Unravelling the Influence of Extraction Techniques on Protein Yield and Nutritional Value in Lesser Mealworm Larvae.

In the present work, chemical and enzymatic assisted techniques were compared for protein extraction from lesser mealworm larvae (LM, Alphitobius diaperinus), recently approved as a novel food in the European Union. All extracts showed appreciable nutritional quality, with quantities of essential amino acids above the reference standard. Conventional alkali extraction allowed the isolation of only 73% of the protein, preserving the amino acid composition but potentially causing denaturation or racemisation. The "stepwise" method, following the Osborne fractionation, improved protein recovery to 91% by isolating four fractions with different solubility properties. Additionally, enzymatic hydrolysis using Bacillus licheniformis proteases was also tested, and it provided hydrolysates with an average degree of hydrolysis of 14%, making them a potential hypoallergenic solution. Overall, these findings indicate the ability to tailor the composition of LM protein to meet specific needs, offering promising prospects for the use of insect protein ingredients in various applications.

Thermophilic Hemicellulases Secreted by Microbial Consortia Selected from an Anaerobic Digester.

The rise of agro-industrial activities over recent decades has exponentially increased lignocellulose biomasses (LCB) production. LCB serves as a cost-effective source for fermentable sugars and other renewable chemicals. This study explores the use of microbial consortia, particularly thermophilic consortia, for LCB deconstruction. Thermophiles produce stable enzymes that retain activity under industrial conditions, presenting a promising approach for LCB conversion. This research focused on two microbial consortia (i.e., microbiomes) that were analyzed for enzyme production using a cheap medium, i.e., a mixture of spent mushroom substrate (SMS) and digestate. The secreted xylanolytic enzymes were characterized in terms of temperature and pH optima, thermal stability, and hydrolysis products from LCB-derived polysaccharides. These enzymes showed optimal activity aligning with common biorefinery conditions and outperformed a formulated enzyme mixture in thermostability tests in the digestate. Phylogenetic and genomic analyses highlighted the genetic diversity and metabolic potential of these microbiomes. Bacillus licheniformis was identified as a key species, with two distinct strains contributing to enzyme production. The presence of specific glycoside hydrolases involved in the cellulose and hemicellulose degradation underscores these consortia's capacity for efficient LCB conversion. These findings highlight the potential of thermophilic microbiomes, isolated from an industrial environment, as a robust source of robust enzymes, paving the way for more sustainable and cost-effective bioconversion processes in biofuel and biochemical production and other biotechnological applications.

Bacillus licheniformis suppresses Clostridium perfringens infection via modulating inflammatory response, antioxidant status, inflammasome activation and microbial homeostasis in broilers.

Pathogenic bacteria infection, especially Clostridium perfringens (C. perfringens), markedly threatened the health of animals, and further caused huge economic loss. In this study, Bacillus licheniformis HJ0135 (BL) was used. Oxford cup bacteriostatic test and inhibitory rate test were conducted to evaluate the antibacterial ability of BL. Results showed the strongest inhibitory role of BL on C. perfringens (P < 0.05). Afterwards, 540 one-day-old yellow-feather broilers (32.7 ± 0.2 g) were randomly allocated into 3 groups, including CON group (basal diet), CP group (basal diet + 1 × 109 CFU C. perfringens in gavage), and BL + CP group (basal diet containing 7.5 × 106 CFU/g BL + 1 × 109 CFU C. perfringens in gavage). At d 70, broilers in the CP and BL + CP groups were treated with C. perfringens by continuously oral administration for 5 d. The experiment lasted for 75 d. The serum, immune organs, jejunal mucosa, and cecal contents were collected for analysis. In vivo experiment showed that BL supplementation markedly improved (P < 0.05) BW, ADG, thymus index, serum immunoglobins and antioxidases, reduced feed conversion ratio (FCR) and serum pro-inflammatory cytokines of C. perfringens-infected broilers. Furthermore, the increased jejunal injury and levels of pro-inflammatory cytokines, decreased gene expressions of tight junction proteins in the jejunal mucosa were significantly alleviated (P < 0.05) by BL. More importantly, the activation of NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome was inhibited (P < 0.05) by BL to further attenuate jejunal damage. Besides, BL supplementation markedly increased (P < 0.05) the cecal isobutyric acid and isovaleric acid. Microbial analysis showed that BL changed the composition and relative abundances of microbiota in the cecal contents (P < 0.05), especially the short chain fatty acids (SCFAs)-producing bacteria including Eubacterium_coprostanoligenes_group, Megamonas, Faecalibacterium, and Lactobacillus, which further protected against C. perfringens-induced jejunal inflammation in broilers. Our study laid a theoretical basis for the application of probiotics in lessening C. perfringens-related diseases in poultry farming.

