Measuring the oral bioavailability of protein hydrolysates derived from food sources: A critical review of current bioassays.

BACKGROUND: Food proteins are a source of hydrolysates with potentially useful biological attributes. Bioactive peptides from food-derived proteins are released from hydrolysates using exogenous industrial processes or endogenous intestinal enzymes. Current in vitro permeability assays have limitations in predicting the oral bioavailability (BA) of bioactive peptides in humans. There are also difficulties in relating the low blood levels of food-derived bioactive peptides detected in preclinical in vivo models to pharmacodynamic read-outs relevant for humans. SCOPE AND APPROACH: In this review, we describe in vitro assays of digestion, permeation, and metabolism as indirect predictors of the potential oral BA of hydrolysates and their constituent bioactive peptides. We discuss the relationship between industrial hydrolysis processes and the oral BA of hydrolysates and their peptide by-products. KEY FINDINGS: Hydrolysates are challenging for analytical detection methods due to capacity for enzymatic generation of peptides with novel sequences and also new modifications of these peptides during digestion. Mass spectrometry and peptidomics can improve the capacity to detect individual peptides released from complex hydrolysates in biological milieu.

Plasma concentrations and ACE-inhibitory effects of tryptophan-containing peptides from whey protein hydrolysate in healthy volunteers.

PURPOSE: The tryptophan-containing dipeptides isoleucine-tryptophan (IW) and tryptophan-leucine (WL) are angiotensin-converting enzyme (ACE)-inhibitors in vitro. These peptides are released by enzymatic hydrolysis of bovine whey protein. To exhibit ACE inhibition in vivo, peptides need to be absorbed into the circulatory system. This study aimed to determine the in vivo ACE-inhibitory potency of a whey protein hydrolysate (MPH), containing IW and WL, and to quantify plasma concentrations of these peptides after oral administration of MPH in healthy volunteers. Additionally, changes in blood pressure were investigated. RESULTS: After intake of 5 and 50 g MPH, plasma ACE activity was reduced to 86.4 ± 5.9 and 75.1 ± 6.9% of baseline activity, respectively. Although a clear ACE inhibition was measured, no effect on blood pressure was seen. Basal plasma concentrations of the tryptophan-containing dipeptides were 2.8 ± 0.7 nM for IW and 10.1 ± 1.8 nM for WL. After intake of 5-50 g MPH, peptide concentrations were dose dependently elevated to values between 12.5 ± 8.4 and 99.1 ± 58.7 nM for IW and 15.0 ± 4.3-34.9 ± 19.4 nM for WL. Administration of intact whey protein showed a minor ACE inhibition, probably caused by release of inhibitory peptides during gastrointestinal digestion. The increase of WL in plasma after intake of intact protein was similar to that determined after intake of MPH. In contrast, resulting IW concentrations were much lower after intake of intact whey protein when compared to MPH administration. CONCLUSION: After intake of MPH, plasma ACE activity decreased in parallel to the increase of IW and WL plasma concentrations. However, the resulting peptide concentrations cannot fully explain the reduction of ACE activity in plasma with a direct enzyme inhibition. Therefore, this study points to a gap in the understanding of the inhibitory action of these peptides in vivo. Thus, to further develop innovative food additives with ACE activity diminishing capabilities, it appears mandatory to better characterize the mode of action of these peptides.

Non-targeted and targeted analysis of collagen hydrolysates during the course of digestion and absorption.

Protein hydrolysates are an important part of the human diet. Often, they are prepared from milk, soy, or collagen. In the present study, four different collagen hydrolysates were tested, varying in the average molecular weight and the animal source. Three types of samples, the dissolved start products, in vitro generated dialysates (containing the digested components that are potentially available for small intestinal absorption), and human serum collected after product ingestion, were analyzed using LC-MS to compare the state of the hydrolysates before and after absorption, i.e., uptake into the blood. It was found that the composition of the collagen hydrolysates prior to and after ingestion was highly complex and dynamic, which made it challenging to predefine a strategy for a targeted analysis. Therefore, we implemented a new analytical approach to first map hydrolysate data sets by performing non-targeted LC-MS analysis followed by non-targeted and targeted data analysis. It was shown that the insight gained by following such a top down (data) analytical workflow could be crucial for defining a suitable targeted setup and considering data trends beyond the defined targets. After having defined and performed a limited targeted analysis, it was found that, in our experimental setup, Hyp-Gly and especially Pro-Hyp contributed significantly as carrier to the total Hyp increase in blood after ingestion of collagen hydrolysate. Graphical abstract.

