Hydrogen Bonding Drives Helical Chirality via 10-Membered Rings in Dipeptide Conjugates of Ferrocene-1,1'-Diamine.

Considering the enormous importance of protein turns as participants in various biological events, such as protein-protein interactions, great efforts have been made to develop their conformationally and proteolytically stable mimetics. Ferrocene-1,1'-diamine was previously shown to nucleate the stable turn structures in peptides prepared by conjugation with Ala (III) and Ala-Pro (VI). Here, we prepared the homochiral conjugates of ferrocene-1,1'-diamine with l-/d-Phe (32/35), l-/d-Val (33/36), and l-/d-Leu (34/37) to investigate (1) whether the organometallic template induces the turn structure upon conjugation with amino acids, and (2) whether the bulky or branched side chains of Phe, Val, and Leu affect hydrogen bonding. Detailed spectroscopic (IR, NMR, CD), X-ray, and DFT studies revealed the presence of two simultaneous 10-membered interstrand hydrogen bonds, i.e., two simultaneous ß-turns in goal compounds. A preliminary biological evaluation of d-Leu conjugate 37 showed its modest potential to induce cell cycle arrest in the G0/G1 phase in the HeLa cell line but these results need further investigation.

Lipid Composition of Sheffersomyces stipitis M12 Strain Grown on Glycerol as a Carbon Source.

RESEARCH BACKGROUND: In this study the content and composition of lipids in ergosterol-reduced Sheffersomyces stipitis M12 strain grown on glycerol as a carbon source is determined. Blocking the ergosterol synthesis route in yeast cells is a recently proposed method for increasing S-adenosyl-l-methionine (SAM) production. EXPERIMENTAL APPROACH: The batch cultivation of M12 yeast was carried out under aerobic conditions in a laboratory bioreactor with glycerol as carbon source, and with pulsed addition of methionine. Glycerol and SAM content were monitored by high-performance liquid chromatography, while fatty acid composition of different lipid classes, separated by solid phase extraction, was determined by gas chromatography. RESULTS AND CONCLUSION: Despite the reduced amount of ergosterol in yeast cells, thanks to the reorganized lipid metabolism, M12 strain achieved high biomass yield and SAM production. Neutral lipids prevailed (making more than 75% of total lipids), but their content and composition differed significantly in the two tested types of yeast. Unsaturated and C18 fatty acids prevailed in both the M12 strain and wild type. In all fractions except free fatty acids, the index of unsaturation in M12 strain was lower than in the wild strain. Our tested strain adjusts itself by changing the content of lipids (mainly phospholipids, sterols and sterol esters), and with desaturation adjustments, to maintain proper functioning and fulfil increased energy needs. NOVELTY AND SCIENTIFIC CONTRIBUTION: Reorganization of S. stipitis lipid composition caused by blocking the metabolic pathway of ergosterol synthesis was presented. A simple scheme of actual lipid metabolism during active SAM production in S. stipitis, grown on glycerol was constructed and shown. This fundamental knowledge of lipid metabolic pathways will be a helpful tool in improving S. stipitis as an expression host and a model organism, opening new perspectives for its applied research.

Properties and Fermentation Activity of Industrial Yeasts Saccharomyces cerevisiae, S. uvarum, Candida utilis and Kluyveromyces marxianus Exposed to AFB1, OTA and ZEA.

In this paper the effect of aflatoxin B1, ochratoxin A and zearalenon on morphology, growth parameters and metabolic activity of yeasts Saccharomyces cerevisiae, Saccharomyces uvarum, Candida utilis and Kluyveromyces marxianus was determined. The results showed that the three mycotoxins affected the morphology of all these yeasts, primarily the cell diameter, but not their final cell count. Fourier transform infrared spectroscopy showed that the yeast membranes bound the mycotoxins, C. utilis in particular. The cell membranes of most yeasts underwent denaturation, except S. uvarum exposed to ochratoxin A and zearalenone. In the early stage of fermentation, all mycotoxin-exposed yeasts had lower metabolic activity and biomass growth than controls, but fermentation products and biomass concentrations reached the control levels by the end of the fermentation, except for C. utilis exposed to 20 µg/mL of zearalenone. The adaptive response to mycotoxins suggests that certain yeasts could be used to control mycotoxin concentrations in the production of fermented food and beverages.

