Gastric devolution of transglutaminase-induced acid and rennet-induced casein gels using dynamic DIDGI® and static COST action INFOGEST protocols.

Limited studies in the literature have compared in vitro dynamic and in vitro static protocols for modelling the gastric digestive process of food systems. This experiment explores the differences between two different in vitro approaches to the devolution of a transglutaminase-induced acid gel (TG, pH 5.1-5.3) and rennet-induced gel (RG, pH 6.5-6.7). Gels were exposed to a simulated oral phase, followed by either the dynamic DIDGI® or static COST action INFOGEST protocol to simulate gastric conditions. Protein hydrolysis was evident from 15 min onwards for TG exposed to the dynamic protocol where levels continued to increase at a steady rate. In contrast, RG exhibited a notable lag-phase before levels increased from around 60 min onwards. Under the static protocol, protein hydrolysis was observed for both TG and RG upon exposure to the gastric environment which continued to increase over time. Despite these differences, similar levels of protein hydrolysis were found for TG and RG at the gastric endpoint using either protocol demonstrating that both the dynamic DIDGI® and static COST action INFOGEST methods provide a suitable and comparable environment for the in vitro digestion of casein protein under simulated gastric conditions.

Assessment of A1 and A2 variants in the CNS2 gene of some cattle breeds by using ACRS-PCR method.

The aim of this study is to reveal ß-casein polymorphism of some cattle breeds and also the potential to produce A2 milk from existing animals and to develop strategies in this area. Therefore, a total of 400 cattle, 100 animals from each breed of Holstein, Brown Swiss, Jersey and Simmental raised commonly in Turkey, were obtained, and C > A polymorphism in 67th amino acid in the 7th exons of ß-casein gene was determined by TaqI enzyme with PCR-ACRS method. Blood samples were collected from dairy cattle farms raising Holstein, Brown Swiss and Jersey breeds from Konya province and Simmental breed from Kütahya province in Turkey. A1 and A2 allele frequencies in Holstein, Brown Swiss, Jersey and Simmental cattle breeds were determined as 0.475/0.525, 0.370/0.630, 0.215/0.785 and 0.440/0.560, respectively. The highest A2 allele frequency (0.785) was found in Jersey breed. A1A1 genotypes in Holstein, Brown Swiss, Jersey and Simmental breeds were 0.240, 0.150, 0.030 and 0.160, respectively; A1A2 genotypes were 0.470, 0.440, 0.370 and 0.560, respectively; A2A2 genotypes were determined as 0.290, 0.410, 0.600 and 0.280, respectively. In these breeds, the highest A2A2 genotype frequency was found in Jersey (0.600), the lowest A1A1 genotype frequency (0.030) was found in Jersey and the highest A1A2 genotype frequency (0.560) was found in Simmental. Holstein, Brown Swiss, Simmental and Jersey populations were at the level of Hardy-Weinberg in terms of ß-casein gene (p > 0.05). The average Ho, He and PIC values were calculated as 0.460, 0.469 and 0.605, respectively. In conclusion, the frequency of commonly reared cattles in Turkey especially Brown Swiss, and Jersey breeds in A2A2 genotype are satisfactory, but it can be said that the use of animals with A2 allele in selection is extremely important for increasing A2 milk producing potential in the future.

Multi-criteria assessment of pea protein quality in rats: a comparison between casein, gluten and pea protein alone or supplemented with methionine.

The objective of this study was to assess the nutritional quality of pea protein isolate in rats and to evaluate the impact of methionine (Met) supplementation. Several protein diets were studied: pea protein, casein, gluten, pea protein-gluten combination and pea protein supplemented with Met. Study 1: Young male Wistar rats (n 8/group) were fed the test diets ad libitum for 28 d. The protein efficiency ratio (PER) was measured. Study 2: Adult male Wistar rats (n 9/group) were fed the test diets for 10 d. A protein-free diet group was used to determine endogenous losses of N. The rats were placed in metabolism cages for 3 d to assess N balance, true faecal N digestibility and to calculate the Protein Digestible-Corrected Amino Acid Score (PDCAAS). They were then given a calibrated meal and euthanised 6 h later for collection of digestive contents. The true caecal amino acid (AA) digestibility was determined, and the Digestible Indispensable Amino Acid Score (DIAAS) was calculated. Met supplementation increased the PER of pea protein (2·52 v. 1·14, P < 0·001) up to the PER of casein (2·55). Mean true caecal AA digestibility was 94 % for pea protein. The DIAAS was 0·88 for pea protein and 1·10 with Met supplementation, 1·29 for casein and 0·25 for gluten. Pea protein was highly digestible in rats under our experimental conditions, and Met supplementation enabled generation of a mixture that had a protein quality that was not different from that of casein.

