Protein Hydrolysates From Achatina Fulica(Giant African Snail) Mucus And Determination Of Antioxidant And Antimicrobial Activities

In recent years the microbial resistance is increasing gradually and it's an alarming situation for the world.Most conventional drugs like antibiotics are losing their resistance against pathogenic bacteria.On the other hand,oxidation is also a serious issue and most food industries are using synthetic antioxidants which could be harmful or may occur severe side effects on human health.Hence,both pharmaceutical and food companies are looking for alternative ways to produce bioactive peptides from natural resources such as animals and plants which is considered safe,with low toxicity and high specificity.These peptides are inactive since they are fused in their parent protein.Therefore,they must be cleaved from the encrypted protein by different methods such as microbial fermentation,chemical hydrolysis,and enzymatic hydrolysis.To produce bioactive peptides from protein,enzymatic hydrolysis is preferred because chemical hydrolysis can destroy some amino acids during the reaction and microbial fermentation can produce inactive peptides because of changes in microbial activity.Enzymatic hydrolysis is an effective,competent method with milder conditions that do not destroy the amino acids during the reaction.It has some significance over the traditional methods of generating bioactive peptides from various protein by-products.Achatina Fulica,the African giant snail,is a pest for vegetable fields and is also used for food purposes.This special species can survive in tough environmental conditions and produce a kind of by-product known as mucus or slime which protects its body from different infective microbes and could heal minor injuries.usually,snail mucus is used in the cosmetics industry as an anti-aging compound.Furthermore,this mucus revealed many bioactivities against free radicals and pathogenic bacteria.The mucus contains protein which helps in different bioactivities.On account of its contribution,recently many researchers have focused to use snail mucus as a bioactive compound.In this study,the protein content in snail mucus was determined by the Lowry method and the Kjeldahl method was applied to know the total nitrogen content.On the other hand,the Formol titration method was developed to measure the total amino nitrogen content,which further helped to determine the degree of hydrolysis.The enzymatic hydrolysis technique was first applied to generate protein hydrolysates from snail mucus protein solution.The protein hydrolysates were obtained by three different enzymes which significantly hydrolyzed the protein in snail mucus solution.During enzymatic hydrolysis,the major optimal conditions like temperature,p H,and enzyme concentration were maintained.The progress of hydrolysis was observed in terms of the degree of hydrolysis and protein hydrolysates were collected after a specific time interval.The antioxidant activities of the different protein hydrolysates were evaluated using in vitro antioxidant assays.Furthermore,protein hydrolysate activity was investigated against pathogenic gram-positive and negative bacteria.The primary research contents are as follows:(1)The snail mucus solution was first employed to determined protein content by three different techniques,including Lowry,Kjeldahl,and Formol titration method.Based on experimental results,the Lowry method showed that snail mucus solution has 51.23%w/w high protein content.The Kjeldahl method was used to digest the snail mucus with sulfuric acid to determine the total nitrogen content of about 0.16 g N/g.The Formol titration method was used to estimate the total amino nitrogen.The degree of hydrolysis was calculated by the number of hydrolyzed peptide bonds and the total number of peptide bonds.(2)The snail mucus protein solution was employed to generate hydrolysates using three enzymes.At first,the protein solution was subjected to hydrolysis by papain with optimum conditions of temperature 60?,p H 6.0,phosphate buffer solution with an enzyme concentration of 2%w/v.The progress of hydrolysis was observed in terms of the degree of hydrolysis(DH).The hydrolysis progress was increasing gradually from 0.5 to 4 h with DH 4.2%-17.5%.After that,rapid DH38.38%was observed at 6 h and the highest DH 44.1%was detected at 8 h.The pepsin was utilized for hydrolysis with an ideal temperature of 37?,glycine-HCl buffer with p H 2.0,and 2%w/v enzyme concentration.The rapid hydrolysis was monitored with DH 15%in early 0.5 h.Subsequently,the reaction exhibited the DH 17.1%-21.5%from 1 to 5 h.The highest DH 24.5%was observed at 6 h.The alcalase with a concentration of 0.5%w/v was employed for hydrolysis reaction with key conditions of temperature 55?,glycine-Na OH buffer at p H 8.5.It was noted that the enzymatic hydrolysis rate was high during the first 10-30 minutes with DH 14.1%-26.3%.The DH 28.01%-30.5%was observed from 60 to120 minutes.The highest DH 34.5%was detected at180 minutes.