Irineu Batista

Impact of ultrafiltration and nanofiltration of an industrial fish protein hydrolysate on its bioactive properties

BACKGROUND Numerous studies have demonstrated that in vitro controlled enzymatic hydrolysis of fish and shellfish proteins leads to bioactive peptides. Ultrafiltration (UF) and/or nanofiltration (NF) can be used to refine hydrolysates and also to fractionate them in order to obtain a peptide population enriched in selected sizes. This study was designed to highlight the impact of controlled UF and NF on the stability of biological activities of an industrial fish protein hydrolysate (FPH) and to understand whether fractionation could improve its content in bioactive peptides. RESULTS The starting fish protein hydrolysate exhibited a balanced amino acid composition, a reproducible molecular weight (MW) profile, and a low sodium chloride content, allowing the study of its biological activity. Successive fractionation on UF and NF membranes allowed concentration of peptides of selected sizes, without, however, carrying out sharp separations, some MW classes being found in several fractions. Peptides containing Pro, Hyp, Asp and Glu were concentrated in the UF and NF retentates compared to the unfractionated hydrolysate and UF permeate, respectively. Gastrin/cholecystokinin-like peptides were present in the starting FPH, UF and NF fractions, but fractionation did not increase their concentration. In contrast, quantification of calcitonin gene-related peptide (CGRP)-like peptides demonstrated an increase in CGRP-like activities in the UF permeate, relative to the starting FPH. The starting hydrolysate also showed a potent antioxidant and radical scavenging activity, and a moderate angiotensin-converting enzyme (ACE)-1 inhibitory activity, which were not increased by UF and NF fractionation. CONCLUSION Fractionation of an FPH using membrane separation, with a molecular weight cut-off adapted to the peptide composition, may provide an effective means to concentrate CGRP-like peptides and peptides enriched in selected amino acids. The peptide size distribution observed after UF and NF fractionation demonstrates that it is misleading to characterize the fractions obtained by membrane filtration according to the MW cut-off of the membrane only, as is currently done in the literature.

HPLC PREPARATION OF FISH WASTE HYDROLYSATE FRACTIONS. EFFECT ON GUINEA PIG ILEUM AND ACE ACTIVITY

ABSTRACT The effect of RP-HPLC-purified fractions of fish waste hydrolysates issued from three fish industries was tested on guinea pig ileum in order to examine the presence of opioid molecules. The evaluation of anti-hypertensive activities of whole hydrolysates and fractions were also tested, monitoring the ability of the fraction to inhibit the activity of angiotensin I-converting enzyme involved in hypertension regulation. Sardine autolysate and cod head hydrolysate powder (50 µg) were able to inhibit near 30% of ACE activity, whereas 50 µg of shrimp hydrolysate allows the inhibition of 57% of ACE activity. HPLC fractionation of cod head hydrolysate and sardine autolysate was necessary to evidence biological activity, whereas HPLC separation of shrimp hydrolysate exhibited low biological activity fractions. Further studies are necessary to characterise bioactive molecules from cod head alcalase hydrolysate and from sardine autolysate.

Evaluation of the risk/benefit associated to the consumption of raw and cooked farmed meagre based on the bioaccessibility of selenium, eicosapentaenoic acid and docosahexaenoic acid, total mercury, and methylmercury determined by an in vitro digestion model.

The bioaccessibility of total lipids, EPA, DHA, Se, Hg, and MeHg in raw and cooked meagre (Argyrosomus regius) was studied by using an in vitro digestion method. A risk-benefit assessment of raw and cooked meagre on the basis of the bioaccessibility data was carried out. The bioaccessibility of total lipids was generally high in raw and cooked meagre with exception of grilled fish. For EPA and DHA, bioaccessibility percentages were low never surpassing the 50% in raw, boiled, and grilled meagre. The bioaccessibility percentage of Se was equal or higher than 82% (grilling treatment). Likewise, for Hg and MeHg, high bioaccessibility values were determined with exception of grilled meagre, displaying lower values of 54% and 64%, respectively. The risk-benefit probabilistic assessment brought about a recommendation of a maximum consumption of two weekly meals for boiled or roasted meagre and three weekly meals for grilled meagre.

Extraction of Sardine Proteins by Acidic and Alkaline Solubilisation

Muscle sardine (Sardina pilchardus) proteins were extracted using acidic or alkaline-aided solubilisation followed by isoelectric protein precipitation and compared to the traditional surimi process. This work reports the results obtained with unwashed and washed sardine mince. The highest solubilisation of sardine miofibrillar proteins was recorded at pH 2.5 (85%) and 11.5—12 (80%) and the isoelectric point was around pH 5—5.5. The global yields achieved were 77% and 73% for the alkaline and acidic processes respectively, which are considerably higher than that of the traditional surimi process (ca. 28%). The fat content reduction was 65.3% and 51.0% for the proteins recovered after alkaline and acidic solubilisation respectively. A reduction of 91.1% was obtained in the surimi process. However, higher fat elimination was achieved when washed mince was used (95.3% and 99.0% for acidic and alkaline processes, respectively). The protein recovered after both solubilisation processes showed poor gelling properties than surimi but the acidic recovered proteins had the lowest gel strength. The washing of mince did not influence the whiteness of acidic protein recovered but the proteins recovered in the alkaline-aided solubilisation process from the washed mince were whiter.

