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Journal Cover Sosyoloji Konferansları (Istanbul Journal of Sociological Studies)
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  This is an Open Access Journal Open Access journal
   ISSN (Print) 1304-0243
   Published by İstanbul Üniversitesi Homepage  [10 journals]
  • Investigation of the Potential of Hemp, Pea, Rice and Soy Protein
           Hydrolysates as a Source of Dipeptidyl Peptidase IV (DPP-IV) Inhibitory
           Peptides

    • Abstract: Abstract Food protein hydrolysates contain peptide sequences with dipeptidyl peptidase IV (DPP-IV) inhibitory properties which may find use to improve serum glucose regulation in type 2 diabetics. Four plant protein isolates from hemp (H), pea (P), rice (R) and soy (S) were hydrolysed with three enzyme preparations: Corolase L10 (Cor), Promod 144MG (Prom) and Protamex (Prot). From the 12 hydrolysates generated after 4-h incubation, 7 yielded DPP-IV inhibitory activity <2.0 mg protein equivalent mL−1. Their IC50 values ranged from 0.73 ± 0.11 to 3.54 ± 0.24 mg dry weight (dw) hydrolysate mL−1 for the 4 h P_Prot and 4 h H_Cor hydrolysates, respectively. Simulated gastrointestinal digestion (SGID) of the intact proteins yielded DPP-IV IC50 values between 1.85 ± 0.34 and 4.50 ± 0.55 mg dw hydrolysate mL−1 for the R and H, respectively. The DPP-IV inhibitory potency of the 4 h hydrolysates subjected to SGID (DPP-IV IC50 ranging from 1.00 ± 0.42 to 3.83 ± 0.36 mg dw hydrolysate mL−1 for the SGID of the 4 h S_Prom and 4 h H_Prot, respectively) was generally higher than that of the corresponding intact protein subjected to SGID, with the exception of the SGID of R and that of the 4 h R_Prot hydrolysate which had similar IC50 values (P > 0.05). To our knowledge, this is the first study reporting that H and P protein hydrolysates inhibit DPP-IV in vitro. This study also demonstrates the potential benefit, in some instances, of hydrolysing plant protein substrates prior to oral ingestion with the view of releasing DPP-IV inhibitory peptides.
      PubDate: 2015-12-01
       
  • A Better Understanding of the Factors Affecting In vitro Lipolysis Using
           Static Mono-compartmental Models

    • Abstract: Abstract In vitro lipolysis studies are the most common approach to investigate digestion profiles and microstructure breakdown of emulsion-based functional foods after ingestion. This study investigated the difference between two static mono-compartmental models, the pH stat titration and a jar digestion model, as tools to evaluate lipolysis in vitro. Two oil-in-water emulsions, consisting of long- or medium-chain triglycerides, were used in the study. Factors essential to the pH-stat model were evaluated, including calcium concentration and mode of addition, i.e. initial or continuous. Continuous addition of calcium resulted in improved control of free fatty acid release kinetics. Results also indicated that titration at pH 9 alleviates the underestimation of free fatty acid release with the pH-stat model. The research clearly highlighted the differences between the models, and the results will help researchers identify the most appropriate model to use for in vitro digestion of emulsions.
      PubDate: 2015-12-01
       
  • A Size Exclusion Chromatography-Based In Vitro Examination of Some Aspects
           of Bread Digestion

    • Abstract: Abstract An in vitro digestion–size exclusion chromatography set-up has been used as to study transfer of macromolecules from bread to simulated gastrointestinal (GI) juices and their subsequent breakdown along the GI tract. Bread samples underwent mechanical processing in a sequence of simulated GI environments, namely in simulated oral, gastric, then intestinal fluids. In the absence of GI enzymes, transfer of starch from the bolus to the GI fluids has been monitored to occur without any apparent hydrolysis. α-Amylase incorporation in the simulated oral fluids resulted in hydrolysis further down the GI tract, namely in the duodenum, suggesting a pH-dependent enzyme deactivation in the stomach and reactivation in the simulated intestinal area. Pepsin in the simulated gastric fluids influenced the release of protein in the simulated stomach environment.
      PubDate: 2015-12-01
       
  • Closing Editorial for Food Digestion

    • PubDate: 2015-12-01
       
  • Rheological Characterization of Gastric Juices from Bread with Different
           Amylose/Amylopectin Ratios