Serine Protease Zymography: Low-Cost, Rapid, and Highly Sensitive RAMA Casein Zymography.

To detect serine protease activity by zymography, casein and CBB stain have been used as a substrate and a detection procedure, respectively. Casein zymography has been using substrate concentration at 1 mg/mL and employing conventional CBB stain. Although ordinary casein zymography provides reproducible results, it has several disadvantages including time-consuming and relative low sensitivity. Improved casein zymography, RAMA casein zymography, is rapid and highly sensitive. RAMA casein zymography completes the detection process within 1 h after incubation and increases the sensitivity at least by tenfold. In addition to serine protease, the method also detects metalloprotease 7 (MMP7, Matrilysin) with high sensitivity.

Functional assessment of CTCF sites at cytokine-sensing mammary enhancers using CRISPR/Cas9 gene editing in mice.

The zinc finger protein CTCF has been invoked in establishing boundaries between genes, thereby controlling spatial and temporal enhancer activities. However, there is limited genetic evidence to support the concept that these boundaries restrict the search space of enhancers. We have addressed this question in the casein locus containing five mammary and two non-mammary genes under the control of at least seven putative enhancers. We have identified two CTCF binding sites flanking the locus and two associated with a super-enhancer. Individual deletion of these sites from the mouse genome did not alter expression of any of the genes. However, deletion of the border CTCF site separating the Csn1s1 mammary enhancer from neighboring genes resulted in the activation of Sult1d1 at a distance of more than 95 kb but not the more proximal and silent Sult1e1 gene. Loss of this CTCF site led to de novo interactions between the Sult1d1 promoter and several enhancers in the casein locus. Our study demonstrates that only one out of the four CTCF sites in the casein locus had a measurable in vivo activity. Studies on additional loci are needed to determine the biological role of CTCF sites associated with enhancers.

Assessment of IgE Reactivity of ß-Casein by Western Blotting After Digestion with Simulated Gastric Fluid.

Cow's milk allergy is defined as an immunologically mediated adverse reaction to cow's milk proteins and it is usually, along with hen's egg allergy, the first food allergy identified in childhood.One of the main aspects to consider when evaluating the allergenic potential of food proteins is the effect of gastric digestion. It is known that allergens are usually able to survive the harsh acidic environment of the stomach, tolerate the presence of surfactants, and resist digestion by pepsin. They might also be digested into high molecular weight peptide fragments, which retain the same, or sometimes increased, IgE-binding. In this respect, western blotting is a highly sensitive and efficient technique that we have used to detect IgE-binding to the digests of milk and egg proteins. Given the importance of the resistance of food proteins to gastric digestion in their capacity to modulate the immune response, we describe in this chapter the assessment of IgE reactivity of a relevant cow's milk allergen, ß-casein, by western blotting after simulated digestion under relevant physiological conditions.

An assessment of the influence of macronutrients on growth performance and nutrient utilisation in broiler chickens by nutritional geometry.

The right-angled triangle mixture experiment was designed to include fourteen diets with different concentrations of starch, protein and lipid. Experimental diets were offered to male Ross 308 broiler chickens from 10 to 23 d after hatching, and response curves and surfaces were generated to illustrate the influence of macronutrients on growth performance and nutrient utilisations. Despite the primary function of macronutrients, especially protein, may not be providing energy, macronutrients were expressed as energy derived from starch, protein and fat for statistical purposes in the mixture design. Energy derived from lipid had a greater impact on feed intake than energy derived from starch and protein. When we compared the influence of starch and protein on feed intake, 'equal distance rule' was observed, which means the animal consumes feed to the point on its respective nutritional rails where the shortage of starch exactly equals the surplus of consumed protein. Increasing the protein-derived energy intake increased weight gain in broiler chickens, whereas energy intake derived from starch and lipid had little impact on weight gain. Feed conversion ratio (FCR) may be reduced by either increasing protein energy intake or decreasing starch energy intake. As the slope of the contours was less than 1, the influence of starch energy intakes on FCR exceeded that of protein energy intakes. In conclusion, energy derived from protein is more important than non-protein energy in terms of weight gain, and a balance between protein and energy supplies is required for efficient muscle protein deposition.

