Endogenous Trypsin Triggers Free Fluoride Release from Antarctic Krill(Euphausia superba)Cuticle

During postmortem storage,fluoride in Antarctic krill can be enriched in the muscle.Trypsin,as the most important digestive enzyme in Antarctic krill with a high activity in low temperature,plays a potential role in this process.In this study,endogenous trypsin was purified and its properties were investigated.The involvement of trypsin in the generation of free fluoride from Antarctic krill cuticle was explored.Cuticle microstructure before and after hydrolysis was compared with scanning electron microscopy,and the ash samples of the hydrolyzed Antarctic krill cuticle were analyzed with X-ray diffraction,Fourier transform infrared spectroscopy,and electron dispersive spectroscopy,respectively.Mass spectrometry analysis and inhibition tests confirmed that the purified enzyme was endogenous trypsin.Results of the present study indicated that trypsin digestion caused the increases of the concentrations of both fluoride ions and free amino N simultaneously,while the protein coated on the cuticle surface was dissolved too.However,no compositional change was detected in the cuticle inorganic salts.These findings suggest that trypsin triggered free fluoride release from Antarctic krill cuticle.In addition,the kinetics of free fluoride release could be described by the equation C_(W)=(1-0.97^(-0.006t)-0.03e^(0.0558t))×337.53+10.50.The present study improved the understanding of the role of trypsin in free fluoride release from Antarctic krill cuticle,facilitating future studies aimed at reducing the fluoride content in krill protein during Antarctic krill processing.

Quality Changes and Safety Evaluation of Ready-to-Eat Roasted Antarctic Krill(Euphausia superba)During Storage at Room Temperature(25℃)

The objective of this study was to determine the quality changes and shelf life of ready-to-eat roasted Antarctic krill,either untreated(CT)or treated with sodium diacetate(SD)or sodium diacetate and a deoxidizer(SDD),during storage at room temperature(25℃)by using sensory,physiochemical,and microbial assessment.Additionally,fluoride accumulation in this food product was used to evaluate its safety.Analysis showed that the addition of SD and SDD resulted in better sensory scores compared of that of CT samples(P<0.05).Accordingly,microorganism growth and total basic nitrogen(TVB-N)were maintained at a low level for the duration of storage with SD and SDD treatments.The total viable counts of SD and SDD reached(4.58±0.08)log(CFU g^(−1))and(4.20±0.11)log(CFUg^(−1)),respectively.The mold was found after 6 and 18 days for SD and SDD treatment,and the numbers reached 5.2×10^(6) and 8.5×10^(4) respectively at the end of shelf life.The TVB-N values from CT continuously increased during the whole storage.While TVB-N values from SD and SDD remained significantly less change(P<0.05)during the early 20 days of the storage,and the values reached(12.11±0.07)mg(100 g)^(−1) and(10.88±0.15)mg(100 g)^(−1) on day 33 and day 70,respectively.Importantly,SDD treatment effectively minimized the oxidation values and retained the color of ready-to-eat roasted Antarctic krill.Our results showed that the shelf life of ready-to-eat roasted Antarctic krill treated with SDD was extended by up to 52 days.Additionally,rats fed ready-to-eat roasted Antarctic krill showed accumulation of fluoride exclusively in the thighbone.The accumulation of fluoride residues in the thighbone showed concentration-dependent.The concentrations of fluoride residues in rats were(1760.03±38.21),(2371.52±42.15)mgkg^(−1) and(3615.44±30.53)mgkg^(−1),which were less than sodium fluoride feeding group(4621.01±28.67)mgkg^(−1).The results suggested that the SD and SDD treatments led to better quality and shelf life extension of ready-to-eat roasted Antarctic krill during storage at room temperature(25℃).Therefore,the ready-to-eat roasted Antarctic krill can be of great interest to the seafood industry.

