Search Results (55)

Geobacillus and Parageobacillus spores are major spoilage agents in thermally treated, shelf-stable foods, particularly milk products, due to their high heat resistance. This study aimed to investigate how spore purification, maturation time, and sporulation temperature influence the germination and heat resistance of P. thermoglucosidasius, G. thermodenitrificans, and G. stearothermophilus spores, with the goal of improving the reliability of microbial risk assessment. All three species germinate efficiently in milk, likely triggered by lactose and glucose. Ethanol-treated spores during purification germinated without heat activation, while water-washed spores required it. At least four days of maturation were needed for efficient germination, though extending maturation to seven days led to strain-dependent changes in heat resistance: it increased in G. thermodenitrificans, decreased in P. thermoglucosidasius, and remained stable in G. stearothermophilus. Sporulation at 55 °C consistently favored germination at the same revival temperature. G. stearothermophilus reached the highest heat resistance at 55 °C, whereas the other species were more resistant when sporulated at 60 °C. These findings underscore the importance of standardizing spore-preparation protocols, as key parameters such as purification, maturation time, and sporulation temperature critically affect spore properties relevant to food stability. Full article

Background/Objectives: During the COVID-19 pandemic, the importance of disinfection and quarantine significantly increased, particularly in situations of staff shortages. Automated disinfection methods, such as hydrogen peroxide vaporization (HPV), are increasingly considered as alternatives to traditional manual disinfection. This study aimed to evaluate the efficacy of HPV compared to standard disinfection practices. Methods: Experiments were conducted at the Infectious Disease Clinical Research Simulation Center of Soonchunhyang University Hospital using Geobacillus stearothermophilus spores as biological indicators. The spores were inoculated on various hospital surfaces and allowed to dry for 120 min. Three disinfection methods were tested: (1) scrubbing with a disposable towel soaked in sodium hypochlorite; (2) placing sodium hypochlorite-soaked towels on the surface for one minute; and (3) HPV alone. Samples were collected post-disinfection and incubated at 55–60 °C. Bacterial cultures were assessed after 24, 48, and 168 h. Results: After 24 h of incubation, sterilization rates were 0% for the scrubbing method, 27% for sodium hypochlorite towels, 68% for HPV alone, and 95% for the combination of sodium hypochlorite and HPV. HPV alone demonstrated statistically greater efficacy compared to standard disinfection practices (p = 0.03). Conclusions: HPV alone may serve as a viable disinfection method in clinical environments, particularly during pandemics when staffing limitations hinder thorough manual cleaning. Further clinical trials are warranted to validate these findings and improve disinfection methods for challenging materials such as fabrics. Full article

The increasing antibiotic resistance among bacteria challenges the biotech industry to search for new antibacterial molecules. Endolysin TP84_28 is a thermostable, lytic enzyme, encoded by the bacteriophage (phage) TP-84, and it effectively digests host bacteria cell wall. Biofilms, together with antibiotic resistance, are major problems in clinical medicine and industry. The challenge is to keep antibacterial molecules at the site of desired action, as their diffusion leads to a loss of efficacy. The TP84_28 endolysin gene was cloned into an expression-fusion vector, forming a fusion gene cbd_tp84_28_his with a cellulose-binding domain from the cellulase enzyme. The Cellulose-Binding Thermostable TP84_Endolysin (CBD_TP84_28_His) fusion protein was biosynthesized in Escherichia coli and purified. Thermostability and enzymatic activities against various bacterial species were measured by a turbidity reduction assay, a spot assay, and biofilm removal. Cellulose-binding properties were confirmed via interactions with microcellulose and cellulose paper-based immunoblotting. The high affinity of the CBD allows for a high concentration of the fusion enzyme at desired target sites such as cellulose-based wound dressings, artificial heart valves and food packaging. CBD_TP84_28_His exhibits a lytic effect against thermophilic bacteria Geobacillus stearothemophilus, Thermus aquaticus, Bacillus stearothermophilus, and Geobacillus ICI and minor effects against mesophilic Bacillus cereus and Bacillus subtilis. CBD_TP84_28_His retains full activity after preincubation in the temperatures of 30–65 °C and exhibits significant activity up to its melting point at 73 °C. CBD_TP84_28_His effectively reduces biofilms. These findings suggest that integrating CBDs into thermostable endolysins could enable the development of targeted antibacterial recombinant proteins with diverse clinical and industrial applications. Full article

