What have we known so far for fluorescence staining and quantification of microplastics: A tutorial review

Understanding the fate and toxicity of microplastics (MPs, < 5 mm plastic particles) is limited by quantification methods. This paper summarizes the methods in use and presents new ones. First, sampling and pretreatment processes of MPs, including sample collection, digestion, density separation, and quality control are reviewed. Then the promising and convenient staining procedures and quantification methods for MPs using fluorescence dyes are reviewed. The factors that influence the staining of MPs, including their physicochemical properties, are summarized to provide an optimal operation procedure. In general, the digestion step is crucial to eliminate natural organic matter (NOM) to avoid interference in quantification. Chloroform was reported to be the most appropriate solvent, and 10–20 μg/mL are recommended as optimal dye concentrations. In addition, a heating and cooling procedure is recommended to maintain the fluorescence intensity of MPs for two months. After staining, a fluorescence microscope is usually used to characterize the morphology, mass, or number of MPs, but compositional analysis cannot be determined with it. These fluorescence staining methods have been implemented to study MP abundance, transport, and toxicity and have been combined with other chemical characterization techniques, such as Fourier transform infrared spectroscopy and Raman spectroscopy. More studies are needed to focus on the synthesis of novel dyes to avoid NOM’s interference. They need to be combined with other spectroscopic techniques to characterize plastic composition and to develop image-analysis methods. The stability of stained MPs needs to be improved.

Salt-Mediated Organic Solvent Precipitation for Enhanced Recovery of Peptides Generated by Pepsin Digestion

Conventional solvent-based precipitation makes it challenging to obtain a high recovery of low mass peptides. However, we previously demonstrated that the inclusion of salt ions, specifically ZnSO4, together with high concentrations of acetone, maximizes the recovery of peptides generated from trypsin digestion. We herein generalized this protocol to the rapid (5 min) precipitation of pepsin-digested peptides recovered from acidic matrices. The precipitation protocol extended to other organic solvents (acetonitrile), with high recovery from dilute peptide samples permitting preconcentration and purification. Mass spectrometry profiling of pepsin-generated peptides demonstrated that the protocol captured peptides as small as 800 u, although with a preferential bias towards recovering larger and more hydrophobic peptides. The precipitation protocol was applied to rapidly quench, concentrate, and purify pepsin-digested samples ahead of MS. Complex mixtures of yeast and plasma proteome extracts were successfully precipitated following digestion, with over 95% of MS-identified peptides observed in the pellet fraction. The full precipitation workflow—including the digestion step—can be completed in under 10 min, with direct MS analysis of the recovered peptide pellets showing exceptional protein sequence coverage.

Processing Hundreds of SARS-CoV-2 Samples with an In-House PCR-Based Method without Robotics

The SARS-CoV-2 pandemic has required the development of multiple testing systems to monitor and control the viral infection. Here, we developed a PCR test to screen COVID-19 infections that can process up to ~180 samples per day without the requirement of robotics. For this purpose, we implemented the use of multichannel pipettes and plate magnetics for the RNA extraction step and combined the reverse transcription with the qPCR within one step. We tested the performance of two RT-qPCR kits as well as different sampling buffers and showed that samples taken in NaCl or PBS are stable and compatible with different COVID-19 testing systems. Finally, we designed a new internal control based on the human RNase P gene that does not require a DNA digestion step. Our protocol is easy to handle and reaches the sensitivity and accuracy of the standardized diagnostic protocols used in the clinic to detect COVID-19 infections.

Effects of recombinant human gastric lipase and pancreatin during in vitro pediatric gastro-intestinal digestion

The aim of this study was to implement a gastric digestion step using recombinant human gastric lipase (rHGL) in an in vitro pediatric gastro-intestinal digestion model to achieve a physiologically...

Fungal Pretreatments on Non-Sterile Solid Digestate to Enhance Methane Yield and the Sustainability of Anaerobic Digestion

Fungi can run feedstock pretreatment to improve the hydrolysis and utilization of recalcitrant lignocellulose-rich biomass during anaerobic digestion (AD). In this study, three fungal strains (Coprinopsis cinerea MUT 6385, Cyclocybe aegerita MUT 5639, Cephalotrichum stemonitis MUT 6326) were inoculated in the non-sterile solid fraction of digestate, with the aim to further (re)use it as a feedstock for AD. The application of fungal pretreatments induced changes in the plant cell wall polymers, and different profiles were observed among strains. Significant increases (p < 0.05) in the cumulative biogas and methane yields with respect to the untreated control were observed. The most effective pretreatment was carried out for 20 days with C. stemonitis, causing the highest hemicellulose, lignin, and cellulose reduction (59.3%, 9.6%, and 8.2%, respectively); the cumulative biogas and methane production showed a 182% and 214% increase, respectively, compared to the untreated control. The increase in AD yields was ascribable both to the addition of fungal biomass, which acted as an organic feedstock, and to the lignocellulose transformation due to fungal activity during pretreatments. The developed technologies have the potential to enhance the anaerobic degradability of solid digestate and untap its biogas potential for a further digestion step, thus allowing an improvement in the environmental and economic sustainability of the AD process and the better management of its by-products.

