Influence of sonication pretreatment on antiradical and anti-ACE activity of protein hydrolysates from fermented pork loins.

The aim of this study was to assess whether ultrasound treatment (sonification time: 5, 15, and 30 min; constants: ∼40 kHz, ∼2.5 W cm2) can be applied prior to hydrolysis to enhance the anti-radical and angiotensin converting enzyme inhibiting (anti-ACE) effect of the hydrolysates from fermented pork loins. Enzymatic hydrolysis was performed using pepsin, followed by pancreatin. The influence of meat matrix on the course of hydrolysis, shaped using a lactic acid bacteria (LAB)-based starter culture, was also analyzed. It was found that proteases caused a systematic increase in the content of peptides, while pancreatin limited the peptide content in the protein hydrolysate from the loins subjected to spontaneous fermentation. Moreover, for these tests, sonication time had a negligible effect on the peptides content of the hydrolysates. On the other hand, for the sample of LAB-fermented products, both sonication time and stage of hydrolysis promoted the biological activity of the hydrolysates. Samples from the LAB-fermented meat had more peptides at the stage of digestion with pepsin and pancreatin, exhibiting much faster antiradical and anti-ACE activity compared to the control sample. The obtained results suggest that the use of LAB promotes the release of antiradical peptides during the two-step enzymatic hydrolysis, the duration of which can be shortened to achieve satisfactory biofunctionalities. Additional application of sonication pretreatment allows controlling the course of the hydrolysis, as the pro-health, biological effect of some protein-derived sequences is associated with the content of peptides.

Biological Activity of Canned Pork Meat Fortified Black Currant Leaf Extract: In Vitro, In Silico, and Molecular Docking Study.

The aim of this study was to assess the antioxidant and inhibiting (ACE-I, DPP IV, and alpha-glucosidase) potential of canned meat featuring reduced sodium nitrate content (50 mg/kg) and fortified with freeze-dried currant leaf extract. Research indicates that employing a lyophilizate dose of 150 mg/kg yields optimal benefits in terms of the antioxidant activity of the meat product. Additionally, three highly promising sequences for canned meat were identified via analysis in the BIOPEP database. These sequences are RPPPPPPPPAD, exhibiting DPP-IV inhibiting activity; ARPPPGPPPLGPPPPGP, demonstrating ACE-I inhibiting activity; and PPGPPPPP, displaying alpha-glucosidase inhibiting activity. Using bioinformatics tools, molecular docking was performed by pairing the selected peptides with protein receptors 2QT9, 1O86, and 5NN8, respectively (PDB ID). The examination of the potential of these selected sequences to manifest specific biological activities toward enzymes was based on the free energy value (∆Gbinding). This knowledge can be harnessed for designing functional foods, thereby contributing to the safeguarding of consumer health.

Nutrition Value of Baked Meat Products Fortified with Lyophilized Dragon Fruit (Hylocereus undatus).

This study evaluates the nutritional value of a baked pork meat product containing lyophilized dragon fruit pulp. The selected nutritional properties of a baked pork meat product fortified with lyophilized Hylocereus undatus pulp in doses of 0.5%, 1.5%, 2.5%, and 4% were evaluated. For this assessment, changes in the basic chemical composition of the products, the content of calcium, magnesium, potassium, iron, and phosphorus, and the profile of fatty acids were considered. Additionally, characteristics typical for meat products, such as pH, water activity, oxidation-reduction potential or thiobarbituric acid reactive substances, and antioxidant properties of the product during 21 days of refrigerated storage, were assessed. The findings indicate that the use of higher doses of lyophilizate, i.e., in the amounts of 2.5% and 4%, significantly (p < 0.05) increases the nutritional value of meat products, leading to an increase in the concentration of essential minerals important for the proper functioning of the human body (calcium, magnesium, potassium, and iron). These changes occurred without affecting the basic chemical composition (except for an increase in the content of fat and carbohydrates in the sample with the addition of 4% lyophilizate). The introduction of the fortification treatment improved the fatty acid profile, resulting in an increase in the content of C14:0, C16:0, C20:0, and C20:5n3. In addition, in the variant with a 4% dosage, there was an increased content of C8:0, C10:0, C16:1n7, C18:0, C18:1n9C + C18:1n9t, and C18:2n6C + C18:2n6t, C18:3n3 (alpha), C20:1n15, and C20:1n9. In this particular variant, an increase in saturated-, monounsaturated-, and polyunsaturated fatty acids was also observed, which was associated with an increased level of TBARS in meat products. However, the increase in the dose of lyophilizate caused an increase in the antiradical effect of meat extracts. Based on the results obtained, it seems reasonable to use a plant additive in the form of lyophilized dragon fruit pulp in the amount of 4.0% in the production of pork meat products.