Cellular and chemical antioxidant activities of chicken blood hydrolysates as affected by in vitro gastrointestinal digestion.

The effects of in vitro gastrointestinal (GI) digestion on the antioxidative activity of hydrolysates prepared from chicken blood plasma and red blood cell (RBC) by pepsin and thermolysin were investigated. The pepsin-hydrolyzed plasma (PHP) showed the highest scavenging activity of ABTS radicals (P < 0.05). RBC and plasma hydrolysates prepared by pepsin were hydrolyzed by GI proteases to a greater extent than hydrolysates prepared by thermolysin as evidenced by MALDI-TOF mass spectra. The antioxidative activity of all digesta increased compared to their respective parent hydrolysates, and PHP digesta showed the highest activity (P < 0.05). The digesta of PHP and thermolysin-hydrolyzed plasma showed cytoprotective properties in a dose-dependent manner, and 100 µg/mL of PHP digesta exhibited the highest protection of HepG2 cells against tert-butyl hydroperoxide (P < 0.05). Based on dichloro-dihydro-fluorescein diacetate assay, PHP digesta exhibited the greatest intracellular reactive oxygen species scavenging activity of approximately 71% at 100 µg/mL (P < 0.05). The peptide sequencing of PHP digesta revealed that they contained less than 10 amino acid residues, with an average hydrophobicity of 18.6. Chicken blood plasma is a better protein source for protein hydrolysates, and their digesta showed higher antioxidant activity compared to RBCs.

A Pilot Study for the Detection of Cyclic Prolyl-Hydroxyproline (Pro-Hyp) in Human Blood after Ingestion of Collagen Hydrolysate.

Levels of short linear hydroxyproline (Hyp)-containing peptides, such as prolyl-hydroxyproline (Pro-Hyp), increase in human blood after the ingestion of collagen hydrolysate, which has been associated with beneficial effects for human skin and joints. The present study demonstrates the presence of a novel food-derived collagen peptide, cyclic Pro-Hyp, in human blood after the ingestion of collagen hydrolysate. The cyclic Pro-Hyp levels in plasma samples were estimated by liquid chromatography mass spectrometry (LC-MS). Cyclic Pro-Hyp levels significantly increased in the plasma after ingestion of collagen hydrolysate, reaching a maximum level after 2 h and then decreasing. The maximum level of cyclic Pro-Hyp in plasma ranged from 0.1413 to 0.3443 nmol/mL, representing approximately 5% of linear Pro-Hyp in plasma after ingestion of collagen hydrolysate. Addition of cyclic Pro-Hyp in medium at 7 nmol/mL significantly enhanced the growth rate of mouse skin fibroblasts on collagen gel more extensively compared to linear Pro-Hyp.

Limited hydrolysis combined with controlled Maillard-induced glycation does not reduce immunoreactivity of soy protein for all sera tested.

Combining proteolysis and Maillard-induced glycation was investigated to reduce the immunoreactivity of soy protein. Soy protein was hydrolyzed by Alcalase following response surface methodology utilizing three variables, temperature, time, and enzyme:substrate ratio, with the degree of hydrolysis (DH) and percent reduction in immunoreactivity as response variables. Western blots and ELISA were used to evaluate immunoreactivity using human sera. Data were fitted to appropriate models and prediction equations were generated to determine optimal hydrolysis conditions. The hydrolysate produced under optimized conditions was subjected to glycation with dextran. Hydrolysate produced under optimal conditions had 7.8% DH and a percent reduction in immunoreactivity ranging from 20% to 52%, depending on the sera used. Upon glycation, immunoreactivity was further reduced only when using serum that had the highest soy-specific IgE. This work revealed limitations and provided premises for future studies intended to prove the potency of the combined modification approach to produce a hypoallergenic protein ingredient.