Improved Properties and Microbiological Safety of Novel Cottage Cheese Containing Spices.

The study focuses on developing novel cottage cheese containing spices with acceptable sensory properties, increased biological value and extended shelf life. Thirty types of cheese with added fresh or dried parsley, dill, pepper, garlic and rosemary were produced. Characterisation of phenolic compounds, antioxidant capacity and antibacterial activity of spices and cheese samples were evaluated. The cheese containing fresh pepper and fresh and dried herbs showed excellent sensory properties, with the best results obtained with fresh sweet red pepper. Dry rosemary had the highest antioxidant and antibacterial activity due to high mass fractions of caffeic and rosmarinic acids as well as high mass fractions of flavones and phenolic diterpenes. The plant extracts examined in vitro and in situ effectively reduce numbers of foodborne pathogens like Salmonella typhimurium, Escherichia coli, Staphylococcus aureus and Listeria monocytogenes, and therefore have potential as natural preservatives and antioxidants.

A Predictive Assessment of Ochratoxin A's Effects on Oxidative Stress Parameters and the Fermentation Ability of Yeasts Using Neural Networks.

The aim of this paper was to examine the effect of different OTA concentrations on the parameters of oxidative stress (glutathione (GSH) and malondialdehyde (MDA) concentrations) and glucose utilization in ethanol production by wine yeasts. In addition to the above, artificial neural networks (ANN) were used to predict the effects of different OTA concentrations on the fermentation ability of yeasts and oxidative stress parameters. The obtained results indicate a negative influence of OTA (4 µg mL-1) on ethanol production after 12 h. For example, K. marxianus produced 1.320 mg mL-1 of ethanol, while in the control sample 1.603 µg mL-1 of ethanol was detected. However, after 24 h, OTA had no negative effect on ethanol production, since it was higher (7.490 and 3.845 mg mL-1) in comparison to control samples. Even low concentrations of OTA affect GSH concentrations, with the highest being detected after 12 and 24 h (up to 16.54 µM), while MDA concentrations are affected by higher OTA concentrations, with the highest being detected at 24 h (1.19 µM). The obtained results with the use of ANNs showed their potential for quantification purposes based on experimental data, while the results of ANN prediction models have shown to be useful for predictions of what outcomes different concentrations of OTA that were not part of experiment will have on the fermentation capacity and oxidative stress parameters of yeasts.

The effect of starvation stress on Lactobacillus brevis L62 protein profile determined by de novo sequencing in positive and negative mass spectrometry ion mode.

RATIONALE: We describe a novel negative chemically activated fragmentation/positive chemically activated fragmentation (CAF-/CAF+) technique for protein identification. The technique was used to investigate Lactobacillus brevis adaptation to nutrient deprivation. METHODS: The CAF-/CAF+ method enables de novo sequencing of derivate peptides with negative and positive ion mode matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS). Peptide sequences obtained from MS/MS spectra were matched against the National Center for Biotechnology Information (NCBI) non-redundant (nr) database and confirmed by the mass spectrometry data of elucidated peptide mass sequences derived from the annotated genome. This improved protein identification method highlighted 36 differentially expressed proteins in the proteome of L. brevis after 75 days of starvation. RESULTS: The results revealed the key differences in the metabolic pathways that are responsible for the survival of L. brevis in a hostile environment. Proteomics analysis demonstrated that numerous proteins engaged in glucose and amino-acid catabolizing pathways, glycerolipid metabolizing pathways, and stress-response mechanisms are differentially expressed after long-term starvation. Amino acid and proteomics analysis indicated that starved L. brevis metabolized arginine, glycine, and histidine from dead cells as alternative nutrient sources. The production of lactic acid also varied between the parent cells and the starved cells. CONCLUSIONS: Differentially expressed proteins identified exclusively by peptide sequence reading provided promising results for CAF-/CAF+ implementation in a standard proteomics workflow (e.g., biomarker and mutation discovery and biotyping). The practical performance of a reliable de novo sequencing technique in routine proteomics analysis is emphasized in this article.

