Protein Hydrolysis as a Way to Valorise Squid-Processing Byproducts: Obtaining and Identification of ACE, DPP-IV and PEP Inhibitory Peptides.

The industrial processing of Argentine shortfin squid to obtain rings generates a significant amount of protein-rich waste, including the skin, which is rich in collagen and attached myofibrillar proteins. This waste is generally discarded. In this study, skin was used as a source of proteins that were hydrolysed using Trypsin, Esperase® or Alcalase®, which released peptides with antioxidant potential and, in particular, antihypertensive (ACE inhibition), hypoglycemic (DPP-IV inhibition) and/or nootropic (PEP inhibition) potential. Among the three enzymes tested, Esperase® and Alcalase produced hydrolysates with potent ACE-, DPP-IV- and PEP-inhibiting properties. These hydrolysates underwent chromatography fractionation, and the composition of the most bioactive fractions was analysed using HPLC-MS-MS. The fractions with the highest bioactivity exhibited very low IC50 values (16 and 66 µg/mL for ACE inhibition, 97 µg/mL for DPP-IV inhibition and 55 µg/mL for PEP inhibition) and were mainly derived from the hydrolysate obtained using Esperase®. The presence of Leu at the C-terminal appeared to be crucial for the ACE inhibitory activity of these fractions. The DPP-IV inhibitory activity of peptides seemed to be determined by the presence of Pro or Ala in the second position from the N-terminus, and Gly and/or Pro in the last C-terminal positions. Similarly, the presence of Pro in the peptides present in the best PEP inhibitory fraction seemed to be important in the inhibitory effect. These results demonstrate that the skin of the Argentine shortfin squid is a valuable source of bioactive peptides, suitable for incorporation into human nutrition as nutraceuticals and food supplements.

Identification of Polyphenols in Sea Fennel (Crithmum maritimum) and Seaside Arrowgrass (Triglochin maritima) Extracts with Antioxidant, ACE-I, DPP-IV and PEP-Inhibitory Capacity.

Sea fennel and seaside arrowgrass are two abundant but underutilized halophytes along the Atlantic and Mediterranean coasts. This study investigated the antioxidant capacity and the potential antihypertensive (Angiotensin Converting Enzyme I, ACE-I inhibition), hypoglycaemic (Dipeptidyl Peptidase IV, DPP-IV inhibition), and nootropic (Prolyl Endopeptidase, PEP inhibition) activity of their polyphenol extracts. They had a high phenol content (21-24 mEq GA/g), antioxidant capacity evaluated using the ABTS (17-2 mg ascorbic acid/g) and FRAP (170-270 mM Mohr's salt/g) assays, and effective ACE-inhibiting properties (80-90% inhibiting activity at final concentration of 0.5 mg/mL). Additionally, the sea fennel extract displayed high DPP-IV inhibitory capacity (73% at 1 mg/mL), while the seaside arrowgrass extract exhibited potent Prolyl endopeptidase inhibitory capacity (75% at 1 mg/mL). Fractionation by HPLC concentrated the bioactive molecules in two fractions, for which the composition was analyzed by LC-MS/MS. Different chlorogenic acids seemed to play an important role in the bioactivity of sea fennel extract, and different flavonoids, mainly apigenin, luteolin and chrysoeriol, in the bioactivity of the seaside arrowgrass extract. Given their potential health benefits, these extracts could serve as valuable bioactive ingredients and could potentially encourage the cultivation of these species in regions where traditional crops face challenges in growth.

Evaluation of by-products from agricultural, livestock and fishing industries as nutrient source for the production of proteolytic enzymes.

This study presents an approach that utilizes low-value agro-industrial by-products as culture media for producing high-value proteolytic enzymes. The objective was to assess the impact of six agro-industrial by-products as culture media on the production of proteolytic enzymes. Bacillus subtilis strains, confirmed through comprehensive biochemical, morphological, and molecular analyses, were isolated and identified. Enzymatic activity was evaluated using azocasein and casein substrates, and the molecular sizes of the purified extract components were determined. The results demonstrated that the isolated bacteria exhibited higher metabolic and enzymatic activity when cultured in media containing 1 % soybean oil cake or feather meal. Furthermore, higher concentrations of the culture media were found to hinder the production of protease. Optimal protease synthesis on soybean oil cake and feather meal media was achieved after 4 days, using both the azocasein and casein methods. Semi-purification of the enzymatic extract obtained from Bacillus subtilis in feather meal and soybean oil cake resulted in a significant increase in azocaseinolytic and caseinolytic activities. Gel electrophoresis analysis revealed multiple bands in the fractions with the highest enzymatic activity in soybean oil cake, indicating the presence of various enzymes with varying molecular sizes. These findings highlight the potential of utilizing low-value agro-industrial by-products as efficient culture media for the sustainable and economically viable production of proteolytic enzymes with promising applications in various industries.

