Isolation And Characterization Of Hydrolyzable Peptides From Nannochloropsis Gaditan And Study On Antibacterial Activity

Nowadays,the extensive use of preservatives in the food industry and the abuse of antibiotics has highly adverse effects on humans and environment.Therefore,developing an efficient,safe and green natural antibacterial agent as a new food preservative and antibiotic replacement has become the focus of attention.In this paper,a selected heterotrophic microalgae strain,Nannochloropsis gaditana was cultivated via fermentation in batch stirring tank.The microalgae cells can be harvested by centrifugation for protein extraction after cellular disruption.A diversity of peptides can be obtained by hydrolyzing microalgae proteins catalyzed by papain.The minimum inhibition concentration(MIC)experiment was designed and performed to determine the antibacterial activities of peptides attained.As a result,a mixture of two peptides(P7)exhibit comparatively high antibacterial activity.Then,the first-order mass spectrometry(MS)was applied to determine the molecular weights of two peptides.After both derivation via the second-order MS and analysis from amino acid auto-analyzer,the amino acid sequences of the novel antibacterial peptides are HGAG and RWKKF,respectively.Finally,in order to validate their antibacterial activities,two peptides were artificially synthesized,and the antibacterial activity was retested.The comparison analysis via general antibacterial peptide database shows that they are two new antibacterial peptides and have potentially promising application for food preservative.The major research works are mentioned as follows:(1)The growth characteristic and single cell protein(SCP)production of a heterotrophic microalgal species,Nannochloropsis gaditana in air-sparging batch culture were studied in detail in the first part.The effect of pH on the microalgal growth was investigated in combination with microscopic morphological analysis in different growth stages.It was found that cultivation of microalgae in alkaline culture(pH 9.0)can make the pectic tight-texture of microalgal vesicle loose and can be easily dissociated into single cells.The dissociation of microalgal vesicles may greatly reduce the absorption of nutrients and consequently result in the decline of both the biomass and SCP productivity.Although lower cultivation temperature can extend the existence of the vesicle,the single cellular propagation rate inside the vesicle may drop significantly.Higher temperature also results in the inactivation of intracellular enzymes as well as the denaturation of tight structure of microalgal vesicle has a negative contribution in dropping process of protein productivity.Generally,at higher agitation level,the greater volumetric mass transfer coefficient,kLa can be achieved that highly improve the uptake of the oxygen.However,when the agitation rate is over 300 rpm,the damage caused by hydrodynamic stress should not be negligible because the turbulent eddy length scale will approach the average diameter of the vesicle.In conclusion,since the heterotrophic microalgal cultivation is efficient and highly-productive compared with the traditional autotrophic modes,it can be considered as a promising alternative for production of the quality SCP.(2)Cell disruption is a key step for recovery of microalgae proteins in the downstream stage.Four methods including high-pressure homogenization,repeating freeze-thaw,ultrasonication and enzymatical hydrolysis were employed to disrupt microalgae cell.The influence of different cellular disruption ways on the protein recovery as well as the microscopic state were investigated systematically.The results showed that the protein recovery reached 81.2%using the two cycle homogenization at 1000 bar.Also,repeating freeze-thaw is not suitable for the cell disruption of the microalgae due to its time-consuming and less than 70%protein recovery.The thermo effictiveness induced by ultrasound-assisted disruption may have a negative effect on the properties of the protein extracted.In addition,the enzyme-assisted method is not recommended since the introduction of exogenetic enzyme,i.e.cellulase can apparently vary the composition of microalgae endogenic proteins,although this method can attain a relatively high recovery of more than 80%.(3)In order to attain potentially bioactive peptides from Nannochloropsis gaditana,proteins recovered from the prior procedures were hydrolyzed by papain,combined with timing detection of the diversity of peptides.The peptides can be fractionated by RP-HPLC from protein hydrolysates.Furthermore,the peptides fractions(F1-F5)were collected by fractionating microalgae hydrolysates using the Prep-C18 semi-preparative column at time of 2h.Then,after further purification using Sepax-Bio-C18 column,the pure peptides(P1-P8)were obtained from antibacterial active fractions,F4 and F5.The final result showed that P7 had significant antibacterial activity.P7 was purified again and found that it was unable to be separated by the chromatographic methods.(4)The antibacterial assay used the broth dilution method to determine the minimum inhibitory concentration(MIC).The test bacterial stains include two Gram-positive(Staphylococcus aureus and Bacillus subtilis)and two Gram-negative(Escherichia coli and Pseudomonas aeruginosa).As a result,for protein hydrolysates at time of 2h,the remarkable activity against Staphylococcus aureus and Escherichia coli was observed.The antibacterial assay demonstrated that F4 and F5 had relatively high activities.Particularly,it was noted that the MICs of P7 against Staphylococcus aureus,Bacillus subtilis and Escherichia coli were 1.25 mg/mL,5 mg/mL and 0.625 mg/mL,respectively.Mass spectrometry was finally employed to analyze the chemical structures and molecular weights of P7.The results showed P7 was a mixture of tetrapeptide(HGAG,MW 340.90 Da)and pentapeptide(RWKKF,MW 764.01 Da)respectively.(5)Comparing with protein database and antibacterial peptide database,concluded to the result that the two novel antibacterial peptides.Then the MICs of the two artificially synthetical peptides were validated.The results showed that the MIC of HGAG was much higher than the P7,while the MIC of RWKKF was lower than the P7.HGAG was the shortest-sequence in the currently known antibacterial peptides of all microalgal,which greatly reduces the cost of artificial synthesis,and has significantly antibacterial activity.It can be considered as a promising alternative for production of the food preservatives and bacteriostatic agent.There is no report about antibacterial peptides of Nannochloropsis gaditana.This is the first time that antibacterial peptides from Nannochloropsis gaditana protein hydrolysates were isolated and characterized.