Tracking bioactive peptides and their origin proteins during the in vitro digestion of meat and meat products.

Existing reviews address bioactive peptides of meat proteins; however, comprehensive reviews summarizing the released sequences and their corresponding parent meat proteins in the digesta are limited. This review explores the bioactive peptides released during the in vitro gastrointestinal (GI) digestion of meat, connecting with parent proteins. The primary bioactivities of meat-derived peptides include angiotensin-converting enzyme (ACE) and dipeptidyl peptidase (DPP)-IV inhibition and antioxidant effects. Myofibrillar, sarcoplasmic, and stromal proteins play a significant role in peptide release during digestion. The release of bioactive peptides varies according to the parent protein and cryptides had short chains, non-toxicity, and great bioavailability and GI absorption scores. Moreover, the structural stability and bioactivities of peptides can be influenced by the digestive properties and amino acid composition of parent proteins. Investigating the properties and origins of bioactive peptides provides insights for enhancing the nutritional quality of meat and understanding its potential health benefits.

Effect of varying pH on solution interactions of soluble meat proteins with different plant proteins.

The exchange of animal-based for plant-based proteins is becoming more and more popular due to an increasing demand for alternative and more sustainable protein sources. In this study, solubilized water- (ws) or salt-and-water (sws) meat proteins were evaluated in their pH-dependent interactions with soluble protein fractions from wheat, pumpkin, sunflower, rapeseed, or potato proteins. For this purpose, 1 : 1 (v/v) mixtures of 1.0 wt% meat (ws or sws) and plant proteins were prepared at a sodium chloride concentration of 1.8 wt% (ionic strength: 0.31 mol L-1) and adjusted to different pH-values in between 4.5-7.0. While only slight differences were found upon comparison of interactions of ws and sws batches (p > 0.05), interactions among these animal-based and soluble plant proteins took place. First, optical observations, light microscopy, and SDS-PAGE revealed increasing protein solubility with increasing pH. Second, particle size distributions (PSDs) revealed a shift towards slightly larger particle sizes e.g. at pH 5.3 and 7.0 with d4,3 of 43.2 and 21.3 µm (sws) to 45.4 and 23.9 µm (sws + potato), respectively. Furthermore, heat-induced gel formation was improved at pH > 6.0, in particular in mixtures of meat and wheat or rapeseed proteins that formed a homogenous gel structure. Based on the obtained results, protein-protein complexations mainly by electrostatic forces are suggested which occur due to various pI of meat and plant proteins e.g. pH 7.5 (wheat), 7.2 (potato), and 6.6 (rapeseed) in comparison to 5.1 (ws) and 5.6 (sws). The filamentous microstructure of some gels (soluble fraction of rapeseed, potato and wheat proteins) led to the assumption that meat proteins, mainly at pH values greater than 5.8 (optimally ≥6.5), had a structuring effect on plant proteins.

Effect of combined treatment of L-arginine and transglutaminase on the gelation behavior of freeze-damaged myofibrillar protein.

This research focused on the effects of L-arginine (Arg, 5 mM), transglutaminase (TG, E : S = 1 : 500), and the combination (Arg + TG) on the physicochemical properties and heat-induced gel performance of freeze-damaged myofibrillar protein (MP). The incorporation of Arg decreased the α-helix percentage (48.4%) and the mean particle size of freeze-damaged MP, as well as cooking loss (46.5%) and the overall textural characteristics of MP gels. The addition of TG reduced the α-helix content by 10.7% but significantly enhanced the crosslinking and heat-induced gel behavior of freeze-damaged MP, resulting in a slight reduction of cooking loss (17.7%) and the most ideal textural properties of MP gels. Although the presence of Arg remarkably suppressed the heat-induced development of storage modulus (G') and reduced the hardness of MP gels (by 13.4%), the combination (Arg + TG) showed the lower cooking loss and the improved textural characteristics, with the set gel displaying the most delicate and compact microstructure. These findings indicated that the combination of Arg and TG could be a potential strategy to enhance the gelling performance of freeze-damaged meat proteins.

Proteolysis and protein oxidation throughout the smoked dry-cured ham process.

