Gas Chromatography-Mass Spectrometry Profiling of Volatile Metabolites Produced by Some Bacillus spp. and Evaluation of Their Antibacterial and Antibiotic Activities.

Bacillus species produce different classes of antimicrobial and antioxidant substances: peptides or proteins with different structural compositions and molecular masses and a broad range of volatile organic compounds (VOCs), some of which may serve as biomarkers for microorganism identification. The aim of this study is the identification of biologically active compounds synthesized by five Bacillus species using gas chromatography coupled to mass spectrometry (GC-MS). The current study profoundly enhances the knowledge of antibacterial and antioxidant metabolites ensuring the unambiguous identification of VOCs produced by some Bacillus species, which were isolated from vegetable samples of potato, carrot, and tomato. Phylogenetic and biochemical studies were used to identify the bacterial isolates after culturing. Phylogenetic analysis proved that five bacterial isolates BSS12, BSS13, BSS16, BSS21, and BSS25 showed 99% nucleotide sequence similarities with Bacillus safensis AS-08, Bacillus cereus WAB2133, Bacillus acidiproducens NiuFun, Bacillus toyonesis FORT 102, and Bacillus thuringiensis F3, respectively. The crude extract was prepared from bacterial isolates to assess the antibiotic resistance potency and the antimicrobial potential against various targeted multidrug-resistant strains, including yeast strains such as Candida albicans, Candida krusei, and bacterial strains of Enterococcus hirae, Escherichia coli, Klebsiella aerogenes, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus group B, Streptococcus mutans, Shigella sonnei, Salmonella enteritidis, Serratia marcescens, Pseudomonas aeruginosa, and Proteus vulgaris. GC-MS analysis of bacterial strains found that VOCs from Bacillus species come in a variety of chemical forms, such as ketones, alcohols, terpenoids, alkenes, etc. Overall, 69 volatile organic compounds were identified from five Bacillus species, and all five were found to share different chemical classes of volatile organic components, which have a variety of pharmacological applications. However, eight antibacterial compounds with different concentrations were commonly found in all five species: acetoin, acetic acid, butanoic acid, 2-methyl-, oxime-, methoxy-phenyl, phenol, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, nonanoic acid, and hexadecanoic acid, methyl. The present study has demonstrated that bacterial isolates BSS25, BSS21, and BSS16 display potent inhibitory effects against Candida albicans, while BSS25, BSS21, and BSS13 exhibit the ability to restrain the growth and activity of Candida krusei. Notably, BSS25 and BSS21 are the only isolates that demonstrate substantial inhibitory activity against Klebsiella aerogenes. This disparity in inhibitory effects could be attributed to the higher concentrations of acetoin in BSS25 and BSS21, whereas BSS16 and BSS13 have relatively elevated levels of butanoic acid, 2-methyl-. Certainly, the presence of acetoin and butanoic acid, 2-methyl-, contributes to the enhanced antibacterial potential of these bacterial strains, in conjunction with other organic volatile compounds and peptides, among other factors. The biology and physiology of Bacillus can be better understood using these results, which can also be used to create novel biotechnological procedures and applications. Moreover, because of its exceptional ability to synthesize and produce a variety of different antibacterial compounds, Bacillus species can serve as natural and universal carriers for antibiotic compounds in the form of probiotic cultures and strains to fight different pathogens, including mycobacteria.

Antagonistic and protective activity of Lactobacillus plantarum strain 17 M against E. amylovora.

The subject of the study was to identify the antagonism of the Lactobacillus plantarum strain 17 M against the causative agent of fire blight, the bacterium Erwinia amylovora, and to evaluate its protective capabilities on apple blossoms against this disease. For comparison 9 strains of lactic acid bacteria from the LLP "SPC of Microbiology and Virology" collection were included in the study. Strain 17 M appeared to be superior in limiting the growth of the pathogen on all 3 liquid media tested. Its maximum inhibitory activity was proved on MRS Broth medium. The analysis of the secondary metabolites produced by strain 17 M in liquid medium revealed that it consisted mainly of acetic acid (53.2 ± 4.3%), lactic acid (16.3 ± 2.3%) and 2,3-butanedione (14.84 ± 4.1%). The presence of other organic compounds was also detected but in a smaller amount. Study on influence of those compounds on growth of E. amylovora showed that lactic acid at concentration of 5% showed inhibitory activity but it was not toxic to apple flowers. The effectiveness of strain 17 M culture liquid diluted with sterile water and added to E. amylovora inoculum at 10% or 20% was 76.7 ± 5.8% and 88.3 ± 12.6%, respectively. This study confirms the potential use of strain 17 M as an active microbial agent to combat fire blight of fruit crops in Kazakhstan.

