Digestion Of Casein Research Articles

Unveiling the Potential Bioactive Peptides Derived From Goat Casein Hydrolysates Based on In Silico Analyses

ABSTRACTBioactive peptides (BAPs) have attracted considerable interest in scientific research due to their heterogeneity in sequence and structure, which underpins various biological functionalities. In this context, goat casein, an abundant by‐product of the dairy industry, emerges as a valuable source of BAPs. The present study undertook a meticulous evaluation of the bioactive potentials of goat casein‐derived peptides through an integrated approach combining computational simulations, high‐throughput screening, and molecular docking techniques. The initial phase involved the enzymatic digestion of goat milk casein using trypsin, followed by the identification of peptides via liquid chromatography–tandem mass spectrometry (LC–MS/MS), uncovering a total of 597 peptides. Subsequent prioritization using the PeptideRanker algorithm identified 70 peptides exhibiting potential bioactivity, as denoted by scores above 0.8. Advanced screening employing the BIOPEP database and the AutoDock and CAMPR4 tools facilitated the elucidation of 16 antioxidant, 59 hypotensive, 63 hypoglycemic, 70 hypolipidemic, and 25 antimicrobial peptides. Molecular docking studies further elucidated the spontaneous nature of the interactions between the peptides and their respective receptors, predominantly mediated by hydrogen bonding and hydrophobic interactions. Four peptides specifying all activities simultaneously were synthesized, and their activities were verified by in vitro experiments. These results not only highlight an effective strategy for the high‐throughput screening of goat casein‐derived peptides but also underscore the potential of utilizing casein as a viable source of functional food ingredients. This study thereby contributes significantly to the expanding field of functional food research, suggesting a sustainable approach to explore the potential of dairy by‐products.

Influence of bile salts on the gastrointestinal digestion of agar-casein hybrid systems and the nanoassembly of their digestion products

This manuscript reports on the effect of different bile salts concentration on the gastrointestinal digestion of casein and casein-agar hybrid systems and evaluates the effect on the nanostructural assembly of the digestion products through the application of advanced small angle X-ray scattering analysis (SAXS).The results showed that bile salts promote the hydrolysis of micellar casein upon in vitro gastrointestinal digestions. It is noteworthy that in the presence of agar, bile salts interact with the polysaccharide, leading to a reduction in their proteolytic activity. While structured agar-casein hydrogels were shown to be able to limit the proteolysis during the gastric phase, increasing concentrations of bile salts promoted the diffusion of casein from the gel network during the intestinal phase, hence leading to higher degree of hydrolysis. The released digestion products were seen to interact with bile salts, forming ordered lamellar/micellar nanostructures. In particular, the presence of solubilized agar in the digesta was seen to promote the formation of these nanostructures. These nanostructures were formed at 10 mM bile salts for the hydrogels, and across all concentrations for agar-casein blends. Thus, this work demonstrates the great relevance of bile salts in the nanostructural assembly of protein digestion products, which is expected to have a great relevance in bioavailability and metabolic responses induced by protein-rich foods.

Comparative Investigation of pH-Dependent Availability of Pancreatic Enzyme Preparations In Vitro.

This study aimed to compare different pancreatic enzyme preparations (PEPs) available in Germany regarding particle geometry and size, and to evaluate enzyme activity under physiologically relevant conditions in vitro. Pancreatic endocrine insufficiency is characterized by deficiency of pancreatic enzymes resulting in maldigestion. It is orally treated by pancreatic enzyme replacement therapy. The formulations differ in their physical properties and enzyme release behavior, potentially resulting in inconsistent dosages and poor interchangeability of products. A total of 25 products were analyzed for particle size and number of particles per capsule. Enzyme activities of lipase, amylase, and protease were measured by digestion of olive oil emulsion, starch, and casein, respectively. To analyze enzyme release, gastric environments were simulated by incubating PEPs at pH 1, 4, or 5. Duodenal conditions were simulated by subsequent incubation at pH 6. Regarding physical properties and enzyme release kinetics, considerable differences between different PEPs were found. Furthermore, compared to the label claim, excess lipase activity was observed for most products, reaching up to 148%. These in vitro results suggest poor interchangeability of PEPs, potentially explained by physical and release characteristics. Physicians and patients should be aware of the potential gap between label claims and the real-life performance of different PEPs.