During the five stages of smoked dry-cured ham processing, proteolysis and protein oxidation were simultaneously detected in the Biceps femoris (BF) and Semimembranosus (SM) muscles. Proteolysis was more advanced in BF than in SM throughout the process of production. The total FAA increased significantly (p < 0.05) throughout the processing, resulting in higher total FAA content in BF than in SM muscle. SDS-PAGE revealed progressive degradation of sarcoplasmic proteins of investigated muscles, with the pronounced changes for the 69.9-41.7 kDa region. SDS-PAGE of BF showed more intense degradation of myofibrillar proteins due to greater proteolysis in BF. Electrophoresis of myofibrillar proteins evidenced the marked degradation of 130 kDa, 96.7 kDa and 27-20.7 kDa bands in both muscles. A similar trend was observed for protein oxidation in BF and SM, with the final values of 26.36 and 23.7 nmol carbonyls/mg proteins, respectively. The Pearson correlation revealed a strong relationship between protein oxidation and proteolysis.

Bioactivities generated from meat proteins by enzymatic hydrolysis and the Maillard reaction.

Bioactive peptides are released from meat proteins by enzymatic hydrolysis (i.e., gastrointestinal digestion, aging/storage, fermentation, and protease treatment). Such peptides attribute physiological functions to meat and meat products and are promising food ingredients for developing functional foods. Meat by-products (e.g., blood and collagen) are also good sources for generating bioactive peptides, since they are produced in large quantities and are rich in proteins. Although protein-derived bioactive peptides are attractive ingredients, their changes by the Maillard reaction during processing, cooking, and storage should be investigated. This article briefly reviews the production of bioactive peptides from meat and meat by-products. Such diverse peptides affects circulatory, nervous, alimentary, and immune systems. Then, the bioactivities of Maillard reaction products (MRPs) generated from protein hydrolysates are discussed. Special attention is paid to bioactivities of 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) inhalation. As such activities, we have evaluated the impact of DMHF on blood pressure, moods, brainwaves, and dietary intake. Our efforts for understanding various aspects and implication of peptides and MRPs from meat proteins would open new avenues in the meat and food industry.

Synergistic recovery and enhancement of gelling properties of oxidatively damaged myofibrillar protein by l-lysine and transglutaminase.

The influence of combined Lysine (Lys) and transglutaminase (TG) on the conformation and gelling properties of oxidatively damaged myofibrillar protein (MP) was investigated. The addition of Lys (5 mM) significantly increased the α-helix content (by 47.8%) and decreased the particle size of oxidatively damaged MP, and improved the cooking yield (by 16.8%) and the breaking strength of MP gels (by 65.5%). The treatment with TG (E:S = 1:500) led to a slightly reduced α-helix content but improved breaking strength (by 41.8%) and cooking loss (by 13.3%) of the gels. Their combination (Lys + TG) showed the greatest and synergistic overall improvement, with the set gel displaying a fine, smooth and compact network structure. Notably, the gelling ability of oxidatively damaged MP upon Lys + TG treatment was significantly stronger than that of non-oxidized MP far exceeding its recovery. Therefore, significantly enhanced gelling properties of oxidatively damaged MP can be attained through the combination Lys and TG.

Biochemical properties of extracellular protease from Staphylococcus carnosus RT6 isolated from Harbin dry sausages, and its hydrolysis of meat proteins.

The characteristics of the extracellular protease, produced by Staphylococcus carnosus RT6 isolated from Harbin dry sausages, and its hydrolysis of meat proteins were investigated. The protease was purified by ammonium sulfate, ion exchange, and gel filtration chromatography to obtain a 20.0 kDa extracellular protease. The protease reached maximal activity at pH 9.0 and 50 °C and was stable at pH 7.0 to 11.0 and 20 to 40 °C. Its protease activity was easily inhibited in the presence of Zn2+ , Fe2+ , and Fe3+ . The enzymatic characterization of the protease revealed a Vmax 49.50 U/ml·min, Km 8.19 mg/ml, and the half-life = 28.06 min, ΔH* d  = 114.11 kJ/mol, ΔG* d  = 89.24 kJ/mol, and ΔS* d  = 77.00 J/mol·K at 50 °C. In addition, the protease hydrolyzed meat protein into small particles and produced soluble peptides. This study provides a basis for understanding the biochemical characteristics of the S. carnosus RT6 protease and its future application for fermented meat products.

