Fresh-Keeping Effect Of Melanin-Free Extract From Sepia Ink Towards White Snapper

Objective: Our aquaculture industry had a rapid development during these years; itsannual production was up to45,000,000tons and took the first place in the world for15years. Aquatic products, especially fish, with low fat, low heat but high protein, havebecome an indispensable part of reasonable diet structure. However, during the advance ofliving standard, the fresh of aquatic production has higher requirements. Then thepreservation methods and antistaling agents turned into focus. Sepia ink is a naturalmaterial which has multiple functions; also there are many reports on melanin andproteoglycan complex. Our previous work found that the polysaccharide in sepia ink hasthe fresh keeping effect on Loligo japonica. So, we take the white snapper as target todiscuss the preservation of sepia ink by determines the preservation indexes and isolate thespoilage bacteria classes in white snapper to explore the bacteriostatic effect. Finally weget the bacteriostatic material from sepia ink and investigate the mechanization inbacteriostatic effect of sepia ink.Method:1、Cleaned fish were divided into groups randomly, treated with five differentconcentrations and four different pH values of sepia ink, the control group was treated withphosphate buffer. All the samples were kept in storage on7days at4℃, and weredetermined the indexes of samples everyday. The indexes include sensory analyses, pHvalue, bacterial count, total volatile basic nitrogen, K value and Ca²⁺-ATPase value. Inaddition, two aspects of preservation—bacteriostasis and antioxidant were researched byvarious indexes.2、separate the bacteria from white snapper, observe the morphological character ofbacteria and individual character of single bacteria. Then use the appropriate physiologicaland biochemical tests to ensure the spoilage bacteria classes in white snapper and the PCRtechnique was used to confirm the dominated spoilage bacteria.3、The bacteriostatic activity and minimal inhibitory concentration （MIC） of sepia inkwere measured by using the antibacterial circle method and colonies notation.4、The chemical composition of squid ink, include the moisture content, crude proteincontent, crude polysaccharide content, crude fat content, ash and salt content, amino acidand metal element content were measured. After that the squid ink was separated by DEAE-cellulose ion-exchange and sepharose4B chromatography, andidentified which substance was the antibiotic substance among the substance separatedfrom sepia ink by using the dominated spoilage bacteria as index. Further identification ofantibacterial substance was done by determination of molecular weight, ultravioletspectrophotometry（UV） and infrared spectrophotometry（IR） spectrum scanning.Results:1、The squid ink has the fresh keeping effects, the higher concentration, themore preservation effects, and the pH value could enhance the effects, the best effects wasobserved when the pH value was8.5. The squid ink has the bacteriostasis and antioxidantactivity to the white snapper.2、22bacteria strains were isolated from fish, most bacteria were roundness, colonysurface were smooth, and colony edge was regular and opaque. A little quantity of thebacteria was irregular, colony surface were rough, and colony edge was irregular andtransparent. The colors of the colony were various, and the white and yellow are dominant.After physiological and biochemical tests and PCR technique, it included five categorieswhich were Aeromonas, Enterobacter, Flavobacterium, Bacillus, shewanella. Then thebacterial phase analysis showed that the number of Aeromonas is the most which accountfor76.77%, followed by the Enterobacter, Flavobacterium and Bacillus, which theproportions were11.94%,7.10%,5.19%,0.03%respectively. In addition, a few ofshewanella also could be found, which accounts for0.03%. So the optimal corruptionbacteria were Aeromonas, Enterobacter, Flavobacterium and Bacillus.3、The squid ink has the inhibition effect on the dominated spoilage bacteria in whitesnapper, especially on Aeromonas and Flavobacterium. When the pH value reached8.5,the inhibition effect was enhanced obviously, especially to Aeromonas and Bacillus.Through the colonies notation, the MIC of squid ink was16mg/mL.4、Chemical composition of squid ink found that the water content was78.93%, thecrude protein content was15.52%, the crude polysaccharide content was0.46%, the crudefat content was1.24%, the ash content was16.15%and salt content was2.89%. The aminoacid was16species （essential amino acid was7species and semiessential amino acid was1species） and it is rich in mineral elements including Ca、K、Mg、Na、S、P andmicroelement like Al、Cr、Cu、Fe、Mn、Zn. Besides, the molecular weight of the inhibitorymaterial was about83,589Da, and the materialwas mainly appraised to be polyose by UV and IR spectrum scanning.Conclusion: The squid ink has fresh-keeping effect on white snapper, not only has inhibitory activity on optimal corruption bacteria of white snapper, but also haveantioxidant activity. The experiment proved that the inhibitory material was polyose whichmolecular weight was about83,589Da.