Characterization of polyphenol oxidase from butter lettuce (Lactuca sativa var. capitata L.)

Polyphenol oxidase (PPO) was isolated from butter lettuce (Lactuca sativa var. capitata L.) grown in Poland and its biochemical characteristic were studied. PPO from butter lettuce showed a higher affinity to 4-methylcatechol than to catechol. The K_M and V_max values were: 3.20 ± 0.01 mM and 4081 ± 8 U/ml min⁻¹ for catechol and 1.00 ± 0.09 mM and 5405 ± 3 U/ml min⁻¹ for 4-methylcatechol. The optimum pHs of the enzyme were found to be 5.5 using catechol and 6.8 using 4-methylcatechol as substrate. The enzyme had a temperature optimum of 35 °C. The enzyme was relatively stable at 30 °C and 40 °C. The times required for 50% inactivation of activity at 50 °C, 60 °C and 70 °C were found to be about 30, 20 and 5 min, respectively. Inhibitors used for investigation in this study were placed in relative order of inhibition: p-hydroxybenzoic acid > glutathione ≈ ascorbic acid > l-cysteine > EDTA > citric acid. The enzyme eluted in the chromatographic separations was analyzed electrophoretically under denaturating conditions. The analysis revealed a single band on the SDS–PAGE which corresponded to a molecular weight of 60 kDa.     

Antioxidant activity of lettuce extract (Lactuca sativa) and synergism with added phenolic antioxidants

Combined antioxidative effects of lettuce extract and α-tocopherol, quercetin or ascorbic acid (AA) were investigated for peroxidation of l-α–phosphatidylcholine liposomes with oxidation initiated by lipophilic or by hydrophilic azo-initiators. Lettuce extract had a clear antioxidative effect as evidenced by a lag phase for formation of conjugated dienes, and α-tocopherol and especially quercetin acted synergistically in prolongation of the lag phase both following initiation in the lipid phase and in the aqueous phase. Combination of AA with lettuce extract showed in contrast a lag phase that was similar to that observed for AA alone. Storage of lettuce extract for 24 h at refrigerator or room temperatures resulted in a decreasing lag phase with increasing storage time for both storage temperatures, an effect not counteracted by addition of quercetin or α-tocopherol. Heating of lettuce extract for 10 min at 80 °C did not affect the lag phase and heating of lettuce extract resulted in an increasing synergism for added quercetin and α-tocopherol indicating that thermal inhibition of polyphenoloxidase (PPO) increases antioxidant potential and interaction.     

Total phenolic content and DPPH radical scavenging activity of lettuce (Lactuca sativa L.) grown in Colorado

Total phenolic content (TPC) and antioxidant capacity of lettuce were evaluated using the Folin-Ciocalteu method and DPPH assay, respectively, on 25 cultivars of lettuce, including leaf, romaine, crisphead, and butterhead types, cultivated over two harvest periods. Leaf lettuce possessed the highest TPC and highest DPPH scavenging ability, followed by romaine, butterhead and Batavia (crisphead subtype). Within a lettuce type, red pigmented lettuce cultivars had higher TPC and antioxidant capacity than did green cultivars grown under the same conditions. In addition, lettuce harvested in July possessed higher TPC and antioxidant capacity than did lettuce harvested in September, suggesting that environmental conditions could influence the phenolic content and antioxidant activity of lettuce. These results suggest that Colorado grown lettuce may serve as potential dietary sources of natural phenolic antioxidants.     

Effect of various anti-browning agents on phenolic compounds profile of fresh lettuce (L. sativa)

High performance liquid chromatography (HPLC) was used to characterise phenolic profile and to select the most effective anti-browning compound(s) on fresh lettuce. Four anti-browning agents, ascorbic acid, cysteine, citric acid and oxalic acid were tested for their effectiveness on preventing loss of phenolic compounds in lettuce during processing and storage. Aliquots of the reaction mixture were withdrawn at different times varying from 0 to 24 h, and directly analysed by HPLC. Protocatechuic acid, chlorogenic acid, ferulic acid, caffeic acid, p-coumaric acid and phloridzin were identified in fresh lettuce. Degradation of phenolic compounds followed a first-order kinetic pattern. The effect of anti-browning agents on first-order degradation rates of phenolic compounds was determined. Lettuce treated with oxalic acid and ascorbic acid maintained a higher level of phenolic compounds than citric acid and cysteine. Interestingly, cysteine had no positive effect for the prevention of oxidation of phenolic compounds even though it prevented browning in lettuce.     