Effects of a Bacillus-based direct-fed microbial on performance, blood parameters, fecal characteristics, rumen morphometrics, and intestinal gene expression in finishing beef bulls.

We evaluated the effects of supplementing direct-fed microbials (DFM), containing Bacillus licheniformis and Bacillus subtilis, on performance, rumen morphometrics, intestinal gene expression, and blood and fecal parameters in finishing bulls. Nellore × Angus bulls (n = 144; initial BW = 401 ±â€…45.5 kg) were distributed at random in 36 pens (4 bulls/pen and 18 pens/treatment), following a completely randomized design. A ground corn-based finishing diet was offered for ad libitum intake twice a day for 84 d, containing the following treatments: 1) control (without DFM); 2) DFM (B. licheniformis and B. subtilis) at 6.4 × 109 CFU (2 g) per animal. The data were analyzed using the MIXED procedure of SAS, with a pen representing an experimental unit, the fixed effect of the treatment, and the random effect of pen nested within the treatment. For fecal parameters (two collections made), the collection effect and its interaction with the treatment were included in the model. Bulls that received the DFM had a decreased dry matter intake (P ≤ 0.01), did not differ in average daily gain (2.05 kg; P = 0.39), and had a 6% improvement in gain:feed (P = 0.05). The other performance variables, final BW, hot carcass weight, and hot carcass yield, did not differ (P > 0.10). Plasma urea-N concentration decreased by 6.2% (P = 0.02) in the bulls that received DFM. Glucose, haptoglobin, and lipopolysaccharides were not different between treatments (P > 0.10). Ruminal morphometrics were not affected by the treatment (P > 0.10). The use of DFM tended to reduce fecal starch (P = 0.10). At slaughter, bulls fed DFM had an increased duodenal gene expression of tryptophan hydroxylase-1 (P = 0.02) and of superoxide dismutase-1 (P = 0.03). Overall, supplementation with DFM based on B. licheniformis and B. subtilis to Nellore × Angus bulls in the finishing phase decreased dry matter intake, did not influence ADG, improved gain:feed, and increased the expression of genes important for duodenal function.

One of the main alternatives of additives to modulate the microbial population in the gastrointestinal tract (GIT), especially in the intestine, is the use of direct-fed microbials (DFM). This class of additives comprises all the feed products that contain a live or naturally occurring source of microorganism. The inclusion of DFM in diets of ruminants in the finishing phase may improve gain:feed by modifying the composition of the microbial community in the GIT to bring about a better symbiotic relationship with the host. These effects may be achieved with the use of Bacillus spp. bacteria, such as Bacillus licheniformis and Bacillus subtilis. Mixtures of these bacteria are able to foster positive effects in the finishing phase of beef cattle fed high-energy diets, which reinforces the need for studies that examine the effects and mechanisms of these species. In this study, feedlot Nellore × Angus bulls that received a DFM composed of B. licheniformis and B. subtilis had decreased dry matter intake, no influence on average daily gain, improved gain:feed, and an increase in expression of genes important for duodenal function.

Cloning, characterization and functional analysis of lichenase produced by Bacillus licheniformis RB16 isolated from cattle faeces.

Lichenan, 1,3-1,4-ß-Glucan, a linear polysaccharide exists in the cell walls of various cereals, has garnered attention for its industrial applications due to its enzymatic breakdown by lichenase enzymes. In this study, Bacillus licheniformis strain RB16, isolated from cattle faeces, was identified as a robust lichenase producer. The lichenase gene, licA, was successfully cloned and characterized. The cloned RB16 lichenase (LicA) demonstrated its highest activity level at pH 7.5. It also retained over 50% of its activity within the pH range of 6.0-8.5 but experienced a decline to 40% at pH 9.0. LicA was active at temperatures ranging from 25 to 65 °C with an optimum at 45 °C. LicA exhibited more than 60% of its activity at the temperature range of 35-55 °C. Zymogram analysis confirmed LicA's lichenan-degrading ability and structural analysis revealed a stable enzyme structure primarily composed of random coils and extended strands. Although LicA exhibited low thermostability, consistent with its relatively low α-helix content, it demonstrated promising industrial potential. Evolutionary analysis placed LicA within a cluster of closely related Bacillus lichenases, particularly B. halotolerans, B. atrophaeus, and B. spizizenii. These findings expand our understanding of lichenases of Bacillus and underscore its potential for various industrial applications.

