Antioxidative, Glucose Management, and Muscle Protein Synthesis Properties of Fish Protein Hydrolysates and Peptides

Shekoohi N., Naik A.S., Amigo-Benavent M., Harnedy-Rothwell P.A., Carson B.P., FitzGerald R.J.

IntroductionSprat (Sprattus sprattus) is an underutilized fish species that may act as an economic and sustainable alternative source of protein due to its good amino acid (AA) profile along with its potential to act as a source of multiple bioactive peptide sequences.Method and resultsThis study characterized the physicochemical, technofunctional, and in vitro antioxidant properties along with the AA profile and score of a sprat protein enzymatic hydrolysate (SPH). Furthermore, the impact of the SPH on the growth, proliferation, and muscle protein synthesis (MPS) in skeletal muscle (C2C12) myotubes was examined. The SPH displayed good solubility and emulsion stabilization properties containing all essential and non-essential AAs. Limited additional hydrolysis was observed following in vitro-simulated gastrointestinal digestion (SGID) of the SPH. The SGID-treated SPH (SPH-SGID) displayed in vitro oxygen radical antioxidant capacity (ORAC) activity (549.42 μmol TE/g sample) and the ability to reduce (68%) reactive oxygen species (ROS) production in C2C12 myotubes. Muscle growth and myotube thickness were analyzed using an xCELLigence™ platform in C2C12 myotubes treated with 1 mg protein equivalent.mL−1 of SPH-SGID for 4 h. Anabolic signaling (phosphorylation of mTOR, rpS6, and 4E-BP1) and MPS (measured by puromycin incorporation) were assessed using immunoblotting. SPH-SGID significantly increased myotube thickness (p < 0.0001) compared to the negative control (cells grown in AA and serum-free medium). MPS was also significantly higher after incubation with SPH-SGID compared with the negative control (p < 0.05).ConclusionsThese preliminary in situ results indicate that SPH may have the ability to promote muscle enhancement. In vivo human studies are required to verify these findings.

Zhu X., Wang J., Lu Y., Zhao Y., Zhang N., Wu W., Zhang Y., Fu Y.

Shekoohi N., Amigo-Benavent M., Wesley Peixoto da Fonseca G., Harnedy-Rothwell P.A., FitzGerald R.J., Carson B.P.

Blue whiting (BW) represents an underutilised fish species containing a high-quality protein and amino acid (AA) profile with numerous potentially bioactive peptide sequences, making BW an economic and sustainable alternative source of protein. This study investigated the impact of three different BW protein hydrolysates (BWPH-X, Y and Z) on growth, proliferation and muscle protein synthesis (MPS) in skeletal muscle (C2C12) myotubes. BWPHs were hydrolysed using different enzymatic and heat exposures and underwent simulated gastrointestinal digestion (SGID), each resulting in a high degree of hydrolysis (33.41–37.29%) and high quantities of low molecular mass peptides (86.17–97.12% <1 kDa). C2C12 myotubes were treated with 1 mg protein equivalent/mL of SGID-BWPHs for 4 h. Muscle growth and myotube thickness were analysed using an xCelligence™ platform. Anabolic signalling (phosphorylation of mTOR, rpS6 and 4E-BP1) and MPS measured by puromycin incorporation were assessed using immunoblotting. BWPH-X significantly increased muscle growth (p < 0.01) and myotube thickness (p < 0.0001) compared to the negative control (amino acid and serum free media). Muscle protein synthesis (MPS), as measured by puromycin incorporation, was significantly higher after incubation with BWPH-X compared with the negative control, but did not significantly change in response to BWPH-Y and Z treatments. Taken together, these preliminary findings demonstrate the anabolic potential of some but not all BWPHs on muscle enhancement, thus providing justification for human dietary intervention studies to confirm and translate the results of such investigations to dietary recommendations and practices.

Pei J., Gao X., Pan D., Hua Y., He J., Liu Z., Dang Y.