Molecular Characterization of Some Bacillus Species from Vegetables and Evaluation of Their Antimicrobial and Antibiotic Potency.

Numerous natural habitats, such as soil, air, fermented foods, and human stomachs, are home to different Bacillus strains. Some Bacillus strains have a distinctive predominance and are widely recognized among other microbial communities, as a result of their varied habitation and physiologically active metabolites. The present study collected vegetable products (potato, carrot, and tomato) from local markets in Almaty, Kazakhstan. The bacterial isolates were identified using biochemical and phylogenetic analyses after culturing. Our phylogenetic analysis revealed three Gram-positive bacterial isolates BSS11, BSS17, and BSS19 showing 99% nucleotide sequence similarities with Bacillus subtilis O-3, Bacillus subtilis Md1-42, and Bacillus subtilis Khozestan2. The crude extract was prepared from bacterial isolates to assess the antibiotic resistance potency and the antimicrobial potential against various targeted multidrug-resistant strains, including Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus group B, Streptococcus mutans, Candida albicans, Candida krusei, Pseudomonas aeruginosa, Shigella sonnei, Klebsiella pneumoniae, Salmonella enteritidis, Klebsiella aerogenes, Enterococcus hirae, Escherichia coli, Serratia marcescens, and Proteus vulgaris. This study found that the species that were identified have the ability to produce antibiotic chemicals. Additionally, the GC-MS analysis of three bacterial extracts revealed the presence of many antibiotic substances including phenol, benzoic acid, 1,2-benzenedicarboxylic acid and bis(2-methylpropyl), methoxyphenyl-oxime, and benzaldehyde. This work sheds light on the potential of Bacillus to be employed as an antimicrobial agent to target different multidrug-resistant bacterial strains. The results indicate that market vegetables may be a useful source of strains displaying a range of advantageous characteristics that can be used in the creation of biological antibiotics.

Miniaturized solid-phase microextraction coupled with gas chromatography-mass spectrometry for determination of endocrine disruptors in drinking water.

A simple and rapid method based on miniaturized solid-phase microextraction (mini-SPME) followed by gas chromatography-mass spectrometry was developed to identify eight endocrine disruptors (atrazine, diethylstilbestrol, hexestrol, estrone, estradiol, ethinylestradiol, norgestrel, and megestrel) in drinking water samples. Extraction parameters was optimized and further analyses was performed using them. The optimum temperature for the determination of endocrine disruptors in water was 80 °C; the optimum extraction and preincubation times were 60 and 20 min, respectively. The studied linear range of endocrine disruptors was 10.0-1000 µg mL-1. The limit of detection ranged from 0.020 to 0.087 µg mL-1. The correlation coefficient (r2) was 0.96-0.99. This research introduces a novel method for detecting analytes at extremely low concentrations, as well as the possibility of combining several detection technologies to give high-accuracy qualitative and quantitative determination of endocrine disruptors in aqueous samples.

Modern Analytical Methods for the Analysis of Pesticides in Grapes: A Review.

