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This study was conducted to develop an immobilized-enzyme system to entrap lipase in chitosan-alginate-CaCl(2) beads for the purpose of concentrating n-3 polyunsaturated fatty acids (n-3 PUFAs) from sardine oil. Lipase was immobilized by an ionotropic gelatin method analyzed for characteristics. Optimum pH of immobilized lipase shifted from pH 7.0 to 6.0 and immobilized lipase showed higher stability against pH and temperature changes. Original sardine oil contained 38.1% n-3 PUFAs (25.2% 20:5n3 and 7.20% 22:6n3), and the concentration was significantly increased to 65.3% (40.2% 20:5n3 and 15.5% 22:6n3) with free lipase and to 64.8% (39.6% 20:5n3 and 15.3% 22:6n3) with immobilized lipase after 90 min of repeated hydrolysis. Fatty acid content of the free fatty acid (FFA) fraction of hydrolyzed oil showed that lipase preferably hydrolyzed 16:0, 16:1n7 and 18:0 accounting for 76.6% and 69.5% of total FFAs (after 1st and 2nd hydrolysis, respectively). This study shows that use of immobilized lipase systems for increasing n-3 PUFA concentration in sardine oil provides new processing opportunities for the industry.

A new oil extraction method involving pH adjustment was developed and compared with the traditional heat extraction method. Sardine oil was obtained by adjusting the pH to the isoelectric point (pI) of sardine muscle (pH 5.5) using HCl or food-grade organic acids [with/without calcium (Ca)] followed by centrifugation. Oil extracted with citric acid plus Ca showed the best quality, along with good recovery, the lowest haze value, and the highest n-3 polyunsaturated fatty acid content. All oils extracted by pH adjustment exhibited high stability against oxidation supported by low conjugated dienes and thiobarbituric acid reactive substance level with fewer impurities compared to the heat process. There was a significant beneficial effect of Ca addition prior to pH adjustment in terms of lipid oxidation stability and color of the final products. The pH adjustment method is a novel process for oil extraction and a promising method that can be utilized for high-oil pelagic species such as Pacific sardines. Key...

The production of n − 3 polyunsaturated fatty acids (n − 3 PUFAs) concentrate from oil extracted from Pacific sardines (Sardinops sagax) was studied using lipase-catalyzed hydrolysis. Commercially available microbial lipases, from Candida Rugosa (CR), Candida cylindracea (CC), Mucor javanicus (MJ), and Aspergillus niger (AN) were used for enzymatic hydrolyses with extracted sardine oil, run at 37 °C with constant stirring for 1.5, 3, 6, and 9 h. Fatty acid composition analysis by gas chromatography showed that the refined unhydrolyzed oil contained 26.86% of eicosapentaenoic acid (EPA) and 13.62% of docosahexaenoic acid (DHA) (wt/wt%). CR lipase was the most effective in concentrating n − 3 PUFA. Hydrolysis with 250 U CR lipase increased EPA concentration to a relatively constant level of 33.74% after 1.5 h. DHA levels were also significantly increased from 13.62% to 29.94% with 500 U after 9 h. Compared to CR and CC lipases, MJ and AN lipases resulted in low n − 3 PUFA concentration. Triacylglycerol levels decreased significantly as reaction time progressed.

Recovery of bioactive compounds, such as proteolytic enzymes, from waste streams is a means to both recuperate value and reduce environmental pollution. Previously optimized lab-scale parameters for the recovery of a stable crude protease fraction from Pacific whiting (Merluccius productus) surimi wash water were tested using pilot plant equipment. Pretreatment of surimi wash water with 60 degrees C heat, acidification to pH 6, and centrifugation doubled ultrafiltration membrane flux and significantly improved protease purity by reducing a majority of the 35-205 kDa proteins. Concentrated crude protease obtained from wash water contained predominantly cathepsin L activity. Enzyme purity was increased about 100-fold, and yield was approximately 80%. Stability (frozen and freeze-dried protease) was maintained for 9 weeks at -80 degrees C. Freeze-dried preparations were also stable for 9 weeks at 4 and -15 degrees C. Successful application of pilot plant conditions allows for sufficient production of protease for further investigations into their applicability.

Proteases are important bioactive compounds that have many applications in food processing. In this study, laboratory scale experiments were performed to establish conditions for recovery of a heat stable, acid protease from Pacific whiting ( Merluccius productus ) surimi process water. Reduction of proteinaceous solids and recovery of protease activity was maximized when process water was pre-treated with acid (pH 4) followed by heat (60 °C). In addition, acid plus heat treatment of process water appeared to improve membrane flux and concentration of protease activity (10-fold) was achieved in half as much time as treatments using acidification but not heat. Neither purification nor concentration of protease was effective using either 300 and 1000 kDa ultrafiltration or 0.3 μm microfiltration membranes. However, concentration of protease using 50 kDa ultrafiltration membranes was successful in recovering about 80% of original protease activity. Results provide conditions for further investigations into pilot plant recovery of protease from surimi process water.