Novel α-Glucosidase Inhibitory Peptides Identified In Silico from Dry-Cured Pork Loins with Probiotics through Peptidomic and Molecular Docking Analysis.

Diabetes mellitus is a serious metabolic disorder characterized by abnormal blood glucose levels in the body. The development of therapeutic strategies for restoring and maintaining blood glucose homeostasis is still in progress. Synthetic alpha-amylase and alpha-glucosidase inhibitors can improve blood glucose control in diabetic patients by effectively reducing the risk of postprandial hyperglycemia. Peptides of natural origin are promising compounds that can serve as alpha-glucosidase inhibitors in the treatment of type 2 diabetes. Potential alpha-glucosidase-inhibiting peptides obtained from aqueous and saline extracts from dry-cured pork loins inoculated with probiotic LAB were evaluated using in vitro and in silico methods. To identify the peptide sequences, liquid chromatography-mass spectrometry was used. For this purpose, in silico calculation methods were used, and the occurrence of bioactive fragments in the protein followed the ADMET approach. The most promising sequences were molecularly docked to test their interaction with the human alpha-glycosidase molecule (PDB ID: 5NN8). The docking studies proved that oligopeptides VATPPPPPPPK, DIPPPPM, TPPPPPPG, and TPPPPPPPK obtained by hydrolysis of proteins from ripening dry-cured pork loins showed the potential to bind to the human alpha-glucosidase molecule and may act effectively as a potential antidiabetic agent.

Reduction of Nitrite in Canned Pork through the Application of Black Currant (Ribes nigrum L.) Leaves Extract.

Sodium nitrite is a multifunctional additive commonly used in the meat industry. However, this compound has carcinogenic potential, and its use should be limited. Therefore, in this study the possibility of reducing the amount of sodium(III) nitrite added to canned meat from 100 to 50 mg/kg, while enriching it with freeze-dried blackcurrant leaf extract, was analyzed. The possibility of fortification of canned meat with blackcurrant leaf extract was confirmed. It contained significant amounts of phenolic acids and flavonoid derivatives. These compounds contributed to their antioxidant activity and their ability to inhibit the growth of selected Gram-positive bacteria. In addition, it was observed that among the three different tested doses (50, 100, and 150 mg/kg) of the blackcurrant leaf extract, the addition of the highest dose allowed the preservation of the antioxidant properties of canned meat during 180 days of storage (4 °C). At the end of the storage period, this variant was characterized by antiradical activity against ABTS (at the level of 4.04 mgTrolox/mL) and the highest reducing capacity. The addition of 150 mg/kg of blackcurrant leaf extract caused a reduction in oxidative transformations of fat in meat products during the entire storage period, reaching a level of TBARS almost two times less than in the control sample. In addition, these products were generally characterized by stability (or slight fluctuations) of color parameters and good microbiological quality and did not contain N-nitrosamines.

Peptides as Potentially Anticarcinogenic Agent from Functional Canned Meat Product with Willow Extract.

The aim of the study was to demonstrate canned pork as a functional meat product due to the presence of potentially anti-cancer factors, e.g., (a) bioactive peptides with potential activity against cancer cells; (b) lowering the content of sodium nitrite and with willow herb extract. In silico (for assessing the anticancer potential of peptides) and in vitro (antiproliferation activity on L-929 and CT-26 cell lines) analysis were performed, and the obtained results confirmed the bioactive potential against cancer of the prepared meat product. After 24 h of incubation with peptides obtained from meat product containing lyophilized herb extract at a concentration of 150 mg/kg, the viability of both tested cell lines was slightly decreased to about 80% and after 72 h to about 40%. On the other hand, after 72 h of incubation with the peptides obtained from the variant containing 1000 mg/kg of freeze-dried willow herb extract, the viability of intestinal cancer cells was decreased to about 40%, while, by comparison, the viability of normal cells was decreased to only about 70%.

