BMC Biotechnology最新文献

Background: Chemical transfection is a widely employed technique in airway epithelium research, enabling the study of gene expression changes and effects. Additionally, it has been explored for its potential application in delivering gene therapies. Here, we characterize the transfection efficiency of EX-EGFP-Lv105, an EGFP-expressing plasmid into three cell lines commonly used to model the airway epithelium (1HAEo-, 16HBE14o-, and NCI-H292).

Results: We used six common and/or commercially available reagents with varying chemical compositions: Lipofectamine 3000 (L3000), FuGENE HD, ViaFect, jetOPTIMUS, EndoFectin, and calcium phosphate. Using L3000, 1HAEo- exhibited the highest transfection efficiency compared to 16HBE14o- and NCI-H292 (1HAEo-: 76.1 ± 3.2%, 16HBE14o-: 35.5 ± 1.2%, NCI-H292: 28.9 ± 2.23%). L3000 yielded the greatest transfection efficiency with the lowest impact on cellular viability, normalized to control, with a 11.3 ± 0.16% reduction in 1HAEo-, 16.3 ± 0.08% reduction in 16HBE14o-, and 17.5 ± 0.09% reduction in NCI-H292 at 48-hour post-transfection. However, jetOPTIMUS had a similar transfection efficiency in 1HAEo- (90.7 ± 4.2%, p = 0.94), but had significantly reduced cellular viability of 37.4 ± 0.11% (p < 0.0001) compared to L3000. In 16HBE14o-, jetOPTIMUS yielded a significantly higher transfection efficiency compared to L3000 (64.6 ± 3.2%, p < 0.0001) but significantly reduced viability of 33.4 ± 0.09% (p < 0.0001) compared to L3000. In NCI-H292, jetOPTIMUS yielded a lower transfection efficiency (22.6 ± 1.2%) with a significant reduction in viability (28.3 ± 0.9%, p < 0.0001). Other reagents varied significantly in their efficiency and impact on cellular viability in other cell lines. Changing the transfection mixture-containing medium at 6-hour post-transfection did not improve transfection efficiency or viability. However, pre-treatment of cell cultures with two rinses of 0.25% trypsin-EDTA improved transfection efficiency in 1HAEo- (85.2 ± 1.1% vs. 71.3 ± 1.0%, p = 0.004) and 16HBE14o- (62.6 ± 4.3 vs. 35.5 ± 1.2, p = 0.003).

Conclusions: Transfection efficiencies can differ based on airway epithelial cell line, reagents, and optimization techniques used. Consideration and optimization of cell line and transfection conditions may be useful for improving nonviral genetic techniques in vitro.

Background: Food safety is a significant global study subject that is strongly intertwined with human life and well-being. The utilization of DNA-based methods for species identification is a valuable instrument in the field of food inspection and regulation. It is particularly significant for traceability purposes, as it enables the monitoring of a specific item at every level of the food chain regulation. However, obtaining amplifiable genomic DNA in this process is a significant obstacle in gene studies. To date, there is a lack of literature on DNA extraction from processed juice or beverages, and no data exist on simultaneous comparisons of various extraction processes. This study aimed to optimize and compare four DNA extraction methods for Chestnut rose juices and beverages. Furthermore, we also conducted a comparison and analysis of the extent of DNA degradation in Chestnut rose juice or beverage by utilizing the amplicon size.

Methods: The quantity and quality of the extracted DNA were assessed using NanoDrop One spectrophotometer, gel electrophoresis, and real-time polymerase chain reaction (real-time PCR or qPCR) assays. An assessment was conducted on the processing time, labor intensity, and cost associated with each approach. The degree of DNA degradation in Chestnut rose juice or beverage was also assessed using TaqMan real-time PCR methods.

Results: The non-commercial modified CTAB-based approach yielded a high DNA concentration. However, spectrophotometric results and real-time PCR analysis showed poor DNA quality. The combination approach showed the greatest performance among the extraction methods, while being comparatively time-consuming and costly in contrast to the other methods. Additionally, the analytical findings of DNA degradation suggested that the integrity of sample DNA could be influenced by the intricacy of processing methods used by various manufacturers.

Conclusions: To achieve precise DNA quantification, selecting suitable extraction strategies for the given matrix is necessary. The combination approach was identified as the most effective DNA extraction technique and is suggested for extracting DNA from Chestnut rose juices and beverages. This comparative assessment can be particularly valuable for extracting and identifying processed Juices and Beverages in a diverse range of food compositions.

Background: Egypt has the highest global prevalence of Hepatitis C Virus (HCV) infection, particularly of genotype 4. The development of a prophylactic vaccine remains crucial for HCV eradication, yet no such vaccine currently exists due to the vaccine development challenges. The ability of Virus-Like Particles (VLPs) to mimic the native virus and incorporate neutralizing and conformational epitopes, while effectively engaging both humoral and cellular immune responses, makes them a promising approach to addressing the challenges in HCV vaccine development.

