Beef Extract Research Articles

Optimizing RT-qPCR Detection of Enveloped and Non-enveloped Viruses in Wastewater: A Comparative Study in Grenada

Abstract Introduction/Objective Wastewater-based epidemiology (WBE) is an effective methodology to monitor the community spread of pathogens and has successfully been used to identify potential outbreaks. Most standardized methodologies used in WBE for the detection of viruses have been validated for non-enveloped viruses; therefore, there is a need for the development of a standardized protocol for the detection of both enveloped (such as SARS- CoV-2) and non-enveloped viruses (such as Hepatitis A). Wastewater seeded with Pseudomonas syringae bacteriophage (Φ6), an enveloped virus surrogate, and coliphage MS2, a non-enveloped virus surrogate, were used to evaluate the efficiency of four different aluminium flocculation methods. Untreated wastewater spiked with known amounts of surrogates was used as a control for viral recovery. Methods/Case Report Four different viral concentration methods were used. Method 1: AlCl3 flocculation at pH 3.5 followed by centrifugation, resuspension with 10% beef extract and filtered with a 0.22 μm filter. Method 2: AlCl3 flocculation at pH 6.0 followed by room temperature agitation, centrifugation, and resuspension with 3% beef extract. Method 3: Mixed method protocol-AlCl3 flocculation at pH 3.5 followed by room temperature agitation, centrifugation, resuspension with 3% beef extract. Method 4: Mixed method protocol-AlCl3 flocculation at pH 6.0 followed by centrifugation, resuspension with 10% beef extract filtered with a 0.22 μm filter. Viral RNA was extracted after concentration, and reverse-transcription quantitative polymerase chain reaction (RT- qPCR) was performed. Results (if a Case Study enter NA) The average Ct reduction of untreated wastewater compared to spiked was calculated for each method. The highest recovery of both Φ6 and MS2 was obtained using Method 3 (mixed protocol) with a significant difference at 95% confidence in the recovery of Φ6 (p= 0.0029) and MS2 (p= 0.0225). Conclusion Previous results from our lab demonstrate the importance of pH during the flocculation step of viral concentration using different methods of concentration. Here, the results suggest that the increased contact time between virus and the flocs is also critical. This can facilitate the implementation of WBE in Grenada to monitor and track potential viral outbreaks of both enveloped and non-enveloped viruses such as SARS-CoV-2 and Hepatitis A.

Bacteriophage-linked superparamagnetic nanoparticle for sensitive and simple detection of pathogenic Escherichia coli O157:H7 bacteria

An easy-to-use and highly-sensitive magnetic nanoprobe to detect veratoxin-producing Escherichia coli O157:H7 was developed. The bacteriophage that targets E. coli O157:H7 was chemically linked to chitosan-coated iron oxide nanoparticles (5–50 nm) to form a nanoprobe for the detection of E. coli O157:H7 in aqueous samples. The nanoprobes had a limit of detection of 2 log CFU/mL in samples with pH from 5 to 8 at 25 °C, and temperatures from 25 to 40 °C at pH 7. In the beef and cabbage liquid extracts, the nanoprobe had a detection limit of 2 log CFU/mL at 25 °C and pH 7. The nanoprobe was highly specific and retrieved only E. coli O157:H7 from a sample of bacterial mixture.Gram staining method was used with the nanoprobe to visualize the bacterial cells captured from bacterial suspensions at 7 pH and 25 °C. Visualization of the bacterial cells captured from suspensions with a concentration of 2 log CFU/mL was achieved.To detect the presence of bacteria, the use of the magnetic nanoprobes and Gram staining took less than 30 min to complete. The system developed in this study offers a fast and operator-friendly alternative for highly-specific bacterial detection.

Biological characterization and in vitro fungicide screening of a new causal agent of walnut leaf spot in Guizhou Province, China.