    • Abstract: Abstract The purpose of this paper is to present a methodology for characterizing the rheological properties of heterogeneous gastric contents, for the first time, to our knowledge. Pigs were used as a model because their gastric system is similar to that of humans. Bread was chosen as a high-calorific, solid test meal. The bread was made with flours offering a wide range of amylose/amylopectin ratios. A specific method of extraction and measurement was developed. For the study, the vane test technique was chosen in order to take into account the heterogeneity and short lifetime of the boluses. The variation in viscosity values for each gastric system was compared with their moisture contents, different residence times in the stomach, and various amylose/amylopectin ratios. The viscosities decrease significantly as the residence time increases. Furthermore, we suggest that the amylose/amylopectin ratio (Am/Ap) determines the moisture content, which then provides the viscosity level, since the gastric contents behave as a concentrated suspension. Pigs add variability to the results because the structure of the bolus depends on mastication.
      PubDate: 2015-12-01
       
  • Improved Bioavailability of Supercritical Rosemary Extract Through
           Encapsulation in Different Delivery Systems After In Vitro Digestion

    • Abstract: Abstract Rosmarinus officinalis L. is an aromatic plant with components known to confer health-promoting properties. The present study aimed to enhance the bioavailability of the main compounds, carnosic acid and carnosol, by encapsulation. The rosemary extracts were obtained by supercritical fluid extraction, and then different delivery systems were tested, namely oil emulsion droplets and casein micelles, native protein particles present in milk. Soybean oil in water emulsions and casein dispersions were digested in vitro with simulated gastric and intestinal fluids, and the bioavailability of the extract was tested on Caco-2 cell monolayers. The apical and basolateral compartments and the cell lysates were further analyzed for carnosic acid and carnosol content by means of ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS). The encapsulation in both casein micelles and emulsion droplets significantly protected the rosemary extract from degradation during digestion, resulting in 81 and 97 % recovery of carnosic acid, respectively. This high protection rate prompted an increased bioavailability of carnosic acid and carnosol in Caco-2 monolayers, with up to 10 % carnosic acid recovered in the bioavailable fraction. Encapsulation in delivery systems like casein micelles or emulsion droplets opens new possibilities for successful incorporation of rosemary extracts in healthy food formulations with increased functionality.
      PubDate: 2015-12-01
       
  • Bioefficacy of Tea Catechins Associated with Milk Caseins Tested Using
           Different In Vitro Digestion Models

    • Abstract: Abstract Milk proteins, and in particular caseins, are avid binders of polyphenols. The objective of this work was to understand how the presence of a polyphenol–casein complex may affect the bioefficacy of tea catechins [epigallocatechin-3-gallate (EGCG)]. In vitro digestion was carried out using two different models: a batch model and a dynamic digestion model, using a simulator of the human intestinal microbial ecosystem (SHIME). SDS-PAGE electrophoresis of the digestates showed that the presence of EGCG did not affect hydrolysis of the caseins, regardless of the digestion model used. However, there was a slower digestion of the caseins in the dynamic model (SHIME) compared to the in vitro batch model. The cytotoxic effect of free and complexed EGCG was tested using adenocarcinoma cells (HT-29) and the HT29-MTX subclone. The HT29-MTX closely mimics the presence of the mucus barrier in the intestine. HT-29 cells showed a decrease in cell viability proportional to the concentration of ECGC, free or complexed and after digestion. The proliferation rate of HT29 assessed on digested EGCG-casein complexes indicated that the binding of EGCG and milk proteins does not affect its bioactivity. Antiproliferative activities were comparable to those for free EGCG, in spite of the lower recovery of EGCG after the various stages of digestion. Exposure of EGCG (free or complexed) to mucus-producing cells HT29-MTX did not show a decrease in viability, and the cell response was typical of normal cells. The presence of mucus also protected coculture monolayers of HT29-MTX and Caco-2. All the models demonstrated that the presence of EGCG–casein micelle complex did not affect the bioefficacy of the polyphenol.
      PubDate: 2014-12-01
       
  • Emulsion Flocculation and Stability in a Simple in Vitro Gastrointestinal
           Model