MOLECULAR IDENTIFICATION OF NOVEL PROBIOTIC BACTERIA ISOLATED FROM COW MILK AND ITS ANTIBACTERIAL APPLICATIONS

The purpose of the study conducted in the year 2021 was to isolate and identify Lactobacillus species through biochemical characterization and to find antibacterial potential of the species. Researchers collected milk sample from local milk vendor, Eachanari in December, 2021. In an Enriching MRS broth the colonies were isolated and loop full of isolate were streaked on MRS agar plate. By morphological characteristics determined through gram staining method the isolated bacteria were identified as Lactobacillus species. Through different identification methods suchas casein digestion, acid and salt tolerance the isolate was characterized for probiotic properties. In a skim milk agar plate the isolated strain digested casein. This indicated production of protease enzyme which is a significant probiotic property. In MRS broth, an acid tolerance test was conducted at pH levels 3, 4, 5, 6, 7 and 8. The isolate was able to endure both an extremely acidic pH and alkaline media. NaCl tolerance test was done in MRS broth at concentrations of 3%, 4%, 5%, 6%, 7%, and 8% NaCl. The isolate survived well in solutions containing 3%, 4%, and 6% NaCl. After biochemical studies, molecular identification was carried out to identify the probiotic starin. The isolated probiotic was identified as Limosilactobacillus fermentum strain BTFSN. The isolated strain was then checked for antibacterial activity against three pathogens. Zone of inhibition were observed. The study indicated that Lactobacillus species from milk sample have potential probiotic properties and possess antimicrobial property.

Investigation into potential allergenicity of DBD plasma-treated casein digestion products based on immunoglobulin E linear epitopes and the sensitized-cell model

The study aimed to investigate the allergenicity change in casein treated with dielectric barrier discharge (DBD) plasma during in vitro simulated digestion, focusing on the immunoglobulin E (IgE) linear epitopes and utilizing a sensitized-cell model. Results indicated that prior treatment with DBD plasma treatment (4 min) before simulated digestion led to a 10.5% reduction in the IgE-binding capacity of casein digestion products. Moreover, the release of biologically active substances induced from KU812 cells, including β-HEX release rate, human histamine, IL-4, IL-6, and TNF-α, decreased by 2.1, 28.1, 20.6, 11.6, and 17.3%, respectively. Through a combined analysis of LC-MS/MS and immunoinformatics tools, it was revealed that DBD plasma treatment promoted the degradation of the IgE linear epitopes of casein during digestion, particularly those located in the α-helix region of αs1-CN and αs2-CN. These findings suggest that DBD plasma treatment prior to digestion may alleviate casein allergic reactions.

Enhancing Bromelain Recovery from Pineapple By-Products: A Sustainable Approach for Value Addition and Waste Reduction.

Pineapple by-products are good sources of bromelain, a complex enzyme with commercial applications. This study evaluated the feasibility of producing bromelain powders from pineapple waste using an organic solvent-free approach. Pineapple by-products (from var. MD2), including cores, peels, crowns, stems, and basal stems, were homogenized with deionized water, and the pH of the mixture was adjusted to 4.5 and 9 (isoelectric points of fruit bromelain and stem bromelain), clarified, ultra-filtered, and freeze-dried to produce bromelain powders. The enzymatic activity of the bromelain powders was measured using the gelatin and casein digestion methods. The bromelain powders from the crowns did not show significant enzymatic activity (p < 0.05). Meanwhile, bromelain powders produced from the cores and peels had an enzymatic activity of 694 gelatin digesting units (GDU)/g and 124 casein digesting units (CDU)/mg, and 1179 GDU/g and 217 CDU/mg, respectively. Bromelain powders from the basal stems showed the highest enzymatic activity (2909 GDU/g and 717 CDU/mg). Increasing the pH of the liquid mixture before the purification and freeze drying significantly (p < 0.05) reduced the enzymatic activity of the bromelain powders. Using a practical and organic solvent-free approach, this study demonstrates the feasibility of producing bromelain powders with high enzymatic activity from pineapple waste.