Effects of hydrolysed casein, intact casein and intact whey protein on energy expenditure and appetite regulation: a randomised, controlled, cross-over study.

Casein and whey differ in amino acid composition and in the rate of absorption; however, the absorption rate of casein can be increased to mimic that of whey by exogenous hydrolysis. The objective of the present study was to compare the effects of hydrolysed casein (HC), intact casein (IC) and intact whey (IW) on energy expenditure (EE) and appetite regulation, and thereby to investigate the influence of amino acid composition and the rate of absorption. In the present randomised cross-over study, twenty-four overweight and moderately obese young men and women consumed three isoenergetic dietary treatments that varied in protein source. The study was conducted in a respiration chamber, where EE, substrate oxidation and subjective appetite were measured over 24 h at three independent visits. Moreover, blood and urine samples were collected from the participants. The results showed no differences in 24 h and postprandial EE or appetite regulation. However, lipid oxidation, estimated from the respiratory quotient (RQ), was found to be higher after consumption of IW than after consumption of HC during daytime (P= 0·014) as well as during the time after the breakfast meal (P= 0·008) when the food was provided. Likewise, NEFA concentrations were found to be higher after consumption of IW than after consumption of HC and IC (P< 0·01). However, there was no overall difference in the concentration of insulin or glucagon-like peptide 1. In conclusion, dietary treatments when served as high-protein mixed meals induced similar effects on EE and appetite regulation, except for lipid oxidation, where RQ values suggest that it is higher after consumption of IW than after consumption of HC.

Assessment of the proliferative capacity of the flavanones 8-prenylnaringenin, 6-(1.1-dimethylallyl)naringenin and naringenin in MCF-7 cells and the rat mammary gland.

8-Prenylnaringenin (8-PN) and naringenin (Nar) are phytoestrogens found in food items and nutritional supplements, while 6-(1.1-dimethylallyl)naringenin (6-DMAN) is a component of an African plant. Besides their assumed beneficial effects they may promote mammary and endometrial cancer. We therefore assessed their proliferative and estrogenic potential on the mammary gland in vitro and in vivo. In competitive estrogen receptor (ER) ligand binding assays 8-PN displayed a high relative binding affinity for both ERs with a preference for ERα and had the strongest mitotic effect on MCF-7 cells among the test substances. In a three day exposure in young adult ovariectomized female rats 15 mg/kg 8-PN had the highest capacity to increase the number of terminal end buds (TEB) in the mammary gland and stimulated expression of proliferation markers in epithelial ductal cells, followed by 6-DMAN and Nar, but overall their capacity to stimulate proliferation was weak in comparison to 17ß-Estradiol (E2).

Review: Production and functionality of active peptides from milk.

In recent years, research on the production of active peptides obtained from milk and their potential functionality has grown, to a great extent. Bioactive peptides have been defined as specific protein fragments that have a positive impact on body functions or conditions, and they may ultimately have an influence on health. Individual proteins of casein or milk-derived products such as cheese and yogurt have been used as a protein source to study the isolation and activity of peptides with several applications. Currently, the milk whey waste obtained in the production of cheese also represents a protein source from which active peptides could be isolated with potential industrial applications. The active properties of milk peptides and the results found with regard to their physiological effects have led to the classification of peptides as belonging to the group of ingredients of protein nature, appropriate for use in functional foods or pharmaceutical formulations. In this study, the main peptides obtained from milk protein and the past research studies about its production and biological activities will be explained. Second, an analysis will be made on the methods to determinate the biological activities, the separation of bioactive peptides and its structure identification. All of these form the base required to obtain synthetic peptides. Finally, we explain the experimental animal and human trials done in the past years. Nevertheless, more research is required on the design and implementation of equipment for the industrial production and separation of peptides. In addition, different authors suggest that more emphasis should therefore be given to preclinical studies, proving that results are consistent and that effects are demonstrated repeatedly by several research human groups.