Currently, research on the determination of pesticides in food products is very popular. Information obtained from research conducted so far mainly concerns the development of a methodology to determine the content of pesticides in food products. However, they do not describe the content of the pesticide used in viticulture in the resulting product. Over the past decade, this study has examined analytical methodologies for assessing pesticide residues in grapes. Scopus, Web of Science, Science Direct, PubMed, and Springer databases were searched for relevant publications. The phrases "pesticides" and "grapes" and their combinations were used to search for articles. The titles and annotations of the extracted articles have been read and studied to ensure that they meet the review criteria. The selected articles were used to compile a systematic review based on scientific research and reliable sources. The need to study the detection of pesticide residues in grapes using advanced analytical methods is confirmed by our systematic review. This review also highlights modern methods of sample preparation, such as QuEChERS, SPME, PLE, dLLME, and ADLL-ME, as well as the most used methods of separation and identification of pesticides in grapes. An overview of the countries where residual grape pesticide amounts are most studied is presented, along with the data on commonly used pesticides to control pests and diseases in grape cultivation. Finally, future possibilities and trends in the analysis of pesticide residues in grapes are discussed by various analytical methods.

Investigation of CO2 Extract of Portulaca oleracea for Antioxidant Activity from Raw Material Cultivated in Kazakhstan.

Medicinal plants remain as an important resource in the fight against many diseases, especially in developing countries. Antioxidants are substances capable of delaying, retarding, and preventing the oxidation of lipids or substances that delay or prevent free radical reactions during lipid oxidation. Natural antioxidants such as ascorbic acid, tocopherol, phenolic compounds, and flavonoids are a safe alternative to chemical antioxidants. In present work, results of antioxidant activity of raw materials from the cultivated plant Portulaca oleracea are presented. The extraction time was optimized to 780 minutes; the yield of extractive substances was 1.25% in the production of CO2 extract under subcritical conditions. For the first time, the antioxidant activity of Portulaca oleracea CO2 extract was determined by the amperometric method. Gas chromatography-mass spectrometry (GC-MS) chemical analysis of Portulaca oleracea CO2 extract dissolved in hexane revealed 37 components, including a complex mixture of aldehydes, alkanes, alkenes, esters, diterpenes, steroids, vitamin E, and carbohydrates. The investigation results showed that the Portulaca oleracea CO2 extract was promising for pharmaceutical, cosmetic, and food industries and had great potential for the prevention and treatment of diseases caused by oxidative stress.

Dairy associations for the targeted control of opportunistic Candida.

Antifungal and antibacterial activities of twenty-six combinations of lactic acid bacteria, propionibacteria, acetic acid bacteria and dairy yeasts inoculated in whey and milk were investigated. Associations including acetic acid bacteria were shown to suppress growth of the opportunistic yeast Candida albicans in well-diffusion assays. The protective effect of milk fermented with the two most promising consortia was confirmed in Caco-2 cell culture infected with C. albicans. Indeed, these fermented milks, after heat-treatment or not, suppressed lactate dehydrogenase release after 48 h while significant increase in LDH release was observed in the positive control (C. albicans alone) and with fermented milk obtained using commercial yogurt starter cultures. The analysis of volatile compounds in the cell-free supernatant using solid phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS) showed accumulation of significant amount of acetic acid by the consortium composed of Lactobacillus delbrueckii 5, Lactobacillus gallinarum 1, Lentilactobacillus parabuchneri 3, Lacticaseibacillus paracasei 33-4, Acetobacter syzygii 2 and Kluyveromyces marxianus 19, which corresponded to the zone of partial inhibition of C. albicans growth during well-diffusion assays. Interestingly, another part of anti-Candida activity, yielding small and transparent inhibition zones, was linked with the consortium cell fraction. This study showed a correlation between anti-Candida activity and the presence of acetic acid bacteria in dairy associations as well as a significant effect of two dairy associations against C. albicans in a Caco-2 cell model. These two associations may be promising consortia for developing functional dairy products with antagonistic action against candidiasis agents.

Component Composition and Antimicrobial Activity of CO2 Extract of Portulaca oleracea, Growing in the Territory of Kazakhstan.