Quantitative mass spectrometry with ¹⁸O labelling as an alternative approach for determining protease activity: an example of trypsin

SCIENTIFIC RELEVANCE. In the quality control of proteolytic enzyme components of medicinal products, the activity of proteases is determined by spectrophotometry, which involves mea­suring the amidase or esterase activity using a synthetic substrate and the proteolytic activity using the Anson method. These methods require special substrates and have low sensitivity; their specificity may be insufficient, which may lead to serious errors. Quantitative mass spectrometry is an alternative approach to protease activity assays, which involves adding an isotope-labelled peptide to hydrolysates of the test enzyme. This approach allows determining the activity of proteases, notably, by the hydrolysis of specific peptide bonds, while simulta­neously confirming the identity and specificity of the test sample. Quantitative mass spectrometry has high sensitivity and does not require special substrates.AIM. This study aimed to investigate the possibility of enzymatic activity assay and enzyme identification by quantitative mass spectrometry with ¹⁸O labelling through an example of trypsin with casein.MATERIALS AND METHODS. The study used trypsin, casein, and H₂¹⁸O (Izotop, Russia). Peptide separation was performed using an Agilent 1100 HPLC system; mass spectra were obtained using a Bruker Ultraflex II MALDI-TOF/TOF mass spectrometer. Quantitative mass spectrometry was performed using a standard peptide, which was obtained from casein by tryptic digestion and HPLC purification. For ¹⁸O labelling, the authors dried the peptide and incubated it in H₂¹⁸О water. The quantitative analysis of the product was carried out using MALDI-TOF mass spectrometry. The authors used quantitative mass spectrometry with ¹⁸O labelling to determine enzymatic activity and calculate the Michaelis constant (KM).RESULTS. Following the tryptic digestion of casein, the authors identified the fragments corre­sponding to casein chains. The authors produced the isotope-labelled standard peptide and calculated its concentration using mass spectrometry. The authors determined the rate of casein digestion by trypsin and calculated the KM for trypsin, which was 13.65±0.60 μM. The standard deviation for repeated measurements showed that the mass-spectrometric method had a lower error of measurement than the spectrophotometric method. The sensitivity threshold for the mass-spectrometric method was 0.50±0.08 μM.CONCLUSIONS. The results obtained with trypsin confirm the possibility of enzymatic activity determination by the proposed method of quantitative mass spectrometry with ¹⁸O labelling. According to the sensitivity evaluation results, this method can be used for the simultaneous determination of enzyme activity, identity, and specificity. The proposed mass spectrometry approach is universal, it does not require expensive materials and reagents, and it can be easily adapted to determine the activity of virtually any protease.

A toolbox for manipulating the genome of the major goat pathogen, Mycoplasma capricolum subsp. capripneumoniae.

