Applied Biochemistry and Microbiology最新文献

We have recently identified S-adenosyl-L-homocysteine hydrolase (SAHH) as a novel banana allergen with a potentially decisive role in the development of cross-reactivity between plant-derived food and respiratory allergens. The recalcitrant nature of banana pulp and intrinsically low abundance of SAHH limit its production from natural sources. Our objective was to optimize production and biochemically characterize recombinant banana SAHH (rSAHH) with implications for the food safety industry, food allergy diagnosis and treatment, and basic research in molecular allergology. rSAHH with C-terminal 6His tag was successfully expressed in Escherichia coli BL21(DE3) cells, and purified to homogeneity by immobilized metal affinity chromatography (IMAC), yielding 10 mg of rSAHH/L of cell culture under optimized conditions. It is functional as a monomer with an approximate molecular weight of 55 kDa and pI of 5.83. Structural integrity, IgE reactivity, and biological activity of rSAHH were confirmed by Western blot and standard colorimetric SAHH assay with Ellman’s reagent, respectively. As a mesophilic enzyme with wide pH stability and high 8-month storage stability, rSAHH obtained in this study is the promising candidate for further diagnostic and therapeutic applications. To the author’s best knowledge, rSAHH is the only recombinantly produced plant-derived SAHH thus far.

Drug-resistant microbes are evolving as a result of the overuse of antibiotics in recent years leading to an increase in the prevalence of drug-resistant infections. Thus, the antibiotics that are currently in development are far from sufficient to treat illnesses and their problems brought on by multi-drug resistance. Antimicrobial peptides (AMPs), which are widely found in the biological world, may help solve this problem. AMPs are highly effective at inhibiting the growth of drug-resistant bacteria via a variety of complex action mechanisms and also have high target specificity. Conjugation to molecules such as antibiotics, proteins, polymers, and nanoparticles are strategies under investigation to boost the therapeutic efficacy of AMPs. As such, they are widely expected to be the basis of a new way of fighting infection in place of antibiotics. This review primarily focuses on these action mechanisms and highlights the biological characteristics and promising applications of AMPs. It also evaluates their prospects and difficulties moving forward. In addition, the antimicrobial efficacies of the design and application of AMPs’ conjugates are summarized to provide a useful reference for future research on AMPs and their application.

Microalgae immobilisation is an attractive alternative for wastewater treatment. These microorganisms have high nutrient absorption capability, and when immobilised, are easy to apply and collect into the treatment, although the viability of cells and effectiveness of this process under these conditions still needs to be explored. This study aims to evaluate the viability of Chlorella vulgaris cells taken at 3 initial concentrations, C1, 5.93 × 10⁵ ± 8.84 × 10⁴, C2, 1.75 × 10⁶ ± 5.09 × 10⁵ and C3, 2.95 × 10⁷± 9.81 × 10⁵ cells/mL, immobilised in the calcium alginate beads for the nutrients removal in Jaworski’s modified medium. The cell growth of immobilised and suspended cells was analysed by cell counting and chlorophyll content by fluorescence spectroscopy in an excitation-emission matrix. The results revealed that the immobilised microalgae grew and removed nutrients regardless of initial concentration. The most concentrated immobilised C. vulgaris (C3) had the highest concentration of cells, 1.67 × 10⁸ cells/mL, and removed more nutrients (100% of nitrate and 96.33% of phosphate) with chlorophyll fluorescence of C3, 22.99 RFU at the end of experiment.

Fluorescence microscopy is a highly sensitive imaging technique used in various scientific fields such as cellular biology, environmental sciences, medicine, and pharmacy. It offers the advantage of using multiple fluorescent labels to visualize different biomolecules and generate multicolored images for identifying specific components within complex biomolecular structures and studying their interactions. These fluorescent labels create chemically stable and minimally disruptive bioconjugates. Amino-reactive fluorescent labels, due to their ease of incorporation into biomolecules, are commonly used in applications like fluorescence in situ hybridization, histochemistry, cell tracing, receptor binding, and immunochemistry. However, the existing popular fluorescent labels are expensive, making coumarin derivatives a potential cost-effective solution for developing bright fluorophores. In this study, the fluorescent 4-styrylcoumarin derivative labels were synthesized and evaluated as potentially effective fluorescent labels for biomolecules. Twelve new fluorescent oligonucleotide probes have been obtained, 6 directed to the rRNA region of eukaryotic cells (EUK516) and 6 directed to the rRNA region of prokaryotic cells (EUB338). The developed fluorescent probes were tested on microorganisms belonging to the culture collection of the Laboratory of Biodegradation and Biotechnology of the HERCULES Laboratory (University of Évora, Portugal), showing effective performance as RNA-FISH probes. These findings evidenced the applicability of the new 4-styrylcoumarin derivatives in labeling of biomolecules and bioimaging.

