Comparative study of proteins recovered from whole North Pacific krill Euphausia pacifica by acidic and alkaline treatment during isoelectric solubilization/precipitation

North Pacific krill is widely distributed in the waters around Japan, however its utilization for human consumption has been limited due to its small size. We have investigated the proximate composition, the amino acid, free fatty acid, mineral and protein composition, respectively, as well as the residual proteolytic activity of krill protein, as recovered by acidic- and alkaline-aided isoelectric solubilization/precipitation, in order to facilitate effective utilization of this krill species. Krill protein yield was 31.7 and 73.4 % by acidic and alkaline treatment, respectively. Both treatments resulted in a comparable higher content of essential amino acids than that in whole krill, accompanied with effective removal of insoluble materials. Three major proteolytic active bands with molecular weights of approximately 28, 18 and 16 kDa, as estimated by gelatin-based zymography, were detected. Pronounced residual proteolytic activity was observed in the krill protein after alkaline processing, implying the possible involvement of proteinase(s) in protein degradation during alkaline solubilization. Based on these results, we suggest that inhibition of proteolysis during isoelectric solubilization/precipitation is required for the effective use of North Pacific krill.     

Preparation,Gel Electrophoresis Analysis,and Nutritional Evaluation of a Functional Krill Protein Concentrate with Low Fluoride Level from Antarctic krill (Euphausia superba)

A functional krill protein concentrate (FKPC) with low level of fluoride was prepared from frozen Antarctic krill (Euphausia superba) using isoelectric precipitation and phosphoric acid treatment. Variations in proteins and nutritional properties of the FKPC were analyzed. Gel electrophoresis analysis indicated that some variations in protein composition were introduced by autolysis and pH shift during the process of isoelectric precipitation and fluoride removal. The FKPC was composed of 81.01% ± 0.45% crude protein (dry basis) and 16.78% ± 0.26% total lipid (dry basis), exhibited excellent amino-acid composition in all the essential amino acids, exceeding the requirements of the FAO/WHO/UNU for food protein, and was rich in omega-3 polyunsaturated fatty acids, especially eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids present in phospholipids. The fluoride level of FKPC was 79.79 ± 2.10 μg/g (dry basis), which met the USFDA requirements. The surimi yield reached 43.74%, and the mean recovery rate of protein and fat was 52.68% and 41.44%, respectively. The nutritional value, safe level of fluoride, and high yield rate provide evidence that FKPC has a promising future in the food industry as a new aquatic protein and lipid resource.     

Extracting Protein from Antarctic Krill (Euphausia superba)

ABSTRACT

The isoelectric solubilization/precipitation (ISP) process was applied to Antarctic krill protein extraction. The solubility of Antarctic krill proteins and their fluoride content were studied between pH 1.00–13.75. The previous krill protein extraction conditions using ISP were improved. Excess fluoride in Antarctic krill was removed using a water-washing method according to its solubility. To save water, a multistage countercurrent system was used to optimize the ISP method. The results showed that the solubility of Antarctic krill proteins reached a minimum at pH 4.5 and a maximum at pH 13.5, while the concentration of dissolved fluoride was relatively unchanged (16.78–20.50 mg/L), with pH 2.00–13.50. Water-washing 4 times could reduce fluoride 98.14 ± 0.01% from krill with about a 5.14 ± 0.21% protein loss. The reduction allows krill protein to meet the Chinese National Standard (GB 2762-2005) for fluoride. Meanwhile, the four-stage countercurrent system achieved almost the same high fluoride removal rate (98.58 ± 0.01%) using only 25% of the water.     

