發(fā)布時間：2018-06-08 16:53

  本文選題：嗜酸耐熱菌 + 獼猴桃汁��； 參考：《西北農林科技大學》2015年博士論文

【摘要】：嗜酸耐熱菌是果汁類產品的一種污染菌,能污染多種果汁,產生令人不快的氣味,世界上關于其污染的事件屢有報道。嗜酸耐熱菌主要指Alicyclobacillus屬細菌,其種類已超過20種,不同種類之間差異很大,只有部分種類具有污染能力。中國是世界上最大的獼猴桃生產國,陜西是中國的獼猴桃主產區(qū),近年來,獼猴桃果汁、果酒等深加工產業(yè)發(fā)展迅速,而隨著其發(fā)展,發(fā)生嗜酸耐熱菌污染事件的概率也在增加。關于獼猴桃汁中嗜酸耐熱菌的分離檢測研究報道尚很少。嗜酸耐熱菌是芽孢桿菌,其芽孢可以通過巴氏滅菌而保持活性,這也是其頻繁造成污染事件的重要原因。本文以陜西眉縣-楊凌-周至沿渭河一線獼猴桃主產區(qū)為樣品采集地區(qū),對該地區(qū)獼猴桃生產線從果園到生產車間再到成品進行了全面采樣。樣品富集培養(yǎng)后分離嗜酸耐熱菌,提取其基因組DNA,通過16S rDNA測序進行鑒定。分離菌株通過構建系統(tǒng)發(fā)育樹和隨機引物擴增多態(tài)性分析,以及代表菌株的API糖醇利用實驗及基因組DNA雜交實驗考察其生物多樣性。然后通過代表菌株的生長曲線實驗、D90℃值測定和產污染物分析考察其污染能力。最后研究了nisin對其增殖能力和抗熱性的抑制情況,并研究了射頻聯(lián)合nisin對其芽孢的殺滅效果。論文的主要研究結果如下:(1)自陜西獼猴桃主產區(qū)的生產線采集了共401個樣品,76個樣品檢出嗜酸耐熱菌(陽性),檢出率(陽性率)19.0%。從中分離出85株嗜酸耐熱菌,其中1株來自獼猴桃產品,16株分離自加工工廠,其余68株分離自果園。85株分離菌中76株為Alicyclobacillus,包括4個種:A.acidoterrestris(46株)、A.contaminans(10株)、A.herbarius(19株)和A.cycloheptanicus(1株)。其中A.contaminans和A.herbarius,在獼猴桃果園內的分離國內屬首次報道。另外還分離到9株Bacillus(分屬3個種)。(2)所有85株分離菌以及4株標準菌的基于16S rDNA的N-J系統(tǒng)發(fā)育樹圖分為7個類群,與隨機引物擴增多態(tài)性分析結果基本吻合,顯示出分離菌具有一定的生物多樣性。代表菌株的API實驗49項糖醇利用反應中,分離菌與本次實驗使用的標準菌株的不同反應結果數目為3項至19項,與文獻報道中標準菌株差異為7項至22項,從代謝方面顯示出分離菌株間的多樣性�；�因組雜交實驗顯示10株代表菌株與相應標準菌株基因組的雜交率為74.75~104.79%,從基因組同源性方面顯示出分離菌株之間的多樣性。(3)代表菌株的生長曲線顯示,在BAM中所有菌株均可生長,生長量最高的2周增加了2.61對數單位。而在獼猴桃汁(pH 4.2)中,只有7株菌可以生長,生長量最高的2周增加了3.23個對數單位。在BAM中,D90℃值在不同菌株之間為16.95 min到45.45 min,在獼猴桃汁中,D90℃值在不同菌株之間為12.50 min到38.46 min。在所有10株受試菌株的GC-MS分析中沒有發(fā)現2,6-DBP和2,6-DCP,只有愈創(chuàng)木酚。愈創(chuàng)木酚產量在2.56 ppb和42.74 ppb之間(靜置21天后)。(4)Nisin對受試菌株的生長和抗熱性均有一定的抑制作用。在BAM及獼猴桃汁中,當nisin濃度超過20 IU/mL時所有菌株均不能生長,最快的在第4天就檢測不到具有活力的細胞。當nisin濃度從0增至100 IU/m L時,BAM在各個菌株的D90℃值的削減量為0.56 min(3.30%)至6.21 min(25.46%),在獼猴桃汁中的范圍則是0.45min(3.60%)到17.11 min(53.04%)。以上結果表明nisin可以抑制Alicyclobacillus營養(yǎng)細胞的增殖,也能削弱其芽孢的抗熱性,但是在菌株間有差異。RF與nisin在殺滅Alicyclobacillus芽孢中有復合作用,90℃處理15 min最高可以使芽孢濃度下降4.17對數單位,添加100 IU/mL nisin時達到5.19對數單位。RF結合nisin比相同條件下普通加熱結合nisin效果更好,同樣90℃處理15 min,在20 IU/mL nisin情況下最大差距2.08對數單位,這表明RF結合nisin是一種較傳統(tǒng)加熱方法更有效的殺滅Alicyclobacillus芽孢的方法。
[Abstract]:Acidophilic thermophilic bacteria are a kind of pollute bacteria of fruit juice products, which can pollute a variety of fruit juices and produce unpleasant odors. The events about its pollution in the world have been reported repeatedly. The acidophilic thermophilic bacteria mainly refer to the Alicyclobacillus genus, which have more than 20 species, different species are very different, only some species have the ability to pollute. China is The largest kiwi fruit producing country in the world, Shaanxi is the main producing area of Chinese kiwi fruit. In recent years, the deep processing industry, such as Kiwi fruit juice and fruit wine, has developed rapidly. With its development, the probability of the occurrence of acidophilic thermophilic bacteria is also increasing. There are few reports on the isolation and detection of acidophilic bacteria in kiwi juice. Bacillus is a bacillus, and its spore can be kept active by pasteurization, which is also an important reason for its frequent pollution events. This article takes Shaanxi Meixian - Yangling - Zhouzhi as the sample area along the main production area of kiwi fruit along Weihe line, and samples the kiwi fruit production line from the orchard to the production workshop and then to the finished product. The genomic DNA was extracted and identified by 16S rDNA sequencing. The isolated strains were amplified by the phylogenetic tree and random primers to amplify the polymorphism analysis, as well as the API sugar alcohol use experiment and the genomic DNA hybridization experiment to investigate the biological diversity. Curve experiment, D90 C value determination and pollutant