Cell detachment from culture surfaces is an essential routine step that can influence cell viability, surface marker expression, membrane integrity, and downstream experimental outcomes. This mini review summarizes the responses of human and selected animal-derived stem cell cultures, particularly MSC-based systems, to trypsin, recombinant trypsin, Accutase, EDTA, and mechanical scraping. It also discusses general approaches for cell-to-cell detachment. Trypsin enables rapid, reproducible, and cost-effective cell recovery; however, its broad proteolytic activity may cleave sensitive membrane proteins and should therefore be used with carefully controlled exposure when phenotypic analysis, migration assays, or functional readouts are required. Recombinant trypsin provides a defined animal-origin-free alternative that may support standardized and xeno-free workflows, although validation in animal stem cell systems remains limited. Accutase offers a gentler enzymatic detachment approach that may improve viability and preserve many membrane markers, but marker-specific and cell-type-specific effects can still occur. EDTA avoids direct proteolysis and may preserve selected antigens, although incomplete detachment may occur in highly adherent cultures. Mechanical scraping avoids enzymatic exposure but may induce shear stress, reduce cell viability, compromise membrane integrity, and alter paracrine signaling or secretome activity. No single method works best for all cases, and this review does not propose a universal ranking. Selection should depend on cell type, species origin, tissue source, culture conditions, and intended downstream applications. More comparative studies, especially in animal-derived primary stem cells, are needed to improve reproducibility and standardisation of detachment protocols.
Introduction: Enterococcus spp. are Gram-positive commensals of the gastrointestinal tract and comprise native plasmids that are commonly used for the development of shuttle expression vectors for the heterologous expression of various proteins. Methods: The Enterococcus faecium native plasmid pRGK7d was molecularly characterized using various methods involving sequence analysis and promoter motif prediction, and was subsequently utilized for the development of an E. coli-Enterococcus shuttle expression vector through PCR-based molecular cloning methods and various transformation procedures for heterologous protein expression. Results: The shuttle plasmid vector previously constructed from the E. faecium plasmid pRGK7d was reduced in size by cloning only the Rep protein, ORFA (Open Reading Frame A). Protein BLAST analysis of ORFA confirmed it to be a replication protein, showing 100% homology with the replication initiation factor of Enterococcus faecium. Sequence analysis of ORFA using Ori-Finder revealed the presence of two DnaA box-like sequences, and promoter motif prediction of ORFA using BPROM revealed the presence of -10 box- and -35 box-like sequences, suggesting a putative promoter upstream of the Rep protein. To construct an expression cassette in the shuttle vector, downstream of the Rep protein, the Ery promoter was PCR-cloned from the plasmid pTRKH3. Downstream of the Ery promoter, a reporter gene, GFP, was first PCR-cloned and subsequently replaced with an MCS (Multiple Cloning Site). The construct was thoroughly sequenced and successfully transformed into both E. coli and the host E. faecium strain RGK7. The 588 bp codonoptimized h-IL10 (human Interleukin-10) sequence for Enterococcus, including the signal peptide from the Enterocin P protein of E. faecium, was de novo synthesized and subsequently PCR-cloned into the MCS for expression in E. faecium and immunoblotted with an anti-h-IL10 antibody. The SignalP 4.1 server predicted the same cleavage site between amino acids 27 and 28 in both the chimeric EntPSP-hIL10 and the precursor Enterocin P protein sequence. Discussion: These shuttle expression vectors can be used for various purposes, including the expression of heterologous proteins, and further studies may reveal their potential as vehicles for the delivery of significant prophylactic and therapeutic molecules. Conclusion: An E. coli-Enterococcus shuttle expression vector containing an Ery promoter and an MCS was developed from a native plasmid of E. faecium and was utilized for the cloning and heterologous expression of the anti-inflammatory cytokine IL-10. The construct containing the signal peptide and codon-optimized h-IL10 was successfully transformed into both E. coli and the host E. faecium strain.
Introduction/Objective: Soy-based beverages are recognised as a nutritious alternative to dairy products. High in protein, they are suitable for individuals with lactose intolerance, milk allergies, or a vegetarian diet. However, they lack omega-3 fatty acids, and probiotics often display instability when incorporated into these matrices. This study formulated and characterised a functional soy-based beverage enriched with omega-3 fatty acids from fish by-products, and containing the probiotic Lactiplantibacillus plantarum. Methods: Fish oil was incorporated via emulsions or calcium-alginate beads (3 mm diameter, sphericity index 0.97). L. plantarum was encapsulated to enhance stability. Formulations were evaluated for proximate composition, omega-3 recovery, probiotic viability during 15 days of storage at 4 °C, simulated gastrointestinal tolerance (pH 2.0–4.0 and 0.1–0.6% bile salts), and microbiological quality. Results: Formulated beverages exhibited a high protein content (4.87–5.59 g/100 g). Calciumalginate encapsulation achieved an efficiency of 92.56%, yielding omega-3 recovery rates >95% in fresh beverages. A single 240 mL serving provides 420–438 mg of EPA+DHA (about 85% of the recommended daily intake). Encapsulated L. plantarum maintained viability above the therapeutic threshold throughout 15 days of refrigerated storage, decreasing from an initial 7.7 ± 0.1 log CFU/mL to 6.2 ± 0.1 log CFU/mL on day 15. Discussion: Protein density exceeded typical commercial plant-based beverages (1–3 g/100 g) and bovine milk averages. The probiotic strain showed survival at pH ≥2.5 and tolerance across all bile concentrations (up to 0.6%). Low oxidation levels in the refined oil (TOTOX 17.72) prevented offflavors, and all formulations complied with microbiological safety criteria after 15 days. Conclusion: Combining microencapsulation strategies allows soy-based beverages to serve as an effective dual-delivery vector for omega-3 fatty acids and viable probiotics, offering a sustainable, high-protein functional food.