Light exposure during storage preserving soluble sugar and l-ascorbic acid content of minimally processed romaine lettuce (Lactuca sativa L.var. longifolia)

Minimally processed romaine lettuce (MPRL) leaves were stored in light condition (2500 lux) or darkness at 4 °C for 7 d. Light exposure significantly delayed the degradation of chlorophyll and decrease of glucose, reducing sugar, and sucrose content, and thus preserved more total soluble solid (TSS) content at the end of storage in comparison with darkness. While, it did not influenced starch content that progressively decreased over time. The l-ascorbic acid (AA) accumulated in light-stored leaves, but deteriorated in dark-stored leaves during storage. The dehydroascorbic acid (DHA) increased in all leaves stored in both light and dark condition, of which light condition resulted in less DHA than darkness. In addition, the fresh weight loss and dry matter significantly increased and these increases were accelerated by light exposure. Conclusively, light exposure in applied intensity effectively alleviated MPRL quality deterioration by delaying the decreases of pigments, soluble sugar, TSS content and accumulating AA.     

Effect of whey protein concentrate on phenolic profile and browning of fresh-cut lettuce (Lactuca Sativa)

The in vitro kinetics of lettuce PPO with respect to dissolved oxygen using catechin, chlorogenic acid, caffeic acid and gallic acid has been examined. In-vitro lettuce polyphenol oxidase (PPO) activity was determined by measuring the consumption of oxygen during the oxidation reaction. The effect of whey protein concentrate (WPC) was tested on the inhibition of lettuce PPO comparing with ascorbic acid (AA) and cysteine. A competitive model that considered inhibitors was the most appropriate model to explain reaction kinetics. Browning of lettuce was also monitored during storage for 24 h. Addition of WPC prevented loss of lightness in lettuce. Loss of identified phenolic compounds in lettuce was measured during the enzymatic browning process by high-performance liquid chromatography. Degradation of identified phenolic compounds followed first order kinetics during storage. Combination of WPC with cysteine was proposed for the protection of phenolics compounds against PPO-catalysed oxidation.     

Effect of various inhibitors on enzymatic browning,antioxidant activity and total phenol content of fresh lettuce (Lactuca sativa)

In this study, polyphenol oxidase (PPO) was isolated from fresh lettuce. Its optimum temperature and pH were found to be 40 °C and 7.0, respectively. Lettuce PPO was shown to use catechin, catechol, chlorogenic acid, caffeic acid and gallic acid as substrates. Among the substrates used, the greatest substrate specificity was observed with chlorogenic acid. Lettuce PPO was sensitive to some inhibitors. Ascorbic acid, cysteine, oxalic acid and citric acid were tested as potential inhibitors of lettuce PPO. Cysteine was the most effective inhibitor. Total phenol and total antioxidant activity contents were also determined in the presence of these inhibitors at room and refrigerator temperatures. Ascorbic acid and cysteine increased the total antioxidant activity of lettuce while citric and oxalic acids had no effect on the total antioxidant activity. Lettuce phenolics were protected from oxidation by ascorbic acid and cysteine.     

