Abelmoschus esculentus L. Moench (okra) is a commonly consumed vegetable that consists of the seeds and peel component which are rich in polyphenolic compounds. The aim of this study is to utilize pressurized hot water extraction (PHWE) for the extraction of bioactive phytochemicals from different parts of okra. A single step PHWE was performed at various temperatures (60 °C, 80 °C, 100 °C and 120 °C) to determine which extraction temperature exhibits the optimum phytochemical profile, antioxidant and antidiabetic activities. The optimum temperature for PHWE extraction was determined at 80 °C and the biological activities of the different parts of okra (Inner Skin, Outer Skin and Seeds) were characterized using antioxidant (DPPH and ABTS), α-glucosidase and vasoprotective assays. Using PHWE, the different parts of okra displayed distinct phytochemical profiles, which consist of primarily polyphenolic compounds. The okra Seeds were shown to have the most antioxidant capacity and antidiabetic effects compared to other okra parts, likely to be attributed to their higher levels of polyphenolic compounds. Similarly, okra Seeds also reduced vascular inflammation by downregulating TNFα-stimulated VCAM-1 and SELE expression. Furthermore, metabolite profiling by LC/MS also provided evidence of the cytoprotective effect of okra Seeds in endothelial cells. Therefore, the use of PHWE may be an alternative approach for the environmentally friendly extraction and evaluation of plant extracts for functional food applications.

The broadly conserved cyclic di-AMP (c-di-AMP) is a conditionally essential bacterial second messenger. The pool of c-di-AMP is fine-tuned through diadenylate cyclase and phosphodiesterase activities, and direct binding of c-di-AMP to proteins and riboswitches allows the regulation of a broad spectrum of cellular processes. c-di-AMP has a significant impact on intrinsic β-lactam antibiotic resistance in Gram-positive bacteria; however, the reason for this is currently unclear. In this work, genetic studies revealed that suppressor mutations that decrease the activity of the potassium (K+) importer KupB or the glutamine importer GlnPQ restore cefuroxime (CEF) resistance in diadenylate cyclase (cdaA) mutants of Lactococcus lactis. Metabolite analyses showed that glutamine is imported by GlnPQ and then rapidly converted to glutamate, and GlnPQ mutations or c-di-AMP negatively affects the pools of the most abundant free amino acids (glutamate and aspartate) during growth. In a high-c-di-AMP mutant, GlnPQ activity could be increased by raising the internal K+ level through the overexpression of a c-di-AMP-insensitive KupB variant. These results demonstrate that c-di-AMP reduces GlnPQ activity and, therefore, the level of the major free anions in L. lactis through its inhibition of K+ import. Excessive ion accumulation in cdaA mutants results in greater spontaneous cell lysis under hypotonic conditions, while CEF-resistant suppressors exhibit reduced cell lysis and lower osmoresistance. This work demonstrates that the overaccumulation of major counter-ion osmolyte pools in c-di-AMP-defective mutants of L. lactis causes cefuroxime sensitivity. IMPORTANCE The bacterial second messenger cyclic di-AMP (c-di-AMP) is a global regulator of potassium homeostasis and compatible solute uptake in many Gram-positive bacteria, making it essential for osmoregulation. The role that c-di-AMP plays in β-lactam resistance, however, is unclear despite being first identified a decade ago. Here, we demonstrate that the overaccumulation of potassium or free amino acids leads to cefuroxime sensitivity in Lactococcus lactis mutants partially defective in c-di-AMP synthesis. It was shown that c-di-AMP negatively affects the levels of the most abundant free amino acids (glutamate and aspartate) in L. lactis. Regulation of these major free anions was found to occur via the glutamine transporter GlnPQ, whose activity increased in response to intracellular potassium levels, which are under c-di-AMP control. Evidence is also presented showing that they are major osmolytes that enhance osmoresistance and cell lysis. The regulatory reach of c-di-AMP can be extended to include the main free anions in bacteria.

Orange peel waste (OPW) is known to contain an abundant amount of polyphenols compounds such as flavonoids, well-reported for their antioxidant and anti-inflammatory properties. While OPW is generally regarded as a food waste, the opportunity to extract bioactive compounds from these “wastes” arises due to their abundance, allowing the investigation of their potential effects on endothelial cells. Hence, this study aims to use a green extraction method and pressurized hot water extraction (PHWE) to extract bioactive compounds from OPW. Liquid chromatography with UV detection (LC/UV) and liquid chromatography mass spectrometry (LC/MS) were subsequently used to identify the bioactive compounds present. Through the optimization of the extraction temperature for PHWE, our results demonstrated that extraction temperatures of 60 °C and 80 °C yield distinct bioactive compounds and resulted in better antioxidant capacity compared to other extraction temperatures or organic solvent extraction. Despite having similar antioxidant capacity, their effects on endothelial cells were distinct. Specifically, treatment of endothelial cells with 60 °C OPW extracts inhibited TNFα-induced vascular inflammation and endothelial dysfunction in vitro, suggesting that OPW possess vasoprotective effects likely mediated by anti-inflammatory effects.

Orange peels are often discarded as food waste despite being a nutritious source of vitamins and antioxidants. These orange peel wastes (OPW) are produced in millions of tons globally every year; discarding them results in detrimental environmental and economical impacts. This paper discusses the application of 3D printing technology to effectively upcycle the OPW into edible, healthy snacks for consumption. We aimed to develop a method to enable OPW to formulate 3D-printable inks for direct ink writing (DIW). Using DIW 3D printing, we successfully created edible constructs of rheologically modified inks containing OPW. The formulated ink possessed an initial viscosity of 22.5 kPa.s, a yield stress of 377 Pa, and a storage modulus of 44.24 kPa. To validate the method, we conducted a biochemical analysis of the OPW at each stage of the fabrication process. This study suggested that our ink formulation and 3D printing process did not affect the content of bioflavonoids and antioxidants of the OPW. The cell viability test using human dermal microvascular endothelium (HMEC-1) suggested that the OPW did not exhibit cytotoxicity throughout the entire process of the ink manipulation. Overall, this study has highlighted a potential scenario to revalorize food waste into the food value chain using 3D printing toward more sustainable and circular food manufacturing and consumption.