Isolation,Purification And Inhibitory Activity Against Citrus Spoilage Fungi Of Antifungal Peptide From Spirulina Platensis

In the process of storage,transportation and sale of citrus,it is easy to be infected by pathogenic microorganisms due to mechanical damage,which resulting in serious resource waste and economic loss.Moreover,most infected pathogenic microorganisms can produce toxins,cause food safety problems and be harm to human health.Compared with traditional chemical preservatives,natural preservatives,especially natural bacteriostatic peptides,have become a research hotspot in food antisepsis,health care and other industries due to their broad-spectrum antibacterial properties,non-resistance to drug resistance,and non-toxicity.Spirolina is a good source of bfoactive peptide due to its high growth rate,high biomass and abundant protein.In this study,Spirulina dry powder was used as raw material to prepare Spirulioa peptides by enzymatic hydrolysis,and the antifungi peptide components were isolated,purified,identified and analyzed.The main research results are as follows(1)Five different pathogenic fungi were isolated from citrus with different musty symptoms.The combination of traditional morphological observation and rDNA-ITS sequence analysis confirmed that the five pathogenic strains were Penicillium digitatum,Peoicillium expaosum,Penicillium glabrum,Trichoderma atroviride and Aspergillus flavus(2)Spirulina protein was extracted from Spirulioa dry powder by repeated freeze-thaw and ultrasonic disruption.The degree of hydrolysis of the Spi,-ulina hydrolysate was determined using the o-phthalaldehyde(OPA)method by comparing with the methods of phthalaldehyde titration,ninhydrin colorimetry and trichloroacetic acid precipitation.By comparing the the degree of hydrolysis by single enzymatic hydrolysis of four proteases with the degree of hydrolysis by stepwise dual-enzymatic hydrolysis of alkaline protease and papain,it was found that the hydrolysis degree of Spirulina hydrolysate can be improved by the stepwise dual-enzymatic hydrolysis and the degree of hydrolysis was as high as 42.19%.Then,the optimal condition for papain was determined by L9(34)orthogonal test:temperature 65℃,pH 5.5,enzyme and substrate ratio(E/S)5%,and the hydrolysis degree 32.01%;the optimal condition of alkaline was determined:temperature 55℃,pH 8.5,E/S 3%,and the hydrolysis degree 38.97%.After that,the optimal condition of pepsin was determined:temperature 40℃,pH 1.5,E/S 5%,and the hydrolysis degree 24.54%;and the optimal conditions for trypsin was determined:temperature 45℃,PH 6.5,E/S 3%,and the hydrolysis degree 26.08%,all these can be applied to other studies of proteolysis.(3)The antifungal activities of peptide components with four different molecular weight of<3KD,3-5KD,5-10KD and>1 0KD against Peoicillium digitatum,Penicillium expansum,Peoicillium glabrum,Trichodeivna atroviride were determined by the diameter of inhibition zone.It was found that the smaller the molecular weight of the peptide component,the stronger the antifungal activity,and the peptide component of<3KD showed the strongest antifungal activity.And then,the peptide component of<3KD was purified by Sephadex G-25 column chromatography.The elution conditions were as follows:the loading volume was 2 mL;the flow rate was 1 mL/min;the detection wavelength was 280 nm;the eluent was 0.01 mol/mLTris-Hcl buffer;and the column parameter was 1.6×40 cm.The eluate was collected in the order of peaks’ appearing,and two peaks were collected,which were named as component A and component B,respectively.The antifungal activities against Pemcillium digitatum,Penicillium expansum,Penicillium glabrum,Tirichhodrma atroviride and Aspergillus flavus of the two peptide components were compared by the diameter of inhibition zone.The results showed that component B showing stronger antifungal activity and the minimum inhibitory concentration(MIC)against the four fungi were 6.25,3.13,12.50 and]2.50 mg/mL,respectively.(4)The primary structure of Spirulina antifungal peptide component B was determined by tandem mass spectrometry LC-MS/MS,and then four unique peptides(RGQGGICY,AKALTEETG,KPTPLCTEFVA and SVAVGVGKM)from highly reliable protein source were identified by the uniprot protein database search.These four unique peptides played a certain role in the antifungal activity of Spirulina antifungal peptide component B.Finally,according to the physiological activity of C-phycocyanin and the typical structural features of antibacterial peptides,SP-4 was found to be the most important peptide when exerting antifungal activity in the Spirulina antifungal peptide component B.Its molecular weight was 629.40Da,and amino acid sequence was SVAVGVGKM,corresponding to the N-terminal 126-135 amino acid residues of the C-phycocyanin beta subunit.These results laid an important foundation for subsequent ·esearch and application.