Identification of bacilysin, chlorotetaine, and iturin a produced by Bacillus sp. strain CS93 isolated from pozol, a Mexican fermented maize dough

Three antimicrobial compounds produced by Bacillus sp. strain CS93 isolated from pozol were identified by using high-performance liquid chromatography and mass spectrometry. The three compounds were iturin, bacilysin, and chlorotetaine. Production of these compounds by CS93 could account for the medicinal properties attributed to pozol.     

Polyphasic Study of the Spatial Distribution of Microorganisms in Mexican Pozol, a Fermented Maize Dough, Demonstrates the Need for Cultivation-Independent Methods To Investigate Traditional Fermentations

The distribution of microorganisms in pozol balls, a fermented maize dough, was investigated by a polyphasic approach in which we used both culture-dependent and culture-independent methods, including microbial enumeration, fermentation product analysis, quantification of microbial taxa with 16S rRNA-targeted oligonucleotide probes, determination of microbial fingerprints by denaturing gradient gel electrophoresis (DGGE), and 16S ribosomal DNA gene sequencing. Our results demonstrate that DGGE fingerprinting and rRNA quantification should allow workers to precisely and rapidly characterize the microbial assemblage in a spontaneous lactic acid fermented food. Lactic acid bacteria (LAB) accounted for 90 to 97% of the total active microflora; no streptococci were isolated, although members of the genus Streptococcus accounted for 25 to 50% of the microflora. Lactobacillus plantarum and Lactobacillus fermentum, together with members of the genera Leuconostoc and Weissella, were the other dominant organisms. The overall activity was more important at the periphery of a ball, where eucaryotes, enterobacteria, and bacterial exopolysacharide producers developed. Our results also showed that the metabolism of heterofermentative LAB was influenced in situ by the distribution of the LAB in the pozol ball, whereas homolactic fermentation was controlled primarily by sugar limitation. We propose that starch is first degraded by amylases from LAB and that the resulting sugars, together with the lactate produced, allow a secondary flora to develop in the presence of oxygen. Our results strongly suggest that cultivation-independent methods should be used to study traditional fermented foods.     

Cloning with Mud-P22 hybrid prophages: mapping of IS200 elements on the chromosome of Salmonella typhimurium LT2

Specific DNA fragments from the chromosome of Salmonella typhimurium LT2 were packaged in P22 capsids by induction of “locked-in” Mud-P22 hybrid prophages. High yields of the packaged DNA were obtained upon capsid disruption. DNA hybridization using a fragment of insertion sequence IS200 as probe permitted physical mapping of IS200 elements on the chromosome of S. typhimurium LT2 within?±1 centisome (CS). IS200 copies were found at the following locations: CS 24 (copy VI), CS 53 (copy V), CS 63 (copy I), CS 80 (copy II) and CS 93 (copy III). Copy IV, previously mapped near fliA (CS 42), was not included in our study.     

Microbial and Physiological Characterization of Weakly Amylolytic but Fast-Growing Lactic Acid Bacteria: a Functional Role in Supporting Microbial Diversity in Pozol, a Mexican Fermented Maize Beverage

Pozol is an acid beverage obtained from the natural fermentation of nixtamal (heat- and alkali-treated maize) dough. The concentration of mono- and disaccharides from maize is reduced during nixtamalization, so that starch is the main carbohydrate available for lactic acid fermentation. In order to provide some basis to understand the role of amylolytic lactic acid bacteria (ALAB) in this fermented food, their diversity and physiological characteristics were determined. Forty amylolytic strains were characterized by phenotypic and molecular taxonomic methods. Four different biotypes were distinguished via ribotyping; Streptococcus bovis strains were found to be predominant. Streptococcus macedonicus, Lactococcus lactis, and Enterococcus sulfureus strains were also identified. S. bovis strain 25124 showed extremely low amylase yield relative to biomass (139 U g [cell dry weight]⁻¹) and specific rate of amylase production (130.7 U g [cell dry weight]⁻¹ h⁻¹). In contrast, it showed a high specific growth rate (0.94 h⁻¹) and an efficient energy conversion yield to bacterial cell biomass (0.31 g of biomass g of substrate⁻¹). These would confer on the strain a competitive advantage and are the possible reasons for its dominance. Transient accumulation of maltooligosaccharides during fermentation could presumably serve as energy sources for nonamylolytic species in pozol fermentation. This would explain the observed diversity and the dominance of nonamylolytic lactic acid bacteria at the end of fermentation. These results are the first step to understanding the importance of ALAB during pozol fermentation.     