Insights into the genomic and phenotypic characteristics of Bacillus spp. strains isolated from biofilms in broiler farms.

The characterization of surface microbiota living in biofilms within livestock buildings has been relatively unexplored, despite its potential impact on animal health. To enhance our understanding of these microbial communities, we characterized 11 spore-forming strains isolated from two commercial broiler chicken farms. Sequencing of the strains revealed them to belong to three species Bacillus velezensis, Bacillus subtilis, and Bacillus licheniformis. Genomic analysis revealed the presence of antimicrobial resistance genes and genes associated with antimicrobial secretion specific to each species. We conducted a comprehensive characterization of the biofilm formed by these strains under various conditions, and we revealed significant structural heterogeneity across the different strains. A macro-colony interaction model was employed to assess the compatibility of these strains to coexist in mixed biofilms. We identified highly competitive B. velezensis strains, which cannot coexist with other Bacillus spp. Using confocal laser scanning microscopy along with a specific dye for extracellular DNA, we uncovered the importance of extracellular DNA for the formation of B. licheniformis biofilms. Altogether, the results highlight the heterogeneity in both genome and biofilm structure among Bacillus spp. isolated from biofilms present within livestock buildings.IMPORTANCELittle is known about the microbial communities that develop on farms in direct contact with animals. Nonpathogenic strains of Bacillus velezensis, Bacillus subtilis, and Bacillus licheniformis were found in biofilm samples collected from surfaces in contact with animals. Significant genetic and phenotypic diversity was described among these Bacillus strains. The strains do not possess mobile antibiotic resistance genes in their genomes and have a strong capacity to form structured biofilms. Among these species, B. velezensis was noted for its high competitiveness compared with the other Bacillus spp. Additionally, the importance of extracellular DNA in the formation of B. licheniformis biofilms was observed. These findings provide insights for the management of these surface microbiota that can influence animal health, such as the use of competitive strains to minimize the establishment of undesirable bacteria or enzymes capable of specifically deconstructing biofilms.

Biosynthesis of silver nanoparticle using Bacillus licheniformis culture-supernatant for combating pathogenic biofilms.

Bacterial biofilms pose a significant threat to healthcare due to their recalcitrance to antibiotics and disinfectants. This study explores the anti-biofilm potential of Bacillus licheniformis cell-free culture supernatant (CFS) and its derived silver nanoparticles (bSNPs) against Staphylococcus aureus and Pseudomonas aeruginosa. The CFS exhibited potent anti-biofilm activity against both bacterial species, even at low concentrations, while devoid of significant bactericidal effects, mitigating resistance risks. Characterization studies revealed the non-proteinaceous nature and thermal stability of the CFS's anti-biofilm agent, suggesting a robust and heat-resistant structure. Green synthesis of bSNPs from CFS resulted in nanoparticles with significant anti-biofilm properties, particularly against P. aeruginosa, indicating differences in susceptibility between the bacterial species. Epifluorescence microscopy confirmed bSNPs' ability to inhibit and partially disrupt biofilm formation without inducing cellular lysis. The study highlights the potential of B. licheniformis CFS and bSNPs as promising biofilm control agents, offering insights into their mechanisms of action and broad-spectrum efficacy. Further research elucidating the underlying molecular mechanisms and identifying specific bioactive compounds is warranted for the translation of these findings into clinically relevant applications for combating biofilm-associated infections.

Isolation of Bacillus licheniformis and its protective effect on liver oxidative stress and apoptosis induced by aflatoxin B1.