    • Abstract: Abstract A simple in vitro model of swallowing–progression from mouth to stomach was used in order to study the phase behavior/aggregation of BSA-stabilized model oil-in-water emulsions. The latter were first dispersed into simulated mouth fluid (SMF); then, their droplets were collected and redispersed into simulated gastric fluid (SGF), either in the presence or not of a model gastric mucin (pig gastric mucin, PGM). Emulsions flocculate weakly due to electrostatic effects upon exposure to simulated mouth fluid (SMF). Upon progression of the droplets to the stomach-like environment, enhancement of flocculation (but no coalescence) is observed. The presence of PGM in this environment reduces the extent of flocculation, suggesting that mucins could be acting as regulators of flocculation in the gastrointestinal tract.
      PubDate: 2014-12-01
       
  • Erratum to: The Role of the Mucus Barrier in Digestion

    • PubDate: 2014-12-01
       
  • Fucoidan from Cladosiphon okamuranus Tokida Added to Food Has No Adverse
           Effect on Availability for Absorption of Divalent Minerals in the Dynamic
           Multicompartmental Model of the Upper Gastrointestinal Tract

    • Abstract: Abstract This study aimed to investigate whether the addition of fucoidan (Yakult Fucoidan) prepared from Cladosiphon okamuranus Tokida to food affects the availability of divalent minerals for intestinal absorption. After coadministration with a test meal into a dynamic multicompartmental model of the upper gastrointestinal tract (TIM-1), the availability for intestinal absorption (bioaccessibility) of minerals was evaluated. Fucoidan (0.033 and 0.1 g, corresponding to approximately 1.7 and 5.0 mg/kg body weight (BW) in humans) or two alginates (Cargill AlgogelTM and SatialgineTM, 3.0 g, corresponding to approximately 150 mg/kg BW in humans) suspended in 200 g water and added to 100 g test meal did not significantly decrease the bioaccessibility of calcium, magnesium, iron, zinc, manganese, copper, and cobalt in comparison to the test meal without these gelling agents, except for calcium by 3.0 g Satialgine (p < 0.05). Positive control experiments with an unrealistically high dosage of fucoidan (3.0 g) significantly decreased the bioaccessibility of calcium, iron, zinc, and manganese and significantly enhanced the bioaccessibility of copper and cobalt (p < 0.05). It is concluded that the oral intake of the tested fucoidan at standard and high dosages in food has no adverse effect on divalent mineral absorption. Based on TIM-1 validation studies with minerals and metals in comparison to human studies, it is expected that these results may be extrapolated to the human situation.
      PubDate: 2014-12-01
       
  • Markers of Cardiovascular Risk and Metabolism Assessed on Multiple
           Baseline Occasions and in Response to a Single Fatty Meal in Healthy Young
           Adults

    • Abstract: Abstract The possible etiological role of C-reactive protein (CRP) has become a focus of cardiovascular and metabolic disease research. Very little is known regarding the cardiovascular and metabolic disease biomarker data in young healthy individuals, and the relationship between these and the postprandial metabolic responses has not been systematically studied. We determined the hsCRP, triglyceride (TAG), cholesterol, glucose, and insulin baseline values in 20 healthy young subjects (ten males, ten females, 22–29 years old) on four distinct occasions each approximately 1 week apart and assessed physical activity score and BMI. There was greater intra-subject variability for hsCRP (18 % of total) than for components of the blood lipid profile (2–4 %). Baseline hsCRP level was directly related to fasting TAG level (P < 0.001), and BMI (P = 0.011), and inversely to physical activity score (P = 0.002), although there were some sex-dependent effects. In response to a high fat meal (~800 total kcal, ~430 fat kcal from margarine), the acute (up to 6 h postprandial) elevation in serum TAG was directly related to the baseline hsCRP level (P = 0.026), while the insulin response was directly related to the BMI (P = 0.01). Thus, a potential relationship has been demonstrated between hsCRP and TAG levels both at baseline and in response to an acute fatty meal in young healthy individuals. It remains to be substantiated whether the scaling of hsCRP with an exaggerated TAG response to a meal challenge contributes to future development of insulin resistance and metabolic and cardiovascular disease.
      PubDate: 2013-12-01
       