In the medicine of many countries, the use of herbal healing agents included a significant contribution to improving human health and well-being. Many antibiotics have been widely used to treat infectious diseases caused by various pathogenic bacteria. However, increased multidrug resistance has led to increased severity of diseases caused by bacterial pathogens. Bacteria remain the main causative agents of diseases that cause human death, even in the present day. This cause prompted scientists to investigate alternative new molecules against bacterial strains. The significant interest for the study is Portulaca oleracea L. (family Portulacaceae), a widespread annual plant used in folk medicine. Thus, the production and study of CO2 extract of Portulaca oleracea is an actual problem. Methods. Raw materials were collected from Almaty and Zhambyl regions (Southeast and South Kazakhstan) in phase flowering. Portulaca oleracea herb's CO2 extract was obtained by subcritical carbon dioxide extraction (installation of carbon dioxide flow-through extraction- 5L). The Wiley 7th edition and NIST'02 library were used to identify the mass spectra obtained. The antimicrobial activity study was conducted by the micromethod of serial dilution and disco-diffuse method. Standard test strains of microorganisms were used: Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538-P, Candida albicans ATCC 10231, and Escherichia coli ATCC 8739. Results. The use of carbon dioxide extraction (further CO2 extract) is a promising direction of obtaining total medicinal substances containing biologically active substances, from fractions of volatile esters of various composition and functional purpose until a fraction of fatty acids and fat-soluble vitamins. In the current study, we obtained CO2 extract at subcritical conditions from aboveground organs of Portulaca oleracea and investigated the component composition for the first time. From 41 to 66 components were identified in the composition of Portulaca oleracea's CO2 extract. Studies of antimicrobial activity showed that CO2 extract of Portulaca oleracea had the expressed effect against clinically significant microorganisms such as Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Candida albicans. Conclusions. This study showed that CO2 extract of Portulaca oleracea's raw material contained biological active compounds exhibiting a significant antimicrobial effect.

Determination of semi-volatile additives in wines using SPME and GC-MS.

Parameters of headspace solid-phase microextraction, such as fiber coating (85µm CAR/PDMS), extraction time (2min for white and 3min for red wines), temperature (85°C), pre-incubation time (15min) were optimized for identification and quantification of semi-volatile additives (propylene glycol, sorbic and benzoic acids) in wines. To overcome problems in their determination, an evaporation of the wine matrix was performed. Using the optimized method, screening of 25 wine samples was performed, and the presence of propylene glycol, sorbic and benzoic acids was found in 22, 20 and 6 samples, respectively. Analysis of different wines using a standard addition approach showed good linearity in concentration ranges 0-250, 0-125, and 0-250mg/L for propylene glycol, sorbic and benzoic acids, respectively. The proposed method can be recommended for quality control of wine and disclosing adulterated samples.

Transformation products of 1,1-dimethylhydrazine and their distribution in soils of fall places of rocket carriers in Central Kazakhstan.

In our research, three fall places of first stages of Proton rockets have been studied for the presence and distribution of transformation products of 1,1-dimethylhydrazine (1,1-DMH). Results of identification of transformation products of 1,1-DMH in real soil samples polluted due to rocket fuel spills allowed to detect 18 earlier unknown metabolites of 1,1-DMH being formed only under field conditions. According to the results of quantitative analyses, maximum concentrations of 1-methyl-1H-1,2,4-triazole made up 57.3, 44.9 and 13.3 mg kg(-1), of 1-ethyl-1H-1,2,4-triazole - 5.45, 3.66 and 0.66 mg kg(-1), of 1,3-dimethyl-1H-1,2,4-triazole - 24.0, 17.8 and 4.9 mg kg(-1) in fall places 1, 2 and 3, respectively. 4-Methyl-4H-1,2,4-triazole was detected only in fall places 2 and 3 where its maximum concentrations made up 4.2 and 0.66 mg kg(-1), respectively. The pollution of soils with transformation products of 1,1-DMH was only detected in epicenters of fall places having a diameter of 8 to10 m where rocket boosters landed. The results of a detailed study of distribution of 1,1-DMH transformation products along the soil profile indicate that transformation products can migrate down to the depth of 120 cm, The highest concentrations of 1,1-DMH transformation products were detected, as a rule, at the depth 20 to 60 cm. However, this index can vary depending on the compound, humidity and physical properties of soil, landscape features and other conditions. In the surface layer, as a rule, only semi-volatile products of transformation were detected which was caused by fast evaporation and biodegradation of volatile metabolites.