Production of bioactive peptide hydrolysates from deer, sheep and pig plasma using plant and fungal protease preparations.

Plasma separated from deer, sheep and pig blood, obtained from abattoirs, was hydrolysed using protease preparations from plant (papain and bromelain) and fungal (FP400 and FPII) sources. Antioxidant and antimicrobial activities of the peptide hydrolysates obtained after 1, 2, 4 and 24h of hydrolysis, were investigated. The release of trichloroacetic acid-soluble peptides over the hydrolysis period was monitored using the o-phthaldialdehyde (OPA) assay, while the hydrolysis profiles were visualised using SDS-PAGE. The major plasma proteins in the animal plasmas were identified using MALDI-TOF-TOF MS. Hydrolysates of plasma generated with fungal proteases exhibited higher DPPH radical-scavenging, oxygen radical-scavenging capacity (ORAC) and ferric reducing antioxidant power (FRAP) than those generated with plant proteases for all three animal plasmas. No antimicrobial activity was detected in the hydrolysates. The results indicated that proteolytic hydrolysis of animal blood plasmas, using fungal protease preparations in particular, produces hydrolysates with high antioxidant properties.

Highly accurate quantification of hydroxyproline-containing peptides in blood using a protease digest of stable isotope-labeled collagen.

Collagen-derived hydroxyproline (Hyp)-containing dipeptides and tripeptides, which are known to possess physiological functions, appear in blood at high concentrations after oral ingestion of gelatin hydrolysate. However, highly accurate and sensitive quantification of the Hyp-containing peptides in blood has been challenging because of the analytical interference from numerous other blood components. We recently developed a stable isotope-labeled collagen named "SI-collagen" that can be used as an internal standard in various types of collagen analyses employing liquid chromatography-mass spectrometry (LC-MS). Here we prepared stable isotope-labeled Hyp-containing peptides from SI-collagen using trypsin/chymotrypsin and plasma proteases by mimicking the protein degradation pathways in the body. With the protease digest of SI-collagen used as an internal standard mixture, we achieved highly accurate simultaneous quantification of Hyp and 13 Hyp-containing peptides in human blood by LC-MS. The area under the plasma concentration-time curve of Hyp-containing peptides ranged from 0.663 ± 0.022 nmol/mL·h for Pro-Hyp-Gly to 163 ± 1 nmol/mL·h for Pro-Hyp after oral ingestion of 25 g of fish gelatin hydrolysate, and the coefficient of variation of three separate measurements was <7% for each peptide except for Glu-Hyp-Gly, which was near the detection limit. Our method is useful for absorption/metabolism studies of the Hyp-containing peptides and development of functionally characterized gelatin hydrolysate.

Mast cell activation syndrome as a significant comorbidity in sickle cell disease.

Some sickle cell anemia (SCA) patients suffer significantly worse phenotypes than others. Causes of such disparities are incompletely understood. Comorbid chronic inflammation likely is a factor. Recently, mast cell (MC) activation (creating an inflammatory state) was found to be a significant factor in sickle pathobiology and pain in a murine SCA model. Also, a new realm of relatively noncytoproliferative MC disease termed MC activation syndrome (MCAS) has been identified recently. MCAS has not previously been described in SCA. Some SCA patients experience pain patterns and other morbidities more congruent with MCAS than traditional SCA pathobiology (eg, vasoocclusion). Presented here are 32 poor-phenotype SCA patients who met MCAS diagnostic criteria; all improved with MCAS-targeted therapy. As hydroxyurea benefits some MCAS patients (particularly SCA-like pain), its benefit in SCA may be partly attributable to treatment of unrecognized MCAS. Further study will better characterize MCAS in SCA and identify optimal therapy.

