Characterization of Salmonella enterica Biofilms and Antibiofilm Effect of Carvacrol and 2-Aminobenzimidazole.

Biofilm-associated foodborne Salmonella infections in poultry have become increasingly challenging for veterinarians, particularly in developing countries, and warrant thorough investigation. We assessed the biofilm-forming tendency of poultry isolates of Salmonella enterica, namely Salmonella Typhimurium (n = 23), Salmonella Infantis (n = 28), and Salmonella Heidelberg (n = 18), in nutrient-rich Rappaport-Vassiliadis Soya (RVS) peptone broth and nutrient-deficient diluted Tryptone Soya Broth (TSB). Seven of the tested isolates exhibited moderate biofilm formation in diluted TSB, whereas two showed such formation in RVS. In addition, the Congo red agar assay revealed curli and cellulose production in seven isolates. Fourteen specific biofilm-associated genes were analyzed identifying sdiA and seqA to be the most prevalent (100%), and glyA the least prevalent (69.5%). The prevalence of the genes bcsA and csgA was significantly lower in moderate and weak biofilm formers, respectively, as compared with nonbiofilm formers in RVS peptone broth. Furthermore, the compounds carvacrol and 2-aminobenzimidazole (2-ABI) effectively inhibited biofilm formation by Salmonella serovars in RVS peptone and TSB media, respectively. Whereas the antibiofilm activity of 2-ABI against Salmonella has not been reported previously, we determined its most effective concentration at 1.5 mM among tested antibiofilm treatments. These findings indicate that Salmonella strains prevalent in poultry farms have the potential to form biofilms, and the tested compounds should be further explored as supportive or alternative antimicrobials.

Modeling and optimization of antibacterial effect of lichen-associated bacteria, Bacillus subtilis KSRLAB3 strain against marine fouling bacteria, Vibrio alginolyticus.

One of the most commonly occurring bacteria, Bacillus subtilis, can produce a wide variety of secondary metabolites. In this study, the antimicrobial effect of B. subtilis KSRLAB3 against Vibrio alginolyticus was optimized using the Plackett-Burman design (PBD) method, response surface methodology (RSM), and genetic algorithm (GA). Initially, the effects of carbon source, nitrogen source, NaCl concentration, pH, temperature, and incubation time on antimicrobial effects were studied. Among the carbon and nitrogen sources investigated, mannose and peptone elicited maximum antimicrobial effect. Using PBD, the most significant variables that influence the antimicrobial effect were identified, including incubation time, peptone concentration, and temperature. The optimum conditions required for attaining maximum antimicrobial effect was identified using the RSM-GA hybrid method, and the optimum condition includes 49.999 h of incubation time, 4.39 g/L of peptone concentration, and 27.629°C of incubation temperature. The confirmatory experiments performed around the optimum condition showed a zone of inhibition of 35 ± 0.52 mm. Methanolic extract also proved the presence of antibacterial lipopeptide surfactin. Therefore, the RSM-GA hybrid method was successfully used in this study to model the antimicrobial effect of B. subtilis KSRLAB3 against V. alginolyticus. The effective inhibition of V. alginolyticus can be investigated further for the development of antifouling coatings.

Modified Drug-Susceptibility Testing and Screening Culture Agar for Colistin-Susceptible Enterobacteriaceae Isolates Harboring a Mobilized Colistin Resistance Gene mcr-9.

