Modeling and Optimization of Herb-Fortified Fresh Kombucha Cheese: An Artificial Neural Network Approach for Enhancing Quality Characteristics.

In this study, an Artificial Neural Network (ANN) model is used to solve the complex task of producing fresh cheese with the desired quality parameters. The study focuses on kombucha fresh cheese samples fortified with ground wild thyme, supercritical fluid extract of wild thyme, ground sage and supercritical fluid extract of sage and optimizes the parameters of chemical composition, antioxidant potential and microbiological profile. The ANN models demonstrate robust generalization capabilities and accurately predict the observed results based on the input parameters. The optimal neural network model (MLP 6-10-16) with 10 neurons provides high r2 values (0.993 for training, 0.992 for testing, and 0.992 for validation cycles). The ANN model identified the optimal sample, a supercritical fluid extract of sage, on the 20th day of storage, showcasing specific favorable process parameters. These parameters encompass dry matter, fat, ash, proteins, water activity, pH, antioxidant potential (TP, DPPH, ABTS, FRAP), and microbiological profile. These findings offer valuable insights into producing fresh cheese efficiently with the desired quality attributes. Moreover, they highlight the effectiveness of the ANN model in optimizing diverse parameters for enhanced product development in the dairy industry.

Label-Free Direct Detection of Cylindrospermopsin via Graphene-Enhanced Surface Plasmon Resonance Aptasensor.

In this work, we report a novel method for the label-free detection of cyanotoxin molecules based on a direct assay utilizing a graphene-modified surface plasmon resonance (SPR) aptasensor. Molecular dynamic simulation of the aptamer's interaction with cylindrospermopsin (CYN) reveals the strongest binding sites between C18-C26 pairs. To modify the SPR sensor, the wet transfer method of CVD monolayer graphene was used. For the first time, we report the use of graphene functionalized by an aptamer as a bioreceptor in conjunction with SPR for the detection of CYN. In a direct assay with an anti-CYN aptamer, we demonstrated a noticeable change in the optical signal in response to the concentrations far below the maximum tolerable level of 1 µg/L and high specificity.

Antimicrobial potential of kombucha fresh cheese with the addition of sage (Salvia officinalis L.) and its preparations.

One of the main challenges in fresh cheese technology is its rather limited shelf life. Prolongation of the shelf life of fresh cheese has been the focus of numerous research studies and different strategies have been thus used. One of the strategies that could prolong the shelf life of fresh cheese, as well as increase its quality is the application of different starter cultures. As the antimicrobial capacity of sage (Salvia officinalis) has been proven, the possibility of reusing its by-product obtained from a tea factory could be a significant step towards the retention of environmental equilibrium and simultaneous production of food with additional functional value. Therefore, the aim of our research was to examine the antimicrobial potential of kombucha fresh cheese with the addition of ground herbal sage, sage essential oil and sage supercritical fluid extract, and compare it with fresh cheese obtained from a commercial starter culture. In order to examine the antimicrobial activity of kombucha fresh cheese produced with the addition of sage preparations, the produced samples were artificially contaminated with common foodborne contaminants: Listeria monocytogenes, Escherichia coli and Staphylococcus aureus. The obtained results revealed that the addition of sage essential oil and herbal ground sage increased the antimicrobial activity during the 30 days of storage against E. coli in kombucha fresh cheese (decrease of 2.9 and 2.5 log CFU g-1, respectively). Implementation of sage significantly increased the antimicrobial activity of the fresh cheese produced with a commercial XPL-1 starter culture against L. monocytogenes (essential oil - 0.9 log CFU g-1 and ground sage - 1.2 log CFU g-1). In the XPL-1 sample, the growth of S. aureus was inhibited by the addition of ground sage - a decrease of 1.4 log CFU g-1. Analysis of the total phenols revealed their 5-fold higher content in the kombucha fresh cheeses compared to the samples obtained by the XPL-1 starter culture. These results correlate with the higher antimicrobial activity of the kombucha fresh cheese samples compared to the XPL-1 samples. According to our results, industrial waste, obtained as a by-product in sage (Salvia officinalis) filter tea production, can be efficiently used in fresh cheese technology in order to increase the antimicrobial activity against L. monocytogenes, E. coli and S. aureus.