Intragastric infusion of pea-protein hydrolysate reduces test-meal size in rats more than pea protein.

Because protein hydrolysates are digested faster than the corresponding proteins, they may increase or hasten the acute eating-inhibitory effect of protein. Potential mediating mechanisms include accelerated or greater release of satiating gut peptides and activation of metabolic signals that inhibit eating. We tested these hypotheses in adult male rats that were surgically equipped with intragastric (IG) cannulas and adapted to 30-min test meals at dark onset after 14-h food deprivation. Equiosmotic 12 ml loads of saline-urea control (C), 13.6% pea protein (PP), or 13.6% PP hydrolysate (PPH, DSM/DFS, Delft, The Netherlands) solutions were IG infused in 1 min just before test meals. PPH reduced test-meal size compared to C more than PP (-3.8±0.3 g vs. -2.6±0.4 g; P<0.0001). Plasma glutamate increased more after PPH than PP (P<0.0001). Plasma lactate, alanine, insulin, glucagon, GLP-1 and paracetemol (an index of gastric emptying) all increased similarly, and glucose decreased similarly, after PPH or PP. Finally, PPH still reduced test-meal size more than PP (-4.6±0.3 g vs. -3.1±0.4 g; P<0.001) in rats after subdiaphragmatic vagal deafferentation, indicating that abdominal vagal afferents are not necessary for the eating-inhibitory effects of PP and PPH and, by extension, that gut peptides whose satiating effects depend on intact vagal afferents (e.g., CCK and glucagon) do not play crucial roles. Thus, PPH reduced short-term food intake more than PP under our conditions, but the mechanism(s) involved remain unclear.

Absorption and effectiveness of orally administered low molecular weight collagen hydrolysate in rats.

Collagen, a major extracellular matrix macromolecule, is widely used for biomedical purposes. We investigated the absorption mechanism of low molecular weight collagen hydrolysate (LMW-CH) and its effects on osteoporosis in rats. When administered to Wistar rats with either [(14)C]proline (Pro group) or glycyl-[(14)C]prolyl-hydroxyproline (CTp group), LMW-CH rapidly increased plasma radioactivity. LMW-CH was absorbed into the blood of Wistar rats in the peptide form. Glycyl-prolyl-hydroxyproline tripeptide remained in the plasma and accumulated in the kidney. In both groups, radioactivity was retained at a high level in the skin until 14 days after administration. Additionally, the administration of LMW-CH to ovariectomized stroke-prone spontaneously hypertensive rats increased the organic substance content and decreased the water content of the left femur. Our findings show that LMW-CH exerts a beneficial effect on osteoporosis by increasing the organic substance content of bone.

Soy protein isolate and its hydrolysate reduce body fat of dietary obese rats and genetically obese mice (yellow KK)

This study examined in dietary obese and genetically obese rodents the effects of soy protein isolate (SPI) and its hydrolysate (SPI-H) on the rate of body-fat disappearance. Male Sprague-Dawley rats (4-16 wk old) and yellow KK mice (6-10 wk old) were made obese by feeding high-fat diets containing 30% fat. They were then fed energy-restricted, low-fat (5.0%), and high-protein (35% casein, SPI, or SPI-H) diets for 4 wk at 60% of the level of the energy intake of rodents on laboratory chow. The body-fat contents of rats and mice fed a high-fat diet were 27.3 and 33.6 g/100 g body weight, respectively, at the end of the obese period. For rats, the apparent absorbability of dietary energy and fat was significantly lower in the SPI and SPI-H groups than in the casein group, but vice-versa for nitrogen balance. Body-fat content in mice fed SPI and SPI-H diets was significantly lower than in those fed the casein diet. In rats, plasma total cholesterol level was lower with the SPI-H diet, and plasma glucose level was lower with the SPI and SPI-H diets than with the casein diet. These results indicate that SPI and SPI-H are suitable protein sources in energy-restricted diets for the treatment of obesity.