Screening Of Antagonistic Bacteria Against Cutibacterium Acnes And Study On The Antibacterial Mechanism

Acne is a chronic skin inflammation of the hair follicle sebaceous unit,with an incidence rate of up to 85%in adolescents.C.acnes is one of the main factors leading to the onset of acne and is involved in the entire process of acne pathogenesis.Long-term use of antibiotics in acne treatment has led to severe antibiotic resistance of C.acnes to commonly used antibiotics.Therefore,the development of safe,effective,and non-resistant new antibacterial agents for acne treatment is an urgent need.In this study,a highly effective Cutibacterium acnes antagonist,Bacillus haynesii HA2,was screened from sauerkraut and subjected to fermentation optimization and in vitro safety evaluation.The antibacterial substances in the fermentation supernatant of strain HA2 were purified and identified,and their physicochemical properties,antibacterial spectrum,and antibacterial mechanism were studied.The results of this study provide a theoretical basis for the application of strain HA2 and bacteriocin HA2-5 in the treatment of acne,cosmetics,and skincare industries.The main research results are as follows:（1）Screening and identification of strains:Using C.acnes as indicator bacteria,a strain HA2 with strong antibacterial activity was screened from sauerkraut samples by Oxford cup method,and the diameter of its supernatant inhibition zone was 35 mm.By morphological observation,biochemical experiments and 16S r DNA sequence comparison,the strain HA2 was identified as B.haynesii.（2）Fermentation optimization and in vitro safety evaluation:The fermentation optimization results showed that strain HA2 had a stronger inhibitory activity when grown in a medium containing 15 g·L^-1 malt extract,30 g·L^-1 tryptone,and 2.5 g·L^-1 Ca Cl₂,at fermentation temperature of 35℃,initial p H of 5.0,inoculum size of 3%,liquid volume of 50%,and fermented for 72 hours.The diameter of the inhibition zone of the fermented supernatant of strain HA2 increased from 38 mm to 53 mm and the inhibition activity increased by 39.50%.In vitro safety experiments showed that strain HA2 was non-hemolytic,had a low risk of antibiotic resistance gene transfer,and lacked common virulence genes of Bacillus,indicating a certain degree of biological safety.（3）Isolation,purification,and identification of antibacterial substance:A bacteriocin,named HA2-5,was isolated and purified from the fermentation supernatant of strain HA2 by acid precipitation,methanol extraction,and RP-HPLC.SDS-PAGE and MALDI-TOF-MS analyses showed that the molecular mass of HA2-5 was 3255 Da.The amino acid sequence of HA2-5 was determined to be CEWYNLCCQLGNKGQWCLELKNPDWPL by LC-MS/MS and N-terminal sequencing.（4）Characterization of the bacteriostatic spectrum,stability and safety of bacteriocins HA2-5:Bacteriocin HA2-5 was found to effectively inhibit various Gram-positive pathogenic bacteria（C.acnes,S.aureus,L.monocytogenes,B.cereus,and E.faecium）with minimum inhibition concentration of 8μg·m L^-1 to 32μg·m L^-1.It exhibited good stability at different temperatures,p H values,and in the presence of proteases,UV radiation,and chemical reagents,and was tolerant to various metal ions.Bacteriocin HA2-5 also had low hemolytic activity（1.15%hemolysis rate）and low cytotoxicity（97%cell viability）.（5）Antibacterial mechanism study of bacteriocins HA2-5:The results of the growth curve and the bactericidal curve showed that bacteriocin HA2-5 could effectively inhibit the growth of C.acnes and had a bactericidal effect.The results of PI staining and intracellular substance release experiments showed that the bacteriocin HA2-5 could disrupt the integrity of the indicator bacteria cell membrane and led to intracellular substance leakage.SEM and TEM showed that the surface of C.acnes cells treated with bacteriocin HA2-5 was wrinkled,and the cell wall and cell membrane were damaged,and intracellular substances leaked.