Three isolates of the Enterobacter cloacae complex harboring mcr-9, a member of the colistin resistance mcr gene family encoded on plasmids, were susceptible to colistin, with MICs of 0.125 to 0.5 µg/mL in standard broth microdilution (BMD) tests using cation-adjusted Mueller-Hinton broth (CA-MHB) in accordance with European Committee on Antimicrobial Susceptibility Testing guidelines. In contrast, their MICs for colistin were significantly higher (4 to 128 µg/mL) when BMD tests were performed using brain-heart infusion (BHI) medium, Luria-Bertani (LB) broth, tryptic soy broth (TSB), or CA-MHB supplemented with casein, tryptonen or peptone. Colistin significantly induced mcr-9 expression in a dose-dependent manner when these mcr-9-positive isolates were cultured in BHI or CA-MHB supplemented with peptone/casein. Pretreatment of mcr-9-positive isolates and Escherichia coli DH5α harboring mcr-9 with colistin significantly increased their survival rates against LL-37, a human antimicrobial peptide. Electrospray ionization time-of-flight mass spectrometry analysis showed that a lipid A moiety of lipopolysaccharide was partially modified by phosphoethanolamine in E. coli DH5α harboring mcr-9 when treated with colistin. Of 93 clinical isolates of Enterobacteriaceae, only the mcr-9-positive isolates showed MICs to colistin that were at least 32 times higher in BHI than in CA-MHB. These mcr-9-positive isolates grew on a modified BHI agar, MCR9-JU, containing 3 µg/mL colistin. These results suggest that the BMD method using BHI is useful when performed together with the BMD method using CA-MHB to detect mcr-9-positive isolates and that MCR9-JU agar is useful in screening for Enterobacteriaceae isolates harboring mcr-9 and other colistin-resistant isolates.

T7-lac promoter vectors spontaneous derepression caused by plant-derived growth media may lead to serious expression problems: a systematic evaluation.

BACKGROUND: The widespread usage of protein expression systems in Escherichia coli (E. coli) is a workhorse of molecular biology research that has practical applications in biotechnology industry, including the production of pharmaceutical drugs. Various factors can strongly affect the successful construction and stable maintenance of clones and the resulting biosynthesis levels. These include an appropriate selection of recombinant hosts, expression systems, regulation of promoters, the repression level at an uninduced state, growth temperature, codon usage, codon context, mRNA secondary structure, translation kinetics, the presence/absence of chaperons and others. However, optimization of the growth medium's composition is often overlooked. We systematically evaluate this factor, which can have a dramatic effect on the expression of recombinant proteins, especially those which are toxic to a recombinant host. RESULTS: Commonly used animal tissue- and plant-based media were evaluated using a series of clones in pET vector, containing expressed Open Reading Frames (ORFs) with a wide spectrum of toxicity to the recombinant E. coli: (i) gfpuv (nontoxic); (ii) tp84_28-which codes for thermophilic endolysin (moderately toxic); and (iii) tthHB27IRM-which codes for thermophilic restriction endonuclease-methyltransferase (REase-MTase)-RM.TthHB27I (very toxic). The use of plant-derived peptones (soy peptone and malt extract) in a culture medium causes the T7-lac expression system to leak. We show that the presence of raffinose and stachyose (galactoside derivatives) in those peptones causes premature and uncontrolled induction of gene expression, which affects the course of the culture, the stability of clones and biosynthesis levels. CONCLUSIONS: The use of plant-derived peptones in a culture medium when using T7-lac hybrid promoter expression systems, such as Tabor-Studier, can lead to uncontrolled production of a recombinant protein. These conclusions also extend to other, lac operator-controlled promoters. In the case of proteins which are toxic to a recombinant host, this can result in mutations or deletions in the expression vector and/or cloned gene, the death of the host or highly decreased expression levels. This phenomenon is caused by the content of certain saccharides in plant peptones, some of which (galactosides) may act as T7-lac promoter inducer by interacting with a Lac repressor. Thus, when attempting to overexpress toxic proteins, it is recommended to either not use plant-derived media or to use them with caution and perform a pilot-scale evaluation of the derepression effect on a case-by-case basis.

An Evaluation of Different Types of Peptone as Partial Substitutes for Animal-derived Serum in Vero Cell Culture.