Antioxidative capacity of fresh kombucha cheese fortified with sage herbal dust and its preparations.

Recent studies have intensively investigated the possibility of kombucha application as non-conventional starter culture in manufacture of various fermented dairy products. Furthermore, natural extracts from medicinal and aromatic plants contain different biologically active components which often have antioxidant properties. Based on the stated above, the aim of this research was to investigate the possibility of kombucha inoculum application as a new starter culture in fresh cheese technology, as well as to investigate effects of sage (Salvia officinalis) herbal dust (by-product from filter tea factory), its essential oil and supercritical fluid extract on antioxidative activity and sensory characteristics of produced fresh kombucha cheese during 10 days of storage. In all samples, higher ABTS than DPPH radical scavenging activity was determined. Freshly prepared and 10 days stored kombucha cheeses fortified with different types of sage preparations had significantly higher FRAP values than the control sample. All analysed samples had satisfied sensory characteristics and same scores of sensory evaluation after the production. Kombucha fresh cheese with addition of different types of sage preparations can be an innovative and valuable dairy product.

Elaiophylin Is a Potent Hsp90/ Cdc37 Protein Interface Inhibitor with K-Ras Nanocluster Selectivity.

The natural product elaiophylin is a macrodiolide with a broad range of biological activities. However, no direct target of elaiophylin in eukaryotes has been described so far, which hinders a systematic explanation of its astonishing activity range. We recently showed that the related conglobatin A, a protein-protein interface inhibitor of the interaction between the N-terminus of Hsp90 and its cochaperone Cdc37, blocks cancer stem cell properties by selectively inhibiting K-Ras4B but not H-Ras. Here, we elaborated that elaiophylin likewise disrupts the Hsp90/ Cdc37 interaction, without affecting the ATP-pocket of Hsp90. Similarly to conglobatin A, elaiophylin decreased expression levels of the Hsp90 client HIF1α, a transcription factor with various downstream targets, including galectin-3. Galectin-3 is a nanocluster scaffold of K-Ras, which explains the K-Ras selectivity of Hsp90 inhibitors. In agreement with this K-Ras targeting and the potent effect on other Hsp90 clients, we observed with elaiophylin treatment a submicromolar IC50 for MDA-MB-231 and MIA-PaCa-2 3D spheroid formation. Finally, a strong inhibition of MDA-MB-231 cells grown in the chorioallantoic membrane (CAM) microtumor model was determined. These results suggest that several other macrodiolides may have the Hsp90/ Cdc37 interface as a target site.

Novel Small Molecule Hsp90/Cdc37 Interface Inhibitors Indirectly Target K-Ras-Signaling.

The ATP-competitive inhibitors of Hsp90 have been tested predominantly in kinase addicted cancers; however, they have had limited success. A mechanistic connection between Hsp90 and oncogenic K-Ras is not known. Here, we show that K-Ras selectivity is enabled by the loss of the K-Ras membrane nanocluster modulator galectin-3 downstream of the Hsp90 client HIF-1α. This mechanism suggests a higher drug sensitivity in the context of KRAS mutant, HIF-1α-high and/or Gal3-high cancer cells, such as those found, in particular, in pancreatic adenocarcinoma. The low toxicity of conglobatin further indicates a beneficial on-target toxicity profile for Hsp90/Cdc37 interface inhibitors. We therefore computationally screened >7 M compounds, and identified four novel small molecules with activities of 4 µM-44 µM in vitro. All of the compounds were K-Ras selective, and potently decreased the Hsp90 client protein levels without inducing the heat shock response. Moreover, they all inhibited the 2D proliferation of breast, pancreatic, and lung cancer cell lines. The most active compounds from each scaffold, furthermore, significantly blocked 3D spheroids and the growth of K-Ras-dependent microtumors. We foresee new opportunities for improved Hsp90/Cdc37 interface inhibitors in cancer and other aging-associated diseases.

Evolution-guided engineering of non-heme iron enzymes involved in nogalamycin biosynthesis.

