原料乳中嗜冷菌多样性研究及LAMP快速检测方法的构建

发布时间：2018-04-28 18:09

  本文选题：原料乳 + 嗜冷菌　； 参考：《哈尔滨工业大学》2017年博士论文

【摘要】：冷藏贮运是保障原料乳品质的重要环节,但是在低温条件下嗜冷菌仍然能够生长繁殖,随着贮藏时间延长逐渐成为主要微生物。由于一些嗜冷菌能够产生耐热的脂肪酶和蛋白酶,原料乳加工过程中的常规热处理方法不能使其完全灭活,残余的酶会分解乳制品中的脂肪和蛋白质,导致乳制品品质下降。本论文采集中国北方主要产乳区多个牧场的原料乳,分析嗜冷菌的多样性,对优势嗜冷菌所分泌的脂肪酶和蛋白酶的耐热性进行研究,确立产耐热酶的典型嗜冷菌,建立并优化环介导等温扩增(LAMP)检测体系,旨在快速检测对原料乳品质破坏能力强的嗜冷菌,为原料乳品质的监测提供保障。将采集自北京、黑河和哈尔滨三个地区的原料乳样品分为两个部分,分别在0~5°C和5~10°C保存。采用PCR-DGGE方法分析两种温度条件下保存0、1和3 d的原料乳样品中嗜冷菌的多样性,结果发现,不同地区的原料乳菌群结构显著不同,相同储存条件下,同一地区的原料乳样品的相似度高于同一储存时间的原料乳样品。但是来自不同地区的原料乳样品中具有相同的优势菌Pseudomonadales和Lactobacillales。原料乳菌群结构也受到温度影响。5~10°C储存的原料乳中嗜冷菌的增长较0~5°C储存的原料乳中增长显著。随着储存时间的延长,L.lactis所占比例降低,Pseudomonas所占比例提高。从采集的原料乳样品中共分离出具有脂肪水解能力的嗜冷菌21株,具有蛋白水解能力的嗜冷菌26株。经16S r DNA鉴定发现,来自不同地区的原料乳中产脂肪酶/蛋白酶菌株所属菌种不同,但均属于Pseudomonas,其中Pseudomonas fragi的占比最高。优势嗜冷菌及其水解酶的耐热性研究发现嗜冷菌自身不具有耐热性,而其水解酶具有极强耐热性。在分离到的嗜冷菌中,38号嗜冷菌具有最强的产脂肪酶和产蛋白酶能力,所产酶也具有较强的耐热能力,该菌株的16s r DNA鉴定结果为Pseudomonas fluorescens。以NCBI数据库中公布的P.fluorescens和38号嗜冷菌脂肪酶和蛋白酶基因比对获得的保守区为目的基因,建立快速检测嗜冷菌脂肪酶和蛋白酶基因的LAMP检测体系,并从Mg2+、d NTPs和Bst DNA聚合酶的添加量,内外引物浓度比,反应温度和反应时间方面进行优化。优化后的LAMP和实时LAMP(RT-LAMP)检测方法均对P.fluorescens有较强的特异性,对分离得到的其他菌株和原料乳中常见的污染微生物Staphylococcus aureus、Salmonella typhimurium、Escherichia coli、Cronobacte sakazakii、Listeria monocytogenes和Shigella flexneri均无检测信号。在检测纯培养38号嗜冷菌时,以脂肪酶基因为目标的普通LAMP和RT-LAMP的检测限在相同数量级,均为4×102 CFU/mL,作为对照的PCR方法检测限是其10倍;以蛋白酶基因为目标的LAMP和RTLAMP的检测限一致,为3×102 CFU/mL,PCR检测限也是其10倍;在检测接种至巴氏灭菌乳中的38号嗜冷菌时,以脂肪酶基因为检测目标的LAMP和RT-LAMP的检测限一致,为6.1×102 CFU/m L,PCR检测限是其100倍;以蛋白酶基因为检测目标的LAMP和RT-LAMP的检测限一致,为8.9×102 CFU/mL,PCR检测限是其100倍。在用时方面,RT-LAMP的检测用时约为80 min,较LAMP的检测用时缩短约20 min。在实际生产中,脂肪酶和蛋白酶基因的LAMP检测可以同时进行,快速反映原料乳中所含危害嗜冷菌的数量,为乳制品品质提供保障。
[Abstract]:Cold storage and transportation is an important link to ensure the quality of raw milk. But under low temperature, the eosinophils can still grow and reproduce, and gradually become the main microbes with the prolongation of storage time. Because some of the cold bacteria can produce heat resistant lipase and protease, the conventional heat treatment method in the process of raw milk processing can not completely inactivate it. The residual enzymes will decompose the fat and protein in dairy products and lead to the decline in the quality of dairy products. This paper collected raw milk from the main dairy farms in the north of China, analyzed the diversity of the eosinophils, studied the heat resistance of the lipase and protease secreted by the dominant eosinophilic bacteria, and established the typical colophophilic bacteria producing heat resistant enzymes. And to optimize the loop mediated isothermal amplification (LAMP) detection system, the aim is to quickly detect the cold strain of the raw milk substance, and provide a guarantee for the quality monitoring of raw milk. The raw milk samples from three regions of Beijing, Heihe and Harbin are divided into two parts, and the samples are kept in 0~5 C and 5~10 C. The PCR-DGGE method is used for analysis. The diversity of eosinophils in raw milk samples of 0,1 and 3 D were preserved under two temperature conditions. The results showed that the structure of raw milk bacteria in different regions was significantly different. The