Hemoglobin Hydrolyzate Promotes Iron Absorption in the Small Intestine through Iron Binding Peptides.

Hemoglobin is an excellent source of iron supplements, and its hydrolyzate spontaneously binds iron during digestion and promotes iron absorption in vivo. However, the underlying mechanisms of what peptides bind and how they bind iron ions remain unclear. This study prepared the porcine hemoglobin hydrolyzate through enzymatic hydrolysis and acid treatment and investigated the mechanisms of hemoglobin hydrolyzate on iron absorption through the determination of iron levels in dietary intervention mice, iron binding site analyses, peptide digestion analyses, molecular simulation docking, and INT407 cell validation. The results showed that ingestion of the hemoglobin hydrolyzate diets increased iron levels in the blood of mice, accompanied by the upregulation of duodenal iron circulation-related genes such as ferritin, PCBP1, and HP. Carboxyl, imidazole groups, and aromatic amino acid residues were iron binding sites of hemoglobin hydrolyzate during digestion. VDEVGGEA and VDEVGGE were found to involve the spontaneous and efficient binding of hemoglobin hydrolyzate to iron ions in the intestinal cavity. In particular, the DEVGGE peptide was the typical sequence for hemoglobin hydrolytic peptides to exert iron binding activity.

The proteomic evidence on protein oxidation in pea protein concentrate-based low-moisture extrudates and its inhibition by antioxidants derived from plant extracts.

This study aimed to assess the antioxidant activity of golden chlorella (GoC) and grape pomace (GrP) extracts both in vitro and in pea protein-based extrudates. We hypothesized that GoC/GrP would limit oxidation of proteins in the extrudates compared with commercial antioxidants. The results showed that GoC extract was effective in metal chelation and GrP extract possessed excellent radical scavenging activity and reducing power. Protein oxidation inevitably occurred after low-moisture extrusion in terms of elevated level of protein carbonyls and the gradual loss of thiols. LC-MS/MS revealed that the monoxidation and 4-hydroxynonenal adduction were the major oxidative modifications, and legumin was the most susceptible globulin for oxidation. The GoC/GrP extracts effectively retarded the oxidation progress in extrudates by lower intensity of oxidized peptides, whereas protein electrophoretic profiles remained unaffected. This study highlighted the great potential of GoC/GrP as natural antioxidants in plant-based foods.

Generation of kokumi γ-glutamyl short peptides in Spanish dry-cured ham during its processing.

The typical dry-cured ham flavor is rich in umami and brothy perceptions, for which short peptides may contribute. Particularly, γ-glutamyl peptides could be the responsible of these previously reported attributes, as they exert a synergistic interaction with other basic tastes and modify the intensity of salty, sweet, and umami tastes. The content of peptides has been reported to evolve along the processing, but no kokumi γ-glutamyl peptides have been identified in Spanish dry-cured hams yet. In this research, nine γ-glutamyl dipeptides (γ-EA, γ-EC, γ-EE, γ-EF, γ-EL, γ-EM, γ-EV, γ-EW, and γ-EY) and two γ-glutamyl tripeptides (GSH and γ-EVG) have been quantitated at 6, 12, 18 and 24 months of traditional processing of Spanish dry-cured ham by performing a Q Exactive Orbitrap-based tandem mass spectrometry. The results show an increase of γ-EA, γ-EE, γ-EF, γ-EL, γ-EM and γ-EVG, obtaining maximums at 24 months of curing ranging from 0.14 (γ-EVG) to 18.86 (γ-EL) µg/g dry-cured ham. Otherwise, γ-EV, γ-EW and γ-EY accumulated until the 18th month of storage to 15.10, 0.54 and 3.17 µg/g dry-cured ham, respectively; whereas γ-EC and GSH amounts decreased starting from 0.0676 and 4.41 µg/g dry-cured ham, respectively at earlier stages. The concentration dynamics of these compounds may be linked with proteolytic and oxidative reactions during processing. In addition, due to their synergistic effect on kokumi activity, this could constitute insights of the brothy perceptions of dry-cured ham, and these peptides probably contribute to the sensory differences existing in long processed Spanish dry-cured hams.