  • In Vitro Infant Digestion of Whey Protein–Dextran Glycates

    • Abstract: Abstract Whey protein–dextran glycates (WD) made from whey protein isolate (WPI) and dextran (DX) of two different molecular masses (10 and 150 kDa) were subjected to an in vitro infant digestion model. The model included physiologically relevant concentrations of porcine gastrointestinal enzymes, as well as phosphatidylcholine and bile salts. Native α-lactalbumin and bovine serum albumin were rapidly digested during the gastric phase, whereas 93 % of β-lactoglobulin (BLG) passed unaltered into the duodenal phase and was completely digested by pancreatin within 150 min. DX-glycated whey protein (WD10; WD150) also survived gastric digestion intact, but was digested by pancreatin. However, after 180 min of duodenal digestion, some glycate and free BLG remained. Titers of free BLG in the duodenal phase were 10-fold higher for WPI compared to the glycates. Duodenal digestion kinetics of the BLG present in WPI, as well as of the free BLG present in WD10 and WD150 followed first-order kinetics. Digestion rate constants (k) were 0.019, 0.010, and 0.012 min−1, for k WPI, k WD10, and k WD150, respectively. Lower rate constants were observed for BLG digestion in glycates as compared to WPI. In conclusion, BLG was digested in glycates, but its digestion was slower in the glycate form. Avoiding high titers of BLG in the duodenum, as well as successful masking of immunogenic peptides by DX, could help in the development of hypoallergenic foods. This work contributes to an understanding of how protein–polysaccharide glycates impact protein digestion in infants.
      PubDate: 2013-12-01
       
  • Lipid Digestion of Protein Stabilized Emulsions Investigated in a Dynamic
           In Vitro Gastro-Intestinal Model System

    • Abstract: Abstract This study investigated the effect of gastric passage of protein stabilized emulsions, i.e., whey protein isolate (WPI) and lysozyme, under dynamic in vitro conditions on both the gastric and intestinal lipolysis. Emulsions were prepared at neutral pH to enable an opposite surface charge. Experiments were performed in a multi-compartmental digestion model (TNO Gastro-Intestinal Model) including a gastric compartment simulating in vivo conditions, i.e., gradual acidification, mixing, lipolysis and proteolysis. Under gastric conditions, lysozyme-stabilized emulsions remained macroscopically homogenous, whereas WPI-stabilized emulsions separated into a cream and serum layer. Microscopy revealed flocculation of both emulsions, but larger particles were found for the initial negatively charged WPI-stabilized emulsions compared to the positively charged lysozyme-stabilized emulsions. This suggested that creaming was due to larger flocs formation caused by a change from net negative to net neutral charge as an effect of the gradual decreasing pH. Analysis of lipid composition, i.e., free fatty acids (FFA), monoglycerides, diglycerides (DG) and triglycerides revealed mainly FFA and DG in the gastric compartment. As a result of creaming, the entry of lipids into the small intestinal part was delayed for WPI-stabilized emulsions. However, the total amount of FFA released at the end of the experiment was similar for both emulsions. Our results show, that the charge differences affected the creaming behavior, but not the lipase activity, on the two studied emulsions.
      PubDate: 2013-12-01
       
  • Influence of Dietary Spices on Protein Digestibility and Absorption in
           Experimental Rats

    • Abstract: Abstract Since spice diets are known to cause significant reduction in food transit time, an animal study was carried out to verify if there is any undesirable compromise with nutrient bioavailability, especially fat and protein digestion and absorption. Young rats were maintained for 6 weeks on diets containing (in gram percent): ginger (0.05), ajowan (0.2), cumin (1.25), and piperine (0.02). Soya protein and casein were incorporated to the basal semisynthetic diets to make up the sole protein source at 9 % protein level (N × 6.25) in an otherwise isocaloric and isonitrogenous diets. Nitrogen content of urine and feces were determined during the last week of feeding regimen. True protein digestibility, biological value, nitrogen balance, and net protein utilization were calculated. Dietary fat absorption was also determined. It was observed that the test spices reduced food transit time, but there was no undesirable compromise with the protein (casein or soya protein) digestion and absorption. All the four test spices in both the test protein groups showed increased protein and fat absorption and utilization. Thus, dietary intake of spices—ginger, ajowan, piperine, and cumin—enhances fat and protein digestion as well as their absorption despite causing a significant reduction in the ororectal food transit time.
      PubDate: 2013-12-01
       