Dual role (promotion and inhibition) of transglutaminase in mediating myofibrillar protein gelation under malondialdehyde-induced oxidative stress.

This study investigated the effects of transglutaminase (TGase) on the properties of myofibrillar protein (MP) and its heat-induced gels under malondialdehyde (MDA)-induced oxidation. The physicochemical characteristics, protein aggregation and rheological properties of MP were assessed. The gelling behaviours of MP were analysed with measurements of gel strength, cooking loss, microstructure and secondary structure. Under varying degrees of MDA oxidation, the addition of TGase always led to changes in the tertiary structure, loss of free amine and thiol groups, crosslinking of the myosin heavy chain, and decreasing solubility. However, the effect of TGase on MP gel quality differed. At 6 mmol/L MDA, the addition of TGase reduced the quality of MP gels by increasing cooking loss. However, at 12 mmol/L MDA, TGase reduced both the cooking loss and gel strength.

Assessment the influence of salt and polyphosphate on protein oxidation and Nε-(carboxymethyl)lysine and Nε-(carboxyethyl)lysine formation in roasted beef patties.

The impact of NaCl and tripolyphosphate (TPP)/pyrophosphate (PP) on protein oxidation and Nε-(carboxymethyl)lysine (CML) and Nε-(carboxyethyl)lysine (CEL) formation in roasted beef patties was investigated. The content of CML and CEL in patties treated with salts was approximately 1.1-1.7 and 1.2-3.2 times higher than that of the control samples, respectively. An increase in salt content caused higher oxidation of tryptophan and protein carbonylation with a decrease in Schiff bases (P < 0.05) and a slight decrease in lipid oxidation (P < 0.05). Significant correlations (P < 0.05) between CML, CEL, and protein oxidation measurements was found. The higher salts content, causing less cooking loss and higher moisture content, significantly correlated (P < 0.05) with CML, CEL content, and protein oxidation of the patties. The increase in CML and CEL content and protein oxidation in roasted patties with salts might be related to the pro-oxidation of salts, and also partly due to the temperature changes caused by the water-holding capacity of salts.

Effects of temperature and pH on the structure of a metalloprotease from Lactobacillus fermentum R6 isolated from Harbin dry sausages and molecular docking between protease and meat protein.

BACKGROUND: Microbial protease can interact with meat protein in fermented meat products at a certain pH and temperature. To investigate the effects of various pH values and temperatures on the structural characteristics of Lactobacillus fermentum R6 protease, which was isolated from Harbin dry sausages, spectroscopy techniques and molecular dynamics were utilized to evaluate structural changes. RESULTS: The protease exhibited a stable spatial structure at pH 7 and 40 °C, and the extension of the protease structure was also promoted. Although the structure of the protease could be changed or destroyed by pH 8 and 70 °C, it was mainly determined by the changes of secondary and tertiary structures such as α-helix, ß-sheet, ß-turn and random coil. In addition, carbonyl vibration, -NH vibration, C-H stretching vibration and disulphide bonds were present in L. fermentum R6 protease under various pH and temperature conditions. Molecular docking showed that the protease can interact with myosin light chain, myosin heavy chain, actin and myoglobin. CONCLUSION: The protease can maintain stable structure and interact with meat protein, which reflected certain application prospects in the fermentation of Harbin dry sausages. © 2021 Society of Chemical Industry.

Changes in the thermal stability and structure of myofibrillar protein from quick-frozen pork patties with different fat addition under freeze-thaw cycles.

