Study Protocol of a Multicenter Randomized Controlled Trial to Tackle Obesity through a Mediterranean Diet vs. a Traditional Low-Fat Diet in Adolescents: The MED4Youth Study.

Youth obesity is a strong predictor of adult obesity, which has well-known negative health consequences. Thus, addressing adult obesity requires tackling youth obesity. MED4Youth's main objective is to strengthen the link between the Mediterranean Diet (MD) and the health benefits against youth obesity and associated cardiovascular disease (CVD) risk factors, identifying positive effects exerted by an MD including sourdough bread and healthy products from the Mediterranean basis (chickpeas/hummus, nuts, and pomegranate juice). For this purpose, a multicenter randomized controlled trial in which an MD-based intervention will be compared to a traditional low-fat diet intervention will be carried out with 240 overweight and obese adolescents (13-17 years) from Spain, Portugal, and Italy. Both interventions will be combined with an educational web-application addressed to engage the adolescents through a learning-through-playing approach, using both educational materials and games. To assess the interventions, adherence to the MD, dietary records, physical activity, food frequency, sociodemographic, and quality of life questionnaires as well as classical anthropometric and biochemical parameters will be evaluated. Furthermore, an omics approach will be performed to elucidate whether the interventions can shape the gut microbiota and gut-derived metabolites to gain knowledge on the mechanisms through which the MD can exert its beneficial effects.

Consumption of Sourdough Breads Improves Postprandial Glucose Response and Produces Sourdough-Specific Effects on Biochemical and Inflammatory Parameters and Mineral Absorption.

To evaluate responses to different sourdough breads, six groups of rats were fed a conventional refined wheat bread with no sourdough content (C_WhB); a leavened spelt bread baked with Rebola sourdough (Re_SpB); a durum wheat bread with Rebola sourdough (Re_DuB); or a multigrain bread leavened with Rebola (Re_MGB), Carla (Ca_MGB), or San Francisco sourdough (SF_MGB). Compared to C_WhB-fed rats, Re_SpB-, Re_DuB-, and Re_MGB-fed animals showed lower postprandial blood glucose levels, whereas SF_MGB-fed rats displayed a decreased postprandial blood insulin response and glucose and insulin products. The 3 week intake of Ca_MGB decreased blood triacylglycerols and the relative apparent absorption (RAA) of Fe2+, Cu2+, and Zn2+, whereas Re_MGB-fed animals showed lower serum levels of the MCP-1 inflammatory marker and decreased the Fe RAA. The 3 week consumption of the multigrain bread produced sourdough-specific effects. Thus, Re_MGB-fed animals displayed higher insulin concentrations than Ca_MGB- and SF_MGB-fed rats and decreased blood MCP-1 levels compared to those of Ca_MGB-fed animals. In addition, Ca_MGB-fed rats showed lower serum triacylglycerol concentrations than those of Re_MGB- and SF_MGB-fed animals, whereas SF_MGB-fed rats displayed higher RAA values of Ca2+, Cu2+, Fe2+, Mg2+, and Zn2+ than their Re_MGB and Ca_MGB counterparts. These sourdough-specific effects could be related to changes in the contents of sugars and organic acids, acidity, microbial composition, and proteolytic activity among sourdoughs. Hence, the consumption of sourdough breads improved postprandial blood glucose and insulin responses and produced sourdough-specific effects on RAA and serum insulin and triacylglycerol and MCP-1 levels in rats, showing that SF_MGB has the most promising beneficial effects.

C-phycocyanin extraction assisted by pulsed electric field from Artrosphira platensis.