Bioactive peptides are widely used in functional foods due to their remarkable efficacy, selectivity, and low toxicity. However, commercially produced bioactive peptides lack quality stability between batches. Furthermore, the efficacies of bioactive peptides cannot be guaranteed in vivo due to gastrointestinal digestion and rapid plasma, liver, and kidney metabolism. The problem of poor stability has restricted the development of peptides. Bioactive peptide stability assessments use different stability assays, so the results of different studies are not always comparable. This review summarizes the quality stability challenges in the enzymatic hydrolysis production of bioactive peptides and the metabolism stability challenges after oral administration. Future directions on the strategies for improving their stability are provided. It was proposed that we use fingerprinting as a quality control measure using qualitative and quantitative characteristic functional peptide sequences. The chemical modification and encapsulation of bioactive peptides in microcapsules and liposomes are widely used to improve the digestive and metabolic stability of bioactive peptides. Additionally, the establishment of a universal stability test and a unified index would greatly improve uniformity and comparability in research into bioactive peptides. In summary, the reliable evaluation of stability is an essential component of peptide characterization, and these ideas may facilitate further development and utilization of bioactive peptides. • Stability challenges encountered by bioactive peptides were summarized. • Strategies to improve the stability of bioactive peptides were provided. • The establishment of a universal stability test and a unified index would greatly improve uniformity and comparability in research into bioactive peptides. • It was proposed that we use a method of traditional Chinese medicine fingerprinting as a quality control measure.

Cai B., Wan P., Chen H., Huang J., Ye Z., Chen D., Pan J.

Antioxidative peptides that inhibit myeloperoxidase (MPO) enzyme activity can effectively defend against oxidative stress damage. The antioxidant peptides from tuna protein were produced using alcalase hydrolysis and purified by ultrafiltration and Sephadex G-15, and the fractions with the highest free radicals scavenging ability and oxygen radical absorbance capacity (ORAC) values were sequenced using HPLC–MS/MS. Fifty-five peptide sequences were identified, 53 of which were successfully docked into MPO. The representative peptide ACGSDGK had better antioxidant activity and inhibition of MPO chlorination and peroxidation than the reference peptide hLF1-11. The docking model further showed intense molecular interactions between ACGSDGK and MPO, including hydrogen bonds, charge, and salt bridge interactions, which occluded the active site and blocked the catalytic activity of MPO. These results suggested that the antioxidant peptide ACGSDGK has the potential to inhibit oxidative stress and alleviate inflammation in vivo because of its inhibitory effect on the MPO enzyme.

Entezari M., Hashemi D., Taheriazam A., Zabolian A., Mohammadi S., Fakhri F., Hashemi M., Hushmandi K., Ashrafizadeh M., Zarrabi A., Ertas Y.N., Mirzaei S., Samarghandian S.

Diabetes mellitus (DM) is considered as a main challenge in both developing and developed countries, as lifestyle has changed and its management seems to be vital. Type I and type II diabetes are the main kinds and they result in hyperglycemia in patients and related complications. The gene expression alteration can lead to development of DM and related complications. The AMP-activated protein kinase (AMPK) is an energy sensor with aberrant expression in various diseases including cancer, cardiovascular diseases and DM. The present review focuses on understanding AMPK role in DM. Inducing AMPK signaling promotes glucose in DM that is of importance for ameliorating hyperglycemia. Further investigation reveals the role of AMPK signaling in enhancing insulin sensitivity for treatment of diabetic patients. Furthermore, AMPK upregulation inhibits stress and cell death in β cells that is of importance for preventing type I diabetes development. The clinical studies on diabetic patients have shown the role of AMPK signaling in improving diabetic complications such as brain disorders. Furthermore, AMPK can improve neuropathy, nephropathy, liver diseases and reproductive alterations occurring during DM. For exerting such protective impacts, AMPK signaling interacts with other molecular pathways such as PGC-1α, PI3K/Akt, NOX4 and NF-κB among others. Therefore, providing therapeutics based on AMPK targeting can be beneficial for amelioration of DM.