Aflatoxin B1 (AFB1) is one of the most toxic mycotoxins. The use of probiotics is an effective approach to reduce aflatoxins content in foods. To find efficient bacterial species that can eliminate or detoxify AFB1, a bacterial strain S51 capable of degrading AFB1 was isolated from chicken intestine and soil samples by using a culture medium containing coumarin as the sole carbon source. Based on the results of 16S rRNA gene sequence analysis, this isolate (strain S51) was identified as Bacillus licheniformis strain QT338. Further characterization of strain S51 showed that it could degrade AFB1 by 61.3% after incubation at 30°C for 72 h. Additional studies demonstrated that S51 promoted good growth performance of the treated chickens, showed no hemolytic activity, carried few drug resistance genes, and exhibited a certain level of tolerance to acid and bile salts. Furthermore, to verify whether strain S51 exerts a protective effect on AFB1-induced liver injury in chickens and to elucidate the underlying mechanism, a chicken toxicity model was induced with AFB1 (100 µg/kg BW) and treated with S51(1×109CFU/mL) for 12 d. The results showed that S51 decreased the level of alanine transaminase, aspartate transaminase, and total bilirubin (P < 0.05); increased glutathione activity and total antioxidant capacityin the liver induced by AFB1, and decreased malondialdehyde production (P < 0.05). S51 also up-regulated the mRNA expression level of the antioxidant proteins HO-1 and Nrf2 and down-regulated the expression of the oxidation-related factor Keap1 in the Nrf2/Keap1 signaling pathway (P <0.05). S51 inhibited hepatocyte apoptosis induced by AFB1 and decreased the mRNA expression levels of the apoptosis-related genes Bax, caspase-3, caspase-9, and Cyt-C (P < 0.05). These results indicate that S51 regulates apoptosis and alleviates AFB1-induced oxidative stress in chicken liver by controlling the Nrf2/Keap1 signaling pathway.

Machine learning-guided multi-site combinatorial mutagenesis enhances the thermostability of pectin lyase.

Enhancing the thermostability of enzymes is crucial for industrial applications. Methods such as directed evolution are often limited by the huge sequence space and combinatorial explosion, making it difficult to obtain optimal mutants. In recent years, machine learning (ML)-guided protein engineering has become an attractive tool because of its ability to comprehensively explore the sequence space of enzymes and discover superior mutants. This study employed ML to perform combinatorial mutation design on the pectin lyase PMGL-Ba from Bacillus licheniformis, aiming to improve its thermostability. First, 18 single-point mutants with enhanced thermostability were identified through semi-rational design. Subsequently, the initial library containing a small number of low-order mutants was utilized to construct an ML model to explore the combinatorial sequence space (theoretically 196,608 mutants) of single-point mutants. The results showed that the ML-predicted second library was successfully enriched with highly thermostable combinatorial mutants. After one iteration of learning, the best-performing combinatorial mutant in the third library, P36, showed a 67-fold and 39-fold increase in half-life at 75 °C and 80 °C, respectively, as well as a 2.1-fold increase in activity. Structural analysis and molecular dynamics simulations provided insights into the improved performance of the engineered enzyme.

Simultaneous removal of malachite green and lead from water by consortium dry-biomasses of Bacillus licheniformis AG3 and Bacillus cereus M116.

Synthetic textile dye malachite green (MG) and heavy metals present in industrial wastewater are hazardous to the ecosystem. Bioremediation of dyes and heavy metals using dry-biomasses has advantages over chemical methods. This study screened an acclimatized, heavy metal-resistant, and dye-degrading Gram positive Bacillus licheniformis AG3 strain from the textile wastewater near Kolkata, West Bengal. The EDXRF analysis of this colored wastewater effluent showed 36.33 mg/L lead, significantly higher than the WHO recommendation. Previously, Bag et al. showed bioremediation of synthetic dyes using dry-biomass of Bacillus cereus M116 from an aqueous solution (Bag et al. Arch Microbiol 203(7):3811-3823, 2021). Here, a consortium of dry-biomasses of B. licheniformis AG3 and B. cereus M116 strains (1:1 w/w ratio) was prepared for the simultaneous removal of lead and MG from wastewater. Statistical optimization determines that the pH, initial concentration of contaminants, and dry-biomass concentrations are critical for bioremediation under batch procedures. Further, optimization using the response surface methodology showed that 0.01% consortium dry-biomasses eliminated a maximum of 99.35% MG and 96.01% lead (II) within 6 h. SEM-EDS and FTIR confirmed a strong surface biosorption. Furthermore, a fixed-bed biofilter column of the consortium dry-biomasses was prepared, which was able to remove 98.1% MG and 98.5% lead at the 0.5-1 mL/min flow rate. Together, this study developed a biofilter with a consortium dry biomasses of B. licheniformis AG3 and B. cereus M116 for the simultaneous removal of MG and lead from wastewater.

Antioxidant and Emulsifying Activity of the Exopolymer Produced by Bacillus licheniformis.