Autolysis and Characterization of Sarcoplasmic and Myofibril Associated Proteinases of Oxeye Scad (Selar boops) Muscle

ABSTRACT

The autolytic profile of oxeye scad mince was characterized. Mince showed higher proteolytic activity than washed mince. The highest autolysis was observed at 65 and 60°C for mince and washed mince, respectively. Both mince and washed mince showed the optimum pH for autolysis at pH 9.0, and their activities decreased with increasing NaCl concentration (0–3.5%). Autolysis of washed mince was markedly inhibited by soybean trypsin inhibitor (SBTI), suggesting that myofibril-associated proteinase was serine proteinase. Sarcoplasmic proteinase was characterized to be heat-activated alkaline proteinase having the optimal pH and temperature of 9.0 and 60°C, respectively. The activities were stable at pH range of 8.0–11.0 at 20–40°C. The crude proteinase was inhibited by N-p-tosyl-L-lysine chloromethyl ketone, SBTI, and phenylmethanesulfonyl fluoride, suggesting the predominance of serine proteinases, especially trypsin. NaCl suppressed the activity while β-mercaptoethanol, dithiothreitol, and CaCl₂ activated the activity. Therefore, trypsin-like proteinase is a major endogenous proteinase responsible for autolysis in oxeye scad muscle. The present results can be used as scientific guidelines to predict the gel strength of surimi made from oxeye scad muscle.     

Recovery of Proteins from Rohu Processing Waste Using pH Shift Method: Characterization of Isolates

Yield and physicochemical characteristics of protein isolates recovered from rohu processing waste by acid- and alkali-aided protein solubilization methods were studied. pH 2.0 from acidic side and pH 13.0 from alkaline side resulted in maximum protein solubility, whereas minimum solubility was observed at pH 5.5. Except for pH 13.0, theoretical protein recovery was more than the actual recovery after centrifugation. Maximum protein recovery after the first centrifugation cycle was observed for pH 2.0 and pH 13.0, and the maximum protein recovery during the second centrifugation cycle was observed at precipitation pH 5.5 in both acidic and alkaline methods. Acidic and alkaline methods resulted in a total process recovery of 31.81% ± 0.001% and 31.11% ± 0.01%, respectively. Isolates prepared by acidic method had more whiteness and high foaming capacity, whereas isolates prepared using alkaline methods had less whiteness, tough texture, and high foaming stability. No microbial counts were detected in any of the isolates. Acid and alkaline methods described in this study can be used for the recovery of proteins from rohu processing waste with good yields and functionality.     

Purification and characterization of alkaline phosphatase from the gut of sea cucumber Stichopus japonicus

An alkaline phosphatase was purified from the gut of sea cucumber Stichopus japonicus by n-butyl alcohol extract, ammonium sulfate precipitation, ion exchange chromatography with diethylaminoethyl cellulose, gel filtration chromatography with Sephacryl S-200 and preparative electrophoresis with polyacrylamide gel electrophoresis. The native enzyme was estimated to be 166 ± 9 kDa and produced a single predominant band corresponding to active enzyme on nondenaturing electrophoresis, but showed 2 bands of 97 and 35 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the native enzyme is composed of two dissimilar subunits. The enzyme displayed maximum activity at pH 11 and 40 °C, showing narrow pH stability (pH 10–12) and thermal instability at temperature higher than 30 °C. The activity of the purified alkaline phosphatase was enhanced by Mg²⁺, whereas inhibited by Zn²⁺, Ca²⁺ and EDTA at 1 and 10 mM, suggesting its activity is in a magnesium ion-dependent manner. The product-analog WO₄ ^2? and product HPO₄ ^2? showed strong inhibitory effects on the enzyme activity. Using p-nitrophenyl phosphate as substrate, the V _max and K _m values were 24.45 μmol/L min and 5.76 mM, respectively.     

Periodic changes in the growth performance and biochemical composition of juvenile red sea bream <Emphasis Type="Italic">Pagrus major</Emphasis> fed non-heated and heated squid and krill meal-based diets

In finfish aquaculture, fish meal is heated during the manufacturing process, which affects the digestibility and protein absorption by fish. However, manufactured fishmeal that is not heated does not undergo thermal denaturation. Few studies have investigated the effects of non-heated animal protein sources on the growth performance of fish. We investigated the effects of heated and non-heated squid and krill meal as diets for red sea bream. Five test diets were formulated to contain heated squid meal, non-heated squid meal, heated krill meal, non-heated krill meal, and fish meal as a control. Fifty fish (initial mean weight?=?3.5 g) were distributed in ten 100-l experimental tanks. Fish were fed one of the five diets 3 times daily until satiation for 5 weeks. Regarding growth performance, fish fed the krill meal diet exhibited better growth than those fed squid meal during the first week of the rearing period. However, the squid meal diet group showed better performance than the krill meal diet group during the third week. Moreover, differences in body weight among treatments were greater during the fifth week. Better weight gain and thermal growth coefficient were recorded in the non-heated diet groups than in the heated diet groups. Higher feed intake was observed in the non-heated diet groups than in the heated diet groups. These results suggest higher performance of non-heated squid and krill meal as the protein source of the red sea bream diet. Further, the suitability of the diet type (e.g., squid and krill) might depend on the feeding period and/or developmental stage of fish.     

