Search Results (29)

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

A novel cyclic amylopectin (CA) was synthesized from waxy corn starch (WCS) using Bacillus stearothermophilus branching enzyme (BstBE), providing insights into its biosynthesis mechanism and solution properties. During the first 4 h, BstBE partially cyclized WCS, producing 68.20% CA with a significantly reduced molecular weight (M_W), from 8.98 × 10⁶ to 3.19 × 10⁴ g/mol and a lower polymer dispersity index (PDI), decreasing from 1.97 to 1.12. This resulted in a uniform CA structure with shorter chain lengths, particularly increasing DP 3–13, especially DP 7–9. Over the subsequent 4–12 h, the PDI slightly increased to 1.18 as the CA content decreased to 50.48%, with an increase in small ring structures (DP 6–12) of CA, suggesting both ring-opening and ring-downsizing due to continued enzyme catalysis. These results propose a two-stage reaction model: initial cyclization followed bybranching and secondary cyclization. CA exhibited excellent solution properties, with BE-4 and BE-12 samples demonstrating high solubility (≥65 g/100 mL), low viscosity (<0.01 Pa·s), and over 90% light transmittance after 14 days at 4 °C, highlighting its broad application potential. Full article

This study aimed to compare the efficiency of two methods for airborne surface decontamination: hydrogen peroxide vapor (HPV) and aerosolized hydrogen peroxide (aHP). Spores of G. stearothermophilus and B. atrophaeus were exposed to a 35% hydrogen peroxide solution under controlled laboratory conditions, including specific concentrations, exposure durations, humidity levels, and temperatures. Following each decontamination procedure, the spores were incubated for 7 days to evaluate bacterial growth and assess the efficacy of each method. The results indicate that the aHP method achieved biocidal rates of 84.76% for G. stearothermophilus and 89.52% for B. atrophaeus, while the HPV method demonstrated respective rates of 90.95% and 90.48%. These findings suggest that both the aHP and HPV methods are highly effective for microbial decontamination, with HPV showing a slight edge in overall efficacy. However, despite its comparable effectiveness, the HPV method has raised concerns regarding technical and economic factors. Observations highlighted issues such as fluctuations in humidity levels causing surface damage, a problem not encountered with the aHP method. Economically, HPV requires specific devices that can cost up to EUR 50,000, whereas aHP equipment costs do not exceed EUR 10,000. These observations emphasize the importance of critically evaluating the pros and cons of each decontamination method, taking into account factors such as biocidal efficacy, technical feasibility, and the associated costs. Full article

Transfer RNA (tRNA) modifications are essential for the temperature adaptation of thermophilic and psychrophilic organisms as they control the rigidity and flexibility of transcripts. To further understand how specific tRNA modifications are adjusted to maintain functionality in response to temperature fluctuations, we investigated whether tRNA modifications represent an adaptation of bacteria to different growth temperatures (minimal, optimal, and maximal), focusing on closely related psychrophilic (P. halocryophilus and E. sibiricum), mesophilic (B. subtilis), and thermophilic (G. stearothermophilus) Bacillales. Utilizing an RNA sequencing approach combined with chemical pre-treatment of tRNA samples, we systematically profiled dihydrouridine (D), 4-thiouridine (s⁴U), 7-methyl-guanosine (m⁷G), and pseudouridine (Ψ) modifications at single-nucleotide resolution. Despite their close relationship, each bacterium exhibited a unique tRNA modification profile. Our findings revealed increased tRNA modifications in the thermophilic bacterium at its optimal growth temperature, particularly showing elevated levels of s⁴U8 and Ψ55 modifications compared to non-thermophilic bacteria, indicating a temperature-dependent regulation that may contribute to thermotolerance. Furthermore, we observed higher levels of D modifications in psychrophilic and mesophilic bacteria, indicating an adaptive strategy for cold environments by enhancing local flexibility in tRNAs. Our method demonstrated high effectiveness in identifying tRNA modifications compared to an established tool, highlighting its potential for precise tRNA profiling studies. 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