The exopolymer (ESPp) was obtained from Bacillus licheniformis IDN-EC, composed of a polyglutamic acid and polyglycerol phosphate chain O-substituted with αGal moieties (αGal/αGlcNH2 3:1 molar ratio) and with a 5000 Da molecular weight. The cytotoxicity activity of EPSp was determined by reducing the MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide) to formazan on HeLa cells. This EPS did not show cytotoxicity against the tested cell line. The ESPp presented great advantages as an antioxidant with free radical scavenging activities (1,1-diphenyl-2-picryl-hydrazyl radical (DPPH),hydroxyl radical (OH), and superoxide anion (O2-)) (65 ± 1.2%, 98.7 ± 1.9%, and 97 ± 1.7%), respectively. Moreover, EPSp increased the enzyme activity for catalase (CAT) and glutathione peroxidase (GSH-Px) in HeLa cells (CAT, 2.6 ± 0.24 U/mL; and GSH-Px, 0.75 ± 0.3 U/L). The presence of ESPp showed a significant protective effect against H2O2 in the cell line studied, showing great viability (91.8 ± 2.8, 89.9 ± 2.9, and 93.5 ± 3.6%). The EPSp presented good emulsifying activity, only for vegetable oils, olive oil (50 ± 2.1%) and sesame (72 ± 3%). Sesame was effective compared to commercials products, Triton X-100 (52.38 ± 1.6%), Tween 20 (14.29 ± 1.1%), and sodium dodecyl sulphate (SDS) (52.63 ± 1.6%). Furthermore, the EPS produced at 0.6 M has potential for environmental applications, such as the removal of hazardous materials by emulsification whilst resulting in positive health effects such as antioxidant activity and non-toxicity. EPSp is presented as a good exopolysaccharide for various applications.

Bacillus licheniformis Jrh14-10 enhances alkaline tolerance in Arabidopsis thaliana by regulating crosstalk between ethylene and polyamine pathways.

Plant growth-promoting rhizobacteria (PGPR) are known for their role in ameliorating plant stress, including alkaline stress, yet the mechanisms involved are not fully understood. This study investigates the impact of various inoculum doses of Bacillus licheniformis Jrh14-10 on Arabidopsis growth under alkaline stress and explores the underlying mechanisms of tolerance enhancement. We found that all tested doses improved the growth of NaHCO3-treated seedlings, with 109 cfu/mL being the most effective. Transcriptome analysis indicated downregulation of ethylene-related genes and an upregulation of polyamine biosynthesis genes following Jrh14-10 treatment under alkaline conditions. Further qRT-PCR analysis confirmed the suppression of ethylene biosynthesis and signaling genes, alongside the activation of polyamine biosynthesis genes in NaHCO3-stressed seedlings treated with Jrh14-10. Genetic analysis showed that ethylene signaling-deficient mutants (etr1-3 and ein3-1) exhibited greater tolerance to NaHCO3 than the wild type, and the growth-promoting effect of Jrh14-10 was significantly diminished in these mutants. Additionally, Jrh14-10 was found unable to produce 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, indicating it does not reduce the ethylene precursor ACC in Arabidopsis. However, Jrh14-10 treatment increased the levels of polyamines (putrescine, spermidine, and spermine) in stressed seedlings, with spermidine particularly effective in reducing H2O2 levels and enhancing Fv/Fm under NaHCO3 stress. These findings reveal a novel mechanism of PGPR-induced alkaline tolerance, highlighting the crosstalk between ethylene and polyamine pathways, and suggest a strategic redirection of S-adenosylmethionine towards polyamine biosynthesis to combat alkaline stress.

Unveiling the matrix effect on Bacillus licheniformis and Bacillus subtilis spores heat inactivation between plant-based milk alternatives, bovine milk and culture medium.

This study examined the inactivation of spores of Bacillus licheniformis and Bacillus subtilis in four pea-based milk alternatives, semi-skimmed bovine milk and Brain Heart Infusion (BHI) broth to assess the matrix impact on the thermal inactivation of bacterial spores. Heat inactivation was performed with the method of capillary tubes in temperature range 97-110 °C. A four-parameter non-linear model, including initial level, shoulder duration, inactivation rate and tailing, was fitted to the data obtained. D-values were estimated and secondary ZT-value models were developed for both species. A secondary model for the shoulder length of B. licheniformis in a plant-based milk alternative formulation was built too. Models were validated at a higher temperature, 113.5 °C. D-values in the different matrices ranged between 2.3 and 8.2 min at 97 °C and 0.1-0.3 min at 110 °C for B. licheniformis. D-values for B. subtilis ranged between 3.9 and 6.3 min at 97 °C and 0.2-0.3 min at 110 °C. ZT-values in the different matrices ranged between 7.3 and 8.9 °C and 8.9-10.0 °C for B. licheniformis and B. subtilis, respectively. Significant differences in inactivation parameters were found within the pea-based formulations as well as when compared to bovine milk. Heat resistance was higher in pea-based matrices. Shoulders observed were temperature- and matrix-dependent, while no such trend was found for the tailings. These results provide insights, useful on designing safe thermal processing, limiting spoilage in plant-based milk alternatives and thus, reducing global food waste.