Antarctic krill antioxidant peptides show inferior IgE-binding ability and RBL-2H3 cell degranulation

Enzymatic hydrolysis,isolation,and purification might make a great deal of difference in antioxidant activity and antigenicity of peptide components.This study aimed to isolate and purify antioxidant peptide components from Antarctic krill and evaluate their allergenicity of them.Electron paramagnetic resonance(EPR)spectroscopy results indicated 3-10 kDa Antarctic krill hydrolysates(AKHs)had higher DPPH and·OH radical scavenging rates.And the second component(N2-2)purified 3-10 kDa hydrolysate showed better ability to scavenge DPPH and·OH radicals(P<0.05),which were(47.43±2.18)%and(34.33±1.25)%,respectively.Additionally,indirect-ELISA results revealed that N2-1 had a weaker ability to bind specific IgE and that N2-2 had a lower binding capability to specific IgG1(P<0.05).And N2-2 had a higher EC50 value of(5.29±0.95)ng/mL(P<0.05)in cell degranulation assay,which was about 13.80 times that of Antarctic krill.Therefore,N2-2 might be the potential source of the antioxidant peptides with lower allergenicity.

Nutritional Metabolism of Antarctic Krill Product Protein in Rats

This work conducted a four-week metabolism test on rats to study the digestion and absorption characteristics of five protein-based krill products prepared from Antarctic krill as raw material.It aimed to provide theoretical support for the effective use of Antarctic krill protein and the development of novel protein resources.The results showed that the weight gain and true digestibility of the rats fed with krill meat,surimi and ordinary krill powder were significantly higher(P<0.05)than those of the rats fed with traditional casein.Compared to casein,proteins from the five Antarctic krill products were found to significantly improve the net protein utilization(P<0.05),and reduce the total cholesterol and triglycerides in the serum of rats(P<0.05).In summary,the Antarctic krill protein-based products with high nutritional values can be used as a potential novel protein resource in the food industry.

Purification of Antimicrobial Peptide from Antarctic Krill (Euphausia superba) and its Function Mechanism

The preliminary purification and antimicrobial mechanism of antimicrobial peptide from Antarctic Krill were studied in this paper. The results showed that the molecular weight range of antimicrobial polypeptide （CMCC-1） obtained by cation exchange chromatography was between 245-709D as detected by molecular sieve chromatography, and the minimum inhibition concentration （MIC） of CMCC-1 against Staphylococcus aureus was 5.0mgmL^-1. The antimicrobial mechanism of CMCC-1 was studied with S. aureus as indicator bacterium. Compared with control group, the results of the experimental group in which S. aureus was treated with CMCC-1 were as follows： l） CMCC-1 could inhibit cell division at logarithmic phase. 2） The protein and reducing sugar con- tent, and the conductivity of culture medium increased, and the activity of alkaline phosphatase and [3-galactosidase could be detected in the culture medium. 3） Observation under scanning electron microscope revealed that somatic morphology became irregular, and then somatic surface became coarse. The cell became much smaller, and most somatic ceils gathered. The boundary between cells became dim and finally fused as a whole. 4） Observation under transmission electron microscope showed that the surface of S. aureus became rough and the reproducing ability was restrained. The cell wall became thin and the cytoplasm shrunk. Substances inside cell leaked out, which caused cells death. 5） SDS-PAGE analysis showed that some bands disappeared, and the residual bands became vague. 6） The genomic DNA electrophoresis results showed that the genomic DNA bands ofS. aureus were not degraded but the brightness significantly reduced. Thus, it is supposed that CMCC-1 could destroy the cell wall and membrane of S. aureu, increase the cell membrane permeability and the leaking-out of intracellular substances, and thus cause the death ofS. aureu.

Study on chemical forms of fluorine enrichment of Antarctic krill

The different forms fluoine in different parts of molting process of krill was analyzed by culture of Antarctic krill (Euphausia superba). The results shaw that fluorine amount in cuticle is up to 90% of total in the krill. During the krill molting process, the fluorine content in cuticle and muscle varies alternatively. This is to say, the fluorine content in new cuticle and muscle is much lower after molted, the fluorine in krill comes from seawater by biological absorption of the krill, and the fluorine absorbed by krill lost with the old cuticle at the end stage of molting process. Meanwhile, the new molting process starts again. The evidence is proved by the change of different forms fluorine content in cuticle and muscle during krill's molting process. At last the author proposes the fluorine enriching process model of Antarctic krill.

