Squid By-products Research Articles

Optimisation of Low-Toxicity Solvent Employment for Total Lipid and Tocopherol Compound Extraction from Patagonian Squid By-Products.

The extraction of total lipids and tocopherol compounds from Patagonian squid (Doriteuthis gahi) by-products (viscera, heads, skin, etc.), resulting from squid mantel commercialisation, was studied. An optimisation simplex-lattice design by employing low-toxicity solvents (ethanol, acetone, and ethyl acetate) was carried out taking into account their relative concentrations. The variance analysis of data showed that the quadratic model was statistically significant (p < 0.05); empirical coded equations were obtained as a function of the low-toxicity solvent ratios. The optimised lipid extraction was obtained by employing the 0.642/0.318/0.040 (ethanol/acetone/ethyl acetate) solvent ratio, respectively, leading to an 84% recovery of the total lipids extracted by the traditional procedure. In all extracting systems tested, the presence of α-, γ-, and δ-tocopherol compounds was detected, α-tocopherol being the most abundant. For α-, γ-, and δ-tocopherol compounds, the optimisation process showed that acetone extraction led to the highest concentrations in the lipid extract obtained (2736.5, 36.8, and 2.8 mg·kg-1 lipids, respectively). Taking into account the recovery yield on a by-product basis, the values obtained for the three tocopherols were included in the 88.0-97.7%, 80.0-95.0%, and 25-75% ranges, respectively, when compared to the traditional extraction. This study provides a novel and valuable possibility for α-tocopherol extraction from marine by-products.

Comparative Study of Bioactive Lipid Extraction from Squid (Doryteuthis gahi) by-Products by Green Solvents.

A novel approach of bioactive lipid extraction by different green solvents was carried out on squid (Doryteuthis gahi) by-products. By-products (viscera, heads, skin, tails, etc.), considered as a single product, were subjected to the following solvent systems: ethanol, acetone, ethyl acetate, 1/1 ethanol/acetone, 1/1 ethanol/ethyl acetate, and 1/1 acetone/ethyl acetate. Analyses carried out included lipid yield, lipid class content, and fatty acid (FA) composition. Results were compared to the lipid extract obtained by the traditional procedure (1/1 chloroform/methanol). Lipid yields obtained by green solvents led to a 33.4–73.2% recovery compared to traditional extraction; the highest values (p < 0.05) were obtained by ethanol-containing systems. Compared to the traditional procedure, ethanol systems showed an 85.8–90.3% recovery of phospholipid compounds and no differences (p > 0.05) in the ω3/ω6 ratio. Green-extracting systems led to higher average values for eicosapentaenoic acid content (15.66–18.56 g·100 g−1 total FAs) and polyene index (1.93–3.29) than chloroform/methanol extraction; differences were significant (p < 0.05) for systems including acetone and ethyl acetate. No differences (p > 0.05) were detected for docosahexaenoic acid content between the traditional procedure and green systems, with all values being included in the 31.12–32.61 g·100 g−1 total FA range. The suitability of EtOH-containing green systems for extraction of bioactive lipid compounds from squid by-products was concluded.

Threadfin bream surimi gel containing squid fin protein hydrolysate: Textural properties, acceptability, and volatile profile.

Gel properties of threadfin bream surimi gels with squid fin protein hydrolysate (SFPH) at various concentrations (0, 1, 2, 3, and 4%; w/w) were determined. The gel without SFPH (CON) had the highest breaking force (BrF) and deformation (DeF) as compared to those with SFPH (p<0.05). Among the gel with SFPH, gel containing 1 and 2% SFPH (SFPH-1 and SFPH-2, respectively) had the highest BrF, whereas the lowest value was obtained when 4% SFPH was used (SFPH-4) (p<0.05). The whiteness of all samples was slightly decreased with an upsurging amount of SFPH. However, water holding capacity was increased with augmenting levels of SFPH (p<0.05). Sensory analysis revealed that SFPH-2 had a higher squid odor and flavor likeness score than CON (p<0.05). Textural properties, especially hardness, were decreased with increasing SFPH, except for SFPH-1 and SFPH-2 (p>0.05). In addition, the rheological properties, microstructure, and volatile profile of the selected surimi gel were also studied. The storage modulus (G') of SFPH added samples was decreased as compared to the CON sample. The CON gel had a finer and more compact network as compared to SFPH-2 and SFPH-4 samples. Volatile profiles indicated that alcohols, carboxylic acids, ketones, and ether were the major volatile compounds present in both gel samples. Furthermore, thiophene, 3-methyl-, contributing to squid flavor, was found in the SFPH-2 sample. Overall, SFPH at 2% could act as a flavorant in the threadfin bream surimi gel without markedly negative impact on gelling and textural properties, while providing squid odor and flavor to the resulting gel. PRACTICAL APPLICATION: The various low-valued byproducts generated from the squid processing industry could result in various environmental problems. Those byproducts are rich in various biomolecules such as proteins, fats, enzymes, and so forth, which could be utilized to produce value-added products. Among them, protein hydrolysate (PH) rich in amino acids with excellent antioxidant properties could serve as an alternative flavorant. Therefore, PH from squid byproducts, especially fins, could be applied in foods such as fish balls, surimi gels, and so forth to enhance the nutritional and flavoring profile of a finished product. Moreover, bioactive peptides in PH with antioxidant potential could retard the oxidation of proteins and lipids.

