Buchovec Irina, Lukseviciute Viktorija, Marsalka Arunas, Reklaitis Ignas, Luksiene Zivile
Vilnius University, Institute of Applied Research, Sauletekio 10, 10223, Vilnius, Lithuania.
Photochem Photobiol Sci. 2016 Apr;15(4):506-16. doi: 10.1039/c5pp00376h. Epub 2016 Mar 7.
This study is focused on the novel approaches to enhance the inactivation of the Gram (-) food pathogen Salmonella enterica and harmful molds in vitro and on the surface of strawberries using the chlorophyllin-chitosan complex. Salmonella enterica (∼1 × 10(7) CFU mL(-1)) was incubated with chlorophyllin 1.5 × 10(-5) M (Chl, food additive), chitosan 0.1% (CHS, food supplement) or the chlorophyllin-chitosan complex (1.5 × 10(-5) M Chl-0.1% CHS) and illuminated with visible light (λ = 405 nm, light dose 38 J cm(-2)) in vitro. Chlorophyllin (Chl)-based photosensitization inactivated Salmonella just by 1.8 log. Chitosan (CHS) alone incubated for 2 h with Salmonella reduced viability 2.15 log, whereas photoactivated Chl-CHS diminished bacterial viability by 7 log. SEM images indicate that the Chl-CHS complex under these experimental conditions covered the entire bacterial surface. Significant cell membrane disintegration was the main lethal injury induced in Gram (-) bacteria by this treatment. Analysis of strawberry decontamination from surface-inoculated Salmonella indicated that photoactivated Chl-CHS (1.5 × 10(-5) M Chl-0.1% CHS, 30 min incubation, light dose 38 J cm(-2)) coatings diminished the pathogen population on the surface of strawberries by 2.2 log. Decontamination of strawberries from naturally distributed yeasts/molds revealed that chitosan alone reduced the population of yeasts/molds just by 0.4 log, Chl-based photosensitization just by 0.9 log, whereas photoactivated Chl-CHS coatings reduced yeasts/molds on the surface of strawberries by 1.4 log. Electron paramagnetic resonance spectroscopy confirmed that no additional photosensitization-induced free radicals have been found in the strawberry matrix. Visual quality (color, texture) of the treated strawberries was not affected either. In conclusion, photoactive Chl-CHS exhibited strong antimicrobial action against more resistant to photosensitization Gram (-) Salmonella enterica in comparison with Gram (+) bacteria in vitro. It reduced significantly the viability of strawberry surface-attached yeasts/molds and inoculated Salmonella without any negative impact on the visual quality of berries. Experimental data support the idea that photoactivated Chl-CHS can be a useful tool for the future development of edible photoactive antimicrobial coatings which can preserve strawberries and prolong their shelf-life according to requirements of "clean green technology".
本研究聚焦于利用叶绿素铜钠盐 - 壳聚糖复合物，在体外及草莓表面增强对革兰氏阴性食源性病原体肠炎沙门氏菌和有害霉菌的灭活新方法。将肠炎沙门氏菌（约1×10⁷ CFU mL⁻¹）与1.5×10⁻⁵ M的叶绿素铜钠盐（Chl，食品添加剂）、0.1%的壳聚糖（CHS，食品补充剂）或叶绿素铜钠盐 - 壳聚糖复合物（1.5×10⁻⁵ M Chl - 0.1% CHS）一起孵育，并在体外以可见光（λ = 405 nm，光剂量38 J cm⁻²）照射。基于叶绿素铜钠盐（Chl）的光致敏仅使肠炎沙门氏菌灭活1.8个对数级。壳聚糖（CHS）单独与肠炎沙门氏菌孵育2小时可使活菌数降低2.15个对数级，而光活化的Chl - CHS可使细菌活菌数降低7个对数级。扫描电子显微镜图像表明，在这些实验条件下，Chl - CHS复合物覆盖了整个细菌表面。显著的细胞膜解体是该处理对革兰氏阴性菌诱导的主要致死损伤。对表面接种肠炎沙门氏菌的草莓去污分析表明，光活化的Chl - CHS（1.5×10⁻⁵ M Chl - 0.1% CHS，孵育30分钟，光剂量38 J cm⁻²）涂层可使草莓表面的病原体数量减少2.2个对数级。对自然分布的酵母/霉菌污染的草莓去污显示，单独的壳聚糖仅使酵母/霉菌数量减少0.4个对数级，基于Chl的光致敏仅减少0.9个对数级，而光活化的Chl - CHS涂层可使草莓表面的酵母/霉菌减少1.4个对数级。电子顺磁共振光谱证实，在草莓基质中未发现额外的光致敏诱导自由基。处理后的草莓视觉品质（颜色、质地）也未受影响。总之，与革兰氏阳性菌相比，光活性Chl - CHS在体外对更耐光致敏的革兰氏阴性肠炎沙门氏菌表现出强大的抗菌作用。它显著降低了草莓表面附着的酵母/霉菌和接种的肠炎沙门氏菌的活力，且对浆果的视觉品质没有任何负面影响。实验数据支持这样的观点，即光活化的Chl - CHS可成为未来开发可食用光活性抗菌涂层的有用工具，根据“清洁绿色技术”的要求，该涂层可保存草莓并延长其保质期。