Methods: Lentiviral-based vectors were constructed and employed to integrate the full-length sequence of Core, E1, E2, and P7 genes of HCV genotype 4 into the genome of Human Embryonic Kidney cells (HEK293T). Upon the expression, HCV structural proteins can oligomerize and self-assemble into VLPs mimicking the structure of HCV native virus. VLPs were purified and characterized for the development of a potential VLPs-based vaccine.

Results: In this study, mammalian cells were successfully engineered to stably express HCV structural proteins and generate non-infectious VLPs for HCV genotype 4. The expression of HCV-integrated genes resulted in a successful production of HCV structural proteins, which oligomerized and self-assembled into two layers enveloped VLPs. Electron microscopy analysis of purified VLPs revealed spherical particles with an average diameter of 60-65 nm, closely resembling mature HCV virions. These results highlighted the potential of these VLPs as a vaccine candidate for HCV genotype 4.

Conclusions: HCV genotype 4 remains an underexplored target in vaccine development, despite its significant public health burden, especially in Egypt. The successful generation of VLPs for this genotype represents a promising avenue for further vaccine development. The established system provides a robust platform for the production and study of VLP-based vaccines targeting HCV genotype 4.

Purpose: To study the potential of a candidate probiotic strain belonging to the Enterococcus durans species in alleviating hypercholesterolemia and improving the microbial milieu of rat gut.

Methods: A previously isolated and characterized E. durans strain NPL 1334 was further screened in vitro for its bile salt hydrolyzation and cholesterol assimilation ability. An in vivo trial using diet-induced hypercholesterolemic rats was conducted to evaluate the effects of the administered test probiotic strain on the animal's blood biochemical parameters such as total cholesterol (TC), high-density lipopolysaccharides (HDL), low-density lipopolysaccharides (LDL), triglycerides (TG), on body weight, oxidative stress markers, and its impact on intestinal and fecal microbiota as well as a histopathological examination of the test animal's livers.

Results: E. durans strain showed good bile salt hydrolyzing ability and ample cholesterol assimilation in vitro. Probiotic-fed hypercholesterolemic rats showed significantly lowered cholesterol, triglyceride and LDL levels. The body weight of probiotic-fed rats was reduced as compared to the control. E. durans also stimulated the growth of beneficial LAB in the intestine of experimental rats and did not harm the liver of the experimental rats.

Conclusion: E. durans can be a natural therapeutic alternative to manage diet-induced hypercholesterolemia and may eventually enhance anti-cholesterolemic therapies.

Background: This study employed melanin synthesized by Aspergillus flavus and Aspergillus carbonarius to inhibit the production of mycotoxins and bioremediation of heavy metals (HMs).

Methods: First, twenty fungal isolates were obtained from soil samples, and were evaluated to produce melanin. The melanin of the most potent producers has undergone several confirmatory experiments, including, Dihydroxyphenylalanine (DOPA)-inhibitor-kojic acid pathway detection, High-performance liquid chromatography (HPLC), Fourier-transform infrared (FTIR) and Nuclear magnetic resonance (NMR). Additionally, the melanin production culture conditions were optimized. The antioxidant activity of melanin was detected with 1,1-Diphenyl-2-picrylhydrazyl (DPPH). HPLC was used to measure the mycotoxins produced in culture media supplemented with melanin. Molecular docking study investigated molecular interactions between melanin and mycotoxins through in silico approaches. FTIR and Energy-dispersive X-ray spectroscopy (EDX) were utilized to determine the percentage of melanin-chelated HMs, and an atomic absorption spectrophotometer (AAS) was used to detect HMs removal efficiency.

Results: The melanin-enriched medium (0.3% and 0.4%) exhibited complete inhibition of aflatoxin B1 (AF-B1) by A. flavus and ochratoxin A (OTA) by A. carbonarius, respectively. Furthermore, melanin showed effective HM removal efficiency, increasing with melanin concentration. The removal efficiency of Cd⁺² and Cr⁺⁶ by 1 mg/mL melanin was 49% and 63%, respectively. When the concentration of melanin was increased to 15 mg/mL, the removal efficiency of Cd⁺² and Cr⁺² increased to 60% and 77%, respectively.

Conclusion: The study exhibited a natural approach for melanin production, using melanin as a heavy metal-chelating agent and capability to inhibit the production of aflatoxin B1 and ochratoxin A. Further, the study provides significant evidence regarding the bioremediation pipeline, for melanin production through biotechnological processes by filamentous fungi.