Walnut (Juglans regia L.) is a widely grown nut plant worldwide, including in Guizhou Province, located in southwest China. The high quality and special taste make Guizhou walnuts, particularly those produced in Hezhang County, a "Chinese National Geographical Indication Product" that substantially contributes to the local economy and grower's income. In July 2022, a serious occurrence of leaf spot disease was observed in a walnut plantation area, Shuitang Town, Hezhang County, Guizhou Province, China (27°07'67″N, 104°64'61″E). The causal agent was identified as Didymella segeticola through morphological characterization and amplification and sequencing of the internal transcribed spacer (ITS) region, beta-tubulin (TUB) gene, and glyceraldehyde-3-phosphate dehydrogenase (G3PD) gene. Koch's postulates, including re-isolation and identification, were performed to confirm its pathogenicity on healthy leaves. To our knowledge, this is the first report of D. segeticola causing leaf spot on walnuts worldwide. Further, to determine its biological characteristics, which could be utilized for future disease management, the effects of temperature, light, and carbon and nitrogen resources on mycelial growth, conidia production, and conidia germination and the effects of humidity on conidia germination were studied. The optimum temperature for mycelial growth of representative strain D. segeticola C27 was 20°C. Increasing the light period significantly decreased conidia production and conidia germination. Maltose and beef extract were the best carbon and nitrogen sources, respectively, for the pathogen. Conidia germination was enhanced at 90% humidity. In vitro screening of effective fungicides was conducted. Among the 20 screened fungicides, difenoconazole showed the best inhibition rate, with an EC50 (concentration for 50% of the maximal effect) of 0.0007 μg/mL. Tetramycin also showed sufficient inhibitory effects against D. segeticola, with an EC50 value of 0.0009 μg/mL. Our study provides new insights into the causal agent of walnut leaf spot in Guizhou, China, as well as the first pathogen characteristics and promising candidate fungicides for its management.

Production and Optimization of Xylanase Enzyme from Bacillus cereus by Submerged Fermentation Method

Three strains of Bacillus cereus were isolated from soil samples in this study in an effort to enhance the circumstances for the production of xylanase by the bacteria. The bacteria released extracellular xylanase throughout the submerged fermentation process. In a 250 ml conical flask, the whole fermentation process was conducted at 150 rpm stirring speed. The highest amount of xylanase synthesis was demonstrated by the isolated strains at 45°C, 48 hours of incubation, pH 5, 1.5% oat splet xylan base concentration, lactose carbon source, and beef extract source.

Production, Characterization, Kinetics, and Thermodynamics Analysis of Amyloglucosidase from Fungal Consortium.

The current study aimed to produce an amyloglucosidase enzyme from the fungal consortium. The best amylolytic fungal consortia were identified as Alternaria alternata and Aspergillus niger through the 18S rDNA technique. Fermentation kinetics and various nutritional and cultural parameters were analyzed. Maximum production was obtained in M4 media, pH 5.5, 30°C, and 4mL inoculum at 150rpm after 72h of incubation. Along with that, sodium nitrate at 2.5%, maltose, beef extract 1%, zinc sulfate (0.1%), and Tween 80 (0.1%) supported the maximum amyloglucosidase production. Amyloglucosidase was partially purified up to 1.6 purification fold with a specific activity of 1.84 Umg-1 in a stepwise manner by ammonium sulfate purification, dialysis, and ion exchange chromatography. The AMG enzyme also revealed maximum activity at 50°C with 5.0 pH. Upon the kinetic analysis, the specific yield coefficient Yp/x and volumetric rates Qp and Qx were also found to be significant in the above optimized conditions. The Km value 0.33mgmL-1 and Vmax 26.31 U mL-1 were obtained at 1% soluble starch substrate. Thermodynamic parameters for soluble starch hydrolysis were as follows: ΔH = 48.78kJmol-1, (Ea) = - 46.0kJmol-1, and ΔS = - 43.10Jmol-1K-1. This finding indicates the indigenously isolated fungal consortium can be the best candidate for industrial applications.

Oxyplasma meridianum gen. nov., sp. nov., an extremely acidophilic organotrophic member of the order Thermoplasmatales.

A mesophilic, hyperacidophilic archaeon, strain M1T, was isolated from a rock sample from Vulcano Island, Italy. Cells of this organism were cocci with an average diameter of 1 µm. Some cells possessed filaments. The strain grew in the range of temperatures between 15 and 52 °C and pH 0.5-4.0 with growth optima at 40 °C and pH 1.0. Strain M1T was aerobic and chemoorganotrophic, growing on complex substrates, such as casamino acids, trypticase, tryptone, yeast and beef extracts. No growth at expenses of oxidation of elemental sulphur or reduced sulphur compounds, pyrite, or ferrous sulphate was observed. The core lipids were glycerol dibiphytanyl glycerol tetraether lipids (membrane spanning) with 0 to 4 cyclopentane moieties and archaeol, with trace amounts of hydroxy archaeol. The dominant quinone was MK-7 : 7. The genome size of M1T was 1.67 Mbp with a G+C content of 39.76 mol%, and both characteristics were well within the common range for Thermoplasmatales. The phylogenetic analysis based on 16S rRNA gene sequence placed the strain M1T within the order Thermoplasmatales with sequence identities of 90.9, 90.3 and 90.5% to the closest SSU rRNA gene sequences from organisms with validly published names, Thermoplasma acidophilum, Thermoplasma volcanium and Thermogymnomonas acidicola, respectively. Based on the results of our genomic, phylogenetic, physiological and chemotaxonomic studies, we propose that strain M1T (=DSM 116605T=JCM 36570T) represents a new genus and species, Oxyplasma meridianum gen. nov., sp. nov., within the order Thermoplasmatales.

