Loop-mediated isothermal amplification (LAMP) assay coupled with gold nanoparticles for colorimetric detection of Trichoderma spp. in Agaricus bisporus cultivation substrates.

One of the significant challenges in organic cultivation of edible mushrooms is the control of invasive Trichoderma species that can hinder the mushroom production and lead to economic losses. Here, we present a novel loop-mediated isothermal amplification (LAMP) assay coupled with gold nanoparticles (AuNPs) for rapid colorimetric detection of Trichoderma spp. The specificity of LAMP primers designed on the tef1 gene was validated in silico and through gel-electrophoresis on Trichoderma harzianum and non-target soil-borne fungal and bacterial strains. LAMP amplification of genomic DNA templates was performed at 65 °C for only 30 min. The results were rapidly visualized in a microplate format within less than 5 min. The assay is based on salt-induced aggregation of AuNPs that is being prevented by the amplicons produced in case of positive LAMP reaction. As the solution color changes from red to violet upon nanoparticle aggregation can be observed with the naked eye, the developed LAMP-AuNPs assay can be easily operated to provide a simple initial screening for the rapid detection of Trichoderma in button mushroom cultivation substrate.

Low-cost goldleaf electrode as a platform for Escherichia coli immunodetection.

Gold electrodes are one of most prevalent substrates in electrochemical biosensors because they can be easily and highly efficiently functionalized with thiolated biomolecules. However, conventional methods to fabricate gold electrodes are costly, time-consuming and require onerous equipment. Here, an affordable method for rapid fabrication of an electrochemical immunosensor for Escherichia coli detection is presented. The gold electrode was generated using 24-karat gold leaves and lowcost polyvinyl chloride adhesive sheets covered with an insulating PTFE layer. The goldleaf electrode (GLE) was patterned using laser ablation and characterized by cyclic voltammetry, electrochemical impedance spectroscopy, scanning electronic microscopy, contact angle and 3D profiling. The GLEs were modified by a self-assembled mercaptopropionic monolayer, followed by surface activation to allow binding of the specific anti-E. coli antibody via carbodiimide linking. The biosensor showed a detection limit of 2 CFU/mL and a linear dynamic range of 10-107 CFU/mL for E. coli cells. No false positive signals were obtained from control bacteria. The obtained results demonstrated suitability of GLE for use in biosensors with high reliability and reproducibility. It is foreseeable that our work will inspire design of point-of-need biosensors broadly applicable in low-resource settings.

Mammalian Cell-Growth Monitoring Based on an Impedimetric Sensor and Image Processing within a Microfluidic Platform.

In recent years, advancements in microfluidic and sensor technologies have led to the development of new methods for monitoring cell growth both in macro- and micro-systems. In this paper, a microfluidic (MF) platform with a microbioreactor and integrated impedimetric sensor is proposed for cell growth monitoring during the cell cultivation process in a scaled-down simulator. The impedimetric sensor with an interdigitated electrode (IDE) design was realized with inkjet printing and integrated into the custom-made MF platform, i.e., the scaled-down simulator. The proposed method, which was integrated into a simple and rapid fabrication MF system, presents an excellent candidate for the scaled-down analyses of cell growths that can be of use in, e.g., optimization of the cultivated meat bioprocess. When applied to MRC-5 cells as a model of adherent mammalian cells, the proposed sensor was able to precisely detect all phases of cell growth (the lag, exponential, stationary, and dying phases) during a 96-h cultivation period with limited available nutrients. By combining the impedimetric approach with image processing, the platform enables the real-time monitoring of biomasses and advanced control of cell growth progress in microbioreactors and scaled-down simulator systems.

Molecular Approaches for Detection of Trichoderma Green Mold Disease in Edible Mushroom Production.

Due to the evident aggressive nature of green mold and the consequently huge economic damage it causes for producers of edible mushrooms, there is an urgent need for prevention and infection control measures, which should be based on the early detection of various Trichoderma spp. as green mold causative agents. The most promising current diagnostic tools are based on molecular methods, although additional optimization for real-time, in-field detection is still required. In the first part of this review, we briefly discuss cultivation-based methods and continue with the secondary metabolite-based methods. Furthermore, we present an overview of the commonly used molecular methods for Trichoderma species/strain detection. Additionally, we also comment on the potential of genomic approaches for green mold detection. In the last part, we discuss fast screening molecular methods for the early detection of Trichoderma infestation with the potential for in-field, point-of-need (PON) application, focusing on isothermal amplification methods. Finally, current challenges and future perspectives in Trichoderma diagnostics are summarized in the conclusions.

Using Vertebrate Stem and Progenitor Cells for Cellular Agriculture, State-of-the-Art, Challenges, and Future Perspectives.