Background: In the fermentation industry, the demand to replace expensive complex media components is increasing for alternative nutrient sources derived from waste or side streams, such as corn steep liquor (CSL). However, the use of CSL is associated with common problems of side products, such as batch-to-batch variations and compositional inconsistencies. In this study, to detect batch-to-batch variations in CSL for Ogataea polymorpha cultivations, a "fingerprinting" system was developed by employing the Respiration Activity Monitoring System designed for shake flasks (RAMOS) and 96-well microtiter plates (µTOM).

Results: At 2.5 g d.s./L CSL and 5 g/L glucose, a limitation by a secondary substrate, other than the carbon source, was observed. For this specific CSL medium, this limitation was caused by ammonium nitrogen and could be removed through targeted supplementation of ammonium sulphate. Under ammonium nitrogen limitation, O. polymorpha showed a change in morphology and developed a different cell size distribution. Increasing CSL storage times impaired O. polymorpha cultivation results. It was speculated that this observation is caused by micronutrient precipitation as sulfide salts. Through targeted nutrient supplementation, these limiting microelements were identified to be copper, iron and zinc.

Conclusions: This study shows the versatility of CSL as an alternative nutrient source for O. polymorpha cultivations. "Fingerprinting" of CSL batches allows for early screening. Fermentation inconsistencies can be eliminated by selecting the better performing CSL batches or by supplementing and improving an inferior CSL prior to large-scale productions.

The increasing demand for sustainable alternatives to conventional antifungal agents has prompted extensive research into the antifungal properties of plant essential oils (EOs). This study investigates the use of EOs mixture (Origanum vulgare, Moringa oleifera, and Cinnamomum verum) for controlling fungal deterioration in wall paintings at the archaeological Youssef Kamal Palace in Nag Hammadi, Egypt. Fungal isolates were collected from deteriorated wall paintings and identified using phenotypic and genotypic analyses. Aspergillus sp. was found to be the predominant species (50%), followed by Penicillium sp. (16.7%), Fusarium sp. (16.7%), and others. They were genetically identified to be Aspergillus oryzae, Aspergillus niger, Penicillium chrysogenum, Fusarium solani, Alternaria alternata, Botrytis cinerea, and Trichoderma viride. The antifungal activity of three individual oils (oregano, moringa and cinnamon) was evaluated against the most predominant A. niger strain. Out of the three oils, oregano oil showed the strongest antifungal effect with an inhibition zone diameter (IZD) of 4.5 cm followed by moringa (3.5 cm) and cinnamon (3.2 cm). A mixture design approach optimized the EOs combination, with the most effective composition being (44% oregano, 46% moringa, 10% cinnamon), yielding an IZD of 6.5 cm. The optimized EOs mixture demonstrated complete inhibition against all tested fungal strains. The minimal inhibitory concentration tests showed varying efficacies against different fungal strains, with MIC values ranging from 125 to 500 µg/mL. GC-MS analysis identified the major bioactive compounds: carvacrol (83.25%) in oregano, trans-13-octadecenoic acid (22.62%) in moringa, and cinnamaldehyde (24.42%) in cinnamon. Cytotoxicity testing on human skin fibroblasts (HSF) showed minimal toxicity of EOs mixture with 87.64% cell viability at 100 µg/ml. Colorimetric measurements revealed some colour changes in experimental painting samples, particularly with cinnamon oil on white pigment (ΔE = 9.64) and moringa oil on a yellow pigment (ΔE = 16.31). However, oregano oil consistently showed the least impact across all pigments. These findings demonstrate the potential of the EOs combination as an effective, eco-friendly approach to mitigating fungal deterioration in wall paintings, contributing to sustainable conservation strategies for cultural heritage preservation.

Background: Successful treatment of pathogenic bacteria like Enterobacter Cloacae with bacteriophage (phage) counteract some hindrance such as phage stability and immunological clearance. Our research is focused on the encapsulation of phage HK6 within chitosan nanoparticles.

Result: Encapsulation significantly improves stability, efficacy, and delivery of phages. Chitosan nanoparticles (CS-NPs) achieve a phage entrapment efficiency of 97%. Fourier-transform infrared spectroscopy (FT-IR) reveals shifts towards higher wavenumbers and a new peak, indicating amide bond formation and successful phage encapsulation. The average particle sizes for CS-NP and phage HK6 encapsulated CS-NPs were 180 ± 10 nm and 297 ± 18 nm, respectively. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) analyses reveal that phage HK6 encapsulated CS-NPs are larger on average than CS-NPs, highlighting successful phage encapsulation. Encapsulated bacteriophages maintain its effectiveness at higher pH levels of 11 and 12. Both encapsulated and free bacteriophages are thermostable between 25 and 60 °C; while at higher temperatures (up to 80 °C), the encapsulated phage is thermally stable. Over four days, 70.57% of phages were released from encapsulated CS-NPs. Encapsulation of bacteriophage HK6 in CS-NPs enhances antibacterial activity within the first 2 h, compared to phage or nanoparticles alone.