  • A Double-Blind Clinical Study to Investigate the Effects of a Fungal
           Protease Enzyme System on Metabolic, Hepato-renal, and Cardiovascular
           Parameters Following 30 Days of Supplementation in Active, Healthy
           Men

    • Abstract: Abstract Research on the role of digestion in overall health has driven increasing interest in the use of digestive enzymes, which may improve nutrient absorption and reduce gastrointestinal symptoms. Sales of digestive aids and enzymes have grown over 8% in 2009, with enzymes accounting for $69 million of this growing category. Recent clinical research reported that acute dosing of Aminogen®, a patented blend of digestive protease enzymes isolated from Aspergillus and blended with whey protein concentrate, increased the rate of protein absorption. The results indicated a faster rate of amino acid absorption reflected in significantly higher blood levels of amino acids, increased nitrogen retention, and significantly reduced levels of C-reactive protein. Few studies, however, have examined the safety of repeated dosing of oral enzymes with an appropriate substrate. The purpose of this study, therefore, was to evaluate basic measures of clinical safety during 30 days of continuous, repeated dosing of Aminogen® and whey protein supplementation in healthy, active men maintaining a regimen of resistance training. Parameters evaluated include various markers of general physical health, metabolic function, hepato-renal function, and cardiovascular health including fasting blood lipids. Forty healthy, resistance-trained men (27.1 ± 7.9 years) were recruited for this double-blind, randomized study. Group A ingested two 40-g doses of whey protein per day containing Aminogen®. Group B ingested two 40-g doses of whey protein per day. No significant changes were noted in measures of general physical health, metabolic function, cardiovascular health, and hepato-renal function within or between groups. However, total cholesterol, LDL cholesterol, and serum calcium significantly increased (P < 0.05) in group B. In group A, whey protein containing Aminogen® was well tolerated with no adverse reactions reported. No differences in serum markers of clinical safety and an improved blood lipid profile are also reported.
      PubDate: 2013-05-01
       
  • Fast and Slow Proteins: Modulation of the Gastric Behavior of Whey and
           Casein In Vitro

    • Abstract: Abstract Ingestion of whey protein rather than casein is associated with a number of enhanced physiological benefits including satiety, glycemic control, and muscle synthesis. Many of these differences can be attributed to apparent differences in digestion and absorption kinetics between these proteins. By simulation of gastric phase digestion, we show that casein coagulates and forms a protein network upon gastric acidification, resulting in an enhanced time viscosity profile, whereas whey proteins stay within solution indicating that altered gastric behavior of these proteins underlies the apparent differences in digestion and absorption kinetics. Moreover, by applying industrially relevant protein modification strategies, the behavior during the gastric phase of these protein sources could be modified. By hydrolysis and Maillard-based modification, protein network formation of casein and concomitantly increased viscosity under simulated gastric conditions could be prevented, likely resulting in faster digestion and absorption kinetics in vivo. On the other hand, cold gelling whey protein aggregates could be prepared that coagulated and formed a temporary protein network under simulated gastric conditions, possibly delaying the overall digestion and absorption kinetics in vivo. Furthermore, combinations of whey proteins or whey protein aggregates and polysaccharides displayed an altered behavior under simulated gastric conditions, most likely as a result of complex coacervation. Overall, the results from this study demonstrate the possibilities within food technology to control the gastric behavior of whey and casein determining the overall digestion and absorption kinetics of these protein sources.
      PubDate: 2013-05-01
       
  • Venturing into In Vitro Physiological Upper GI System Focusing on the
           Motility Effect Provided by a Mechanised Rat Stomach Model

    • Abstract: Abstract It is desirable to possess an in vitro digestion track model that can mimic to the fullest possible extent what is going on in a targeted animal system. Presently, there is still a long way before such a system becomes a reality despite a few attempts having been made in recent years to realise such a ‘dream’. Here, the design and laboratory experiences on a newly constructed in vitro rat stomach system are reported. Fine rice starch particles (obtained from cooked rice) have been used as the model food fed into the model stomach. Since the focus was on the motility effect, the microbial effect is neglected. The effects of motility rate and amplitudes of mechanical operation automated in the current rig have been focused upon and examined. The results are illustrative for a near-future development of a more effective construction of in vitro systems.
      PubDate: 2013-05-01
       