A novel thermoactive phosphatidylcholine-specific phospholipase C (PC-PLC_Bs) was identified from Bacillus stearothermophilus isolated from a soil sample from an olive oil mill. Enhanced PLC_Bs production was observed after 10 h of incubation at 55 °C in a culture medium containing 1 mM of Zn²⁺ with an 8% inoculum size and 6 g/L glucose and 4/L yeast extract as the preferred carbon energy and nitrogen sources, respectively. PLC_Bs was purified to homogeneity by heat treatment, ammonium sulfate fractionation, and anion exchange chromatography, resulting in a purification factor of 17.6 with 39% recovery. Interestingly, this enzyme showed a high specific activity of 8450 U/mg at pH 8–9 and 60 °C, using phosphatidylcholine PC as the substrate, in the presence of 9 mM sodium deoxycholate and 0.4 mM Zn²⁺. Remarkable stability at acidic and alkali pH and up to 65 °C was also observed. PLC_Bs displayed a substrate specificity order of phosphatidylcholine > phosphatidylethanolamine > phosphatidylserine > sphingomyelin > phosphatidylinositol > cardiolipin and was classified as a PC-PLC. In contrast to phospholipases C previously isolated from Bacillus strains, this PLC_Bs substrate specificity was correlated to its hemolytic and anti-bacterial potential against erythrocytes and Gram-positive bacterial membranes, which are rich in glycerophospholipids and cardiolipin. An evaluation of PLC_Bs soybean degumming process efficiency showed that the purified enzyme reduced the phosphorus content to 35 mg/kg and increased the amount of diacylglycerols released, indicating its ability to hydrolyze phospholipids in the crude soybean oil. Collectively, PLC_Bs could be considered as a potential catalyst for efficient industrial oil degumming, advancing the edible oil industry by reducing the oil gum volume through transforming non-hydratable phospholipids into their hydratable forms, as well as through generating diacylglycerols, which are miscible with triacylglycerols, thereby reducing losses. 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

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

The genus Geobacillus comprises thermophilic gram-positive bacteria which are widely distributed, and their ability to withstand high temperatures makes them suitable for various applications in biotechnology and industrial production. Geobacillus stearothermophilus H6 is an extremely thermophilic Geobacillus strain isolated from hyperthermophilic compost at 80 °C. Through whole-genome sequencing and genome annotation analysis of the strain, the gene functions of G. stearothermophilus H6 were predicted and the thermophilic enzyme in the strain was mined. The G. stearothermophilus H6 draft genome consisted of 3,054,993 bp, with a genome GC content of 51.66%, and it was predicted to contain 3750 coding genes. The analysis showed that strain H6 contained a variety of enzyme-coding genes, including protease, glycoside hydrolase, xylanase, amylase and lipase genes. A skimmed milk plate experiment showed that G. stearothermophilus H6 could produce extracellular protease that functioned at 60 °C, and the genome predictions included 18 secreted proteases with signal peptides. By analyzing the sequence of the strain genome, a protease gene gs-sp1 was successfully screened. The gene sequence was analyzed and heterologously expressed, and the protease was successfully expressed in Escherichia coli. These results could provide a theoretical basis for the development and application of industrial strains. Full article

