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BioTech, Volume 13, Issue 4 (December 2024) – 6 articles

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13 pages, 1793 KiB

Open AccessArticle

Microbial Protein and Metabolite Profiles of Klebsiella oxytoca M5A1 in a Bubble Column Bioreactor

by Tawakalt Ayodele, Musiliu Liadi, Abodunrin Tirmidhi Tijani, Kudirat Alarape, Christiana Bitrus, Clairmont L. Clementson and Ademola Hammed

BioTech 2024, 13(4), 43; https://doi.org/10.3390/biotech13040043 (registering DOI) - 19 Oct 2024

Abstract

The production of microbial proteins (MPs) has emerged as a critical focus in biotechnology, driven by the need for sustainable and scalable alternatives to traditional protein sources. This study investigates the efficacy of two experimental setups in producing MPs using the nitrogen-fixing bacterium Klebsiella oxytoca M5A1. K. oxytoca M5A1, known for its facultative anaerobic growth and capability to fix atmospheric nitrogen, offers a promising avenue for environmentally friendly protein production. This research compares the performance of a simple bubble column (BC) bioreactor, which promotes efficient mixing and cross-membrane gas transfer, with static fermentation, a traditional method lacking agitation and aeration. The study involved the parallel cultivation of K. oxytoca M5A1 in both systems, with key parameters such as microbial growth, glucose utilization, protein concentration, and metabolite profiles monitored over a 48 h period. The results indicate that the BC bioreactor consistently outperformed static fermentation regarding the growth rate, protein yield, and glucose utilization efficiency. The BC exhibited a significant increase in protein production, reaching 299.90 µg/mL at 48 h, compared to 219.44 µg/mL in static fermentation. The organic acid profile reveals both synthesis and utilization regimes of varying patterns. These findings highlight the advantages of the BC bioreactor for MP production, particularly its ability to maintain aerobic conditions that support higher growth and yield. Full article

(This article belongs to the Section Industrial Biotechnology)

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18 pages, 3640 KiB

Open AccessArticle

Phytochemical Composition, Antioxidant, Anti-Helicobacter pylori, and Enzyme Inhibitory Evaluations of Cleistocalyx operculatus Flower Bud and Leaf Fractions

by Doan Thien Thanh, Mai Thanh Tan, Nguyen Thi My Thu, Pham Nhat Phuong Trinh, Pham Thi Hoai Thuong, Pham Thi Giang Tuyet, Luong Thi My Ngan and Tran Trung Hieu

BioTech 2024, 13(4), 42; https://doi.org/10.3390/biotech13040042 - 11 Oct 2024

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Abstract

Six solvent fractions isolated from flower bud and leaf ethanolic extracts of Cleistocalyx operculatus were analyzed for their phytochemical contents, including phenolics, flavonoids, saponins, tannins, and alkaloids. Antioxidant activities were measured using the ABTS, DPPH, and FRAP assays. The results showed that the flower bud aqueous fraction (BAF) and the leaf aqueous fraction (LAF) rich in phenolic content (768.18 and 490.74 mg GAE/g dry extract, respectively) exhibited significantly higher antioxidant activities than the other fractions. The flower bud hexane fraction (BHF) had remarkably high flavonoid and saponin contents (134.77 mg QE/g and 153.33 mg OA/g dry extract, respectively), followed by that of the leaf hexane fraction (LHF) (76.54 mg QE/g and 88.25 mg OA/g dry extract, respectively). The BHF and LHF were found to have extremely high antibacterial activity against two H. pylori strains, ATCC 51932 and 43504 (MICs of 125 µg/mL). Interestingly, DMC (2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone) isolated from the BHF displayed greater antibacterial activity against the bacterial strains (MICs of 25–50 µg/mL) than those of the fractions. In addition, DMC presented potent inhibitory effects on H. pylori urease (IC₅₀ of 3.2 µg/mL) and α-amylase (IC₅₀ of 83.80 µg/mL), but no inhibition against α-glucosidase. It was also demonstrated that DMC showed pronounced inhibitory effects on the urease activity and biofilm formation of H. pylori, and could increase the membrane permeability of the bacterial cells. Scanning electron micrographs depicted that the BHF and DMC had strong effects on the cell shape and significantly induced the distortion and damage of the cell membrane. The fractions and DMC showed no significant toxicity to four tested human cell lines. Efforts to reduce antibiotic use indicate the need for further studies of the flower buds and DMC as potential products to prevent or treat gastric H. pylori infections. Full article