Effect of willow herb (Epilobium angustifolium L.) extract addition to canned meat with reduced amount of nitrite on the antioxidant and other activities of peptides.

The present study aimed to evaluate the effect of the addition of various amounts (50, 100, 150 and 1000 mg kg-1) of E. angustifolium L. extracts on the biological activity of peptides in canned meat with reduced amount of sodium nitrite and their stability during 180 days of storage (4 °C). The initial peptide data were collected by LC/MS. Antioxidant activities of peptide extracts were detected on the basis of ABTS˙*, FRAP, and iron(II) chelating activity in in vitro tests. A computational study (based on the BIOPEP-UWM database and INNOVAGEN, PeptideRanker and PROTPARAM tools) was also performed to assist in the interpretation of results. The addition of E. angustifolium L. extracts has a positive effect on the peptide profile and various biological activities, the results of which depend on the amount of the extract added to the meat product. However, it should be remembered that the differences between in silico and in vitro experimental environments necessitate further research to confirm the antioxidant behavior of canned meat products supplemented with E. angustifolium L. extract under physiological conditions. Other interactions between the peptide and the food matrix should also be considered as these can lead to chemical and structural modifications that can affect the bioavailability of the bioactive peptides.

Dipeptidyl Peptidase IV Inhibitory Peptides Generated in Dry-Cured Pork Loin during Aging and Gastrointestinal Digestion.

The ability of peptides from an aqueous and salt-soluble protein extract of dry-cured pork loins to inhibit the action of dipeptidyl peptidase IV was determined. This activity was assessed at different times of the production process, i.e., 28, 90, 180, 270 and 360 days. The resistance of the biological property during the simulated digestive process was also assessed. For this, the extracts were hydrolyzed with pepsin and pancreatin as a simulated digestion step of the gastrointestinal tract and fractionated (>7 kDa) as an intestinal absorption step. The results indicate that dried-pork-loin peptides may have potential as functional food ingredients in the prevention and treatment of type 2 diabetes mellitus. In particular, the APPPPAEV, APPPPAEVH, KLPPLPL, RLPLLP, VATPPPPPPK, VPIPVPLPM and VPLPVPVPI sequences show promise as natural food compounds helpful in maintaining good health.

Potential DPP IV Inhibitory Peptides from Dry-Cured Pork Loins after Hydrolysis: An In Vitro and In Silico Study.

Peptidyl peptidase IV (DPP-IV) is a pharmacotherapeutic target in type 2 diabetes, and inhibitors of this enzyme are an important class of drugs for the treatment of type 2 diabetes. In the present study, peptides (<7 kDa) isolated from dry-cured pork loins after pepsin and pancreatin hydrolysis were identified by mass spectrometry and tested as potential inhibitors of DPP-IV by the in silico method. Two peptides, namely WTIAVPGPPHS from myomesin (water-soluble fraction, A = 0.9091) and FKRPPL from troponin (salt-soluble fraction, A = 0.8333), were selected as the most promising inhibitors of DPP-IV. Both peptides were subjected to ADMET analysis. Fragments of these peptides showed promising drug-likeness properties as well as favorable absorption, distribution, metabolism, excretion, and toxicity functions, suggesting that they are novel leads in the development of DPP-IV inhibitors from food.

Influence of Sonication and Taraxacum Officinale Addition on the Antioxidant and Anti-ACE Activity of Protein Extracts from Sous Vide Beef Marinated with Sour Milk and after In Vitro Digestion.