Mycoplasma capricolum subspecies capripneumoniae (Mccp) is the causative agent of contagious caprine pleuropneumonia (CCPP), a devastating disease listed by the World Organisation for Animal Health (WOAH) as a notifiable disease and threatening goat production in Africa and Asia. Although a few commercial inactivated vaccines are available, they do not comply with WOAH standards and there are serious doubts regarding their efficacy. One of the limiting factors to comprehend the molecular pathogenesis of CCPP and develop improved vaccines has been the lack of tools for Mccp genome engineering. In this work, key synthetic biology techniques recently developed for closely related mycoplasmas were adapted to Mccp. CReasPy-Cloning was used to simultaneously clone and engineer the Mccp genome in yeast, prior to whole-genome transplantation into M. capricolum subsp. capricolum recipient cells. This approach was used to knock out an S41 serine protease gene recently identified as a potential virulence factor, leading to the generation of the first site-specific Mccp mutants. The Cre-lox recombination system was then applied to remove all DNA sequences added during genome engineering. Finally, the resulting unmarked S41 serine protease mutants were validated by whole-genome sequencing and their non-caseinolytic phenotype was confirmed by casein digestion assay on milk agar. The synthetic biology tools that have been successfully implemented in Mccp allow the addition and removal of genes and other genetic features for the construction of seamless targeted mutants at ease, which will pave the way for both the identification of key pathogenicity determinants of Mccp and the rational design of novel, improved vaccines for the control of CCPP.

Different influences of dietary fiber from various sources on the in vitro digestibility of casein as uncovered by the study of protein-dietary fiber interactions

How different dietary fibers including pectin, cellulose and lignin affect casein digestibility was studied using in vitro static protocols. Peptides’ profile, free amino acids (AAs) content, casein-DF interactions and their influences on enzymatic activities of proteolytic enzymes were studied using combined techniques. Under gastric and intestinal digestive conditions, while pectin could reduce casein digestibility (with an averaged decrease of 12.15% and 7.83, respectively) through both depletion flocculation and hydrogen-binding interactions, lignin inhibited the digestion of casein straightly through reducing the enzymatic activity of proteolytic enzymes, thereby altering the production of free AAs. Although cellulose showed the least detrimental effects, it still significantly reduced the content of Thr, Glu, Val, Leu, Phe, Lys, and no Arg was released. Deeper insight into casein-DF interactions and their influences on casein digestibility improves the development of more effective forms of DF for improving AA homeostasis in individuals.

Escherichia coli Cellular Activity and Frontal Trizonal Evaluation of Microspace Between Implants and Abutments Under Calibrated Cyclic Stress.

To evaluate microspace and microleakage between implant and abutments subjected to pre- and post-calibrated cyclic stress. Twelve screw-retained implant prostheses with BioHPP polyetheretherketone (PEEK) abutment (Noris Dental Implant System Ltd.,Nesher, Israel) (Group I) and 12 screw-retained implant prostheses with computer-assisted design/computer-assisted manufacturing (CAD/CAM) milled zirconia abutment (DentGallop, Houston, TX, USA) (Group II) were connected to their respective implant, and the prosthetic screw was torqued to 30N/cm (Noris). The microspace was evaluated using scanning electron microscopy (SEM; TeScan, Brno, Czech Republic). Twenty-four samples were then induced to cyclic stress (Lokesh Industries, Pune, India) simulating 180 days duration of oral stress. The microspaces (Group IA and Group IIB) were measured post-cyclic stress. Group I and II were again renamed into Group Ia and Group IIb for microbial study. Both implant assemblies were immersed in fresh soybean casein digest broth (SCDB) (Himedia, Mumbai, India) and subsequently inoculated with 1.0µL E. coli suspension (Himedia) at the open end and incubated at 37ºC for seven days. After the incubation period, cellular activity was determined by the spread plate method, and total colony-forming units (CFU) were calculated. The results were evaluated using independent Tand Mann-Whitney tests. Average and microspace at the implant-abutment junction of Group I samples in the front right was 12.98µm, center 13.76µm, front left 13.22µm, and in Group II samples, the front right was 18.52µm, center 17.84µm, front left 18.58µm.After being subjected to cyclic loading, the mean levels of the vertical microgap for Group IA samples were: in the front right region 10.37µm, in the center 9.34µm, in the front left 10.51µm and in Group IIB samples front right was 14.59µm, center 13.39µm, front left 13.8µm. Independent t-tests showed insignificant differences between the two groups. The median value of microbial leakage of Group Ia samples after cyclic loading was 30 x 103 CFU/ml, and Group IIb samples were 42 x 103 CFU/ml and were significant. There was minimal variation in the mean microspace between the BioHPP PEEK abutment and CAD/CAM milled zirconia abutment, and it was insignificant before and after cyclic stress. BioHPP PEEK abutment-titanium implant interfaces showed significantly decreased microbial leakage than CAD/CAM milled zirconia abutment-titanium implant interfaces after cyclic stress.