(3)The protein hydrolysates(Pap-H 0.5,Pap-H 1,Pap-H 2,Pap-H 4,Pap-H 6 and Pap-H 8)obtained by Papain,protein hydrolysates(Pep-H 0.5,Pep-H 1,Pep-H 2,Pep-H 3,Pep-H 4,Pep-H 5,and Pep-H 6)generated by Pepsin,protein hydrolysates(Alc-H 10,Alc-H 20,Alc-H 30,Alc-H 60,Alc-H 120 and Alc-H 180)produced by Alcalase were subjected to antioxidative measurements based on 2,2-diphenyl-1-picrylhydrazyl(DPPH·),2,2'-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid(ABTS^·+)scavenging and Ferric ion reducing antioxidant power(FRAP)ability.The DPPH free radical scavenging assay revealed that Alc-H 180 and Pap-H 8 showed the lowest IC₅₀ values of 21.3mg/m L and 21.5 mg/m L respectively.Pep-H 6 showed an IC50 value of 30.5 mg/m L.The ABTS^·+free radical scavenging presented that Alc-H 180,Pep-H 6,and Pap-H 8 exhibited the lowest IC₅₀values of 17.1 mg/m L,19.1 mg/m L,and 27.9 mg/m L respectively.The FRAP assay displayed that Pep-H 6 had the highest reducing power at 1.6 mmol/L.In papain-derived hydrolysates,Pap-H 8 had strong reducing power at 1.1 mmol/L.Alcalase-derived hydrolysate,Alc-H 180 had reducing power at 0.27 mmol/L.(4)The antimicrobial abilities of protein hydrolysates were evaluated by the microdilution method,against Gram-negative bacteria Escherichia coli(ATCC NO.8739)and Pseudomonas aeruginosa(ATCC NO.27853),as well as Gram-positive bacteria Bacillus subtilis(ATCC NO.14579)and Staphylococcus aureus(ATCC NO.6538).The results showed that Alcalase-derived hydrolysates(Alc-H 10,Alc-H 20,Alc-H 30,Alc-H 60,Alc-H 120,and Alc-H 180)exhibited an antimicrobial ability against two Gram-negative bacteria(Escherichia coli and Pseudomonas aeruginosa).The minimum inhibitory concentration(MIC)was 10000 ug/m L.MIC is the lowest concentration of an antimicrobial agent that is bacteriostatic.The inhibition percent of bacterial growth was determined.Alc-H 180 after 8 hours incubation showed the strongest inhibition percentage of 38.46%and 66.66%against E.coli and P.aeruginosa respectively.Papain-derived hydrolysates(Pap-H 0.5,Pap-H 1,Pap-H 2,Pap-H 4,Pap-H 6,and Pap-H 8)exhibited the activity against gram-positive bacteria Bacillus subtilis.All hydrolysates showed a MIC of 2500 mg/m L.The Pap-H 8 hydrolysates showed a great inhibition of 68.75%against Bacillus subtilis in 4 hours incubation.Pepsin-derived hydrolysates(Pep-H 0.5,Pep-H 1,Pep-H 2,Pep-H 3,Pep-H 4,Pep-H 5,and Pep-H 6)inhibited another gram-positive bacterium.Staphylococcus aureus.The MIC 2500ug/m L presented strong resistance and Pep-H 6 exhibited a high inhibition of 68.57%against Staphylococcus aureus after 8 hours of incubation.In summary,the enzymatic hydrolysis was applied to generate protein hydrolysates from snail mucus protein by three different enzymes(Papain,Pepsin,and Alcalase).This technique is significant to produce hydrolysates using different time intervals with increasing DH%.The bioactivity improves when DH%increase.To investigate the bioactivity of protein hydrolysates,free radical assays and pathogenic microbes were employed.As expected,high DH exhibited more strong activities.The higher DH%have been shown by Pap-H 8,Pep-H 6,and Alc-H 180.The control sample could not show significant activity in comparison with protein hydrolysates.In Alcalase-derived hydrolysates,Alc-H 180 displayed higher scavenging ability and contributed to inhibiting two-gram negative bacteria.While Pep-H 6 presented high reducing power and displayed a strong inhibition percentage against gram-positive bacteria.Papain-derived hydrolysate Pap-H 8 displayed remarkable inhibition against gram-positive bacteria.The enzymatic hydrolysis is an effective and promising technique to generate protein hydrolysates or peptides from parent protein.The protein hydrolysates Alc-H 180,Pep-H 6,and Pap-H 8 presented an outstanding ability against free radicals and resistive microbes.