The present study assessed the effect of pretreating beef as a raw material for sous vide steak preparation. The pretreatment involved maceration of a batch of meat in sour milk with the simultaneous use of ultrasound (250 or 500 W) as well as the addition of Taraxacum officinale. The biological activity profile of the peptides was assessed in terms of their antioxidant activity and inhibiting activity against angiotensin-converting enzyme (ACE). Changes in the biological activity of peptides under the influence of hydrolysis by gastrointestinal enzymes, i.e., pepsin and pancreatin, were also considered. There was no significant effect of T. officinale addition and sonication of beef batches on the protein content (except for lot S6, after sonication at 500 W as acoustic power and with the addition of dandelion). It was observed that the interaction of maceration in sour milk with simultaneous ultrasound treatment as the initial production step of sous vide beef steak generates the formation of peptides with antioxidant properties. Moreover, peptide formation can be further enhanced by adding dandelion (based on the results of antiradical and chelating activity tests). In addition, the progression of hydrolysis under the influence of gastrointestinal enzymes promotes the release of peptides with antioxidant and anti-ACE activity.

Peptides from Different Carcass Elements of Organic and Conventional Pork-Potential Source of Antioxidant Activity.

The growing consumer interest in organic foods, as well as, in many cases, the inconclusiveness of the research comparing organic and conventional foods, indicates a need to study this issue further. The aim of the study was to compare the effects of meat origin (conventional vs. organic) and selected elements of the pork carcass (ham, loin, and shoulder) on the meat proteome and the antioxidant potential of its peptides. The peptidomic approach was used, while the ability of antioxidants to scavenge 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS), to chelate Fe(II) ions, and to reduce Fe(III) was determined. Most peptides were derived from myofibrillary proteins. The meat origin and the element of the pork carcass did not have a significant effect on the proteome. On the other hand, the pork origin and the carcass element significantly affected the iron ion-chelating capacity (Fe(II)) and the reducing power of peptides. In particular, pork ham from conventional rearing systems had the best antioxidant properties in relation to potential antioxidant peptides. This could be a factor for human health, as well as for stabilized meat products (e.g., toward lipid oxidation).

Structure-activity relationships study on biological activity of peptides as dipeptidyl peptidase IV inhibitors by chemometric modeling.

The aim of this study is to identify the potential descriptors affecting the inhibitory activity of the peptides inhibiting dipeptidyl peptidase IV (DPP-IV). This study provides important information for assessing the biological activity of the new peptide sequences of food origin or making structural modifications to the current inhibitors to improve their performance. For this purpose, the chemometric method describing the relationship between the structure of food peptides and their biological activity (structure-activity relationship [SAR]) was used to theoretically predict the potential of bioactivity of peptides. Data on the physicochemical properties of amino acids in the dipeptides acting as inhibitors of DPP-IV were collected and analyzed for using these properties as descriptors in further analysis. A total of 252 dipeptide sequences with confirmed DPP-IV inhibitory activity available in the BIOPEP-UWM database were included in the analysis, and 16 descriptors defining individual amino acids (such as molecular weight, polarity, hydropathicity, bulkiness, buried residue, and acceptable and normalized frequency of alpha-helix and beta-sheet) were identified. Based on this information, a data matrix was constructed and used in the chemometric analysis (principal component analysis and multiple linear regression). From the SAR model created, a multiple regression equation was derived to predict the biological activity of the dipeptide DPP-IV inhibitors.

Effect of Marination Time on the Antioxidant Properties of Peptides Extracted from Organic Dry-Fermented Beef.

In this study, we evaluated the effect of marination time on changes in the antioxidant properties of peptides extracted from bovine semimembranosus muscle. We measured antiradical scavenging capacity and reducing power of the peptides using a spectrophotometric decolorization method; inhibition of lipid oxidation was also assessed by estimating the level of malondialdehyde formed. According to our results, there was no benefit from the doubling of marinating time (from 24 to 48 h) as part of the preprocessing of beef. Samples from S1 batch (24 h marination) showed better antioxidant properties than those from S2 batch. We also tested various color parameters as a reflection of the inhibition of oxidative processes, in which case, the most favorable parameters from the consumer point of view were found to be lightness and redness. The effect of marination time on the degree of proteolytic changes was estimated using peptidomic approach. The degradation of myoglobin, hemoglobin, creatine kinase-type M, and beta-enolase-as the most sensitive proteins to proteolytic degradation-was observed during the 62 days of processing. It seems that the prolongation of marination time as a preprocessing step intensifies the hydrolytic degradation of proteins and peptides during the processing step. This results in the loss (or it has no effect) of antioxidative properties in organic dry-fermented beef.