Microbes are competent chemists that are able to generate thousands of chemically complex natural products with potent biological activities. The key to the formation of this chemical diversity has been the rapid evolution of secondary metabolism. Many enzymes residing on these metabolic pathways have acquired atypical catalytic properties in comparison with their counterparts found in primary metabolism. The biosynthetic pathway of the anthracycline nogalamycin contains two such proteins, SnoK and SnoN, belonging to nonheme iron and 2-oxoglutarate-dependent mono-oxygenases. In spite of structural similarity, the two proteins catalyze distinct chemical reactions; SnoK is a C2-C5″ carbocyclase, whereas SnoN catalyzes stereoinversion at the adjacent C4″ position. Here, we have identified four structural regions involved in the functional differentiation and generated 30 chimeric enzymes to probe catalysis. Our analyses indicate that the carbocyclase SnoK is the ancestral form of the enzyme from which SnoN has evolved to catalyze stereoinversion at the neighboring carbon. The critical step in the appearance of epimerization activity has likely been the insertion of three residues near the C-terminus, which allow repositioning of the substrate in front of the iron center. The loss of the original carbocyclization activity has then occurred with changes in four amino acids near the iron center that prohibit alignment of the substrate for the formation of the C2-C5″ bond. Our study provides detailed insights into the evolutionary processes that have enabled Streptomyces soil bacteria to become the major source of antibiotics and antiproliferative agents. ENZYMES: EC number 1.14.11.

In vitro antitumor activity, ADME-Tox and 3D-QSAR of synthesized and selected natural styryl lactones.

Prostate cancer is a common cause of death in men and a novel treating methods should be developed. In order to find a new drug for prostate cancer, a series of novel conformationally constrained analogues of (+)-goniofufurone and 7-epi-(+)-goniofufurone, as well as the newly synthesized styryl lactones containing the cinnamic acid ester groups were evaluated for in vitro cytotoxicity against prostate cancer cell (PC-3). Furthermore, prediction of physicochemical characteristics and drugability as well as in silico ADME-Tox tests of investigated compounds were performed. The 3D-QSAR model was established using the comparative molecular field analysis method. According to obtained results, the tricyclic compounds 9 and 10 had the highest potency with IC50 < 20 µM. Evaluation of structural features through 3D-QSAR model identified steric field feature on the cinnamic acid ester groups at C-7 as a crucial for the cytotoxic activity. This research suggests that most of the analysed compounds have desirable properties for drug candidates and high potential in drug development, which recommend them for further research in treatment of prostate cancer. Furthermore, obtained 3D-QSAR model is able to successfully identify styryl lactones that have significant cytotoxic activity and provide information for screening and design of novel inhibitors against PC-3 cell line that could be used as drugs in treatment of the prostate cancer.

Toward steroidal anticancer drugs: Non-parametric and 3D-QSAR modeling of 17-picolyl and 17-picolinylidene androstanes with antiproliferative activity on breast adenocarcinoma cells.

The present study is aimed to analyze lipophilicity and ADMET profiles, and to develop field based 3D-QSAR and ligand-based pharmacophore hypothesis for a series of 17α-picolyl and 17(E)-picolinylidene androstane derivatives in order to give detailed structural insights and to highlight important binding features of novel androstane derivatives, as compounds with antiproliferative activity toward breast adenocarcinoma cells. This study can provide guidelines for the rational design of novel potent compounds. Sum of ranking differences (SRD), as a non-parametric method, was applied for compounds ranking. 3D-QSAR methods, including comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA), were applied to predict the antiproliferative effect on breast adenocarcinoma cells and provide the regions in space where interactive fields may influence the activity. The compounds are ranked so the compounds with the most favorable ADME and lipophilicity features together with significant anticancer activity can be distinguished. The established 3D-QSAR model could be used for design of new compounds with antiproliferative activity on the human ER- breast adenocarcinoma cells. The pharmacophore model is able to accurately predict antiproliferative activity. Generally, the present study provides significant guidelines for further selection, synthesis and rational design of new highly potential androstane derivatives as anticancer drugs.

Kinetics of lactose fermentation in milk with kombucha starter.

