Extraction and fractionation of lipolytic enzyme from viscera of Monterey sardine (Sardinops sagax caerulea)

In this study, lipolytic activity of a semi-purified lipolytic enzyme (SLE) from the viscera of sardine ( Sardinops sagax caerulea ) was screened on the lipolysis of olive, Menhaden and sardine oil. A lipolytic enzyme was partially purified from the crude extract of sardine viscera by fractional precipitation followed by ultrafiltration (30 kDa). The main tissues found in sardine viscera were pyloric caeca (19.0% w/w), digestive tract (13.0% w/w), liver (4.8% w/w) and pancreas (1.5% w/w). Results show that pancreas had the highest lipolytic activity. There were no significant differences in lipolytic activity between pyloric caeca, intestine and liver ( P  < 0.05). Specific activity of the SLE increased 47.0-fold after extraction and fractionation, with a yield of 0.34% calculated for the whole viscera weight. Lipolytic activity of SLE from sardine viscera increased threefold when sardine oil was used as substrate. The results of this study confirm the potential importance of lipases from marine sources.     

Production of n-3 polyunsaturated fatty acid concentrate from sardine oil by immobilized Candida rugosa lipase

ABSTRACT:  This study was conducted to develop an immobilized-enzyme system to entrap lipase in chitosan-alginate-CaCl₂ 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.     

酶法提取沙丁鱼内脏鱼油工艺优化及其品质分析

为提高沙丁鱼加工副产物的利用率,以沙丁鱼内脏为原料,研究5种蛋白酶（胃蛋白酶、木瓜蛋白酶、中性蛋白酶、胰蛋白酶和碱性蛋白酶）对沙丁鱼内脏鱼油提取率的影响,并优选1种蛋白酶作为鱼油提取酶,以鱼油提取率为指标,通过单因素试验和正交试验确定沙丁鱼内脏鱼油提取的最佳工艺条件。对酶法提取的粗鱼油进行精制,对精制鱼油的理化指标和脂肪酸组成进行分析。结果表明：采用中性蛋白酶时,鱼油提取率最高;中性蛋白酶酶解提取沙丁鱼内脏鱼油的最佳工艺条件为料液比1∶ 1、加酶量1%、酶解时间2 h、酶解pH 7、酶解温度50 ℃,在此条件下沙丁鱼内脏鱼油提取率可达67.86%;粗鱼油经精制后,达到SC/T 3502—2016精制鱼油的二级标准;精制鱼油中DHA含量为26.57%,EPA含量为2.64%,营养价值较高。     

The effects of the combination of freezing and the use of natural antioxidant technology on the quality of frozen sardine fillets (Sardinella aurita)

The effects of combination of freezing and the use of antioxidant technology on the quality of frozen sardine fillets were investigated in terms of sensory, biochemical [thiobarbituric acid (TBA), total volatile basic nitrogen (TVB‐N), peroxide value (PV) and free fatty acids (FFA)] and microbiological analyses [total viable count (TVC)]. Fish were filleted and divided into three groups. The first group was used as the control (C) without rosemary extract, the second group was treated with 1% rosemary extracts for 2 min (R1) and the third was treated with 2% rosemary extracts for 2 min (R2). All groups were frozen at ?18 °C over the storage period of 6 months. The results obtained from this study showed that the combination of antioxidant and frozen storage resulted in significant reduction of bacterial growth and stabilised the biochemical characteristics, especially for R2. However, the use of antioxidant at the level of 2% (R2) gave a bitter taste according to sensory assessment whereas the panellists mostly preferred R1.     