This paper assesses the application of pulsed electric fields (PEF) to the fresh biomass of Artrhospira platensis in order to enhance the extraction of C-phycocyanin into aqueous media. Electroporation of A. platensis depended on both electric field strength and treatment duration. The minimum electric field intensity for detecting C-phycocyanin in the extraction medium was 15kV/cm after the application of a treatment time 150µs (50 pulses of 3µs). However higher electric field strength were required when shorter treatment times were applied. Response surface methodology was used in order to investigate the influence of electric field strength (15-25kV/cm), treatment time (60-150µs), and temperature of application of PEF (10-40°C) on C-phycocyanin extraction yield (PEY). The increment of the temperature PEF treatment reduced the electric field strength and the treatment time required to obtain a given PEY and, consequently decreased the total specific energy delivered by the treatment. For example, the increment of temperature from 10°C to 40°C permitted to reduce the electric field strength required to extract 100mg/g dw of C-phycocyanin from 25 to 18kV/cm, and the specific energy input from 106.7 to 67.5kJ/Kg. Results obtained in this investigation demonstrated PEF's potential for selectively extraction C-phycocyanin from fresh A. platensis biomass. The purity of the C-phycocyanin extract obtained from the electroporated cells was higher than that obtained using other techniques based on the cell complete destruction.

The effect of temperature and bacterial growth phase on protein extraction by means of electroporation.

Different chemical and physical methods are used for extraction of proteins from bacteria, which are used in variety of fields. But on a large scale, many methods have severe drawbacks. Recently, extraction by means of electroporation showed a great potential to quickly obtain proteins from bacteria. Since many parameters are affecting the yield of extracted proteins, our aim was to investigate the effect of temperature and bacterial growth phase on the yield of extracted proteins. At the same time bacterial viability was tested. Our results showed that the temperature has a great effect on protein extraction, the best temperature post treatment being 4°C. No effect on bacterial viability was observed for all temperatures tested. Also bacterial growth phase did not affect the yield of extracted proteins or bacterial viability. Nevertheless, further experiments may need to be performed to confirm this observation, since only one incubation temperature (4°C) and one incubation time before and after electroporation (0.5 and 1h) were tested for bacterial growth phase. Based on our results we conclude that temperature is a key element for bacterial membrane to stay in a permeabilized state, so more proteins flow out of bacteria into surrounding media.

A Comparative Study on the Effects of Millisecond- and Microsecond-Pulsed Electric Field Treatments on the Permeabilization and Extraction of Pigments from Chlorella vulgaris.

The interdependencies of the two main processing parameters affecting "electroporation" (electric field strength and pulse duration) while using pulse duration in the range of milliseconds and microseconds on the permeabilization, inactivation, and extraction of pigments from Chlorella vulgaris was compared. While irreversible "electroporation" was observed above 4 kV/cm in the millisecond range, electric field strengths of ≥10 kV/cm were required in the microseconds range. However, to cause the electroporation of most of the 90 % of the population of C. vulgaris in the millisecond (5 kV/cm, 20 pulses) or microsecond (15 kV/cm, 25 pulses) range, the specific energy that was delivered was lower for microsecond treatments (16.87 kJ/L) than in millisecond treatments (150 kJ/L). In terms of the specific energy required to cause microalgae inactivation, treatments in the microsecond range also resulted in greater energy efficiency. The comparison of extraction yields in the range of milliseconds (5 kV, 20 ms) and microseconds (20, 25 pulses) under the conditions in which the maximum extraction was observed revealed that the improvement in the carotenoid extraction was similar and chlorophyll a and b extraction was slightly higher for treatments in the microsecond range. The specific energy that was required for the treatment in the millisecond range (150 kJ/L) was much higher than those required in the microsecond range (30 kJ/L). The comparison of the efficacy of both types of pulses on the extraction enhancement just after the treatment and after a post-pulse incubation period seemed to indicate that PEF in the millisecond range created irreversible alterations while, in the microsecond range, the defects were a dynamic structure along the post-pulse time that caused a subsequent increment in the extraction yield.

Effect of pulsed electric field treatments on permeabilization and extraction of pigments from Chlorella vulgaris.