Stability of blueberry polyphenols subjected to in vitro digestion with human gastrointestinal enzymes

Blueberries (Vaccinium corymbosum, L.) are a valuable source of health-promoting bioactive compounds, particularly polyphenols. The beneficial effects of polyphenols on health depend not only on food sources but also on their stability, which can vary depending on the post-harvest processing methods, the matrix in which they are incorporated, and endogenous factors such as microbiota and digestive enzymes. The aim of this study was to investigate the influence of high-intensity ultrasound treatment on in vitro gastrointestinal stability of the total polyphenol content and antioxidant capacity of blueberries. Blueberry puree was treated with high-intensity ultrasound using different amplitudes (30, 45 and 60μm) and treatment times (3, 6 and 9 min), and a part of sample was used as a control (untreated sample). Total phenolic content (TPC) and antioxidant capacity (by DPPH and FRAP methods) were determined prior to and after two-phase in vitro digestion process (gastric and duodenal) using human gastrointestinal enzymes. The results showed a high stability of total blueberry polyphenols and antioxidant activity during simulated gastric digestion step, both in untreated and ultrasound treated samples. The use of high-intensity ultrasound had a significant effect on the stability of polyphenols and their antioxidant activity after simulated digestion in duodenum. Significant increase in polyphenol content after duodenal digestion step was determined in three ultrasound treated samples with longer treatment time (6 and 9 minutes), and only sample with significant reduction of TPC was untreated sample. Modification of the food matrix as a result of cavitation from ultrasound treatment may be the reason for higher stability of polyphenol during in vitro digestion. After in vitro digestion blueberry polyphenols still possessed antioxidant activity that may be directly related to their beneficial effects on health. The highest antioxidant activity measured with DPPH and FRAP method was determined for sample treated with maximum amplitude (60μm) and longest time (9 minutes). Determination of the stability of phenolic compounds is important as it affects their bioavailability for uptake as well as their possible beneficial effects on intestinal epithelial tissue such as reduction of oxidative stress and cancer prevention. Our results showed that ultrasound treatment can preserve the blueberry phenolics and their antioxidant characteristics after in vitro digestion.

Bioavailability and Bioactivity of Encapsulated Phenolics and Carotenoids Isolated from Red Pepper Waste

In order to deactivate the health properties of bioactive compounds, they need to withstand the effects of food processing, their potential release from the food matrix, and remain bio-accessible in the gastrointestinal tract. Bio-actives from different plants are prone to oxidative degradation, and encapsulation is an effective method in improving their stability. In the present study, red pepper waste (RPW), a by-product of vegetable processing industry, was encapsulated in whey protein using spray and freeze-drying techniques. The aim was to evaluate the effects of in vitro gastrointestinal digestion on the release and bioactivity of encapsulated bio-actives, after each digestion step. The results showed that the release of phenolics and carotenoids, as well as antioxidants, anti-hyperglycemic, and anti-inflammatory activities are influenced by pH and intestinal fluid, with pH 7.5 exhibited at higher levels. There was a rapid initial release of carotenoids from whey protein matrices, while a more gradual increase of phenolics was observed, reaching around 50% for both encapsulates first at 6 h and 37 °C, and small intestine conditions. The encapsulation of RPW demonstrated a protective effect against pH changes and enzymatic activities along digestion, and contributed to the increase in bio-accessibility in the gut. Also, the results suggest that encapsulation is an efficient method for valorization of bio-actives from RPW, with improvements in nutrition, color, and bioactive properties.

The Optimized Workflow for Sample Preparation in LC-MS/MS-Based Urine Proteomics

The sample condition is an important factor in urine proteomics with stability and accuracy. However, a general protocol of urine protein preparation in mass spectrometry analysis has not yet been established. Here, we proposed a workflow for optimized sample preparation based on methanol/chloroform (M/C) precipitation and in-solution trypsin digestion in LC-MS/MS-based urine proteomics. The urine proteins prepared by M/C precipitation showed around 80% of the protein recovery rate. The samples showed the largest number of identified proteins, which were over 1000 on average compared with other precipitation methods in LC-MS/MS-based urine proteomics. For further improvement of the workflow, the essences were arranged in protein dissolving and trypsin digestion step for the extraction of urine proteins. Addition of Ethylene diamine tetraacetic acid (EDTA) dramatically enhanced the dissolution of protein and promoted the trypsin activity in the digestion step because the treatment increased the number of identified proteins with less missed cleavage sites. Eventually, an optimized workflow was established by a well-organized strategy for daily use in the LC-MS/MS-based urine proteomics. The workflow will be of great help for several aims based on urine proteomics approaches, such as diagnosis and biomarker discovery.