Acid pH Tolerance Research Articles

PROBIOTIC AND NUTRITIONAL CONTENT OF SOURO, AN IRANIAN TRADITIONAL FOOD

Probiotic foods are products containing adequate amounts of live and specific microorganisms that confer benefit to human health. This research was performed to spotlight on the nutritional value evaluation as well as isolation and probiotic characterization of lactic acid bacteria (LAB) isolated from native food "Souro" belonging to Iran. The isolated LABs were investigated in vitro for potential probiotic features based on their acidic pH tolerance, bile-salt resistance, hydrophobicity, autoaggragation, production of amylase and protease as well as antibiotic susceptibility test. Results showed that Souro had high content of protein (51 ± 0.16%,) and total lipid was 11.18 ± 0.38%. Most of isolates were able to produce protease and amylase. From the best potential probiotic isolates, 16S rRNA gene sequencing disclosed Lactobacillus plantarum, L. hilgardii, L. casei, L. fermentum and Weissella paramesenteroides. Thus, good physiological traits and safety evaluation of the isolated LAB emphasized the benefits of Souro for further use in the human diet as well as the preparation of diverse probiotic food formulations.

THE PROBIOTIC AND NUTRITIONAL CONTENT OF SOURO, AN IRANIAN TRADITIONAL FOOD

Probiotic foods are products containing adequate amounts of live and specific microorganisms that confer benefit to human health. This research was performed to spotlight on the nutritional value evaluation as well as isolation and probiotic characterization of lactic acid bacteria (LAB) isolated from native food belonging to Iran. The isolated LABs were investigated in vitro for potential probiotic features based on their acidic pH tolerance, bile-salt resistance, hydrophobicity, autoaggragation, production of amylase and protease as well as antibiotic susceptibility test. Results showed that Souro had high content of protein (51 ± 0.16%,) and total lipid was 11.18 ± 0.38%. Most of isolates were able to produce protease and amylase. From the best potential probiotic isolates, 16S rRNA gene sequencing disclosed Lactobacillus plantarum, L. hilgardii, L. casei, L. fermentum and Weissella paramesenteroides. Thus, good physiological traits and safety evaluation of the isolated LAB emphasized the benefits of Souro for further use in the human diet as well as the preparation of diverse probiotic food formulations.

Development and characterization of acidic-pH-tolerant mutants of Zymomonas mobilis through adaptation and next-generation sequencing-based genome resequencing and RNA-Seq

BackgroundAcid pretreatment is a common strategy used to break down the hemicellulose component of the lignocellulosic biomass to release pentoses, and a subsequent enzymatic hydrolysis step is usually applied to release hexoses from the cellulose. The hydrolysate after pretreatment and enzymatic hydrolysis containing both hexoses and pentoses can then be used as substrates for biochemical production. However, the acid-pretreated liquor can also be directly used as the substrate for microbial fermentation, which has an acidic pH and contains inhibitory compounds generated during pretreatment. Although the natural ethanologenic bacterium Zymomonas mobilis can grow in a broad range of pH 3.5 ~ 7.5, cell growth and ethanol fermentation are still affected under acidic-pH conditions below pH 4.0.ResultsIn this study, adaptive laboratory evolution (ALE) strategy was applied to adapt Z. mobilis under acidic-pH conditions. Two mutant strains named 3.6M and 3.5M with enhanced acidic pH tolerance were selected and confirmed, of which 3.5M grew better than ZM4 but worse than 3.6M in acidic-pH conditions that is served as a reference strain between 3.6M and ZM4 to help unravel the acidic-pH tolerance mechanism. Mutant strains 3.5M and 3.6M exhibited 50 ~ 130% enhancement on growth rate, 4 ~ 9 h reduction on fermentation time to consume glucose, and 20 ~ 63% improvement on ethanol productivity than wild-type ZM4 at pH 3.8. Next-generation sequencing (NGS)-based whole-genome resequencing (WGR) and RNA-Seq technologies were applied to unravel the acidic-pH tolerance mechanism of mutant strains. WGR result indicated that compared to wild-type ZM4, 3.5M and 3.6M have seven and five single nucleotide polymorphisms (SNPs), respectively, among which four are shared in common. Additionally, RNA-Seq result showed that the upregulation of genes involved in glycolysis and the downregulation of flagellar and mobility related genes would help generate and redistribute cellular energy to resist acidic pH while keeping normal biological processes in Z. mobilis. Moreover, genes involved in RND efflux pump, ATP-binding cassette (ABC) transporter, proton consumption, and alkaline metabolite production were significantly upregulated in mutants under the acidic-pH condition compared with ZM4, which could help maintain the pH homeostasis in mutant strains for acidic-pH resistance. Furthermore, our results demonstrated that in mutant 3.6M, genes encoding F1F0 ATPase to pump excess protons out of cells were upregulated under pH 3.8 compared to pH 6.2. This difference might help mutant 3.6M manage acidic conditions better than ZM4 and 3.5M. A few gene targets were then selected for genetics study to explore their role in acidic pH tolerance, and our results demonstrated that the expression of two operons in the shuttle plasmids, ZMO0956–ZMO0958 encoding cytochrome bc1 complex and ZMO1428–ZMO1432 encoding RND efflux pump, could help Z. mobilis tolerate acidic-pH conditions.ConclusionAn acidic-pH-tolerant mutant 3.6M obtained through this study can be used for commercial bioethanol production under acidic fermentation conditions. In addition, the molecular mechanism of acidic pH tolerance of Z. mobilis was further proposed, which can facilitate future research on rational design of synthetic microorganisms with enhanced tolerance against acidic-pH conditions. Moreover, the strategy developed in this study combining approaches of ALE, genome resequencing, RNA-Seq, and classical genetics study for mutant evolution and characterization can be applied in other industrial microorganisms.