Nutritional and Healthy Value of Chemical Constituents Obtained from Patagonian Squid (Doryteuthis gahi) By-Products Captured at Different Seasons.

This study focuses on the extraction of nutritional and healthy constituents of whole by-products resulting from the commercialisation of Patagonian squid (Doriteuthis gahi). By-products corresponding to squid individuals captured at different seasons were comparatively analysed for proximate composition, lipid classes content, fatty acid (FA) profile, and macroelement and trace element composition. As a result, moisture, lipid, protein, and ash values were included in the ranges 829.0–842.8, 17.5–21.8, 106.0–123.7, and 9.3–13.3 g·kg−1 by-products, respectively. Phospholipids showed to be the most abundant lipid class (359.2–463.5 g·kg−1 lipids), while triacylglycerols were only present in a 9.5–13.1 g·kg−1 lipids range. Valuable levels were detected for α-tocopherol (539.6–973.3 mg·kg−1 lipids), polyunsaturated fatty acids (PUFA; 50.5–52.6 g·100 g−1 FA), ω3 PUFA (47.0–48.6 g·100 g−1 FA), PUFA/saturated FA ratio (1.4–1.6), and ω3/ω6 ratio (12.1–13.4). Among macroelements, S, P, and Na showed to be more abundant than K, Mg, and Ca. Profitable levels of Co, Cu, Fe, Mn, Se, and Zn were detected in all kinds of individuals. In spite of content variations found as a result of the capture season of Patagonian squid individuals, whole by-products of this cephalopod species can be considered as a profitable source to provide the food and pharmaceutical industries with useful value-added constituents.

Characterization and bioactivities of phospholipids from squid viscera and gonads using ultra-performance liquid chromatography-Q-exactive orbitrap/mass spectrometry-based lipidomics and zebrafish models.

There has been great interest in phospholipids (PLs) from marine by-products due to their long-chain polyunsaturated fatty acids with unique health and functional properties. Here, marine PLs from squid viscera and gonads were comprehensively characterized and compared by UPLC-Q-Exactive Orbitrap/MS-based lipidomics analysis. A total of thirteen phospholipid classes including 1223 molecular species were identified and quantified in both resources. PC, PE and SM were further isolated from the total PLs of squid viscera and gonads, respectively. All isolated squid PL components were first evaluated for anti-inflammatory, antioxidant and cardiovascular effects using in vivo zebrafish models. Our results showed the diversity, content and physiological functions of PLs from squid by-products, which provided a basis for their future application in the nutritional and pharmaceutical industry.

Recovery of Proteins from Squid By-Products with Enzymatic Hydrolysis and Increasing the Hydrolysate’s Bioactivity by Maillard Reaction

ABSTRACTThe Maillard reaction is an effective method for enhancing the activity of bioactive peptides. In this study, squid by-product hydrolytic peptides (SPHPs) were obtained. The effects of the Maillard reaction on the antioxidant and antibacterial activities of the SPHPs were explored. The results showed most of the peptides in the < 500 Da fraction of SPHPs reacted with D-arabinose to form the 500–1000 Da fraction (89.60%). The Maillard reaction products (MRPs) possessed more potent antioxidant activities, including free radical scavenging activity, ferrous chelating capacity, and reducing power, than the SPHPs. Although both the SPHPs and MRPs had broad-spectrum antibiotic activities and could inhibit the growth of both gram-negative and gram-positive bacteria, the antibacterial activities of the MRPs were higher than those of the SPHPs. Furthermore, the efficacy of the MRPs lasted significantly longer than that of ampicillin and could last for more than 30 days. In addition, both the SPHPs and MRPs had good functional properties and many potential applications. The results suggested that the Maillard reaction has potential to be used to improve the activities and physicochemical properties of bioactive peptides.