Process Optimization for Biosurfactant Production by Bacillus aryabhattai SPS1001 using Taguchi Method.

This study employs Taguchi design of experiments (DOE) to optimize biosurfactant yield by analyzing the impact of various input parameters. Signal-to-noise ratio analysis was utilized for optimization, corroborated by ANOVA findings. Regression equations depicted response behaviour and are validated through a confirmation test. Taguchi methodology identified optimal conditions for maximum biosurfactant yield: agitation (180rpm), inoculum size (2%), beef extract (5g/L), diesel (20ml/L), peptone (5g/L), NaCl (7g/L), incubation time (4days), pH (7.9), and yeast extract (6g/L). This yielded an 8.33% increase to 1.53g/L, with initial optimum parameters projecting 1.41g/L. ANOVA ranked and quantified control factor contributions, revealing agitation's significant (31.41%) impact on yield. The study underscores the viability of Taguchi's optimal conditions for substantial yield improvement within specific ranges. The strong alignment between expected and experimental yields affirmed the reliability of developed models for optimal yield selection. This study underscores the power of statistical techniques like Taguchi DOE and ANOVA in systematically enhancing biosurfactant production by Bacillus aryabhattai SPS1001 and paves the way for future advancements in bioprocess optimization.

Isolation and Characterization of Staphylococcus saprophyticus from Textile Industry Soil for Decolorization of Crystal Violet Dye

Textile dyes are an important class of synthetic organic compounds and are, therefore, common industrial pollutants. They are produced in large scale and may enter the environment during production or later on during fiber dying. Thus, there is a need for developing treatment methods that were more effective in eliminating dyes from textile waste soil as its source. Staphylococcus saprophyticus was isolated from soil of a textile plant and selected as the most active dye degrader of 11 isolates. The important parameters including temperature, pH, carbon and nitrogen source on crystal violet decolorization were investigated. Under the optimum conditions, dye decolorization (92.35%) was successfully achieved within 120 h. at 30°C, pH 8 with sucrose and beef extract as the energy source.

Isolation, identification, biological characteristics, and pathogenicity of an entomogenous fungus against the Egyptian mealybug, Icerya aegyptiaca (J.) (Hemiptera: Monophlebidae)

BackgroundIn this study, an entomogenous, fungus was isolated from the Egyptian mealybug, Icerya aegyptiaca (J.) (Hemiptera: Monophlebidae) on the parasol leaf tree, Macaranga tanarius, in China where evaluated as a biocontrol fungus to reduce the population of the target insect. The strain was identified as Aspergillus parasiticus by morphological and phylogenetic analysisand named ZHKUAP1. The biological characteristics, pathogenicity, and field control effect of the strain were determined.ResultsThe most suitable medium for the mycelial growth of strain ZHKUAP1 was PPDA medium, with an optimum temperature of 30 °C and pH 7, in addition to glucose and peptone as carbon and nitrogen sources. The optimum sporulation conditions were the PPDA medium at 30 °C and pH 6, using the soluble starch and beef extract as carbon and nitrogen sources. The mycelial growth and spore production of strain ZHKUAP1 were stopped at 70 °C and above, indicating that it was not resistant to high temperatures. High concentrations of spore suspension, against young insect age, resulted high corrected mortality, as well as decreased the median lethal time. When the spore concentration was 1 × 108 cfu/ml, the corrected mortality of the second nymph was 88.33%, and the LT50 was 0.66 day. After 10 days of inoculation, the LC50 of the second instar nymph was the smallest, reaching 4.07 × 104 cfu/ml. On the 10th day of the field experiment, the corrected mortality was 76.45%, indicating that the A. parasiticus strain ZHKUAP1 had strong pathogenicity on I. aegyptiaca population.ConclusionsThe indoor toxicity of the strain to I. aegyptiaca was determined, and the field control effect of the pathogen was explored on this basis. The results have important application prospects in the biological control of I. aegyptiaca.