Partial purification and characterisation of cysteine protease in wheat germ.

BACKGROUND: Proteases have become an essential part of the modern food and feed industry, being incorporated in a large and diversified range of products for human and animal consumption. The objective of this study was to purify and characterise a protease from wheat germ. RESULTS: After purification a single protease of molecular weight 61-63 kDa (determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis) was obtained. The purified protease had optimal activity at 50 °C and maintained its activity completely after incubation at 30 °C for 30 min, while over 47% of the activity was lost after incubation at 80 °C for 30 min. The purified protease had optimal activity and maintained maximum stability at pH 5.5, while the activity decreased after incubation for 30 min at other pH values. The protease was inhibited by Mg(2+), Mn(2+), Ba(2+) and iodoacetic acid and stimulated by Li(+), Ca(2+), Cu(2+), ß-mercaptoethanol and dithiothreitol, while Zn(2+), L-cysteine and glutathione had no significant effect on its activity. At pH 5.5 the enzyme had a K(m) of 0.562 mg mL(-1) with casein as substrate and showed higher affinity to casein than to bovine serum albumin, ovalbumin and gelatin. CONCLUSION: The purified enzyme from wheat germ was identified as a cysteine protease.

Quantitative assessment of multi-laboratory reproducibility of SDS-PAGE assays: Digestion pattern of beta-casein and beta-lactoglobulin under simulated conditions.

A digestion protocol was applied in triplicate by ten laboratories, simulating in vivo gastric and duodenal conditions. The intra- and inter-laboratory variability in the kinetics of protein degradation was quantified, focussing on the digestion of beta-casein under gastric conditions, and of beta-lactoglobulin (beta-Lg) under duodenal conditions. The addition of surfactants such as phosphatidylcholine (PC) in the digestion mix was also evaluated. Identification and quantification of peptide bands on SDS-PAGE gels formed the basis for analysis. An average intensity loss of 69% (SD=13.5) at 5 min (89% at 10 min, with SD=5.5) was observed for beta-casein, whereas the beta-Lg duodenal digestion showed an 82% loss at 30 min (SD=14.2). Constant rates of first-order reactions showed that for fast reactions, inaccuracies in the time of first sampling contributed to the variability, which were also affected by image quality, saturation, and the splitting of time courses across gels. Breakdown products for beta-casein included ten other polypeptides, with four detected in all and two in most gels, and for beta-Lg ten polypeptides, with five detected in most, and two in two-third of the cases. Addition of PC in the gastric phase led to beta-Lg intensity loss only a quarter as large as without PC and altered beta-Lg proteolysis in the duodenal compartment.

Digestibility and allergenicity assessment of enzymatically crosslinked beta-casein.

Crosslinking enzymes are frequently used in bioprocessing of dairy products. The aim of this study was to examine the effects of enzymatic crosslinking on IgE binding, allergenicity and digestion stability of beta-casein (CN). beta-CN was crosslinked by transglutaminase, tyrosinase, mushroom tyrosinase/caffeic acid and laccase/caffeic acid. The IgE binding to beta-CN was compared in vitro by CAP inhibition assay, ELISA inhibition as well as ex vivo by basophil activation assay. Crosslinked CNs were digested by simulated gastric fluid for 15 and 60 min and obtained digests analyzed for their ability to inhibit IgE binding by CAP inhibition assay and SDS-PAGE. The ability of crosslinked CNs to activate basophils was significantly reduced in seven patients in the case of CN crosslinked by laccase and moderately reduced in the case of tyrosinase/caffeic acid crosslinked CN (in two cow's milk allergy patients tested with different allergen concentrations). The response to various crosslinked CNs differed individually among patients' sera tested by ELISA inhibition assay. The presence of caffeic acid hampered digestion by pepsin, and this effect was most pronounced for the tyrosinase/caffeic acid crosslinked CN. The laccase/caffeic acid and mushroom tyrosinase/caffeic acid had the highest potential in mitigating IgE binding and allergenicity of the beta-CN out of all investigated enzymes. The presence of a small phenolic compound also increased digestion stability of beta-CN.

Bovine chymosin: a computational study of recognition and binding of bovine kappa-casein.