Carnosic acid and carnosol inhibit adipocyte differentiation in mouse 3T3-L1 cells through induction of phase2 enzymes and activation of glutathione metabolism

In the previous studies, we reported that carnosic acid (CA) and carnosol (CS) originating from rosemary protected cortical neurons by activating the Keap1/Nrf2 pathway, which activation was initiated by S-alkylation of the critical cysteine thiol of the Keap1 protein by the “electrophilic” quinone-type of CA or CS. Here, we found that CA and CS inhibited the in vitro differentiation of mouse preadipocytes, 3T3-L1 cells, into adipocytes. In contrast, other physiologically-active and rosemary-originated compounds were completely negative. These actions seemed to be mediated by activation of the antioxidant-response element (ARE) and induction of phase2 enzymes. This estimation is justified by our present findings that only CA and CS among rosemary-originated compounds significantly activated the ARE and induced the phase2 enzymes. Next, we performed cDNA microarray analysis in order to identify the gene(s) responsible for these biological actions and found that phase2 enzymes (Gsta2, Gclc, Abcc4, and Abcc1), all of which are involved in the metabolism of glutathione (GSH), constituted 4 of the top 5 CA-induced genes. Furthermore, CA and CS, but not the other compounds tested, significantly increased the intracellular level of total GSH. Thus, we propose that the stimulation of GSH metabolism may be a critical step for the inhibition of adipocyte differentiation in 3T3-L1 cells and suggest that pro-electrophilic compounds such as CA and CS may be potential drugs against obesity-related diseases.     

Green synthesis,characterization, enhanced functionality and biological evaluation of silver nanoparticles based on Coriander sativum

The present study focused on the green synthesis of silver nanoparticles from Coriander sativum (CS) containing structural polymers, phenolic compounds and glycosidic bioactive macromolecules. Plant phenolic compounds can act as antioxidants, lignin, and attractants like flavonoids and carotenoids. Henceforth, silver nanoparticles (AgNPs) were prepared extracellularly by the combinatorial action of stabilizing and reduction of the CS leaf extract. The biologically synthesized CS-AgNPs were studied by UV-spectroscopy, zeta potential determination, scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analysis to characterize and confirm the formation of crystalline nanoparticles. The synthesized nanoparticles demonstrated strong antimicrobial activity against all microbial strains examined with varying degrees. The scavenging action on free radicals by CS-AgNPs showed strong antioxidant efficiency with superoxide and hydroxyl radicals at different concentrations as compared with standard ascorbic acid. The presence of in vitro anticancer effect was confirmed at different concentrations on the MCF-7 cell line as revealed with decrease in cell viability which was proportionately related to the concentration of CS-AgNPs illustrating the toxigenic nature of synthesized nanoparticles on cancerous cells.     

Caloric Restriction Shortens Lifespan through an Increase in Lipid Peroxidation,Inflammation and Apoptosis in the G93A Mouse,an Animal Model of ALS