Flavored-functional protein hydrolysates from enzymatic hydrolysis of dried squid by-products: Effect of drying method

Dried squid head is an abundant–low value by product from dried squid snack industry. The head is consisted of high protein content with the abundant sweet-umami amino acid, glutamic acid (7.45 mg/100 mg). The major volatile compounds found are trimethylamine and toluene along with the 15 important compounds exhibited dried squid flavor. Enzymatic hydrolysis with Flavourzyme® was performed at the optimal pH and temperature in order to produce flavored-functional protein hydrolysate. The supernatant was collected at 0, 30, 60, 90, 120, 150, 180 and 210 min of hydrolysis. The sweet-umami hydrolysate solution with the highest liking score at 6.64 was obtained after 180 min of hydrolysis. This sample was subjected to dry using freeze drying and foam-mat drying and investigated the changes of flavor compounds and functional properties. The freeze dried sample had umami taste and light brown color, and possessed the various types of volatile compounds. It contained protein at 76.42%, and EAI at 22.00, while the foam-mat dried sample had the highest ESI (15.27) and FS (29.87). The antioxidant property of the freeze dried sample (ABTS scavenging activity at 14.77 mg/g, FRAP scavenging activity at 5.93 mmol Fe2SO4 7H2O/g and DPPH scavenging activity at 19.51%, respectively) was higher than that of the foam-mat dried sample. Hence, it could be used as flavored-functional ingredient in foods.

Squid by-product gelatines: Effect on oxidative stress biomarkers in healthy rats

Chan-Higuera J.E., Robles-Sanchez R.M., Burgos-Hernandez A., Marquez-Rios E., VelazquezContreras C.A., Ezquerra-Brauer J.M. (2016): Squid by-product gelatines: Effect on oxidative stress biomarkers in healthy rats. Czech J. Food Sci., 34: 105–110. Gelatines from three different jumbo squid (dosidicus gigas) by-products (fins, arms, and skin) were compared based on their chemical and biochemical properties (amino acid composition, molecular weight distribution, and in vitro digestibility), antioxidant capacity (ABTS+ and ORAC assays) and effect on oxidative stress biomarkers (serum antioxidant capacity (TAC) and serum malondialdehyde levels (MDA)) in healthy rats. Gelatine from the skin showed the highest polar and imino amino acid contents and a higher proline hydroxylation degree. Gelatine β-component was not detected in either fins or arms. These differences may explain the higher in vitro digestibility and higher antioxidant capacity (before and after digestibility) of the skin gelatine. Fin gelatines decreased TAC-ORAC values. All obtained gelatines decreased the MDA levels. Jumbo squid gelatine, administered during feeding, may help decrease a breakdown product of spontaneous lipid peroxidation in serum.

In vitro antioxidant, antimutagenic and antiproliferative activities of collagen hydrolysates of jumbo squid (Dosidicus gigas) byproducts

Hydrolysates from two different jumbo squid byproducts (fins and arms), produced by trypsin and protease type XIV were compared on the basis of their antioxidant (DPPH and ABTS radical scavenging assays), antimutagenic (Ames test) and antiproliferative (Transformation cell proliferation in M12.C3F6 murine cells) activities. Jumbo squid arms had higher content of collagen than fins, and their hydrolysates had the highest antioxidant activity. Also, jumbo squid arm-derived collagen hydrolyzed with protease XIV showed the highest antimutagenic activity. The four hydrolysates obtained showed low antiproliferative activity, however they are susceptible for further studies to be applied as food additives.