Identification of ACE I-Inhibitory Peptides Released by the Hydrolysis of Tub Gurnard (Chelidonichthys lucerna) Skin Proteins and the Impact of Their In Silico Gastrointestinal Digestion.

Tub gurnard is a highly abundant fishery species caught as a discard in the Mediterranean Sea. This work proposes its valorisation through the release of potential antihypertensive peptides and glycosaminoglycans (GAGs) through the controlled hydrolysis of tub gurnard skin proteins. Four proteases (Esperase, Alcalase, Trypsin and Pronase E) were used to obtain potent angiotensin converting enzyme I (ACE)-inhibitory hydrolysates. Peptides and GAGs were separated and evaluated for their antihypertensive potential by fluorometry. The peptide-rich fractions derived from the Esperase and Alcalase hydrolysates showed very low IC50 values (47 and 68 µg/mL, respectively). Only the GAGs from the Trypsin and Esperase hydrolysates were relevant ACE inhibitors (63 and 52% at 1 mg/mL, respectively). The peptide composition of the most potent ACE-inhibitory fractions derived from the Esperase and Alcalase hydrolysates (IC50 values of 33 and 29 µg/mL, respectively) was analysed by RP-LC-ESI-MS/MS. The analysis suggests that the ACE-inhibitory activity is related to the peptide hydrophobicity, as well as to the presence of specific residues at any of the last four C-terminal positions. The in silico gastrointestinal digestion of these fractions yielded small peptides with antihypertensive potential.

Antioxidant, Antihypertensive, Hypoglycaemic and Nootropic Activity of a Polyphenolic Extract from the Halophyte Ice Plant (Mesembryanthemum crystallinum).

This study aims to determine the potential antioxidant, antihypertensive, hypoglycaemic and nootropic activity of a purified polyphenolic extract from the halophyte ice plant (Mesembryanthemum crystallinum). The ice plant extract showed good antioxidant activity measured by DPPH, ORAC, TEAC, FRAP and ferrous ion chelating activity. Moreover, the extract showed potent ACE, DPP-IV and PEP-inhibitory activity (90.5%, 98.6% and 73.1%, respectively, at a final concentration of 1 mg/mL). The extract was fractionated and the fraction with the highest content of total phenolic compounds showed the highest bioactivity, suggesting that polyphenols could be mainly responsible for the abovementioned activities. The tentative polyphenol identification by HPLC-ESI-QTOF-MS in this fraction revealed that flavones (>65%) are the major group, with apigenin (38%) predominating, followed by diosmin (17.7%) and luteolin (11.9%). They could presumably be the main elements responsible for the enzymatic inhibition activity. Additionally, 4-hydroxybenzoic acid, p-coumaric acid and a hydroxycinnamic acid derivative (2-O-(p-cumaroyl)-l-malic acid) were found in the extract. To our knowledge, this is the first time that some of these activities have been reported for halophyte extracts.

Protein Hydrolysis and Glycosylation as Strategies to Produce Bioactive Ingredients from Unmarketable Prawns.

The present work shows a procedure to valorize non-commercial boiled shrimp to produce functional ingredients, using a combined treatment based on enzymatic hydrolysis and subsequent glycation under mild conditions. Antioxidant and prolyl endopeptidase-inhibiting activities were determined as a function of hydrolysis and glycation times (0-120 min and 0-180 min, respectively). The reaction products were characterized by determining the degree of hydrolysis, browning, fluorescent compounds, free amino acids, phenol content, Fourier transform infrared spectroscopy (FTIR), and molecular weight of the different fractions obtained. Enzymatic hydrolysis generated hydrolysates with significant antioxidant and prolyl endopeptidase-inhibiting activities. Glycation under mild conditions was used as a strategy to improve the antioxidant and potential nootropic properties of the hydrolysates. During glycation, the free amino acid content decreased, total phenols and fluorescent compounds increased significantly, and low molecular weight melanoidins were formed. The presence of peptide-glucose conjugates was also confirmed by FTIR. Glycation increased the antioxidant activities of the hydrolysates; however, their prolyl-endopeptidase-inhibiting activity was lost. Results showed that compounds with promising antioxidant (hydrolysis and glycation) and potential nootropic (hydrolysis) activities and applications in food systems were obtained from the biotechnological strategy used.

