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.

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.

Advanced mesoporous silica nanocarriers in cancer theranostics and gene editing applications.

Targeted nanomaterials for cancer theranostics have been the subject of an expanding volume of research studies in recent years. Mesoporous silica nanoparticles (MSNs) are particularly attractive for such applications due to possibilities to synthesize nanoparticles (NPs) of different morphologies, pore diameters and pore arrangements, large surface areas and various options for surface functionalization. Functionalization of MSNs with different organic and inorganic molecules, polymers, surface-attachment of other NPs, loading and entrapping cargo molecules with on-desire release capabilities, lead to seemingly endless prospects for designing advanced nanoconstructs exerting multiple functions, such as simultaneous cancer-targeting, imaging and therapy. Describing composition and multifunctional capabilities of these advanced nanoassemblies for targeted therapy (passive, ligand-functionalized MSNs, stimuli-responsive therapy), including one or more modalities for imaging of tumors, is the subject of this review article, along with an overview of developments within a novel and attractive research trend, comprising the use of MSNs for CRISPR/Cas9 systems delivery and gene editing in cancer. Such advanced nanconstructs exhibit high potential for applications in image-guided therapies and the development of personalized cancer treatment.

Cultivating Multidisciplinarity: Manufacturing and Sensing Challenges in Cultured Meat Production.

Meat cultivation via cellular agriculture holds great promise as a method for future food production. In theory, it is an ideal way of meat production, humane to the animals and sustainable for the environment, while keeping the same taste and nutritional values as traditional meat and having additional benefits such as controlled fat content and absence of antibiotics and hormones used in the traditional meat industry. However, in practice, there is still a number of challenges, such as those associated with the upscale of cultured meat (CM). CM food safety monitoring is a necessary factor when envisioning both the regulatory compliance and consumer acceptance. To achieve this, a multidisciplinary approach is necessary. This includes extensive development of the sensitive and specific analytical devices i.e., sensors to enable reliable food safety monitoring throughout the whole future food supply chain. In addition, advanced monitoring options can help in the further optimization of the meat cultivation which may reduce the currently still high costs of production. This review presents an overview of the sensor monitoring options for the most relevant parameters of importance for meat cultivation. Examples of the various types of sensors that can potentially be used in CM production are provided and the options for their integration into bioreactors, as well as suggestions on further improvements and more advanced integration approaches. In favor of the multidisciplinary approach, we also include an overview of the bioreactor types, scaffolding options as well as imaging techniques relevant for CM research. Furthermore, we briefly present the current status of the CM research and related regulation, societal aspects and challenges to its upscaling and commercialization.

Antimicrobial nanoparticles and biodegradable polymer composites for active food packaging applications.

The food industry faces numerous challenges to assure provision of tasty and convenient food that possesses extended shelf life and shows long-term high-quality preservation. Research and development of antimicrobial materials for food applications have provided active antibacterial packaging technologies that are able to meet these challenges. Furthermore, consumers expect and demand sustainable packaging materials that would reduce environmental problems associated with plastic waste. In this review, we discuss antimicrobial composite materials for active food packaging applications that combine highly efficient antibacterial nanoparticles (i.e., metal, metal oxide, mesoporous silica and graphene-based nanomaterials) with biodegradable and environmentally friendly green polymers (i.e., gelatin, alginate, cellulose, and chitosan) obtained from plants, bacteria, and animals. In addition, innovative syntheses and processing techniques used to obtain active and safe packaging are showcased. Implementation of such green active packaging can significantly reduce the risk of foodborne pathogen outbreaks, improve food safety and quality, and minimize product losses, while reducing waste and maintaining sustainability.