Transcription factor AbrB regulates ROS generation and clearance in Bacillus licheniformis.

Oxidative damage caused by the accumulation of reactive oxygen species (ROS) is one of the main obstacles to the improvement of microbial cell growth and fermentation characteristics under adverse environments. And the antioxidant capacity of cells will increase with the cell growth. Here, we found that a transition state transcription factor AbrB related to changes in cell growth status could regulate the accumulation of ROS and antioxidant capacity in Bacillus licheniformis. The results showed that the accumulation of intracellular ROS was reduced by 23.91 % and the cell survival rates were increased by 1.77-fold under 0.5 mM H2O2 when AbrB was knocked out. We further mapped regulatory target genes of AbrB related to ROS generation or clearance based on our previously analyzed transcriptome sequencing. It proved that AbrB could promote ROS generation via upregulating the synthesis of oxidase and siderophores, and negatively regulating the synthesis of iron chelators (pulcherriminic acid, and H2S). Additionally, AbrB could inhibit ROS clearance by negatively regulating the synthesis of antioxidase (superoxide dismutase, catalase, peroxidase, thioredoxin, thioredoxin reductase) and cysteine. Those results illustrated that the inactivation of AbrB during the stationary phase, along with its control over ROS generation and clearance, might represent a vital self-protection mechanism during cell evolution. Overall, the systematic investigation of the multi-pathway regulation network of ROS generation and clearance highlights the important function of AbrB in maintaining intracellular redox balance.

Engineering Bacillus licheniformis as industrial chassis for efficient bioproduction from starch.

Starch is an attractive feedstock in biorefinery processes, while the low natural conversion rate of most microorganisms limits its applications. Herein, starch metabolic pathway was systematically investigated using Bacillus licheniformis DW2 as the host organism. Initially, the effects of overexpressing amylolytic enzymes on starch hydrolysis were evaluated. Subsequently, the transmembrane transport system and intracellular degradation module were modified to accelerate the uptake of hydrolysates and their further conversion to glucose-6-phosphate. The DW2-derived strains exhibited robust growth in starch medium, and productivity of bacitracin and subtilisin were improved by 38.5% and 32.6%, with an 32.3% and 22.9% increase of starch conversion rate, respectively. Lastly, the employment of engineering strategies enabled another B. licheniformis WX-02 to produce poly-γ-glutamic acid from starch with a 2.1-fold increase of starch conversion rate. This study not only provided excellent B. licheniformis chassis for sustainable bioproduction from starch, but shed light on researches of substrate utilization.

Optimization of fermentation parameters to improve the biosynthesis of selenium nanoparticles by Bacillus licheniformis F1 and its comprehensive application.

BACKGROUND: Selenium nanoparticles (SeNPs) are increasingly gaining attention due to its characteristics of low toxicity, high activity, and stability. Additionally, Bacillus licheniformis, as a probiotic, has achieved remarkable research outcomes in diverse fields such as medicine, feed processing, and pesticides, attracting widespread attention. Consequently, evaluating the activity of probiotics and SeNPs is paramount. The utilization of probiotics to synthesize SeNPs, achieving large-scale industrialization, is a current hotspot in the field of SeNPs synthesis and is currently the most promising synthetic method. To minimize production costs and maximize yield of SeNPs, this study selected agricultural by-products that are nutrient-rich, cost-effective, and readily available as culture medium components. This approach not only fulfills industrial production requirements but also mitigates the impact on downstream processes. RESULTS: The experimental findings revealed that SeNPs synthesized by B. licheniformis F1 exhibited a spherical morphology with diameters ranging from 110 to 170 nm and demonstrating high stability. Both the secondary metabolites of B. licheniformis F1 and the synthesized SeNPs possessed significant free radical scavenging ability. To provide a more robust foundation for acquiring large quantities of SeNPs via fermentation with B. licheniformis F1, key factors were identified through single-factor experiments and response surface methodology (RSM) include a 2% seed liquid inoculum, a temperature of 37 ℃, and agitation at 180 rpm. Additionally, critical factors during the optimization process were corn powder (11.18 g/L), soybean meal (10.34 g/L), and NaCl (10.68 g/L). Upon validating the optimized conditions and culture medium, B. licheniformis F1 can synthesize nearly 100.00% SeNPs from 5 mmol/L sodium selenite. Subsequently, pilot-scale verification in a 5 L fermentor using the optimized medium resulted in a shortened fermentation time, significantly reducing production costs. CONCLUSION: In this study, the efficient production of SeNPs by the probiotic B. licheniformis F1 was successfully achieved, leading to a significant reduction in fermentation costs. The exploration of the practical applications of this strain holds significant potential and provides valuable guidance for facilitating the industrial-scale implementation of microbial synthesis of SeNPs.