similarity of raw milk samples in the same area was higher than that of the raw milk samples with the same storage time. The structure of the same dominant bacteria Pseudomonadales and Lactobacillales. Lactobacillus group was also increased significantly in raw milk of raw milk stored in.5~10 degree C than that stored in 0~5 C. With the prolongation of storage time, the proportion of L.lactis decreased and the proportion of Pseudomonas increased. From the sample of raw milk collected from the collected raw milk. 21 strains of adipophilic bacteria with the ability of fatty hydrolysis and 26 strains of proteolytic bacteria were isolated. It was found by 16S R DNA that the strains of lipase / protease strains belonging to the raw milk from different regions were different, but all belonged to Pseudomonas, and the proportion of Pseudomonas fragi was the highest. The heat resistance study found that eosinophilia itself does not have heat resistance, but its hydrolase has a very strong heat resistance. In the isolated eosinophilia, No. 38 eosinophilia has the strongest lipase production and protease producing ability, and the enzyme produced by the strain is also highly resistant to heat. The 16S R DNA identification result of the strain is Pseudomonas fluorescens. with NCBI database A LAMP detection system for rapid detection of lipase and protease genes was established to detect the lipase and protease genes of the eosinophilia bacteria, which was obtained by the comparison of the lipase and protease gene of P.fluorescens and 38. It was optimized from the addition of Mg2+, D NTPs and Bst DNA polymerase, the concentration ratio of internal and external primers, reaction temperature and reaction time. The optimized LAMP and real time LAMP (RT-LAMP) detection methods have strong specificity to P.fluorescens, and there are no tests on the contaminated microorganisms, such as Staphylococcus aureus, Salmonella typhimurium, Escherichia coli, Cronobacte sakazakii, etc., which are isolated from other strains and raw milk. The detection limit of LAMP and RT-LAMP with lipase gene as the target was 4 x 102 CFU/mL, and the detection limit of the PCR method as the control was 10 times, and the detection limit of LAMP and RTLAMP with the target of protease gene was 3 x 102 CFU/mL and the PCR detection limit was 10 times; The detection limit of LAMP and RT-LAMP for lipase gene was the same as the detection limit of LAMP and RT-LAMP in the pasteurized milk. The detection limit was 6.1 x 102 CFU/m L, and the detection limit of PCR was 100 times. The detection limit of LAMP and RT-LAMP with protease gene as the target was 8.9 x 102 CFU/mL, and the PCR detection limit was 100 times. In time of use, RT The detection time of -LAMP is about 80 min, which is about 20 min. shorter than that of LAMP. The LAMP detection of lipase and protease gene can be carried out at the same time, which can quickly reflect the number of harmful eosinophils contained in raw milk and provide a guarantee for the quality of dairy products.

【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS252.7
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本文编号：1816325