【作者】 董周永；

【导师】 刘兴华；

【作者基本信息】 西北农林科技大学 ， 食品科学， 2008， 博士

【摘要】 石榴（Punica granatum L．）为石榴科石榴属植物，原产于伊朗、阿富汗等中亚地区，西汉时引入中国，至今已有2000多年栽培历史。石榴果实具有很高的食用价值和药用价值，研究发现石榴具有抗氧化、抗癌、抗病毒、降血脂等多种生理活性功能。目前，对石榴果皮的生物活性研究主要集中在抗氧化活性方面，对其抑茵活性方面的研究相对较少，而从食品安全保藏学角度对其抑菌活性研究更是鲜见报道。本研究以石榴加工中的副产物——石榴果皮为试材，以食品中的常见污染菌为供试菌，以抑菌圈直径为活性追踪指标，对石榴果皮中抑菌活性物质提取的工艺条件、抑菌特性、抑菌机理及对冷却猪肉的保鲜作用进行了系统地研究，研究结果为石榴果皮工业化开发利用提供了理论和技术依据。主要研究结果如下：（1）对石榴果皮提取物抑菌活性初步研究结果表明，石榴果皮甲醇提取物的抑菌活性最高，对供试菌中细菌的抑制效果最好，其中对革兰氏阳性菌的抑制效果优于革兰氏阴性菌：对酵母菌的抑制效果较弱；对霉菌几乎没有抑制作用。对各供试菌的抑制效果依次为：金黄色葡萄球菌＞李斯特菌＞巨大芽孢杆菌、枯草芽孢杆菌＞沙门氏菌＞大肠杆菌＞解脂假丝酵母。（2）在供试的10个石榴品种中，‘峄城软籽’石榴果皮提取物的抑菌活性最高。石榴表果皮提取物的抑菌活性高于锥形横膜和萼筒的提取物。（3）在单因素试验的基础上，通过二次回归正交旋转组合设计进行提取工艺参数优化。结果表明，对石榴果皮抑菌活性物质提取效果影响的大小顺序为：乙醇浓度、提取温度、液固比、提取时间；其最佳提取工艺条件为：乙醇浓度60％，液固比10：1，提取温度60℃，提取时间4h。在此优化条件下，石榴果皮提取液抑菌圈直径为16．30mm。（4）抑菌试验结果表明，在优化条件下得到的石榴果皮提取物的抑菌谱与甲醇提取物相似，但活性更高；其对金黄色葡萄球菌的MIC、MBC分别为1．562 mg／mL、3．125mg／mL，抑菌活性与Nisin接近，而远高于苯甲酸钠和山梨酸钾。（5）石榴果皮提取物的抑菌活性具有较好的热稳定性，对紫外线照射和光照稳定；在介质pH6时活性最高；盐和糖对其抑菌活性具有一定的增效作用；Na⁺、Ca²⁺、K⁺、Fe²⁺、Fe³⁺均能提高石榴果皮提取物的抑菌活性，其中Fe²⁺的效果最为明显。（6）石榴果皮提取物经石油醚、氯仿、乙酸乙酯、正丁醇依次萃取后，各萃取部分经抑菌活性比较，表明正丁醇部抑菌活性最高；对其化学成分分析结果表明抑菌活性物质主要为石榴多酚。采用硅胶柱层析法对正丁醇部进行进一步分离纯化，得到25个不同的部分；抑菌试验结果表明，几乎所有部分均具有一定的抑菌作用，但其活性均略低于正丁醇部，大部分与石榴果皮粗提物抑菌活性相近。（7）呼吸抑制试验结果表明，石榴果皮提取物主要是通过抑制HMP途径，导致还原力NADPH缺乏，引起代谢功能障碍，达到抑菌的目的；加药组和正常组金黄色葡萄球菌生长曲线的对比结果表明，石榴果皮提取物能够抑制金黄色葡萄球菌对数生长期的菌体分裂；SDS-聚丙烯酰胺凝胶电泳分析表明，石榴果皮提取物能够抑制菌细胞蛋白合成，尤其对菌体内大分子量蛋白作用最明显。通过扫描电镜、透射电镜观察，经石榴果皮提取物作用后菌体表面和内部结构均发生改变，细胞壁被破坏，出现质壁分离现象。综上所述，石榴果皮提取物的抑菌机理是其抑制了菌体的HMP途径，引起代谢功能障碍，使调控菌体生长、分裂相关蛋白的合成受到抑制，引起细胞质固缩，最终使菌体的生长繁殖受到抑制，甚至死亡。（8）通过对接种于冷却猪肉上的金黄色葡萄球菌的抑制试验研究表明，各抑菌剂均具有很好地抑制金黄色葡萄球菌的活性，其中1％石榴果皮提取物处理组和Fe²⁺+0．5％石榴果皮提取物处理组的效果优于Nisin处理组。通过对冷却猪肉贮藏中的pH值、TVB-N值、细菌总数及感官评价等指标的综合分析表明，处理组中1％石榴果皮提取物对冷却猪肉的保鲜效果最好。本研究的创新点：（1）从食品安全保藏学的角度，采用生物活性示踪法，对石榴果皮的抑菌活性进行了较为系统地研究，表明石榴果皮中抑菌活性物质主要为石榴多酚。（2）首次对石榴果皮提取物抑菌机理进行了较为系统地研究。（3）首次将石榴果皮提取物应用到冷却猪肉的保鲜中，并取得了较好的效果。更多还原