Decolorization of porcine hemoglobin hydrolysates: The role of peptide characteristics and pH values.

The unpleasant color caused by heme limits the utilization of hemoglobin as a food ingredient. Enzymatic hydrolysis has been used to decolorize hemoglobin, but the underlying mechanisms are poorly understood. The aim of this study was to investigate the decolorization efficiency of porcine hemoglobin using different enzymes and final pH values, and to elucidate their influence on decolorization. Based on higher yields and better decolorization, hemoglobin hydrolysates produced by papain, bromelain, savinase, and protease A were further studied. Compared to hydrolysates by savinase and protease A, a higher proportion of histidine-containing peptides was responsible for better decolorization by papain and bromelain. For all hydrolysates, a moderate reduction in pH to 4.0-5.0 facilitated decolorization of the hydrolysates. Similar peptide profiles of hydrolysates from the same enzyme treatment reflected that pH mainly affected the precipitation of the heme-containing fraction through heme-heme interaction rather than heme-peptide interaction. Overall, this study sheds light on the use of enzymatic hydrolysis to remove the heme group from hemoglobin. PRACTICAL APPLICATION: Slaughterhouses produce tons of protein-rich blood each year. Due to the presence of the heme group in hemoglobin, blood has a dark red color and metallic taste, making it generally unacceptable for consumers. This study provided information on the decolorization of porcine hemoglobin by removing the heme fraction, which should facilitate the utilization of decolored hemoglobin hydrolysates as nutritional food ingredients.

Physicochemical properties, texture, and in vitro protein digestibility in high-moisture extrudate with different oil/water ratio.

Oil addition is challenging during high-moisture extrusion due to the negative fiber formation effects. A previous study found that oil-in-water (O/W) emulsions could significantly increase the oil content in high-moisture extrudates, but the molecular mechanism remained unclear. This study aimed to determine O/W emulsion influence on protein physicochemical properties in SPI extrudates during high-moisture extrusion. O/W emulsions were mixed with soy protein isolates (SPI) to prepare extrudates with oil/water ratios of 0/65, 4/61, and 8/57 (w/w). SDS-PAGE and ATR-FTIR analysis showed that higher oil/water ratios enhanced protein aggregation and promoted alteration from ß-sheet to random coil in SPI extrudates, which could be correlated to the reduction of protein solubility. The color was altered to lighter and yellow, and hardness, chewiness, and fiber degree decreased with increased oil/water ratios in SPI extrudates. In addition, in vitro digestion analyses showed that higher oil content contributed to improved protein digestibility.

Protein phosphorylation profile of Atlantic cod (Gadus morhua) in response to pre-slaughter pumping stress and postmortem time.

This study aimed to evaluate the effect of pumping stress (pumping and pumping-resting) and postmortem time (before and after rigor mortis) on phosphorylation profiles of myofibrillar protein (MP) and sarcoplasmic protein (SP) of Atlantic cod (Gadus morhua) fillets. The result showed that MP had higher global phosphorylation levels than SP regardless of stress condition and postmortem time. The pumping process resulted in significant changes in phosphorylation of structural proteins including myosin heavy and light chains. Pumping also affected the phosphorylation status of heat shock proteins and metabolic enzymes involved in the glycolytic pathways, indicating the possible role of phosphorylation in regulating energy hemostasis of fish under stressful conditions. The pumping-induced phosphorylation changes mainly occurred before rigor mortis, and postmortem time affected the phosphorylation status to a less extent. This work contributes to a deeper understanding on protein phosphorylation affected by pre-slaughter stress and postmortem time of fish.

Increase of Kokumi γ-Glutamyl Peptides in Porcine Hemoglobin Hydrolysate Using Bacterial γ-Glutamyltransferase.