Ultraviolet (UV)-C irradiation is a promising method for microbial eradication on surfaces. Major developments have taken place in UV-C light-emitting diodes (LEDs) technology. In this study, we examined the suitability of UV-C LED-based surface disinfection in hospitals. We tested the efficacy of UV-C LED surface treatment on different microorganisms dried on a carrier surface or in a liquid solution. The influences of soiling, shading, surface material, radiation wavelength, microbial load and species on the disinfection performance were investigated. UV-C LED caused a reduction of >5 log₁₀ levels of E. coli, S. aureus and C. albicans, whereas 3 log₁₀ reduction was observed for G. stearothermophilus spores. The components of the medium led to a reduced UV-C LED efficiency compared to buffered solutions. We observed that the microbial load and the roughness of the carrier surface had a major influence on the UV-C LED disinfection efficiencies, whereas shading had no impact on inactivation. This study showed that UV-C is suitable for surface disinfection, but only under certain conditions. We showed that the main factors influencing microbial inactivation through UV-C light (e.g., intrinsic and extrinsic factors) had a similar impact when using a UV-C LED radiation source compared to a conventional UV-C lamp. However, the potential of LEDs is contributed by their adjustable wavelength and customizable geometry for the decontamination of medical devices and surfaces, and thereby their ability to overcome shading effects. Full article

Thermoduric sporeformers survive heat treatment and can form biofilm on contact food surfaces that is difficult to clean and may cause cross contamination to milk products. It was hypothesized that cavitation would influence sporeformers’ ability to attach to contact surfaces and form biofilm. Common dairy sporeformers of Geobacillus stearothermophilus, Bacillus licheniformis, and Bacillus sporothermodurans were individually inoculated in sterile skim milk at the levels of 6.0 log CFU/mL. Inoculated samples were treated by cavitation at 80% amplitudes for 10 min each. Pre and post samples were used to develop biofilms on stainless steel coupons under static conditions. Scanning electron micrograph was used to observe the developed biofilms. All the experiments were conducted in triplicate and were statistically analyzed using a t test. The average counts of spiked milk samples were 7.2, 8.0, and 7.7 logs CFU/mL, respectively, for the three sporeformers. Post-cavitation counts were reduced significantly to 3.4, 4.2, and 3.7 logs CFU/mL, respectively. Pre-cavitation biofilm counts of the three sporeformers were 5.35, 6.42, and 6.5 logs CFU/ cm², respectively in 72 h. The three sporeformers’ biofilm showed significantly (p < 0.05) lower counts after cavitation of 4.39, 5.44, and lower counts of 4.39 logs CFU/cm², respectively, for the three organisms. The result showed that G. stearothermophilus formed the least biofilms among others after cavitation. Although the ultrasonication treatment reduced the number of sporeformer bacteria, the survivors still retained the ability to attach to the stainless-steel food contact surfaces. Full article

Photocatalytic inactivation of pathogens in aqueous waste is gaining increasing attention. Several homogeneous and heterogeneous photocatalytic protocols exist using the Fenton’s reagent and TiO₂, respectively. A comprehensive study of homogeneous and heterogeneous photocatalysis on a range of microorganisms will significantly establish the most efficient method. Here, we report a comparative study of TiO₂- and Fe⁺³-based photocatalytic inactivation under UV-A of diverse microorganisms, including Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria, bacterial spores (Bacillus stearothermophilus spores) and viruses (MS2). We also present data on the optimization of TiO₂ photocatalysis, including optimal catalyst concentration and H₂O₂ supplementation. Our results indicate that both photo-Fenton and TiO₂ could be successfully applied for the management of microbial loads in liquids. Efficient microorganism inactivation is achieved with homogeneous photocatalysis (7 mg/L Fe⁺³, 100 mg/L H₂O₂, UV-A) in a shorter processing time compared to heterogeneous photocatalysis (0.5 g/L TiO₂, UV-A), whereas similar or shorter processing is required when heterogenous photocatalysis is performed using microorganism-specific optimized TiO₂ concentrations and H₂O₂ supplementation (100 mg/L); higher H₂O₂ concentrations further enhance the heterogenous photocatalytic inactivation efficiency. Our study provides a template protocol for the design and further application for large-scale photocatalytic approaches to inactivate pathogens in liquid biomedical waste. Full article

