODS-A C₁₈ column with a gradient elution of acetonitrile and 0.1% formic acid. Curcumin, demethoxycurcumin and bisdemethoxycurcumin showed good linearity (r > 0.9998) in the concentration ranges of 4.88–625, 4.29–550 and 3.98–510 μg/mL, respectively. The results suggested that the contents of three major curcuminoids in different herbal medicines varied significantly. Curcuminoids were only detected in Jianghuang, HuangsiYujin, and PengEzhu. Amongst them, Jianghuang contained the highest amounts of curcuminoids (40.36 mg/g), which were almost 20 times higher than HuangsiYujin (1.94 mg/g) and 400 times higher than PengEzhu (0.098 mg/g). Furthermore, amongst the Jianghuang samples collected from different areas, samples from Sichuan Province contained remarkably higher amounts of curcuminoids (22.21–40.36 mg/g) than other cultivation regions.     

Antioxidant properties of different cultivars of Portulaca oleracea

Methanolic extracts of six cultivars of Portulaca oleracea were analyzed for their total phenol content (TPC) using the Folin–Ciocalteu method. The antioxidant activity was measured using the 1,1-diphenyl-2-picrylhydrazyl, ferric-reducing antioxidant power (FRAP) and β-carotene bleaching (BCB) assays. The iodine titration method was used to determine the ascorbic acid content (AAC). The TPC of the cultivars of P. oleracea ranged from 127 ± 13 to 478 ± 45 mg GAE/100 g of fresh weight of plant. There was good correlation between the TPC value and its AEAC, IC₅₀ and FRAP values (r² > 0.9) for all the cultivars. The AAC for the cultivars ranged from 38.5 ± 0.6 to 73.0 ± 17.5 mg/100 g. The TPC value of the common variety PO1, was the lowest compared to the ornamental cultivars (PO2–PO6). The BCB assay showed that all cultivars were capable of inhibiting lipid peroxidation and the inhibition power did not correlate with TPC value.     

Organic acids,antioxidant capacity,phenolic content and lipid characterisation of Georgia-grown underutilized fruit crops

Four underutilized Georgia-grown fruit crops, namely loquat (Eriobotrya japonica), mayhaw (Crataegus sp.), fig (Ficus carica), and pawpaw (Asimina triloba), and their leaves were analysed for total polyphenols by Folin–Ciocalteau method, and antioxidant capacity by ferric-reducing antioxidant power (FRAP) and Trolox-equivalent antioxidant capacity (TEAC) assays. Organic acids and phenolic compounds were identified by RP-HPLC. For lipid profile, fruits were separated into two fractions – seed and fruit (i.e., without seed); lipid was extracted using the Folch method and analysed for fatty acids, phytosterols, tocopherols, and phospholipids. The major organic acid identified in all samples was malic acid (177–1918 mg/100 g FW). The predominant phenolic acids in all the fruits were gallic (1.5–6.4 mg/100 g FW) and ellagic (0.2–33.8 mg/100 g FW), and the most abundant flavonoid was catechin (12.2–37.8 mg/100 g FW). Total lipid content varied from 0.1% in mayhaw fruit to 21.5% in pawpaw seed. Linoleic acid was the predominant fatty acid in all of the samples (28.2–55.7%).     

Exploration of Vanilla pompona from the Peruvian Amazon as a potential source of vanilla essence: Quantification of phenolics by HPLC-DAD

This study provides the first chemical investigation of wild-harvested fruits of Vanilla pompona ssp. grandiflora (Lindl.) Soto-Arenas developed in their natural habitat in the Peruvian Amazon. Flowers were hand-pollinated and the resulting fruits were analysed at different developmental stages using an HPLC-DAD method validated for the quantification of glucovanillin and seven other compounds. The method showed satisfactory linearity (r² > 0.9969), precision (coefficient of variation <2%), recoveries (70–100%), limit of detection (0.008–0.212 μg/ml), and limit of quantification (0.027–0.707 μg/ml). The evaluation of crude and enzyme-hydrolyzed Soxhlet-extracted samples confirmed the leading role of glucosides in fruit development. LC–ESI-MS studies corroborated the identities of four glucosides and seven aglycones, among them vanillin (5.7/100 g), 4-hydroxybenzyl alcohol (3.6/100 g), and anisyl alcohol (7.1/100 g) were found in high concentrations. The attractive flavor/aroma profile exhibited by wild V. pompona fruits supports studies focused on the development of this species as a specialty crop.     