Halophytes as a potential source of melanosis-inhibiting compounds. Mechanism of inhibition of a characterized polyphenol extract of purslane (Portulaca oleracea).

The market value of crustaceans depreciates during storage due to the appearance of melanosis caused by polyphenol oxidases. Sulfite derivatives are used as melanosis-inhibiting agents, but their unhealthy effects make it preferable to replace them with natural preservatives. In this work, a crude enzymatic extract from whiteleg shrimp (Penaeus vannamei) was characterized and used to test the diphenol oxidase-inhibiting activity of polyphenol extracts of five underutilized halophyte plants, namely crystalline ice plant, seaside arrowgrass, purslane, sea fennel, and seashore aster. The extracts inhibited diphenol oxidase activity more efficiently than sodium sulfite. The purslane extract was rich in isoorientins, isovitexin, and apigenin, and showed the highest inhibiting effect, being this classified as mixed or non-competitive. Hydroxyl groups in the phenyl B ring could be responsible for the inhibitory activity of the extract. The polyphenol extracts tested in this work could be promising melanosis-inhibiting agents of interest for seafood industries.

Use of Sea Fennel as a Natural Ingredient of Edible Films for Extending the Shelf Life of Fresh Fish Burgers.

The growing interest from consumers toward healthy and nutritious products and their benefits for health has increased the consumption of whole and processed fish. One of the main problems of fish is the short shelf life, especially when it is processed as in the case of burgers. The use of edible coating is an interesting strategy to extend the quality and safety of the product, reducing the need for artificial preservatives. This study evaluated the use of chitosan-based edible film formulated with sea fennel plant and sea fennel extracts. The analyses showed than the use of edible film extended the shelf life of fish burgers regardless of the incorporation of sea fennel mainly associated to the gas barrier properties and selective permeability of the film applied to the fish surface. The incorporation of sea fennel in the films did not produce any antimicrobial enhancement, although sea fennel (mostly extract) produced a better pH and enhanced the antioxidant properties and lipid oxidation of fish burgers. However, sensory analyses showed than fish burgers coated with sea fennel film plant had better acceptability than those with sea fennel extracts, probably due to the better odour and colour of the whole plant during storage. The study showed that the use of sea fennel plant at 12.5% extended the shelf life of fish burgers using a safe and clean label strategy.

Exploring the potential of common iceplant, seaside arrowgrass and sea fennel as edible halophytic plants.

The recent interest in diversification in food consumption and the current salinization and desertification processes of farmland have placed the focus on halophytic plants as new food, making necessary the characterization of their biochemical composition and the identification of possible bioactive compounds. In this work, three edible halophytic plants were characterized: common iceplant (Mesembryanthemum crystallinum), sea fennel (Crithmum maritimum), and seaside arrowgrass (Triglochin maritima). The plants studied were a good source of minerals. Sea fennel showed high contents of dietary fibre and calcium (8.5 ppm, wet weight), common iceplant had a high potassium content (6500 ppm, wet weight), while seaside arrowgrass presented high levels of iron (62 ppm, wet weight). The glucose content of the three species was below 30 mg/g per dried weight. The Sb, Pb, Cr, As, Cd, and Hg content was negligible. Polyunsaturated fatty acids, mainly α-linolenic and linoleic acid, prevailed in the three species analyzed. Hydroxycinnamic and hydroxybenzoic acids predominated in common iceplant and sea fennel. Glycosylated flavones, especially isoorientin, prevailed in seaside arrowgrass. These plants present a relevant nutritional profile for which their use as foods or ingredients should be promoted.

Recent Advances in Astaxanthin Micro/Nanoencapsulation to Improve Its Stability and Functionality as a Food Ingredient.

