Novel pulsed electric field (PEF) process conditions at moderate electric field strength and long pulse duration have recently been established to obtain microbial inactivation. In this study, the ...effect of these PEF conditions (E = 0.9 and 2.7 kV/cm, with pulse duration 1000 µs) at variable maximum temperatures was evaluated on quality attributes of freshly squeezed orange juice. Results were compared to orange juice that received no treatment or a mild or severe thermal pasteurization treatment. No differences for pH and soluble solids were found after application of any treatment, and only small differences were observed for color and vitamin C content (ascorbic acid and dehydroascorbic acid) after processing, mainly for conditions applied at higher temperature. Variations in the maximum temperatures of the PEF and thermal processes led to differences in flavor compounds and the remaining activity of pectinmethylesterase (PME). At PEF conditions with a maximum temperature of 78 °C or higher, PME activity levels were below a critical value, meaning that the cloud is stable. At this temperature volatiles associated with fresh juice (such as octanal and nonanal) are statistically identical to untreated juice, while they are statistically distinguishable from thermal treated. This papers demonstrates the potential of using moderate intensity PEF as an adequate alternative to thermal pasteurization of orange juice with a better retention of the fresh flavor.
Thiamine is an essential vitamin for most living organisms, of which yeasts are a rich nutritional source. In this study we developed a thiamine extraction and determination method to detect thiamine ...in fresh yeast biomass. The thiamine determination method combines the derivatization of thiamine to a highly fluorescent product, with chromatographic separation (HPLC) and fluorescence detection. The method specifically detects free thiamine (T), thiamine phosphate (TP), and thiamine pyrophosphate (TPP). It has a high sensitivity of 2 ng/ml for TPP and TP, and 1 ng/ml for T, excellent instrumental repeatability, and low day-to-day variation in retention time of the different phosphate forms. We demonstrated the robustness of the method by proving that the fluorescence signals of the derivatised samples are stable for at least 82 h after derivatization, and by showing that the final pH of the samples does not influence the fluorescent response. In addition, we developed and validated a thiamine extraction method consisting of beads beating the fresh yeast biomass in 0.1 M HCl using a lysing matrix composed of 0.1 mm silica spheres. The performance of this method was compared to extraction via heat treatment at 95 °C for 30 min, and a combination of beads beating and heat treatment carried out in different order. We demonstrated that thiamine extraction via beads beating is the only method that prevents the biologically active form thiamine pyrophosphate to be degraded to thiamine phosphate, therefore, the extraction method developed and described in this study is preferred when the different thiamine vitamers need to be detected in their actual proportions. The combination of the extraction via beads beating, the conversion of all vitamers to the thiochrome derivatives, and the separation of these compounds on the reversed phase HPLC with a fluorescence detector, yielded a sensitive, specific, repeatable, and robust method for extraction and determination of vitamin B
in fresh yeast biomass.
Evasion of apoptosis is critical for the development and growth of tumors. The pro-survival protein myeloid cell leukemia 1 (Mcl-1) is an antiapoptotic member of the Bcl-2 family, associated with ...tumor aggressiveness, poor survival, and drug resistance. Development of Mcl-1 inhibitors implies blocking of protein–protein interactions, generally requiring a lengthy optimization process of large, complex molecules. Herein, we describe the use of DNA-encoded chemical library synthesis and screening to directly generate complex, yet conformationally privileged macrocyclic hits that serve as Mcl-1 inhibitors. By applying a conceptual combination of conformational analysis and structure-based design in combination with a robust synthetic platform allowing rapid analoging, we optimized in vitro potency of a lead series into the low nanomolar regime. Additionally, we demonstrate fine-tuning of the physicochemical properties of the macrocyclic compounds, resulting in the identification of lead candidates 57/59 with a balanced profile, which are suitable for future development toward therapeutic use.
Human saliva contains numerous salivary components that are fundamental for a healthy oral environment and the oral processing of foods. To study a possible differential influence of orosensory ...stimulation and metabolic activation on salivary composition, human parotid salivary flow, pH, A280, and α-amylase activity were measured before, during and after real or sham (sip-and-spit) sucrose intakes. Variations in these salivary characteristics were related to perceived satiety. Sucrose, as either real or sham intake, increased salivary flow and pH and decreased A280 before returning to pre-intake levels. Increased salivation was dependent on the sucrose concentration and was accompanied with a higher pH and lower A280. After sucrose ingestion, the salivary α-amylase activity increased, while no increase occurred after sham sucrose intake. Similarly, rated satiety increased with real but not by sham sucrose intake. This indicated that salivary α-amylase is associated with perceived satiety controlled by caloric perception downstream of the oral cavity.
A mild and efficient method for the conversion of C-terminal esters of side-chain protected peptides into an amide function via enzyme-catalysed ammonolysis in organic media with low water content is ...described. Subtilisin A, the alkaline serine protease from
Bacillus licheniformis, was used as biocatalyst and ammonium carbamate as source of ammonia. Response surface methodology (RSM) and central composite design were employed to estimate the effects of reaction parameters such as molar ratio of ammonia source to peptide methyl ester (2:1–10:1), composition of the solvent system (Bu
t
OH/DMF, % v/v, 70:30–95:5) and water concentration (0.2–0.8%) at different temperatures (30–50
°C) for the preparation of Z-Ala-Phe-NH
2 starting from Z-Ala-Phe-OMe. Optimum reaction conditions for maximum amide yield and minimum secondary hydrolysis were determined from cross-section analysis: temperature 30
°C, solvent composition Bu
t
OH/DMF 82.5:17.5 (v/v) containing 0.2% water (v/v) and molar ratio of ammonia source to peptide methyl ester of 10:1. The maximum yield of Z-Ala-Phe-NH
2 was 87% after 21
h for a quantitative substrate conversion. The method proved to be generally applicable for the synthesis of C-terminal amides of dipeptides with different terminal amino acids and sequence.