Bovine chymosin is an aspartic protease that selectively cleaves the milk protein kappa-casein. The enzyme is widely used to promote milk clotting in cheese manufacturing. We have developed models of residues 97-112 of bovine kappa-casein complexed with bovine chymosin, using ligand docking, conformational search algorithms, and molecular dynamics simulations. In agreement with limited experimental evidence, the model suggests that the substrate binds in an extended conformation with charged residues on either side of the scissile bond playing an important role in stabilizing the binding pose. Lys111 and Lys112 are observed to bind to the N-terminal domain of chymosin displacing a conserved water molecule. A cluster of histidine and proline residues (His98-Pro99-His100-Pro101-His102) in kappa-casein binds to the C-terminal domain of the protein, where a neighboring conserved arginine residue (Arg97) is found to be important for stabilizing the binding pose. The catalytic site (including the catalytic water molecule) is stable in the starting conformation of the previously proposed general acid/base catalytic mechanism for 18 ns of molecular dynamics simulations.

A binary matrix for improved detection of phosphopeptides in matrix-assisted laser desorption/ionization mass spectrometry.

Application of matrix-assisted laser-desorption/ionization mass spectrometry (MALDI MS) to analysis and characterization of phosphopeptides in peptide mixtures may have a limitation, because of the lower ionizing efficiency of phosphopeptides than nonphosphorylated peptides in MALDI MS. In this work, a binary matrix that consists of two conventional matrices of 3-hydroxypicolinic acid (3-HPA) and alpha-cyano-4-hydroxycinnamic acid (CCA) was tested for phosphopeptide analysis. 3-HPA and CCA were found to be hot matrices, and 3-HPA not as good as CCA and 2,5-dihydroxybenzoic acid (DHB) for peptide analysis. However, the presence of 3-HPA in the CCA solution with a volume ratio of 1:1 could significantly enhance ion signals for phosphopeptides in both positive-ion and negative-ion detection modes compared with the use of pure CCA or DHB, the most common phosphopeptide matrices. Higher signal intensities of phosphopeptides could be obtained with lower laser power using the binary matrix. Neutral loss of the phosphate group (-80 Da) and phosphoric acid (-98 Da) from the phosphorylated-residue-containing peptide ions with the binary matrix was decreased compared with CCA alone. In addition, since the crystal shape prepared with the binary matrix was more homogeneous than that prepared with DHB, searching for 'sweet' spots can be avoided. The sensitivity to detect singly or doubly phosphorylated peptides in peptide mixtures was higher than that obtained with pure CCA and as good as that obtained using DHB. We also used the binary matrix to detect the in-solution tryptic digest of the crude casein extracted from commercially available low fat milk sample, and found six phosphopeptides to match the digestion products of casein, based on mass-to-charge values and LIFT TOF-TOF spectra.

In vitro assessment of intestinal IGF-I stability.

Insulin-like growth factor (IGF-I) is a 7648-Da polypeptide consisting of 70 amino acids. Clinically, IGF-I might be used in type II diabetes, which requires a life-long treatment. Therefore, delivery routes other than parenteral injections are highly desirable. For convenience, the peroral route is the most attractive. Therefore, in an attempt to answer the feasibility of oral delivery of IGF-I we examined the metabolism of this polypeptide in the gut in the presence of crude porcine pancreatic enzymes (CPPE) and flushings of the small and large intestine from pig, rat, and dog. Moreover, incubation studies with purified pancreatic enzymes that are present in the intestine were performed to determine the most active enzymes responsible for the intestinal cleavage of IGF-I. IGF-I was mainly degraded by chymotrypsin (t(1/2) = 2.7 min) and trypsin (t(1/2) = 34.6 min), whereas in the presence of aminopeptidase M and carboxypeptidase A IGF-I was stable up to 90 min. IGF-I was degraded in flushings from the jejunum, ileum, and colon. However, there were no significant differences in the stability of IGF-I between the examined intestinal segments. The addition of serine protease inhibitors such as a combination of aprotinin, soybean trypsin inhibitor, and Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), as well as casein profoundly improved the stability. Because we were able to improve the stability of IGF-I in vitro in all species at the same degree we speculate that a similar extension of half-life might also be possible in the human intestinal system.