There is currently increased interest in the use of alternatives to autoclaved culture media, in order to maintain the properties of the media, while saving energy and time. In this study, we assess a new system for culture media preparation, using a conventional microwave with a water bath and a glass bottle with a rubber cap that allows depressurization. Sterilization, using the proposed system (1000 W, 3 to 20 min), was compared with autoclaving for the preparation of tryptone soy agar (TSA), tryptone soy broth (TSB), Sabouraud 4% dextrose agar (SDA), and violet red bile glucose agar (VRBG). Microwave exposure for 7 min yielded sterile TSA plates. The productivity of both sterilization methods was assessed using the pour plate method, and significant increases in the growth of certain micro-organisms after using a microwave were observed for every culture medium, especially those that were sterilized by boiling (VRBG). The kinetics of microbial destruction showed that Escherichia coli and Bacillus subtilis spores were destroyed after 3 and 7 min in a microwave, respectively, while three decimal reductions were obtained for Geobacillus stearothermophilus spores after 15 min in an autoclave. This new sterilization method could be a feasible, rapid, and economical method to prepare microbiological media, with a quality similar to that obtained through autoclaving. Full article

Bacteriophages associated with thermophiles are gaining increased attention due to their pivotal roles in various biogeochemical and ecological processes, as well as their applications in biotechnology and bionanotechnology. Although thermophages are not suitable for controlling bacterial infections in humans or animals, their individual components, such as enzymes and capsid proteins, can be employed in molecular biology and significantly contribute to the enhancement of human and animal health. Despite their significance, thermophages still remain underrepresented in the known prokaryotic virosphere, primarily due to limited in-depth investigations. However, due to their unique properties, thermophages are currently attracting increasing interest, as evidenced by several newly discovered phages belonging to this group. This review offers an updated compilation of thermophages characterized to date, focusing on species infecting the thermophilic bacilli. Moreover, it presents experimental findings, including novel proteomic data (39 proteins) concerning the model TP-84 bacteriophage, along with the first announcement of 6 recently discovered thermophages infecting Geobacillus thermodenitrificans: PK5.2, PK2.1, NIIg10.1, NIIg2.1, NIIg2.2, and NIIg2.3. This review serves as an update to our previous publication in 2021. Full article

The TP-84 bacteriophage, which infects Geobacillus stearothermophilus strain 10 (G. stearothermophilus), has a genome size of 47.7 kilobase pairs (kbps) and contains 81 predicted protein-coding ORFs. One of these, TP84_26 encodes a putative tail fiber protein possessing capsule depolymerase activity. In this study, we cloned the TP84_26 gene into a high-expression Escherichia coli (E. coli) system, modified its N-terminus with His-tag, expressed both the wild type gene and His-tagged variant, purified the recombinant depolymerase variants, and further evaluated their properties. We developed a direct enzymatic assay for the depolymerase activity toward G. stearothermophilus capsules. The recombinant TP84_26 protein variants effectively degraded the existing bacterial capsules and inhibited the formation of new ones. Our results provide insights into the novel TP84_26 depolymerase with specific activity against thermostable G. stearothermophilus and its role in the TP-84 life cycle. The identification and characterization of novel depolymerases, such as TP84_26, hold promise for innovative strategies to combat bacterial infections and improve various industrial processes. Full article

