Towards understanding the antagonistic activity of phytic acid against common foodborne bacterial pathogens using a general linear model.

The increasing challenge of antibiotic resistance requires not only the discovery of new antibiotics, but also the development of new alternative approaches. Therefore, in the present study, we investigated for the first time the antibacterial potential of phytic acid (myo-inositol hexakisphosphate, IP6), a natural molecule that is 'generally recognized as safe' (FDA classification), against the proliferation of common foodborne bacterial pathogens such as Listeria monocytogenes, Staphylococcus aureus and Salmonella Typhimurium. Interestingly, compared to citric acid, IP6 was found to exhibit significantly greater inhibitory activity (P<0.05) against these pathogenic bacteria. The minimum inhibitory concentration of IP6 varied from 0.488 to 0.97 mg/ml for the Gram-positive bacteria that were tested, and was 0.244 mg/ml for the Gram-negative bacteria. Linear and general models were used to further explore the antibacterial effects of IP6. The developed models were validated using experimental growth data for L. monocytogenes, S. aureus and S. Typhimurium. Overall, the models were able to accurately predict the growth of L. monocytogenes, S. aureus, and S. Typhimuriumin Polymyxin acriflavine lithium chloride ceftazidime aesculin mannitol (PALCAM), Chapman broth, and xylose lysine xeoxycholate (XLD) broth, respectively. Remarkably, the early logarithmic growth phase of S. Typhimurium showed a rapid and severe decrease in a period of less than one hour, illustrating the bactericidal effect of IP6. These results suggest that IP6 is an efficient antibacterial agent and can be used to control the proliferation of foodborne pathogens. It has promising potential for environmentally friendly applications in the food industry, such as for food preservation, food safety, and for prolonging shelf life.

Optimization of ß-mannanase production by Bacillus subtilis US191 using economical agricultural substrates.

The Bacillus subtilis US191 strain producing highly thermostable ß-mannanase was previously selected as potential probiotic candidate for application as feed supplement in poultry industry. Initially, the level of extracellular ß-mannanase production by this strain was 1.48 U ml-1 . To improve this enzyme titer, the present study was undertaken to optimize the fermentation conditions through experimental designs and valorization of agro-industrial byproducts. Using the Plackett-Burman design, in submerged fermentation, a set of 14 culture variables was evaluated in terms of their effects on ß-mannanase production. Locust bean gum (LBG), soymeal, temperature, and inoculum size were subsequently optimized by response surface methodology using Box-Behnken design. Under optimized conditions (1 g L-1 LBG, 8 g L-1 soymeal, temperature of 30°C and inoculum size of 1010 CFU ml-1 ), a 2.59-fold enhancement in ß-mannanase titer was achieved. Next, to decrease the enzyme production cost, the effect of partial substitution of LBG (1 g L-1 ) by agro-industrial byproducts was investigated, and a Taguchi design was applied. This allowed the attaining of a ß-mannanase production level of 8.75 U ml-1 in presence of 0.25 g L-1 LBG, 5 g L-1 of coffee residue powder, 5 g L-1 of date seeds powder, and 5 g L-1 of prickly pear seeds powder as mannans sources. Overall, a 5.91-fold improvement in ß-mannanase production by B. subtilis US191 was achieved.

Selection of Bacillus subtilis US191 as a mannanase-producing probiotic candidate.

ß-Mannanases are crucial enzymes for the breakdown of mannans. As Mannans are being considered as antinutritional factors in poultry production, the search for mannanase-producing probiotic bacteria is now attracting considerable attention as a strategy to enhance nutrients bioavailability. Five soil born Bacilli (US134, US150, US176, US180, and US191) were selected for their ability to produce extracellular ß-mannanases that were biochemically characterized. The probiotic properties of these strains were determined to assess their potential as animal feed supplements. Bacillus subtilis US191 was shown to be sensitive to all antibiotics tested, to inhibit growth of the bacterial pathogens tested, and to produce a thermostable ß-mannanase. It exhibited a notable acid and bovine bile tolerance and high ability to form biofilm. These features may favor its adherence to the intestinal epithelial cells allowing its survival and persistence in the digestive tract. Furthermore, our study revealed that US191 was among the strains showing the highest ability to digest wheat dry matter in vitro when compared to the commercial feed additive Rovabio® Max. Altogether, our findings suggest that the ß-mannanase producer B.subtilis US191 is a promising probiotic candidate for the feed industry.