Prediction of coliforms and Escherichia coli on tomato fruits and lettuce leaves after sanitizing by using Artificial Neural Networks

The objectives of this study were to investigate the efficacy of two sanitizers, i.e. hypochlorous and peracetic acids, in reducing coliforms and Escherichia coli levels on tomato fruits and lettuce leaves, and to mathematically predict the relationship among the initial bacterial load, type of vegetable/fruit, types and concentration of sanitizer and residual microorganism levels after the sanitizing, by applying artificial neural networks (ANNs). The E. coli and coliforms used in this study were isolated from the two food types, and their cultures were activated in Tryptic Soy Broth (ca. 6-7 log₁₀ cfu/ml) before inoculating onto the fruit and vegetable. Both sanitizers reduced the number of the micro-organisms. However, as the hypochlorous acid concentration was increased, the level of viable coliforms and E. coli on the tomato fruits was reduced around 2-3 log₁₀ cfu/g (p ≤ 0.05), compared to only about 1 log₁₀ cfu/g reduction on lettuce leaves (p ≤ 0.05). Conversely, when the peracetic acid concentration was increased, the coliforms and E. coli levels on tomato fruits were reduced by some 3-4 log₁₀ cfu/g (p > 0.05) compared to only about 2 log₁₀ cfu/g on lettuce leaves (p > 0.05).The best sum square error from the neural prediction of residual coliforms and E. coli were 0.50 and 0.84, respectively, and the maximum R² of residual coliforms and E. coli were 0.85 and 0.72, respectively. Only one hidden layer with three hidden neurons for coliforms and five for E. coli, were required to model this data.     

Electron-beam irradiation of fresh broccoli heads (Brassica oleracea L. italica)

Broccoli is a popular item in the diet of the US population, commonly found in salads ready-to-eat. The recent recalls of fresh produce due to contamination with Escherichia coli and other pathogens emphasize the need to find effective means to treat minimally processed fresh foods. Our study assessed the effect of ionizing radiation using electron beams on the shelf-life, physicochemical properties, and consumer acceptability of broccoli florets. One-hundred broccoli heads were irradiated at 1, 2, and 3 kGy with a 10 MeV linear accelerator at 22 °C. We monitored pH, color, texture, respiration rate, weight loss, chlorophyll, total carotenoids, and vitamin C of irradiated and non-irradiated samples at 5-day intervals up to 14 days at 4 °C and 95% RH. Fifty consumer panelists scored the samples using a nine-point hedonic scale.Irradiation did not affect color, firmness, pH, and weight loss of the samples. Both irradiated samples and controls showed a slight change in color during storage, though the effect was not dose-dependent. Irradiation affected (P < 0.05) the respiration rates on the first 5 days of storage (higher CO₂ levels) but all samples, including the controls, attained the same equilibrium value. Vitamin C content of all samples decreased (P < 0.05) with storage time. Chlorophyll and total carotenoids content followed the same trend. In terms of overall acceptability, color, odor, and texture, all irradiated samples were highly accepted by the panelists with scores of 5 and above. By the end of shelf-life, only the controls showed significant quality decline (yellow color, off-odor) due to microbial spoilage. In summary, electron-beam treatment up to 3 kGy maintains the overall quality of fresh broccoli.     

Anthocyanin composition of cauliflower (Brassica oleracea L. var. botrytis) and cabbage (B. oleracea L. var. capitata) and its stability in relation to thermal treatments

Violet cauliflower and red cabbage were analysed for their anthocyanin profiles before and after thermal treatments. Anthocyanins are well-noted as healthy compounds due to their antioxidant properties. Samples were analysed for total anthocyanin content by using a spectrophotometric differential pH method. An MS-based method, combining high-performance liquid chromatography (HPLC) with quadrupole tandem mass spectrometry (HPLC–MS/MS) was developed, aimed to separate, identify and quantify the main anthocyanin forms. The procedure involves a rapid and efficient pre-treatment of the samples by solid-phase extraction, followed by selective determination of all compounds in a single run analysis using HPLC–MS/MS. Structural information for the identification of compounds was obtained from their fragmentation patterns (MS/MS spectra). The compounds were separated by HPLC and detected in the multiple reaction monitoring mode (MRM), which provides a high level of selectivity for targeting the analytes in vegetables. Cauliflower and red cabbage showed differences in their anthocyanin profiles: cyanidin-3,5-diglucoside was absent in cauliflower, while it was well represented in red cabbage, together with the characteristic anthocyanin of Brassica genus, cyanidin-3-sophoroside-5-glucoside. The p-coumaryl and feruloyl esterified forms of cyanidin-3-sophoroside-5-glucoside were predominant in cauliflower, while the sinapyl one was mostly present in red cabbage. Besides, the stability of cauliflower’s anthocyanin profile was evaluated in relation to thermal pre-treatments. All thermal treatments, except microwave heating, drastically reduced total cauliflower anthocyanin content. The amount of individual anthocyanins was expressed as the percentage with respect to total anthocyanin amount, spectrophotometrically measured. Significant individual changes were observed after different thermal treatment with an isomer formation.     