(This article belongs to the Special Issue Natural Antioxidants: Determination in Food and Nutraceuticals and Implications on Human Health)

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16 pages, 2581 KiB

Open AccessArticle

Loss of Cell-Cell Contact Inhibits Cellular Differentiation of α-Catenin Knock Out P19 Embryonal Carcinoma Cells and Their Colonization into the Developing Mouse Embryos

by Masahiro Sato, Emi Inada, Naoko Kubota and Masayuki Ozawa

BioTech 2024, 13(4), 41; https://doi.org/10.3390/biotech13040041 - 3 Oct 2024

Viewed by 637

Abstract

Cadherin−catenin cell−cell adhesion complexes, composed of cadherin, β-catenin or plakoglobin, and α-catenin (α-cat) molecules, are crucial for maintaining cell−cell contact and are commonly referred to as “adherens junctions (AJs).” Inactivating this system leads to loss of cell−cell contact and developmental arrest in early embryos. However, it remains unclear whether the loss of cell−cell contact affects the differentiation of embryonic cells. In this study, we explored the use of a murine embryonal carcinoma cell line, P19, as an in vitro model for early embryogenesis. P19 cells easily form embryoid bodies (EBs) and are susceptible to cellular differentiation in response to retinoic acid (RA) and teratoma formation. Using CRISPR/Cas9 technology to disrupt the endogenous α-cat gene in P19 cells, we generated α-cat knockout (KO) cells that exhibited a loss of cell−cell contact. When cultivated on non-coated dishes, these α-cat KO cells formed EBs, but their structures were labile. In the RA-containing medium, the α-cat KO EBs failed to produce differentiated cells on their outer layer and continued to express SSEA-1, an antigen specific to pluripotent cells. Teratoma formation assays revealed an absence of overt differentiated cells in tumors derived from α-cat KO P19 cells. Aggregation assays revealed the inability of the KO cells to colonize into the zona pellucida-denuded 8-cell embryos. These findings suggest that the AJs are essential for promoting the early stages of cellular differentiation and for the colonization of early-developing embryos. Full article

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16 pages, 2552 KiB

Open AccessArticle

Structural Analysis and Substrate Specificity of D-Carbamoylase from Pseudomonas

by Marina Paronyan, Haykanush Koloyan, Hovsep Aganyants, Artur Hambardzumyan, Tigran Soghomonyan, Sona Avetisyan, Sergey Kocharov, Henry Panosyan, Vehary Sakanyan and Anichka Hovsepyan

BioTech 2024, 13(4), 40; https://doi.org/10.3390/biotech13040040 - 3 Oct 2024

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Abstract

The synthesis of enantiomeric forms of D-amino acids can be achieved by a two-step “hydantoinase process” based on the sequential catalysis of substrates by specific enzymes, D-carbamoylase and D-hydantoinase. Here, we describe the structural features of D-carbamoylase from Pseudomonas, the encoded gene of which was chemically synthesized and cloned into Escherichia coli. A significant fraction of the overexpressed recombinant protein forms insoluble inclusion bodies, which are partially converted to a soluble state upon treatment with N-lauroylsarcosine or upon incubation of cells at 28 °C. Purified His-tagged protein exhibits the highest activity towards N-carbamoyl-D-alanine and N-carbamoyl-D-tryptophan. Comprehensive virtual analysis of the interactions of bulky carbamylated amino acids with D-carbamoylase provided valuable information. Molecular docking analysis revealed the location of the substrate binding site in the three-dimensional structure of D-carbamoylase. Molecular dynamics simulations showed that the binding pocket of the enzyme in complex with N-carbamoyl-D-tryptophan was stabilized within 100 nanoseconds. The free energy data showed that Arg176 and Asn173 formed hydrogen bonds between the enzyme and substrates. The studies of D-carbamoylases and the properties of our previously obtained D-hydantoinase suggest the possibility of developing a harmonized biotechnological process for the production of new drugs and peptide hormones. Full article