Meat Proteins as Dipeptidyl Peptidase IV Inhibitors and Glucose Uptake Stimulating Peptides for the Management of a Type 2 Diabetes Mellitus In Silico Study.

Diabetes mellitus is a non-communicable disease entity currently constituting one of the most significant health problems. The development of effective therapeutic strategies for the prevention and/or treatment of diabetes mellitus based on the selection of methods to restore and maintain blood glucose homeostasis is still in progress. Among the different courses of action, inhibition of dipeptidyl peptidase IV (DPP-IV) can improve blood glucose control in diabetic patients. Pharmacological therapy offering synthetic drugs is commonly used. In addition to medication, dietary intervention may be effective in combating metabolic disturbances caused by diabetes mellitus. Food proteins as a source of biologically active sequences are a potential source of anti-diabetic peptides (DPP-IV inhibitors and glucose uptake stimulating peptides). This study showed that in silico pork meat proteins digested with gastrointestinal enzymes are a potential source of bioactive peptides with a high potential to control blood glucose levels in patients with type 2 diabetes mellitus. Analysis revealed that the sequences released during in silico digestion were small dipeptides (with an average weight of 270.07 g mol-1), and most were poorly soluble in water. The selected electron properties of the peptides with the highest bioactivity index (i.e., GF, MW, MF, PF, PW) were described using the DFT method. The contribution of hydrophobic amino acids, in particular Phe and Trp, in forming the anti-diabetic properties of peptides released from pork meat was emphasized.

Stability of Antiradical Activity of Protein Extracts and Hydrolysates from Dry-Cured Pork Loins with Probiotic Strains of LAB.

The application of starter cultures to improve quality and safety has become a very common practice in the meat industry. Probiotic strains of lactic acid bacteria (LAB) can also bring health benefits by releasing bioactive peptides. The aim of this work was to evaluate the stability of antiradical activity of protein extracts from LAB-inoculated dry-cured pork loins during long-term aging and evaluate their hydrolysates after simulated gastrointestinal digestion. Analyses of hydrolysates by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) were strengthened with in silico analysis. The highest antiradical activity of the protein extracts was observed after 180 days of aging. The influence of the strain used (LOCK, BAUER, or BB12) on the inactivation ability of ABTS radicals varied during long-term aging. The IC50 values indicated the higher antiradical properties of salt-soluble (SSF) compared to water-soluble fraction (WSF) of proteins. The peptides generated by in vitro digestion have MW between 700 and 4232 Da and their length ranged from 5 to 47 amino acids in a sequence where Leu, Pro, Lys, Glu, and His had the largest share. This study demonstrates that the degradation of pork muscle proteins during gastrointestinal digestion may give rise to a wide variety of peptides with antiradical properties.

The influence of acid whey on the antioxidant peptides generated to reduce oxidation and improve colour stability in uncured roast beef.

BACKGROUND: The effect of marinating beef in acid whey on the antioxidant peptides generated, and their influence on lipid oxidation, colour stability, sensory analysis and protein degradation products in uncured roasted beef stored 6 weeks in vacuum conditions (T = 4 °C) were determined. Measurements of pHe, water activity, oxidation-reduction potential, colour, TBARS, the cutting force, texture and amino acid profile, the total content of peptides, and antioxidant activity of isolated peptides were conducted immediately after production and during 42 days of storage. RESULTS: The non-nitrite control batch (C) was characterized by a lower a* value (6.33-6.70) during the whole storage period compared to the non-nitrite sample with acid whey (W). It also appears that meat with the worst colour stability has the poorest oxidative stability (C = 1.57 mg MDA kg-1 , W = 0.76 mg MDA kg-1 ). Activity against reactive forms of oxygen to fraction A and fraction B (P < 0.05) showed an increasing trend with time and it was the largest in sample C and W. CONCLUSION: The results indicated that bioactive peptides could be generated in uncured roasted beef. The <3.5 kDa peptides have strong antioxidant activity, as a result of which they function as inhibitors of lipid oxidation and colour discoloration during prolonged storage. © 2018 Society of Chemical Industry.