Extracellular DNA controls expression of Pseudomonas aeruginosa genes involved in nutrient utilization, metal homeostasis, acid pH tolerance and virulence.

Introduction. Pseudomonas aeruginosa grows in extracellular DNA (eDNA)-enriched biofilms and infection sites. eDNA is generally considered to be a structural biofilm polymer required for aggregation and biofilm maturation. In addition, eDNA can sequester divalent metal cations, acidify growth media and serve as a nutrient source.Aim. We wanted to determine the genome-wide influence on the transcriptome of planktonic P. aeruginosa PAO1 grown in the presence of eDNA.Methodology. RNA-seq analysis was performed to determine the genome-wide effects on gene expression of PAO1 grown with eDNA. Transcriptional lux fusions were used to confirm eDNA regulation and to validate phenotypes associated with growth in eDNA.Results. The transcriptome of eDNA-regulated genes included 89 induced and 76 repressed genes (FDR<0.05). A large number of eDNA-induced genes appear to be involved in utilizing DNA as a nutrient. Several eDNA-induced genes are also induced by acidic pH 5.5, and eDNA/acidic pH promoted an acid tolerance response in P. aeruginosa. The cyoABCDE terminal oxidase is induced by both eDNA and pH 5.5, and contributed to the acid tolerance phenotype. Quantitative metal analysis confirmed that DNA binds to diverse metals, which helps explain why many genes involved in a general uptake of metals were controlled by eDNA. Growth in the presence of eDNA also promoted intracellular bacterial survival and influenced virulence in the acute infection model of fruit flies.Conclusion. The diverse functions of the eDNA-regulated genes underscore the important role of this extracellular polymer in promoting antibiotic resistance, virulence, acid tolerance and nutrient utilization; phenotypes that contribute to long-term survival.

Probiotic effects of marine Debaryomyces hansenii CBS 8339 on innate immune and antioxidant parameters in newborn goats.

Several marine Debaryomyces hansenii strains have shown probiotic effects on aquatic animals, and D. hansenii-derived β-glucans have recently provided immunostimulant effects on goat leukocytes. This study assessed the probiotic effects of live yeast D. hansenii CBS 8339 on newborn goats administered orally, and subsequently challenged in vitro with Escherichia coli. D. hansenii CBS 8339 demonstrated the capacity to survive gastrointestinal tract conditions (bile salts and acid pH tolerance) and adhere to goat intestine. Twelve Saanen × Nubian crossbred newborn goats (2.9 ± 0.47kg) were fed with a controlled diet or D. hansenii (0.7g/kg body weight per day)-supplemented milk for 30days. Blood samples of newborn goats were taken at days 15 and 30, and peripheral blood leukocytes were isolated for bacterial challenge, and immunological and antioxidant analyses. Despite cell viability was higher in leukocytes of goat kids fed with the yeast supplement, protection against E. coli challenge was not significantly affected. On the other hand, at day 15, oral administration of D. hansenii enhanced respiratory burst and catalase activity and increased superoxide dismutase activity after challenge. In contrast, at day 30, administration of the yeast supplement increased peroxidase activity and enhanced nitric oxide production and catalase activity after challenge. Finally, the yeast-supplemented diet upregulated the expression of the receptor genes TLR (2, 4, 6), modulator genes Raf.1, Syk, and Myd88, transcription factor gene AP-1, and cytokine genes IL-1β and TNF-α only at day 15 in leukocytes from unchallenged goat kids. These results demonstrated that a short time (15days) of orally administering the probiotic D. hansenii CBS 8339 to newborn goats stimulated innate immune and antioxidant parameters and the expression of immune-related gene signaling pathways.