Astaxanthin is a carotenoid produced by different organisms and microorganisms such as microalgae, bacteria, yeasts, protists, and plants, and it is also accumulated in aquatic animals such as fish and crustaceans. Astaxanthin and astaxanthin-containing lipid extracts obtained from these sources present an intense red color and a remarkable antioxidant activity, providing great potential to be employed as food ingredients with both technological and bioactive functions. However, their use is hindered by: their instability in the presence of high temperatures, acidic pH, oxygen or light; their low water solubility, bioaccessibility and bioavailability; their intense odor/flavor. The present paper reviews recent advances in the micro/nanoencapsulation of astaxanthin and astaxanthin-containing lipid extracts, developed to improve their stability, bioactivity and technological functionality for use as food ingredients. The use of diverse micro/nanoencapsulation techniques using wall materials of a different nature to improve water solubility and dispersibility in foods, masking undesirable odor and flavor, is firstly discussed, followed by a discussion of the importance of the encapsulation to retard astaxanthin release, protecting it from degradation in the gastrointestinal tract. The nanoencapsulation of astaxanthin to improve its bioaccessibility, bioavailability and bioactivity is further reviewed. Finally, the main limitations and future trends on the topic are discussed.

Glycosaminoglycans from grey triggerfish and smooth hound skins: Rheological, Anti-inflammatory and wound healing properties.

The present study aims to evaluate for the first time the wound healing and in vivo anti-inflammatory effects of glycosaminoglycans from skins of smooth hound (SHSG) and grey triggerfish (GTSG). Thermal analysis of GTSG and SHSG was evaluated using differential scanning calorimetry (DSC). The rheologie properties and water absorption capacity of two gels prepared from SHSG and GTSG were also studied. The application of GTSG and SHSG based gels on dermal full-thickness excision wounds in a mouse model, enhanced significantly wound healing activity and a total closure was achieved after eleven days of wound induction for SHSG. Further, histological examination of biopsies showed advanced tissue regeneration, characterized by the presence of well-organized stratum of both derma and epidermis. The anti-inflammatory evaluation of GTSG and SHSG in mice showed a significant inhibition of edema paw, after 5 h of carrageenan injection. The edema inhibition was 91.6% and 90% for SHSG and GTSG, respectively at the dose of 50 mg/kg. Furthermore, the histological evaluation and the superoxide dismutase, catalase and malondialdehyde level in muscle tissue were investigated. In summary, this work demonstrates that both GTSG and SHSG could be promising drugs with good wound healing and anti-inflammatory effects in animal model.

Fish skin gelatin hydrolysates produced by visceral peptidase and bovine trypsin: Bioactivity and stability.

The peptidase from the viscera of farmed giant catfish was used for producing gelatin hydrolysates (HG) and compared with those produced from commercial bovine trypsin (HB). The degree of hydrolysis (DH) observed suggests that proteolytic cleavage rapidly occurred within the first 120min of incubation, and there was higher DH in HG than in HB. HG demonstrated the highest ACE-inhibitory activity, DPPH, ABTS radical scavenging activity, and FRAP. HB showed the highest FRAP activity. The DPPH radical scavenging activity of HG was quite stable over the pH range of 1-11, but it increased slightly when the heating duration time reached 240min at 100°C. The ACE-inhibitory activity of HG showed the highest stability at a pH of 7, and it remained very stable at 100°C for over 15-240min. The visceral peptidase from farmed giant catfish could be an alternative protease for generating protein hydrolysates with desirable bioactivities. The resulting hydrolysates showed good stability, making them potential functional ingredients for food formulations.

Enhancement of ACE and prolyl oligopeptidase inhibitory potency of protein hydrolysates from sardine and tuna by-products by simulated gastrointestinal digestion.

This work was focused on the study of the bioactive potential of three fish protein hydrolysates, one of them prepared from industrial sardine by-products (head and viscera) and the others from tuna by-products (head, and muscle and viscera). These protein hydrolysates exhibited moderate ability to inhibit Angiotensin Converting Enzyme or ACE (IC50 between 0.24-1.16 mg dry weight per ml) and prolyl oligopeptidase or PO (IC50 between 3.30-9.57 mg ml(-1)), those obtained from tuna by-products being the most effective. Overall, ACE- and PO-inhibiting activities were enhanced by sequential nanofiltration through 3 and 1 kDa MWCO membranes (IC50 between 0.02-0.16 mg ml(-1) (ACE) and 1.10-4.21 mg ml(-1) (PO)). The inhibitory properties of the hydrolysates were greatly improved by in vitro gastric digestion, and were barely affected by further intestinal digestion. The digested tuna hydrolysates, mainly that from heads, proved to be the best source of PO- and ACE- inhibiting molecules (IC50 = 0.16 mg ml(-1) (ACE) and 1.04 mg ml(-1) (PO)) and could be potential new ingredients in food with interest in the prevention or treatment of cardiovascular and neurological diseases.

