Study On The Forms Of Fluoride In The Integument Of Antarctic Krill And The Mechanism Of Free Fluoride Release

Antarctic krill is one of the most abundant animal species on Earth,with a biomass of6.5-10.0 billion tons,and is a promising protein source for alleviation of food shortage.Fluoride content in Antarctic krill is high,with an uneven distribution though.It is reported that 99%of the fluoride is localized in the integument and only 1%in the muscle.However,fluoride migrates from the integument to the muscle during postmortem storage,along with the muscle autolysis,resulting in fluoride contamination of krill meat which is then considered unsafe to eat.High fluoride is one of the key issues restricting the utilization of Antarctic krill.Disclosure of the forms of fluoride in the integument of Antarctic krill and the mechanism of free fluoride release would promote a further research on obstruction of fluoride migration and subsequently suppress the increase of fluoride content in krill meat.In this study,the micro-structure of Antarctic krill integument and the forms of fluoride were analyzed firstly,and then the factors inducing the release of free fluoride from integument were screened and identified by investigation of the p H of krill’s body,microorganisms and endogenous enzymes.It turned out that N-acetyl-beta-D-aminoglucosidase（NAGase）and trypsin-like protease might work up as potential inducers.At last,the purified NAGase and trypsin-like protease were used as models to investigate the mechanism of the free fluoride release from Antarctic krill integument.The changes of the microstructure of chitin and protein in the integument were observed.The main research contents and results were as follows:1.The microstructure of Antarctic krill integument was observed by scanning electron microscope（SEM）,and the organic and inorganic fluoride in the integument were characterized by nuclear magnetic resonance（NMR）,fourier transform infrared spectroscopy（FTIR）,X-ray diffraction（XRD）etc.The results showed that:（1）Protein,chitin and inorganic salts were the dominant components of Antarctic krill integument.The integument presented a typical layered structure consisting of a dense outer layer and a loose inner,withα-crystalline chitin fibers extending out of the channels localized in the integument and expanding to be layers which stacked together forming the main structure of the integument.There were regular nodules in the chitin fiber structure,filled with inorganic salts sediments.The sites were coated with proteins,and eventually comprised the integument.（2）The predominant form of the inorganic fluoride in Antarctic krill integument was fluorapatite.The rest of the fluoride bounded to weak organic and form a complex matrix.（3）Basically,the fluoride in the Antarctic krill integument existed in two forms:free state and bound state.The bounded fluoride had two patterns:fluorapatite and chitin-protein-fluoride complex.The content of total fluoride in Antarctic krill integument was estimated to be 3140±86 ppm,containing 722±35 ppm free fluoride,1272±44 ppm fluorapatite-bound fluoride and 1146±8 ppm chitin-protein-bound fluoride,which accounting for 23.0%,40.5%and 36.5%of total fluoride respectively.2.p H value of Antarctic krill changed from 7.42 to 7.95 after 24-hour storage at 10℃.Within this p H range,the amount of free fluoride released from the integuments remained unchanged.After being preserved at-30℃for 7 months,the total number of colonies in Antarctic krill remained unchanged,while the free fluoride released from the integument increased from 156.2 ug/g to 220.5 ug/g.Therefore,p H and microorganisms were not the key factors related to the free fluoride releasing from Antarctic krill integument.Antarctic krill integument treated with crude NAGase,trypsin-like protease and alkaline phosphatase（ALP）solution at 20℃for 10 h,the release of free fluoride increased from 35.0 ug/g to 58.7ug/g,27.0 ug/g to 48.9 ug/g,29.0 ug/g to 37.5 ug/g,and the increase was 23.7,21.9 and 8.5ug/g,respectively.Crude NAGase and trypsin-like proteases accelerated the release of free fluoride from the integument and might play a key role in the release of free fluoride.3.NAGase was isolated and purified by ammonium sulfate fractional precipitation,ion exchange chromatography and gel chromatography sequentially.Its purity and molecular weight were examined by SDS-PAGE.The purified enzyme was identified by MALDI-TOF-MS and its enzymatic properties were studied.The results showed that the purification fold of NAGase was 102.94,the yield was 16.11%,and its molecular weight was 59.0 k Da.The amino acid sequence of the peptide segment was similar to that of chitinase precursor（ACG60513.1）of Penaeus vannamei（protein score 132）,indicating that the purified enzyme was chitinase.The optimal p H of NAGase was 6.5,and the enzyme maintained stable in a p H range from 5.0-6.0.The enzyme showed the highest activity at 45℃and kept stable were 0.41 mmol/L and 2.66μmol/（min·m L）,respectively.4.Trypsin-like protease was isolated and purified by ammonium sulfate fractional precipitation,ion exchange chromatography and gel chromatography as above.Its purity and molecular weight were examined by SDS-PAGE.The purified enzyme was identified by MALDI-TOF-MS and its enzymatic properties were studied.The results showed that the purification fold of trypsin-like protease was 26.97,the yield was 9.32%,and the molecular weight was 25.9 k Da.The amino acid sequence of the peptide segment was similar to that of the Antarctic krill trypsin-like protease（AOW41610.1）（the protein score was 126 points）,indicating that the purified enzyme was trypsin-like protease.The optimal p H was 7.2,stable under neutral and weak alkaline condition.The optimal reaction temperature was 40℃and stable below 35℃.Mg²⁺,Ba²⁺and Ca²⁺activated trypsin-like protease.The kinetic parameters₈)and(1₈（6）were 0.31 mmol/L and 222μmol/（m L·min）,respectively.5.The effects of purified NAGase and trypsin-like proteases on the release of free fluoride were studied and the changes of the microstructure of chitin and protein in the integument were monitored.The regularity of free fluoride released from Antarctic krill integument was evaluated from the changes of the integument microstructure.A model was established to look into the mechanism of free fluoride releasing from Antarctic krill integument under the action of NAGase and trypsin-like proteases.The results demonstrated that the purified NAGase and trypsin-like proteases induced free fluoride release.Their respectively.The mechanisms of free fluoride releasing from Antarctic krill integument were as follows:NAGase and trypsin-like proteases degraded the chitin and protein,damaged the structure of Antarctic krill integument,and eventually leaded to the transformation of fluoride from organic binding state to free state.Heat treatment inactivated the endogenous enzymes of Antarctic krill,consequently obstructed the release of free fluoride from the integument and suppressed the increase of fluoride content in krill meat.