Vero cells are one of the most frequently used cell types in virology. They can be used not only as a vehicle for the replication of viruses, but also as a model for investigating viral infectivity, cytopathology and vaccine production. There is increasing awareness of the need to limit the use of animal-derived components in cell culture media for a number of reasons, which include reducing the risk of contamination and decreasing costs related to the downstream processing of commercial products obtained via cell culture. The current study evaluates the use of protein hydrolysates (PHLs), also known as peptones, as partial substitutes for fetal bovine serum (FBS) in Vero cell culture. Eleven plant-based, two yeast-based, and three casein-based peptones were assessed, with different batches evaluated in the study. We tested the effects of three concentration ratios of FBS and peptone on Vero cell proliferation, four days after the initial cell seeding. Some of the tested peptones, when in combination with a minimal 1% level of FBS, supported cell proliferation rates equivalent to those achieved with 10% FBS. Collectively, our findings showed that plant-based peptones could represent promising options for the successful formulation of serum-reduced cell culture media for vaccine production. This is especially relevant in the context of the current COVID-19 pandemic, in view of the urgent need for SARS-CoV-2 virus production for certain types of vaccine. The current study contributes to the Three Rs principle of reduction, as well as addressing animal ethics concerns associated with FBS, by repurposing PHLs for use in cell culture.

Investigation on mycelial growth requirements of Cantharellus cibarius under laboratory conditions.

The golden chanterelle represents one of the commonly found, edible mushrooms that is highly valued in various cuisines. The present study focused on assessing the requirements of Cantharellus cibarius such as pH, temperature, as well as the carbon and nitrogen sources for mycelial growth. Optimization of the growth parameters was carried out by one-factor-at-a-time method. The optimal pH and temperature were determined to be 6.0 and 22.5 °C, respectively. Among the various carbon sources studied, sucrose at a concentration of 2% gave maximum mycelial growth and proved to be the most suitable one. Amongst the nitrogen sources studied, peptone, ammonium sulphate, and sodium nitrate, gave the maximum mycelial growth at an optimized concentration of 0.5%. In the presence of beef extract and yeast extract, a change in colony pigmentation from yellow to dark grey was observed. Finally, the carbon to nitrogen ratio of 2:0.5 proved to be optimal for mycelial growth. This study is the first report on the optimisation of in vitro growth requirements of C. cibarius.

Aspergillus oryzae-Fermented Wheat Peptone Enhances the Potential of Proliferation and Hydration of Human Keratinocytes through Activation of p44/42 MAPK.

Identifying materials contributing to skin hydration, essential for normal skin homeostasis, has recently gained increased research interest. In this study, we investigated the potential benefits and mechanisms of action of Aspergillus oryzae-fermented wheat peptone (AFWP) on the proliferation and hydration of human skin keratinocytes, through in vitro experiments using HaCaT cell lines. The findings revealed that compared to unfermented wheat peptone, AFWP exhibited an improved amino acid composition, significantly (p < 0.05) higher DPPH scavenging capability and cell proliferation activity, and reduced lipopolysaccharide-induced NO production in RAW 264.7 cells. Furthermore, we separated AFWP into eleven fractions, each ≤2 kDa; of these, fraction 4 (AFW4) demonstrated the highest efficacy in the cell proliferation assay and was found to be the key component responsible for the cell proliferation potential and antioxidant properties of AFWP. Additionally, AFW4 increased the expression of genes encoding natural moisturizing factors, including filaggrin, transglutaminase-1, and hyaluronic acid synthase 1-3. Furthermore, AFW4 activated p44/42 MAPK, but not JNK and p38 MAPK, whereas PD98059, a p44/42 MAPK inhibitor, attenuated the beneficial effects of AFW4 on the skin, suggesting that the effects of AFW4 are mediated via p44/42 MAPK activation. Finally, in clinical studies, AFW4 treatment resulted in increased skin hydration and reduced trans-epidermal water loss compared with a placebo group. Collectively, these data provide evidence that AFW4 could be used as a potential therapeutic agent to improve skin barrier damage induced by external stresses.

Production of Valuable Compounds and Bioactive Metabolites from By-Products of Fish Discards Using Chemical Processing, Enzymatic Hydrolysis, and Bacterial Fermentation.