Physicochemical Characterization of Protein Hydrolysates Produced by Autolysis of Jumbo Squid (Dosidicus gigas) Byproducts

The physicochemical characteristics of protein hydrolysates of jumbo squid (Dosidicus gigas) byproducts (JSBP) produced by endogenous proteases at two different pH values (5.0 and 7.0) were studied using destructive and nondestructive methods. Reaction mixture samples were collected at different interval times during hydrolysis to monitor changes in the level of hydrolysis (DH) using the OPA method, the protein peptide molecular masses (MM), and SDS-PAGE. The DH increased from 3.5 to 11.2 % at pH 5.0 and from 4.8 to 17.5 % at pH 7.0. Both pH treatments exhibited similar degradation patterns with progressive proteolysis and, after 120 min of hydrolysis, yielded hydrolysates that contained MM <45 kDa proteins. It was detected lower hydrophobic amino acid exposure for the protein hydrolysates prepared at pH 5.0 compared with the hydrolysates at pH 7.0. In several wavebands, higher wavenumbers were observed in the FT-IR spectra for the pH 5.0 hydrolysates. Nine different equivalent protons were observed in the NMR spectra for both hydrolysates; these protons might belong to amino acid side chains. SEM showed substantially lower particle size for the JSBPs after hydrolysis at pH 7.0. The hydrolysate zeta potentials were −29.4 mV at pH 7.0 and −10.5 mV at pH 5.0. The pH 5.0 hydrolysates exhibited lower endothermic resistance and hydrophobicity (SoANS) compared with the pH 7.0 hydrolysates. This biophysical characterization enhances the understanding of jumbo squid byproduct hydrolysate physiochemical properties, which will aid in determining the proper use of these hydrolysates.

Evaluation of jumbo squid (Dosidicus gigas) byproduct hydrolysates obtained by acid-enzymatic hydrolysis and by autohydrolysis in practical diets for Pacific white shrimp (Litopenaeus vannamei)

The marine bioprocessing industry offers great potential to utilize byproducts for fish meal replacement in aquafeeds. Jumbo squid is an important fishery commodity in Mexico, but only the mantle is marketed. Head, fins, guts and tentacles are discarded in spite of being protein-rich byproducts. This study evaluated the use of two jumbo squid byproduct hydrolysates obtained by acid-enzymatic hydrolysis (AEH) and by autohydrolysis (AH) as ingredients in practical diets for shrimp. The hydrolysates were included at levels of 2.5 and 5.0% of the diet dry weight in four practical diets, including a control diet without hydrolysate. Shrimp growth and survival were not significantly affected by the dietary treatments. Postharvest quality of abdominal muscle was evaluated in terms of proximate composition and sensory evaluation. Significantly higher crude protein was observed in the muscle of shrimp fed the highest hydrolysate levels, AH 5% (204.8 g kg - 1) or AEH 5% (201.3 g kg - 1). Sensory analysis of cooked muscle showed significant differences for all variables evaluated: color, odor, flavor, and firmness. It was concluded that Jumbo squid byproducts can be successfully processed by autohydrolysis or acid-enzymatic hydrolysis, and that up to 5.0% of the hydrolysates can be incorporated into shrimp diets without affecting growth or survival.

Effectiveness of Squid Hydrolysate as a Home Lawn Fertilizer

Consumer demand for cleaned squid generates a substantial amount of waste that must be properly disposed of, creating an economic burden on processors. A potential solution to this problem involves converting squid byproducts into an organic fertilizer, for which there is growing consumer demand. Organic fertilizers are reputed to offer advantages that synthetic fertilizers cannot provide, such as increasing soil water- and nutrient-holding capacity and promoting the growth of beneficial soil organisms. To evaluate the effectiveness of hydrolyzed squid waste as an organic fertilizer, we quantified soil fertility and turfgrass quality on perennial ryegrass turf (Lolium perenne L.) amended with two types of fertilizer: squid-based (SQ) or synthetic (SY). Field plots were established on an Enfield silt loam in Kingston, RI, and liquid (L) or granular (G) fertilizer formulations of squid or synthetic fertilizers were applied at 0, 48, 146, and 292 kg nitrogen/ha/year. Soil physical, chemical, and biological properties were determined monthly and turfgrass quality was determined periodically during the growing season in 2008 and 2009. Squid hydrolysate applied as a liquid (SQL) and granular (SQG) fertilizer consistently provided high-quality, uniform turf when compared with synthetic fertilizer applied at the same rate. Soil concentrations of NO3, NH4, PO4, pH, moisture, soil organic matter, C:N ratio, and levels of trace metals were unaffected by fertilizer type, formulation, or rate throughout the two-year study. Both squid-based organic fertilizer formulations gave significantly higher microbial activity rates than their synthetic counterparts regardless of application rate. Conversion of squid processing byproducts into fertilizer has the potential to improve turfgrass quality while providing a sustainable solution to waste disposal problems in the seafood processing industry.