Influence of Casein and Milk Phospholipid Emulsifiers on the Digestion and Self-Assembled Structures of Milk Lipids

Interfacial compositions of fat globules modulate the digestion behaviour of milk triglycerides in the gastrointestinal tract, thereby affecting lipid metabolism and delivery of nutrients. In this study, we aim to understand the impact of emulsifiers on lipid digestibility and the self-assembled liquid crystal structures formed by anhydrous milk fat (AMF) during digestion. AMF was emulsified with casein and milk phospholipids, and digestion was performed in both gastric and small intestinal conditions to account for changes at the oil/water interface following enzymatic digestion in the gastric phase. Small angle X-ray scattering was used to characterise the self-assembled structures of the digestion products, while coherent anti-Stokes Raman scattering microscopy was utilised to probe changes in lipid distribution at the single droplet level during digestion. Our findings confirmed that emulsifiers play a key role in the digestion of AMF. Milk phospholipids exhibited a protective effect on milk triglycerides against pancreatic lipase digestion by slowing digestion, but this effect was slightly negated in emulsions pre-digested under gastric conditions. The overall types of liquid crystal structures formed after digestion of casein- and milk phospholipids-emulsified AMF were comparable to commercial bovine milk irrespective of gastric pre-treatment. However, emulsification of AMF with milk phospholipids resulted in changes in the microstructures of the liquid crystal phases, suggesting potential interactions between the digested products of the fat globules and milk phospholipids. This study highlights the importance of emulsifiers in regulating lipid digestion behaviour and lipid self-assembly during digestion.

Gastrointestinal digestibility of micellar casein dispersions: Effects of caprine vs bovine origin, and partial colloidal calcium depletion using in vitro digestion models for the adults and elderly.

In vitro coagulation and digestion of caprine and bovine micellar casein concentrate (MCC) with or without partial colloidal calcium depletion (deCa) were studied under simulated adult and elderly conditions. Gastric clots were smaller and looser for caprine than bovine MCC, and were further looser with deCa and under elderly condition for both caprine and bovine MCC. Casein hydrolysis and concomitant formation of large peptides was faster for caprine than bovine MCC, and with deCa and under adult condition for caprine and bovine MCC. Formation of free amino groups and small peptides were faster for caprine MCC, and with deCa and under adult condition. Upon intestinal digestion, proteolysis occurred rapidly, and was faster under adult condition, but showed less differences with increasing digestion between caprine and bovine MCC, and with and without deCa. These results suggested weakened coagulation and greater digestibility for caprine MCC and MCC with deCa under both conditions.

Impact of Physicochemical Modifications in Casein Promoted by UV-C on the Peptide Profile of Gastric Digestion and the Transepithelial Transport of Peptides.

Caseins are the main proteins in milk, and their structure and spatial conformation are responsible for their slow digestion rate. The release of bioactive and β-casomorphin peptides from casein digestion may induce allergic responses during consumption. Spectroscopic techniques were used to observe the structural changes in casein conformation induced by Ultraviolet light irradiation (UV-C). Raman spectroscopy results showed more pronounced peaks at 618 and 640 cm-1 for phenylalanine and tyrosine moieties of the photolyzed micellar casein, respectively, suggesting changes in the micelle structure. The decrease in the intensity of Raman signals for tryptophan and tyrosine corroborates to the UV-C-induced modifications of the micelle structure. Particle size distribution showed a decrease in the average micelle size after 15 min of UV-C exposure, while low-temperature, long-time (LTLT) pasteurization led to the formation of large aggregates, as observed by atomic force microscopy. UV-C did not impact the formation or transport of peptides, as observed by using the Caco-2 cell as a model for peptide absorption. However, the absence of the opioid peptide SRYPSY from κ-casein and only 20% of the concentration of opioid peptide RYLGY were noted. This work demonstrated that UV-C can be utilized to induce the physicochemical modification of dairy products, promoting a higher digestion rate and reducing allergenicity.