Rosettibacter primus gen. nov., sp. nov., and Rosettibacter firmus sp. nov., facultatively anaerobic moderately thermophilic bacteria of the class Ignavibacteria from hot springs of North Ossetia

A novel facultatively anaerobic moderately thermophilic bacteria, strains 4137-MeT and 4148-MeT, were isolated from hot springs of Karmadon and Ursdon, respectively (North Ossetia, Russian Federation). Gram-negative, motile rods were present singly, in pairs, rosettes, and aggregates, or formed biofilms. Both strains grew optimally at 50–55 °C, pH 7.0 and did not require sodium chloride or yeast extract for growth. They were chemoorganoheterotrophs, growing on mono-, di- and polysaccharides (cellulose, starch, xylan, lichenan, galactan, xyloglucan, mannan, xanthan gum, guar gum) as well as proteinaceous substrates (gelatin, peptone, beef and yeast extract). Growth under anaerobic conditions was observed in presence and absence of external electron acceptors. Sulfur, thiosulfate, arsenate, Fe-citrate, and ferrihydrite were reduced with acetate, starch, or yeast extract as electron donors. The respiratory quinone was MK-7. Major cellular fatty acids of both strains were iso-C15:0, anteiso-C17:0, C15:0, iso-C16:0 and additionally iso-C17:0 for strain 4137-MeT. The size of the genome and genomic DNA G + C content of strain 4137-MeT were 3.24 Mb. and 29.9 %, respectively; for strain 4148-MeT – 3.33 Mb and 30.7 %. According to the 16S rRNA gene sequence and conserved protein sequences phylogenies, strains 4137-MeT and 4148-MeT represented a distinct lineage of the family Melioribacteraceae within the class Ignavibacteria. Based on phylogenetic analysis and phenotypic features, the novel isolates were assigned to a novel genus, for which the name Rosettibacter gen. nov. is proposed. Strain 4148-MeT represents its type species Rosettibacter primus sp. nov., while strain 4137-MeT represents a new species Rosettibacter firmus sp. nov.

Determination of fermentation conditions of Streptomyces sp. VNUA116 to improve the antifungal activities against Fusarium oxysporum f.sp. cubense causing Panama wilt disease on banana plants

This study aims to identify medium components (carbon source, carbon concentration, nitrogen source) and fermentation conditions (temperature, pH) to enhance the resistance against Fusarium oxysporum f. sp. cubenseTropical Race 4 (Foc Tr4), causing Panama wilt disease of Streptomyces sp. VNUA116 (VNUA116). By using the agar well diffusion method, it was determined that strain VNUA116 has strong anti-fungal pathogen activities FocTr4 under fermentation nutrients: YIM 301 medium, carbon source CB1 (soluble starch 40 g/l: D-glucose 1 g/l, and nitrogen source N1 beef extract 3 g/l: yeast extract 5 g/l: peptone 3 g/l). Additionally, optimal fermentation conditions were established, including pH 7, and temperature 30oC. Strain VNUA116 exhibited the highest antifungal activity, reaching a diameter of 30.67±0.58 mm after 10 days of cultivation. Furthermore, under these optimised fermentation conditions, Streptomycessp. VNUA116 demonstrated an impressive inhibitory capability against Foc-Tr4, reaching up to 93.20%, equivalent to the concentration of the fungicide Ridomil gold 68WG at 3 g/l in laboratory conditions. This affirms the considerable potential of the VNUA116 strain for applications in the production of biofertilizers for the biological control of Panama wilt disease in banana plants.