Assessment of the potential of the multi-enzyme producer Bacillus amyloliquefaciens US573 as alternative feed additive.

BACKGROUND: Recently, probiotics have increasingly been used as feed additives in poultry diets as an alternative to antibiotic growth promoters fostering resistance development. RESULTS: This study was aimed at assessing the potential of Bacillus amyloliquefaciens US573 as a direct-fed microbial. The US573 strain was found to be free of harmful enzymatic activities and sensitive to antibiotics. In addition, it showed a good acid and bovine bile tolerance, high adhesion efficacy to chicken enterocytes, and an ability to form biofilms, which may favor its survival and persistence in the animal gastrointestinal tract. Moreover, besides the previously described extremely salt-tolerant and highly thermostable phytase, the US573 strain secretes xylanase, ß-glucanase and amylase activities useful in neutralizing antinutritional factors and maximizing the absorption of nutrients. The secretion of such enzymes may be responsible for the good performance of the US573 isolate in the digestibility of wheat in vitro. Indeed, using the vegetative cells, a yield of wheat dry matter digestibility of approximately 48% was achieved, which is slightly lower than the commercial feed additive Rovabio used as a reference (56.73% digestibility). CONCLUSION: The obtained results illustrate the potential of US573 strain as a promising direct-fed microbial candidate for application in the poultry industry. © 2017 Society of Chemical Industry.

Cloning and characterization of the first actinomycete ß-propeller phytase from Streptomyces sp. US42.

A gene encoding an extracellular phytase was cloned for the first time from an Actinomycete, Streptomyces sp. US42 and sequenced. The sequence of this gene revealed an encoded polypeptide (PHY US42) exhibiting one and six residues difference with the putative phytases of Streptomyces lividans TK24 and Streptomyces coelicolor A3(2), respectively. The molecular modeling of PHY US42 indicated that this phytase belongs to the group of ß-propeller phytases that are usually calcium-dependent. PHY US42 was purified and characterized. Its activity was calcium-dependent and maximal at pH 7 and 65 °C. The enzyme was perfectly stable at pH ranging from 5 to 10 and its thermostability was greatly enhanced in the presence of calcium. Indeed, PHY US42 maintained 80% of activity after 10 min of incubation at 75 °C in the presence of 5 mM CaCl2 . PHY US42 was also found to exhibit high stability after incubation at 37 °C for 1 h in the presence of bovine bile and digestive proteases like of pepsin, trypsin, and chymotrypsin. Considering its biochemical properties, PHY US42 could be used as feed additive in combination with an acid phytase for monogastric animals.

Evidence for the negative regulation of phytase gene expression in Streptomyces lividans and Streptomyces coelicolor.

Sco7697, a gene encoding a phytase, enzyme able to degrade phytate (myo-inositol 1,2,3,4,5,6-hexakis phosphate), the most abundant phosphorus storing compound in plants is present in the genome of S. coelicolor, a soil born bacteria with a saprophytic lifestyle. The expression of this gene was previously shown to be induced in conditions of Pi limitation by the response regulator PhoP binding to an operator sequence, the PHO box, located upstream of the -35 promoter sequence. A close examination of the promoter region of sco7697 revealed the presence of another putative operator site, a Direct Repeat (DR), located downstream of the -10 promoter sequence. In order to determine whether this DR played a role in regulation of sco7697 expression, different variants of the phytase gene promoter region were transcriptionally fused to the ß-glucuronidase reporter gene (GUS). As expected, deletion of the PHO box led to abolition of sco7697 induction in conditions of Pi limitation. Interestingly, alteration of the DR correlated with a dramatic increase of GUS expression but only when PhoP was present. These results demonstrated that this DR is the site of strong negative regulation by an unknown repressor. The latter would impede the necessary activation of phytase expression by PhoP.

Characterization of an extremely salt-tolerant and thermostable phytase from Bacillus amyloliquefaciens US573.