Guaiacol, 4-vinylguaiacol, 4-methylguaiacol and 4-ethylguaiacol, being guaiacol compounds characterized by spicy and smoky flavor, are important functional factors and key aroma components in sesame flavore Baijiu. In this study, to enhance the content of guaiacol compounds in this product, a strain of Pseudomonas azotoformans YF-58 which produced guaiacol compounds was added to the Zaopei (fermented grains) in the form of bacterial Fuqu. Subsequently, the effect of YF-58 strain on the fermentation system was explored from multiple perspectives, including microbial community composition, volatile flavor substance content and the expression levels of functional enzymes. The results showed that the addition of YF-58 strain altered the relative abundance of microorganisms such as Lactobacillus, Aspergillus, Pseudomonas, Staphylococcus, Weiniella, Bacillus and Corynebacterium in Zaopei. Moreover, the contents of acids, esters, alcohols and guaiacol compounds in Zaopei increased. Additionally, key enzymes associated with guaiacol compounds formation, such as 4-hydroxy-3-polyprenylbenzoate decarboxylase (EC 4.1.1.98), cellulase (EC 3.2.1.4) and catechol O-methyltransferase (EC2.1.1.6), were up-regulated. As a result, the contents of guaiacol, 4-methyl guaiacol, 4-ethylguaiacol and 4-vinylguaiacol in sesame flavor Baijiu were significantly increased by 193.1, 11.6, 95.4 and 105.2%, respectively. The research findings provided some theoretical guidance for enhancing the content of volatile flavor substances in Baijiu.

Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen that seriously threats the public and animal health. The major virulence of STEC is Shiga toxin (Stx) which is encoded by Stx prophage. Stx prophage induction leads to the production of Stx. Our recent research has shown that dietary functional sugars can differently regulate Shiga toxin type 2 (Stx2) prophage induction. However, high-throughput assays are required to find more dietary inhibitors for Stx prophage induction. This study aims to establish the recA dual-luciferase reporter system for rapidly screening the functional sugars that can inhibit Stx prophage induction. Results showed that the recA dual-luciferase reporter plasmid was successfully constructed as confirmed by DNA sequencing. Established recA dual-luciferase reporter system showed similar screening results when compared with traditional temperate phage determination method (i.e., qPCR) and suggested that L-arabinose is a dietary functional Stx prophage induction inhibitor. Supplementation with L-arabinose in culture medium reduces Stx production in STEC isolated from fecal samples of diarrhea piglets. Collectively, the recA dual-luciferase reporter system is a promising tool for convenient and rapid screening of dietary Stx prophage induction inhibitor, especially suitable for large-scale screening experiments.

Acidification is a crucial preservation technique that affects the survival and infection of foodborne pathogens. In this study, influence of the acidic growth medium on the resistance pattern of the pathogenic Escherichia coli in terms of changes in fatty acid composition as well as expression of virulent genes (eae, stx1, stx2 and hlyA) was investigated. It was revealed that lemon juice mediated acidic pH induced the transcription of stx2, with 3.1- and 2.5-fold increase of survived cells at pH 3.5 and 4.5, respectively, while the transcription level of stx1 in cells grown at pH 3.5 and 4.5 were 0.57 and 0.71-fold lower compared to control. Highest transcription level was observed in the cells grown at pH 3.5 and containing hlyA (4.6-fold), which was much higher than that at pH 5.5 and 4.5. However, pH had no effect on the transcription of eae gene. The results showed that isolated pathogenic E. coli cells from meat were susceptible to lemon juice mediated stress possibly due to the increased level of unsaturated fatty acid (USFA) and decreased level cyclic fatty acid (CFA). The fluidity of the membranes increased as the pH of the media dropped. The highest USFA/SFA ratio was observed in strains incubated at pH 3.5 with ratio of 0.31-0.38 and the value decreased as the pH increased, reaching a minimum value of 0.03 at pH 7.0. This study emphasizes on the importance of establishing effective strategies to combat acid-resistant pathogenic E. coli in food matrices.