The kokumi sensation of protein hydrolysates could be enhanced by γ-glutamylation through forming a series of γ-glutamyl di- and tri-peptides. In this study, porcine hemoglobin hydrolysate was γ-glutamylated using enzymes from Bacillus amyloliquefaciens (Ba) or Bacillus licheniformis (Bl), which are sold as glutaminases but identified as γ-glutamyltransferases (GGTs). To yield more γ-glutamyl peptides, reaction conditions were optimized in terms of GGT source (BaGGT and BlGGT), substrate concentration (10, 20, and 40%), reaction time (3, 6, 12, and 24 h), and glutamine supplementation (20, 40, and 80 mM). Results showed that both the GGTs had the highest transpeptidase activity at similar pH values but different temperatures. In addition, BaGGT had stronger catalytic ability to form γ-glutamyl dipeptides, while BlGGT was more capable to generate γ-Glu-Val-Gly. Adding glutamine was more efficient to obtain more target peptides than adjusting the hydrolysate concentration and reaction time. This study contributes to the valorization of animal side streams.

Structural characteristics of high-moisture extrudates with oil-in-water emulsions.

There is an increasing demand to produce high-quality plant-based meat analogs rich in tenderness and juiciness, presenting a significant challenge in creating oil-containing fibrous structures. A novel oil addition approach was developed by adding oil-in-water (O/W) emulsion during high-moisture extrusion processing. The current study investigated the effect of oil content using O/W emulsion on high-moisture extrudates prepared from soy protein isolate (SPI) and wheat gluten (WG) (SPI-WG). The oil content in fibrous SPI-WG extrudate could be up to 8.0% using O/W emulsion, whereas only 4.0% was possible by direct oil addition. O/W emulsion addition significantly decreased the extrusion response parameters of die pressure and specific mechanical energy. Confocal laser scanning microscopy indicated that oil was distributed to small droplets (0.5-15.0 µm) within the protein matrix in SPI-WG extrudates. Oil-free SPI-WG extrudate presented a close-meshed protein network. In contrast, higher oil contents led to more porous structures in SPI-WG extrudates with 3.0-8.0% oil. O/W emulsion addition reduced the rubber-like texture and rheological properties of SPI-WG extrudates, which improved the textural attributes (e.g., chewiness), making them similar to those of cooked chicken breast. In addition, the mobility of water and oil protons increased with increasing oil contents in SPI-WG extrudates, indicating the water and oil binding properties were reduced. Overall, this study demonstrated the potential of using O/W emulsions to promote fibrous structures of high-moisture extrudates.

Current progress in kokumi-active peptides, evaluation and preparation methods: a review.

Kokumi is a complex sensation characterized by thickness, mouthfulness and continuity. Kokumi-active peptides, which are distributed in many kinds of food, induce a rich and long-lasting mouthfeel of food. Aimed to provide a comprehensive overview of kokumi peptides, this review covers the aspects of preparation and evaluation methods for kokumi peptides, kokumi receptor calcium-sensing receptor (CaSR), as well as structural features of kokumi peptides and derivatives. Apart from extraction and separation from natural and fermented food, preparation of kokumi peptides can be effectively obtained from enzymatic generation. Kokumi peptides are perceived by CaSR in taste cells and the proposed transduction pathway has been described. The evaluation on kokumi-inducing effect of peptides has employed a combination of sensory assessment and CaSR method. The discovered kokumi peptides mainly comprise glutamyl peptides, leucyl peptides and other peptides without specific features. Derivatives of amino acids and peptides including sulphur-containing amino acids, N-acyl-Tyr derivatives, N-acetylated amino acids and Maillard reaction products (MRPs) also work as kokumi enhancers. Based on the summarized developments, great sensory properties and bioactivities enable kokumi peptides as promising protein ingredients in future application.

Transglutaminase-Mediated Caseinate Oligochitosan Glycation Enhances the Effect of Caseinate Hydrolysate to Ameliorate the LPS-Induced Damage on the Intestinal Barrier Function in IEC-6 Cells.