Changes in thermal stability and structure of myofibrillar protein from pork patties with different fat addition (0%, 5%, 10%, 15% and 20%) under freeze-thaw (F-T) cycles were discussed. The results showed that the total sulfhydryl, reactive sulfhydryl, free amino group, α-helix and ß-sheet contents, fluorescence intensity (FI), and protein thermal stability (Tmax, ∆Htotal) of samples with the same fat content were significantly decreased, while the ß-turn and random-coil content and the maximum fluorescence emission wavelength (λmax) were significantly increased with increasing F-T cycles (P < 0.05). These changes in samples with 20% fat at the 5th F-T cycle were obvious and were verified by the decreases in ∆Htotal (26.1%), reactive sulfhydryl (16.1%), and FI (16.8%) compared with the patties without fat. Therefore, repeated F-T cycles could decline the thermal stability of protein, destroy the protein structure of patty, and the changes were positively correlated with fat content of patty.

Structural characteristics and emulsifying properties of myofibrillar protein-dextran conjugates induced by ultrasound Maillard reaction.

In this study, we investigated the effect of the ultrasound-assisted Maillard reaction on the structural and emulsifying properties of myofibrillar protein (MP) and dextran (DX) conjugates with different molecular weights (40, 70 and 150 kDa). Compared with classical heating, mild and moderate ultrasound-assisted methods (100-200 W) could accelerate the later stage of the Maillard reaction, which increased the degree of graft (DG) and the content of advanced Maillard reaction products (MPRs). Structural analysis revealed conjugates obtained by Maillard reaction induced the loss of ordered secondary structures (α-helix, ß-sheets) and red-shift of maximum emission wavelength of intrinsic fluorescence spectrum. The conjugate containing 40 kDa DX exhibited higher extent of Maillard reaction compared to those containing 70 kDa and 150 kDa DX under various treating methods. Moreover, the ultrasound-assisted Maillard reaction could effectively improve the emulsifying behaviors. 100 W ultrasound-induced conjugates grafted by 70 kDa DX produced the smallest emulsion size with optimum storage stability. Confocal laser scanning microscopy and analytical centrifugal analyzer further confirmed MP grafted by 70 kDa DX with the assistance of 100 W ultrasound field could produce the smallest and most homogeneous MP-base emulsion with no flocculation. Our study demonstrated that mild ultrasound treatment resulted in well-controlled Maillard reaction, and the related glycoconjugate grafted with 70 kDa DX showed the greatest improvements in emulsifying ability and stability. These findings provided a theoretical foundation for the development of emulsion-based foods with excellent characteristics.

Inert hydrophilic particles enhance the thermal properties and structural resilience of meat protein gels during heating.

Meat protein gels are present in a variety of foods and are frequently filled with fat particles. This study set out to elucidate the effect of replacing hydrophobic fat-based particles with hydrophilic inert glass particles on thermal and structural properties during heating. Meat protein gels were prepared with different diameters (60 to 90 mm) according to a typical emulsified sausage recipe and fat-based particles as well as inert glass particles were incorporated at concentrations from 10 to 40% and heated to 85 °C, while thermal as well as structural properties were monitored. The results revealed two main effects. First, due to the higher thermal conductivity, the lower heat capacity and the absence of extensive phase transitions, the time to reach the final temperature was reduced with increasing glass particle content (e.g. from 118 ± 2 min with 40% fat particles to 86 ± 1 min for gels with 40% glass particles at a depth of 40 mm). Second, volume change and temperature sweep measurements revealed that glass particles fostered the protein gel formation and enhanced the resilience against structural breakdown during heating. This was evident during small-amplitude shear experiments that showed an almost twofold increase in the storage modulus when 10% fat-based particles were replaced with 10% glass particles from G'85 °C = 223.4 ± 14.8 kPa to G'85 °C = 431.0 ± 16.6 kPa, respectively. Overall, these findings might be of interest to meat-product manufacturers that seek to lower heating times to reach the core temperature necessary for protein denaturation and ensure microbial safety with additional holding times and modify the structural properties of foods while replacing fat particles.

Mimicking myofibrillar protein denaturation in frozen-thawed meat: Effect of pH at high ionic strength.