Quantitative analysis of phenolic compounds in Chinese hawthorn (Crataegus spp.) fruits by high performance liquid chromatography–electrospray ionisation mass spectrometry

Eleven major phenolic compounds (hyperoside, isoquercitrin, chlorogenic acid, ideain, epicatechin, two procyanidin (PA) dimers, three PA trimers and a PA dimer-hexoside) were quantified in the fruits of 22 cultivars/origins of three species of the Chinese hawthorn (Crataegus spp.) by HPLC–ESI-MS-SIR. Hyperoside (0.1–0.8 mg/g dry mass [DM]), isoquercitrin (0.1–0.3 mg/g DM), chlorogenic acid (0.2–1.6 mg/g DM), epicatechin (0.9–11.7 mg/g DM), PA B2 (0.7–12.4 mg/g DM), PA dimer II (0.1–1.5 mg/g DM), PA trimer I (0.1–2.7 mg/g DM), PA trimer II (0.7–6.9 mg/g DM), PA trimer III (0.01–1.2 mg/g DM) and a PA dimer-hexoside (trace–1.1 mg/g DM) were detected in all the samples. Ideain (0.0–0.7 mg/g DM) was found in all the samples except Crataegus scabrifolia. Significant correlations between the contents of individual PA aglycons were observed (r > 0.9, P < 0.01). A strong correlation between flavonols was also shown (r = 0.71, P < 0.01). Fruits of Crataegus pinnatifida var. major had higher contents of PAs but lower contents of flavonols compared with Crataegus brettschneideri. The fruits of C. scabrifolia contained the highest level of PA dimer-hexoside, which was present in trace amounts in the fruits of C. pinnatifida.     

Raw Millefiori honey is packed full of antioxidants

Total polyphenols, flavonoids and antioxidant power of raw honey samples from two of the most common Italian varieties, i.e., Millefiori and Acacia, were evaluated. Phenolic content, expressed as caffeic acid equivalents, ranged from 12.5 to 17.5 mg/100 g and from 3 to 11 mg/100 g in Millefiori and Acacia honeys, respectively. All Millefiori samples exhibited the highest flavonoid concentration being between 1.23 and 2.93 mg catechin equivalents (CE)/100 g honey. Total flavonoids in 100 g Acacia honeys were in the range of 0.45–1.01 mg CE. Acacia honeys had lower total antioxidant power, as assessed by ferric reducing/antioxidant power assay, than Millefiori. The relationship between phenolic content and antioxidant power was discussed. Comparative experimental analysis was performed with an artificial honey and processed honeys. Raw Millefiori honey is rich in both amount and variety of antioxidant substances, and its inclusion in the diet may be recommended to complement other polyphenol sources.     

Characterisation of a β-N-acetylhexosaminidase from a commercial papaya latex preparation

A β-N-acetylhexosaminidase (β-NAHA) (EC 3.2.1.52) with molecular mass of 64.1 kDa and isoelectric point of 5.5 was purified from a commercial papaya latex preparation. The optimum pH for p-nitrophenyl-N-acetyl-β-d-glucosaminide (pNP-β-GlcNAc) hydrolysis was five; the optimum temperature was 50 °C; the K_m was 0.18 mM, V_max was 37.6 μmol min⁻¹ mg⁻¹ and activation energy (E_a) was 10.3 kcal/mol. The enzyme was thermally stable after holding at 30–45 °C for 40 min, but its activity decreased significantly when the temperature exceeded 50 °C. Heavy metal ions, Ag⁺ and Hg²⁺, at a concentration of 0.25 mM and Zn²⁺ and Cu²⁺, at a concentration of 0.5 mM, significantly inhibited enzyme activity. The β-NAHA had only one active site for binding both pNP-β-GlcNAc and p-nitrophenyl-N-acetyl-β-d-galactosaminide (pNP-β-GalNAc). A prototropic group with pKa value of about five on the enzyme may be involved in substrate binding and transformation, as examined by Dixon–Webb plots.     