Characterization of Lactobacillus brevis L62 strain, highly tolerant to copper ions.

Lactic acid bacteria (LAB) as starter culture in food industry must be suitable for large-scale industrial production and possess the ability to survive in unfavorable processes and storage conditions. Approaches taken to address these problems include the selection of stress-resistant strains. In food industry, LAB are often exposed to metal ions induced stress. The interactions between LAB and metal ions are very poorly investigated. Because of that, the influence of non-toxic, toxic and antioxidant metal ions (Zn, Cu, and Mn) on growth, acid production, metal ions binding capacity of wild and adapted species of Leuconostoc mesenteroides L3, Lactobacillus brevis L62 and Lactobacillus plantarum L73 were investigated. The proteomic approach was applied to clarify how the LAB cells, especially the adapted ones, protect themselves and tolerate high concentrations of toxic metal ions. Results have shown that Zn and Mn addition into MRS medium in the investigated concentrations did not have effect on the bacterial growth and acid production, while copper ions were highly toxic, especially in static conditions. Leuc. mesenteroides L3 was the most efficient in Zn binding processes among the chosen LAB species, while L. plantarum L73 accumulated the highest concentration of Mn. L. brevis L62 was the most copper resistant species. Adaptation had a positive effect on growth and acid production of all species in the presence of copper. However, the adapted species incorporated less metal ions than the wild species. The exception was adapted L. brevis L62 that accumulated high concentration of copper ions in static conditions. The obtained results showed that L. brevis L62 is highly tolerant to copper ions, which allows its use as starter culture in fermentative processes in media with high concentration of copper ions.

Alcoholic Fermentation as a Source of Congeners in Fruit Spirits.

Fermentation is a crucial process in the production of alcoholic beverages such as spirits, which produces a number of volatile compounds due to the metabolic activities of yeast. These volatile compounds, together with the volatile components of the raw materials and the volatile compounds produced during the distillation and aging process, play a crucial role in determining the final flavor and aroma of spirits. In this manuscript, we provide a comprehensive overview of yeast fermentation and the volatile compounds produced during alcoholic fermentation. We will establish a link between the microbiome and volatile compounds during alcoholic fermentation and describe the various factors that influence volatile compound production, including yeast strain, temperature, pH, and nutrient availability. We will also discuss the effects of these volatile compounds on the sensory properties of spirits and describe the major aroma compounds in these alcoholic beverages.

Interaction of lactic acid bacteria with metal ions: opportunities for improving food safety and quality.

Certain species of lactic acid bacteria (LAB), as well as other microorganisms, can bind metal ions to their cells surface or transport and store them inside the cell. Due to this fact, over the past few years interactions of metal ions with LAB have been intensively investigated in order to develop the usage of these bacteria in new biotechnology processes in addition to their health and probiotic aspects. Preliminary studies in model aqueous solutions yielded LAB with high absorption potential for toxic and essential metal ions, which can be used for improving food safety and quality. This paper provides an overview of results obtained by LAB application in toxic metal ions removing from drinking water, food and human body, as well as production of functional foods and nutraceutics. The biosorption abilities of LAB towards metal ions are emphasized. The binding mechanisms, as well as the parameters influencing the passive and active uptake are analyzed.

Physicochemical and aromatic characterization of carob macerates produced by different maceration conditions.