Relation between in vitro and in vivo assessment of amino acid availability.

Even though the availability of dietary amino acids is the result of integrated phenomena of digestion, absorption and transport, it may be mainly affected by the stage of luminal digestion. In this case, amino acid availability could be predicted by an in vitro method designed to reproduce in vivo proteolysis conditions. In order to check this hypothesis, the essential amino acid (EAA) profiles of digesta collected at 8 intervals during a 24-h in vitro enzymatic proteolysis of casein and rapeseed proteins were compared to the pattern of appearance of dietary EAA in portal vein of pigs fed the same proteins, determined at each hour over a 8-h postprandial period by coupling blood flow rate with porto-arterial differences in plasma EAA concentrations. Comparisons of in vitro and in vivo data first bore on overall EAA profiles measured at each interval, and then on the individual kinetics of each EAA. Regarding total profiles, the highest correlations for casein (r: 0.80-0.98) were found when comparing EAA patterns determined during the first half of in vitro digestion and in vivo absorption periods. Similar r values were obtained with rapeseed proteins, but over longer periods of measurement. Concerning individual kinetics, the highest correspondences were found with rapeseed proteins, with 5 out of 9 EAA (methionine, isoleucine, leucine, phenylalanine and arginine) having their in vitro sequence of release significantly correlated with their in vivo sequence of absorption. With casein, correlations were significant for threonine, valine, isoleucine and leucine. These results suggest that sequential hydrolysis in the digestive tract, as reproduced by the in vitro technique, is a key determinant of amino acid appearance in the portal blood to a degree varying with the protein source and with the nature of the amino acid.

[Assessment of the in vitro digestibility of dietary proteins and of the degree of the breakdown of their antigenic structures by using polyenzyme systems]. / Otsenka perevarivaemosti in vitro pishchevykh belkov i stepeni rasshchepleniia ikh antigennykh struktur s ispol'zovaniem polifermentnykh sistem.

A comparative assessment was made of the digestion of bovine serum albumin (BSA), chicken ovalbumin (OVA), and casein by means of the gastric juice--duodenal contents floccular gel structures (FGS) system and a four-enzymic system including trypsin, chymotrypsin, peptidase, and bacterial protease preparations. Decomposition of the BSA and OVA antigenic structures with the use of the two systems was also studied. Significant differences in BSA and OVA digestion by the gastric juice--FGS system were detected both with respect to amino nitrogen content and to the degree of their antigenic structure decomposition, whereas no such differences were observed when the four-enzymic system was used. The systems most accurately simulating the 'proteolytic conveyer' conditions of the gastrointestinal tract are preferable for the studies. The developed method is recommended for use in comparative assessment of the nutrient protein sensitizing properties.

[Pathology and physiology of microbes. IV. Economic and metabolic coefficients in assessing the functional states of Salmonella typhi]. / Patologiia i fiziologiia mikrobov. Soobshchenie IV. Ekonomicheskii i metabolicheskii koéffitsienty v otsenke funktsional'nykh sostoianii Salmonella typhi.

The study of economic and metabolic coefficients under different conditions of batch and continuous cultivation with the use of S. typhi, a facultative anaerobe, as a model has demonstrated that the functional state of the microbial population is determined by the phase of the development of the culture, the composition of the culture medium, the conditions of aeration and stirring, the concentration of the energy-providing substrate and the dilution rate in continuous cultivation. The economic coefficient with respect to the energy-providing substrate has been found to be the main characteristic of the functional state of the population: the high values of this characteristic correspond to the physiological state of the population. The decrease of the economic coefficient below a certain critical value is indicative of the pathological state of the population.

Protein quality and the cost of selected commercial protein supplements.

The biologic value and the cost of three commercial protein supplements were compared using casein as the control. Each product was fed to rats as the sole protein source supplying 10 percent of the energy in a semipurified diet. Nonfat dry milk was equal to casein in PER, but the PERs of low-fat soy powder and high-protein supplement were significantly lower than the PER of casein. Nitrogen balance was positive for all protein supplements, but low-fat soy powder was significantly lower than the others. cost comparison per gram of protein showed low-fat soy powder to be the least expensive, nonfat dry milk to be slightly higher in cost, and the high-protein supplement to be the most expensive.