Chemical and antioxidant properties of pomegranate cultivars grown in the Mediterranean region of Turkey

Arils from six pomegranate (Punica granatum L.) cultivars obtained from various sites from the Mediterranean region of Turkey were evaluated for their chemical and antioxidant properties. These properties included total phenolics (TP), total monomeric anthocyanins (TMA), soluble solids (TSS), titratable acidity (TA), individual sugars and organic acids. Antioxidant capacities of arils were determined by both the ferric reducing antioxidant power (FRAP) and trolox equivalent antioxidant capacity (TEAC) assays. The antioxidant capacities averaged 5.60 and 7.35 mmol TE/l by the TEAC and FRAP methods. Variability among cultivars was greatest for TMA content (CV 132%); individuals ranged from 6.1 to 219 mg cy3-Gluc l⁻¹. TP means averaged 1507 mg GAE/l. Levels of FRAP, TEAC, TP, and TMA were strongly correlated (r = 0.82–0.96). The major sugars were fructose (6.4 g/100 ml) and glucose (6.8 g/100 ml), the major acids were citric (1.78 g/100 ml) and malic (0.12 g/100 ml).     

Optimisation of the solvent extraction of bioactive compounds from Parkia speciosa pod using response surface methodology

Central composite design was employed to optimise the buffer-to-solids ratio (X₁: 20–50 ml/g), incubation temperature (X₂: 35–55 °C) and time (X₃: 100–200 min), obtaining extracts from Parkia speciosa pod with high total phenolic and flavonoid contents and high antioxidant activities. Analysis of variance showed that the contribution of a quadratic model was significant for the responses. An optimisation study using response surface methodology was performed and 3D response surfaces were plotted from the mathematical models. The optimal conditions based on combination responses were: X₁ = 20 ml/g, X₂ = 35–36 °C and X₃ = 100–102 min. These optimum conditions yielded total phenolic contents of 664–668 mg gallic acid equivalents/100 g, total flavonoid contents of 47.4–49.6 mg pyrocatechol equivalents/100 g, %DPPH_sc of 81.2–82.1%, %ABTS_sc of 78.2–79.8% and FRAP values of 3.2–3.3 mM. Close agreement between experimental and predicted values was found. This methodology could be applied in the extraction of bioactive compounds in the natural product industry.     

Gas chromatographic–mass spectrometric characterisation of triterpene alcohols and monomethylsterols in developing Olea europaea L. fruits

Five triterpene alcohols and four 4-monomethylsterols were identified by GC–MS during the ripening of Picholine olive. The quantitative characterisation of these compounds was performed using GC–FID. The results showed that the maximum level of total triterpene alcohols (263.68 mg/100 g oil) was reached at 26th week after the flowering date (WAF) of olive; whilst the highest level of total 4-monomethylsterols (234 mg/100 g oil) was attained at 24th WAF of fruit. The percentage of these two classes represented 20–33% of total phytosterols during olive maturity. 24-Methylene cycloartenol (12–207 mg/100 g oil) and cycloartenol (27–198 mg/100 g oil) were the predominant triterpene alcohols during the ripening of Picholine olive; whereas citrostadienol (30–161 mg/100 g oil) and cycloeucalenol (11–74 mg/100 g oil) were the main 4-monomethylsterol compounds followed by obtusifoliol and gramisterol. β-Amyrin, δ-amyrin and traroxerol were less present in Picholine olive and they accounted for 14% of total triterpene alcohols at complete maturity of fruit. The level of these methylsterols was overwhelmed by the amount of 4-desmethylsterols at each stage of Picholine olive maturity.     