Heffernan S., Harnedy-Rothwell P.A., Gite S., Whooley J., Giblin L., Fitzgerald R.J., O’Brien N.M.

This study investigated the antioxidant and immunomodulatory potential of six blue whiting soluble protein hydrolysates (BWSPHs, BW-SPH-A to -F) and their simulated gastrointestinal digests (SGID, BW-SPH-A-GI to -F-GI) in murine RAW264.7 macrophages. Hydrolysate BW-SPH-A, both pre- and post-SGID, increased endogenous antioxidant glutathione (GSH) in tert-butylhydroperoxide (tBOOH)-treated cells and reduced reactive oxygen species (ROS) in H2O2-challenged RAW264.7 cells compared with treated controls in the absence of BWSPHs (p < 0.05). BW-SPH-A-GI also exhibited higher ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) activities than the other BWSPHs tested (p < 0.05). All BWSPHs and SGID BWSPH samples induced immunostimulating effects in lipopolysaccharide (LPS)-activated RAW264.7 macrophages through the upregulation of NO production. BW-SPH-F-GI increased IL-6 and TNF-α levels compared with the LPS controls indicating the liberation of immunomodulatory peptide/amino acids during the SGID process. Therefore, BW-SPH-A and BW-SPH-F may have potential use against oxidative stress and immunosuppression-related diseases, respectively.

Gui M., Gao L., Rao L., Li P., Zhang Y., Han J., Li J.

Recent studies demonstrate that fish byproducts can be used as sources of bioactive peptides for functional foods. Sturgeon skin contains abundant proteins but it has commonly been discarded during sturgeon processing. The objective of the present work was to identify and characterize the bioactive peptides from protein hydrolysates of sturgeon skin.Sturgeon skin protein extract (SKPE) hydrolyzed by flavourzyme for 60 min exhibited high antioxidant activity, dipeptidyl peptidase IV (DPP-IV) and angiotensin converting enzyme (ACE) inhibitory activity. The sequences of peptides from flavourzyme hydrolysates were identified using high-performance liquid chromatography-tandem mass spectrometry. Gly-Asp-Arg-Gly-Glu-Ser-Gly-Pro-Ala (P1) showed the highest DPPH radical scavenging activity (DPPH IC50 = 1.93 mmol L-1 ). Gly-Pro-Ala-Gly-Glu-Arg-Gly-Glu-Gly-Gly-Pro-Arg (P11) (DPP-IV IC50 = 2.14 mmol L-1 ) and Ser-Pro-Gly-Pro-Asp-Gly-Lys-Thr-Gly-Pro-Arg (P12) (DPP-IV IC50 = 2.61 mmol L-1 ) exhibited the strongest DPP-IV inhibitory activity. Gly-Pro-Pro-Gly-Ala-Asp-Gly-Gln-Ala-Gly-Ala-Lys (P6) displayed the highest ACE inhibitory activity (ACE IC50 = 3.77 mmol L-1 ). The molecular docking analysis revealed that DPP-IV inhibition of P11 and P12 are mainly attributed to hydrogen bonds and hydrophobic interactions, whereas ACE inhibition of P6 is mainly attributed to strong hydrogen bonds.These results indicate that SKPE hydrolysates generated by flavourzyme are potential sources of bioactive peptides that could be used in the health food industry. © 2021 Society of Chemical Industry.

Chelliah R., Wei S., Daliri E.B., Elahi F., Yeon S., Tyagi A., Liu S., Madar I.H., Sultan G., Oh D.