Carob liqueur is an alcoholic drink (minimum 15% v/v of ethanol and 100 g/L of sugar) typical for the Mediterranean countries. In the current work, carob macerate produced by maceration of carob pods in hydroalcoholic base at different maceration conditions was characterized for the first time based on its aroma compounds/profile, physicochemical parameters, and chromatic characteristics. The results confirm the migration process of bioactive compounds, aroma compounds, and sugars flowing from the carob pod to the hydroalcoholic base. Changes in ethanol concentration modify the physical properties of the solvent and influence the phenolic and aroma compounds extraction, color, and acidity of the obtained samples. The higher content of phenolic compounds was determinate in the samples obtained in the darkness. The amounts of phenols were in the range of some red fruit liqueurs or walnut liqueurs, and sugars (mostly sucrose) ranging between 96 and 107 g/L. Twenty-six (out of total 94) aroma compounds were detected in all samples, of which 17 esters, 3 alcohols, 4 ketones, and 2 acids. Low molecular weight ethyl esters, ethyl hexanoate, ethyl 2-methyl propanoate, ethyl octanoate, ethyl benzoate, ethyl butanoate, and ethyl cinnamate, were the most abundant. Carob pod maceration in 50% v/v hydroalcoholic base (1:5 solid to liquid ratio) in darkness at room temperature during 8 weeks can be recommended as optimal maceration conditions for production of the aromatic carob macerate with functional properties.

Optimization of bioprocess for production of copper-enriched biomass of industrially important microorganism Saccharomyces cerevisiae.

The production of Saccharomyces cerevisiae cells enriched with copper and the effects of adding copper ions to different media on yeast cell growth and ethanol production were studied. In the media Cu(2+) concentrations of up to 0.094 mM had no effect on alcoholic fermentation, whereas higher Cu(2+) concentrations markedly decreased yeast cell growth rate and ethanol production. Under static conditions, the maximum amounts of copper uptake (i.e., 1.16 mg/g, 1.2 mg/g and 0.81 mg/g dry matter yeast biomass for glucose, sucrose and molasses media, respectively) were obtained after 8 h of fermentation, whereas under dynamic conditions smaller amounts of copper uptake (i.e., 0.98 mg/g, 1.02 mg/g and 0.7 mg/g dry matter yeast biomass for glucose, sucrose and molasses media, respectively) were obtained after 6 h of fermentation.

Characterization of a S-adenosyl-l-methionine (SAM)-accumulating strain of Scheffersomyces stipitis.

S-adenosyl-l-methionine (SAM) is an important molecule in the cellular metabolism of mammals. In this study, we examined several of the physiological characteristics of a SAM-accumulating strain of the yeast Scheffersomyces stipitis (M12), including SAM production, ergosterol content, and ethanol tolerance. S. stipitis M12 accumulated up to 52.48 mg SAM/g dry cell weight. Proteome analyses showed that the disruption of C-24 methylation in ergosterol biosynthesis, a step mediated by C-24 sterol methyltransferase (Erg6p), results in greater SAM accumulation by S. stipitis M12 compared to the wild-type strain. A comparative proteome-wide analysis identified 25 proteins that were differentially expressed by S. stipitis M12. These proteins are involved in ribosome biogenesis, translation, the stress response, ubiquitin-dependent catabolic processes, the cell cycle, ethanol tolerance, posttranslational modification, peroxisomal membrane stability, epigenetic regulation, the actin cytoskeleton and cell morphology, iron and copper homeostasis, cell signaling, and energy metabolism.

Antifungal and antipatulin activity of Gluconobacter oxydans isolated from apple surface.

Fungicides are the most common agents used in postharvest treatment of fruit and are the most effective against blue mould, primarily caused by Penicillium expansum. Alternatively, blue mould can be treated with antagonistic microorganisms naturally occurring on fruit, such as the bacterium Gluconobacter oxydans. The aim of this study was to establish the antifungal potential of the G. oxydans 1J strain isolated from apple surface against Penicillium expansum in culture and apple juice and to compare it with the efficiency of a reference strain G. oxydans ATCC 621H. The highest antifungal activity of G. oxydans 1J was observed between days 3 and 9 with no colony growth, while on day 12, P. expansum colony diameter was reduced to 42.3% of the control diameter. Although G. oxydans 1J did not fully inhibit mould growth, it showed a high level of efficiency and completely prevented patulin accumulation in apple juice.