The absorption kinetics of Antarctic krill oil phospholipid liposome in blood and the digestive tract of healthy mice by single gavage

Antarctic krill(Euphausia superba)oil has been gaining increasing attention due to its nutritional and functional potentials.Krill oil usually contains a high concentration(about 50%)of phospholipids(AKOP)rich in DHA and EPA accompanied with 30%–40%triacylglycerols.Phospholipids can be made into liposomes without emulsifiers due to its amphiphilic characteristics.However,the absorption kinetics of AKOP liposome in vivo is not clear,which restrict the molecular mechanism analysis related to its distinct bioactivities.The lipid analysis in serum,small intestinal content and wall was carried out after oral administration of AKOP liposome to illustrate its absorption kinetics in blood and the digestive tract of healthy mice by single gavage.The major type of the obtained AKOP was phosphatidylcholine,and the total contents of the DHA and EPA were 29.31%.AKOP liposome was almost completely digested in the small intestine in 1 h and the hydrolysis products could be quickly absorbed by intestinal enterocytes.The DHA in serum peaked at 2 h after administration of AKOP liposome.AKOP liposome could be quickly digested and absorbed in vivo.The obtained results might provide a scientific basis for the molecular mechanism analysis related to distinct bioactivities of Antarctic krill oil phospholipid.

Purification and Characterization of a Novel Lipase from Antarctic Krill

Lipase from Antarctic krill,with a molecular weight of 71.27kDa,was purified with ammonium sulfate precipitation and a series of chromatographic separations over ion exchange(DEAE)and gel filtration columns(Sephacryl S-100),resulting in 5.2%recovery with a 22.4-fold purification ratio.The optimal pH and temperature for enzyme activity were 8.0 and 45℃,respectively.Purified lipase had Km and Vmax values of 3.27mmolL−1 and 2.4Umg−1,respectively,using p-nitrophenyl laurate as the substrate.Lipase activity was enhanced by adding Ca2+and Mg2+ions in the concentration ranges of 0–0.5mmolL−1 and 0–0.3mmolL−1,respectively,while the activity was inhibited by a further increase in these ion concentrations.Fe3+and Cu2+ions showed obvious inhibitory effects on enzyme activity,and the inhibition rates were 71.8%and 53.3%when the ion concentrations were 0.5mmolL−1.

Evaluation of the toxicity of fluorine in Antarctic krill on soft tissues of Wistar rats

Antarctic krill are a potential food source for humans and animals, but krill are known to contain high levels of fluorine （F）. In this study, we investigated the toxicity of F in Antarctic krill using Wistar rats. There were three experimental groups： The control group were fed a basal diet, the krill treatment group were fed the same basal diet mixed with krill powder （150 mg＇kg-~ F）, and the sodium fluoride （NaF） treatment group were fed the basal diet with added NaF （150 mg.kg1 F）. General toxicity indicators including body weight and food intake were measured during the experiment. After three months the rats were dissected and tissue samples were collected from the liver, kidney, spleen, brain, and testis. Morphological changes in the cells of these tissues were assessed using HE staining. There were no significant differences in the body weight, the food intake, or the viscera coefficients among the three groups. In both treatment groups some pathological changes were observed in all soft tissue samples except the testis, although there were fewer and less severe pathological changes in the krill treatment group than in the NaF treatment group. The results showed that the toxicity of F in Antarctic krill was lower than for an equivalent amount of F in NaF, but it was still toxic to rats consuming large quantities of krill. The findings of this study highlight the need for further investigation into potential F toxicity if krill is to be used for human consumption.

