Martina B. O'Keeffe

Peptide identification in a salmon gelatin hydrolysate with antihypertensive, dipeptidyl peptidase IV inhibitory and antioxidant activities

Salmon gelatin (Salmo salar, SG) enzymatic hydrolysates were generated using Alcalase 2.4L, Alcalase 2.4L in combination with Flavourzyme 500L, Corolase PP, Promod 144MG and Brewers Clarex. The hydrolysate generated with Corolase PP for 1h (SG-C1) had the highest angiotensin converting enzyme (ACE, IC50=0.13±0.05mgmL-1) and dipeptidyl peptidase IV (DPP-IV, IC50=0.08±0.01mgmL-1) inhibitory activities, and oxygen radical absorbance capacity (ORAC, 540.94±9.57μmolTEg-1d.w.). The in vitro bioactivities of SG-C1 were retained following simulated gastrointestinal digestion. Administration of SG and SG-C1 (50mgkg-1 body weight) to spontaneously hypertensive rats (SHR) lowered heart rate along with systolic, diastolic and mean arterial blood pressure. The SG-C1 hydrolysate was fractionated using semi-preparative RP-HPLC and the fraction with highest overall in vitro bioactivity (fraction 25) was analysed by UPLC-MS/MS. Four peptide sequences (Gly-Gly-Pro-Ala-Gly-Pro-Ala-Val, Gly-Pro-Val-Ala, Pro-Pro and Gly-Phe) and two free amino acids (Arg and Tyr) were identified in this fraction. These peptides and free amino acids had potent ACE and DPP-IV inhibitory, and ORAC activities. The results show that SG hydrolysates have potential as multifunctional food ingredients particularly for the management of hypertension.

Fractionation and identification of antioxidant peptides from an enzymatically hydrolysed Palmaria palmata protein isolate

Proteins derived from the macroalgal species Palmaria palmata have emerged as potential substrates for the generation of bioactive peptides. The aim of this study was to fractionate, identify and characterize antioxidant peptides from a P. palmata protein hydrolysate. The P. palmata protein hydrolysate generated with the food-grade proteolytic enzyme Corolase PP was sequentially fractionated using solid phase extraction and semi-preparative (SP) RP-HPLC. The most active SP-RP-HPLC peptide fraction (SP-RP-HPLC-30-F26) was analysed by ESI-MS/MS. Seventeen novel peptide sequences were identified in this fraction. Of the peptides selected for synthesis, Ser-Asp-Ile-Thr-Arg-Pro-Gly-Gly-Asn-Met, showed the highest oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) activity with values of 152.43±2.73 and 21.23±0.90nmolTE/μmol peptide, respectively. The results presented herein indicate that P. palmata derived peptides may have potential applications as health enhancing ingredients and as food preservatives due to their antioxidant activity.

Atlantic salmon (Salmo salar) co-product-derived protein hydrolysates: a source of antidiabetic peptides

Large quantities of low-value protein rich co-products, such as salmon skin and trimmings, are generated annually. These co-products can be upgraded to high-value functional ingredients. The aim of this study was to assess the antidiabetic potential of salmon skin gelatin and trimmings-derived protein hydrolysates in vitro. The gelatin hydrolysate generated with Alcalase 2.4L and Flavourzyme 500L exhibited significantly higher (p < 0.001) insulin and GLP-1 secretory activity from pancreatic BRIN-BD11 and enteroendocrine GLUTag cells, respectively, when tested at 2.5 mg/mL compared to hydrolysates generated with Alcalase 2.4L or Promod 144MG. The gelatin hydrolysate generated with Alcalase 2.4L and Flavourzyme 500L showed significantly more potent (p < 0.01) DPP-IV inhibitory activity than those generated with Alcalase 2.4L or Promod 144MG. No significant difference was observed in the insulinotropic activity mediated by any of the trimmings-derived hydrolysates when tested at 2.5 mg/mL. However, the trimmings hydrolysate generated with Alcalase 2.4L and Flavourzyme 500L exhibited significantly higher DPP-IV inhibitory (p < 0.05:Alcalase 2.4L and p < 0.01:Promod 144MG) and GLP-1 (p < 0.001, 2.5 mg/mL) secretory activity than those generated with Alcalase 2.4L or Promod 144MG. The salmon trimmings hydrolysate generated with Alcalase 2.4L and Flavourzyme 500L when subjected to simulated gastrointestinal digestion (SGID) was shown to retain its GLP-1 secretory and DPP-IV inhibitory activities, in addition to improving its insulin secretory activity. However, the gelatin hydrolysate generated with Alcalase 2.4L and Flavourzyme 500L was shown to lose GLP-1 secretory activity following SGID. A significant increase in membrane potential (p < 0.001) and intracellular calcium (p < 0.001) by both co-product hydrolysates generated with Alcalase 2.4L and Flavourzyme 500L suggest that both hydrolysates mediate their insulinotropic activity through the KATP channel-dependent pathway. Additionally, by stimulating a significant increase in intracellular cAMP release (p < 0.05) it is likely that the trimmings-derived hydrolysate may also mediate insulin secretion through the protein kinase A pathway. The results presented herein demonstrate that salmon co-product hydrolysates exhibit promising in vitro antidiabetic activity.

The effect of a caseinate hydrolysate on cytokine release and RNA expression in TNF-α stressed 3T3-L1 adipocytes

Obesity and diabetes are linked to chronic inflammation and elevated pro-inflammatory cytokine release. The use of antiinflammatory agents to reduce this inflammation may help to prevent or alleviate these conditions. Bioactive peptides with various physiological functions, including anti-inflammatory activity, have been isolated from bovine casein following hydrolysis by plant, mammalian and microbial-derived proteinases. The objective of this study was to determine the effect of intact caseinate, a caseinate hydrolysate and their simulated gastrointestinal digests (SGID) on cytokine production and RNA expression in Tumour Necrosis Factor-α (TNF-α) stressed adipocytes. A 5 kDa permeate of a Flavourzyme hydrolysate of sodium caseinate was generated at laboratory scale. The unhydrolysed caseinate (UH) and the 5 kDa permeate of the hydrolysate (H) were subjected to SGID. Pre-adipocytes (3T3-L1) were differentiated to adipocytes over three weeks in media containing 1μg/ml insulin, 0·25μM dexamethasone, 0·5 mM 3-isobutyl-1-methyxanthine (IBMX) and 2μM rosiglitazone. After differentiation, adipocytes were pre-treated with test samples at 0·05 % (w/v) for 24 hours, following which stress was induced using TNF-α for 24 hours. The production of pro-inflammatory cytokines; Interleukin (IL)-6 and Monocyte Chemotactic Protein-1 (MCP-1) along with the anti-inflammatory adipokine: adiponectin, were then measured using ELISA. The RNA expression of IL-6 was also measured using quantitative RT-PCR. Data was expressed as a percentage of the TNF-α stressed control.