Exploring the decolorization efficiency and biodegradation mechanisms of different functional textile azo dyes by Streptomyces albidoflavus 3MGH

Efficiently mitigating and managing environmental pollution caused by the improper disposal of dyes and effluents from the textile industry is of great importance. This study evaluated the effectiveness of Streptomyces albidoflavus 3MGH in decolorizing and degrading three different azo dyes, namely Reactive Orange 122 (RO 122), Direct Blue 15 (DB 15), and Direct Black 38 (DB 38). Various analytical techniques, such as Fourier Transform Infrared (FTIR) spectroscopy, High-Performance Liquid Chromatography (HPLC), and Gas Chromatography-Mass Spectrometry (GC-MS) were used to analyze the degraded byproducts of the dyes. S. albidoflavus 3MGH demonstrated a strong capability to decolorize RO 122, DB 15, and DB 38, achieving up to 60.74%, 61.38%, and 53.43% decolorization within 5 days at a concentration of 0.3 g/L, respectively. The optimal conditions for the maximum decolorization of these azo dyes were found to be a temperature of 35 °C, a pH of 6, sucrose as a carbon source, and beef extract as a nitrogen source. Additionally, after optimization of the decolorization process, treatment with S. albidoflavus 3MGH resulted in significant reductions of 94.4%, 86.3%, and 68.2% in the total organic carbon of RO 122, DB 15, and DB 38, respectively. After the treatment process, we found the specific activity of the laccase enzyme, one of the mediating enzymes of the degradation mechanism, to be 5.96 U/mg. FT-IR spectroscopy analysis of the degraded metabolites showed specific changes and shifts in peaks compared to the control samples. GC-MS analysis revealed the presence of metabolites such as benzene, biphenyl, and naphthalene derivatives. Overall, this study demonstrated the potential of S. albidoflavus 3MGH for the effective decolorization and degradation of different azo dyes. The findings were validated through various analytical techniques, shedding light on the biodegradation mechanism employed by this strain.

A feasible approach for azo-dye (methyl orange) degradation by textile effluent isolate Serratia marcescens ED1 strain for water sustainability: AST identification, degradation optimization and pathway hypothesis

Methyl orange (MO) is a dye commonly used in the textile industry that harms aquatic life, soil and human health due to its potential as an environmental pollutant. The present study describes the dye degradation ability of Serratia marcescens strain ED1 isolated from textile effluent and characterized by 16S rRNA gene sequence analysis. The laccase property of bacterial isolate was confirmed qualitatively. The effects of various factors (pH, temperature, incubation time, and dye concentration) were evaluated using Response Surface Methodology (RSM). The maximum dye (MO) degradation was 81.02 % achieved at 37 °C temperature and 7.0 pH with 200 mg/L dye concentration after 48 h of incubation. The beef extract, ammonium nitrate and fructose supplementation showed better response during bioremediation among the different carbon and nitrogen sources. The degree of pathogenicity was confirmed through the simple plate-based method, and an antibiotic resistance profile was used to check the low-risk rate of antibiotic resistance. However, the fate and extinct of degraded MO products were analysed through UV–Vis spectroscopy, FT-IR, and GC-MS analysis to confirm the biodegradation potential of the bacterial strain ED1 and intermediate metabolites were identified to propose metabolic pathway. The phytotoxicity study on Vigna radiata L. seeds confirmed nontoxic effect of degraded MO metabolites and indicates promising degradation potential of S. marcescens strain ED1 to successfully remediate MO dye ecologically sustainably.

Optimization of Culturing Conditions for the Production of Antimicrobial Metabolite by Bacillus Carotarum

Forest soil antagonistic bacteria Bacillus carotarum was isolated and tested for optimum antimicrobial metabolite production. Maximum antimicrobial metabolite production was found at temperature 30°C and pH 7.0 and on Ist day of incubation at stationary condition. The medium consisting of beef extract - 0.3%, peptone - 0.5% and NaCl - 0.05%, was screened out as a suitable medium for maximum antimicrobial production supplemented individually with four carbon sources of which starch was found as the best carbon source. The active agent was best extracted with petroleum ether. Homogeneity of the crude bioactive metabolites were also tested by paper chromatographic analysis where a single type of bioactive metabolite was found with a Rf value of 0.94 in the solvent system n- butanol: acetic acid : water = 4 : 1 : 5 . The antimicrobial spectrum of the metabolite was wide and showed activity against Shigella dysenteriae (AE14612), Salmonella typhi(AE14296), Vibrio cholerae (A214748), Pseudomonas aeruginosa (CRL, ICDDR’ B), Bacillus cereus (BTCC19), and Bacillus subtilis (BTTC17). The Chittagong Univ. J. B. Sci. 8(1&2): 01-10, 2013

Biodegradation of Keratin Waste by Bacillus velezensis HFS_F2 through Optimized Keratinase Production Medium.