Here, we present an immobilized enzyme cascade in a basket-type reactor allowing a one-pot, two-step enzymatic synthesis of L-erythrulose from D-serine and glycolaldehyde. Three enzymes, D-amino acid oxidase from Rhodotorula gracilis (DAAO_Rg), catalase from bovine liver (CAT), and transketolase from Geobacillus stearothermophilus (TK_gst) were covalently immobilized on silica monolithic pellets, characterized by an open structure of interconnected macropores and a specific surface area of up to 300 m²/g. Three strategies were considered: (i) separate immobilization of enzymes on silica supports ([DAAO][CAT][TK]), (ii) co-immobilization of two of the three enzymes followed by the third ([DAAO+CAT][TK]), and (iii) co-immobilization of all three enzymes ([DAAO+CAT+TK]). The highest L-erythrulose concentrations were observed for the co-immobilization protocols (ii) and (iii) (30.7 mM and 29.1 mM, respectively). The reusability study showed that the best combination was [DAAO + CAT][TK], which led to the same level of L-erythrulose formation after two reuse cycles. The described process paves the way for the effective synthesis of a wide range of α-hydroxyketones from D-serine and suitable aldehydes. Full article

Traditional wastewater treatments involve expensive mechanical and physiochemical methods, so researchers have been developing cost-effective, sustainable technologies that use enzymes to produce higher quality effluents and recover more energy and nutrients from wastewater. A thermostable, alkaline, and solvent-tolerant lipase was partially purified from thermophilic Bacillus stearothermophilus. The lipase displayed maximum activity at 50 °C and pH 11.0 and catalyzed both short- and long-chain triacylglycerols at similar rates. B. stearothermophilus lipase also exhibited high stability when incubated at 40 °C for 1 h with anionic and non-ionic surfactants. Studies show that thermostable enzymes can be improved through immobilization and modification of other reaction conditions. Therefore, B. stearothermophilus lipase was immobilized through adsorption on CaCO₃, Celite 545, and silica gel with the CaCO₃ support producing the best adsorption rate (89.33%). The optimal initial lipase activity was approximately 4500 U.g⁻¹ after 60 min. Interestingly, 93% of the initial lipase activity was retained after six cycles, and almost 50% of the initial activity remained after 12 cycles. Furthermore, immobilization improved storage stability with 98.85% of the initial lipase activity retained after 60 days of storage at 4 °C. The biochemical characteristics of immobilized lipase shifted toward a slightly alkaline region, reaching maximum activity at pH 12. The optimal temperature of immobilized lipase was 60 °C. Immobilization also improved enzymatic stability by widening the pH range from 5–9 (for free lipase) to 4–11, and thermostability by reaching 65 °C. The application of immobilized lipase in wastewater treatment was observed through oil layer biodegradation. Notably, treating wastewater for 10 days with immobilized lipase almost removed the chemical oxygen demand (COD) from 1950.1 down to 4.04 mg.L⁻¹. Similarly, lipid content was almost removed from 15,500 ± 546 mg.L⁻¹ down to 12 mg.L⁻¹. All results highlight the potential value of CaCO₃-immobilized lipase as an effective biocatalyst for hydrolyzing wastewater. Full article

In this study, a new device was used to inactivate G. stearothermophilus spores in ready-to-eat (RTE) poached spicy pork slices (PSPS) applying radio frequency (RF) energy (27.12 MHz, 6 kW) and superheated water (SW) simultaneously. The cold spot in the PSPS sample was determined. The effects of electrode gap and SW temperature on heating rate, spore inactivation, physiochemical properties (water loss, texture, and oxidation), sensory properties, and SEM of samples were investigated. The cold spot lies in the geometric center of the soup. The heating rate increased with increasing electrode gap and hit a peak under 190 mm. Radio frequency combined superheated water (RFSW) sterilization greatly decreased the come-up time (CUT) compared with SW sterilization, and a 5 log reduction in G. stearothermophilus spores was achieved. RFSW sterilization under 170 mm electrode gap reduced the water loss, thermal damage of texture, oxidation, and tissues and cells of the sample, and kept a better sensory evaluation. RFSW sterilization has great potential in solid or semisolid food processing engineering. Full article