The objective of this report was to investigate the isolation and recovery of different biocompounds and bioproducts from wastes (skins and heads) that were obtained from five species discarded by fishing fleets (megrim, hake, boarfish, grenadier, and Atlantic horse mackerel). Based on chemical treatments, enzymatic hydrolysis, and bacterial fermentation, we have isolated and produced gelatinous solutions, oils that are rich in omega-3, fish protein hydrolysates (FPHs) with antioxidant and antihypertensive activities, and peptones. FPHs showed degrees of hydrolysis higher than 13%, with soluble protein concentrations greater than 27 g/L and in vitro digestibilities superior to 90%. Additionally, amino acids compositions were always valuable and bioactivities were, in some cases, remarkable. Peptones that were obtained from FPHs of skin and the heads were demonstrated to be a viable alternative to expensive commercial ones indicated for the production of biomass, lactic acid, and pediocin SA-1 from Pediococcus acidilactici.

Influence of medium composition and physical factors on enhanced production of endoglucanase by locally isolated fungal strain in solid state fermentation.

Endoglucanase is one of the most important enzymes of the cellulase group.  Endoglucanase are involved in the catalytic hydrolysis of cellulose and plays a pivotal role in different sectors like pharmaceutical, textile, detergent, and food processing as well as paper and pulp industry. With consumers getting more and more aware of environmental issues, industries find enzymes as a better option over other chemical catalysts. In the current research different thermophilic fungal strains were isolated from the different sources. Qualitative screening was carried out on the basis of cellulose hydrolysis zone. The quantitative screening was carried out employing solid state fermentation.  The fungal culture, showing highest EG potential was selected identified and assigned the code Aspergillus fumigatus BBT2. Different fermentation media were evaluated and M 2 containing wheat bran gave maximum EG production. The maximal enzyme productivity was recorded in 72 hours, 40°C, pH 5, inoculum size 1.5ml, and moisture content (1:1). Glucose (1%) and peptone (1%) were optimized as best carbon and nitrogen sources, respectively.

Tryptone-stabilized gold nanoparticles target tubulin and inhibit cell viability by inducing an unusual form of cell cycle arrest.

Gold nanoparticles have been investigated extensively for their molecular mechanisms of action and anticancer potential. We report a novel, tubulin-targeted antiproliferative mechanism of action of tryptone-stabilized gold nanoparticles (TsAuNPs). TsAuNPs, synthesized using HAuCl4·3H2O and tryptone and characterized by a variety of spectroscopic methods and transmission electron microscopy, were found to be inhibitory to viability of human pancreatic (PANC-1), cervical (HeLa), and breast (MDA-MB-231) cancer cell lines in a concentration-dependent manner, with highest efficacy against PANC-1 cells. The particles strongly inhibited the clonogenic propagation of PANC-1 cells. TsAuNPs-mediated inhibition of cell viability involved an unusual mode of cell cycle arrest (arrest at both G0/G1 phase and S-phase) followed by apoptosis. In vitro, TsAuNPs bound purified tubulin, competitively inhibited anilinonaphthalene sulfonate binding to tubulin, and suppressed tubulin assembly. In cells, tubulin-TsAuNPs interactions were manifested as a disrupted microtubule network, defective reassembly of cold-disassembled microtubules, and induction of tubulin acetylation. Our data indicate that TsAuNPs inhibit cell viability by inducing differential cell cycle arrest possibly through disrupted dynamicity of cellular microtubules.

Composition of the Stabilizer and Conditions of Lyophilization for Preserving Infectious Activity of Influenza Virus.

For stabilization of vaccine preparations, they are lyophilized. The composition of the protective medium is an important parameter affecting the quality of the vaccine after drying. In view of the risk of spreading prion diseases, the use of media containing animal proteins is not recommended. In this study, protective media containing no animal proteins and lyophilization regimen were determined. The optimum lyophilization regimen consisted of three stages: freezing at -70°C, main stage at -35°C, and drying at 24°C. Protective medium containing 4% trehalose or protective medium with 10% sucrose and 5% soy peptone ensured highest stability of the lyophilized vaccine preparation in temperature range of 4-24°C. This can help to overcome possible break in the cold chain, which is important during transporting or storage of vaccine preparations.

Natural Mutagenesis-Enabled Global Proteomic Study of Metabolic and Carbon Source Implications in Mutant Thermoacidophillic Archaeon Sulfolobus solfataricus PBL2025.