Some food components can regulate the intestinal barrier function. Herein, the effect of transglutaminase-type oligochitosan glycation on caseinate hydrolysate for its ability to maintain intestinal epithelial integrity and the tight junction (TJ) structure was investigated by assessing and comparing the bioactivities of glycated caseinate hydrolysate and caseinate hydrolysate against the lipopolysaccharide-induced barrier damage in the model cells (rat intestinal epithelial IEC-6 cells). The results from liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis demonstrated that oligochitosan glycation occurred at the Gln residues of α-S1-casein and α-S2-casein. The two hydrolysates retarded the lipopolysaccharide cytotoxicity toward IEC-6 cells and enhanced the barrier integrity by increasing the transepithelial electrical resistance or decreasing the paracellular permeability. In addition, these two hydrolysates could upregulate both mRNA and protein expression of three TJ proteins in IEC-6 cells. More importantly, the glycated caseinate hydrolysate had higher potential than caseinate hydrolysate to protect IEC-6 cells against the lipopolysaccharide-induced barrier damage, suggesting that the transglutaminase-mediated oligochitosan glycation of proteins is a useful approach to enforce protein biofunctions in the intestine.

Proteolytic activity of selected commercial Lactobacillus helveticus strains on soy protein isolates.

Soy protein isolates were fermented by three commercial Lactobacillus helveticus strains for a maximum of seven days to investigate the resulting proteolysis. The proteolytic activity of the most active strain (LH88) was further analyzed (LC-MS/MS and GC-MS) and it was shown that the ß-conglycinin α subunit 1, ß-conglycinin α' subunit, glycinin G1, and 2S albumin were specifically degraded. Peptigram analysis and visualization of the crystal structure showed that the hydrolysis sites of ß-conglycinin α subunit, α' subunit, and the glycinin G1 were located on the surface of the molecule or at the mobile disordered region, hence being highly accessible for the proteinase of LH88. The proteins were partially further degraded to free amino acids, and subsequently catabolized to volatile compounds. However, most of the proteins remained native, even after seven days of fermentation, thus additional modification of protein structure or adjustment of fermentation conditions are required for effective generation of flavor compounds.

Plastein from hydrolysates of porcine hemoglobin and meat using Alcalase and papain.

Plastein is defined as a protease-induced peptide aggregate and has been explored for over a century. This study investigated the effects of Alcalase and papain on plastein formation in protein hydrolysates of porcine hemoglobin and meat by measuring turbidity, particle size distribution, free amino groups and chemical interactions, as well as identifying the soluble peptides remaining in solution by LC-MS/MS. The results showed that Alcalase induced more peptide aggregation than papain in terms of increases in turbidity and particle size. Porcine hemoglobin was better than meat in inducing plastein formation in a short reaction time. Besides, covalent bonds involving peptide bonds and disulfide bonds were not crucial in the plastein reaction, instead a high proportion of hydrophobic interactions dominated the plastein. Not all peptides of both hydrolysates took part in plastein formation, and the regions of sequence that were prone to aggregation were visualized by Peptigram.

Quantitation of Protein Cysteine-Phenol Adducts in Minced Beef Containing 4-Methyl Catechol.

Thiol groups of cysteine (Cys) residues in proteins react with quinones, oxidation products of polyphenols, to form protein-polyphenol adducts. The aim of the present work was to quantify the amount of adduct formed between Cys residues and 4-methylcatechol (4MC) in minced beef. A Cys-4MC adduct standard was electrochemically synthesized and characterized by liquid chromatography-mass spectrometry (LC-MS) as well as NMR spectroscopy. Cys-4MC adducts were quantified after acidic hydrolysis of myofibrillar protein isolates (MPIs) and LC-MS/MS analysis of meat containing either 500 or 1500 ppm 4MC and stored at 4 °C for 7 days under a nitrogen or oxygen atmosphere. The concentrations of Cys-4MC were found to be 2.2 ± 0.3 nmol/mg MPI and 8.1 ± 0.9 nmol/mg MPI in meat containing 500 and 1500 ppm 4MC, respectively, and stored for 7 days under oxygen. The formation of the Cys-4MC adduct resulted in protein thiol loss, and ca. 62% of the thiol loss was estimated to account for the formation of the Cys-4MC adduct for meat containing 1500 ppm 4MC. Furthermore, protein polymerization increased in samples containing 4MC as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the polymerization was found to originate from protein-polyphenol interactions as evaluated by a blotting assay with staining by nitroblue tetrazolium.

