本文選題：耐冷菌 + 乳酸菌抗菌肽�。� 參考：《哈爾濱工業(yè)大學(xué)》2016年博士論文

【摘要】：食品腐敗是指食品品質(zhì)發(fā)生變化,而無法被人們所食用。引起食品腐敗的原因多種多樣,其中最主要的原因是由微生物引起的食品腐敗。微生物污染不僅可以出現(xiàn)在食品原料的初期,還可能伴隨著成品或半成品的加工和運(yùn)輸過程中。人們通常選擇低溫保藏或者冷凍的方法,用于控制微生物的生長和繁殖,防止微生物引起的食品腐敗。但是耐冷菌的發(fā)現(xiàn)打破了人們對低溫或冷凍可以保證食品質(zhì)量的認(rèn)識。乳酸菌抗菌肽作為一種天然防腐劑,在抑制耐冷菌方面具有很大潛力。本研究以乳酸菌為研究對象,旨在篩選出可以抑制假單胞菌的乳酸菌抗菌肽,并對抗菌肽的結(jié)構(gòu)和抑菌機(jī)理及其理化性質(zhì)進(jìn)行研究,為抑制食品中耐冷菌的研究提供理論依據(jù)和基礎(chǔ)。通過96-孔瓊脂擴(kuò)散法,對實(shí)驗(yàn)室保存的325株乳酸菌進(jìn)行篩選。獲得兩株抑制菌耐冷菌的乳酸菌T1和Q7,通過對兩株發(fā)酵液中和有機(jī)酸實(shí)驗(yàn)、過氧化氫排除實(shí)驗(yàn)、蛋白酶驗(yàn)證實(shí)驗(yàn)以及Tricine-SDS-PAGE實(shí)驗(yàn),確定兩株菌發(fā)酵液中的抑菌物質(zhì)為抗菌肽。通過對兩種抗菌肽性質(zhì)研究,表明抗菌肽Enterocin T1和Plantaricin Q7可以有效抑制耐冷菌Pseudomonas flurescens AS1.1802,Pseudomonas putida AS1.1819,Pseudomonas aeruginosa CICC21636,Listeria monocytogenes ATCC19111和致病菌Escherichia coli ATCC25922,Salmonella typhimurium ATCC14028,Shigella flexneri ATCC1202 2,Shigella sonnei ATCC25931,Staphylococcus aureus。在不同pH值和溫度下,抗菌肽Enterocin T1和Plantaricin Q7都具有穩(wěn)定的抑菌活性。蛋白酶E、蛋白酶K、胃蛋白酶、胰蛋白酶以及木瓜蛋白酶都可以破壞兩種抗菌肽的活性。兩種抗菌肽對于不同的化學(xué)試劑都有很好的耐受性。通過16Sr DNA和API種屬鑒定,確定產(chǎn)抗菌肽的二株菌種屬:菌株T1為屎腸球菌(Enterococcus faecium)和菌株Q7植物乳酸桿菌(Lactobacillus plantarum)。以Enterococcus faecium T1和Lactobacillus plantarum Q7的發(fā)酵液為原料,依次采用SP-sepharose、CM-FF、Superdex Peptide 10/300 GL對乳酸菌發(fā)酵液進(jìn)行分離純化,最終獲得高純度的抗菌肽T1和抗菌肽Q7。LC-ESI/MS測定抗菌肽Enterocin T1分子量為4629 Da、抗菌肽Plantaricin Q7的分子量為4624 Da。通過指紋圖譜分析兩種抗菌肽序列,并未發(fā)現(xiàn)與兩種抗菌肽含有相同的氨基酸序列。通過對兩種抗菌肽的氨基酸成份和序列的分析,抗菌肽Enterocin T1含有41個(gè)氨基酸,部分氨基酸序列為:M-A-E-T-F-A-P�？咕腜lantaricin Q7含有40個(gè)氨基酸,部分氨基酸序列為V-H-A-K-I-K-F-D。采用熒光探針標(biāo)記法研究抗菌肽Enterocin T1和Plantaricin Q7對單增李斯特菌和熒光假單胞菌的抑菌機(jī)理,結(jié)果表明抗菌肽Enterocin T1和Plantaricin Q7都是通過吸附于單增李斯特菌細(xì)胞膜表面后,插入細(xì)胞膜的內(nèi)部的作用方式抑制單增李斯特菌。抑制熒光假單胞菌的作用方式是以螯合Zn2+后,破壞熒光假單胞菌的外膜,最后作用細(xì)胞膜。研究還發(fā)現(xiàn)抗菌肽Enterocin T1破壞細(xì)胞胞上跨膜電位和pH梯度,抗菌肽Plantaricin Q7只能破壞細(xì)胞上的pH梯度。兩種抗菌肽都能引起單增李斯特菌和熒光假單胞菌細(xì)胞內(nèi)K+和磷酸鹽離子的泄露。選取牛奶、豬肉、帶魚作為食品模型,研究抗菌肽Enterocin T1和Plantaricin Q7在食品防腐中的應(yīng)用。并以感觀評價(jià)、菌落總數(shù)、TVB-N、pH值為主要指標(biāo)反應(yīng)抗菌肽Enterocin T1和Plantaricin Q7的保鮮效果。結(jié)果表明兩種抗菌肽可以有效的抑制熒光假單胞菌,對不同類型的食品起到良好的保鮮效果,并且保鮮效果隨著抗菌肽活力的增加而增強(qiáng)。
[Abstract]:Food corruption means that the quality of food has changed and can not be eaten by people. There are many reasons for food corruption. The main reason is the food corruption caused by microbes. Microbial pollution can not only appear in the early stage of food raw materials, but also may accompany the processing and transportation process of finished products or semi finished products. They usually choose low temperature preservation or cryosurgery to control the growth and reproduction of microorganisms to prevent food corruption caused by microorganisms. But the discovery of cold resistant bacteria breaks people's understanding of the quality of food at low temperature or freezing. In this study, lactic acid bacteria were studied in this study. The aim of this study was to screen out the antibacterial peptide of lactic acid bacteria that could inhibit Pseudomonas, and to study the structure and antibacterial mechanism and physicochemical properties of bacteriocin, and provide theoretical basis and basis for the study of the inhibition of cold resistant bacteria in food. Through the 96- pore agar diffusion method, the 325 strains of milk preserved in the laboratory were stored in the laboratory. Acid bacteria were screened. Two strains of lactic acid bacteria T1 and Q7 were obtained. Through the experiments on two strains of fermentation