The effect of pulsed electric field (PEF) treatments of different intensities on the electroporation of the cytoplasmatic membrane of Chlorella vulgaris, and on the extraction of carotenoids and chlorophylls were investigated. Staining the cells with propidium iodide before and after the PEF treatment revealed the existence of reversible and irreversible electroporation. Application of PEF treatments in the range of 20-25 kV cm(-1) caused most of the population of C. vulgaris to be irreversibly electroporated even at short treatment times (5 pulses of 3 µs). However, at lower electric field strengths (10 kV cm(-1)), cells that were reversibly electroporated were observed even after 50 pulses of 3 µs. The electroporation of C. vulgaris cells by PEF higher than 15 kV cm(-1) and duration is higher than 15 µs increased significantly the extraction yield of intracellular components of C. vulgaris. The application of a 20 kV cm(-1) for 75 µs increased the extraction yield just after the PEF treatment of the carotenoids, and chlorophylls a and b 0.5, 0.7, and 0.8 times, respectively. However, further increments in electric field strength and treatment time did not cause significant increments in the extraction yield. The extraction of carotenoids from PEF-treated C. vulgaris cells after 1 h of the application of the treatment significantly increased the extraction yield in comparison to the yield obtained from the cells extracted just after the PEF treatment. After PEF treatment at 20 kV cm(-1) for 75 µs, extraction yield for carotenoids, and chlorophylls a and b increased 1.2, 1.6, and 2.1 times, respectively. A high correlation was observed between irreversible electroporation and percentage of yield increase when the extraction was conducted after 1 h of the application of PEF treatment (R: 0.93), but not when the extraction was conducted just after PEF treatment (R: 0.67).

Improving carotenoid extraction from tomato waste by pulsed electric fields.

In this investigation, the influence of the application of pulsed electric fields (PEFs) of different intensities (3-7 kV/cm and 0-300 µs) on the carotenoid extraction from tomato peel and pulp in a mixture of hexane:acetone:ethanol was studied with the aim of increasing extraction yield or reducing the percentage of the less green solvents in the extraction medium. According to the cellular disintegration index, the optimum treatment time for the permeabilization of tomato peel and pulp at different electric field strengths was 90 µs. The PEF permeabilization of tomato pulp did not significantly increase the carotenoid extraction. However, a PEF treatment at 5 kV/cm improved the carotenoid extraction from tomato peel by 39% as compared with the control in a mixture of hexane:ethanol:acetone (50:25:25). Further increments of electric field from 5 to 7 kV/cm did not increase significantly the extraction of carotenoids. The presence of acetone in the solvent mixture did not positively affect the carotenoid extraction when the tomato peels were PEF-treated. Response surface methodology was used to determine the potential of PEF for reducing the percentage of hexane in a hexane:ethanol mixture. The application of a PEF treatment allowed reducing the hexane percentage from 45 to 30% without affecting the carotenoid extraction yield. The antioxidant capacity of the extracts obtained from tomato peel was correlated with the carotenoid concentration and it was not affected by the PEF treatment.

Pulsed-electric-field-assisted extraction of anthocyanins from purple-fleshed potato.

The influence of pulsed electric field (PEF) treatment on the anthocyanin extraction yield (AEY) from purple-fleshed potato (PFP) at different extraction times (60-480 min) and temperatures (10-40°C) using water and ethanol (48% and 96%) as solvents has been investigated. Response surface methodology was used to determine optimal PEF treatment and optimise anthocyanin extraction. A PEF treatment of 3.4 kV/cm and 105 µs (35 pulses of 3 µs) resulted in the highest cell disintegration index (Z(p)=1) at the lowest specific energy requirements (8.92 kJ/kg). This PEF treatment increased the AEY, the effect being higher at lower extraction temperature with water as solvent. After 480 min at 40°C, the AEY obtained for the untreated sample using 96% ethanol as the solvent (63.9 mg/100 g fw) was similar to that obtained in the PEF-treated sample using water (65.8 mg/100 g fw). Therefore, PEF was possible with water, a more environmental-friendly solvent than ethanol, without decreasing the AEY from PFP.