Lipases can catalyze synthesis reactions in a micro aqueous system, producing useful partial glycerides (mono- and diglycerides), and these compounds are commonly utilized in different products as surfactants. Depending on the microbial sources for lipases, immobilization conditions, and starting substrates for synthesis reaction, the composition and yields of the resulting partial glycerides could be variable. These differences could lead to the final efficacy of partial glycerides as surfactants in targeted products. Therefore, it is necessary to establish a group of immobilized lipases from different microbial sources with information about substrate specificity to produce effective partial glycerides for various product types. Here, lipases from thermophilic Geobacillus stearothermophilus and Anoxybacillus flavithermus were prepared with a simple partial purification method, and after immobilization, these lipases were tested to synthesize partial glycerides using different types of decanoic acids. The distinct product patterns were analyzed using HPLC. Both immobilized lipases showed the highest substrate selectivity to decanoic acids in common, producing mainly glyceryl monodecanoate. However, commercial immobilized lipases from Thermomyces lanuginosus produced the largest glyceryl monodecanoate from methyl decanoate. These results indicate the importance of immobilization conditions like different microbial sources and substrates and the need for their optimal combination. Full article

β-xylosidases (4-β-d-xylan xylohydrolase, E.C. 3.2.1.37) are glycoside hydrolases (GH) catalyzing the hydrolysis of (1→4)-β-d-xylans, allowing for the removal of β-d-xylose residues from its non-reducing termini. Together with other xylan-degrading enzymes, β-xylosidases are involved in the enzymatic hydrolysis of lignocellulosic biomass, making them highly valuable in the biotechnological field. Whereas different GH families are deeply characterized from a structural point of view, the GH52 family has been barely described. In this work, we report the 2.25 Å resolution structure of Geobacillus stearothermophilus CECT43 XynB2, providing the second structural characterization for this GH family. A plausible dynamic loop closing the entrance of the catalytic cleft is proposed based on the comparison of the available GH52 structures, suggesting the relevance of a dimeric structure for members of this family. The glycone specificity at the −1 site for GH52 and GH116 members is also explained by our structural studies. Full article

Poultry production faces several challenges, with feed efficiency being the main factor that can be influenced through the use of different nutritional strategies. Xylooligosaccharides (XOS) are functional feed additives that are attracting growing commercial interest due to their excellent ability to modulate the composition of the gut microbiota. The aim of the study was to apply crude and purified fungal xylanases, from Trichoderma harzianum, as well as a recombinant glycoside hydrolase family 10 xylanase, derived from Geobacillus stearothermophilus T6, as additives to locally produced chicken feeds. A Box–Behnken Design (BBD) was used to optimize the reducing sugar yield. Response surface methodology (RSM) revealed that reducing sugars were higher (8.05 mg/mL, 2.81 mg/mL and 2.98 mg/mL) for the starter feed treated with each of the three enzymes compared to the treatment with grower feed (3.11 mg/mL, 2.41 mg/mL and 2.62 mg/mL). The hydrolysis products were analysed by thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC) analysis and showed that the enzymes hydrolysed the chicken feeds, producing a range of monosaccharides (arabinose, mannose, glucose, and galactose) and XOS, with xylobiose being the predominant XOS. These results show promising data for future applications as additives to poultry feeds. Full article

Purification of bacteriophage-expressed proteins poses methodological difficulties associated with the need to process entire culture medium volume upon bacteriophage-induced bacterial cell lysis. We have used novel capsule glycosylase-depolymerase (TP84_26 GD) from bacteriophage TP-84, infecting thermophilic Geobacillus stearothermophilus bacteria, as a representative enzyme to develop a method for rapid concentration and purification of the enzyme present in diluted crude host cell lysate. A novel variant of the polyethyleneimine (PEI)-based purification method was devised that offers a fast and effective approach for handling PEI-facilitated purification of bacteriophage-expressed native proteins. Due to the very basic nature of PEI, the method is suitable for proteins interacting with nucleic acids or acidic proteins, where either mixed PEI-DNA or RNA–protein complexes or PEI–acidic protein complexes are reversibly precipitated. (i) The method is of general use, applicable with minor modifications to a variety of bacteriophage cell lysates and proteins. (ii) In the example application, TP84_26 GD was highly purified (over 50%) in a single PEI step; subsequent chromatography yielded a homogeneous enzyme. (iii) The enzyme’s properties were examined, revealing the presence of three distinct forms of the TP84_26 GD. These forms included soluble, unbound proteins found in host cell lysate, as well as an integrated form within the TP-84 virion. Full article