Studies were conducted to investigate the development of membrane fouling in the microfiltration of protein solutions such as surimi wash water, containing both soluble and suspended proteinaceous solids. A laboratory scale plate-and-frame crossflow membrane filtration unit was fabricated and installed. Continuous filtration was performed to recover the suspended myofibrillar proteins from the wash water at refrigerated temperatures (12–15°C) using polysulfone membranes. The transmembrane pressure was controlled at a constant pressure of 34.5±2.2 KPa, and the feed was pumped to the filtration unit at a crossflow velocity of 0.262±0.026 m/s. It was hypothesized that the development of membrane fouling was a dynamic process of two distinctive stages. The pore blocking resistance was the dominant resistance at the initial period of filtration, and the cake resistance began to dominate following the initial pore blocking. Experimental results showed that the initial membrane fouling process could be modeled by the standard pore blocking law, and the development of fouling continued with a continuous process of cake formation. A new concentration-dependent power law cake resistance model was found. The filtration resistance of the cake layer increased with the feed concentration and was found to be one order in magnitude higher than the initial dominant pore blocking resistance.

Pacific whiting surimi wash water (SWW) proteinase was recovered by ohmic heating, ultrafiltration, and freeze-drying. By these processes, 5.9-fold purification was achieved. The most efficient step was ohmic heating, which concentrated the proteinase by 4.8 fold. Specific activity of the recovered SWW proteinase on casein and Z-Phe-Arg-NMec was 28.2 and 0.17 U/mg protein, respectively. The SWW proteinase showed good hydrolytic activity towards casein, acid-denatured hemoglobin and myofibrils. Acidification increased specific activity on all substrates tested but reduced thermal stability. β-Mercaptoethanol, dithiothreitol and urea enhanced activity against Z-Phe-Arg-NMec. Proteinase activity on Z-Phe-Arg-NMec showed an optimum pH of 4.0. The recovered proteinase showed 18.5% residual activity after 7 week storage at 4C.

Alcalase and Neutrase showed optimum activity against Pacific whiting solid wastes (PWSW) at pH 9.5, 60 °C and pH 7.0, 55 °C, respectively. Alcalase had a higher proteolytic activity than Neutrase. Enzyme concentration, reaction time, and waste/buffer ratio significantly affected the hydrolysis and nitrogen recovery (NR) (p < 0.05). Optimum conditions for PWSW hydrolysis were 20 AU Alcalase/kg, 1 h reaction time, waste/buffer ratio of 1:1 (w/v). Correlation between the degree of hydrolysis (DH) and NR (R2 = 0.970−0.978) was high. Freeze-dried hydrolysate was brownish yellow in color (L* = 54.59, a* = 6.70, b* = 27.89) and contained 2.77% moisture, 79.97% protein, 13.44% ash, and 3.83% lipid. Amino acid composition of freeze-dried hydrolysate was similar to that of PWSW and Pacific whiting muscle but tryptophan was reduced to 21.50% and 14.74%, respectively. Keywords: Waste; Pacific whiting; hydrolysate; Alcalase; Neutrase

Gel strength enhancement of Pacific whiting surimi was studied by using an α2-macroglobulin (α2-M)-enriched plasma fraction (FIV-1) and a transglutaminase (TG)-enriched plasma fraction (FI-S). Fluorescent amine-incorporating activity was detected in FIV-1, FI-S, and bovine plasma protein (BPP), indicating potential protein cross-linking activity by α2-M and TG. Inhibition of autolytic activity was observed with FIV-1 and BPP. FIV-1 in combination with bovine serum albumin, fibrinogen, or FI-S enhanced gelation more than FIV-1 alone. These results indicate various components in BPP function both to enhance gelation of Pacific whiting surimi and to inhibit proteolysis. Keywords: Plasma protein; transglutaminase; α2-macroglobulin; surimi; gelation

A proteinase from Pacific whiting surimi wash water (SWW) was recovered by ohmic heating, ultrafiltration and freeze drying with overall yield of 0.83 g protein/L SWW and 78% recovery of activity. Ohmic heating conditions were optimized for the maximum recovery of the enzyme. Different applied voltages (50, 70, 90 V) showed no differences in efficiency for removing protein and retaining cathepsin L activity. Cathepsin L activity reached its maximum after ohmic heating to 55C whereas cathepsin B activity decreased constantly with increased temperature. A constant reduction in protein content was observed with the increase in temperatures from 45C to 60C and holding time up to 5 min. The highest retention of both total and specific activity of cathepsin L was obtained with the treatment at 55C for 3 min. Under these conditioner, 193% activity was recovered from SWW although a large amount of the activity was lost by the subsequent steps of ultrafiltration and freeze drying.

A proteinase in Pacific whiting surimi wash water (SWW) was characterized to be cathepsin L based on molecular mass (M r ), substrate specificity, and SDS-substrate gel electrophoresis. The proteinase was highly active on Z-Phe-Arg-NMec, and the native M r was 27,400 based on size exclusion (SEC)-HPLC. Acidification of the SEC-HPLC fractions showed a two-fold increase in activity on Z-Phe-Arg-NMec. SWW proteolytic activity was found at M r 54,200 on SDS-substrate gel. However, acidification shifted the activity zone to M r 39,500 corresponding to cathepsin L. No evidence of activity by calpain or cathepsin B or H was found in Pacific whiting SWW.