Structure (micro, ultra, nano), color and mechanical properties of Vitis labrusca L. (grape berry) fruits treated by hydrogen peroxide, UV–C irradiation and ultrasound

Main structural (micro, ultra, and nano) alterations occurred in the outer tangential epidermal cell wall of Vitis labrusca L. fruits (grape berry) due to hydrogen peroxide, UV–C irradiation and ultrasound treatments were examined, and described using light microscopy, scanning electron microscopy, transmission electron microscopy and atomic force microscopy. Changes in mechanical properties and surface color were also evaluated. In general, decontamination treatments caused epicuticular wax pattern alteration, epicarp disruptions, cell plasmolysis, and mesocarp collapse. Major observed ultrastructural and nanostructural changes were increased demarcation between cellulose layer and cuticular membrane, alteration of cellulose aggregates pattern, and presence of nanofractures, variables in shape and size. All treatments, mainly ultrasound, provoked significant but small differences in color parameters compared to untreated fruit. On contrast, mechanical properties determined by puncture test did not vary.     

Colour stability of shisonin,red pigment of a Perilla (Perilla ocimoides L. var.crispa Benth)

The colour stability of shisonin, a red pigment isolated from the leaves of a perilla (Perilla ocimoidesL. var. crispaBenth),was studied in model systems. At pH less than 6, the absorbance of shisonin in solution decreases with increase in pH. The degradation of shisonin in buffers at both pH 3 and 4 followed first-order reaction kinetics. Fructose, sucrose, metal cations Cu⁺⁺ and Fe⁺⁺⁺, hydrogen peroxide, oxygen and daylight affected the degradation rate of shisonin, while glucose, lactose, glycine and leucine had essentially no effect on the degradation rate.     

In vitro antioxidant activity and inhibitory effect,on oleic acid-induced hepatic steatosis,of fractions and subfractions from oat (Avena sativa L.) ethanol extract

Oats (Avena sativa L.) were extracted with 80% aqueous ethanol and the extract was successively isolated by liquid–liquid partition to yield n-hexane, ethyl acetate, n-butanol and water layers. Among them, the ethyl acetate (EA) layer exhibited the highest total phenolic content (TPC), the strongest DPPH radical-scavenging activity and an inhibitory effect on an oleic acid-induced (OA-induced) fatty liver model in vitro. Thus, it was further fractionated by a Sephadex LH-20 column into three subfractions (SF1–SF3). SF3 was the most active subfraction in all the assays above, the yield being 1.70% of the dry weight of the EA fraction. The major components in SF3 were identified as avenanthramides Bc, Bp and Bf by HPLC analysis, with contents of about 5.20%, 9.19% and 8.06% of the dry weight of SF3, respectively. Also, the avenanthramides Bc, Bp and Bf all had significant inhibitory effects on oleic acid-induced (OA-induced) fatty liver.     

Effect of ultrasound treatment on water holding properties and microstructure of beef (m. semimembranosus) during ageing

The purpose of this study was to investigate the structural, water holding capacity (WHC) and water compartmentalization changes in meat samples sonicated at 24 h after slaughter. Analyses were carried out at the following fixed intervals post mortem: 24, 48, 72 and 96 h on the beef muscle (m. semimembranosus) taken from carcasses chilled at 2 °C. Muscle was divided into eight parts. Four of them were subjected to ultrasound treatment within 2 min with frequency of 45 kHz (sample S). The other four were regarded as control samples. Water compartmentalization was assessed by NMR relaxation time measurements. Water holding capacity as well as acidity of meat was measured. More advanced post mortem changes in sonicated sample than in the control sample support the hypothesis that application of ultrasound treatment can cause acceleration of the ageing process.     