Enormous aggregates of keratinous wastes are produced annually by the poultry and leather industries which cause environmental degradation globally. To combat this issue, microbially synthesized extracellular proteases known as keratinase are used widely which is effective in degrading keratin found in hair and feathers. In the present work, keratinolytic bacteria were isolated from poultry farm soil and feather waste, and various cultural conditions were optimized to provide the highest enzyme production for efficient keratin waste degradation. Based on the primary and secondary screening methods, the potent keratinolytic strain (HFS_F2T) with the highest enzyme activity 32.65 ± 0.16 U/mL was genotypically characterized by 16S rRNA sequencing and was confirmed as Bacillus velezensis HFS_F2T ON556508. Through one-variable-at-a-time approach (OVAT), the keratinase production medium was optimized with sucrose (carbon source), beef extract (nitrogen source) pH-7, inoculum size (5%), and incubation at 37 °C). The degree of degradation (%DD) of keratin wastes was evaluated after 35days of degradation in the optimized keratinase production medium devoid of feather meal under submerged fermentation conditions. Further, the deteriorated keratin wastes were visually examined and the hydrolysed bovine hair with 77.32 ± 0.32% degradation was morphologically analysed through Field Emission Scanning Electron Microscopy (FESEM) to confirm the structural disintegration of the cuticle. Therefore, the current study would be a convincing strategy for reducing the detrimental impact of pollutants from the poultry and leather industries by efficient keratin waste degradation through the production of microbial keratinase.

Antibacterial Activity of Oregano (Origanum vulgare L.) Essential Oil Vapors against Microbial Contaminants of Food-Contact Surfaces.

The antimicrobial effect of eight essential oils' vapors against pathogens and spoilage bacteria was assayed. Oreganum vulgare L. essential oil (OVO) showed a broad antibacterial effect, with Minimum Inhibitory Concentration (MIC) values ranging from 94 to 754 µg cm-3 air, depending on the bacterial species. Then, gaseous OVO was used for the treatment of stainless steel, polypropylene, and glass surfaces contaminated with four bacterial pathogens at 6-7 log cfu coupon-1. No viable cells were found after OVO treatment on all food-contact surfaces contaminated with all pathogens, with the exception of Sta. aureus DSM 799 on the glass surface. The antimicrobial activity of OVO after the addition of beef extract as a soiling agent reduced the Sta. aureus DSM 799 viable cell count by more than 5 log cfu coupon-1 on polypropylene and glass, while no viable cells were found in the case of stainless steel. HS-GC-MS analysis of the headspace of the boxes used for the antibacterial assay revealed 14 different volatile compounds with α-Pinene (62-63%), and p-Cymene (21%) as the main terpenes. In conclusion, gaseous OVO could be used for the microbial decontamination of food-contact surfaces, although its efficacy needs to be evaluated since it depends on several parameters such as target microorganisms, food-contact material, temperature, time of contact, and relative humidity.

Effectiveness of attractants and bait for Iberian wolf detection: captivity-based and free-ranging trials

Monitoring large carnivores requires substantial effort, which is why indirect methodologies such as camera trapping with attractants or baits are commonly employed. The Iberian wolf (Canis lupus signatus) is one of the top predators in the Iberian Peninsula, and monitoring its packs is essential to understand its distribution and mitigate conflicts arising from livestock predation. We performed a captivity-based study evaluating the effectiveness of five attractants (beef extract, cadaverine, Fatty Acid Scent (FAS), lynx urine and valerian extract) on wolf detection. To accomplish this objective, Jacobs selectivity index and generalized linear models were employed to assess the attractiveness and induced behaviour of each attractant. Subsequently, the three most effective attractants, combined or not with a bait, were tested in the field and analyzed using generalized linear mixed models. The five attractants tested elicited different behavioural responses in the wolves in captivity, including smelling, rubbing, rolling, marking and licking. Among the captive wolves, cadaverine, FAS and lynx urine emerged as the top three preferred attractants. In the field tests with these three attractants cadaverine remained the most preferred option. The inclusion of bait did not have any significant effect on the wolf’s visitation rates. Our results show that employing species-specific attractants can significantly improve the efficiency of carnivore surveys conducted in the field. Specifically, cadaverine was the most effective attractant for wild Iberian wolves. Consequently, the careful selection of an appropriate attractant becomes crucial to attain the precise objectives of the study, such as camera trapping, bait deployment or DNA sampling.

Evaluation of the efficiency of thermostable L-asparaginase from B. licheniformis UDS-5 for acrylamide mitigation during preparation of French fries.