Bioactive peptides are present in most soy products and eggs and have essential protective functions. Infection is a core feature of innate immunity that affects blood pressure and the glucose level, and ageing can be delayed by killing senescent cells. Food also encrypts bioactive peptides and protein sequences produced through proteolysis or food processing. Unique food protein fragments can improve human health and avoid metabolic diseases, inflammation, hypertension, obesity, and diabetes mellitus. This review focuses on drug targets and fundamental mechanisms of bioactive peptides on metabolic syndromes, namely obesity and type 2 diabetes, to provide new ideas and knowledge on the ability of bioactive peptide to control metabolic syndromes.

Henaux L., Pereira K.D., Thibodeau J., Pilon G., Gill T., Marette A., Bazinet L.

Natural bioactive peptides are suitable candidates for preventing the development of Type 2 diabetes (T2D), by reducing the various risk factors. The aim of this study was to concentrate glucoregulatory and anti-inflammatory peptides, from salmon by-products, by electrodialysis with ultrafiltration membrane (EDUF), and to identify peptides responsible for these bioactivities. Two EDUF configurations (1 and 2) were used to concentrate anionic and cationic peptides, respectively. After EDUF separation, two fractions demonstrated interesting properties: the initial fraction of the EDUF configuration 1 and the final fraction of the EDUF configuration 2 both showed biological activities to (1) increase glucose uptake in L6 muscle cells in insulin condition at 1 ng/mL (by 12% and 21%, respectively), (2) decrease hepatic glucose production in hepatic cells at 1 ng/mL in basal (17% and 16%, respectively), and insulin (25% and 34%, respectively) conditions, and (3) decrease LPS-induced inflammation in macrophages at 1 g/mL (45% and 30%, respectively). More impressive, the initial fraction of the EDUF configuration 1 (45% reduction) showed the same effect as the phenformin at 10 μM (40%), a drug used to treat T2D. Thirteen peptides were identified, chemically synthesized, and tested in-vitro for these three bioactivities. Thus, four new bioactive peptides were identified: IPVE increased glucose uptake by muscle cells, IVDI and IEGTL decreased hepatic glucose production (HGP) of insulin, whereas VAPEEHPTL decreased HGP under both basal condition and in the presence of insulin. To the best of our knowledge, this is the first time that (1) bioactive peptide fractions generated after separation by EDUF were demonstrated to be bioactive on three different criteria; all involved in the T2D, and (2) potential sequences involved in the improvement of glucose uptake and/or in the regulation of HGP were identified from a salmon protein hydrolysate.

Hu J., Liu R., Yu X., Li Z., Liu X., Hao Y., Zhu N., Kang J., Li Y.

Peptic ulcer has a serious impact on people’s health around the world, and traditional medicines can cause adverse reactions. This study investigated the protective effects of tilapia collagen oligopeptides (TCOPs) on gastroduodenal injury. Seventy-two specific pathogen-free (SPF) male Sprague Dawley (SD) rats were randomly divided into six groups according to body weight: normal control group, ethanol group, whey protein group (500 mg/kg BW), and three TCOPs dose groups (250, 500, 1000 mg/kg BW). After intragastric administration for 30 days, the acute gastroduodenal injury was induced by anhydrous ethanol (5 mL/kg, intragastrically) in all groups except the normal control group. Biomarkers in gastric and duodenal tissue and serum were measured. Furthermore, western blot was used to detect the expression of apoptosis-related proteins. The results showed that the administration with TCOPs significantly reduced gastric and duodenal ulcer index, increased gastric juice pH, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities, along with the reduction of malondialdehyde (MDA) contents. TCOPs decreased tumor Necrosis Factor-α (TNF-α), interleukin-1β (IL-1β), and myeloperoxidase (MPO) levels, while interleukin– 10 (IL-10) levels were increased. Furthermore, pepsinogens 1 (PG1), pepsinogens 2 (PG2), gastrin (GAS), and the pepsinogen ratio (PGR) were decreased, the prostaglandin E2 (PGE2) and NO contents were increased after TCOPs intervention. Moreover, TCOPs up-regulated the expression of Bcl-2 and inhibited the expression of Bax and Caspase-3. In conclusion, TCOPs have protective effects on ethanol-induced gastroduodenal injury through gastrointestinal mucosal microcirculation promotion, antioxidation, anti-inflammation, and anti-apoptosis mechanisms.