Two immunochromatographic assays based on hematite (α-Fe₂O₃) nanoparticles were developed for rapid detection of aflatoxin M1 (AFM1) and Escherichia coli O157:H7 in the milk. Hematite nanoparticles were prepared and utilized as colored signal labels. The competitive format was applied for AFM1 detection, while the sandwich format for E. coli O157:H7 determination. Under optimal conditions, the assay exhibited a visual detection limit of 0.10 ng/mL and quantitative determination limit of 0.017 ng/mL for AFM1. For E. coli O157:H7, a visual detection limit of 2.9 × 10⁵ CFU/mL was achieved. The entire determination process could be accomplished within 15 min. Compared with traditional nanolabels, hematite nanoparticles showed advantages of facile preparation, high stability and easy bioconjugation. The developed assays provided effective and rapid screening strategies for AFM1 and E. coli O157:H7 in milk samples with high sensitivity and specificity.

The black soldier fly (BSF, Hermetia illucens) is a well-known insect with promising potential in transforming organic wastes into protein-rich biomass. The BSF larvae (BSFL) can replace expensive sources of protein used in aquaculture and have antimicrobial characteristics that are recognized to inhibit pathogenic bacteria in humans, plants, poultry, and livestock. However, its potential against pathogenic bacteria in aquaculture remains largely unexplored. To address this gap, BSFL in different instar stages were analyzed for their proximate, mineral, and fatty acid contents to determine their nutritional composition. Additionally, disk diffusion and minimum inhibitory concentration assays were used to evaluate the antibacterial activity of the crude methanolic extracts of the BSFL against Aeromonas veronii, Edwardsiella tarda, Streptococcus agalactiae, and Vibrio harveyi. Results showed high nutritional composition across instar stages but the highest level was revealed in 2- and 3-instar stages (crude protein: 36.17, 36.96%, and crude fat: 33.84, 40.90%, respectively). Crude methanolic extract of BSFL inhibited the growth and proliferation of all tested bacteria, with the highest inhibition observed in Streptococcus agalactiae. Moreover, the extracts obtained at 5-instar exhibited the highest antibacterial activity compared to other extracts. This study showed that the BSFL have the potential to serve as a sustainable protein source in aquafeeds, and the crude methanolic extracts from later-stage BSFL can be used as antibacterial agents in aquaculture.

Plant growth-promoting microbes (PGPM) are a group of beneficial microorganisms that stimulate plant growth directly by improving nitrogen fixation, phosphate solubilization, mineral uptake/transport, plant growth hormone/regulator metabolism, and indirectly by systemic induced/acquired resistance by salicylic/jasmonic acid production. These microorganisms can act against plant pathogens by exoenzymes, secondary metabolite production, osmolyte production in abiotic stress. Pseudomonas/Bacillus spp. are the most versatile plant growth-promoting bacteria (PGPB). The PGPMs can be used as rhizome-remediators, phytostimulators, biofertilizers, and stress controllers. The PGPMs’ treatment promotes the growth of several plants such as lentils, peas, cucumbers, and soybeans. Their availability depends largely on interactions between microorganisms in soil and the ecto- and endophytes of root, stem, and leaf. PGPMs affect positively to plant growth, nutrient uptake, biotic resistance, and disease-causing pathogens, and contributes to an increase in yield in several crop plants. This review extensively covers all these aspects of various ways adapted by the PGPM to stimulate positively plant growth effects. PGPB influence phosphorus (P) homeostasis to induce beneficial effects in plants. The review introduces the topic from the perspectives of P and their relation with PGPB. It interlinks the entomopathogens and endophytes with the PGPBs’ regulation of plant growth. Different mechanisms and techniques of P solubilization by the PGPB are discussed. Followingly, the genetics and metagenomics of P solubilization are explained. This review opens various perspective points for further research for enhancing plant growth, strengthening sustainable agriculture of food crops.