正交实验优化南极磷虾蛋白肽的纳滤脱盐工艺

为建立南极磷虾(Euphausia superba)蛋白肽纳滤脱盐工艺,以脱盐率和蛋白损失率为评价指标,通过单因素实验和正交实验对影响南极磷虾蛋白肽纳滤脱盐效果的主要因素(蛋白肽浓度、压力、循环次数)进行优化。结果显示,采用纳滤技术对南极磷虾蛋白肽进行脱盐处理的最佳工艺条件：蛋白肽浓度为3%、纳滤压力为1.2 MPa、循环次数3次,在该条件下,南极磷虾蛋白肽的脱盐率达到(86.35±2.11)%、蛋白损失率为(9.10±0.35)%。采用优化工艺获得的南极磷虾蛋白肽的盐分含量为(1.14±0.12)%,蛋白质含量为(92.73±2.29)%,相对分子质量主要分布于3000 Da以下,氨基酸组成合理且符合联合国粮农组织/世界卫生组织规定的标准。研究将为高品质南极磷虾蛋白肽产品开发提供技术支撑。     

The replacement of fish meal with Antarctic krill, Euphausia superba in diets for Atlantic salmon, Salmo salar

A total of six isoprotein and isolipid diets for Atlantic salmon, Salmo salar L., were prepared substituting from 0 to 100% of fish meal protein (0–68% of diet by dry weight) with meal from Antarctic krill (Euphausia superba). The feed produced from high inclusion levels of krill meal had lower ability to absorb lipid during vacuum coating than fish meal. Both amino acid and fatty acid compositions of the diets were fairly similar. The experiment commenced using salmon averaging 500 g and ended at a mean weight of 1500–1800 g (140 days of feeding). Moderate amounts of krill meal (20–60% of krill protein) in the diets increased growth during the first 71 days of feeding compared with the fish meal control, while no growth difference was observed during the last 69 days of feeding. This may, at least in parts, be explained by a feed‐attractant function of the krill meal. Muscle dry weight and lipid concentrations were unaffected by the diet. Feed conversion rate increased with high levels of krill meal in the diets (e.g. for the last period from 0.94 in the 0% diet to 1.26 in the 100% diet). This indicates that the fish were able to compensate by eating more to maintain growth. The apparent digestibility coefficients of dry matter and protein were not influenced by diet, but both faecal moisture and lipid had a tendency to increase at the highest inclusion level (all protein from krill meal). This may be related to chitin in the krill diet that is known to decrease lipid absorption and induce diarrhoea (increased water content in faeces). Chitin was not utilized to any major extent. Welfare parameters such as blood haemoglobin, red blood cell counts, plasma protein, cholesterol, triacylglycerols and glucose levels were unaffected by diets. Clinical indicators of cellular damage (alanine aminotransferase and aspartate aminotransferase) were similar indicating no diet‐induced tissue damage during the trial.     

南极磷虾主要过敏原的分离纯化及性质研究

为了探究南极磷虾致敏问题,从南极磷虾中筛选、鉴定、分离纯化其主要过敏原,并对过敏原的性质进行研究,实验通过缓冲盐溶液提取南极磷虾蛋白;采用SDS-PAGE和Western blot(WB)筛选南极磷虾过敏原;使用高效液相色谱-串联质谱鉴定过敏蛋白;通过等电点沉淀、硫酸铵盐析、阴离子交换柱层析分离纯化过敏原;采用SDS-...     