Isolation of Proteolytic Enzyme from Pineapple Crown

The pineapple waste from the pineapple industry has contributed to an increase in waste in Malaysia and worldwide every year. A major type of endopeptidase enzymes found in pineapple is fruit bromelain, stem bromelain, ananain, and comasain. This study aims to extract and purify protease from the crown of MD2 pineapple. Protease was extracted and purified using anion exchange chromatography, gel filtration, and desalting before being identified using liquid chromatography-mass spectrometry (LC-MS). Proteolytic activity was determined using the well diffusion method and Casein Digestion Unit. In the present study, the proteolytic assay showed that 1 kg crown of MD2 cultivar produced an activity of 126.0 ± 3.86 U/ml, a specific activity of 3937.50 U/mg. In the present study, the proteolytic assay showed that 1 kg crown of MD2 cultivar produced an activity of 126.0 ± 3.86 U/mL, a specific activity of 3937.50 U/mg and the total activity of 3.94 × 109 U. The molecular weight of the purified enzyme was in the range of 25 to 35 kDa under the optimum condition of pH 7 and 37°C. Purification of the extract yielded a band at the molecular weight of 20–25 kDa at the optimum pH of 3 and 9 at 60°C. From LC-MS analysis, the purified enzyme from the crown extract was similar to ananain under accession number A0A199VSS3 (according to Uniprot). It had five unique peptides and covered 97/356 amino acids (44.9% coverage). The ananain (EC 3.4.22.31) is classified in the subfamilies of cysteine protease C1A (clan CA, family C1), a peptidase family related to papain. In conclusion, protease was extracted and identified as an ananain-like protease from the crown.

Coagulation of model infant formulae: Impact on their in vitro dynamic gastric digestion

The rheological properties and the in vitro dynamic gastric digestion behaviours of model infant formulae containing various ratios of casein to whey protein (0:100, 40:60, 60:40 and 80:20) were investigated using a rheometer and an infant human gastric simulator. The pH profiles and the viscoelastic moduli were determined during acidification by glucono-δ-lactone and at different pepsin concentrations (0, 0.3, 1.0, 1.7 and 2.5 U/mL). The infant formula containing whey protein only formed a weak gel at about pH 5. In the presence of pepsin, all casein-containing infant formulae formed gels at pH ∼6.0 and only low pepsin activity (>0.3 U/mL) was required for the gelation. The strength of the gel/coagulum was dependent mainly on the casein content. Coagulation of protein and flocculation of oil droplets, induced by both pepsin proteolysis and the low pH under gastric conditions, were observed in the infant formulae during dynamic digestion in the human gastric simulator. Confocal images showed that the infant formula containing whey protein only formed smaller flocs of aggregated protein and oil droplets, which may have led to their observed faster protein digestion, than the infant formulae containing a combination of casein and whey protein. The casein-dominant infant formulae had greater aggregation than the whey-protein-dominant infant formulae during gastric digestion, which led to their lower rate of casein digestion. These results suggest that the extent of coagulation and the gastric digestion of the proteins in infant formulae can be controlled by the rational selection of the protein components and the ratio of casein to whey protein, such that different nutritional requirements of infants can be met.

Peptidomic Characterization and Amino Acid Availability after Intake of Casein vs. a Casein Hydrolysate in a Pig Model.