An extra-cellular alkaline metallolipase from Bacillus licheniformisMTCC 6824: Purification and biochemical characterization

An extra-cellular lipase produced by Bacillus licheniformis MTCC 6824 was purified to homogeneity by ammonium sulphate fractionation, ethanol/ether precipitation, dialysis, followed by anion-exchange chromatography on Amberlite IRA 410 (Cl⁻ form) and gel exclusion chromatography on Sephadex G 100 using Tris–HCl buffer (pH 8.0). The crude lipase extract had an activity of 41.7 LU/ml of culture medium when the bacterium was cultured for 48 h at 37 °C and pH 8.0 with nutrient broth supplemented with sardine oil as carbon source. The enzyme was purified 208-fold with 8.36% recovery and a specific activity of 520 LU/mg after gel exclusion chromatography. The pure enzyme is a monomeric protein and has an apparent molecular mass of 74.8 kDa. The lipase had a V_max and K_m of 0.64 mM/mg/min and 29 mM, respectively, with 4-nitro phenylpalmitate as a substrate, as calculated from the Lineweaver–Burk plot. The lipase exhibited optimum activity at 45 °C and pH 8.0, respectively. The enzyme had half-lives (T_1/2) of 82 min at 45 °C, and 48 min at 55 °C. The catalytic activity was enhanced by Ca²⁺ (18%) and Mg²⁺ (12%) at 30 mM. The lipase was inhibited by Co²⁺, Cu²⁺, Zn²⁺, Fe² even at low concentration (10 mM). EDTA, at 70 mM concentration, significantly inhibited the activity of lipase. Phenyl methyl sulfonyl fluoride (PMSF, 70 mM) completely inactivated the original lipase. A combination of Ca²⁺ and sorbitol induced a synergistic effect on the activity of lipase with a significantly high residual activity (100%), even after 45 min, as compared to 91.5% when incubated with Ca²⁺ alone. The lipase was found to be hydrolytically resistant toward triacylglycerols with more double bonds.     

Saponins in Ipomoeabatatas tubers: Isolation,characterization, quantification and antioxidant properties

Triterpene saponins are a class of plant natural products with a wide range of bioactivities, which makes them an interesting research subject. This work reports, for the first time, the isolation and characterization of saponins in Ipomoeabatatas tuber flour, their quantification and antioxidant properties. Their structures were characterized on the basis of UV, FAB–MS, ESI–MS, GC–MS, polarimetry and NMR data, as: oleanolic acid-3-O-[β-d-glucopyranosyl-(1→2)-β-d-galactopyranosyl-(1→2)-β-d-glucuronopyranosyl]-28-O-β-d-glucopyranoside (sandrosaponin IX) (1) and oleanolic acid-3-O-[β-d-galactopyranosyl-(1→3)-β-d-glucuronopyranosyl]-28-O-β-d-glucopyranoside (2). A new quantitative HPLC–DAD method for saponin content determination in this tuber was developed and validated. Their total content was 200.01 mg/100 g dry weight (RSD = 7.2%; p < 0.001). The single saponin contents were: 161.20 mg/100 g dry weight (RSD = 0.58%; p < 0.001) for saponin 1 and 14.67 mg/100 g dry weight (RSD = 0.41%; p < 0.001) for saponin 2. The antioxidant activities, tested by DPPH and FRAP assay, of total phytochemical fraction and of single saponins were moderate in relation to commercial standards.     

Quantification of main phenolic compounds in sweet and bitter orange peel using CE–MS/MS

The food and agricultural products processing industries generate substantial quantities of phenolics-rich subproducts, which could be valuable natural sources of polyphenols. In oranges, the peel represents roughly 30% of the fruit mass and the highest concentrations of flavonoids in citrus fruit occur in peel. In this work we have carried out the characterisation and quantification of citrus flavonoids in methanolic extracts of bitter and sweet orange peels using CE–ESI–IT–MS. Naringin (m/z 579.2) and neohesperidin (m/z 609.2) are the major polyphenols in bitter orange peels and narirutin (m/z 579.2) and hesperidin (m/z 609.2) in sweet orange peels. The proposed method allowed the unmistakable identification, using MS/MS experiments, and also the quantification of naringin (5.1 ± 0.4 mg/g), neohesperidin (7.9 ± 0.8 mg/g), narirutin (26.9 ± 2.1 mg/g) and hesperidin (35.2 ± 3.6 mg/g) in bitter and sweet orange peels. CE coupled to MS detection can provides structure-selective information about the analytes. In this work we have developed a CE–ESI–IT–MS method for the analysis and quantification of main phenolic compounds in orange peels.     