Autolysis of Anchovy (Engraulis japonicus) Protein: Evaluation of Antioxidant Properties and Nutritional Quality of Resulting Hydrolysates

Protein hydrolysis of underutilized marine organisms is a method to improve added value. In the present article, anchovy protein was autolyzed at pH 8.5 and 50°C. After autolysis processing for 24 h, total nitrogen recovery reached 92.1%. The protein hydrolysates had antioxidant properties on reducing power, 1, 1-diphenyl-2-picryhydrazyl radical, and hydroxyl radical scavenging activity. More than 79% of the peptides in the hydrolysates had a molecular weight of lower than 3 kDa. Protein hydrolysates had balanced amino acid compositions, and total content of essential amino acids had no significant difference after hydrolysis. Protein hydrolysates from anchovy autolysis have potential in nutritional and functional foods.     

Detection of cathepsin L in red cell membranes from fish blood

Cathepsin L, one of the cysteine proteases found in fish muscle, is considered to be the main cause of post-mortem autolysis of fish muscle. We have determined the presence of cathepsin L in the membranes of red blood cells of carp, amberjack, and red sea bream and measured its activity. Immunoblotting of an extract of the red blood cell membranes from these three fish species using human anti-cathepsin L antibody revealed the presence of cathepsin L of different molecular masses. The molecular masses of cathepsin L was estimated to be 120 and 85 kDa in the amberjack and 75 and 70 kDa in the carp. These proteins have higher molecular masses than the mature form of cathepsin L, suggesting that they are precursor forms. In contrast, the protein in the red sea bream was estimated to have a molecular mass of 30 kDa, suggesting that this cathepsin L is a mature form. The specific activity of cathepsin L was highest in the red blood cell membranes of the amberjack, followed by the carp and the red sea bream in descending order.     

Growth, feed conversion and chemical composition of Atlantic salmon (Salmo salar L.) and Atlantic halibut (Hippoglossus hippoglossus L.) fed diets supplemented with krill or amphipods

The effects of partial replacement of fish meal (FM) with meal made from northern krill (Thysanoessa inermis), Antarctic krill (Euphausia superba) or Arctic amphipod (Themsto libellula) as protein source in the diets for Atlantic salmon (Salmo salar L.) and Atlantic halibut (Hippoglossus hippoglossus L.) on growth, feed conversion, macro‐nutrient utilization, muscle chemical composition and fish welfare were studied. Six experimental diets were prepared using a low‐temperature FM diet as control. The other diets included northern krill where 20, 40 or 60% of the dietary FM protein was replaced with protein from northern krill, and two diets where the FM protein was replaced with protein from Antarctic krill or Arctic amphipod at 40% protein replacement level. All diets were iso‐nitrogenous and iso‐caloric. Atlantic salmon grew from 410 g to approximately 1500 g during the 160 day experiment, and Atlantic halibut grew from 345 g to 500–600 g during the 150 day experiment. Inclusion of krill in the diets enhanced specific growth rate in salmon, especially during the first 100 days (P < 0.01), and in a dose–response manner in halibut for over the 150 day feeding period (P < 0.05). Feed conversion ratio did not differ between dietary treatments, and no difference was found in dry matter digestibility, protein digestibility and fish muscle composition. Good growth rates, blood parameters within normal ranges and low mortalities in all experimental treatments indicted that fish health was not affected either Atlantic salmon or Atlantic halibut fed the various zooplankton diets.     

Physicochemical and Functional Properties of Protein Recovered from Fish Waste

ABSTRACT

This work aimed to evaluate the physicochemical and functional properties of proteins recovered from whitemouth croaker (Micropogonias furnieri) and Argentine anchovy (Engraulis anchoita) residues obtained by the process of acid solubilization using HCl at pH = 2 and alkaline solubilization using NaOH at pH = 12 and isoelectric precipitation (pH = 5.5). The proximate composition and functional properties were determined. The solubilization and water holding capacity (WHC) of the recovered protein were studied in the pH range of 3, 5, 7, 9, and 11. The high protein content and lowest lipid reduction presented by the protein recovered from the whitemouth croaker residue and obtained by the acid solubilizing process was 97.1 and 98.4%, respectively. The maximum solubilized protein was at pH 3 and 11, and the minimum solubilized protein was at pH 5 for both species. The greater ability to retain oil was observed for the alkaline solubilization process (8.3 and 7.3). The lower WHC was at pH 5. The acid and alkali residue concentrates of the whitemouth croaker presented higher WHC at pH 3 and 11. These proteins can be used in preparing edible films and have been used to produce bioactive compounds for supplements or functional ingredients.     