Potential of bacteriocins from lab to improve microbial quality of dry-cured and fermented meat products.

Meat and meat products are an important component of the daily diet. Nevertheless, they are perishable goods and are prone to microbial contamination, which leads to an increased risk to the health of consumers as well as economic losses in the meat industry. Fermentation has been used for thousands of years to preserve meat. As a result of extensive biochemical reactions occurring in meat during fermentation and ripening, the condi- tions inhibiting the growth of pathogenic and spoilage bacteria are formed. These changes are catalyzed by endogenous meat enzymes and exogenous enzymes derived from natural contaminating bacteria or starter cultures applied. In dry-cured and fermented meat products they are represented mainly by lactic acid bacte- ria (LAB) that produce a wide range of compounds, such as bacteriocins, directed against other microorgan- isms. The use of bactericidal peptides does not affect the sensory quality of foodstuffs, so that they attract attention as alternative means of preserving the stability and safety of dry-cured products.

Antimicrobial Peptides of Meat Origin - An In silico and In vitro Analysis.

The aim of this study was to evaluate the antimicrobial activity of meat protein-derived peptides against selected Gram-positive and Gram-negative bacteria. The in silico and in vitro approach was combined to determine the potency of antimicrobial peptides derived from pig (Sus scrofa) and cow (Bos taurus) proteins. The in silico studies consisted of an analysis of the amino acid composition of peptides obtained from the CAMPR database, their molecular weight and other physicochemical properties (isoelectric point, molar extinction coefficient, instability index, aliphatic index, hydropathy index and net charge). The degree of similarity was estimated between the antimicrobial peptide sequences derived from the slaughtered animals and the main meat proteins. Antimicrobial activity of peptides isolated from dry-cured meat products was analysed (in vitro) against two strains of pathogenic bacteria using the disc diffusion method. There was no evidence of growthinhibitory properties of peptides isolated from dry-cured meat products against Escherichia coli K12 ATCC 10798 and Staphylococcus aureus ATCC 25923.

Porcine myofibrillar proteins as potential precursors of bioactive peptides - an in silico study.

Selected porcine myofibrillar proteins have been assessed as potential precursors of bioactive peptides based on in silico analysis. The potential of protein sequences for releasing peptides was evaluated by determining the profile of their potential biological activity and the frequency of occurrence of fragments with a given activity using the BIOPEP database. Digestive enzymes: pepsin, trypsin and chymotrypsin have been used for the in silico proteolysis with the use of the "Enzyme(s) action" tool in BIOPEP. After simulated gastrointestinal digestion the tested sequences of pig myofibrillar proteins are a potential source of a total of 399 peptides with activities such as enzyme inhibition, antioxidative, hypotensive, stimulating or regulating various body functions and antiamnestic activities. Within the intact proteins and after simulated gastrointestinal digestion, dipeptidyl peptidase IV inhibitory peptide sequences were the most frequently observed. The results indicate that pork myofibrillar proteins are a promising source of peptides with biological activity.

Meat and fermented meat products as a source of bioactive peptides.

Bioactive peptides are short amino acid sequences, that upon release from the parent protein may play different physiological roles, including antioxidant, antihypertensive, antimicrobial, and other bioactivities. They have been identified from a range of foods, including those of animal origin, e.g., milk and muscle sources (with pork, beef, or chicken and various species of fish and marine organism). Bioactive peptides are encrypted within the sequence of the parent protein molecule and latent until released and activated by enzymatic proteolysis, e.g. during gastrointestinal digestion or food processing. Bioactive peptides derived from food sources have the potential for incorporation into functional foods and nutraceuticals. The aim of this paper is to present an overview of the muscle-derived bioactive peptides, especially those of fermented meats and the potential benefits of these bioactive compounds to human health.