It is known that casein hydrolysis accelerates gastrointestinal transit in comparison to intact casein, although the effect of the protein hydrolysis on the composition of the digests is not fully understood. The aim of this work is to characterize, at the peptidome level, duodenal digests from pigs, as a model of human digestion, fed with micellar casein and a previously described casein hydrolysate. In addition, in parallel experiments, plasma amino acid levels were quantified. A slower transit of nitrogen to the duodenum was found when the animals received micellar casein. Duodenal digests from casein contained a wider range of peptide sizes and a higher number of peptides above five amino acids long in comparison with the digests from the hydrolysate. The peptide profile was markedly different, and although β-casomorphin-7 precursors were also found in hydrolysate samples, other opioid sequences were more abundant in the casein digests. Within the same substrate, the evolution of the peptide pattern at different time points showed minimal changes, suggesting that the protein degradation rate relies more on the gastrointestinal location than on digestion time. Higher plasma concentrations of methionine, valine, lysine and amino acid metabolites were found in animals fed with the hydrolysate at short times (<200 min). The duodenal peptide profiles were evaluated with discriminant analysis tools specific for peptidomics to identify sequence differences between both substrates that can be used for future human physiological and metabolic studies.

Physicochemical Properties Analysis and Bromelain Activity of Three Indexes Pineapple (Ananas Comosus) Peel Extract Variety Josapine

Industrial waste such as pineapple peels that accumulate in massive quantities contributes to environmental pollution. However, there is limited data on the physicochemical properties of pineapple peel for several ripening stages. The aim of this study was to determine the physicochemical properties and bromelain activity of three indexes of pineapple peel extract specifically for the variety Josapine. The method used for the analysis of physicochemical properties includes total soluble solid (TSS), pH, titratable acidity (TA), pulp content, sugar content and the casein digestion unit (CDU) method for bromelain activity. Significant differences at p&lt;0.05 were found between TSS, pulp, pH, titratable acidity and TSS/TA ratio among an index of maturity. It was found that pineapple peel extract at maturity index 7 contained significantly the highest in titratable acidity, TSS and total sugar. From this present study, it was observed that bromelain activity in pineapple peel extract was significantly higher in maturity index 5 as compared to maturity indices 2 and 7.

Controlled gastrointestinal digestion of micellar casein loaded anthocyanins: The chelating and complexing effect of dextran sulfate

Casein micelle is known to be a CCP-mediated tight structure that often shows unique stomach curd behavior and rapid intestinal hydrolysis. These inherent properties make it difficult for casein micelles-formed powders called micellar casein (MC) to achieve high loading capacity and controlled release of loaded bioactive compounds. In this study, dextran sulfate (DS) was used as a calcium chelator to combine with MC, and spray-dried microparticles were prepared to load blueberry anthocyanins (ACNs). In vitro gastrointestinal digestion was carried out to investigate the relationship between the structural changes of MC induced by DS as well as the dynamic release process of ACNs. The results indicated that the encapsulation efficiency of microparticles increased from 52.76% ± 2.36%–91.86% ± 0.87% with the addition of DS. During in vitro digestion, the different CCP dissociation degrees induced by DS resulted in different dissolution and curdling abilities of microparticles in the stomach, which thus precisely controlled the release process of ACNs. Simultaneously, the combination with DS could delay the disintegration of the structures of microparticles in the small intestine, thereby hindering the degradation of ACNs. This study will contribute to the design of casein-based delivery systems and the development of related functional products. • MC formed curds in the stomach and hydrolyzed quickly in the small intestine. • The digestion properties made MC fail to achieve controlled release of ACNs. • DS with calcium chelating ability could adjust the digestion properties of MC. • The less release of ACNs was achieved by the structure barrier of DS.