This study aims at providing new insight on protein denaturation in freezing-thawing. Freezing-thawing minced pork reduced water-holding of myofibrils and increased surface hydrophobicity. One additional freezing-thawing cycle at slow freezing rate caused appearance of a 160 kDa myosin-4 fragment in SDS-PAGE, further decreased water-holding of myofibrils and increased surface hydrophobicity. Fresh minced pork was exposed to either high salt (2 M KCl) only or high salt with lower pH to mimic conditions in freezing. Exposure to high salt only increased water-holding of myofibrils and hence did not reproduce myofibrillar protein changes in freezing. Exposure to combinations of lower pHs and high salt decreased water-holding and increased surface hydrophobicity, suggesting myofibrillar protein denaturation occurred by a comparable mechanism as in freezing-thawing. We propose that exposure to decreased pH combined with high solute concentrations in the unfrozen water of frozen meat is the primary cause of myofibrillar protein denaturation in frozen-thawed meat.

The gelation properties of myofibrillar proteins prepared with malondialdehyde and (-)-epigallocatechin-3-gallate.

Impact of malondialdehyde (MDA) and (-)-Epigallocatechin-3-gallate (EGCG) on gelling properties of myofibrillar proteins (MPs) was investigated. Addition of 6 mM MDA enhanced molecular interactions of proteins, thus the strength and elastic modulus (G') of gel were improved. EGCG addition aggravated gel quality deterioration due to further modification of MPs induced by EGCG. Addition of 12 mM MDA jeopardized gel quality according to the increasing of strength and G', but the decreasing of water-holding capacity (WHC), and the collapse of microstructure. Nevertheless, EGCG reacted with MDA forming EGCG-MDA adducts, hence improved gel quality, which was supported by the decreasing of strength, but the increasing of WHC, and the repaired microstructure of gel at 12 mM MDA. Addition of 24 mM MDA severely jeopardized gel quality, which became even worse due to EGCG addition. This work is helpful to understand the impact of MDA and polyphenols on the gel-forming capacity of MPs.

Application of temperature-controlled ultrasound treatment and its potential to reduce phosphate content in frankfurter-type sausages by 50.

The objective of this study was to evaluate the effect of ultrasound treatments with different durations (15, 20, 25, 30, and 35 min) at a low static temperature (12 °C) controlled by an intelligent temperature control and monitoring system on the quality of 50% reduced-phosphate frankfurters. The results show that without ultrasound treatment, phosphate reduction caused some obvious deficits in the textural properties, sensorial parameters, and oxidative stability of frankfurters. Moreover, 25-min ultrasound treatment could significantly lower the cooking loss and enhance emulsion stability, textural properties, and sensorial parameters of reduced phosphate frankfurters, which was also verified by dynamic water distribution analysis and microstructural observation. Additionally, low constant temperature during ultrasound treatment was another crucial factor in retarding lipid oxidation during storage. Therefore, ultrasound treatment with moderate duration and stable low temperature could be considered a successful approach to obtain healthier reduced-phosphate frankfurters under the "clean label" concept.

Effect of freeze-thaw cycles on the quality of quick-frozen pork patty with different fat content by consumer assessment and instrument-based detection.

The change in quality of quick-frozen patties containing different amounts of added fat (0%, 5%, 10%, 15%, and 20%) under different freeze-thaw (F-T) cycles (a F-T cycle was performed by freezing at -18 °C and thawing at 4 °C) was evaluated. The results showed that the a*-values of samples were significantly decreased, while L*-values, b*-values, thawing loss, and cooking loss were notably increased after 3 F-T cycles. Low-field nuclear magnetic resonance (LF-NMR) results showed that the water mobility of patties was enhanced. Textural properties (hardness, springiness, cohesiveness, and chewiness) of patties were significantly decreased after 5 F-T cycles (P < 0.05). Lipid and protein oxidation were aggravated with increasing fat content and number of F-T cycles, as confirmed by the increase in lipid peroxides, TBARS, and carbonyl content. Therefore, the results from instrument-based detection and consumer scores indicated that repeated F-T cycles accelerated the quality deterioration of quick-frozen pork patties, and rendered them unacceptable after 3 F-T cycles.

Effect of ice structuring protein on the quality of quick-frozen patties subjected to multiple freeze-thaw cycles.