Global food systems are under significant pressure to provide enough food, particularly protein-rich foods whose demand is on the rise in times of crisis and inflation, as presently existing due to post-COVID-19 pandemic effects and ongoing conflict in Ukraine and resulting in looming food insecurity, according to FAO. Cultivated meat (CM) and cultivated seafood (CS) are protein-rich alternatives for traditional meat and fish that are obtained via cellular agriculture (CA) i.e., tissue engineering for food applications. Stem and progenitor cells are the building blocks and starting point for any CA bioprocess. This review presents CA-relevant vertebrate cell types and procedures needed for their myogenic and adipogenic differentiation since muscle and fat tissue are the primary target tissues for CM/CS production. The review also describes existing challenges, such as a need for immortalized cell lines, or physical and biochemical parameters needed for enhanced meat/fat culture efficiency and ways to address them.

Bioengineering Outlook on Cultivated Meat Production.

Cultured meat (also referred to as cultivated meat or cell-based meat)-CM-is fabricated through the process of cellular agriculture (CA), which entails application of bioengineering, i.e., tissue engineering (TE) principles to the production of food. The main TE principles include usage of cells, grown in a controlled environment provided by bioreactors and cultivation media supplemented with growth factors and other needed nutrients and signaling molecules, and seeded onto the immobilization elements-microcarriers and scaffolds that provide the adhesion surfaces necessary for anchor-dependent cells and offer 3D organization for multiple cell types. Theoretically, many solutions from regenerative medicine and biomedical engineering can be applied in CM-TE, i.e., CA. However, in practice, there are a number of specificities regarding fabrication of a CM product that needs to fulfill not only the majority of functional criteria of muscle and fat TE, but also has to possess the sensory and nutritional qualities of a traditional food component, i.e., the meat it aims to replace. This is the reason that bioengineering aimed at CM production needs to be regarded as a specific scientific discipline of a multidisciplinary nature, integrating principles from biomedical engineering as well as from food manufacturing, design and development, i.e., food engineering. An important requirement is also the need to use as little as possible of animal-derived components in the whole CM bioprocess. In this review, we aim to present the current knowledge on different bioengineering aspects, pertinent to different current scientific disciplines but all relevant for CM engineering, relevant for muscle TE, including different cell sources, bioreactor types, media requirements, bioprocess monitoring and kinetics and their modifications for use in CA, all in view of their potential for efficient CM bioprocess scale-up. We believe such a review will offer a good overview of different bioengineering strategies for CM production and will be useful to a range of interested stakeholders, from students just entering the CA field to experienced researchers looking for the latest innovations in the field.

Coprinus comatus filtrate extract, a novel neuroprotective agent of natural origin.

In vitro acetylholinesterase (AChE) inhibitory activity of an autochthonous sample of the mushroom Coprinus comatus (encompassing fruiting body FB, mycelia M and filtrate F from the submerged cultivation) was the subject of this study. C. comatus F extract exhibited rather potent anti-AChE activity (73.0 ± 1.5%) at in liquid conditions, comparable to those of the conventional drug donepezil (80.6 ± 1.4%). Also, the same extract exhibited high anti-AChE activity (1 µg) in solid. While its FTIR spectrum indicated the presence of phenolic compounds, quercetin (28.1 µg g-1 d.w.) was found to affect the observed bioactivity (59.8 ± 0.9%). This is the first report of profound anti-AChE activity of any C. comatus extract, a medicinal mushroom that has been successfully cultivated in P.R. China, due to the demanding needs of food industry.

Anti-hydroxyl radical activity, redox potential and anti-AChE activity of Amanita strobiliformis polysaccharide extract.

This study outlines antioxidant and anti-AChE activities of the polysaccharide (PSH) extract from the mushroom species Amanita strobiliformis. Both the presence of α and ß glucans within the aforementioned extract was recorded. PSH extract displayed a profound scavenging activity of OH radicals (IC50 value, 11.86 ± 0.59 µg/mL) and high potential for reduction of Fe3+ ions (174.11 ± 8.70 mg eq. AA/g d.w.) being almost 48- and 5-fold more effective than mannitol and butylated hydroxytoluene used as a positive control, respectively. Compared with galanthamine (0.001 µg), the same extract exhibited a moderate anti-AChE activity (10 µg) in solid. Since purified PSH extract exhibited higher bioactivity (IC50 value 7.27 ± 0.31 µg/mL, 197.68 ± 9.47 mg eq. AA/g d.w. and 0.1 µg, respectively), it can be predominantly ascribed to the polysaccharide compounds. A. strobiliformis PSH extract may be considered as a promising resource of potent bioactive polysaccharides of natural origin successfully addressing both oxidative stress and lack of acetylcholine.

Trametes versicolor ethanol extract, a promising candidate for health-promoting food supplement.

