A streamlined workflow for a fast and cost-effective count of tyndallized probiotics using flow cytometry.

The use of dead probiotics and their cellular metabolites seems to exhibit immunomodulatory and anti-inflammatory properties, providing protection against pathogens. These inanimate microorganisms, often referred to as tyndallized or heat-killed bacteria, are a new class of probiotics employed in clinical practice. Safety concerns regarding the extensive use of live microbial cells have increased interest in inactivated bacteria, as they could eliminate shelf-life problems and reduce the risks of microbial translocation and infection. Culture-dependent methods are not suitable for the quality assessment of these products, and alternative methods are needed for their quantification. To date, bacterial counting chambers and microscopy have been used for tyndallized bacteria enumeration, but no alternative validated methods are now available for commercial release. The aim of the present study is to design a new method for the qualitative and quantitative determination of tyndallized bacterial cells using flow cytometric technology. Using a live/dead viability assay based on two nucleic acid stains, thiazole orange (TO) and propidium iodide (PI), we optimized a workflow to evaluate bacterial viability beyond the reproduction capacity that provides information about the structural properties and metabolic activities of probiotics on FACSVerse without using beads as a reference. The data obtained in this study represent the first analytical application that works effectively both on viable and non-viable cells. The results provided consistent evidence, and different samples were analyzed using the same staining protocol and acquisition settings. No significant discrepancies were highlighted between the declared specification of commercial strain and the analytical data obtained. For the first time, flow cytometry was used for counting tyndallized bacterial cells as a quality control assessment in probiotic production. This aspect becomes important if applied to medical devices where we cannot boast metabolic but only mechanical activities.

Verification of a Rapid Analytical Method for the Qualitative Detection of Listeria spp. and Listeria monocytogenes by a Real-Time PCR Assay according to EN UNI ISO 16140-3:2021.

Microbial contamination and foodborne infections are a significant global public health concern. For this reason, the detection, monitoring, and characterization of pathogens represent a significant challenge in quality control settings. Standard approaches, such as culture methods and biochemical tests, are known to be very time-consuming and intensive. Conversely, molecular technologies based on the genomic identification of bacteria are quick and low-cost. Listeria monocytogenes is an opportunistic pathogen and a major concern especially in food industries. It is important to understand and implement multiple quality control measures to control Listeria infection risk and prevent the contamination of products. Standardized detection and confirmation tests such as the API Listeria test, MALDI-TOF MS, and PCR analysis are available. The aim of our work is to provide a specific molecular method, designed according to the EN UNI ISO 16140-3:2021, for the specific detection, monitoring, and characterization of Listeria spp. and Listeria monocytogenes contamination. The verification of this new rapid approach by real-time PCR (qPCR) overcomes the limitations of culture-based techniques, meeting all the verification criteria required by ISO guidelines, including implementation and item confirmation. This system offers a powerful approach to the real-time assessment of food safety, useful for industry self-monitoring and regulatory inspection.

An Integrated Analytical Approach for the Characterization of Probiotic Strains in Food Supplements.

Research surrounding health benefits from probiotics is becoming popular because of the increasing demand for safer products with protective and therapeutic effects. Proven benefits are species- or genus-specific; however, no certified assays are available for their characterization and quantification at the strain level in the food supplement industry. The objective of this study was to develop a strain-specific Real-time quantitative polymerase chain reaction (RT-qPCR)-based method to be implemented in routine tests for the identification and quantification of Bifidobacterium longum, Bifidobacterium animalis spp. lactis, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus casei, Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus helveticus, starting from a powder mixture of food supplements. The method optimization was carried out in combination with flow cytometry to compare results between the two strategies and implement the analytical workflow with the information also regarding cell viability. These assays were validated in accordance with the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) criteria using the plate count enumeration as the gold standard reference. Briefly, probiotic DNAs were extracted from two powder food supplements. Strain-specific primers targeting unique sequence regions of 16S RNA were identified and amplified by RT-qPCR. Primers were tested for specificity, sensitivity, and efficiency. Both RT-qPCR and flow-cytometry methods described in our work for the quantification and identification of Lactobacillus and Bifidobacterium strains were specific, sensitive, and precise, showing better performances with respect to the morphological colony identification. This work demonstrated that RT-qPCR can be implemented in the quality control workflow of commercial probiotic products giving more standardized and effective results regarding species discrimination.

Intra-Laboratory Validation of Alpha-Galactosidase Activity Measurement in Dietary Supplements.