Exopeptidase treatment combined with Maillard reaction modification of protein hydrolysates derived from porcine muscle and plasma: Structure-taste relationship.

Reduction of bitter taste in protein hydrolysates is a challenging task. The aim of this study was to apply a simple two-step approach to prepare low bitter hydrolysates and investigate the influence of peptide modifications on taste characteristics. Protein hydrolysates were prepared from porcine muscle and plasma through simultaneous hydrolysis using endo- and exo-peptidases combined with peptide glycation by glucosamine (GlcN). Spectroscopic analysis and quantification of major alpha-dicarbonyl compounds (α-DCs) indicated the relatively low extent of Maillard reaction in GlcN-glycated protein hydrolysates. Thermal degradation of high MW peptides (>10 kDa) might play a major role in Maillard reaction, reflected by the formation of more Maillard reacted peptides (1-5 kDa), especially in plasma samples. Sensory evaluation indicated that glycation by GlcN can alter taste profiles of protein hydrolysates, which may be attributed to the formation of Maillard reacted peptides and peptide modifications revealed by LC-MS/MS analysis.

Carprofen-induced depletion of proton motive force reverses TetK-mediated doxycycline resistance in methicillin-resistant Staphylococcus pseudintermedius.

We previously showed that doxycycline (DOX) and carprofen (CPF), a veterinary non-steroidal anti-inflammatory drug, have synergistic antimicrobial activity against methicillin-resistant Staphylococcus pseudintermedius (MRSP) carrying the tetracycline resistance determinant TetK. To elucidate the molecular mechanism of this synergy, we investigated the effects of the two drugs, individually and in combination, using a comprehensive approach including RNA sequencing, two-dimensional differential in-gel electrophoresis, macromolecule biosynthesis assays and fluorescence spectroscopy. Exposure of TetK-positive MRSP to CPF alone resulted in upregulation of pathways that generate ATP and NADH, and promote the proton gradient. We showed that CPF is a proton carrier that dissipates the electrochemical potential of the membrane. In the presence of both CPF and DOX, the energy compensation strategy was attenuated by downregulation of all the processes involved, such as citric acid cycle, oxidative phosphorylation and ATP-providing arginine deiminase pathway. Furthermore, protein biosynthesis inhibition increased from 20% under DOX exposure alone to 75% upon simultaneous exposure to CPF. We conclude that synergistic interaction of the drugs restores DOX susceptibility in MRSP by compromising proton-motive-force-dependent TetK-mediated efflux of the antibiotic. MRSP is unable to counterbalance CPF-mediated PMF depletion by cellular metabolic adaptations, resulting in intracellular accumulation of DOX and inhibition of protein biosynthesis.

Exploration of collagen recovered from animal by-products as a precursor of bioactive peptides: Successes and challenges.

A large amount of food-grade animal by-products is annually produced during industrial processing and they are normally utilized as animal feed or other low-value purposes. These by-products are good sources of valuable proteins, including collagen or gelatin. The revalorization of collagen may lead to development of a high benefit-to-cost ratio. In this review, the major approaches for generation of collagen peptides with a wide variety of bioactivities were summarized, including antihypertensive, antioxidant and antidiabetic activities, and beneficial effects on bone, joint and skin health. The biological potentials of collagen peptides and their bioavailability were reviewed. Moreover, the unique advantages of collagen peptides over other therapeutic peptides were highlighted. In addition, the current challenges for development of collagen peptides as functional food ingredients were also discussed. This article discusses the opportunity to utilize collagen peptides as high value-added bio-functional ingredients in the food industry.

Investigation of nitrite alternatives for the color stabilization of heme-iron hydrolysates.