Effect of ice storage on the functional properties of pink perch and oil sardine meat

Ice storage of dressed pink perch (Nemipterus japonicus) and Indian oil sardine (Sardinella longiceps) for 16 and 20 days, respectively, resulted in a decrease in emulsifying capacity (EC), protein solubility (PS), relative viscosity (RV) of salt‐soluble proteins (SSP) and water‐soluble proteins (WSP), water binding capacity (WBC) in terms of absorbed moisture in water (AM_w), absorbed moisture in brine lpar;AM_b), retained moisture in water (RM_w) and retained moisture in brine (RM_b), WSP and SSP, and increase in cook loss (CL). Decrease in protein solubility influenced the EC, RV, CL and WBC in both the species of fish. Significant (P<0.05) correlations existed among various functional properties analysed, in both the fishes during the ice storage. © 1999 Society of Chemical Industry     

Purification and biochemical characterization of chymotrypsin from the viscera of Monterey sardine (Sardinops sagax caeruleus)

Chymotrypsin was isolated from the viscera of Monterey sardine by ammonium sulphate fractionation, gel filtration, and ionic exchange chromatography. The approximate molecular weight was 26,000 and its isoelectric point was about 5. Identity as chymotrypsin was established by its catalytic specificity for amide or ester bonds on the synthetic substrates succinyl-l-ala-ala-pro-l-pheilalanine-p-nitroanilide and benzoyl-l-tyrosine-ethyl-ester, showing esterase activity 3.2-fold higher than amidase. It was inhibited by phenylmethylsulfonyl-fluoride and soybean trypsin inhibitor, partly inhibited by the specific chymotrypsin inhibitor N-toluenesulfonyl-l-phenylalanine chloromethyl-ketone, but not inhibited by EDTA or Benzamidine. Chymotrypsin showed its maximum activity at pH 8.0 and 50 °C for the hydrolysis of SAAPNA. The Michaelis–Menten constant was 0.074 mM with a catalysis constant of 18.6 seg⁻¹, and catalytic efficiency of 252 seg⁻¹ mM⁻¹. Results indicated that Monterey sardine chymotrypsin is a good catalyst and could be used as a biotechnological tool in food processing and using sardine industry wastes as a material for production of fine reagents.     

Laundry detergent-stable lipase from Pacific white shrimp (Litopenaeus vannamei) hepatopancreas: Effect of extraction media and biochemical characterization

Extraction and biochemical properties of a new lipase from the hepatopancreas of Pacific white shrimp were studied. Recovery of the hepatopancreas powder with 50 mM Tris-HCl, pH 7.0 containing 0.2% (v/v) Brij35 gave a higher recovery of lipase activity than other extractants tested (p < 0.05). The optimal pH and temperature for lipase activity were 8.5 and 60°C, respectively, when p-nitrophenyl palmitate was used as a substrate. The enzyme was stable to heat treatment up to 40°C and over a pH range of 7.0–10.0 for 30–120 min. Lipase activities continuously decreased as the sodium deoxycholate (NaDC) concentration increased, but activities increased as NaCl concentration increased up to 3.0 M. Hydrolytic activity was enhanced by NaN₃, but strongly inhibited by Hg²⁺, Cu²⁺, Al³⁺, and phenylmethanesulfonyl fluoride. The lipase was evaluated as highly stable against surfactants (Tween 20, Tween 80, Triton X-100, and gum arabic). However, the enzyme was unstable against sodium dodecyl sulphate. Stability of the lipase with commercial liquid and solid detergents (Attack^®, Bres^®, Omo^®, and Pao^®) was also investigated. The lipase exhibited substantial stability and compatibility with tested commercial liquid and solid laundry detergents for 30–60 min. The overall properties of the lipase from Pacific white shrimp hepatopancreas, thus leading us to propose that it is an excellent candidate for use as biocatalysts for better detergent formulation.     