오징어 부산물의 급여가 한우암소 산육 및 도체특성에 미치는 영향

본 연구는 한우 암소의 산육 및 도체특성에 있어 오징어 부산물의 급여 효과를 알아보기 위해 수행되었다. 한우 16두(나이 7세, 체중<TEX>$484.1{\pm}63.6$</TEX> kg)를 대조구 및 처리구(오징어 부산물 첨가), 등 2그룹(각각 8두)으로 완전임의 배치하였다. 사료급여는 두 그룹 모두 자유채식토록 하여 150일간 시험을 수행하였다. 일당증체량은 처리구가 대조구 보다 유의적으로 높았다(p<0.05). 시험기간 내 사료섭취량 및 사료요구율은 처리구가 대조구 보다 유의적으로 낮았다(p<0.05). 육량특성에 있어서, 등지방두께, 등심단면적 및 육량지수는 두 처리구간 비슷한 경향을 나타내었고, 도체중은 처리구가 363.4 kg 대조구 353.1 kg으로 처리구간 유의적 차이는 없었다. 육질특성에서는 육색, 지방색, 조직감 및 성숙도는 두 그룹간 유의적 차이가 없었다. 그러나 근내지방도 및 육질 1등급 이상 출현율은 대조구 보다 처리구가 높았다. 그러므로 본 연구의 결과 오징어부산물의 급여는 한우 암소의 산육 및 도체특성을 향상시키는 것으로 생각된다. This study was conducted to evaluate the effect of feeding squid by-products on the growth performance and carcass characteristics of Hanwoo cows. Sixteen Hanwoo cows, aged 7 years and weighing <TEX>$484.1{\pm}63.6$</TEX> kg, were randomly allocated to 2 feeding groups (8 animals per group): control and treatment (fed squid by-products). Both groups were fed ad libitum for 150d. Average daily gain was significantly higher in the treatment group than in the control group (p<0.05). During the experimental period, DMI and feed conversion ratios were significantly lower in the treatment group than in the control group (p<0.05). The results of yield traits, back fat thickness, rib eye area, and yield index were similar between the two groups. Carcass weights were 363.4 kg and 353.1 kg for treatment and control, respectively, but these differences were not statistically significant. Quality traits such as meat color, fat color, texture, and maturity were also not significantly different between the two groups. However, marbling score and rate of appearance of high quality grade (<TEX>$1^{++}$</TEX>, <TEX>$1^+$</TEX>, and 1) were higher in the treatment group than in the control group. These results suggest that feeding squid by-products may improve the growth performance and carcass characteristics of Hanwoo cows.

Nitrate and Phosphate Leaching under Turfgrass Fertilized with a Squid-based Organic Fertilizer

Consumer demand for cleaned squid generates a substantial amount of waste that must be properly disposed of, creating an economic burden on processors. A potential solution to this problem involves converting squid by-products into an organic fertilizer, for which there is growing demand. Because fertilizer application to lawns can increase the risk of nutrient contamination of groundwater, we quantified leaching of NO3–N and PO4–P from perennial ryegrass turf (Lolium perenne L.) amended with two types of fertilizer: squid-based (SQ) and synthetic (SY). Field plots were established on an Enfield silt loam, and liquid (L) and granular (G) fertilizer formulations of squid and synthetic fertilizers were applied at 0, 48, 146, and 292 kg N ha−1 year−1. Levels of NO3–N and PO4–P in soil pore water from a depth of 60 cm were determined periodically during the growing season in 2008 and 2009. Pore water NO3–N levels were not significantly different among fertilizer type or formulation within an application rate throughout the course of the study. The concentration of NO3–N remained below the maximum contaminant level (MCL) of 10 mg L−1 until midSeptember 2009, when values above the MCL were observed for SQG at all application rates, and for SYL at the high application rate. Annual mass losses of NO3–N were below the estimated inputs (10 kg N ha−1 year−1) from atmospheric deposition except for the SQG and SYL treatments applied at 292 kg N ha−1 year−1, which had losses of 13.2 and 14.9 kg N ha−1 year−1, respectively. Pore water PO4–P levels ranged from 0 to 1.5 mg P L−1 and were not significantly different among fertilizer type or formulation within an application rate. Our results indicate that N and P losses from turf amended with squid-based fertilizer do not differ from those amended with synthetic fertilizers or unfertilized turf. Although organic in nature, squid-based fertilizer does not appear to be more—or less—environmentally benign than synthetic fertilizers.

