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

Use of time-resolved fluorescence to monitor bioactive compounds in plant based foodstuffs

The study of compounds that exhibit antioxidant activity has recently received much interest in the food industry because of their potential health benefits. Most of these compounds are plant based, such as polyphenolics and carotenoids, and there is a need to monitor them from the field through processing and into the body. Ideally, a monitoring technique should be non-invasive with the potential for remote capabilities. The application of the phenomenon of fluorescence has proved to be well suited, as many plant associated compounds exhibit fluorescence. The photophysical behaviour of fluorescent molecules is also highly dependent on their microenvironment, making them suitable probes to monitor changes in pH, viscosity and polarity, for example. Time-resolved fluorescence techniques have recently come to the fore, as they offer the ability to obtain more information, coupled with the fact that the fluorescence lifetime is an absolute measure, while steady state just provides relative and average information. In this work, we will present illustrative time-resolved measurements, rather than a comprehensive review, to show the potential of time-resolved fluorescence applied to the study of bioactive substances. The aim is to help assess if any changes occur in their form, going from extraction via storage and cooking to the interaction with serum albumin, a principal blood transport protein

<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

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

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

Effect of ultrasound on the activity of mushroom (<i>Agaricus bisporous</i>) polyphenol oxidase and observation of structural changes using time-resolved fluorescence

Polyphenol oxidase (PPO) is an enzyme associated with the browning process that can occur from mechanical injury and postharvest storage. Thus, its inactivation to inhibit this process is of great interest to the food industry. Recently a nonthermal technology, high frequency ultrasound (100–1 MHz), has found usage in this aspect. In this work, the application of high-frequency (378 kHz, 583 kHz, 1144 kHz, and 1175 kHz) and low frequency (20 kHz) treatment on a PPO extract (from mushrooms) by monitoring the residual enzymatic activity is described. A control thermal treatment at 40 °C was also performed for comparison purposes. High-frequency inactivation data fitted well using the Weibull model, whereas those obtained upon low frequency followed first-order kinetics. The inactivation rate constant obtained ranged from 0.0054 (20 kHz) to 0.028 min−1 (at 583 kHz). To elucidate changes in the enzyme structure time-resolved spectroscopy of a commercial PPO enzyme model was employed. Results indicated that ultrasound-induced structural changes in PPO, in keeping with the activity behaviour upon sonication

Phenolic content and potential bioactivity of apple juice as affected by thermal and ultrasound pasteurization

Thermal (T) and ultrasound (US) pasteurization processes were applied to apple juice and the phenolic compounds (TPC) were quantified before and after in vitro digestion by HPLC-DAD-ESI-MSn, with their bioaccessibility ascertained. Digested samples were analysed for their inhibitory capacity against α-glucosidase. Since some of the compounds exhibit fluorescence, both steady state and time-resolved fluorescence methods were used to investigate the binding to a blood transport protein, human serum albumin (HSA). It was found that processing induced an increase in the TPC content, which was more pronounced when US was applied. In contrast, digestion reduced the TPC content, evening out the overall effect. Still T and US pasteurized juices exhibited a higher quantity of TPC upon digestion as compared to the raw sample. No correlation was found between the TPC content and α-glucosidase inhibition, as the T and US pasteurized juices showed the highest and lowest inhibitory capacities against the enzyme, respectively. This is indicative that other compounds, such as those formed upon thermal treatment, may be involved in the antidiabetic effect of apple juice. The fluorescence study showed that binding occurred to HSA, at slightly different rates for different species present in the US treated extract. Considering energy consumption, US pasteurization is the most power consuming treatment despite its shorter duration. Overall, no univocal indication on the best pasteurization process can be gathered. Thus, it is necessary to define the desired target in order to drive technological interventions by a customized approach.</p