A protease-resistant α-galactosidase characterized by relatively acid pH tolerance from the Shitake Mushroom Lentinula edodes.

A monomeric α-galactosidase with a molecular weight of 64 kDa was purified from fresh fruiting bodies of Lentinula edodes. The purification protocol involved ion-exchange chromatography on DEAE-cellulose, CM-cellulose and Q-Sepharose and a final gel-filtration on Superdex 75. The purified α-galactosidase (LEGI) was identified by LC-MS/MS. It demonstrated the optimum pH of 5.0 and temperature optimum of 60 °C towards pNPGal. It was inhibited by Cd2+, Fe3+, Pb2+, Zn2+, Al3+, Hg2+, Cr2+, Ba2+. The LEGI activity was strongly abolished by the chemical modification N-bromosuccinimide (NBS) at 1 mM, while significantly enhanced by the thiol-reducing agents dithiothreitol (DTT). Moreover, LEGI showed strong resistance to protease pepsin, papain, acid protease and neutral protease. LEGI demonstrated hydrolysis towards melibiose (13.27%), raffinose (4.75%), stachyose (2.58%), locust bean gum (0.82%) and guar gum (1.29%). The Km values of LEGI for pNPGal, stachyose, raffinose, and melibiose were found to be 1.08, 17.24, 13.80 and 8.05 mM, respectively. Results suggest that LEGI demonstrates potential for elimination of indigestible oligosaccharides.

Cell Surface Display of Salmonella Outer Membrane Protein A on Lactobacillus salivarius: A First Step Towards Food-Grade Live Vaccine Against Salmonella Infections

As one of the most important pathogens in both humans and animals, Salmonella is a significant health problem, especially in the developing countries. Outer membrane protein A (OmpA) is among the major outer membrane proteins of Salmonella with strong immunogenicity. Vaccination is an effective tool for the prevention of Salmonella infections. Lactic acid bacteria are the normal flora of gastrointestinal (GI) tract. In this research, lactobacilli from different parts of chicken GI tract were isolated and evaluated for bile salts and acidic pH tolerance. One strain isolated from the small intestine was identified as Lactobacillus salivarius by carbohydrate fermentation test and 16SrRNA partial sequencing. To address the need for safe oral vaccine to control Salmonella infection, we engineered this natural chicken isolate of L. salivarius to express OmpA under promotion of Lactate dehydrogenase (ldh) promoter. The surface displayed OmpA reacted with anti-OmpA in ELISA, Immunoblotting and flow cytometry assays, suggesting that the expressed protein adopted a native conformation. Strong agglutination reaction of the recombinant lactobacilli with sera of the naturally Salmonella infected chickens showed the multivalency and overexpression of the OmpA on the surface of L. salivarius. Experimental infection assay confirmed that the engineered commensal bacteria could be an attractive candidate as food-grade live vaccine against Salmonella infections.

SPECIES DISTRIBUTION AND PHYSIOLOGICAL CHARACTERIZATION OF ACINETOBACTER GENOSPECIES FROM HEALTHY HUMAN SKIN OF TRIBAL POPULATION IN INDIA

Background: Various reports on distribution of Acinetobacter spp. from healthy human skin restricted to urban population. However, no such data is available from healthy human skin of tribal population not exposed to modern antibiotics during their life time. Purpose: Isolation, biotyping, distribution and physiological characterisation of Acinetobacter spp. from healthy human skin of tribal population. Methods: Tribal population of Toranmal area of Satpuda Ranges, Maharashtra, India were sampled for ten body sites. Tentative Acinetobacter isolates were confirmed to the genus level by chromosomal DNA transformation assay and to species level using Bouvet and Grimont system. Novel physiological characteristics like pH, temperature and salt tolerance were studied. All strains were screened for production of various enzymes. Results: One hundred and eighteen strains were isolated, which belonged to nine Acinetobacter genospecies. A. haemolyticus was most abundant followed by A. calcoaceticus and A. genospecies 1-3. Higher percentage of Acinetobacter was recovered from skin of nose, Pawara tribe and female volunteers. They showed wide variation in temperature, salt and pH tolerance. Most of the strains could produce enzymes viz, lipase, esterase, urease and amylase. Conclusions:Acinetobacter spp. belonging to nine genospecies were obtained in the present study. Physiological characteristics including high salt, temperature and acidic pH tolerance were helpful to differentiate between the commensal and pathogenic species of Acinetobacter genus.