The thermoacidophilic crenarchaeon Sulfolobus solfataricus has been widely used as a model organism for archaeal systems biology research. Investigation using its spontaneous mutant PBL2025 provides an effective metabolic baseline to study subsequent mutagenesis-induced functional process shifts as well as changes in feedback inhibitions. Here, an untargeted metabolic investigation using quantitative proteomics and metabolomics was performed to correlate changes in S. solfataricus strains P2 against PBL2025 and under both glucose and tryptone. The study is combined with pathway enrichment analysis to identify prominent proteins with differential stoichiometry. Proteome level quantification reveals that over 20% of the observed overlapping proteome is differentially expressed under these conditions. Metabolic-induced differential expressions are observed along the central carbon metabolism, along with 12 other significantly regulated pathways. Current findings suggest that PBL2025 is able to compensate through the induction of carbon metabolism, as well as other anabolic pathways such as Val, Leu and iso-Leu biosynthesis. Studying protein abundance changes after changes in carbon sources also reveals distinct differences in metabolic strategies employed by both strains, whereby a clear down-regulation of carbohydrate and nucleotide metabolism is observed for P2, while a mixed response through down-regulation of energy formation and up-regulation of glycolysis is observed for PBL2025. This study contributes, to date, the most comprehensive network of changes in carbohydrate and amino acid pathways using the complementary systems biology observations at the protein and metabolite levels. Current findings provide a unique insight into molecular processing changes through natural (spontaneous) metabolic rewiring, as well as a systems biology understanding of the metabolic elasticity of thermoacidophiles to environmental carbon source change, potentially guiding more efficient directed mutagenesis in archaea.

Urea enhances cell lysis of Schizosaccharomyces pombe ura4 mutants.

Cell lysis is induced in Schizosaccharomyces pombe ∆ura4 cells grown in YPD medium, which contains yeast extract, polypeptone, and glucose. To identify the medium components that induce cell lysis, we first tested various kinds of yeast extracts from different suppliers. Cell lysis of ∆ura4 cells on YE medium was observed when yeast extracts from OXOID, BD, Oriental, and Difco were used, but not when using yeast extract from Kyokuto. To determine which compounds induced cell lysis, we subjected yeast extract and polypeptone to GC-MS analysis. Ten kinds of compounds were detected in OXOID and BD yeast extracts, but not in Kyokuto yeast extract. Among them was urea, which was also present in polypeptone, and it clearly induced cell lysis. Deletion of the ure2 gene, which is responsible for utilizing urea, abolished the lytic effect of urea. The effect of urea was suppressed by deletion of pub1, and a similar phenotype was observed in the presence of polypeptone. Thus, urea is an inducer of cell lysis in S. pombe ∆ura4 cells.

A Simple Defined Medium for the Production of True Diketopiperazines in Xylella fastidiosa and Their Identification by Ultra-Fast Liquid Chromatography-Electrospray Ionization Ion Trap Mass Spectrometry.

Diketopiperazines can be generated by non-enzymatic cyclization of linear dipeptides at extreme temperature or pH, and the complex medium used to culture bacteria and fungi including phytone peptone and trypticase peptone, can also produce cyclic peptides by heat sterilization. As a result, it is not always clear if many diketopiperazines reported in the literature are artifacts formed by the different complex media used in microorganism growth. An ideal method for analysis of these compounds should identify whether they are either synthesized de novo from the products of primary metabolism and deliver true diketopiperazines. A simple defined medium (X. fastidiosa medium or XFM) containing a single carbon source and no preformed amino acids has emerged as a method with a particularly high potential for the grown of X. fastidiosa and to produce genuine natural products. In this work, we identified a range of diketopiperazines from X. fastidiosa 9a5c growth in XFM, using Ultra-Fast Liquid Chromatography coupled with mass spectrometry. Diketopiperazines are reported for the first time from X. fastidiosa, which is responsible for citrus variegated chlorosis. We also report here fatty acids from X. fastidiosa, which were not biologically active as diffusible signals, and the role of diketopiperazines in signal transduction still remains unknown.

Statistical optimization of pigment production by Monascus sanguineus under stress condition.