Conclusion: This suggests that the phage HK6 encapsulated CS-NPs exhibit potentiality as biocontrol agents against resistant microorganisms offering an alternative to phage alone.

Background: With the growing interest in applying fermentation to seaweed biomasses, there is a need for fast and efficient selection of microbial strains that have the ability to 1) acidify quickly, 2) utilize seaweed constituents and c) exhibit some proteolytic activity. The present study aims to provide a fast methodology to screen large bacterial collections for potential applications in optimized seaweed fermentations, as well as investigate and assess the performance of a selected bacterial collection of the National Food Institute Culture Collection (NFICC) in seaweed fermentation. This approach is directed toward high-throughput (HT) methodologies, employing microwell assays for different phenotypical characteristics of lactic acid bacteria isolated from different sources. The overarching aim is the deeper understanding of the selection criteria when designing starter cultures for seaweed fermentation.

Results: By employing high-throughput analytical workflows, the screening processing time is minimized, and among the different strains from a well-characterized strain collection, it was possible to distinguish between strong acidifiers and to replicate similar results when the volumes were scaled from 96-well plates to lab-scale fermentations (40 mL) of whole seaweed. Lactiplantibacillus plantarum, Lacticaseibacillus paracasei and, to a lesser extent, Lacticaseibacillus rhamnosus were among the fastest strains to reach the lowest endpoint pH values (< 4.5) in less than 48 h. Although the results regarding proteolytic capacity were not sufficient to prove that the candidates can also provide some flavor generation by the cleavage of proteins, NFICC1746 and NFICC2041 exhibited potential in releasing free alanine, glutamate and asparate as free amino acids.

Conclusions: With the described methodology, a large number of terrestrial lactic acid bacteria (LAB) isolates were screened for their performance and possible application for fermentation of brown sewaeeds. With a a fast conversion of sugars to organic acids, three potential new plant-isolated strains from NFICC, specifically Lactiplantibacillus plantarum ssp. argentoratensis (NFICC983), Lacticaseibacillus paracasei (NFICC1746) and Lacticaseibacillus rhamnosus (NFICC2041), were identified as promising candidates for future synthetic consortia aimed at application in bioprocessed seaweed. The combination of such strains will be the future focus to further optimize robust seaweed fermentations.

Background: In this study, thermophilic pectinase-producing strains were isolated. Among all the isolates, strain No. 4 was identified as Aspergillus fumigatus BT-4 based on its morphology and 18 S rDNA analysis. This strain was employed to screen various fermentation media to enhance pectinase production. Pectinases are crucial enzymes with significant industrial applications, particularly in the food and textile industries. Identifying efficient pectinase producers and optimizing their production processes are essential for improving industrial applications.

Results: Maximum pectinase production was observed using 1% grapefruit peel in M5 media. Shake flask kinetics demonstrated the highest values of specific rate constant (qp), specific growth rate (µ), product yield coefficient (Y_p/x), volumetric rate of product formation (Q_p), and biomass formation (Q_x) after 72 h of incubation. Furthermore, Optimization of fermentation components via Response Surface Methodology (RSM) improved pectinase production by 50%, showcasing the effectiveness of factorial and central composite designs in fine-tuning parameters. The use of agricultural waste (grapefruit peel) significantly reduced production costs, offering an economically viable substrate alternative. The pectinase enzyme was purified through ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography, resulting in a 2.3-fold purification. The molecular weight of the purified enzyme was determined to be 48 kDa. Enzyme kinetics, determined using a Lineweaver-Burk plot at various pectin concentrations, showed a V_max of 32.7 UmL^- 1 and a K_m of 0.3 mg mL^- 1. Thermodynamic parameters, including activation energy (Ea), enthalpy (ΔH), and entropy (ΔS), were measured at 41.74 kJmol^- 1, 39.53 kJmol^- 1, and 46.9 kJmol^- 1, respectively.

Conclusions: The study successfully isolated and identified Aspergillus fumigatus BT-4 as a potent thermophilic pectinase producer. Optimization of the fermentation process using 1% grapefruit peel in M5 media significantly enhanced pectinase production. Using grapefruit peel as an agricultural waste in pectinase production reduces costs by eliminating the need for expensive raw materials and utilizing a low-cost, sustainable, and locally available substrate. This approach also minimizes waste disposal expenses, making the process more economical. The enzyme was effectively purified, and its kinetic and thermodynamic properties were thoroughly characterized, revealing its potential for industrial applications. The comprehensive analysis of production kinetics and optimization strategies provides a robust foundation for scaling up pectinase production, contributing to more efficient and cost-effective industrial processes.