INTRODUCTION: Alpha-galactosidase (α-Gal) is an enzyme responsible for the hydrolyzation of glycolipids and glycoprotein commonly found in dietary sources. More than 20% of the general population suffers from abdominal pain or discomfort caused by intestinal gas and by indigested or partially digested food residuals. Therefore, α-Gal is used in dietary supplements to reduce intestinal gases and help complex food digestion. Marketed enzyme-containing dietary supplements must be produced in accordance with the Food and Drug Administration (FDA) regulations for Current Good Manufacturing Practice (cGMPs). AIM: in this work we illustrated the process used to develop and validate a spectrophotometric enzymatic assay for α-Gal activity quantification in dietary supplements. METHODS: The validation workflow included an initial statistical-phase optimization of materials, reagents, and conditions, and subsequently a comparative study with another fluorimetric assay. A final validation of method performance in terms of specificity, linearity, accuracy, intermediate-precision repeatability, and system precision was then executed. RESULTS AND CONCLUSIONS: The proven method achieved good performance in the quantitative determination of α-Gal activity in commercial food supplements in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals (ICH) guidelines and is suitable as a rapid in-house quality control test.

Rationale and evidence for the incorporation of heparin into the diclofenac epolamine medicated plaster.

OBJECTIVE: The nonsteroidal anti-inflammatory drug (NSAID) diclofenac epolamine (DHEP) formulated as a topical patch has demonstrated efficacy and safety in the localized treatment of acute pain from minor strains, sprains and contusions, and for epicondylitis and knee osteoarthritis. The glycosaminoglycan heparin enhances the activity of topical NSAIDs formulated as a medicated plaster, even in the absence of any significant release of heparin. Therefore, DHEP plus, a new formulation of the DHEP medicated plaster containing a small amount of heparin sodium as excipient, has been developed. METHODS: We reviewed the pivotal and supportive studies of the clinical development program of the new patch and evaluated the role of heparin as an enhancer in the treatment of localized pain/inflammation of musculoskeletal structures, associated with post-traumatic and/or rheumatic conditions. RESULTS: The data was consistent with the concept that heparin increased the clinical activity of the DHEP plus medicated plaster versus the reference DHEP medicated plaster through improved bioavailability due to enhanced movement of diclofenac from the plaster. Both DHEP formulations have the same dissolution profile, indicating that heparin does not change the physical and chemical characteristics of the plaster. Permeation testing showed that heparin is not released from the DHEP plus medicated plaster. Efficacy studies showed that the DHEP plus medicated plaster was significantly more effective in reducing pain than the reference marketed DHEP medicated plaster. CONCLUSIONS: The benefit/risk assessment of DHEP plus 180 mg medicated plaster is favorable, with a safety profile equal to placebo and improved efficacy over the reference marketed DHEP medicated plaster.

Validated high-performance anion-exchange chromatography with pulsed amperometric detection method for the determination of residual keratan sulfate and other glucosamine impurities in sodium chondroitin sulfate.

An efficient and sensitive analytical method based on high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was devised for the determination of glucosamine (GlcN) in sodium chondroitin sulfate (CS). Glucosamine (GlcN) is intended as marker of residual keratan sulfate (KS) and other impurities generating glucosamine by acidic hydrolyzation. The latter brings CS and KS to their respective monomers. Since GlcN is present only in KS we developed a method that separates GlcN from GalN, the principal hydrolytic product of CS, and then we validated it in order to quantify GlcN. Method validation was performed by spiking CS raw material with known amounts of KS. Detection limit was 0.5% of KS in CS (corresponding to 0.1µg/ml), and the linear range was 0.5-5% of KS in CS (corresponding to 0.1-1µg/ml). The optimized analysis was carried out on an ICS-5000 system (Dionex, Sunnyvale, CA, USA) equipped with a Dionex Amino Trap guard column (3mm×30mm), Dionex CarboPac-PA20 (3mm×30mm) and a Dionex CarboPac-PA20 analytical column (3mm×150mm) using gradient elution at a 0.5ml/min flow rate. Regression equations revealed good linear relationship (R2=0.99, n=5) within the test ranges. Quality parameters, including precision and accuracy, were fully validated and found to be satisfactory. The fully validated HPAEC-PAD method was readily applied for the quantification of residual KS in CS in several raw materials and USP/EP reference substance. Results confirmed that the HPAEC-PAD method is more specific than the electrophoretic method for related substance reported in EP and provides sensitive determination of KS in acid-hydrolyzed CS samples, enabling the quantitation of KS and other impurities (generating glucosamine) in CS.