Introduction/Objective: Medicinal plants constitute a major reservoir of biologically active compounds with applications in medicine, nutrition, and biotechnology. Among them, the family Asteraceae— comprising nearly 23,000 species worldwide—represents one of the most diverse botanical groups with significant ethnopharmacological value. Numerous species are traditionally used for therapeutic purposes and increasingly exploited in the food, cosmetic, and pharmaceutical industries. Patent systems play a central role in documenting technological progress and protecting intellectual property in this domain. The present study aims to analyze the evolution and technological dynamics of innovations related to medicinal plants of the Asteraceae family through a comprehensive patent landscape assessment spanning 2000–2025. Methods: A systematic patent landscape analysis was conducted on November 1, 2025, using The Lens database (https://www.lens.org). Targeted keyword strategies related to the Asteraceae family and selected medicinal species were used to retrieve patent records, with searches restricted to the title, abstract, and claims fields. Publication date and document type filters were applied to improve search precision and ensure reproducibility. The final dataset comprised 130,605 patent documents (103,676 simple patent families), including patent applications, granted patents, plant patents, amended applications, and patents of addition, published between January 1, 2000 and November 1, 2025. The dataset was analyzed by International Patent Classification (IPC) codes, geographical jurisdictions, leading applicants, application domains, and temporal trends in patent activity. Results: Patent activity demonstrated a steady growth from the early 2000s, reaching a maximum in 2016 with 11,499 documents, followed by a gradual decline after 2020. Therefore, the observed decrease during 2023-2025 is likely attributable, at least in part, to publication lag rather than to a true reduction in research and development or patenting efforts. Geographically, China dominated the innovation landscape, accounting for 61,494 patent documents, highlighting its strong investment in plant-based biotechnology. The most frequent IPC classifications were associated with genetic engineering, cosmetic formulations, and nutraceutical compositions. The analyzed patents reveal extensive technological diversity. Agricultural innovations include genetic improvement of sunflower cultivars for early flowering, development of disease- resistant lettuce varieties, and the development of high-performance ornamental cultivars, such as Zinnia elegans. Concurrently, phytopharmaceutical developments involve bioactive extracts from species, such as Cynara scolymus with hypoglycemic activity, Smallanthus sonchifolius for diabetes management, Stevia rebaudiana for noncaloric sweeteners, Tanacetum vulgare in anti-aging formulations, and Echinacea purpurea for immunostimulatory preparations. discussion: The analyzed patents showcase diverse innovations across horticulture and phytopharmacy. Agricultural developments range from the genetic improvement of sunflowers for early flowering to the creation of disease-resistant lettuce and high-performance Zinnia elegans varieties. Simultaneously, significant advances are evident in botanical extracts, including artichoke with hypoglycemic activity, Smallanthus sonchifolius for diabetes management, Stevia rebaudiana-based sweeteners, Tanacetum vulgare for anti-aging, and standardized Echinacea purpurea for immunostimulation. These results highlight the multifunctionality of Asteraceae species and their strategic role in biotechnology, cosmetics, and functional foods. Discussion: The patent landscape highlights the multifunctional potential of Asteraceae species across several industrial sectors. Innovations span agricultural biotechnology, functional foods, phytotherapy, and cosmetic formulations. The coexistence of traditional botanical knowledge with modern biotechnological approaches demonstrates a strong convergence between ethnopharmacology and industrial innovation. However, challenges remain regarding regulatory approval, sustainable resource management, and efficient technology transfer from research institutions to commercial applications. Conclusion: Patent analysis provides valuable insights into technological trajectories and innovation dynamics within plant-based industries. The results confirm the strategic importance of Asteraceae species as versatile bioresources for biotechnology, nutraceuticals, and pharmaceutical development. Despite strong global innovation momentum, further efforts are required to ensure sustainable exploitation, regulatory compliance, and effective valorization of plant biodiversity. Consequently, patent landscape studies represent a powerful analytical tool for guiding research priorities and promoting responsible innovation in medicinal plant research.
Callus culture is a technique that is used in plant biotechnology. For the controlled production of important secondary metabolites with medicinal value, the callus culture technique is widely utilized. Callus is an unorganized cellular mass of totipotent plant cells with the ability of biosynthesizing bioactive secondary metabolites, such as alkaloids, phenolics, flavonoids, terpenoids, and steroids. This review emphasizes the historical developments in callus culture and recent advances in optimizing culture conditions, plant growth regulators, and elicitors to enhance metabolite accumulation. The callus induction by phytohormones, followed by stimulation of secondary metabolite production by biotic and abiotic elicitors are extensively discussed in this review. Further, the molecular mechanisms and insights underlying elicitor perception and signal transduction, including Ca²⁺ fluxes, ROS bursts, MAPK activation, and transcriptional regulation of genes involved in biosynthesis, are emphasized. This review also focuses on applications, limitations, and the possibilities for scaling up metabolite production using the callus culture technique. In conclusion, this review highlights that callus culture paves the way for a sustainable platform for the production of medicinally important secondary metabolites.