Supplementing a Bacillus-based probiotic to high-risk stocker cattle.

This experiment evaluated the performance, health, and physiological responses of high-risk steers receiving a Bacillus-based probiotic during a 90-d grazing period. A total of 240 Angus-influenced steers were used in this experiment that was replicated over 2 yr (120 steers/year). Each year, steers were obtained from an auction yard and transported to the experimental facility (120 km). Steer body weight (BW) was recorded at arrival (day -1), and this value was averaged with BW recorded on day 0 to represent the initial BW (236.6 ± 1.5 kg). On day 0, steers were ranked by BW and allocated to 1 of 12 pastures with stockpiled native grass (4-ha pastures; 10 steers/pasture). Pastures were randomly assigned to receive daily supplementation with dried distillers' grains at 1% of BW containing either: 1) Bacillus subtilis + B. licheniformis probiotic (BOV; 2 g/steer daily of Bovacillus; Novonesis, Horsholm, Denmark) or 2) no feed additive (CON). Cattle received treatments from days 0 to 90, in addition to free-choice access to water and mineral + vitamin mix without ionophore. Steers were assessed for bovine respiratory disease (BRD) signs daily. Blood samples were collected and full BW was recorded on days 0, 14, 28, 56, and 90. Shrunk BW was recorded on day 91 after 16 h of feed and water restriction, and a 4% pencil shrink was used to calculate the final BW. Average daily gain (ADG) was calculated based on initial and final BW. No treatment effects were detected (P ≥ 0.73) for steer final BW and ADG. A treatment × day interaction was detected (P ≤ 0.05) for plasma haptoglobin concentration, which was greater for CON steers on days 14 and 28 (P ≤ 0.02). Incidence of BRD signs did not differ (P = 0.97) between treatments (51.7% and 51.3% for BOV and CON, respectively; SEM = 7.70). However, steer mortality + removals for health complications were greater (P = 0.01) in CON compared to BOV (0.00% vs. 5.04%, respectively; SEM = 1.41). Supplementing BOV improved (P ≤ 0.04) total pasture-based liveweight change (643 vs. 502 kg/pasture, respectively; SEM = 45) and final pasture-based total liveweight (3,007 vs. 2,869 kg/pasture, respectively; SEM = 46). Collectively, supplementation with a probiotic based on B. subtilis and B. licheniformis to high-risk stocker cattle did not alleviate the incidence of BRD signs nor improved ADG, but decreased acute-phase protein response, reduced steer mortality + removal, and increased pasture-based productivity during a 90-d grazing period.

Stocker cattle are exposed to several stressors within a short period of time, which impair their immunity and lead to bovine respiratory disease (BRD). With the increased regulations regarding the use of antimicrobials in cattle nutrition, novel dietary strategies to improve health and productivity of stocker cattle are warranted. One example is supplementing Bacillus-based probiotics, which promote performance and immunity in high-stress cattle. In this study, steers were purchased from a commercial auction yard soon after weaning, transported to the research facility, and assigned initial processing within a 48-h period. Steers were assigned to pastures and were supplemented or not with the Bacillus-based probiotic during a 90-d grazing period. In general, supplementing steers with the Bacillus-based probiotic did not impact growth rates or BRD incidence. However, no steers that received the Bacillus-based probiotic died from BRD consequences nor were removed from the experiment due to health reasons, whereas 5% of unsupplemented steers did not complete the 90-d experiment. Consequently, pasture-based liveweight gain was increased by 28% due to Bacillus-based probiotic supplementation. Results from this study indicate that supplementing a B. subtilis + B. licheniformis probiotic could be an alternative to improve the health and overall productivity of high-risk stocker cattle.