The extracellular phytase produced by the Bacillus amyloliquefaciens US573 strain, isolated from geothermal soil located in Southern Tunisia was purified and characterized. This calcium-dependent and bile-stable enzyme (PHY US573) was optimally active at pH 7.5 and 70 °C. It showed a good stability at pH ranging from 4 to 10, and especially, an exceptional thermostability as it recovered 50 and 62% of activity after heating for 10 min at 100 and 90 °C, respectively. In addition, PHY US573 was found to be extremely salt-tolerant since it preserved 80 and 95% of activity in the presence of 20 g/l of NaCl and LiCl, respectively. The gene corresponding to PHY US573 was cloned. It encodes a 383 amino acids polypeptide exhibiting 99% identity with the highly thermostable phytases from Bacillus sp. MD2 and B. amyloliquefaciens DS11 (3 and 5 residues difference, respectively), suggesting the existence of common molecular determinants responsible for their remarkable heat stability. Overall, our findings illustrated that in addition to its high potential for application in feed industry, the salt tolerance of the PHY US573 phytase, may represent an exciting new avenue for improvement of phosphorus-use efficiency of salt-tolerant plants in soils with high salt and phytate content.

Mineral phosphate solubilization by Streptomyces sp. CTM396 involves the excretion of gluconic acid and is stimulated by humic acids.

The actinomycetes isolates (128) which were taken from agricultural soil samples and collected near a rock phosphate processing unit were screened for mineral phosphate-solubilizing (MPS) ability. A significant MPS activity was observed for 30 isolates on various phosphate sources when grown in the National Botanical Research Institute's phosphate broth. CTM396 and CTM397 strains which showed the highest MPS abilities were identified by 16S rDNA sequencing as members of the genus Streptomyces. Their MPS activity was proved to be concomitant with a drop in pH due to the secretion of gluconic acid (GA). This was correlated with the simultaneous detection by PCR of genes gdh [encoding the glucose dehydrogenase (GDH) responsible for GA production from glucose] and pqq (involved in biosynthesis of the pyrroloquinoline quinone cofactor of GDH), as well as the highlighting of GHD enzyme activity, for the first time in a Streptomyces sp. strain producing GA. Furthermore, the 0.05% of humic acids proved to have a stimulatory effect on the growth and the ability of CTM396 to solubilize Gafsa rock phosphate. According to this study, it is possible to use humic acids and Gafsa rock phosphate in association with spores of ad hoc Streptomyces strains as natural and efficient amendments to improve plant growth with no need of costly and pollutant transformation of Gafsa rock phosphate.

Crucial role of Pro 257 in the thermostability of Bacillus phytases: biochemical and structural investigation.

We have previously cloned and characterized the thermostable phytase (PHY US417) from Bacillus subtilis US417. It differs with PhyC from B. subtilis VTTE-68013 by the R257P substitution. PHY US417 was shown to be more thermostable than PhyC. To elucidate the mechanism of how the Pro 257 changes the thermostability of Bacillus phytases, this residue was mutated to Arg and Ala. The experimental results revealed that the thermostability of the P257A mutants and especially P257R was significantly decreased. The P257R and P257A mutants recovered, respectively, 64.4 and 81.5% of the wild-type activity after incubation at 75 °C for 30 min in the presence of 5mM CaCl(2). The P257R mutation also led to a severe reduction in the specific activity and catalytic efficiency of the enzyme. Structural investigation, by molecular modeling of PHY US417 and PhyC focused on the region of the 257 residue, revealed that this residue was present in a surface loop connecting two of the six characteristic ß sheets. The P257 residue is presumed to reduce the local thermal flexibility of the loop, thus generating a higher thermostability.

Heterologous expression and optimization using experimental designs allowed highly efficient production of the PHY US417 phytase in Bacillus subtilis 168.

To attempt cost-effective production of US417 phytase in Bacillus subtilis, we developed an efficient system for its large-scale production in the generally recognized as safe microorganism B. subtilis 168. Hence, the phy US417 corresponding gene was cloned in the pMSP3535 vector, and for the first time for a plasmid carrying the pAMß1 replication origin, multimeric forms of the resulting plasmid were used to transform naturally competent B. subtilis 168 cells. Subsequently, a sequential optimization strategy based on Plackett-Burman and Box-Behnken experimental designs was applied to enhance phytase production by the recombinant Bacillus. The maximum phytase activity of 47 U ml-1 was reached in the presence of 12.5 g l-1 of yeast extract and 15 g l-1 of ammonium sulphate with shaking at 300 rpm. This is 73 fold higher than the activity produced by the native US417 strain before optimization. Characterization of the produced recombinant phytase has revealed that the enzyme exhibited improved thermostability compared to the wild type PHY US417 phytase strengthening its potential for application as feed supplement. Together, our findings strongly suggest that the strategy herein developed combining heterologous expression using a cloning vector carrying the pAMß1 replication origin and experimental designs optimization can be generalized for recombinant proteins production in Bacillus.