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The food industry sector faces many obstacles all over the world due to biofilm formation causing economical huge losses and serious problems to human health. The present work was designed to discover new natural alternatives of effective antimicrobial peptides from insects against three pathogenic bacterial strains Escherichia coli O157:H7, Bacillus cereus ATCC11778 and Staphylococcus aureus ATCC29737. A total of 150 adult P. americana were reared and hemolymph was extracted to test their inhibitory activities. The adult P. americana hemolymph extract markedly inhibited the growth of B. cereus ATCC11778 and S. aureus ATCC2973 while E. coli O157:H7 was resistant. All cockroaches insects' hemolymph extraction and different concentrations of hemolymph were prepared then tested against the previously mentioned bacterial pathogens applying the Clinical Laboratory Standard Institute (CLSI) methods. Minimum inhibitory concentration of the extracts was also determined with zones of inhibition ranging from 10.0 to 30.0 mm. The highest value of the inhibition zone was for B. cereus, which recorded 30.0 mm for both crude extract and 0.5 mg/ml of hemolymph extraction. Crystal violet staining experiment was carried for testing biofilm inhibition. As a result, the formed biofilm of B. cereus and S. aureus was strongly inhibited by hemolymph and destroyed after 48 hrs of incubation with optical density of 0.388 and 0.0503 compared to control treatments of OD readings reached 1.91, at 590 nm, respectively. The cytotoxicity test against human skin fibroblast indicated no toxicity effect of the hemolymph. The Gas chromatography analysis of the adult Periplaneta americana cockroach hemolymph indicated the presence of antimicrobial peptides and fatty acids. The predominant antimicrobial peptides were 3,5-Triazine, 2,4-Diamine, 6-Chloro-N-Ethyl, N,N,N'-Trimethyl-N'-(4-Meth Oxy-Cis-Cinnamoyl-Putresc, 3',8,8'-Trimethoxy-3-piperidyl-2,2'-b inaphthalene-1,1',4,4'-tetrone at retention time (RT) of 6.99, 27.67 and 36.61 min, respectively. The most abundant Fatty acids were hexadecanoic acid, dodecanoic acid, Octadecanedioic acid at RT of 4.15 min. The inhibitory potency of hemolymph extract from Periplaneta americana as a new insectary alternative against microbial growth and biofilm formation makes this insect a new promising source for effective production of antimicrobial peptides (AMPs).

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