A thermostable L-asparaginase was produced from Bacillus licheniformis UDS-5 (GenBank accession number, OP117154). The production conditions were optimized by the Plackett Burman method, followed by the Box Behnken method, where the enzyme production was enhanced up to fourfold. It secreted L-asparaginase optimally in the medium, pH 7, containing 0.5% (w/v) peptone, 1% (w/v) sodium chloride, 0.15% (w/v) beef extract, 0.15% (w/v) yeast extract, 3% (w/v) L-asparagine at 50 °C for 96h. The enzyme, with a molecular weight of 85kDa, was purified by ion exchange chromatography and size exclusion chromatography with better purification fold and percent yield. It displayed optimal catalysis at 70°C in 20mM Tris-Cl buffer, pH 8. The purified enzyme also exhibited significant salt tolerance too, making it a suitable candidate for the food application. The L-asparaginase was employed at different doses to evaluate its ability to mitigate acrylamide, while preparing French fries without any prior treatment. The salient attributes of B. licheniformis UDS-5 L-asparaginase, such as greater thermal stability, salt stability and acrylamide reduction in starchy foods, highlights its possible application in the food industry.

Optimization of the extracellular secretion of black goat rumen metagenome-derived KG42 xylanase by Bacillus subtilis

Xylanase (E.C. 3.2.1.8) is the enzyme that breaks down β-1,4 xylan by cleaving β-1,4 glycosidic linkages. Production of xylanases is important for various industrial applications. Here, we aimed to determine the optimal incubation conditions for expression and secretion of KG42 xylanase in Bacillus subtilis using response surface methodology based on Box-Behnken design in preparation for industrial applications. Among nine broth media tested in this study, Power Broth was chosen as a basal medium. In addition to the basal medium, the four other independent variables of extra carbon sources (glucose, lactose, mannose, fructose, and sucrose), extra nitrogen sources (beef extract, yeast extract, tryptone, urea, NaNO3, and (NH4)2SO4), isopropyl β-D-1-thiogalactopyranoside concentrations, and induction times were individually tested using one factor at a time in an optimization experiment. Next, a Box-Behnken design-based response surface methodology approach was used to identify and validate the optimized incubation conditions with the four variables in batch culture. The statistically optimized incubation conditions obtained from this study yielded a maximum of approximately 3- to 4-fold increases in the expression and secretion of KG42 xylanase by B. subtilis in comparison with unoptimized medium and incubation conditions.

Transcriptomic profiling of adding cobalt chloride to improve dendrobine-type total alkaloid production.

Trichoderma longibrachiatum UN32 is known for its efficient production of dendrobine-type total alkaloids (DTTAs). This study aimed to determine the optimal medium composition for the UN32 strain using response surface methodology. Key factors, including glucose, beef extract, and CoCl2, were selected through the Plackett-Burman design. Subsequently, a factorial optimization approach was employed using the steepest ascent design, and 17 trial sets were completed via the Box-Behnken design. The optimal medium composition was found to consist of 29.4g/L of glucose, 17.3g/L of beef extract, and 0.28mmol/L of CoCl2. This optimized medium resulted in an impressive 80.8% increase in mycelial dry weight (reaching 12.303g/L) and a substantial 76.4% boost in DTTA production (reaching 541.63 ± 46.95μg). Furthermore, the fermentation process was scaled up to a 5-L bioreactor, leading to a DTTA production approximately 1.95 times than the control. Transcriptome analysis of strain UN32 in response to CoCl2 supplementation revealed significant changes in the expression of critical genes associated with the TCA cycle and L-valine, L-leucine, and L-isoleucine biosynthesis changed. These alterations resulted in a heightened influx of acetyl-CoA into DTTA production. Additionally, the expression of genes related to antioxidant enzymes was modified to maintain homeostasis of reactive oxygen species (ROS). A potential mechanism for the accumulation of DTTAs based on ROS as a signal transduction was proposed. These findings provide valuable insights into the regulatory mechanisms of DTTA biosynthesis, potentially offering a method to enhance the production of secondary metabolites in the UN32 strain. KEY POINTS: • After the RSM optimization, there is a substantial increase of 80.8% in biomass production and a significant 76.4% rise in DTTA production. • Transcriptome analysis revealed that the inclusion of CoCl2 supplements resulted in an enhanced influx of acetyl-CoA. • Proposed a mechanism for the accumulation of DTTAs for the role of ROS as a signal transduction pathway.