Invited review: Role of Bacillus licheniformis in the dairy industry-Friends or foes?

Bacillus licheniformis is one of the major spore-forming bacteria with great genotypic diversity in raw milk, dairy ingredients, and final dairy products; it is found throughout the dairy-processing continuum. Although being widely used as a probiotic strain, this species also serves as a potential risk in the dairy industry based on its roles in foodborne illness and dairy spoilage. Biofilm formation of B. licheniformis, combined with the heat resistance of its spores, make it impossible to prevent the presence of B. licheniformis in final dairy products by using traditional cleaning and disinfection procedures. Despite the extensive efforts to identify B. licheniformis in various dairy samples, no reviews have been written on both hazards and benefits of this sporeformer. This review discusses the prevalence of B. licheniformis from raw milk to commercial dairy products, biofilm formation and spoilage potential of B. licheniformis, and possible prevention methods. In addition, the potential benefits of B. licheniformis in the dairy industry are also summarized.

LC-MS/MS profiling and analysis of Bacillus licheniformis extracellular proteins for antifungal potential against Candida albicans.

Candida albicans, a significant human pathogenic fungus, employs hydrolytic proteases for host invasion. Conventional antifungal agents are reported with resistance issues from around the world. This study investigates the role of Bacillus licheniformis extracellular proteins (ECP) as effective antifungal peptides (AFPs). The aim was to identify and characterize the ECP of B. licheniformis through LC-MS/MS and bioinformatics analysis. LC-MS/MS analysis identified 326 proteins with 69 putative ECP, further analyzed in silico. Of these, 21 peptides exhibited antifungal properties revealed by classAMP tool and are predominantly anionic. Peptide-protein docking revealed interactions between AFPs like Peptide chain release factor 1 (Q65DV1_Seq1: SASEQLSDAK) and Putative carboxy peptidase (Q65IF0_Seq7: SDSSLEDQDFILESK) with C. albicans virulent SAP5 proteins (PDB ID 2QZX), forming hydrogen bonds and significant Pi-Pi interactions. The identification of B. licheniformis ECP is the novelty of the study that sheds light on their antifungal potential. The identified AFPs, particularly those interacting with bonafide pharmaceutical targets SAP5 of C. albicans represent promising avenues for the development of antifungal treatments with AFPs that could be the pursuit of a novel therapeutic strategy against C. albicans. SIGNIFICANCE OF STUDY: The purpose of this work was to carry out proteomic profiling of the secretome of B. licheniformis. Previously, the efficacy of Bacillus licheniformis extracellular proteins against Candida albicans was investigated and documented in a recently communicated manuscript, showcasing the antifungal activity of these proteins. In order to achieve high-throughput identification of ES (Excretory-secretory) proteins, the utilization of liquid chromatography tandem mass spectrometry (LC-MS) was utilized. There was a lack of comprehensive research on AFPs in B. licheniformis, nevertheless. The proteins secreted by B. licheniformis in liquid medium were initially discovered using liquid chromatography-tandem mass spectrometry (LC-MS) analysis and identification in order to immediately characterize the unidentified active metabolites in fermentation broth.

Heterologous synthesis of poly-γ-glutamic acid enhanced drought resistance in maize (Zea mays L.).

Drought stress is the main factor restricting maize yield. Poly-γ-glutamic acid (γ-PGA), as a water-retaining agent and fertilizer synergist, could significantly improve the drought resistance and yield of many crops. However, its high production costs and unclear long-term impact on soil ecology limit its large-scale application. In this study, an environmentally friendly green material γ-PGA was heterologous synthesized in maize for the first time using the synthetic biology method. The genes (PgsA, PgsB, PgsC) participated in γ-PGA synthesis were cloned from Bacillus licheniformis and transformed into maize to produce γ-PGA for the first time. Under drought stress, transgenic maize significantly increased the ear length, ear weight and grain weight by 50 % compared to the control, whereas the yield characteristic of ear weight, grain number per ear, grain weight per ear and 100-grain weight increased by 1.67 %-2.33 %, 3.78 %-13.06 %, 8.41 %-22.06 %, 6.03 %-19.28 %, and 11.85 %-18.36 %, respectively under normal growth conditions. γ-PGA was mainly expressed in the mesophyll cells of maize leaf rosette structure and improved drought resistance and yield by protecting and increasing the expression of genes for the photosynthetic and carbon fixation. This study is an important exploration for maize drought stress molecular breeding and building resource-saving agriculture.