Species distribution and physiological characterization of &lt;i&gt;Acinetobacter &lt;/i&gt;genospecies from healthy human skin of tribal population in India

Various reports on distribution of Acinetobacter spp. from healthy human skin restricted to urban population. However, no such data is available from healthy human skin of tribal population not exposed to modern antibiotics during their life time. Isolation, biotyping, distribution and physiological characterisation of Acinetobacter spp. from healthy human skin of tribal population. Tribal population of Toranmal area of Satpuda Ranges, Maharashtra, India were sampled for ten body sites. Tentative Acinetobacter isolates were confirmed to the genus level by chromosomal DNA transformation assay and to species level using Bouvet and Grimont system. Novel physiological characteristics like pH, temperature and salt tolerance were studied. All strains were screened for production of various enzymes. One hundred and eighteen strains were isolated, which belonged to nine Acinetobacter genospecies. A. haemolyticus was most abundant followed by A. calcoaceticus and A. genospecies 1-3. Higher percentage of Acinetobacter was recovered from skin of nose, Pawara tribe and female volunteers. They showed wide variation in temperature, salt and pH tolerance. Most of the strains could produce enzymes viz, lipase, esterase, urease and amylase. Acinetobacter spp. belonging to nine genospecies were obtained in the present study. Physiological characteristics including high salt, temperature and acidic pH tolerance were helpful to differentiate between the commensal and pathogenic species of Acinetobacter genus.

Acid adaptation and survival of Listeria monocytogenes in Italian-style soft cheeses

The ability of Listeria monocytogenes to survive and grow at high salt concentrations and low pH makes it a potential hazard after the consumption of milk and dairy products, often implicated in severe outbreaks of listeriosis. This study was designed to evaluate the behaviour of L. monocytogenes in traditional acid and salted Italian-style soft cheeses and to investigate whether Listeria occurrence and growth in these environments may represent a potential increase of hazard. A first approach was addressed to in vitro evaluate survival, acid tolerance response, ability to produce biofilm, and capability to invade intestinal-like cells of a L. monocytogenes strain grown under experimental conditions mimicking environmental features that this pathogen encounters in soft cheeses (such as acid pH and high NaCl content). A second set of experiments was performed to monitor, during the storage at 4 degrees C, the survival of acid-adapted and nonadapted Listeriae in artificially contaminated soft cheeses. Both acid tolerance response and invasion efficiency of acid-adapted bacteria resulted in an increase, even when bacteria were simultaneously pre-exposed to increasing salt stress. The contamination of cheeses with acid-adapted and nonadapted bacteria evidenced in all products a good survival. A significant increased survival, the recovery of bacterial cells highly resistant to lethal pH exposure, and the prevalence of filamentous structures were observed in crescenza cheese during the storage. The Listeria survival and acid pH tolerance observed during refrigerated storage are probably related to the intrinsic acid and saline features of soft cheeses analysed. Italian soft cheeses tested may represent a potential hazard for the recovery of acid-adapted L. monocytogenes cells with enhanced ability to adhere to inert surfaces and/or to penetrate host cells.

Lactococcus lactis and stress.