  • The Formation of an Anti-Cancer Complex Under Simulated Gastric Conditions

    • Abstract: Abstract A potent anti-cancer complex has previously been formed from two major components of milk. Human/bovine α-lactalbumin made lethal to tumour cells (H/BAMLET) is a protein–fatty acid complex that has been produced using the whey protein α-lactalbumin (α-LA) and the fatty acid oleic acid (OA). It was shown that it possesses selective anti-tumour and anti-microbial activity, which was first identified in acidic fractions of human breast milk. The aim of this study was to determine whether the two components would form a bioactive complex during simulated gastric (GI) transit. Results showed that a complex consisting of α-LA and OA is formed as the protein unfolds under acidic conditions and subsequently refolds upon pH increase. Analysis of this complex using Nuclear Magnetic Resonance and Fourier Transform Infra-Red (FTIR) spectroscopies estimated a stoichiometry of 4.1 and 4.4 oleic acids per mole of protein, respectively. FTIR and fluorescence spectroscopies showed that the structure was similar to that of BAMLET. Cytotoxicity testing against cancer cell line U937 cells showed that the complex had an LC50 value of 14.08 μM compared to 9.15 μM for BAMLET. These findings suggest that a BAMLET-like complex may be formed under the tested in vitro GI conditions.
      PubDate: 2013-05-01
       
  • An Expanded Finite Element Model of the Intestinal Mixing of Digesta

    • Abstract: Abstract We used a finite element model to simulate flow through the intestine induced by peristalsis and segmentation. These models explored the effects of varying the rheological properties of digesta, incorporating a slip effect at the wall and the presence of an area of lumen distension adjacent to the area of vorticeal mixing that developed during peristalsis. Reynolds numbers were consistently low (<200) throughout all simulations indicating that local conditions were insufficient to bring about turbulence. When the viscosity of the material flowing through a simple tube model was increased, the area of the vortex orad (upstream) to the zone of peristaltic coaptation was relatively reduced, whilst that aborad (downstream) to the zone of peristalsis was increased. This effect was reduced when the material in the lumen had shear thinning properties. When a region of relative distension of the lumen that travelled aborad to the zone of peristalsis was incorporated into the model, the total area of vortex formation was reduced when material of either high or low Newtonian viscosity was in the lumen. However, these reductions were small compared with those obtained when slip at the intestine wall was incorporated into the model. Incorporation of slip resulted in a marked reduction of both orad and aborad vortices, an effect that persisted when the other effects were incorporated into the same model. The authors conclude that the translocation of nutrients from the intestinal lumen to the wall by action of short-lived vortices will be significantly reduced when the apparent viscosity of digesta is high and there is significant slip at the wall, even when digesta is a shear thinning, non-Newtonian fluid. Hence, the consumption of a diet that contains a high proportion of fibre or other insoluble residue will physically impair absorption by reducing vorticeal flow and promoting creep flow.
      PubDate: 2013-05-01
       
  • Absorption of Casein Antihypertensive Peptides through an In Vitro Model
           of Intestinal Epithelium

    • Abstract: Abstract A commercial casein hydrolysate, which contains peptides RYLGY and AYFYPEL as active molecules, has shown antihypertensive effects after acute and long-term administration. This study examines transepithelial absorption of RYLGY and AYFYPEL and derived digestion fragments using Caco-2, HT29-MTX−6 and co-culture 75 % Caco-2/25 % HT29-MTX−6 as predictive models and RP-HPLC–MS as analytical tool. Peptides RYLGY and AYFYPEL were absorbed intact through cell monolayers, although RYLGY was partly hydrolyzed by intestinal peptidases. Co-culture 75 % Caco-2/25 % HT29-MTX−6 exhibited intermediate properties, with regard to transepithelial electrical resistance, peptide hydrolysis, and absorption of the studied peptides, between Caco-2 and HT29-MTX−6 pure cultures. Interestingly, mucus layer that covered completely HT29-MTX−6 and co-culture monolayers was not a barrier for the absorption of the studied peptides. The apparent permeability values for absorptive transport across Caco-2 monolayers for RYLGY (0.22 × 10−6 cm/s) and AYFYPEL (0.26 × 10−6 cm/s) were similar. These findings highlight that in vivo absorption of antihypertensive peptides RYLGY and AYFYPEL may occur partially as intact peptides.
      PubDate: 2012-12-01
       
 
 
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