【Abstract】 The pomegranate （P.granatum L.）, a species belonging to the family Punicaceae, originated from Central Aia Area such as Iran’s, Afghanistan, etc. It has been 2000 years since pomegranate was cultured in China during the Western Han Dynasty. Pomegranate has high nutritional and pharmaceutical value. The researches showed pomegranate has several physiological functions such as antioxidation, anticancer, antivirus and reducing blood lipid. At present, the researches about biological activity of pomegranate peel focus on antioxidation. The studies about antimicrobial activity are relatively scarce （limited）. Moreover, the reports about antimicrobial activity are rare in the terms of food safety and preserving. 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 and the diameter of inhibition zone was used as the activity monitoring parameter. This paper studied the technology for extracting antibacterial substances from pomegranate peel, antagonistic property and mechanism, the antiseptic effect of pomegranate peel extract in chilled pork. These researches would give reference for exploitation and to other researches of pomegranate peel. The results were described as follows:（1） Preliminary results revealed that the antimicrobial activity of methanol extract was the most potent. The antibacterial activity of pomegranate peel extract was better than the yeast-anti activity. It could hardly inhibit the growth of mildew. The antimicrobial activity was in sequence of S.aureas > L.momocytogenes > B.megaterium, B.subtilis > S.typhimurium> E.coil > Candida lipolytica.（2） The methanol extract of ’ Yichengruanzi’ had the highest antibacterial activity in 10 tested varieties. The antibacterial activity of pomegranate external tissue was higher than velamen and calyx.（3） The optimum extraction conditions were obtained by quadric regression orthogonal rotary experiments. The result showed that the main factors influencing the extracting effect from more important to less were as follows: ethanol concentration, extracted temperature, ratio of liquid to solid and extracted time; the optimum extraction conditions were that the concentration of ethanol was 60%, the ratio of liquid to solid was 10:1, the temperature of extraction was 60℃and the time of extraction was 4h. By the above conditions, the inhibition zone diameter was 16.30 mm.（4） The results showed that the antimicrobial spectrum of pomegranate peel extract in optimum conditions was similar to that of methanol extract, but the antimicrobial activity of the pomegranate peel extract was better than methanol extract. The MIC and MBC of pomegranate peel extract to S.aurea were 1.562 mg/mL and 3.125mg/mL respectively. The antibacterial activity of the pomegranate peel extract was similar to that of Nisin and higher than that of Sorbic acid potassium and Sodium benzoate.（5） The antibacterial activity of the pomegranate peel extract was stable to temperature, light and ultraviolet radiation. The pomegranate peel extract had the most powerful antibacterial activity under the medium pH 6. Salt, sugar, Na⁺, Ca²⁺, K⁺, Fe²⁺, and Fe³⁺, could increase the antibacterial activity and Fe²⁺ exhibited best effect in these metal ions.（6） The pomegranate peel extract was successively partitioned with petroleum ether, chloroform, EtOAc, butanol. Butanol extract showed the highest antibacterial activity among these extracts. Chemical composition analysis result showed that the antibacterial substances in pomegranate peel were pomegranate polyphenols. 25 fractions were obtained by separating the butanol extract with silica gel column chromatography. Almost all fractions had inhibition activity against S.aureas. But the inhibition activities of all these fractions were slightly lower than that of butanol extract. The antibacterial activities of most of fractions were similar to the pomegranate peel extract.（7） 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 induced metabolic function dysfunction. Growth curve assay showed that the pomegranate peel extract inhibited cytodieresis in logarithmic growth phase. After exposure to the pomegranate peel extract, bacterial protein was analyzed by SDS-PAGE. The results showed that protein synthesis was inhibited, especially for large molecular weight protein. After treated by the pomegranate peel extract, it was observed by electron microscopy that the surface and inner structure of bacterial cells were destroyed and induced cells plasmolysis. This study suggested that the antibacterial mechanism of the pomegranate peel extract was that it inhibited HMP and leaded to metabolic function dysfunction and inhibited the synthesis of proteins associated with growth and cleavage of bacteria, then blocked their division and development, leaded to cell pyknosis. Eventually growth and reproduction of cells were inhibited and death.（8） Inhibition of S.aureas by pomegranate peel extract was evaluated in chilled pork. The results showed that all treatment groups had good inhibition activity against S.aureas, 1 % pomegranate peel extract and Fe²⁺+0.5 % pomegranate peel extract both were better than Nisin in the aspect of inhibition activity. Preservation effect of pomegranate peel extract on chilled pork was evaluated by investigating pH value, TVB-N value, total bacterial count, sensory evaluation during storage period. The preservation effect of 1 % pomegranate peel extract was best in all treatment groups.The innovation points were as follows:（1） Biological activity was used as the monitoring parameter and the antimicrobial activity was studied systematically in the terms of food safety and preserving. The results showed that the antimicrobial substances of pomegranate peel mainly were pomegranate polyphenolic compounds.（2） The antimicrobial mechanism of pomegranate peel extract was studied systematically for the first time.（3） Pomegranate peel extract was applied to chilled pork preservation, and it achieved a good effect.更多还原