It is now generally recognized that cell growth conditions in nature are often suboptimal compared to controlled conditions provided in the laboratory. Natural stresses like starvation and acidity are generated by cell growth itself. Other stresses like temperature or osmotic shock, or oxygen, are imposed by the environment. It is now clear that defense mechanisms to withstand different stresses must be present in all organisms. The exploration of stress responses in lactic acid bacteria has just begun. Several stress response genes have been revealed through homologies with known genes in other organisms. While stress response genes appear to be highly conserved, however, their regulation may not be. Thus, search of the regulation of stress response in lactic acid bacteria may reveal new regulatory circuits. The first part of this report addresses the available information on stress response in Lactococcus lactis. Acid stress response may be particularly important in lactic acid bacteria, whose growth and transition to stationary phase is accompanied by the production of lactic acid, which results in acidification of the media, arrest of cell multiplication, and possible cell death. The second part of this report will focus on progress made in acid stress response, particularly in L. lactis and on factors which may affect its regulation. Acid tolerance is presently under study in L. lactis. Our results with strain MG1363 show that it survives a lethal challenge at pH 4.0 if adapted briefly (5 to 15 minutes) at a pH between 4.5 and 6.5. Adaptation requires protein synthesis, indicating that acid conditions induce expression of newly synthesized genes. These results show that L. lactis possesses an inducible response to acid stress in exponential phase. To identify possible regulatory genes involved in acid stress response, we determined low pH conditions in which MG1363 is unable to grow, and selected at 37 degrees C for transposition insertional mutants which were able to survive. About thirty mutants resistant to low pH conditions were characterized. The interrupted genes were identified by sequence homology with known genes. One insertion interrupts ahrC, the putative regulator of arginine metabolism; possibly, increased arginine catabolism in the mutant produces metabolites which increase the pH. Several other mutations putatively map at some step in the pathway of (p)ppGpp synthesis. Our results suggest that the stringent response pathway, which is involved in starvation and stationary phase survival, may also be implicated in acid pH tolerance.

Cultivar and pH effects on competition for nodule sites between isolates of Rhizobium in beans

Two experiments were carried out to evaluate the effect of acidity on bean-Rhizobium competition for nodule sites. SevenPhaseolus vulgaris host cultivars differing in acid-pH tolerance were grown in sand culture, and irrigated using a sub-irrigation system and nutrient solutions of pH 4.5, 5.0, 5.5, and 6.0. A mixed inoculant of two antibiotically markedRhizobium leguminosarum bvphaseoli strains CIAT899 (acid-tolerant) and CIAT632 (acid-sensitive) was used. The acid-tolerant CIAT899 dominated CIAT632 in nodule occupancy across all cultivars and pH treatments. Although several of the varieties had previously been identified as PH-tolerant, and these cultivars performed better than those reported to be acid sensitive, all showed a marked increase in nodulation and plant development when the pH was raised from 4.5 to 6.0. The second experiment using a modified Leonard jar system varied the inoculation ratio between CIAT899 and UMR1116 (acid-sensitive, inefficient in N2-fixation) and contrasted nodulation response for the bean varieties ‘Preto 143’ (pH-tolerant) and ‘Negro Argel’ (pH-sensitive) at 3 pH treatments (4.5, 5.5, 6.5). There was a significant effect of host cultivar, ratio of inoculation, and pH on the percentage of nodule occupancy by each strain. At low pH CIAT899 had higher nodule occupancy than UM1116 in the variety ‘Negro Argel’ but had the same percentage of nodulation when the variety was ‘Preto 143’. Increasing the cell concentration of UMR1116 produced more inefficient nodules at all treatment combinations and reduced plant growth for both cultivars used.

Phaseolus vulgaris cultivar and Rhizobium strain variation in acid-pH tolerance and nodulation under acid conditions

Acid-soil pH is a major factor limiting nodulation and N 2 fixation in beans. The experiments reported here sought strains of Rhizobium and cultivars of Phaseolus vulgaris L. tolerant of acid pH, and to study the interaction of strains and cultivars differing in pH tolerance. Seventy-eight of 217 isolates of R. leguminosarum bv. phaseoli grew on modified Keyser-Munns medium, pH 4.5. Of these, 33 grew at pH 4.5 in the presence of 100 μM aluminum and 40 tolerated pH 4.5 medium containing 200 μM manganese. Twenty isolates were both highly efficient in nitrogen (N 2) fixation and acid-pH-tolerant. Marked variation was also evident in the growth and nodulation at pH 4.5 of 126 cultivars of Phaseolus vulgaris. Thus ‘Capixaba precoce’, ‘Preto 143’ and ‘Bico de Ouro’ had high nodule number and nodule mass plant −1 when grown at this pH, whereas BAT115 and BAT165 were essentially without nodules. When host cultivars and Rhizobium strains which differed in acid pH tolerance were used in inoculation experiments in soil of pH 4.5, nodulation and plant growth were most affected when both host and Rhizobium were intolerant of low pH. this was associated with reduced multiplication of Rhizobium in the rhizosphere 2 days after planting and with lack of attachment of the Rhizobium to the host root. Competition for nodule sites at acid pH was also affected. When either the Rhizobium strain or the cultivar was acid-tolerant, nodulation and plant development were similar to that achieved when both host cultivars and Rhizobium strain were acid-pH-tolerant.