Study On The Antibacterial Activity Against Putrefactive Bateria In Food And The Mechanism Of Herbal Pomegranate Peel Extract

According to statistics, about 20% of meat and meat products were polluted by food spoilage every year in the world, which not only causes great economic losses, but also is extremely detrimental to human health. Previous studies showed that a major of food deterioration was caused by microbial contamination. In this work, Pomegranate peel is byproduct in pomegranate juice process, in order to investigate antimicrobial activity of pomegranate peel, common food contaminating microorganisms were used as tested microorganisms, the diameter of inhibition zone and MTT clearance rate were used as the activity monitoring parameter.This paper studied the antagonistic property for extracting antibacterial substances from pomegranate peel, component analysis, the extraction condition and mechanism. These researches would give reference for exploitation and developing new food preservatives. The main results are shown as follows:(1) Preliminary results revealed that the ethanol extract from pomegranate peel have antimicrobial activity for nine microbial contamination, in which Macrococcus caseolyticus,Micrococcuslu teus and Proteusbacillus vulgaris are the mose potent. The antimicrobial activity was in sequence of M. caseolyticus>M.luteus>P.vulgaris >S. saprophyticus>B. subtilis, E. coli>E. aerogenes, P.fluorescens>M. albican.(2) The MIC value of the ethanol extracts from pomegranate peel to the all tested microorganism were further examined. The results showed that the ethanol extracts of pomegranate peel had strong inhibitory effects on Macrococcus caseolyticus, Micrococcus luteus; the MIC of the ethanol extracts from pomegranate peel to Macrococcus caseolyticus and Micrococcus luteus were 12.5 mg/mL respectively; while the MIC of the extract to Staphylococcus saprophyticus, Proteusbacillus vulgaris, Escherichia coli were 25.0 mg/mL; the MIC of the extract to Bacillus subtilis, Enterobacter aerogenes and Pseudomonas fluorescens were 50.0 mg/mL, the MIC of the extract to Monilia albican was 100.0 mg/mL.(3) The inhibition of ethyl acetate, n-butanol and water phases of the ethanol extracts from pomegranate peel on Macrococcus caseolyticus and Micrococcus luteus was further examined, and the results showed that the n-butanol phases had strong inhibition on the two tested microorganisms. The MIC value of pomegranate peel n-butanol phases to Macrococcus caseolyticus and Micrococcus luteus were 12.5 mg/mL, which were similar to the ethanol extracts from pomegranate peel; the MIC values of pomegranate peel ethyl acetate phases were 50.0 mg/mL and 25.0 mg/mL to Macrococcus caseolyticus and Micrococcus luteus respectively, which were higher than the ethanol extracts from pomegranate peel. While the water phases had no antibacterial activity to Macrococcus caseolyticus and Micrococcus luteus. The result showed that the main antimicrobial active substances of the ethanol extracts from pomegranate peel were in n-butanol phases.(4) The effects of heat treatment and UV irradiation on antibacterial activity of pomegranate peel n-butanol phases were examined. The result showed that the antibacterial activity to Macrococcus caseolyticus was stable to high temperature and UV irradiation.(5) The chemical constituent in the n-butanol extract of pomegranate peel was separated and purified by modern chromatographic techniques including macroporous resin column,silica gel, Sephadex LH-20,and HPLC. The compound was purified from the n-butanol extract of pomegranate peel and eventually identified as isoquercitrin.(6) The content isoquercitrin as indicator, using ethanol refluxing extraction technology of isoquercitrin in pomegranate peel single factor experiment, through the orthogonal method to get the main factors influencing the extracting effect from more important to less were as follows:ethanol concentration, extracted temperature, extracted time and ratio of liquid to solid; the optimum extraction conditions were that the concentraction of ethanol was 90%,the temperature of extraction was 80℃,the time of extraction was 2.5h and the ratio of liquid to solid was 15:1. By the above conditions, the isoquercitrin content is 2.36%.(7) Growth curve assay showed that the pomrgranate peel n-butanol extract inhibited Macrococcus caseolyticus in logarithmic growth phase. Respiratory inhibition experiment showed that the respiration of thallus was inhibited mainly through the hexose monophosphate pathway, which leaded to be short of NADPH, and induecd metabolic function dysfunction. The results showed that by measuring alkaline phosphatase AKP, the pomegranate peel n-butanol extract caused Macrococcus caseolyticus increased permeability of cell walls, there by undermining the integrity of the cell structure. After treated by the pomrgranate peel n-butanol extract, it was observed by TEM that the surface and inner structure of bacterial cells were destroyed and induced cells plasmolysis. This study suggested that the antibacterial mechanism of the pomrgranate peel extract was that it inhibited HMP and leaded to metabolic function dysfunction and inhibited Synthesis of respiratory metabolic enzymes, then blocked their division and development, leaded to cell pyknosis. Eventually growth and reproduction of cells were inhibited and death.