Gelatin hydrolysates from farmed Giant catfish skin using alkaline proteases and its antioxidative function of simulated gastro-intestinal digestion.

This work aims to evaluate the ability of different alkaline proteases to prepare active gelatin hydrolysates. Fish skin gelatin was hydrolysed by visceral alkaline-proteases from Giant catfish, commercial trypsin, and Izyme AL®. All antioxidant activity indices of the hydrolysates increased with increasing degree of hydrolysis (P<0.05). The hydrolysates obtained with Izyme AL® and visceral alkaline-proteases showed the highest and lowest radical scavenging capacity, while prepared with commercial trypsin was the most effective in reducing ferric ions and showed the best metal chelating properties. The hydrolysate obtained with Izyme AL® showed the lowest iron reducing ability, but provided the highest average molecular weight (⩾ 7 kDa), followed by commercial trypsin (2.2 kDa) and visceral alkaline-proteases (1.75 kDa). After in vitro gastrointestinal digestion, the hydrolysates showed significant higher radical scavenging, reducing ferric ions and chelating activities. Gelatin hydrolysates, from fish skin, could serve as a potential source of functional food ingredients for health promotion.

Sulfated polysaccharides from Loligo vulgaris skin: potential biological activities and partial purification.

The characteristics, biological properties, and purification of sulfated polysaccharides extracted from squid (Loligo vulgaris) skin were investigated. Their chemical and physical characteristics were determined using X-ray diffraction and infrared spectroscopic analysis. Sulfated polysaccharides from squid skin (SPSS) contained 85.06% sugar, 2.54% protein, 1.87% ash, 8.07% sulfate, and 1.72% uronic acid. The antioxidant properties of SPSS were investigated based on DPPH radical-scavenging capacity (IC50 = 19.42 mg mL(-1)), hydrogen peroxide-scavenging activity (IC50 = 0.91 mg mL(-1)), and ß-carotene bleaching inhibition (IC50 = 2.79 mg mL(-1)) assays. ACE-inhibitory activity of SPSS was also investigated (IC50 = 0.14 mg mL(-1)). Further antimicrobial activity assays indicated that SPSS exhibited marked inhibitory activity against the bacterial and fungal strains tested. Those polysaccharides did not display hemolytic activity towards bovine erythrocytes. Fractionation by DEAE-cellulose column chromatography showed three major absorbance peaks. Results of this study suggest that sulfated polysaccharides from squid skin are attractive sources of polysaccharides and promising candidates for future application as dietary ingredients.

Recovery, viscoelastic and functional properties of Barbel skin gelatine: investigation of anti-DPP-IV and anti-prolyl endopeptidase activities of generated gelatine polypeptides.

The characteristics and functional properties of gelatine from freshwater fish skin (Barbus callensis) were investigated. The gelatine extraction efficiency was improved by an acid-swelling process in the presence of barbel crude acid protease extract. Barbel skin gelatine (BSG) contained 92.15% protein, 0.31% lipid and 0.72% ash. The amino acid profile of BSG showed a high percentage of imino acids. The electrophoretic profile showed that BSG is mainly composed of α- and ß-components. BSG showed an excellent solubility and possessed interfacial properties, which were governed by the protein concentration. Biological activities of the hydrolysates obtained after digestion of BSG with several commercial proteases were evaluated. The results suggested that these hydrolysates are a good source of natural inhibitors of dipeptidyl peptidase-IV and prolyl endopeptidase and could potentially be used as dietary ingredients in the management of type 2-diabetes and/or neuropathological disorders.

Biochemical and antioxidant properties of peptidic fraction of carotenoproteins generated from shrimp by-products by enzymatic hydrolysis.

The composition, functional properties and in vitro antioxidative activity of the peptidic fraction of carotenoproteins from shrimp (Parapenaeus longirostris) by-products generated by enzymatic treatment with Alcalase was evaluated. The peptidic fraction of carotenoproteins (PFCP) contained 80.8 ± 0.21% protein, 2.74 ± 0.3% lipid, 14.4 ± 0.14% ash, 1.13 ± 0.08% chitin and 1.08 ± 0.02 µg total carotenoid/g of sample. The amino acid profile of PFCP showed a high percentage of essential amino acids, such as arginine, lysine, histidine and leucine. Therefore, PFCP had a high nutritional value and could be used as a supplement to poorly balanced dietary proteins. PFCP showed an excellent solubility and possessed interfacial properties, which were governed by their concentrations. The antioxidant activities of PFCP at different concentrations were evaluated using various in vitro antioxidant assays, including the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical method, reducing power, chelating effects assay and ß-carotene bleaching. The antioxidant activity of PFCP, based on their protection of supercoiled DNA strand from scission by peroxyl and hydroxyl radicals into the nicked circular form was also investigated. Results from this study suggest that the peptidic fraction of carotenoproteins is a good source of natural antioxidants and peptides with interesting functionalities.