Natural pigments are produced by the Monascus sp., which are used for coloring food substances. The intent of this study was to optimize the pigment yield and biomass produced from the unexplored Monascus sanguineus in submerged culture under stress conditions. For inducing thermal stress, the spores were incubated at various temperatures at higher ranges. For inducing osmotic stress, varied concentrations of NaCl, glycerol, and peptone were used. The medium components were optimized by response surface methodology (RSM). The combined effects of the four medium constituents mentioned were studied using a 24 full factorial central composite design (CCD). The relationships between the predicted values and actual values, independent variable, and the response were calculated according to a second-order quadratic model. It was deduced that the variable with the leading effect was the linear effect of glycerol concentration. Furthermore, the quadratic effects of peptone and the interactive effects of temperature and glycerol were more noteworthy than other factors. The optimum values for the test variables in coded factors were found to be spores treated with 70°C for temperature, 0.25 M for glycerol, 0.51% (w/v) for peptone, and 1.25% (w/v) for NaCl, corresponding to a maximum red pigment yield of 55.67 color value units (CVU)/mL. With optimized conditions, the pigment yield was almost three times the yield observed with the control.

Synergistic efficacy of ultrasound and ammonium persulfate in inactivating Escherichia coli O157:H7 in buffered peptone water and orange juice.

This study investigated the synergistic effects of ammonium persulfate (PS) and ultrasound (US) on the inactivation of Escherichia coli O157:H7 in buffered peptone water (BPW) and orange juice products. A comprehensive assessment of PS concentrations ranging from 1 to 300 mM, considering not only the statistical significance but also the reliability and stability of the experimental outcomes, showed that 150 mM was the optimal PS concentration for the inactivation of E. coli O157:H7. Additionally, US output intensities varying from 30 % to 60 % of the maximum US intensity were evaluated, and 50 % US amplitude was found to be the optimal US condition. A 50 % amplitude setting on the sonicator corresponds to half of its maximum displacement, approximately 60 µm, based on a maximum amplitude of 120 µm. The inactivation level of E. coli O157:H7 was significantly enhanced by the combined treatment of PS and US, compared to each treatment of PS and US alone. In the BPW, a 10-min treatment with the combination of PS and US resulted in a significant synergistic inactivation, achieving up to a log reduction of 3.86 log CFU/mL. Similarly, in orange juice products, a 5-min treatment with the combination of PS and US yielded a significant synergistic inactivation, with a reduction reaching 5.90 log CFU/mL. Although the treatment caused a significant color change in the sample, the visual differences between the treated and non-treated groups were not pronounced. Furthermore, the combined treatment in orange juice demonstrated significantly enhanced antimicrobial efficacy relative to BPW. Despite identical 5-min treatment periods, the application in orange juice resulted in a substantially higher log reduction of E. coli O157:H7, achieving 7.16 log CFU/mL at a reduced PS concentration of 30 mM, whereas the same treatment in BPW yielded only a 2.89 log CFU/mL reduction at a PS concentration of 150 mM, thereby highlighting its significantly superior antimicrobial performance in orange juice. The mechanism underlying microbial inactivation, induced by the combined treatment of PS and US, was identified as significant cell membrane damage. This damage is mediated by sulfate radicals, generated through the sono-activation of persulfate. In addition, the low pH of orange juice, measured at 3.7, is likely to have further deteriorated the E. coli O157:H7 cells compared to BPW (pH 7.2), by disrupting their cell membranes, proton gradients, and energy metabolism. These findings underscore the effectiveness of PS and US integration as a promising approach for non-thermal pasteurization in the food industry. Further research is needed to optimize treatment parameters and fully explore the practical application of this technique in large-scale food processing operations. Sensory evaluation and nutritional assessment are also necessary to address the limitations of PS.

Sweet potatoes as a basic component in developing a medium for the cultivation of lactobacilli.