The aim of this research was to investigate the effect of new, non-conventional starter culture on the kinetics of the lactose transformation during milk fermentation by kombucha, at pH 5.8; 5.4; 5.1; 4.8; and 4.6, at two different temperatures 37 °C and 42 °C. Milk fermentation at 42 °C lasted significantly shorter (about 5 h, 30 min) compared to the fermentation at 37 °C. Changes of lactose concentration at the both temperatures are consisting of two retaining stages and very steep decline in-between. The analysis of the rate curves showed that the reaction rate passes through the maximum after 9 h, 30 min at 37 °C and after 4 h at 42 °C. The sigmoidal saturation curve indicates a complex kinetics of lactose fermentation by kombucha starter.

Modeling of rheological characteristics of the fermented dairy products obtained by novel and traditional starter cultures.

Tree different fermented dairy products obtained by conventional and non-conventional starter cultures were investigated in this paper. Textural and rheological characteristics as well as chemical composition during 21 days of storage were analysed and subsequent data processing was performed by principal component analysis. The analysis of samples` flow behaviour was focused on their time dependent properties. Parameters of Power law model described flow behaviour of samples depended on used starter culture and days of storage. The Power law model was applied successfully to describe the flow of the fermented milk, which had characteristics of shear thinning and non-Newtonian fluid behaviour.

In silico identification of milk antihypertensive di- and tripeptides involved in angiotensin I-converting enzyme inhibitory activity.

Identification of bioactive milk peptides could improve food technology through improved selection of food supplements with a focus on antihypertensive properties. We hypothesized that angiotensin I-converting enzyme (ACE) inhibitory activities of milk di- and tripeptides could be predicted using 3-dimensional quantitative structure activity relationship methods and that these activities could be explained through evaluation of structural features (hydrogen bond donor/acceptor, hydrophobic, steric, and electrostatic) that are responsible for this bioactivity. We aimed to build comparative molecular field analysis (CoMFA) models combined with in silico digestion to predict the peptide sequences released from enzymatic digestion and to evaluate peptides without experimental data. Furthermore, molecular docking simulation was performed with the aim to evaluate structural features. Molecular docking simulations revealed that the most potent inhibitory peptides contain hydrophobic amino acids that enter deep into the hydrophobic pocket of the ACE active site and make interactions with its residues. CoMFA results point out favorable steric interactions and electronegativity at the C-terminus of the milk dipeptides. The CoMFA model appears to favor electropositive amino acids at the second place in tripeptides and electronegative interaction with Tyr520. Furthermore, predicted values of ACE inhibitory activity of dipeptides obtained by peptide cutter are relatively high, which recommend them for application as functional food supplements and natural alternatives to ACE inhibitory drugs. This research suggests that obtained 3-dimensional quantitative structure activity relationship models are able to successfully identify milk-derived di- and tripeptides that have significant antihypertensive activity and provide information for screening and design of novel ACE inhibitors that could be used as supplements in human nutrition.

Preselection of A- and B- modified d-homo lactone and d-seco androstane derivatives as potent compounds with antiproliferative activity against breast and prostate cancer cells - QSAR approach and molecular docking analysis.

The problem with trial-and-error approach in organic synthesis of targeted anticancer compounds can be successfully avoided by computational modeling of molecules, docking studies and chemometric tools. It has been proven that A- and B- modified d-homo lactone and d-seco androstane derivatives are compounds with significant antiproliferative activity against estrogen-independent breast adenocarcinoma (ER-, MDA-MB-231) and androgen-independent prostate cancer cells (AR-, PC-3). This paper presents the quantitative structure-activity relationship (QSAR) models based on artificial neural networks (ANNs) which are able to predict whether d-homo lactone and/or d-seco androstane-based compounds will express antiproliferative activity against breast cancer cells (MDA-MB-231) or not. Also, the present paper describes the molecular docking study of 3ß-acetoxy-5α,6α-epoxy- (3) and 6α,7α-epoxy-1,4-dien-3-one (24) d-homo lactone androstane derivatives, as well as 4-en-3-one (15) d-seco androstane derivative, which are compounds with strong or moderate antiproliferative activity against prostate cancer cells (PC-3), and compares them with commercially available medicament for prostate cancer - abiraterone. The obtained promising results can be used as guidelines in further syntheses of novel d-homo lactone and d-seco androstane derivatives with antiproliferative activity against breast and prostate cancer cells.