Novel Insights for Metabiotics Production by Using Artisanal Probiotic Cultures.

Wild probiotic consortia of microorganisms (bacteria and yeasts) associated in the artisanal cultures' microbiota (milk kefir grains, water kefir grains and kombucha) are considered valuable promoters for metabiotics (prebiotics, probiotics, postbiotics and paraprobiotics) production. The beneficial effects of the fermented products obtained with the artisanal cultures on human well-being are described by centuries and the interest for them is continuously increasing. The wild origin and microbial diversity of these above-mentioned consortia give them extraordinary protection capacity against microbiological contaminants in unusual physico-chemical conditions and unique fermentative behaviour. This review summarizes the state of the art for the wild artisanal cultures (milk and water kefir grains, respectively, kombucha-SCOBY), their symbiotic functionality, and the ability to ferment unconventional substrates in order to obtain valuable bioactive compounds with in vitro and in vivo beneficial functional properties. Due to the necessity of the bioactives production and their use as metabiotics in the modern consumer's life, artisanal cultures are the perfect sources able to biosynthesize complex functional metabolites (bioactive peptides, antimicrobials, polysaccharides, enzymes, vitamins, cell wall components). Depending on the purposes of the biotechnological fermentation processes, artisanal cultures can be used as starters on different substrates. Current studies show that the microbial synergy between bacteria-yeast and/or bacteria-offers new perspectives to develop functional products (food, feeds, and ingredients) with a great impact on life quality.

Bovine ß-lactoglobulin peptides as novel carriers for flavonoids extracted with supercritical fluids from yellow onion skins.

Our study describes in detail the binding mechanism between the main flavonoids that were extracted from onion skins by supercritical CO2 and peptides from whey proteins, from the perspective of obtaining multifunctional ingredients, with health-promoting benefits. The supercritical CO2 extract had 202.31 ± 11.56 mg quercetin equivalents/g DW as the major flavonoid and antioxidant activity of 404.93±1.39 mM Trolox/g DW. The experiments on thermolysin-derived peptides fluorescence quenching by flavonoids extract allowed estimating the binding parameters, in terms of binding constants, and the number of binding sites. The thermodynamic analysis indicated that the main forces involved in complex formation were hydrogen bonds and van der Waals interactions. Molecular docking tests indicated that peptide fluorescence quenching upon gradual addition of onion skin extract might be due to flavonoids binding by Val15 -Ser21 . All 7 to 14 amino acids long peptides appeared to have affinity toward quercetin-3,4'-O-diglucoside and quercetin-4'-O-monoglucoside. The study is important as a potential solution for reuse of valuable resources, underutilized, such as whey peptides and yellow onion skins flavonoids for efficient microencapsulation, as a holistic approach to deliver healthy and nutritious food. PRACTICAL APPLICATION: A growing interest was noticed in the last years in investigating the interactions between proteins and different biologically active compounds, such as to provide knowledge for efficient development of new food, pharmaceutical, and cosmetic products. Recent studies suggest that flavonoid-protein complexes may be designed to improve the functional performance of the flavonoids. The results obtained in this study bring certain benefits in terms of exploiting the bioactive potential of both flavonoids and bioactive peptides, for developing of formulas with improved functional properties.

Tailoring the Health-Promoting Potential of Protein Hydrolysate Derived from Fish Wastes and Flavonoids from Yellow Onion Skins: From Binding Mechanisms to Microencapsulated Functional Ingredients.

This study focuses on combining different bioprocessing tools in order to develop an in-depth engineering approach for enhancing the biological properties of two valuable food by-products, namely fish waste and yellow onion skins, in a single new bioactive formulation. Bone tissue from phytophagous carp (Hypophthalmichthys molitrix) was used to obtain bioactive peptides through papain-assisted hydrolysis. The peptides with molecular weight lower than 3 kDa were characterized through MALDI-ToF/ToF mass spectrometry and bioinformatics tools. As a prerequisite for microencapsulation, the ability of these peptides to bind the flavonoids extracted from yellow onion skins was further tested through fluorescence quenching measurements. The results obtained demonstrate a considerable binding potency with a binding value of 106 and also the presence of one single or one class of binding site during the interaction process of flavonoids with peptides, in which the main forces involved are hydrogen bonds and van der Waals interactions. In the freeze-drying microencapsulation process, an efficiency for total flavonoids of 88.68 ± 2.37% was obtained, considering the total flavonoids and total polyphenols from the powder of 75.72 ± 2.58 quercetin equivalents/g dry weight (DW) and 97.32 ± 2.80 gallic acid equivalents/g DW, respectively. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test on the L929 cell line cultivated in the presence of different concentrations of microencapsulated samples (0.05-1.5 mg/mL) proved no sign of cytotoxicity, the cell viability being over 80% for all the samples.