BIOGEOCHEMISTRY RESEARCH OF FLUORIDE IN ANTARCTIC OCEAN Ⅰ. THE STUDY OF FLUORIDE ANOMALY IN ANTARCTIC KRILL

The aim of the study is to investigate the existing form and the partitioning pattern of fluoride in krill (Euphusia superba) by analysing the fluoride and other elements in various part of krill, primarily to approach the potential concentrating mechanism and the effect of fluoride in krill on the geochemical characteristics of fluoride in the Antarctic ecoenvironment. The results of the study show that the amount of flouride in various part of krill has a considerable difference. Most of fluoride is concentrated in the carapace, up to 4028 μg/g, and the head and legs, respectively 2724 μg/g and 2828 μg/g. The muscle contains the least flouride with amount of 226 μg/g. The amount of fluoride in whole freeze - dried krill is averagely 1232 μg/g, which indicates that the functional position of fluoride in krill is mainly located at the crust. Only a few of fluoride is found in the chitin of the carapaces (200 μg/g), which exhibits that fluoride in the carapaces exists mostly in the form of the nonchitious constitutent. In addition, the variation and concentration of flouride is related closely to some other elements such as P, Ca. Thus, fluoride in the carapaces is likely to exist as the form of the inorganic salt with P and Ca. It is also estimated frorn the study that a slightly higher concentration of fluoride in seawater and lower in sediment of the area relative to other oceans is possibly affected by the concentration of fluoride in the huge storage of krill in the area. The bioprocesses and precipitation with relation to the activity of krill should be very important and key sectoin to the geochemical cycling of the fluoride in Antarctic ocean.

Study on the distribution,abundance and growth condition of Antarctic Krill(Euphausia superba)in the Prydz Bay region,Antarctica during the Austral summer of 1999/2000

Antarctic krill (Euphausia superba) were collected from the Prydz Bay region during the austral summer of 1999/2000. The sample - collection was made at 15 sites in 3 longitudinal transects. Although krill were encountered at 14 sites, the abundance was comparatively low in general. The main component of the krill population was of post -larval stages. The mean numerical and weight density of krill integrated for all sampling stations in the survey area were 16. 17 ind. 1000 m -3and 12.02 g 1000 m -3 , respectively. In the survey area, stations with larger krill density are mainly located in the slope zone, except that the largest sample was collected in the open sea zone. No krill occurred in the only station of the shelf zone during our investigation. Krill collected from the slope zone were under better growth condition than those from the open sea zone, but when the survey area is considered as a whole, the growth condition is normal. The results of the comprehensive analysis of the environmental factors show that the three large sample stations seem to be related with the cold water mass at 75 m depth and the confluence area between the high and low Chla centers at the depth of 25 m.

BIOGEOCHEMISTRY RESEARCH OF FLUORIDE IN ANTARCTIC OCEAN Ⅱ. THE VARIATION CHARACTERISTICS AND CONCENTRATION CAUSE OF FLUORIDE IN THE CUTICLE OF ANTARCTIC KRILL

The cause of the concentration of fluoride in Antaractic krill is studied by the analysis of the characteristics of fluoride change in the cuticle of the krill before and after moulting. Associated with other related information, the source and accumulating mechanism of fluoride in krill are also disscussed. Results show that as an inorganic medium the cuticle of krill has the second concentrating function and action to fluoride after moulting , which has nothing to do with the biological action of the krill. The fluoride is concentrated from seawater, which is prosecuted mainly in the form of ion exchange.