Partial characterisation of digestive proteases of the Mayan cichlid Cichlasoma urophthalmus

The characterisation of digestive proteases in native freshwater fish such as the Mayan cichlid Cichlasoma urophthalmus provides scientific elements that may be used to design balanced feed that matches with the digestive capacity of the fish. The purpose of this study was to characterise the digestive proteases, including the effect of the pH and the temperature on enzyme activity and stability, as well as the effect of inhibitors using multienzymatic extracts of the stomach and intestine of C. urophthalmus juveniles. Results showed that the optimum activities of the acid and alkaline proteases occurred at pH values of 3 and 9, respectively, whereas their optimum temperatures were 55 and 65 °C, respectively. The acid proteases were most stable at pH values of 2–3 and at temperatures of 35–45 °C, whereas the alkaline proteases were most stable at pH values of 6–9 and at 25–55 °C. The inhibition assays recorded a residual activity of 4 % with pepstatin A for the acid proteases. The inhibition of the alkaline proteases was greater than 80 % with TPCK, TLCK, EDTA and ovalbumin, and of 60 and 43.8 % with PMSF and SBT1, respectively. The results obtained in this study make it possible to state that C. urophthalmus has a sufficiently complete digestive enzyme machinery to degrade food items characteristic of an omnivorous fish species, although specimens showed a tendency to carnivory.     

Amino acids composition and protein quality evaluation of marine species and meals for feed formulations in cephalopods

The amino acid composition and protein levels of three species of cephalopods (Octopus vulgaris, Loligo gahi and Todarodes sagittatus), the natural diets of common octopus (O. vulgaris) and different kinds of meals were determined in order to optimise the content of these nutrients in artificial feeds. Arginine, leucine and lysine were the most abundant essential amino acids in cephalopods, while glutamate and aspartate represented the main non-essential amino acids. Arginine and leucine were the limiting amino acid in most samples, with maximum Chemical Score values for mussel (79–98 %), squid (84 %) and crustaceans (65–91 %); medium for fish (41–70 %); and minimum for meals (29–64 %). Mussel, squid, crustaceans and fish showed a high essential amino acid index according to Oser (OI: 88–99 %) suggesting a suitable amino acid balance. The protein from animal meals (fish and krill) covered all the essential amino acids except arginine and lysine in fish meal. The vegetable meal presented the worst amino acid balance (OI: 74–89 %) with several deficiencies in essential amino acids, including arginine, threonine, lysine and methionine. Supplementation with arginine or leucine and protein complementation of crustaceans and bivalves with fish or animal meal are proposed as alternatives for improving the performance of protein in feed for cephalopods.     

拖网作业参数对南极磷虾捕捞效率的影响

为了解南极磷虾(Euphausia superba Dana)拖网作业中捕捞效果及影响因素,根据 2017 年 12 月 2018 年 6月随“龙腾”轮在南极海域生产时收集的作业参数、网具深度和声学数据等信息,利用声学方法获取磷虾集群质量中心深度与期望入网渔获量,结合网位变化和渔捞统计情况量化捕捞效率;利用广义可加模型(GAM)分析不同拖速和曳纲长度下,网位与磷虾集群质量中心的偏离程度,研究其对网具捕捞效率的影响。结果表明:(1)磷虾集群质量中心水深为(37.29±9.72) m,拖网稳定拖曳期间网位深度为(30.72±10.41) m,网位调整幅度为(11.52±7.09) m。网位与磷虾集群质量中心深度偏离(6.33±3.58) m。(2)网次渔获量为(16.25±6.77) t,拖曳过程中网口始终对准磷虾集群质量中心进入网口的磷虾总量为(27.06±10.19) t,捕捞效率为(63±19)%。(3)网位与磷虾集群质量中心深度差值对捕捞效率影响极显著(P<0.001),两者吻合较好时捕捞效率高;拖速与捕捞效率呈显著相关关系(P<0.05),拖速增大网位上升,捕捞效率越低;曳纲长度增加网位下降,但曳纲长度变化与捕捞效率相关性不明显(P=0.087)。研究结果将有助于南极磷虾拖网作业中有效利用磷虾行为特征,改善网具性能,提高拖网捕捞效率。     