Caloric restriction (CR) extends lifespan through a reduction in oxidative stress, delays the onset of morbidity and prolongs lifespan. We previously reported that long-term CR hastened clinical onset, disease progression and shortened lifespan, while transiently improving motor performance in G93A mice, a model of amyotrophic lateral sclerosis (ALS) that shows increased free radical production. To investigate the long-term CR-induced pathology in G93A mice, we assessed the mitochondrial bioenergetic efficiency and oxidative capacity (CS – citrate synthase content and activity, cytochrome c oxidase - COX activity and protein content of COX subunit- I and IV and UCP3- uncoupling protein 3), oxidative damage (MDA – malondialdehyde and PC – protein carbonyls), antioxidant enzyme capacity (Mn-SOD, Cu/Zn-SOD and catalase), inflammation (TNF-α), stress response (Hsp70) and markers of apoptosis (Bax, Bcl-2, caspase 9, cleaved caspase 9) in their skeletal muscle. At age 40 days, G93A mice were divided into two groups: Ad libitum (AL; n = 14; 7 females) or CR (n = 13; 6 females), with a diet equal to 60% of AL. COX/CS enzyme activity was lower in CR vs. AL male quadriceps (35%), despite a 2.3-fold higher COX-IV/CS protein content. UCP3 was higher in CR vs. AL females only. MnSOD and Cu/Zn-SOD were higher in CR vs. AL mice and CR vs. AL females. MDA was higher (83%) in CR vs. AL red gastrocnemius. Conversely, PC was lower in CR vs. AL red (62%) and white (30%) gastrocnemius. TNF-α was higher (52%) in CR vs. AL mice and Hsp70 was lower (62%) in CR vs. AL quadriceps. Bax was higher in CR vs. AL mice (41%) and CR vs. AL females (52%). Catalase, Bcl-2 and caspases did not differ. We conclude that CR increases lipid peroxidation, inflammation and apoptosis, while decreasing mitochondrial bioenergetic efficiency, protein oxidation and stress response in G93A mice.     

Polyphasic study of the spatial distribution of microorganisms in Mexican pozol, a fermented maize dough, demonstrates the need for cultivation-independent methods to investigate traditional fermentations

The distribution of microorganisms in pozol balls, a fermented maize dough, was investigated by a polyphasic approach in which we used both culture-dependent and culture-independent methods, including microbial enumeration, fermentation product analysis, quantification of microbial taxa with 16S rRNA-targeted oligonucleotide probes, determination of microbial fingerprints by denaturing gradient gel electrophoresis (DGGE), and 16S ribosomal DNA gene sequencing. Our results demonstrate that DGGE fingerprinting and rRNA quantification should allow workers to precisely and rapidly characterize the microbial assemblage in a spontaneous lactic acid fermented food. Lactic acid bacteria (LAB) accounted for 90 to 97% of the total active microflora; no streptococci were isolated, although members of the genus Streptococcus accounted for 25 to 50% of the microflora. Lactobacillus plantarum and Lactobacillus fermentum, together with members of the genera Leuconostoc and Weissella, were the other dominant organisms. The overall activity was more important at the periphery of a ball, where eucaryotes, enterobacteria, and bacterial exopolysacharide producers developed. Our results also showed that the metabolism of heterofermentative LAB was influenced in situ by the distribution of the LAB in the pozol ball, whereas homolactic fermentation was controlled primarily by sugar limitation. We propose that starch is first degraded by amylases from LAB and that the resulting sugars, together with the lactate produced, allow a secondary flora to develop in the presence of oxygen. Our results strongly suggest that cultivation-independent methods should be used to study traditional fermented foods.     

Experimental databases on inhibition of the bacterial mutagenicity of 4-nitroquinoline 1-oxide and cigarette smoke