Evaluation of the biological toxicity of fluorine in Antarctic krill

Antarctic krill is a potentially nutritious food source for humans, but lfuorine （F） toxicity is a matter of concern. To evaluate the toxicity of F in Antarctic krill, 30 Wistar rats were divided into three groups with different dietary regimens：a control group, a krill treatment group （150 mg＆#183;kg-1 F）, and a sodium lfuoride （NaF） treatment group （150 mg＆#183;kg-1 F）. After three months, F concentrations in feces, plasma, and bone were determined, and the degree of dental and skeletal lfuorosis was assessed. The F concentrations in plasma and bone from the krill treatment group were 0.167 0＆#177;0.020 4 mg.L-1 and 2 709.8＆#177;301.9 mg＆#183;kg-1, respectively, compared with 0.043 8＆#177;0.005 5 mg＆#183;L-1 and 442.4＆#177;60.7 mg＆#183;kg-1, respectively, in samples from the control group. Concentrations of F in plasma and bone in the krill treatment group were higher than in the control group, but lower than in the NaF treatment group. The degree of dental lfuorosis in the krill treatment group was moderate, compared with severe in the NaF treatment group and normal in the control group. The degree of skeletal lfuorosis did not change signiifcantly in any group. These results showed that the toxicity of F in Antarctic krill was lower than for an equivalent concentration of F in NaF, but it was toxic for rats consuming krill in large quantities. To conclude, we discuss possible reasons for the reduced toxicity of F in Antarctic krill. The present study provides a direct toxicological reference for the consideration of Antarctic krill for human consumption.

Ontogenetic diet shift in Antarctic krill(Euphausia superba)in the Prydz Bay:a stable isotope analysis

As one of the most common and dominant species in the Southern Ocean, Antarctic krill（Euphausia superba）play a significant role in food web structure and the process of energy flow. The diet of Antarctic krill in the Prydz Bay during austral summer of 2012/2013 was investigated and the ontogenetic shift in krill diet was evaluated using the stable isotope method. The nitrogen stable isotope values（δ^（15） N） of adults（（2.78±0.58）‰） were much higher than those of juveniles（（1.69±0.70）‰）, whereas the carbon stable isotope values（δ^（13） C） of adults（–（28.26±1.08）‰） were slightly lower than those of juveniles（–（27.48±1.35）‰）. Particulate organic matter（POM）from 0, 25, and 50 m depth combined（0/25/50 m） represented phytoplankton food items. The results showed that phytoplankton food items in surface water and mesozooplankton were two essential food items for Antarctic krill in the Prydz Bay during summer. POM（0/25/50 m） contributes 56%–69% and 26%–34% to the diet of juvenile and adult krill, respectively, whereas mesozooplankton composes 13%–34% and 58%–71% of the diet of juvenile and adult krill, respectively. Thus, an ontogenetic diet shift from POM（0/25/50 m）, which consists mainly of phytoplankton, to a higher trophic level diet containing mesozooplankton, was detected. The capacity for adults to consume more zooplankton food items may minimize their food competition with juveniles, which rely mostly on phytoplankton food items. This suggests ＂diet shift with ontogeny＂ which may somehow help krill keep their dietary energy budget balanced and well adapted to the Antarctic marine ecosystem as a dominant species.

Notes Diet analysis of Antarctic krill Euphausia superba Dana

Sampling date and position are shown in Table 1. Complete E. superbasamples with fullstomach were usua1ly used. They were averagely 45 mm long and 1.5 g weight (wet). Beingconcentrated with filtered sea water, every food species from krill stomach was observed andidentified under an optical microscope, and their size was measured. Food species in stomachcontent of E. superbawere divided into five classes, according to the presence of species indi-

A worldwide bibliometric analysis of Antarctic krill research during 1960 to 2015

This study conducted a bibliometric analysis of 1251 articles related to Antarctic krill from 1960 to 2015, based on the Core Collection in Thomson Reuters＇ Web of Science. We analyzed the results from four aspects： annual distribution, Web of Science categories and the major journals, inter- national collaboration and author keywords. The Antarctic krill research has developed rapidly since 1978, with an increasing article output. The studies mostly focused on the fields of marine and freshwater biology, oceanography, and ecology. Polar Biology, Marine Ecology-Progress Series and Deep-Sea Research Part II-Topical Studies in Oceanography, were the top three journals to publish the Antarctic krill-related papers. Although Russia is the earliest state to report Antarctic krill, U.S.A. and England were leaders with the largest output quantity and the highest cooperation frequencies. The researches in PR China have taken a blooming since 2009 and will play a more and more important role in the world. The frequencies of author keywords were counted. The high-frequency keywords were further divided into four groups with cohesive subgroup analysis, which suggested the major themes. This paper depicts the existing publica- tions on Antarctic krill. Our goal is to serve a point of reference and provide the direction for the future work.