This study investigates the potential of novel heme-ligand complexes, derived from heme-iron isolated from porcine hemoglobin by enzymatic hydrolysis, to use as pigments for meat products. Five alternatives to sodium nitrite were identified as possible heme ligands and stabilizing agents of the red conformation of heme. The effects of 4-methylimidazole, methyl nicotinate, pyrrolidine, piperidine, pyrazine and sodium nitrite (as comparative benchmark) on the color of heme-iron extract and pure hemin standard were studied in solution. The ligand affinity and heme-ligand stability was assessed over time in solution by UV-Vis absorbance spectroscopy and CIELAB color space parameters. The CIE redness score a* was used as a single measurement to propose a predictive model based on the following parameters: heme source (heme-iron extract or hemin standard), heme-to-ligand molar ratio (1:20 to 1:300), and storage time (up to 32 days). The optimal concentration at which each ligand can be added to either heme source, as well as the stability of the red color of the formed heme-ligand complexes in-solution was determined. Heme-iron extract-derived samples showed increased redness and color stability as compared to their hemin counterparts. No ligand showed as much affinity for heme as sodium nitrite. As the most promising ligand candidates, methyl nicotinate and 4-methylimidazole started to show color changes at a 1:50 molar ratio, but higher amounts (1:100 and 1:300, respectively) were required to attain the maximum redness possible with the highest stability.

Advanced glycation end products, protein crosslinks and post translational modifications in pork subjected to different heat treatments.

The aim of the study was to characterize Maillard reactions in meat under different cooking treatments. Considered temperature-time combinations included raw samples (control), 58, 80, 98 and 160 °C for 72 min, 118 °C for 8 min and 58 °C for 17 h. Furosine, a marker for heat treatment, was detected in all groups with roasting having a 4-fold increase over the control. Sous-vide treatment at 80 °C, boiling and autoclaving also contribute to a significant increase in furosine. Nɛ-carboxymethyllysine, an indicator for advanced glycation end products, showed negligible amount in control, but increased with cooking temperature, with oven samples showing the highest values. A similar increasing trend was observed in lanthionine, covalently bonded protein crosslinks, which arises due to severe thermal regimes. Simultaneously, glycation and deamidation formation were tracked in meat proteins through peptidomics to highlight residue level changes that might affect nutrient value in processed muscle based foods.

Structural characteristics of low bitter and high umami protein hydrolysates prepared from bovine muscle and porcine plasma.

The aim of this study was to use different enzyme mixtures to investigate the influence of peptide characteristics and taste of protein hydrolysates from bovine muscle and porcine plasma. Minced beef and porcine plasma were hydrolysed using 10 food-grade enzymes, including Protease A, Protease P, ProteAX, Flavourzyme, Alcalase, Papain, Bromelain, Protamex, Neutrase and Sumizyme BNP-L. The relationship between degree of hydrolysis (DH), molecular weight (MW) distribution, enzyme specificity, and sensory characteristics of hydrolysates were investigated. The results demonstrated that Protease A, a mixture of endo- and exo-peptidases, was the optimal protease to generate hydrolysates with low bitterness. Endopeptidases (Papain and Bromelain) elicited bitter taste of protein hydrolysates. A positive correlation was suggested between umami taste and MW distribution (<0.5 kDa), while bitterness was positively correlated with MW distribution (0.5-1 kDa). Overall, hydrolysis with enzyme preparations containing endo- and exo-peptidases was effective to reduce bitterness of hydrolysates.

Proteomic profiling of oxidized cysteine and methionine residues by hydroxyl radicals in myosin of pork.

In order to investigate the effects of hydroxyl radicals on the myosin of pork, with focus on reducible and non-reducible oxidation of specific cysteine and methionine residues, extracted myofibrillar protein from longissimus dorsi of pork was incubated with H2O2 for 24, 48 and 72h, respectively. The thiol contents and crosslinking of myofibrillar protein were analyzed after oxidation of the protein. Moreover, cysteine (labeled with N-ethylmaleimide (NEM) and iodoacetamide (IAM) before and after reduction, respectively) and methionine oxidation were detected by LC/MS using label-free quantitation. The result revealed that cysteine at head of myosin tended to form sulfinic and sulfonic acid, while the cysteine at coiled tail of myosin easily generated disulfide under same condition. Furthermore, it was also revealed that the methionine at the coiled tail of myosin was more easily oxidized than that of the head.