Characterization of polyphenoloxidase (PPO) and total phenolic contents in medlar (Mespilus germanica L.) fruit during ripening and over ripening

Characterization of polyphenoloxidase (PPO) enzyme and determination of total phenolic concentrations during fruit ripening and over ripening in medlar (Mespilus germanica L.) were determined. During ripening, PPO substrate specificity, optimum pH and temperature, optimum enzyme and substrate concentrations were determined. Among the five mono- and di-phenolic substrates examined ((p-hydroxyphenyl) propionic acid, l-3,4-dihydroxyphenylalanine, catechol, 4-methylcatechol and tyrosine), 4-methylcatechol was selected as the best substrate for all ripening stages. A range of pH 3.0–9.0 was also tested and the highest enzyme activity was at pH 7.0 throughout ripening. The optimum temperature for each ripening stage was determined by measuring the enzyme activity at various temperatures over the range of 10–70 °C with 10 °C increments. The optimum temperatures were found to be 30, 20 and 30 °C, respectively, for each ripening stage. Optimum enzyme and substrate concentrations were found to be 0.1 mg/ml and 40 mM, respectively. The V_max and K_m value of the reaction were determined during ripening and found to be 476 U/mg protein and 26 mM at 193 DAFB (days after full bloom) – stage 1, 256 U/mg protein and 12 mM at 207 DAFB – stage 2, 222 U/mg protein and 8 mM at 214 DAFB – stage 3. For all ripening stages sodium metabisulfite markedly inhibited PPO activity. For stage 1 of ripening, Cu²⁺, Hg²⁺ and Al³⁺, for stage 2, Cu²⁺ and Hg²⁺, and for stage 3, Cu²⁺, Hg²⁺, Al³⁺ and Ca²⁺ strongly inhibited diphenolase activity. Accordingly, it can be concluded that as medlar fruit ripen there is no significant changes in the optimum values of polyphenoloxidases, although their kinetic parametres change. As the fruit ripening progressed through ripe to over-ripe, in contrary to polyphenoloxidase activity, there was an apparent gradual decrease in total fruit phenolic concentrations, as determined by using the aqueous solvents and water extractions.     

Influence of technological processing on apple aroma analysed by high resolution gas chromatography–mass spectrometry and on-line gas chromatography-combustion/pyrolysis-isotope ratio mass spectrometry

Extracts obtained by simultaneous distillation extraction (SDE) from industrial raw materials, namely single strength apple juices, and concentrates and aromas made thereof (each n = 31, from one production line; origin Poland, Germany, Turkey, Romania and China), as well as commercially available juices (n = 27), were analysed by standard controlled capillary gas chromatography–mass spectrometry (HRGC–MS). During the technological processing from juice to the aroma, no qualitative changes in the apple aroma profile were observed. Major constituents of the juices and aromas under study were found to be 1-hexanol (juice, 0.06–5.9 mg/l; aroma, 47–685 mg/l), 1-butanol (juice, 0.1–4.7 mg/l; aroma, 17–370 mg/l); E-2-hexenol (juice, 0.01–3.4 mg/l; aroma, 12–300 mg/l); E-2-hexenal (juice, 0–3.0 mg/l; aroma 0–470 mg/l), and butyl acetate (juice, 0–1.7 mg/l; aroma, 0–165 mg/l). By far the major component of the apple juice concentrates under study was furfural (2.4–56 mg/kg). The observed occurrence of 3-methyl-1-butanol (juice, 0.01–2.1 mg/l; aroma, 1.5–134 mg/l) and, in part, its acetate (juice, 0–0.3 mg/l; aroma, 0–3.3 mg/l), both known not to be genuine apple constituents, was obviously caused by fermentative effects in the course of industrial juice production. In addition, on-line capillary gas chromatography–isotope ratio mass spectrometry was used in the combustion (C) and the pyrolysis (P) modes (HRGC–C/P–IRMS) for the determination of δ¹³C_V-PDB and δ²H_V-SMOW values of selected apple flavour constituents to check potential isotope discrimination during distillative aroma production. As shown by means of the representative examples of E-2-hexenal, 1-hexanol and E-2-hexenol, their δ²H_V-SMOW values were slightly depleted. However, authenticity assessment by stable IRMS will not be influenced by this effect.