| Persimmon(Diospyros kaki L.),as one kind of deciduous and medicinal fruit,possesses a wide spectrum of bioactive compounds including polyphenols,tannins,flavonoids,pigments,vitamins and dietary fiber,and consequently multiple nutritional values and health benefits,which is a plant resource with great potential.Persimmon tannin(PT),the unique natural compound in persimmon,is the main contributor to bio-physiological activities of persimmon.Therefore,comprehensive mining the physiological and biochemical characteristics of PT is the first step to promote its application as well as persimmon industry development.Previous studies have reported that PT had antioxidant,anti-arteriosclerosis and anti-radiation activities,and the relative mechanisms have been preliminarily studied.However,studies on the antibacterial activity of PT were usually carried out with crude PT extracts,and the antibacterial mechanism of PT was still unclear.Therefore,in order to clarify the antibacterial activity and antibacterial mechanism of PT,this study firstly screened the appropriate materials for PT extraction and optimized the extraction conditions of ultrasonic-assisted extraction method to obtain PT with high quality and purity.Secondly,the antibacterial activity of PT on methicillin-resistant Staphylococcus aureus(MRSA)isolated from retailed pork was investigated.Thirdly,the effects of PT on MRSA at levels of cell,transcriptiome and metabolome were investigated to analyze the antibacterial mechanism of PT.The results laid a theoretical foundation for comprehensive utilization of persimmon resources and development of novel antibacterial agents.The main results were as follows:1.Changes of physiological activator in persimmon and screening of material for PT extraction.The changes of PT,pigments and antioxidant activity of persimmon pulp and peel from six persimmon varieties(Jinpingshi,Shaguyihao,Huoguan,Zenjimaru,Fuyu and Youhou)were investigated during the whole growth period(30?170 d after bloom).Results showed that PT contents were significantly affected by persimmon varieties,growth periods and fruit parts.PT contents in pulp of different varieties from high to low were pollination constant astringent(PCA)> pollination-variant non-astringent(PVNA)>pollination-constant and non-astringent(PCNA),while PT contents in peel were similar.With the development of fruit,PT contents in pulp and peel decreased gradually.The change of antioxidant capacity of persimmon was similar to that of PT,and positive correlation was detected between them.Chlorophyll contents were mainly influenced by growth period and fruit parts.The contents reached the maximum around 70 days after blooming and were significantly higher in peel than in pulp.For single fruit,PCA type had the highest PT content and reached the maximum at 110?130 d after blooming.Besides,the hardness of persimmon fruit decreased rapidly from 110 d after blooming.To conclude,the pulp of PCA fruit harvested at 110 d after blooming was the most appropriate material for PT extraction.2.Optimization and comparative analysis of PT extraction technology.Ultrasonic-assisted extraction(UAE)and traditional water bath extraction(WBE)methods used for PT extraction were optimized by response surface methodology.Their extraction efficiency as well as effects on extraction material and PT characteristics were comprehensively compared.The maximum yield(33.96 mg/g)extracted by UAE was obtained under conditions of ultrasonic power 207 W,time 30.9 min,L:S ratio 21.1:1 m L:g and 55.0 ?,whereas PT yield reached the maximum(32.39 mg/g)under conditions of73.5?,time 63.0 min,and L:S ratio 32.4:1 m L:g when using WBE.In comparison,the extraction yield of UAE was 4.94% higher than WBE,but the temperature,liquid:solid ratio and time were 75.34%,65.12% and 49.05% of WBE,respectively.Moreover,UAE significantly altered the structure of persimmon pulp and peel than WBE,while the UV,FT-IR spectra and antioxidant activity of PT were not influenced by different extraction methods.These results indicated that UAE was suitable for PT extraction for its low energy consumption,high PT yield and few effects on product properties.After purification by macroporous resin,the purity of PT reached 92.88%.3.The antibacterial activity and antibacterial mechanism of PT on MRSA.Agar dilution assay showed that PT had antibacterial activity against 4 MRSA strains isolated from retailed pork,with minimum antibacterial concentrations of 1000 ?g/m L.Analysis of growth curve showed that PT could significantly inhibit MRSA proliferation,prolong the lag period and reduce the maximum growth rate.A series of biochemical tests indicated that PT treatment significantly reduced cell membrane potential and intracellular ATP content,destroyed the integrity of cell membrane and protein,affected cell cycle and led to S-phase arrested.Moreover,surface hydrophobicity and hemolytic activity of MRSA were significantly reduced.In addition,electron microscopy showed that PT significantly changed the normal physiological morphology of MRSA,resulting in deformation,depression,content overflow and other irreversible damages.It was preliminarily speculated that PT exerted anti-MRSA effect by changing cell membrane permeability and damaging cell function.4.Transcriptome analysis of antibacterial mechanism of PT on MRSA.The influence of PT(500 ?g/m L)on transcriptional level of MRSA was studied using Illumina Hi Seq PE150 sequencing platform.A total of 370 differentially expressed genes(DEGs)were obtained,200 DEGs of which were up-regulated and 170 DEGs were down-regulated.Gene Ontology(GO)analysis showed that DEGs were mainly involved in catalytic activity,binding,cell membrane,cell,metabolic process and cellular process.Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis indicated that DEGs were mainly participated in metabolic pathways including membrane transport,amino acid metabolism,carbohydrate metabolism and energy metabolism.Clue GO+Clue Pedia analysis found that DEGs involved in ABC transport,amino acid metabolism and pyrimidine pathways were mostly up-regulated,while DEGs involved in nitrogen metabolism,glycolysis and TCA cycle pathways were mostly down-regulated.Based on further analysis,PT on one hand caused damages on cell membrane function,resulted in increased membrane permeability and dysfunctional p H regulation,leading to disorder in amino acid metabolism and inhibition on energy metabolism.On the other hand,PT significantly reduced the expression of genes related to nutrient uptake,which induced deficiency of intracellular amino acids and aggravated disorder in intracellulat metabolic,and thereby inhibiting the growth of MRSA.Therefore,it was speculated that membrane damage and nutrient limitation were the main antibacterial mechanism of PT on MRSA.5.Metabolome study and validation for transcriptome.The changes in metabolites of MRSA under PT(500 ?g/m L)treatment were detected using metabolome technology.Principal component analysis and partial least square discriminant analysis showed that metabolic profiles of MRSA in PT group were significantly different from those in control.Nineteen differential metabolites(DMs)were obtained,12 of which were up-regulated and 7were down-regulated.KEGG results showed that DMs were mainly related to amino acid metabolism,TCA cycle and nitrogen metabolism.Based on the results of metabolome and transcriptome,a gene-metabolite regulatory network for amino acid and energy metabolism was constructed.It was found that PT significantly influenced the amino acid and energy metabolism of MRSA.Under PT treatment,aspartic acid,lysine,tyrosine and ornithine were largely consumed,while proline was accumulated which could regulate the osmotic pressure and scavenge free radicals;urea content increased as well.Moreover,the content of succinic acid and citric acid increased,indicating that TCA cycle was arrested.It can be seen that the changes and flow direction of DMs were mostly closely related to cell membrane damage,nutrient deficiency and energy metabolism arrest,further proving the results of transcriptome. |
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