Purification of Two Antimicrobial Substances Produced by Bacillus subtilis Strain B11 and Their Properties

Two antimicrobial substances produced by Bacillus subtilis strain B 11 were purified by boiling, DEAE 52 anion exchange chromatography and aluminum oxide adsorption chromatography, These purified substances showed only one spot on silica gel thin layer chromatography （TLC）. Antimicrobial assays showed that antimicrobial substances A and B inhibited the growth of plant pathogens such as Fusarium oxysporum Schl f.sp. niveum, Rhizoctonia solani, Ralstonia solanacearum and Xanthomonas oryzae pv. oryzae. In addition, antimicrobial substance B could also inhibit the growth of Magnaporthe grisea. These two antimicrobial substances were resistant to proteolytic enzymes and temperature as high as 121℃.

Stricter regulation applies to antimicrobial substances when used as biocides compared to cosmetics under current EU legislation

Antimicrobial substances are substances that have the ability to kill or slow down the growth of microorganisms such as bacteria,algae and fungi.Exposure of microorganisms to low concentrations of antimicrobial substances may lead to the development of antimicrobial resistance.To protect human health and the environment and to limit resistance,the risks for the release of these substances into the environment should be minimized from all sources.Under the current EU legal framework,some specific uses of antimicrobial substances are strictly regulated whereas others are not.When antimicrobial substances are used as active substances in biocidal products,such as disinfectants,they are regulated by Biocidal Products Regulation.When the same substances are used as preservatives in cosmetic products they fall under Cosmetic Products Regulation.In this paper,we investigated how the regulation of antimicrobial substances differ when used in biocidal products compared to cosmetic products.This was achieved by performing a systematic comparison using document analysis with focus on aims,scope,information requirements,and risk assessment procedures for biocidal active substances and cosmetic preservatives.One of the main differences identified is that no environmental data or environmental risk assessment are required for approval of cosmetic preservatives.In contrast,environmental data and risk assessment for both the active substances and one representative product would be required for the approval of the same substance if used as a biocidal active substance.This means first that a substance not approved for use in biocidal products due to its hazardous environmental properties can still be approved as a cosmetic preservative.And second,the environmental release of antimicrobial substances from cosmetic products remains unassessed and uncontrolled,despite the obvious risks of cosmetic ingredients being emitted into the environment via wastewater.From this,we recommend that an environmental risk assessment is added to the requirements for market approval of cosmetic preservatives to achieve the aim of a high level of protection for the environment as set by several EU regulations.This would be in line with the“one substance e one assessment”approach proposed by the European Commission.While the details of“one substance e one assessment”approach are not decided,it is believed to contribute to more coordinated and transparent chemical safety assessments and enhance interlinking between the regulations.

Characterization of a Bacteriocin-Like Substance Produced from a Novel Isolated Strain of Bacillus subtilis SLYY-3

In the present research, the strain SLYY-3 was isolated from sediments of Jiaozhou Bay, Qingdao, China. The strain SLYY- 3, which produced a bacteriocin-like substance （BLS）, was characterized to be a strain of Bacillus subtillis by biochemical profiling and 16S rDNA sequence analysis. It is the first time to report that Bacillus subtilis from Jiaozhou Bay sediments could produce a BLS. The BLS of B. subtillis SLYY-3 exhibited strong inhibitory activity against gram-positive bacteria （including Staphylococcus aureus and B. subtillis） and some fimgi （including Penicillium glaucum, Aspergillus niger and Aspergillus flavus）. The antimicrobial activity was detected from culture in the exponential growth phase and reached its maximum when culture entered into stationary growth phase. It was thermo-tolerant even when being kept at 100~C for 60 min without losing any activity and stable over a wide pH range from 1.0 to 12.0 while being inactivated by proteolytic enzyme and trypsin, indicating the proteinaceous nature of the BLS. The BLS was purified by precipitation with hydrochloric acid （HC1） and gel filteration （Sephadex G-100）. SDS-PAGE analysis of the extracellular peptides of SLYY-3 revealed a bacteriocin-like protein with a molecular mass of 66 kDa. Altogether, these characteristics indicate the potential of the BLS for food industry as a protection against pathogenic and spoilage microorganisms.