Inhibitory Activities of Protein Hydrolysates from Spotted Babylon Snails on Tyrosinase and Melanogenesis

The optimal conditions for protein hydrolysates preparation from spotted babylon Babylonia areolata exhibiting tyrosinase inhibitory activity and antioxidant activity using alkaline protease (Protease G6) were undiluted Protease G6 (5.8 × 10⁵ DU/g) at 60 min of hydrolysis time and eightfold diluted Protease G6 (7.25 × 10⁴ DU/g) at 240 min of hydrolysis time. These two conditions were fractioned using molecular weight (MW) cutoff values of 10, 5, and 3 kDa membranes, and their anti-melanogenic and antioxidant properties were further analyzed. Among the fractions, the MW < 3 kDa fraction exhibited high levels of inhibitory activity toward the mono- and diphenolase activities of tyrosinase, with IC₅₀ values of 1.758 and 8.995 μg/mL, respectively. Kinetic studies revealed that this fraction behaved as an uncompetitive inhibitor. The results demonstrated that the MW < 3 kDa fraction suppressed melanin synthesis and decreased cellular tyrosinase activity with no cytotoxicity to B16F10 melanoma cells.     

饲料中添加南极磷虾粉对银鲳幼鱼生长、非特异性免疫及抗氧化功能的影响

以银鲳(Pampus argenteus)幼鱼为研究对象,设置4组不同南极磷虾粉替代鱼粉水平(0%、10%、20%、40%,其中0%为对照组)的饲料,进行60 d的饲养实验,探讨饲料中添加南极磷虾粉对银鲳幼鱼生长、非特异性免疫及抗氧化功能的影响。结果显示:饲料中添加南极磷虾粉的处理组银鲳增重率与特定生长率(SGR)较对照组均有显著性提高(P <0.05),但在添加不同比例南极磷虾粉的饲料组间(10%、20%、40%),银鲳的增重率与特定生长率无显著性差异(P> 0.05)。血清中碱性磷酸酶(AKP)、酸性磷酸酶(ACP)及溶菌酶(LZM) 20%饲料组活性均为最高,但与对照组无显著差异;肝脏AKP活性、肾脏LZM活性10%饲料组均分别显著高于其它各组(P <0.05);肌肉中超氧化物歧化酶(SOD)活性40%处理组显著高于对照组(P <0.05),20%饲料组也高于对照组,但差异不显著(P> 0.05);添加南极磷虾粉的饲料组血清和肌肉中过氧化氢酶(CAT)的活性均高于对照组,其中40%饲料组血清和肌肉CAT的活性均显著高于对照组(P <0.05)。结果表明,本实验条件下,结合银鲳幼鱼生长性能、非特异性免疫及抗氧化能力的分析,南极磷虾粉替代饲料中鱼粉的适宜比例建议控制在10%～20%。     

Common Kilka Hydrolysis: Investigation of Functional and In Vitro Antioxidant Properties of Hydrolysates Obtained by Kiwifruit and Ginger Proteases

The functional and in vitro antioxidant properties of common kilka fish protein hydrolysates with different degree of hydrolysis (DH) obtained by kiwifruit protease (KP) and ginger protease (GP) were evaluated. The electrophoretic patterns of hydrolysates showed the presence of one major band in different DHs with a molecular weight of less than 25 kDa. Protein solubility was positively correlated with DH, and the solubility of KP and GP hydrolysates (HKP and HGP, respectively) at 15% DH was higher than other DHs (p ≤ 0.05). Higher emulsifying and foaming properties were observed in HKP over a pH range of 2–10 (p ≤ 0.05), and in vitro antioxidant activity was higher in HKP at 15% DH as seen from 2,2-diphenyl-1-picrylhydrazyl radical scavenging and ferric-reducing power. Thus, the results reveal that DH and enzyme type affects functional properties and antioxidant activity of the hydrolysates.