Enhanced recovery of alkaline protease from fish viscera by phase partitioning and its application.

BACKGROUND: Too many different protein and enzyme purification techniques have been reported, especially, chromatographic techniques. Apart from low recovery, these multi-step methods are complicated, time consuming, high operating cost. So, alternative beneficially methods are still required. Since, the outstanding advantages of aqueous two phase system (ATPS) such as simple, low cost, high recovery and scalable, ATPS have been used to purify various enzymes. To improve purification efficiency, parameters affected to enzyme recovery or purity was investigated. The objectives of the present study were to optimize of alkaline protease recovery from giant catfish fish viscera by using ATPS and to study of hydrolytic patterns against gelatin. RESULTS: Using 70% (w/w) crude enzyme extract (CE) in system (15% PEG2000-15% sodium citrate) provided the highest recovery, PF and KE. At unmodified pH (8.5) gave the best recovery and PF with compare to other pHs of the system. The addition of 1% (w/w) NaCl showed the recovery (64.18%), 3.33-fold and 15.09 of KE compared to the system without NaCl. After addition of 10% (w/w) sodium citrate in the second ATPS cycle, the highest protease recovery (365.53%) and PF (11.60-fold) were obtained. Thus, the top phase from the system was subjected to further studied. The protein bands with molecular weights (MWs) of 20, 24, 27, 36, 94 and 130 kDa appeared on the protein stained gel and also exhibited clear zone on casein-substrate gel electrophoresis. The ß, α1, α2 of skin gelatin extensively degraded into small molecules when treated with 10 units of the extracted alkaline protease compared to those of the level of 0.21 units of Flavourzyme. CONCLUSIONS: Repetitive ATPS is the alternative strategy to increase both recovery and purity of the alkaline protease from farmed giant catfish viscera. Extracted alkaline protease exposed very high effectiveness in gelatin hydrolysis. It is suggested that the alkaline protease from this fish viscera can further be used in protein hydrolysate production.

Use of a commercial protease and yeasts to obtain CGRP-like molecules from saithe protein.

Different bioactive molecules, such as CGRP-like peptides, can be found in fish protein hydrolysates. Calcitonin gene-related peptide (CGRP) is a neuropeptide known to act as a potent arterial and venous vasodilator in humans. This study focuses on the industrial obtaining of CGRP-like molecules from saithe (Pollachius virens) byproduct, derived from the filleting process. Protein from P. virens was primarily hydrolyzed with Alcalase and later treated with Saccharomyces cerevisiae live cells. Treatment with Saccharomyces doubled the quantity of bioactive molecules obtained. The CGRP-like molecules were partially purified by chromatography, and the immunoreactive material was further analyzed for its CGRP-like bioactivity, using a specific radioreceptor assay. The concentration of CGRP-like molecules increased over 100-fold after purification. The bioactive molecules were able to induce cyclic AMP stimulation in rat liver membranes. Finally, partial sequencing of the bioactive peptide was performed, showing some homology with alpha-actin and myosin of several fish species.

Evidence of an active laccase-like enzyme in deepwater pink shrimp (Parapenaeus longirostris).

This paper demonstrates the presence of an active laccase-like enzyme from deepwater pink shrimp (Parapenaeus longirostris) using polyacrylamide gel electrophoresis. This enzyme was found in all anatomical parts of the deepwater pink shrimp, but particularly in the cephalothorax, and became active during the course of storage. Gel staining with laccase-specific substrates such as ADA, DMP and DAB was used to characterize a protein of around 44kDa as containing laccase activity. The enzyme was inhibited by a specific inhibitor, CTAB. 4-Hexylresorcinol, a specific inhibitor of polyphenoloxidase (PPO), did not inhibit the laccase-like enzyme. Low concentrations of antioxidants ascorbic acid or sodium metabisulphite were sufficient to inhibit the laccase-like enzyme. ABTS and DMP were subsequently used to characterize the enzyme. Given the evidence of this enzyme in deepwater pink shrimp, new melanosis-inhibiting compounds that are suitable for consumption need to be found to complement specific inhibitors of PPO activity.