Process Optimization and Composition Analysis of Antioxidant Enzymatic Hydrolysate from Squid By-Products by Papain

In order to evaluate the high-value application of squid by-products yielded hydrolysate, the process of preparation and purification technology, chemical composition and in vitro antioxidant activity of the hydrolysate were investigated. The optimal conditions of papain hydrolysis were obtained by single-factor experiments and orthogonal test with the DPPH• scavenging ratio as index, amino acid composition was analysed by automatic amino acid analyzer, the hydrolysate was isolated with a Sephadex G-25 column. Based on single-factor experiments, the hydrolysate with the DPPH• scavenging ratio being 53.96 % was gained under the optimal condition of enzymolysis temperature of 45 °C, enzymolysis time of 3 h, total enzyme dose of 1.2 %, and the pH value of 7. The protein content of the hydrolysate reached up to 17.53 %, and the essential amino acids were accounted for 51.06 % of total amino acids. The largest content amino acid was glutamic acid, which accounted for 10.74 % of total amino acids. Compared with the amino acid profiles recommended by FAO/WHO, the quality of the protein hydrolysate was high, as it was rich in essential amino acids, including isoleucine, leucine, lysine, methionine and cystine, threonine, tryptophan, and valine, which covered 88 %-100 % of the FAO/WHO recommended. The hydrolysate was divided into three fractions (F1-F3) using a Sephadex G-25 column, the F1 possessed the highest antioxidation activity with the reducing power, •OH and DPPH• scavenging ratio being 0.236, 18.13 % and 63.85 % at the concentration of 5 mg/mL. Compared with the retention time of the reduced glutathione chromatomap, the relative molecular mass of F1 was higher than 307, F2 and F3 was lower than 307. The result revealed that the protein hydrolysate from squid by-products by papain had strongly antioxidant capacity in vitro and high nutrition, and this finding provided a new way of advanced exploitation of squid scrap resources.

오징어 가공 부산물의 해양투기에 따르는 주변해양환경의 영향에 대한 검토

Squid by-product like viscera has just been recognized as one of the serious pollutants on the surrounding marine environment in Ulleung island of Korea. The effect as a marine pollutant, however, has not been examined and evaluated properly. It was carried out the comprehensive examination of the marine pollution by disposing squid by-products. The sediments and water qualities were investigated in several positions, including the dumping position around Ulleung island. It is also estimated a spreading of by-product in terms of estimating the sinking speed and evaluating the attracting characteristics as a feed. As a result, There were no significant pollutions of the sediment and water quality. The spreading of by-product by the local current was 2-2.5 times of the depth, and it will become longer in spring tide. It was confirmed that the squid by-product has attracting ability for local fish by acoustical survey. Furthermore, it can be useful for the local fish species as feeds.

Optimization of Enzyme-Assisted Squid Flavor Production Using an Orthogonal Array Method

Squid flavor from denatured or undenatured squid processing by-products was produced by enzymatic hydrolysis using Flavourzyme 1000 MG and Protamex (Novo Nordisk BioChem). In process optimization, an orthogonal array method L4(23) was used to determine the effect of processing variables on total volatile base N, total free amino N, squid flavor, and bitterness. The critical variables in the influential order were denaturation treatment, hydrolysis time, and maturation for flavor development. Enzymatic hydrolysis of undenatured squid by-products increased 24.8% taste-active free amino acids from 8.2% for the control. The most favorable flavor profile was obtained from raw squid by-products after 6 hr hydrolysis at 50_C, followed by 2 hr maturation at 90_C This work was funded by the National Oceanic and Atmospheric Administration (NOAA). Contribution No. 3634 of the College of Resource Development, University of Rhode Island, with support from the Rhode Island Agricultural Experiment Station. Thanks to Seafood Processing of RI and Town Dock, Inc., Narragansett, RI, for supplying squid processing by-products, and NovoNordisk BioChem for providing enzymes.