The Y509E mutant of β-xylosidase from Geobacillus stearothermophilus (XynB2^Y509E) (which also bears xylanase activity) has been immobilized in chitosan spheres through either entrapment or covalent bond formation methods. The maximum immobilization yield by entrapment was achieved by chitosan beads developed using a 2% chitosan solution after 1 h of maturation time in CFG buffer with ethanol. On the other hand, the highest value in covalent bond immobilization was observed when employing chitosan beads that were prepared from a 2% chitosan solution after 4 h of activation in 1% glutaraldehyde solution at pH 8. The activity expressed after immobilization by covalent bonding was 23% higher compared to the activity expressed following entrapment immobilization, with values of 122.3 and 99.4 IU.g⁻¹, respectively. Kinetic data revealed that catalytic turnover values were decreased as compared to a free counterpart. Both biocatalysts showed increased thermal and pH stability, along with an improved storage capacity, as they retained 88% and 40% of their activity after being stored at 4 °C for two months. Moreover, XynB2^Y509E immobilized by covalent binding also exhibited outstanding reusability, retaining 92% of activity after 10 cycles of reuse. In conclusion, our results suggest that the covalent bond method appears to be the best choice for XynB2^Y509E immobilization. Full article

Mesonia algae K4-1 from the Arctic secretes a novel cold-adapted and salt-tolerant protease EK4-1. It has the highest sequence similarity with Stearolysin, an M4 family protease from Geobacillus stearothermophilus, with only 45% sequence identity, and is a novel M4 family protease. Ek4-1 has a low optimal catalytic temperature (40 °C) and is stable at low temperatures. Moreover, EK4-1 is still active in 4 mol/L NaCl solution and is tolerant to surfactants, oxidizing agents and organic solvents; furthermore, it prefers the hydrolysis of peptide bonds at the P1’ position as the hydrophobic residues, such as Leu, Phe and Val, and amino acids with a long side chain, such as Phe and Tyr. Mn²⁺and Mg²⁺ significantly promoted enzyme activity, while Fe³⁺, Co⁺, Zn²⁺ and Cu²⁺ significantly inhibited enzyme activity. Amino acid composition analysis showed that EK4-1 had more small-side-chain amino acids and fewer large-side-chain amino acids. Compared with a thermophilic protease Stearolysin, the cold-adapted protease EK4-1 contains more random coils (48.07%) and a larger active pocket (727.42 ų). In addition, the acidic amino acid content of protease EK4-1 was higher than that of the basic amino acid, which might be related to the salt tolerance of protease. Compared with the homologous proteases EB62 and E423, the cold-adapted protease EK4-1 was more efficient in the proteolysis of grass carp skin, salmon skin and casein at a low temperature, and produced a large number of antioxidant peptides, with DPPH, ·OH and ROO· scavenging activities. Therefore, cold-adapted and salt-tolerant protease EK4-1 offers wide application prospects in the cosmetic and detergent industries. Full article