Preparation of eicosapentaenoic acid concentrates from sardine oil by Bacillus circulans lipase

An extracellular lipase derived from Bacillus circulans, isolated from marine macroalga, Turbinaraia conoides, was used to prepare n-3 polyunsaturated fatty acid (PUFA) concentrates from sardine oil triglycerides. The enzyme was purified 132-fold with specific activity of 386 LU/mg. The purified lipase was able to enrich sardine oil with 37.7 ± 1.98% 20:5n-3 and 5.11 ± 0.14% 18:3n-3 in the triglyceride fraction after 3 h of hydrolysis. Lower hydrophobic constants of n-3 fatty acids (18:3n-3_logP = 5.65; 20:5n-3_logP = 5.85, respectively) than n-6 (20:4n-6_logP = 6.16) resulted in higher hydrolytic resistance of the former toward lipase, leading to their enrichment in the triglyceride fraction. Lipase-catalysed hydrolysis of sardine oil for 3 h, followed by urea complexation, provided free fatty acids containing 51.3 ± 4.65% 20:5n-3. The purified methyl ester of 20:5n-3 (68.29 ± 2.15%) from the urea concentrate was attained by chromatography on argentated neutral alumina.     

Thermoactivity and effects of organic solvents on digestive lipase from hepatopancreas of the green crab

Unlike classical digestive lipases, the crab digestive lipase (CDL) displayed its maximal activity at a high temperature. The CDL activity’s optimal temperature, when using emulsified or monomolecular film as substrate, was 60 °C. To our knowledge, this is the first report of an animal digestive lipase having such an optimal temperature. The maximum activity of CDL appeared at pH 8. Lipase activity was compatible with the presence of organic solvents, except for butanol. Furthermore, the hydrolysis was found to be specifically dependent on the presence of Ca²⁺ ions, since no significant CDL activity was detected in the presence of ion chelators such as EDTA. Nevertheless, the CDL does not require Ca²⁺ to trigger the hydrolysis of tributyrin emulsion. Interestingly, Zn²⁺ and Cu²⁺ ions acted as strong inhibitors of CDL activity when using tributyrin as substrate. Lipase stability in the presence of organic solvents, as well as at high temperatures, makes it a good candidate for application in non-aqueous catalysis.     

Effect of chitooligosaccharide from squid pen on gel properties of sardine surimi gel and its stability during refrigerated storage

Effect of chitooligosaccharide from squid pen prepared using lipase (COS-L) at various concentrations (0–30 g kg⁻¹) on gel properties of sardine surimi gel was investigated. Breaking force (BF) and deformation (DF) of gel were increased, when COS-L level was increased up to 10 g kg⁻¹ (P < 0.05). Water holding capacity and whiteness of gel were improved with the addition of COS-L than those of control. Gel added with 10 g kg⁻¹ COS-L had denser network with higher likeness score for all sensory attributes, compared to control. When gel incorporated with 10 g kg⁻¹ COS-L was stored at 4 °C, BF, DF and whiteness were maintained during 10 days of storage. Textural properties of surimi gel added with COS-L were higher than those of control throughout storage. Thus, incorporation of 10 g kg⁻¹ COS-L could improve gel properties of sardine surimi gel and retarded the deterioration of gel properties during refrigerated storage.     

Extraction of sardine myoglobin and its effect on gelation properties of Pacific whiting surimi

ABSTRACT:  Myoglobin (Mb) was extracted from Pacific sardine and added to Pacific whiting surimi to measure its effects on protein gelation. The purity of Mb extract was determined by SDS-PAGE. Mb extract using ethanol showed higher purity than Mb extract using ammonium sulfate. The addition of 0.2% Mb significantly reduced breaking force. However, a synergistic effect of 1.0% Mb was observed with 1.0% beef plasma protein (BPP) to increase surimi gel strength. The highest storage modulus of gels was observed at 1.0% Mb addition, which corresponded with the nonfracture gel and also supported a synergistic interaction between 1.0% Mb and 1.0% BPP. Differential scanning calorimetry showed Mb addition might have affected myosin denaturation and aggregation.     