This study aimed to estimate antiradical, antioxidant (AO) and cytotoxic activities of the fungus Trametes versicolor ethanol fruiting body extract. The extract was found to effectively scavenge both O2•- and NO• (29.62 and 52.48 µg/mL, respectively). It also showed a good AO activity in the polarographic HPMC assay (950%/mL). p-Hydroxybenzoic acid may be one of the responsible compounds for the afore-mentioned activities. The same extract also exhibited a concentration-dependent cytotoxicity against MCF-7 and HepG2 tumour cell lines reaching IC50 values of 123.51 and 134.29 µg/mL, respectively with no cytotoxic activity against normal MRC-5 cells. Gentisic, syringic and protocatechuic acids may be among the bioactive principles for the observed cytotoxicity. Taken all together, T. versicolor ethanol extract can be considered as a promising candidate for development of health promoting food supplement.

A comparative overview of antioxidative properties and phenolic profiles of different fungal origins: fruiting bodies and submerged cultures of Coprinus comatus and Coprinellus truncorum.

Bioactive properties of fungi considerably differ between the fruiting body (FB) and the submerged culture as regards mycelia (M) and the fermentation broth (F). Antioxidant properties of hot-water extracts obtained from three different fungal origins: FB, M and F of two autochthonous fungal species (Northern Serbia), Coprinus comatus and Coprinellus truncorum were investigated. Free radical scavenging capacity (RSC) was evaluated in vitro by the DPPH assay and reducing power ability (FRAP assay). Considering possible bioactive properties of different compounds present in fungal extracts, the content of total proteins (TP), phenols (TC) and flavonoids (TF) were investigated colorimetrically. The chemical characterisation of the examined extracts was evaluated using the HPLC-MS/MS method. C. comatus showed the strongest RSC activity; more precisely, fermentation broth extract (FCc) on DPPH radicals (IC50 = 5.06 µg mL-1) and fruiting body extract (FBCc) for the FRAP assay (42.86 mg ascorbic acid equivalents (AAE)/g). Submerged M extract of both species showed the highest TC (MCc 81.95 mg gallic acid eq (GAE)/g d.w.; MCt 81.64 mg GAE/g d.w.), while FB extracts contained the highest content of TP. Comparing LC-MS phenolic profiles between species-interspecifically and among different fungal origins-intraspecifically (fruiting bodies and submerged cultures), high variations were noticed. In submerged M or F extracts of C. comatus, vanillic, gallic, gentisic and cinnamic acids were detected, as opposed to FB. Considering that diverse phenolic profiles of detected antioxidant compounds were obtained by submerged cultivation, this type of cultivation is promising for the production of antioxidant substances.

The lignicolous fungus Trametes versicolor (L.) Lloyd (1920): a promising natural source of antiradical and AChE inhibitory agents.

This study aimed to determine antiradical (DPPH• and •OH) and acetylcholinesterase (AChE) inhibitory activities along with chemical composition of autochtonous fungal species Trametes versicolor (Serbia). A total of 38 phenolic compounds with notable presence of phenolic acids were identified using HPLC/MS-MS. Its water extract exhibited the highest antiradical activity against •OH (3.21 µg/mL), among the rest due to the presence of gallic, p-coumaric and caffeic acids. At the concentration of 100 µg/mL, the same extract displayed a profound AChE inhibitory activity (60.53%) in liquid, compared to donepezil (89.05%), a drug in clinical practice used as positive control. The flavonoids baicalein and quercetin may be responsible compounds for the AChE inhibitory activity observed. These findings have demonstrated considerable potential of T. versicolor water extract as a natural source of antioxidant(s) and/or AChE inhibitor(s) to be eventually used as drug-like compounds or food supplements in the treatment of Alzheimer's disease.

Antimicrobial Potential of Rosmarinus officinalis Commercial Essential Oil in the Treatment of Vaginal Infections in Pregnant Women.

Treating inflammatory conditions .such as vaginosis, vaginitis, and vulvovaginitis in pregnancy is- a special problem due to limitations. of available drugs. However, possible treatment options can be found also in naturally originated products, such as essential oils (EOs) of different plants. The aim of this study was to evaluate in -vitro antimicrobial and toxic activities of the commercial EO of Rosmiarinus officinalis L. (Lamiaceae) against five Gram-positive and Gram-negative bacterial strains and two Candida strains obtained from pregnant women with Vaginal infection. Gas chromatography-mass spectrometry of the tested EO revealed oxygenated monoterpenes to be the major ingredients, while microdilution assay showed the highest activity on Staphylococcus aureus II strain at 6.2 mg/mL. After 24 hours, toxicity was determined at 19.4 mg/mL on Artemia salina nauplii. The obtained results show this EO to be a promising alternative therapy for vaginal infections, although further toxicity and safety research is required.