liquid and organic acid, hydrogen peroxide elimination experiment, protease verification experiment and Tricine-SDS-PAGE experiment, the bacteriostat in two strains of bacteria fermentation liquid was determined as antibacterial peptide. Through the study on the properties of two kinds of antimicrobial peptides, the antibacterial peptide En was shown. Terocin T1 and Plantaricin Q7 can effectively inhibit the Pseudomonas flurescens AS1.1802, Pseudomonas putida AS1.1819, Pseudomonas aeruginosa. C25931, Staphylococcus aureus. at different pH and temperature, antibacterial peptide Enterocin T1 and Plantaricin Q7 have stable antibacterial activity. Protease E, proteinase K, pepsin, trypsin and papain can all destroy the activity of two kinds of antimicrobial peptides. Two kinds of antimicrobial peptides are well tolerated to different chemical reagents. Through the identification of 16Sr DNA and API species, the two strains of antimicrobial peptides were identified: the strain T1 was Enterococcus faecium and the strain Q7 plant lactobacillus (Lactobacillus plantarum). 300 GL was isolated and purified for lactic acid bacteria fermentation broth. Finally, high purity antibacterial peptide T1 and antibacterial peptide Q7.LC-ESI/MS were obtained for the determination of Enterocin T1 molecular weight of 4629 Da. The molecular weight of antibacterial peptide Plantaricin Q7 was 4624 Da. by fingerprint analysis, and the same amino acids were not found with the two antimicrobial peptides. By analyzing the amino acid composition and sequence of two antimicrobial peptides, the antibacterial peptide Enterocin T1 contains 41 amino acids. The sequence of partial amino acids is: M-A-E-T-F-A-P. antibacterial peptide Plantaricin Q7 contains 40 amino acids, and some amino acid sequences are V-H-A-K-I-K-F-D. using fluorescence probe labeling method to study antibacterial peptide Enterocin T1 and Plantaric The bacteriostasis mechanism of in Q7 on mono Lester and Pseudomonas fluorescens showed that the antibacterial peptide Enterocin T1 and Plantaricin Q7 were all inhibited by the insertion of the membrane on the surface of the cell membrane of the single increase of the cell membrane of Lester bacteria. The inhibition of Pseudomonas fluorescens was by chelating Zn2+, after the inhibition of Pseudomonas fluorescens. The outer membrane of the bad Pseudomonas fluorescens acted as the membrane. The study also found that the antibacterial peptide Enterocin T1 destroys the cell transmembrane potential and pH gradient, and the antibacterial peptide Plantaricin Q7 can only destroy the pH gradient on the cell. The two antimicrobial peptides can cause the leakage of K+ and phosphate ions in the cells of Lester and Pseudomonas fluorescens. Milk, pork, and fish were used as food models to study the application of antimicrobial peptide Enterocin T1 and Plantaricin Q7 in food preservation. The effect of antimicrobial peptide Enterocin T1 and Plantaricin Q7 on the fresh-keeping effect of antibacterial peptide Enterocin T1 and Plantaricin Q7 was evaluated by sensory evaluation, total colony total, TVB-N and pH value. The results showed that the two antimicrobial peptides could effectively inhibit Pseudomonas fluorescens. Different types of food had good preservation effect, and the fresh-keeping effect increased with the increase of antibacterial peptide activity.

【學(xué)位授予單位】：哈爾濱工業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：TS201.3

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