Two antimutagenicity databases were prepared by applying a co-treatment procedure to the Salmonella reversion assay. Ninety compounds belonging to various chemical classes were quantitatively tested for antimutagenicity towards the direct-acting mutagen 4-nitroquinoline 1-oxide (4NQO) in strain TA100 of S. typhimurium and 63 of them were additionally tested for antimutagenicity towards unfractionated mainstream cigarette smoke (CS) in strain TA98, in the presence of S9 mix. Twelve compounds (13.3%) inhibited 4NQO mutagenicity by at least 50%, with a MID₅₀ (dose inhibiting 50% of mutagenicity) varying over a 1226-fold range. Twenty-six compounds (41.3%) inhibited CS mutagenicity, with a MID₅₀ varying over a 520-fold range. Three compounds only, i.e., bilirubin, curcumin and myricetin, were capable of inhibiting the mutagenicities of both 4NQO and CS. However, myricetin and the other flavonoid rutin were at the same time mutagenic by inducing frameshift mutations following metabolic activation. There was a rather rigorous selectivity of antimutagenicity data depending on the chemical class of inhibitors and it was possible to discriminate protective effects within several pairs or series of structurally related compounds. For instance, all eight thiols and aminothiols inhibited 4NQO mutagenicity, which contrasted with the inactivity of the remaining 17 sulfur compounds tested, all of them lacking a free sulfhydryl group. The mutagenicity of CS was consistently inhibited by the majority of phenols (eight out of 10 tested) and by all two isothiocyanates, two dithiocarbamates, three indole derivatives, three tetrapyrrole compounds and three flavonoids tested. Although the results obtained cannot be extrapolated to other mutagens or test systems, they may provide a useful source of information for research in the area of antimutagenesis and for the development of chemopreventive agents.     

Microbial Community Dynamics during Production of the Mexican Fermented Maize Dough Pozol

The dynamics of the microbial community responsible for the traditional fermentation of maize in the production of Mexican pozol was investigated by using a polyphasic approach combining (i) microbial enumerations with culture media, (ii) denaturing gradient gel electrophoresis (DGGE) fingerprinting of total community DNA with bacterial and eukaryotic primers and sequencing of partial 16S ribosomal DNA (rDNA) genes, (iii) quantification of rRNAs from dominant microbial taxa by using phylogenetic oligonucleotide probes, and (iv) analysis of sugars and fermentation products. A Streptococcus species dominated the fermentation and accounted for between 25 and 75% of the total flora throughout the process. Results also showed that the initial epiphytic aerobic microflora was replaced in the first 2 days by heterofermentative lactic acid bacteria (LAB), including a close relative of Lactobacillus fermentum, producing lactic acid and ethanol; this heterolactic flora was then progressively replaced by homofermentative LAB (mainly close relatives of L. plantarum, L. casei, and L. delbrueckii) which continued acidification of the maize dough. At the same time, a very diverse community of yeasts and fungi developed, mainly at the periphery of the dough. The analysis of the DGGE patterns obtained with bacterial and eukaryotic primers targeting the 16S and 18S rDNA genes clearly demonstrated that there was a major shift in the community structure after 24 h and that high biodiversity—according to the Shannon-Weaver index—was maintained throughout the process. These results proved that a relatively high number of species, at least six to eight, are needed to perform this traditional lactic acid fermentation. The presence of Bifidobacterium, Enterococcus, and enterobacteria suggests a fecal origin of some important pozol microorganisms. Overall, the results obtained with different culture-dependent or -independent techniques clearly confirmed the importance of developing a polyphasic approach to study the ecology of fermented foods.     

High levels of oxysterol sulfates in serum of patients with steroid sulfatase deficiency

Steroid sulfatase (STS) deficiency is the underlying cause of the skin condition known as recessive X-linked ichthyosis (RXLI). RXLI patients show scales on their skin caused by high concentrations of cholesterol sulfate (CS), as they are not capable of releasing the sulfate group from its structure to obtain free cholesterol. CS has been reported, so far, as the sole sulfated steroid with increased concentrations in the blood of RXLI patients. A non-targeted LC-MS approach in negative mode detection (LC-MS precursor ion scan mode) was applied to serum samples of 12 RXLI patients and 19 healthy males. We found that CS was not the only sulfated compound consistently elevated in RXLI patients, because a group of compounds with a m/z of 481 was found in high concentrations too. Further LC-MS/MS demonstrated that the main contributor to the m/z 481 signal in RXLI serum is 27-hydroxycholesterol-3-sulfate (27OHC3S). Accordingly, a new method for 27OHC3S quantification in the context of RXLI has been developed and validated. Other hydroxycholesterol sulfate compounds were elevated as well in RXLI patients.     