Influence of sea ice and sea surface temperature on the abundance of Antarctic krill in Area 48.2

In this paper we examine the relationship between Antarctic krill catch, sea ice concentration, and sea surface tempera- ture （SST）. Data on the Antarctic krill catch from 2003 to 2010 in CCAMLR Area 48.2 were combined with sea ice and SST data. Results showed that krill fishing in Area 48.2 took place from February to August each year but the catch was concentrated from March to July, with production during this period accounting for about 99.3% of the annual catch. Regression analysis showed that the catch per unit effort （CPUE） was clearly related to sea ice concentration and SST intervals. CPUE was negatively correlated with the area of sea ice among years （R2=0.64）, and the correlation was strongest （R2=0.71） when sea ice concentration was greater than 90%. Over the months the CPUE initially increased, then decreased as the area of sea ice increased. The relationship was strongest （R2=0.88） when the concentration of sea ice was 60%--70%. There was no negative correlation among years between CPUE and the ice-free area when S ST was between -2 ℃ and 3 ℃ （R2=0.21）, but there was a significant negative correlation when SST was between 1 ℃ and 2℃ （R2=0.82）. Over the months, CPUE initially increased then decreased with increasing sea ice-free area, and the relationship was strongest （R2=0.94） when SST was between 0℃and 1 ℃. This study shows that sea ice concentra- tion and SST have significant effects on the abundance of krill in Area 48.2, and the findings have practical significance for the use and conservation of Antarctic krill resources.

The distribution and standing crops of Antarctic krill in the Prydz Bay region during the austral summer of 1991/1992 and 1992/1993

The horizontal and vertical distributions of Antarctic krill in the Prydz Bay region were investigated in 1991/1992 and 1992/1993. The result showed that the high density distribution area of Antarctic krill was within the area of 63°～67°S 、68°E and the waters of above 70 m deep. The standing crops of Antarctic kill was 1.699×10 6 t within the surveyed area of 150533.9 km 2 in 1991/1992 and 4.043×10 6 t within the surveyed area of 125740.7 km 2 in 1992/1993.

Reproductive characteristics of Antarctic Krill，Euphausia superba Dana，in the Prydz Bay region

Experiments and observations on reproduction of Antarctic krill Euphausia superba Dana,were conducted on board ship of R/V 'JIDI' during the austral summer of 1993 in the Prydz Bay region. The development of ovaria oocytes in gravid females,spawning epicces, and brood size were studied. The sexual maturity process and spawning season were discussed Fifty-three live gravid females captured at two stations in January, 1993 were cultured in a cool-room on board. They spawned within 1 to 16 days after capture. Most of them spawned only once except two, that spawned secondly on the 5th and 7th day after first spawning. The brood size of first spawning ranged from 225 to 5919 eggs with the average of 2132 eggs. The second brood was rather small,about 500 eggs. There were 0～213 (mean 32) mature oocytes remained in the post-spawn ovaries, and revitellogenesis was not observed. Brood size increased with the increasing of they length, but the correlation was not good. The number of ovarian oocytes in gravid female ranged from 2188 to 9263 with a mean of 5283. The composition of oocytes of different type varied in different samples. That all oocytes become completely matured seems to be an indication of pre-spawning. Spawning could take place through the summer with the peakspawning occurring from late January to late February. Interannual variation in peak spawning season might be significant.

Study on the relationship between the diameter of the compound eye and the growth of the Antarctickrill

Correlation between the body length and the diameter of the compound eyeof the Antarctic krill Euphausis sulfa was examined. From the samples collected inthe late summer, it shows that there is an apparent exponential relationship betweeneye diameter and the body length. From the laboratory population, it seems that whenthe krill shrink, the diameter of the compound eye does not decrease. It is more reliableto use the eye diameter as krill growth index than they length. The ratio of the theylength to the diameter of the compound eye offers another method for detecting the effect of shrinking in natural populations of krill.