PSEUDOMONAS FLUORESCENS IN SHEEP MILK GREEK YOGHURT FROM VLASINA – A BIOCHEMICAL CHARACTERIZATION

Pseudomonas fluorescens is an aerobic, rod-shaped, non-sporulating gram-negative bacteria, mostlyfound in soil, decaying organic matter and feces. This species contaminates the milk mainly through animal feeddust, silage, utensils and polluted water. In addition to casein digestion, this psychotropic and lipolytic speciesgenerates butyric and caproic acids by fermentation of milk fat, which release a strong unpleasant odor and give arancid and bitter taste to dairy products. In this study we tested 48 hours old sheep's sour milk, produced in ahousehold in Vlasina, southeastern Serbia, using traditional method. Standard bacteriological protocols were usedfor isolation and identification Pseudomonas fluorescens strains. Biochemical identification was performed usingthe commercial API 32GN E system, and 60473057073 profile was obtained (Pseudomonas fluorescens, %Id=99.5and T=0.74). Positive biochemical tests were: N-acetyl-glukosamine (NAG), D-Ribose (RIB), Sodium malonate(MNT), Sodium acetate (ACE), Lactic acid (LAT), L-Alanine (ALA), D-Mannitol (MAN), D-Glucose (GLU), LArabinose(ARA), Capric acid (CAP), Valeric acid (VALT), Trisodium citrate (CIT), L-Histidine (HIS), Potasium2-ketogluconate (2KG), 3-Hydroxybutyric acid (3OBU), 4-Hydroxybenzoic acid (pOBE), L-Serine (SER), LProline(PRO) and Oxidase (OX). Negative biochemical tests were: L-Rhamnose (RHA), Inositol (INO), DSaccharose(SAC), D-Maltose (MAL), Itaconic acid (ITA), Suberic acid (SUB), Salicin (SAL), D-Melibose (MEL),L-Fucose (FUC), D-Sorbitol (SOR), Propionic acid (PROP), Potasium 5-ketogluconate (5KG), Glycogen (GLYG)and 3-hydroxybenzoic acid (mOBE). Based on the example of the strain Pseudomonas fluorescens, the API ID 32GN system proved to be precise and very efficient in the identification of this lipolytic type. Given thatpasteurization and cooling processes do not entirely inhibit the enzym activity and growth of this psychotrophicbacteria, informing individual milk and milk product manufacturers in the Vlasina region about good manufacturingpractices would limit contamination and bacteriological deterioration.

Bitter Peptides YFYPEL, VAPFPEVF, and YQEPVLGPVRGPFPIIV, Released during Gastric Digestion of Casein, Stimulate Mechanisms of Gastric Acid Secretion via Bitter Taste Receptors TAS2R16 and TAS2R38.

Eating satiating, protein-rich foods is one of the key aspects of modern diet, although a bitter off-taste often limits the application of some proteins and protein hydrolysates, especially in processed foods. Previous studies of our group demonstrated that bitter-tasting food constituents, such as caffeine, stimulate mechanisms of gastric acid secretion as a signal of gastric satiation and a key process of gastric protein digestion via activation of bitter taste receptors (TAS2Rs). Here, we tried to elucidate whether dietary non-bitter-tasting casein is intra-gastrically degraded into bitter peptides that stimulate mechanisms of gastric acid secretion in physiologically achievable concentrations. An in vitro model of gastric digestion was verified by casein-fed pigs, and the peptides resulting from gastric digestion were identified by liquid chromatography–time-of-flight-mass spectrometry. The bitterness of five selected casein-derived peptides was validated by sensory analyses and by an in vitro screening approach based on human gastric parietal cells (HGT-1). For three of these peptides (YFYPEL, VAPFPEVF, and YQEPVLGPVRGPFPIIV), an upregulation of gene expression of TAS2R16 and TAS2R38 was observed. The functional involvement of these TAS2Rs was verified by siRNA knock-down (kd) experiments in HGT-1 cells. This resulted in a reduction of the mean proton secretion promoted by the peptides by up to 86.3 ± 9.9% for TAS2R16kd (p < 0.0001) cells and by up to 62.8 ± 7.0% for TAS2R38kd (p < 0.0001) cells compared with mock-transfected cells.