Spectroscopic and Molecular Modeling Investigation on the Interaction between Folic Acid and Bovine Lactoferrin from Encapsulation Perspectives.

The impact of thermal treatment on the ability of lactoferrin (FL) to bind folic acid (FA) was investigated by employing fluorescence spectroscopy, molecular dynamics and docking tests. The structural and conformational particularities of LF upon heating at 80 °C and 100 °C were first estimated based on the intrinsic fluorescence changes in respect to the native protein. The emission spectra indicated gradual unfolding events accompanied by Trp exposure with increasing temperature. In agreement with the experimental results, molecular modeling investigations showed that the secondary and tertiary structure of LF are slightly affected by the thermal treatment. Some minor unfolding events related particularly to the α-helical regions of LF were observed when the temperature increased to 100 °C. The LF fluorescence quenching upon FA addition indicated that a static mechanism stands behind LF-FA complex formation. Regardless of the simulated temperature, the hydrogen bonds played an important role in regulating the interaction between the protein and ligand. FA binding to LF equilibrated at different temperatures occurred spontaneously, and all complexes displayed good thermodynamic stability. The obtained results support the suitability of LF as biocompatible material, for obtaining micro- and nanoparticles for delivery of dietary supplements or for enhancing the functionality of target delivery systems.

Cross-Linked Microencapsulation of CO2 Supercritical Extracted Oleoresins from Sea Buckthorn: Evidence of Targeted Functionality and Stability.

Oleoresin supercritical extracts from sea buckthorn were microencapsulated in whey proteins isolate and casein, in two states: native (N) and cross-linked mediated by transglutaminase (TG). The encapsulation efficiency showed values higher than 92% for total carotenoids and lycopene. Phytochemicals content was 352.90 ± 1.02 mg/g dry weight (DW) for total carotenoids in TG and 302.98 ± 2.30 mg/g DW in N, with antioxidant activity of 703.13 ± 23.60 mMol Trolox/g DW and 608.74 ± 7.12 mMol Trolox/g DW, respectively. Both powders had an inhibitory effect on α-glucosidase, of about 40% for N and 35% for TG. The presence of spherosomes was highlighted, with sizes ranging between 15.23-73.41 µm and an agglutination tendency in N, and lower sizes, up to 35 µm in TG. The in vitro digestibility revealed a prolonged release in an intestinal environment, up to 65% for TG. Moisture sorption isotherms were studied at 20 °C and the shape of curves corresponds to sigmoidal type II model. The presence of cross-linked mediated aggregates in TG powders improved stability and flowability. Our results can be used as evidence that cross-linked aggregates mediated by transglutaminase applied for microencapsulation of oleoresins have the potential to become new delivery systems, for carotenoids and lycopene, being valuable in terms of their attractive color and biological and bioaccessibility properties.

Fluorescence spectroscopy and molecular modeling of anthocyanins binding to bovine lactoferrin peptides.

This work was aimed to obtain lactoferrin peptides, with anthocyanins-binding capabilities, by using eggplant peels extract as a source of anthocyanins. The chromatographic analysis of the extract evidenced the presence of five individual anthocyanins, with delfinidin-3-rutinoside being identified as the predominant. 20 small peptides were identified, from which four are containing Trp at C-terminal. By estimating the thermodynamic parameters, van der Waals and hydrogen bonding were found to have important roles in binding of anthocyanins to LF and LF-derived peptides. In order to complement the experimental results, the in silico methods were further employed to add single molecule level details on the potential interactions between different peptides and the main anthocyanins from eggplant peels. The docking tests indicated that the Trp containing peptides can bind, with different affinities either delphynidine-3-glycoside or delphynidine-3-rutinoside, therefore explaining the fluorescence quenching results. Our results have indicated a mechanism for the interactions between anthocyanins and LF and its small molecular weight peptides, whereas providing insights for formulating ingredients and foods with enhanced bioactives-binding properties.

New Functional Ingredients Based on Microencapsulation of Aqueous Anthocyanin-Rich Extracts Derived from Black Rice (Oryza sativa L.).