production analysis to investigate its pollution ability. Finally, the inhibitory effects of nisin on its proliferation and heat resistance were studied, and the effects of radiofrequency combined with nisin on its spores were studied. The main results of this paper were as follows: (1) 401 samples were collected from the production line of Shaanxi macaque peach, 76 85 strains of acidophilic thermophilic bacteria were isolated from 19.0%., of which 1 were from Actinidia, 16 isolated from processing plants, and 76 of the other 68 isolates from the orchard.85 isolates were Alicyclobacillus, including 4 species: A.acidoterrestris (46), A.contaminans (10 strains), A.herbariu S (19 strains) and A.cycloheptanicus (1 strains). Among them, A.contaminans and A.herbarius were first reported in Chinese gooseberry orchard. In addition, 9 strains of Bacillus (3 species) were also separated. (2) all 85 isolates and 4 standard strains of N-J based on 16S rDNA were divided into 7 groups and amplified polymorphic with random primers. The results of the analysis showed that the isolated bacteria had a certain biological diversity. The number of different reaction results of the separate bacteria and the standard strains used in this experiment were 3 to 19, and the difference between the standard strains and the standard strains in the API experiment of the API experiment was 7 to 22. The genomic hybridization experiment showed that the hybridization rate of the genome of 10 representative strains and the corresponding standard strains was 74.75~104.79%, showing the diversity among the isolated strains from the genome homology. (3) the growth curve of the representative strain showed that all strains could grow in BAM, and the highest growth rate increased by 2.61 pairs of 2 weeks. In kiwi juice (pH 4.2), only 7 strains of bacteria can grow, and 3.23 logarithmic units are increased in the 2 week of the highest growth. In BAM, the value of D90 is 16.95 min to 45.45 min between different strains. In the juice of kiwi fruit, the value of D90 is 12.50 min to 38.46 Min. in the GC-MS analysis of all the 10 strains of the strain. No 2,6-DBP and 2,6-DCP were found, only guaiacol. Guaiacol production was between 2.56 ppb and 42.74 ppb (4). (4) Nisin had a certain inhibitory effect on the growth and heat resistance of the tested strains. In BAM and kiwi juice, all strains were unable to grow when the nisin concentration exceeded 20 IU/mL, and the fastest was detected in the fourth days. Less active cells. When nisin concentration increased from 0 to 100 IU/m L, BAM was reduced from 0.56 min (3.30%) to 6.21 min (25.46%) at D90 C value of each strain, and the range in kiwi juice was 0.45min (3.60%) to 17.11 min (53.04%). The above results showed that nisin could inhibit the proliferation of Alicyclobacillus nutrient cells and also weaken the growth of Alicyclobacillus nutrient cells. The heat resistance of the spores was different, but there were differences between.RF and nisin in the killing of Alicyclobacillus spore, and the highest concentration of 15 min at 90 C could decrease the spore concentration by 4.17 logarithm units, and 5.19 logarithm unit.RF combined nisin was better than the common heating and nisin combined with the same strip when adding 100 IU/mL nisin. The maximum gap between 15 min and 20 IU/mL nisin is 2.08 logarithmic units, which indicates that RF combined with nisin is a more effective method for killing Alicyclobacillus spore than the traditional heating method.
【學位授予單位】：西北農林科技大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：TS207.4
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本文編號：1996512