Polyphenolic composition in different parts of some cultivars of globe artichoke (Cynara cardunculus L. var. scolymus (L.) Fiori)

The analysis of polyphenols of leaves and different parts (outer, intermediate and inner bracts, and receptacle) of heads in five globe artichoke cultivars of Campania region (Italy) and one accession of cultivated cardoon was performed. Data obtained suggest that the edible parts (receptacles with inner and intermediate bracts) of these cultivars of artichoke could represent a good source of health-promoting polyphenols and therefore encourage a nutriceutical use of this species, as an alternative to the more traditional phytopharmaceutical applications of leaf extracts. Moreover, it was demonstrated that single polyphenols accumulate preferentially in specific parts of the heads and in specific genotypes.     

Chemical and antioxidative assessment of dietary turnip (Brassica rapa var. rapa L.)

The phenolic compounds and organic acids of turnip (Brassica rapa var. rapa L.) edible parts (leaves and stems, flower buds and roots) were determined by HPLC–DAD and HPLC–UV, respectively. The results revealed a profile composed of 14 phenolics (3-p-coumaroylquinic, caffeic, ferulic and sinapic acids, kaempferol 3-O-sophoroside-7-O-glucoside, kaempferol 3-O-sophoroside-7-O-sophoroside, kaempferol 3-O-(feruloyl/caffeoyl)-sophoroside-7-O-glucoside, kaempferol 3,7-O-diglucoside, isorhamnetin 3,7-O-diglucoside, kaempferol 3-O-sophoroside, 1,2-disinapoylgentiobiose, 1,2′-disinapoyl-2-feruloylgentiobiose, kaempferol 3-O-glucoside and isorhamnetin 3-O-glucoside) and six organic acids (aconitic, citric, ketoglutaric, malic, shikimic and fumaric acids). The quantification of the identified compounds showed kaempferol 3-O-sophoroside-7-O-glucoside, kaempferol 3-O-(feruloyl/caffeoyl)-sophoroside-7-O-glucoside, isorhamnetin 3,7-O-diglucoside and isorhamnetin 3-O-glucoside as the main phenolics, and malic acid as the organic acid present in highest amounts. A screening of the antioxidative potential was also performed by means of the DPPH radical scavenging assay. Turnip flower buds exhibited the strongest antioxidant capacity.     

Uptake of arsenic species by turnip (Brassica rapa L.) and lettuce (Lactuca sativa L.) treated with roxarsone and its metabolites in chicken manure

Roxarsone is an organoarsenic feed additive that can be metabolised to other higher toxic arsenic (As) species in animal manure such as arsenate, arsenite, monomethylarsonic acid, dimethylarsinic acid, 3-amino-4-hydroxyphenylarsonic acid and other unknown As species. The accumulation, transport and distribution of As species in turnip (Brassica rapa L.) and lettuce (Lactuca sativa L.) amended with roxarsone and its metabolites in chicken manure were investigated. Results showed arsenite was the predominant As form, followed by arsenate in turnip and lettuce plants, and a low content of dimethylarsinic acid was detected only in lettuce roots. Compared with the control plants treated with chicken manure without roxarsone and its metabolites, the treatments containing roxarsone and its metabolites increased arsenite content by 2.0–3.2% in turnip shoots, by 6.6–6.7% in lettuce shoots, by 11–44% in turnip tubers and by 18–20% in lettuce roots at two growth stages. The enhanced proportion of arsenate content in turnip shoots, turnip tubers and lettuce roots was 4.3–14%, 20–35% and 70%, respectively, while dimethylarsinic acid content in lettuce roots increased 2.4 times. Results showed that the occurrence of dimethylarsinic acid in lettuce roots might be converted from the inorganic As species and the uptake of both inorganic and organic As compounds in turnip and lettuce plants would be enhanced by roxarsone and its metabolites in chicken manure. The pathway of roxarsone metabolites introduced into the human body via roxarsone → animal → manure → soil → crop was indicated.