Novel, developmentally specific control of Ds transposition in maize

Plants form their gametes late in somatic development and, as a result, often pass somatic mutations on to their progeny. Classic examples of this process are the germinal revertants of unstable, Ac/Ds transposon-induced kernel mutations in maize: frequent and early reversion events during somatic development are generally correlated with a high frequency of revertant gametes. We have characterized a Ds allele of the maize waxy(wx) gene, wx-m5:CS7, for which the correlation between somatic and germinal reversion frequencies no longer holds. The ability of wx-m5:CS7 (CS7) to produce revertant gametes is suppressed ～100-fold in comparison with a second Ds allele, wx-m5:CS8 (CS8), which has an identical insertion at Wx and the same frequent and early somatic reversion pattern in endosperm. The excision of Ds from wx is not reduced 100-fold in the somatic tissues of CS7 plants as compared with CS8 plants. Suppressed formation of CS7 revertant gametes is independent of the Ac transposase source and is heritably passed to the embryos of progeny kernels; however, frequent and early somatic reversion is observed again in endosperms of these progeny kernels. This suppression appears to be caused by a dominant mutation in a trans-acting product that can suppress the germinal reversion of other Ds-induced alleles as well; the mutation is tightly linked to Wx but is not in the CS7 Ds itself. Taken together, the data suggest a novel mode of developmental control of Ac/Ds elements by the host plant, suppressing element excision in the shoot meristem.     

Insecticidal activity and feeding behavior of the green peach aphid,Myzus persicae,after treatment with nano types of pyrifluquinazon

Pyrifluquinazon modifies insect behavior by rapidly stopping feeding so that insects starve to death. In the present study, the effects of two nano types of pyrifluquinazon and a non-nano type of pyrifluquinazon on mortality of the green peach aphid, Myzus persicae, were investigated using three different concentrations (25, 50, and 100 ppm). The nano types of pyrifluquinazon were formulated with a different molecular weight and density than that of chitosan (CS 30,000, 0.1% and CS 3000, 0.3%) for stability of unsteady core materials. According to the bioassay and the increased rate of aphids, both types of pyrifluquinazon at all concentrations were effective for controlling M. persicae. The CS 3000 0.3% nano type pyrifluquinazon exhibited a more effective controlled-release feature than that of the other type. The two nano types and the non-nano type pyrifluquinazon were bioassayed against M. persicae, and feeding behaviors were compared using the electrical penetration graph technique. The non-nano type appeared to have the best lethal efficiency at 2 days after treatment at 50 and 25 ppm according to the non-penetration time; however, both CS 30,000 0.1% and CS 3000 0.3% were effective at 14 days after treatment. Furthermore, reaction time slowed from 14 to 30 days after treatment in the CS 3000 0.3% treated aphids. As a result, CS 3000 0.3% had more effective controlled release at both concentrations.     

Redox-dependent induction of antioxidant defenses by phenolic diterpenes confers stress tolerance in normal human skin fibroblasts: Insights on replicative senescence

Mild stress-induced hormesis represents a promising strategy for targeting the age-related accumulation of molecular damage and, therefore, for preventing diseases and achieving healthy aging. Fruits, vegetables, and spices contain a wide variety of hormetic phytochemicals, which may explain the beneficial health effects associated with the consumption of these dietary components. In the present study, the induction of cellular antioxidant defenses by the phenolic diterpenes carnosic acid (CA) and carnosol (CS) were studied in normal human skin fibroblasts, and insights into the aging process at the cellular level investigated. We observed that CA and CS induced several cytoprotective enzymes and antioxidant defenses in human fibroblasts, whose induction was dependent on the cellular redox state for CS and associated with Nrf2 signaling for both compounds. The stress response elicited by preincubation with CS conferred a cytoprotective action against a following oxidant challenge with tert-butyl hydroperoxide, confirming its hormetic effect. Preincubation of normal fibroblasts with CS also protected against hydrogen peroxide-induced premature senescence. Furthermore, cultivation of middle passage normal human skin fibroblasts in the presence of CS ameliorated the physiological state of cells during replicative senescence. Our results support the view that mild stress-induced antioxidant defenses by CS can confer stress tolerance in normal cells and may have important implications in the promotion of healthy aging.     