精制沙丁鱼油品质及挥发性风味成分分析

为研究精制沙丁鱼油品质及挥发性风味成分,采用气相色谱仪(GC)和顶空固相微萃取-气质联用仪(HS-SPME-GC-MS)对其理化指标、脂肪酸组成及挥发性风味成分进行系统研究。结果表明:精制沙丁鱼油水分及挥发物、酸值、过氧化值均达到SC/T 3502—2016规定的精制鱼油二级标准;精制沙丁鱼油中多不饱和脂肪酸含量为(26.66±0.18)%,其中EPA与DHA总含量为(23.41±0.16)%;精制沙丁鱼油的关键风味成分为壬醛、丁醛、己醛、十一醛、2-壬酮、乙苯,具有甜香、果香等风味的挥发性成分种类较多,如萘、2-十一酮,对鱼油的风味有一定的修饰作用。该研究为精制沙丁鱼油的进一步开发利用提供了一定的理论依据。     

Effectiveness of rutin and its lipophilic ester in improving oxidative stability of sardine oil containing trace water

Poor oxidative stability exhibited by n‐3 polyunsaturated fatty acid rich sardine oil is a major challenge for its utilisation in industry. Considering the fact that water is always present in bulk oil in trace amounts during storage, an effort was made to understand and compare the effectiveness of rutin and its corresponding lipophilic ester in enhancing oxidative stability of refined sardine oil containing trace water (0.16% w/w). Peroxide value, conjugated diene value, p‐anisidine value and thiobarbituric acid reactive substances (TBARS) value were determined during 20 days storage. Rutin fatty ester showed 50% reduction in primary oxidation and 42.46% reduction in secondary oxidation, whereas rutin showed 20.6% and 20.43% reduction in primary and secondary oxidation, respectively, by the end of 20 days storage. Thus, it is clearly established that rutin fatty ester is more effective than hydrophilic rutin in sardine oil containing trace water, which contradicts the polar paradox theory.     

Lipase activity in different tissues of four species of fish: rohu (Labeo rohita Hamilton), oil sardine (Sardinella longiceps Linnaeus), mullet (Liza subviridis Valenciennes) and Indian mackerel (Rastrelliger kanagurta Cuvier)

Lipase activity in stomach and pyloric caeca, liver, intestine and red muscle of rohu (Labeo rohita Hamilton), oil sardine (Sardinella longiceps Linnaeus), mullet (Liza subviridis Valenciennes) and Indian mackerel (Rastrelliger kanagurta Cuvier) was studied. Distinct differences in lipolytic activity in different tissues of these fish were observed. Rohu showed the highest activity in all tissues in comparison with the other three species of fish. Among the three size groups of mullet, medium‐sized mullet showed higher activity than the other two groups in all tissues except intestine. Rohu hepatopancreatic lipase exhibited more hydrolytic activity on fish oil than rohu intestinal lipase. Copyright © 2003 Society of Chemical Industry     

Effect of Calcium Salts on the Properties of Proteins from Oil Sardine (Sardinella longiceps) During Frozen Storage

ABSTRACT:  The effect of calcium sulfate and calcium chloride on the enzymatic and structural properties of actomyosin isolated from sardine was investigated. Mince was prepared from sardine and different concentrations of calcium chloride and calcium sulfate were added to the mince and kept in frozen condition at −20 °C. The physico-chemical and functional properties of proteins from mince were analyzed as a function of time. The solubility of proteins decreased during storage. The reduction in solubility was less for samples treated with calcium chloride. However, sardine mince showed better functionality during storage in the presence of calcium compounds. The ATPase enzyme activity of actomyosin increased with increase in concentration of calcium and decreased after reaching the maximum value. ATPase activity of proteins from mince decreased during storage at low temperature. The reduction in ATPase activity did not correlate with the loss of functionality of proteins. SDS-PAGE did not reveal any major changes in the protein profile during storage as well as in the presence of different concentration of calcium compounds. The secondary structural content of actomyosin was not altered in the presence of both calcium chloride and calcium sulfate as evident from circular dichroic measurements.     

