本文選題：原料乳 + 嗜冷菌　； 參考：《哈爾濱工業(yè)大學(xué)》2017年博士論文

【摘要】：冷藏貯運是保障原料乳品質(zhì)的重要環(huán)節(jié),但是在低溫條件下嗜冷菌仍然能夠生長繁殖,隨著貯藏時間延長逐漸成為主要微生物。由于一些嗜冷菌能夠產(chǎn)生耐熱的脂肪酶和蛋白酶,原料乳加工過程中的常規(guī)熱處理方法不能使其完全滅活,殘余的酶會分解乳制品中的脂肪和蛋白質(zhì),導(dǎo)致乳制品品質(zhì)下降。本論文采集中國北方主要產(chǎn)乳區(qū)多個牧場的原料乳,分析嗜冷菌的多樣性,對優(yōu)勢嗜冷菌所分泌的脂肪酶和蛋白酶的耐熱性進(jìn)行研究,確立產(chǎn)耐熱酶的典型嗜冷菌,建立并優(yōu)化環(huán)介導(dǎo)等溫擴增(LAMP)檢測體系,旨在快速檢測對原料乳品質(zhì)破壞能力強的嗜冷菌,為原料乳品質(zhì)的監(jiān)測提供保障。將采集自北京、黑河和哈爾濱三個地區(qū)的原料乳樣品分為兩個部分,分別在0~5°C和5~10°C保存。采用PCR-DGGE方法分析兩種溫度條件下保存0、1和3 d的原料乳樣品中嗜冷菌的多樣性,結(jié)果發(fā)現(xiàn),不同地區(qū)的原料乳菌群結(jié)構(gòu)顯著不同,相同儲存條件下,同一地區(qū)的原料乳樣品的相似度高于同一儲存時間的原料乳樣品。但是來自不同地區(qū)的原料乳樣品中具有相同的優(yōu)勢菌Pseudomonadales和Lactobacillales。原料乳菌群結(jié)構(gòu)也受到溫度影響。5~10°C儲存的原料乳中嗜冷菌的增長較0~5°C儲存的原料乳中增長顯著。隨著儲存時間的延長,L.lactis所占比例降低,Pseudomonas所占比例提高。從采集的原料乳樣品中共分離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。經(jīng)16S r DNA鑒定發(fā)現(xiàn),來自不同地區(qū)的原料乳中產(chǎn)脂肪酶/蛋白酶菌株所屬菌種不同,但均屬于Pseudomonas,其中Pseudomonas fragi的占比最高。優(yōu)勢嗜冷菌及其水解酶的耐熱性研究發(fā)現(xiàn)嗜冷菌自身不具有耐熱性,而其水解酶具有極強耐熱性。在分離到的嗜冷菌中,38號嗜冷菌具有最強的產(chǎn)脂肪酶和產(chǎn)蛋白酶能力,所產(chǎn)酶也具有較強的耐熱能力,該菌株的16s r DNA鑒定結(jié)果為Pseudomonas fluorescens。以NCBI數(shù)據(jù)庫中公布的P.fluorescens和38號嗜冷菌脂肪酶和蛋白酶基因比對獲得的保守區(qū)為目的基因,建立快速檢測嗜冷菌脂肪酶和蛋白酶基因的LAMP檢測體系,并從Mg2+、d NTPs和Bst DNA聚合酶的添加量,內(nèi)外引物濃度比,反應(yīng)溫度和反應(yīng)時間方面進(jìn)行優(yōu)化。優(yōu)化后的LAMP和實時LAMP(RT-LAMP)檢測方法均對P.fluorescens有較強的特異性,對分離得到的其他菌株和原料乳中常見的污染微生物Staphylococcus aureus、Salmonella typhimurium、Escherichia coli、Cronobacte sakazakii、Listeria monocytogenes和Shigella flexneri均無檢測信號。在檢測純培養(yǎng)38號嗜冷菌時,以脂肪酶基因為目標(biāo)的普通LAMP和RT-LAMP的檢測限在相同數(shù)量級,均為4×102 CFU/mL,作為對照的PCR方法檢測限是其10倍;以蛋白酶基因為目標(biāo)的LAMP和RTLAMP的檢測限一致,為3×102 CFU/mL,PCR檢測限也是其10倍;在檢測接種至巴氏滅菌乳中的38號嗜冷菌時,以脂肪酶基因為檢測目標(biāo)的LAMP和RT-LAMP的檢測限一致,為6.1×102 CFU/m L,PCR檢測限是其100倍;以蛋白酶基因為檢測目標(biāo)的LAMP和RT-LAMP的檢測限一致,為8.9×102 CFU/mL,PCR檢測限是其100倍。在用時方面,RT-LAMP的檢測用時約為80 min,較LAMP的檢測用時縮短約20 min。在實際生產(chǎn)中,脂肪酶和蛋白酶基因的LAMP檢測可以同時進(jìn)行,快速反映原料乳中所含危害嗜冷菌的數(shù)量,為乳制品品質(zhì)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.

【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】：2017
【分類號】：TS252.7
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本文編號：1816325