The aqueous anthocyanin-rich extract derived from black rice (Oryza sativa L.) was encapsulated by freeze drying using milk proteins and peptides as coating materials. The molecular modelling approach indicated that all major casein fractions and whey proteins were able to bind at least one anthocyanin molecule. The hydrophobic interactions and hydrogen bonding across the interfaces appeared to be mainly responsible for the stabilizations of the complexes formed between the coating material and bioactive compounds. Two dark purple colored powders, differentiated by the ratio of the encapsulation materials used, rich in phytochemicals were obtained, with an encapsulation efficiency of up to 99%. The powders were tested for antioxidant activity, cytocompatibility, and thermal stability. The morphological structure of the powders highlighted the presence of encapsulated anthocyanins. Both powders showed a remarkable antioxidant activity of about 46 mM Trolox/g D.W., and cytocompatibility on the L929 fibroblast culture. At certain concentrations, both powders stimulated cell proliferation. The powders showed a good thermal stability between 75 and 100 °C for 15 min. The powders were tested in a food model system and checked for stability of phytochemicals during storage. The added value of the powders was demonstrated throughout the antioxidant activity, which remained unchanged during storage.

Interactions of flavonoids from yellow onion skins with whey proteins: Mechanisms of binding and microencapsulation with different combinations of polymers.

The interaction of flavonoids extracted from yellow onion skins with whey proteins isolate was studied using fluorescence spectroscopy and simulation methods from the perspectives of microencapsulation. The fluorescence spectroscopy revealed a static quenching mechanism and the involvement of van der Waals and H bonding in complexes formation. The in silico methods suggested that the heat treatment of the major whey proteins affected the binding pockets and therefore the affinity for the main flavonoids. The interaction surface decreased and the interaction energy increased, suggesting lower binding strength. Further, the yellow onion skins extract was successfully encapsulated in whey proteins isolate and different combinations of polymers, including chitosan, maltodextrin and pectin by freeze drying. The resulted powder showed a total flavonoid content of 5.84 ±â€¯0.23 mg quercetin equivalents/g DW in whey protein-chitosan combination and 104.97 ±â€¯5.02 mg quercetin equivalents/g DW in whey protein-maltodextrin-pectin combinations, with antioxidant activity of 175.93 ±â€¯1.50 mM mM Trolox/g DW and 269.20 ±â€¯3.59 mM Trolox/g DW, respectively. The confocal microscopy indicated that the flavonoids aggregated inside the matrix formed between the whey proteins and various polymers and irregular and compact clusters. Therefore, a comprehensive approach involving the extraction of flavonoids from underutilized food by-products, such as yellow onion skins, evaluation of binding mechanisms with whey proteins, whereas tailoring their functional benefit through microencapsulation in order to obtain active ingredients are reported.

Effect of buckwheat flour and oat bran on growth and cell viability of the probiotic strains Lactobacillus rhamnosus IMC 501®, Lactobacillus paracasei IMC 502® and their combination SYNBIO®, in synbiotic fermented milk.

Fermented foods have a great significance since they provide and preserve large quantities of nutritious foods in a wide diversity of flavors, aromas and texture, which enrich the human diet. Originally fermented milks were developed as a means of preserving nutrients and are the most representatives of the category. The first aim of this study was to screen the effect of buckwheat flour and oat bran as prebiotics on the production of probiotic fiber-enriched fermented milks, by investigating the kinetics of acidification of buckwheat flour- and oat bran-supplemented milk fermented by Lactobacillus rhamnosus IMC 501®, Lactobacillus paracasei IMC 502® and their 1:1 combination named SYNBIO®. The probiotic strains viability, pH and sensory characteristics of the fermented fiber-enriched milk products, stored at 4 °C for 28 days were also monitored. The results showed that supplementation of whole milk with the tested probiotic strains and the two vegetable substrates results in a significant faster lowering of the pH. Also, the stability of L. rhamnosus IMC 501®, L. paracasei IMC 502® and SYNBIO® during storage at 4 °C for 28 days in buckwheat flour- and oat bran-supplemented samples was remarkably enhanced. The second aim of the study was to develop a new synbiotic product using the best combination of probiotics and prebiotics by promoting better growth and survival and be acceptable to the consumers with high concentration of probiotic strain. This new product was used to conduct a human feeding trial to validate the fermented milk as a carrier for transporting bacterial cells into the human gastrointestinal tract. The probiotic strains were recovered from fecal samples in 40 out of 40 volunteers fed for 4 weeks one portion per day of synbiotic fermented milk carrying about 10(9) viable cells.