Analysis of volatile flavor compounds of sardine (Sardinops melanostica) by solid phase microextraction

ABSTRACT:  Generally the main component of fishy flavor is considered to be trimethylamine. On the other hand, carbonyl compounds, produced from oxidation of polyunsaturated fatty acid by lipoxygenase or by autoxidation, might have some contribution to the fishy flavor. Since sardine skin contains high levels of polyunsaturated fatty acids and lipoxygenase, carbonyl compounds may be generated more easily than trimethylamine. In this study, volatile flavor compounds of sardine were analyzed by gas chromatograph-mass spectrometry and gas chromatograph-olfactometry combined with solid phase microextraction. Then, the flavor components that contribute to fishy flavor were identified. At normal pH (6.2), trimethylamine was not detected or sensed from the fresh sardines. When the pH was raised, the amount of trimethylamine became higher. Trimethylamine flavor was weak at pH 9 and strongly sensed at pH 11 or higher. On the other hand, 33 other compounds were positively or tentatively identified, including 8 hydrocarbons, 5 ketones, 1 furan, 1 sulfur compound, 12 aldehydes, and 6 alcohols in fresh sardines. Among them, 2,3-pentanedione, hexanal, and 1-penten-3-ol were the main components. Forty-seven flavors were detected by gas chromatograph-olfactometry. Among them, paint-like (1-penten-3-one), caramel-like (2,3-pentanedione), green-like (hexanal), shore-like ((Z)-4-heptenal), citrus note (octanal), mushroom-like (1-octen-3-one), potato-like (methional), insect-like ((E,Z)-2,6-nonadienal), and bloody note (not identified) were strongly sensed. From the aforementioned results, it can be concluded that these compounds rather than trimethylamine contributed to fresh sardine flavor.     

变形杆菌属脂肪酶LipK107的分离纯化、结晶及初步晶体学分析

经过异丙基-β-D-硫代吡喃半乳糖苷（IPTG）诱导,重组大肠杆菌高效表达出带有组氨酸标签的可溶性脂肪酶LipK107。通过亲和层析和凝胶层析两步法纯化,得到电泳纯的脂肪酶样品。在获得的纯酶基础上,进行了圆二色谱实验,以分析脂肪酶LipK107的二级结构组成。接着,使用悬滴气相扩散法进行了纯酶的结晶实验,经过晶体生长条件的初步筛选及优化,在0.1mol/L pH6.2柠檬酸钠,10%v/v异丙醇,15%w/v聚乙二醇4000条件下得到高质量的脂肪酶晶体,使用同步辐射光源收集了一套分辨率为2.0的X-射线衍射数据。      

Influence of texture of suwari gels on kamaboko gels made from sardine (Sardina pilchardus) surimi

The textural characteristics and water holding capacity of suwari (set) and kamaboko (set and cooked) sardine surimi gels were examined in order to clarify the influence of the initial network formed in setting conditions (25, 35 and 40°C for 30 and 60 min) on the texture of the kamaboko gels. Although the texture of suwari gels set at 35°C improved with longer setting, both setting times ensured kamaboko gels with the highest gel strength. Suwari gels set at 25°C also improved with longer setting but the gel strength of both suwari and kamaboko gels was lower than at 35°C. For gels set at 40°C prolonged setting weakened the suwari networks formed, leading to kamaboko gels with poorer textural characteristics. ©1997 SCI     

The presence of bioactive peptides in hydrolysates prepared from processing waste of sardine (Sardina pilchardus)

A proteolytic enzyme, Alcalase®, was used to produce partly digested proteins from cooked wastes of sardine (Sardina pilchardus). The presence of biologically active molecules was investigated in these hydrolysates prepared under various conditions of time and enzyme/substrate ratio. By means of radioimmunoassays and mitogenic and radioreceptor assays, the presence of molecules related to secretagogue peptides, growth factors and calcitonin gene‐related peptide (CGRP) respectively was detected in the hydrolysates. Exclusion chromatography permits the conclusion that the biological activity of the different fractions is related to their size. © 2001 Society of Chemical Industry