The most effective inhibition of autolytic proteinase activity was found at 3% whey protein concentrate (WPC) with residual proteinase activity at 14.2%. WPC reduced papain and trypsin activities linearly with lowest residual activities of 8.7% and 41.4%, respectively. An unidentified high-molecular-weight protein was inhibitory for papain and trypsin with no inhibitory components detected at the region

The chemical structure of an antioxidant purified from shrimp shell was analyzed. Positive reactions with both Folin−Ciocalteu's phenol reagent and ferric chloride−potassium ferricyanide reagent in...

Cathepsin L from Pacific whiting was separated into two activity peaks (P-I and P-II) on butyl-Sepharose. Acidification of the fractions at pH 3.3 resulted in an 11-fold increase in activity of P-I and a slight decrease in P-II on Z-Phe-Arg-NMec, suggesting that P-I is complex-formed with an inhibitor while P-II is the free enzyme. Analysis of further purified DEAE P-I fractions showed that only 55% of the activity was titrated by E-64 while low-pH-treated fractions were completely titrated. The DEAE P-I fractions showed highest pH stability at pH 4, while acidified DEAE P-I fractions showed the highest stability at pH 7.0

Micro- and ultrafiltration were utilized for recovery of proteins and for water recycling from commercial surimi processing waste water. The proteins recovered by microfiltration showed highly functional properties and composition comparable with proteins in regular surimi. Surimi with a substitution (10%) of recovered proteins did not diminish in gel hardness, elasticity, color, or water retention. Aerobic plate count, chemical oxygen demand, protease activity, and turbidity in the surimi waste waters were reduced by ultrafiltration. Results indicated a potential for re covering proteins to extend yield and recycling water

Two forms (P-I and P-II) of a protease were purified to 560- and 21.5-fold from the sarcoplasmic fluid of Pacific whiting (Merluccius productus). The enzymes were assayed by hydrolysis of azocasein at 55 degrees C and pH 5.5. Both forms were composed of a single polypeptide of Mr 28 800; however, P-I also contained two low Mr components which were removed by low pH treatment prior to size exclusion chromatography. P-I and P-II showed pH optima at 5.5 and 6.0 and temperature optima of 55 and 60 degrees C, respectively. The enzymes had an identical isoelectric point of 4.91 with a minor band at 4.94. The proteases had high activity toward Z-Phe-Arg-NMec and casein. The activity of both forms was inhibited by all sulfhydryl reagents tested as well as the cathepsin L-specific inhibitor Z-Phe-Phe CHN2 and activated by reducing agents, suggesting the enzymes are forms of cathepsin L.

The effects of varying concentrations of salt and pH on the gel strength of Pacific whiting (Merluccius productus) surimi were investigated. Surimi gels were made with and without the protease inhibitor beef plasma protein (BPP). Gel strength was measured by torsion and reported as shear stress and shear strain. In general, surimi gels increased in gel strength with increased pH. Stress increased to a greater degree than strain about pH 7. Water holding capacity values increased up to pH7 and than leveled off. Surimi gels made with low salt (0.9%) or not salt had greater stress values that the higher salt surimi gels in the alkaline pH range. The results demonstrated that the effects of salt and pH are interactive on gels strength for Pacific whiting surimi

Beef plasma protein (BPP), egg white and potato extract were tested for their ability to inhibit proteolysis in fish mince and surimi made from Pacific whiting (Merluccius productus). Strong inhibition resulted from all three compounds in fish mince when measured by autolysis. However, when tested in surimi significant differences occurred among the compounds. BPP showed strongest inhibition of proteolytic effect followed by egg white and potato extract when measured by autolysis, gel electrophoresis and torsion. BPP was an effective inhibitor in surimi at a concentration as low as 1%.

The effects of high hydrostatic pressure (HHP) on gel strength, microbial numbers, proteolytic activity, color and pH of Pacific whiting surimi gels were investigated. Strong gels without the use of protease inhibitors were formed with HHP treatments from 1–4 kBars. Gel strength was not affected by holding time. Total plate counts showed that destruction of vegetative cells was accomplished with 4 kBar. Proteolytic activity was diminished but not eliminated with pressure treatments. The color of the surimi gels treated were translucent while the heat-treated gels were opaque. The pH was increased slightly with HHP treatment. HHP was an effective processing method for making high quality surimi gels from Pacific whiting.

Protein denaturation in Pacific whiting fillets stored at −8, −20, −34, and −50°C was investigated over a 10-mo period by several methods, including salt-soluble protein extractability, Ca++-ATPase activity, and the torsion test. Changes in quality of fillets stored at −8°C were significantly greater than those stored at lower temperatures. Fillets stored at −34 and −50°C showed no significant advantage over those stored at −20°C as measured by salt-soluble protein extractability and Ca++-ATPase activity. However, fillets stored at the lower temperatures had slightly higher torsion shear strain.