Takahashi Y., Kamata A., Konishi T.

The hydrolysate of Oncorhynchus keta (chum salmon) milt was found to exhibit strong inhibitory activity against dipeptidyl peptidase-IV (DPP-IV), and the hypoglycaemic effect of these salmon milt peptides (SMPs) was confirmed using oral starch tolerance tests in Sprague–Dawley rats. Specifically, the oral administration of SMPs at a dose of 300 mg/kg body weight for 1 week resulted in a significant decrease in the blood glucose level of the rats 60 min after starch consumption compared with the control. The bioassay-guided separation of SMPs, using a combination of column chromatographic techniques, led to the identification of 12 active di-, tri- and tetrapeptides, including four novel DPP-IV inhibitory peptides, namely Phe-Pro-Val-Gly, Leu-Pro-Val-Leu, Val-Pro-Phe-Pro and Ile-Pro-Leu. Among these 12 peptides, Ile-Pro contributed the most (1.3%) to the total DPP-IV inhibitory activity of SMPs. Therefore, SMPs may have the potential to be used as a suppressor of postprandial hyperglycaemia and to be considered a promising functional food material for the prevention of type II diabetes.

Sukkhown P., Pirak T., Jangchud K., Prinyawiwatkul W.

Gao R., Yu Q., Shen Y., Chu Q., Chen G., Fen S., Yang M., Yuan L., McClements D.J., Sun Q.

The health benefits associated with consuming fish products are mainly attributed to their desirable nutrition profiles, including vitamins, minerals, essential amino acids, and polyunsaturated fatty acids. However, large quantities of fish proteins are presently underutilized or discarded as waste. Effective strategies to utilize fish proteins are therefore needed. Recently, researchers have focused on generating and characterizing bioactive fish protein hydrolysates and peptides and then studying their potential health benefits. The major methods of producing, separating, and purifying protein hydrolysates are initially given. Then, the biological activities and potential mechanisms of action of protein hydrolysates and peptides are discussed. Finally, current limitations and future possibilities of fish peptide identification, production, and bioactivity are identified and discussed. Fermentation, chemical synthesis, and enzymatic hydrolysis are effective methods of obtaining hydrolysates from underutilized fish protein by-products. These hydrolysates can then be purified by membrane separation and chromatographic methods to obtain bioactive peptides. The molecular characteristics of the peptides can then be identified using mass spectrometry. Fish hydrolysates/peptides have multiple biological activities, including antioxidative, lipid homeostasis modulation, anti-inflammatory, anticancer, neuroprotective, and antihypertensive activities, which make them promising nutraceutical ingredients for application in foods. Moreover, they often have emulsifying, foaming, and gelling properties, which means they may be suitable as multipurpose functional ingredients. Thus, waste-derived fish by-products may be turned into value-added functional ingredients designed to address chronic diseases. However, further research is required to develop large-scale commercially viable extraction and purification methods, develop robust structure-function relationships for peptides, and perform in vivo human studies of peptide bioactivity. • The production of protein hydrolysates from underutilized fish protein or by-products. • The purification and identification of bioactive peptides. • The main biological activities of fish hydrolysates/peptides. • Current food applications and future perspectives of fish protein hydrolysates/peptides.

Ritian J., Teng X., Liao M., Zhang L., Wei Z., Meng R., Liu N.

Maghaydah S., Alrosan M., Alu’datt M.H., Razzak Mahmood A.A., Gammoh S., Bani-Melhem K., Al Qudsi F.R., Tan T., Almajwal A.M., Rashed Alshammari A.