Antioxidant and antibacterial activity of essential oil and extracts of bay laurel Laurus nobilis Linnaeus (Lauraceae) from Portugal

Laurus nobilis L. is an aromatic plant frequently used as a spice in Mediterranean cookery and as a traditional medicine for the treatment of several infectious diseases. The aim of this study was to characterise the antibacterial and antioxidant activities of bay laurel essential oil (EO), ethanolic extract (EE) and hot/cold aqueous extract (AE). The major components detected in bay laurel EO were eucalyptol (27.2%), α-terpinenyl acetate (10.2%), linalool (8.4%), methyleugenol (5.4%), sabinene (4.0%) and carvacrol (3.2%). The EO exhibited strong antibacterial activity against all tested foodborne spoilage and pathogenic bacteria, whereas this activity was less pronounced or even nonexistent in the EE and AE. In contrast, EO exhibited low antioxidant activity compared to extracts (EX), and among the EX, the hot AE revealed the highest antioxidant ability. The results show that bay laurel EO and its EX have potential as natural alternatives to synthetic food preservatives, in order to enhance food safety and increase food shelf life.

Purification of a functional competitive antagonist for calcitonin gene related peptide action from sardine hydrolysates

Calcitonin gene related peptide (CGRP) related molecules were purified from sardine hydrolysates prepared using 0.1% alcalase and two hours of hydrolysis. Gel exclusion chromatography and HPLC performed purification of these molecules. The purified molecules were characterised using specific CGRP radioimmunoassays and radioreceptoraasays. From 22 mg of crude extract, we obtained 14 µg of CGRP related molecules, the molecular weight determined by mass spectrophotometry was 6000 daltons. The biological activity of these molecules was analysed using the ability of CGRP to stimulate the adenylate cyclase activity in rat liver membranes. The purified molecules induced an inhibition of the CGRP stimulated adenylate cyclase activity, this effect was specific as no such effect was observed on the glucagon stimulated adenylate cyclase activity measured in the same rat liver membrane preparation. These results suggest that the purified molecules may act as antagonists for peptides that bind to CGRP receptors in rat liver membranes. These new antagonists may be of particular importance in various aspects of CGRP action in vertebrates.

Enzymatic hydrolysis of sardine (Sardina pilchardus) by-products and lipid recovery.

Raw and cooked sardine by-products from the canning industry were used to prepare protein hydrolysates and oil using food-grade 0.5% commercial enzymes (Alcalase®, Neutrase®, and ProtamexTM) and a water/fish ratio of 1:1. The highest nitrogen solubilization and degree of hydrolysis were obtained with Alcalase and Protamex both with raw and cooked sardines, but raw by-products were more easily hydrolyzed by these enzymes than the cooked sardine. The peptide profile of the hydrolysates reflected the specificity of each enzyme. The highest percentage of oil released was obtained from raw sardine, and Alcalase and Protamex were the most efficient. The oil released had a dark color and high peroxide value.

Functional and antioxidative properties of protein hydrolysates from Cape hake by-products prepared by three different methodologies.

BACKGROUND The production of fish protein hydrolysates (FPH) is a convenient technology for upgrading fish by-products. The aim of this work was to study three different methods of FPH preparation from Cape hake by-products to improve yield and quality. Functional and antioxidative properties of all FPHs were determined. RESULTS The protein content of hake FPH was in the range 807-860 g kg(-1) and the degree of hydrolysis was between 19% and 22%. The maximum yield (71.9%) was achieved by methodology B but the hydrolysate was darker. The peptide profile of all FPHs was very similar. FPH prepared by methodology C had significantly higher emulsifying activity index and hydrolysate prepared by methodology B had the highest foaming capacity. The solubility of FPH was in the range 71-76% and increased the water-holding capacity of minced fish by about 9%. The fractionation of FPH obtained by methodologies A and B allowed concentrating peptides with higher radical scavenging activity and reducing power. CONCLUSION The properties of the FPH prepared indicated that they can be used in food systems as natural additives, particularly to improve their water-holding capacity.

Effect of a Supplemented Diet with Canned Sardine on the Lipid Fraction of Human Plasma and Erythrocytes

Abstract Over the past 20 years many studies and clinical investigations have addressed the beneficial effects of polyunsaturated fatty acids in general and ω3 fatty acids in particular. It is currently known that ω3 fatty acids play important role in the prevention of several pathologies like the coronary heart disease, hypertension, diabetes and other. The protection played by ω3 fatty acids against coronary heart disease may be related to antiatherogenic effects and/or to the modification of risk factors through mechanisms related to lipid metabolism. The objective of this work was to evaluate, in healthy volunteers, the effect of a diet supplementation with canned sardine, which is rich in co3 fatty acids on the profile of plasma and erythrocytes, lipids, phospholipids and fatty acids. The serum levels of total cholesterol and high density lipoproteins (HDL) did not change during the period of the supplementation, tri-acylglycerol levels showed a trend for a decrease and low density lipoproteins (LDL)-derived thiobarbituric acid-reactive substances (LDL TBARS) levels showed a decrease. Incorporation of eicosapentaenoic and docosahexaenoic fatty acids (EPA and DHA, respectively) into plasma and erythrocyte phospholipids was observed. The results suggest that the undergone dietary supplementation may have allowed the incorporation of co3 polyunsaturated fatty acids into cell membranes and has increased the resistance of LDL to oxidative stress. This may represent protection factors against atherosclerosis and cardiovascular disease.

Preliminary Observations on Spoilage Potential of Flora from Desalted Cod (Gadus morhua)

Abstract Desalted cod products are already marketed, although still presenting some problems. Fresh raw materials and good manufacturing practices during salting, drying and desalting are essential to improve their quality and shelf life. The production of off-odor, hydrogen sulfide, trimethylamine, ornithine, putrescine, cadaverine, histamine, indole, hydrolyzed gelatin and ammonia was investigated in 127 bacterial strains isolated from dried/wet salted cod, later desalted either at 4 or 24°C. Desalting could either introduce or recover off-odor, sulfide hydrogen and/or ornithine producing strains. Cod desalting at 4°C is of great importance in order to improve the shelf life and safety of cod desalted products.