Structure And Cytoprotective Effects Against UVC Radiation Of Exopolysaccharide Produced From Paenibacillus Polymyxa PYQ1

Skin is the first biological barrier for human body to defense various physical and chemical stimuli.Among them,Ultraviolet（UV）radiation is one of the most common harmful factors.Recent studies indicated that overexposure to UV radiation is the main cause of skin photo-damage,which is characterized by pigmentation,erythema,inflammation and even skin cancer.Therefore,the harmful effect of UV on organisms and its protection have become hot issues.According to wavelengths,UV can be classified into three types:long-wave ultraviolet（UVA）,medium-wave ultraviolet（UVB）and short-wave ultraviolet（UVC）.Given that the ozone layer could absorb most of UVC,there are few reports focus on UVC.However,as the ozone in stratospheric decreases,the proportion of shorter wavelengths in ultraviolet light reaching the ground is rising.Meanwhile,due to its unique biological sterilization function,UVC is widely used in daily life and industrial production nowadays.The risk of exposure to UVC is increasing.Therefore,it is necessary to find suitable substances mitigating the adverse effects caused by UVC.Among abundant natural active ingredients,bacterial exopolysaccharides（EPS）have attracted researchers’interest on account of their excellent biological activity,stable quality,and low production cost.In this paper,the EPS with excellent cytoprotective effect was screened using UVC injury model of Human immortalized keratinocyte cells（HaCaT）.The structure and protection mechanism of EPS was explored.Our results would provide new ideas for the development of novel UV-induced damage repair agents as well as the theoretical basis for the application of bacterial EPS in the field of cosmetics.The key research findings are as follows:（1）Seven strains with high production of EPS were isolated and screened from various samples through observing the properties on plate and measuring the yield of EPS.After constructing UVC injury model in HaCaT cells,the protective effect of EPS1 from strain PYQ1 was found to be the best.Therefore,EPS1 produced by strain PYQ1 was selected for subsequent research.After 16S r DNA analysis,the strain PYQ1was identified as Paenibacillus polymyxa.Then the fermentation characteristics of PYQ1 were measured.It was found that PYQ1 entered a stable phase after 24 h of fermentation,when the number of bacterial cells reached at 10⁸ CFU/mL.The polysaccharide yield continued to rise within 0-36 h,and then remained stable.（2）Further purifications of EPS1 using chromatography of DEAE-52 and Sephadex G-100 revealed that EPS1 was a kind of acidic polysaccharide with single fraction.The recovery rate of purified EPS1 was 71.82%.The carbohydrate content in the purified EPS1was 95.21%,and no protein was detected.（3）The structure of purified EPS1 was characterized.UV spectrum showed no absorption at 260 and 280 nm,indicating the absence of nucleic acid or protein in purified EPS1.HPGPC results showed that EPS1 was a homogeneous and narrowly distributed polysaccharide with the Mw of 4.38×10⁶ Da.HPLC analysis demonstrated that EPS1 was mainly composed of mannose（20.40%）and glucose（66.20%）.The FT-IR spectrum of EPS1 showed the existence of typical polysaccharide absorption peaks containingβ-type pyranose specially.¹H and ¹³C NMR spectra illustrated that EPS1was composed of repeat units of six monosaccharide residues,of whichβ-D-Glcp was the main component.NMR results also showed the absence of furanose ring in EPS1,and all sugar residues belong to pyranose ring type.（4）The protective mechanism of EPS1 was explored from the perspective of oxidative stress.Our results showed that under the EPS1 treatment,the viability of HaCaT cells irradiated by UVC was improved.After detecting a series of indicators about oxidative stress,it was found that EPS1 could significantly alleviate UVC-induced ROS burst,GSH depletion,CAT activity decrease,mitochondrial membrane potential（MMP）collapse,membrane integrity decline and nuclear damage.