(This article belongs to the Topic Microbial Biotechnology Products and Biocatalysis Processes for a Sustainable Bioeconomy)

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19 pages, 34131 KiB

Open AccessArticle

Optimization of Sugar Extraction Process from Date Waste Using Full Factorial Design Toward Its Use for New Biotechnological Applications

by Islam Sayah, Mondher Njehi, Nicola Cicero, Vincenzo Nava, Manel Ben M’hadheb, Hatem Majdoub, Sami Achour and Teresa Gervasi

BioTech 2024, 13(4), 39; https://doi.org/10.3390/biotech13040039 - 3 Oct 2024

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Abstract

In Tunisia, the date industry generates a large quantity of waste, raising environmental concerns. However, dates are rich in sugars, which offer a renewable source of nutrients for various applications. In this study, sugar extraction from two low-grade pitted date fruits (Alig and Kentichi) under ultrasound, was optimized using full factorial design. At 40 °C, for20 min, and with a liquid-to-solid ratio of 10 mL/g, the optimum sugar contents were 60.87% and 50.79% for the varieties Alig and Kentichi, respectively. The date extracts were chemically analyzed, revealing low fat and protein contents, but significant polyphenol and mineral contents in both varieties. HPLC-IR analysis revealed more inverted sugars (glucose and fructose) in the Alig variety and more sucrose in the Kentichi variety. FTIR and SEM analysis showed the efficiency of the ultrasonic treatment of the biomass in terms of improving mass transfer diffusion through ultrasonic cavitation. Thus, ultrasound-assisted extraction constitutes an effective method for the recovery of sugar from date waste. Full article

(This article belongs to the Section Agricultural and Food Biotechnology)

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18 pages, 1912 KiB

Open AccessArticle

Chemical, Textural and Antioxidant Properties of Oat-Fermented Beverages with Different Starter Lactic Acid Bacteria and Pectin

by Dmitrii V. Khrundin and Elena V. Nikitina

BioTech 2024, 13(4), 38; https://doi.org/10.3390/biotech13040038 - 25 Sep 2024

Viewed by 409

Abstract

Currently, starter cultures for fermenting plant-based beverages are not widely available commercially, but producers can use starter cultures for dairy products. Therefore, the aim of this study was to determine the physicochemical, rheological, antioxidant and sensory properties of oat beverages with/without pectin fermented by four different dairy starter cultures. The use of a mono-starter with Lactobacillus bulgaricus or Sreptococcus thermophilus allows for the efficient use of glucose, and more lactic acid is accumulated. The beverage with L. bulgaricus is characterised by high adhesion, syneresis and low cohesiveness, and it has high antioxidant activity and a low sensory profile. Using starter with L. bulgaricus, S. thermophilus and some Lactococcus for fermentation yields a product with high sensory capacity, forming a high-viscosity beverage matrix with low syneresis, high water retention, chewy texture and stickiness. It has been observed that the absence of lactococci and the presence of Lactobacillus casei, L. Rhamnosus and L. paracasei in the starter yields a product with high antioxidant activity, especially in the presence of pectin. The use of pectin significantly improves the viscosity and textural properties of oat yoghurt, enhancing the drink’s flavour and giving it body. For many reasons, the use of different commercial starters in the dairy industry results in different viscosities of oat fermented beverages, forming a matrix with different textural, sensory and antioxidant properties. Full article

(This article belongs to the Section Agricultural and Food Biotechnology)

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