Exploring the mechanism of Cassiae semen in regulating lipid metabolism through network pharmacology and experimental validation

Background: Multiple studies have assessed the role of Cassiae semen (CS) in regulating lipid metabolism. However, the mechanism of action of CS on non-alcoholic fatty liver disease (NAFLD) has seen rare scrutiny. Objective: The objective of this study was to explore the regulatory mechanism of CS on lipid metabolism in NAFLD. Methods: Components of CS ethanol extract (CSEE) were analyzed and identified using UPLC-Q-Orbirap HRMS. The candidate compounds of CS and its relative targets were extracted from the Traditional Chinese Medicine Systems Pharmacology, Swiss-Target-Prediction, and TargetNet web server. The Therapeutic Target Database, Genecards, Online Mendelian Inheritance in Man, and DisGeNET were searched for NAFLD targets. Binding affinity between potential core components and key targets was established employing molecular docking simulations. After that, free fatty acid (FFA)-induced HepG2 cells were used to further validate part of the network pharmacology results. Results: Six genes, including Caspase 3 (CASP3), phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α (PIK3CA), epidermal growth factor receptor (EGFR), and amyloid β (A4) precursor protein (APP) were identified as key targets. The mitogen-activated protein kinase (MAPK) signaling pathway was found to associate closely with CS’s effect on NAFLD. Per molecular docking findings, toralactone and quinizarin formed the most stable combinations with hub genes. About 0.1 (vs. FFA, P<0.01) and 0.2 (vs. FFA, P<0.05) mg/ml CSEE decreased lipid accumulation in vitro by reversing the up-regulation of CASP3, EGFR, and APP and the down-regulation of PIK3CA. Conclusion: CSEE can significantly reduce intracellular lipid accumulation by modulating the MAPK signaling pathway to decrease CASP3 and EGFR expression.     

Improved chlamydospore formation in Tricholoma bakamatsutake with addition of amino acids in vitro

Tricholoma bakamatsutake, which forms its fruit body in oak forests, is similar to T. matsutake in shape and odor; therefore, it is expected to have a high economic value similar to T. matsutake. Recently, the fruit bodies of T. bakamatsutake were produced after planting of a Quercus phillyraeoides sapling with a pure culture of the fungal mycelia. This fungus forms pigmented thick-walled spores (chlamydospores [CSs]) terminally on the hyphae in culture and in the field settings. Abundant CS production in vitro seems to be advantageous for cultivating mycorrhizal trees inoculated with T. bakamatsutake CSs. We investigated the effects of adding 16 different nitrogen (N) compounds (three inorganic and 13 organic) to a culture medium on vegetative hyphal growth and CS formation. The growth of T. bakamatsutake was reduced by many of the N sources used. Conversely, the rate of CS formation was improved by the addition of valine or glutamine.     

Novel fluorinated lipopeptides from Bacillus sp. CS93 via precursor-directed biosynthesis

While attempting to improve production of fluoro-iturin A in Bacillus sp. CS93 new mono- and di-fluorinated fengycins were detected in culture supernatants by ¹⁹F NMR and tandem mass spectrometry, after incubation of the bacterium with 3-fluoro-l-tyrosine. The fluorinated amino acid was presumably incorporated in place of one or both of the tyrosyl residues in fengycin. Investigations to generate additional new fluorinated derivatives were undertaken using commercially available fluorinated phenylalanines and 2-fluoro- and 2,3-difluoro-tyrosine that were synthesised by Negishi cross-coupling of iodoalanine and fluorinated bromo-phenols. The anti-fungal activity of the fluorinated lipopeptides was assayed against Trichophyton rubrum and found to be similar to that of the non-fluorinated metabolites.