The antimicrobial and antioxidant effects of plant extracts are well known, but their use is limited because they affect the physicochemical and sensory characteristics of products. Encapsulation presents an option to limit or prevent these changes. The paper presents the composition of individual polyphenols (HPLC–DAD-ESI-MS) from basil (Ocimum basilicum L.) extracts (BE), and their antioxidant activity and inhibitory effects against strains of Staphylococcus aureus, Geobacillus stearothermophilus, Bacillus cereus, Candida albicans, Enterococcus faecalis, Escherichia coli, and Salmonella Abony. The BE was encapsulated in sodium alginate (Alg) using the drop technique. The encapsulation efficiency of microencapsulated basil extract (MBE) was 78.59 ± 0.01%. SEM and FTIR analyses demonstrated the morphological aspect of the microcapsules and the existence of weak physical interactions between the components. Sensory, physicochemical and textural properties of MBE-fortified cream cheese were evaluated over a 28-day storage time at 4 °C. In the optimal concentration range of 0.6–0.9% (w/w) MBE, we determined the inhibition of the post-fermentation process and the improvement in the degree of water retention. This led to the improvement of the textural parameters of the cream cheese, contributing to the extension of the shelf life of the product by 7 days. Full article

The article investigated the antioxidant and antimicrobial activity of extracts from two aromatic plants—Satureja hortensis L. (SE) and Rosmarinus officinalis L. (RE), encapsulated in alginate, on—yogurt properties. The encapsulation efficiency was controlled by FTIR and SEM analysis. In both extracts, the individual polyphenol content was determined by HPLC–DAD–ESI-MS. The total polyphenol content and the antioxidant activity were spectrophotometrically quantified. The antimicrobial properties of SE and RE against gram-positive bacteria (Bacillus cereus, Enterococcus faecalis, Staphylococcus aureus, Geobacillus stearothermophilus), gram-negative bacteria (Escherichia coli, Acinetobacter baumannii, Salmonella abony) and yeasts (Candida albicans) were analyzed in vitro. The encapsulated extracts were used to prepare the functional concentrated yogurt. It was established that the addition of 0.30–0.45% microencapsulated plant extracts caused the inhibition of the post-fermentation process, the improvement of the textural parameters of the yogurt during storage, thus the shelf life of the yogurt increased by seven days, compared to the yogurt simple. Mutual information analysis was applied to establish the correlation between the concentration of the encapsulated extracts on the sensory, physical-chemical, and textural characteristics of the yogurt. Full article

Fungal diseases caused by Alternaria alternata constitute a significant threat to the production and quality of a wide range of crops, including beans, fruits, vegetables, and grains. Traditional methods for controlling these diseases involve synthetic chemical pesticides, which can negatively impact the environment and human health. Biosurfactants are natural, biodegradable secondary metabolites of microorganisms that have also been shown to possibly have antifungal activity against plant pathogenic fungi, including A. alternata being sustainable alternatives to synthetic pesticides. In this study, we investigated the potential of biosurfactants of three bacilli (Bacillus licheniformis DSM13, Bacillus subtilis DSM10, and Geobacillus stearothermophilus DSM2313) as a biocontrol agent against A. alternata on beans as a model organism. For this fermentation, we describe using an in-line biomass sensor monitoring both permittivity and conductivity, which are expected to correlate with cell concentration and products, respectively. After the fermentation of biosurfactants, we first characterised the properties of the biosurfactant, including their product yield, surface tension decrement capability, and emulsification index. Then, we evaluated the antifungal properties of the crude biosurfactant extracts against A. alternata, both in vitro and in vivo, by analysing various plant growth and health parameters. Our results showed that bacterial biosurfactants effectively inhibited the growth and reproduction of A. alternata in vitro and in vivo. B. licheniformis manufactured the highest amount of biosurfactant (1.37 g/L) and demonstrated the fastest growth rate, while G. stearothermophilus produced the least amount (1.28 g/L). The correlation study showed a strong positive relationship between viable cell density VCD and OD600, as well as a similarly good positive relationship between conductivity and pH. The poisoned food approach in vitro demonstrated that all three strains suppressed mycelial development by 70–80% when applied with the highest tested dosage of 30%. Regarding in vivo investigations, B. subtilis post-infection treatment decreased the disease severity to 30%, whereas B. licheniformis and G. stearothermophilus post-infection treatment reduced disease severity by 25% and 5%, respectively. The study also revealed that the plant’s total height, root length, and stem length were unaffected by the treatment or the infection. Full article