The inhibitory effect of ice structuring protein (ISP) on the quality deterioration of quick-frozen pork patties subjected to multiple freeze-thaw (F-T) cycles was investigated. The inhibitory effect of ISP on patty quality deterioration was obvious after five F-T cycles (P < 0.05). The hardness and springiness of patties with 0.20% ISP were 3.84% and 10.61% higher than those of patties without ISP, and the thawing loss of patties with 0.20% ISP was 43.64% lower than that of patties without ISP (P < 0.05). In addition, ISP effectively restrained moisture migration and destruction of pork patty microstructure during F-T cycles. More importantly, thiobarbituric acid reactive substance levels and carbonyl contents in the patties with 0.20% ISP were 25% and 32% lower than those in the control group (no significant difference with patties with 0.30% ISP) after five F-T cycles. Therefore, these results illustrated the potential benefits of ISP in meat products.

Combination of light and oxygen accelerates formation of covalent protein-polyphenol bonding during chill storage of meat added 4-methyl catechol.

Plant polyphenols applied as natural antioxidant ingredients, are known to bind to cysteine residues on meat proteins. The aim of this study was to examine the effect of light exposure on the formation of cysteine-phenol adduct in meat added 4-methylcatechol (4MC), a model polyphenol, during storage through quantitative LC-MS/MS-based analysis. Cysteine-4-methylcatechol adduct (Cys-4MC) formation in meat added 1500 ppm 4-MC increased significantly (by 50%) when stored under light in oxygen at 4 °C for 7 days as compared to storage in the dark. This was reflected by a significant decrease in thiol concentrations in the same sample. Gel electrophoresis showed loss in myosin heavy chain (MHC), and a resulting increase in cross-linked MHC (CL-MHC) and larger protein polymers in samples added 4MC. Protein blots stained with nitroblue tetrazolium (NBT) showed intensive protein-polyphenol binding in the meat samples added 4MC, but no major differences between storage conditions.

Effect of the exposure to oxidation and malondialdehyde on turkey and rabbit meat protein oxidative stability.

The present study aimed at evaluating the effect of the exposure to a strong oxidative environment (100 mM NaClO) and the concurrent incubation with different malondialdehyde (MDA) concentrations (0 to 5 mM) on protein carbonylation, free thiol groups, total heme pigments, and on the relative concentration of the different myoglobin (Mb) derivatives in turkey thigh and rabbit hind leg meat to elucidate their eventual role in inducing oxidative modifications on the protein fraction. With regard to turkey meat, the addition of a strong oxidant resulted in remarkably higher (P < 0.001) carbonyls along with a reduction in free thiol groups (which become undetectable). The relative concentration of MbO2 and MetMb was significantly affected (P < 0.001) and total heme pigment was reduced by 62% when fresh meat is compared to its oxidized counterpart. The addition of MDA 2.5 mM (or greater) resulted in a 1.4-fold increase in carbonyls and a tendency (P = 0.07) has been observed for free thiol groups whose content decreased as the MDA level increased. Overall, the aforementioned results are in agreement with those obtained on rabbit hind leg meat in which a three-fold increase (P < 0.001) in carbonyls and a remarkably higher (P < 0.001) MetMb content coupled with a 53% reduction in free thiol groups were found. No significant differences ascribable to MDA addition were found. In conclusion, exposing turkey and rabbit meat to the same environment resulted in distinctive oxidative changes that might be related to the compositional traits and a species-specific susceptibility of the meat toward oxidation. PRACTICAL APPLICATION: Protein and lipid oxidation have been investigated in different meat types; nevertheless, the knowledge concerning the interaction between these two phenomena is limited. According to their compositional traits (high Polyunsaturated fatty acids (PUFA) and heme pigment content), turkey and rabbit leg meats are particularly susceptible to oxidation. Providing information concerning the oxidative stability of turkey thigh and rabbit hind leg meat exposed to strong oxidative conditions and to a concurrent increasing content of a secondary product of lipid oxidation, the findings of the present study can be useful when proper processing strategies and storage conditions have to be implemented for manufacturing processed products.