A sweet potato medium (SPM) was formed with extract from baked sweet potatoes supplemented with 0, 4, or 8 g/L of each nitrogen source (beef extract, yeast extract, and proteose peptone #3) to form SPM1, SPM2, and SPM3 respectively. Lactobacilli MRS was used as control medium. Ten Lactobacillus strains containing an average of 2.34 ± 0.29 log CFU/mL were inoculated individually into batches of MRS, SPM1, SPM2, and SPM3. The growth patterns for the tested Lactobacillus strains growing in SPM2 and SPM3 were found to be similar to that in MRS. The average final population after 24 h of incubation in MRS, SPM2, and SPM3 reached 10.41 ± 0.35, 10.59 ± 0.27, and 10.72 ± 0.19 log CFU/mL respectively. SPM2 and SPM3 maintained higher pH values throughout the incubation period than MRS. These findings indicate that SPM2 can be a suitable medium for the growth of Lactobacillus and can provide an alternative at low-cost.

Constitutive expression of barley α-amylase in Pichia pastoris by high-density cell culture.

α-amy gene amplified from barley genome was cloned into MCS of pGAP9K to generate pGAP9K-α-amy which was then transformed into Pichia pastoris GS115 by electroporation. Transformants with multi-copies and high expression for the foreign gene were selected on G418 containing plate and expression analysis. The fermentation was carried out in a 50 l bioreactor with 20 l working volume, using a high-density cell culture method by continuously feeding with 50% glycerol-0.8% PTM4 to the growing culture for 54 h at 30°C. Under the control of GAP promoter (pGAP), α-amy gene was constitutively expressed. At the end of the fermentation, the α-AMY expression reached 125 mg/l, while the biomass growth was 186 as measured by absorption of 600 nm. The secreted α-AMY was purified to 97.5% by SP-Sepharose FF ion-exchange chromatography and affinity purification. The recombinant α-AMY showed activity on hydrolysis of starch.

Effect of zeolite (clinoptilolite) as feed additive in Tunisian broilers on the total flora, meat texture and the production of omega 3 polyunsaturated fatty acid.

BACKGROUND: Increasing consumer demand for healthier food products has led to the development of governmental policies regarding health claims in many developed countries. In this context, contamination of poultry by food-borne pathogens is considered one of the major problems facing the progress of the poultry industry in Tunisia. RESULT: Zeolite (Clinoptilolites) was added to chicken feed at concentrations 0,5% or 1% and was evaluated for its effectiveness to reduce total flora in chickens and its effects on performance of the production. The broilers were given free and continuous access to a nutritionally non-limiting diet (in meal form)that was either a basal diet or a' zeolite diet' (the basal diet supplemented with clinoptilolite at a level of 0,5% or 1%). It was found that adding zeolite in the broiler diet significantly (p < 0,05) reduced total flora levels, as compared to the control, on the chicken body. In addition, it was found that zeolite treatment had a positive effect on performance production and organoleptic parameters that were measured and mainly on the increase level of Omega 3 fatty acid. CONCLUSION: This study showed the significance of using zeolite, as a feed additive for broilers, as part of a comprehensive program to control total flora at the broiler farm and to increase level of Omega 3 fatty acid on the chicken body.

Optimization of acid protease production by Aspergillus niger I1 on shrimp peptone using statistical experimental design.

Medium composition and culture conditions for the acid protease production by Aspergillus niger I1 were optimized by response surface methodology (RSM). A significant influence of temperature, KH(2)PO(4), and initial pH on the protease production was evaluated by Plackett-Burman design (PBD). These factors were further optimized using Box-Behnken design and RSM. Under the proposed optimized conditions, the experimental protease production (183.13 U mL(-1)) closely matched the yield predicted by the statistical model (172.57 U mL(-1)) with R(2) = 0.914. Compared with the initial M1 medium on which protease production was 43.13 U mL(-1), a successful and significant improvement by 4.25 folds was achieved in the optimized medium containing (g/L): hulled grain of wheat (HGW) 5.0; KH(2)PO(4) 1.0; NaCl 0.3; MgSO(4)(7H(2)O) 0.5; CaCl(2) (7H(2)O) 0.4; ZnSO(4) 0.1; Na(2)HPO(4) 1.6; shrimp peptone (SP) 1.0. The pH was adjusted at 5 and the temperature at 30°C. More interestingly, the optimization was accomplished using two cheap and local fermentation substrates, HGW and SP, which may result in a significant reduction in the cost of medium constituents.