猕猴桃全产业链嗜酸耐热菌识别分析及射频控制研究

发布时间：2018-06-08 16:53

  本文选题：嗜酸耐热菌 + 猕猴桃汁　； 参考：《西北农林科技大学》2015年博士论文

【摘要】：嗜酸耐热菌是果汁类产品的一种污染菌,能污染多种果汁,产生令人不快的气味,世界上关于其污染的事件屡有报道。嗜酸耐热菌主要指Alicyclobacillus属细菌,其种类已超过20种,不同种类之间差异很大,只有部分种类具有污染能力。中国是世界上最大的猕猴桃生产国,陕西是中国的猕猴桃主产区,近年来,猕猴桃果汁、果酒等深加工产业发展迅速,而随着其发展,发生嗜酸耐热菌污染事件的概率也在增加。关于猕猴桃汁中嗜酸耐热菌的分离检测研究报道尚很少。嗜酸耐热菌是芽孢杆菌,其芽孢可以通过巴氏灭菌而保持活性,这也是其频繁造成污染事件的重要原因。本文以陕西眉县-杨凌-周至沿渭河一线猕猴桃主产区为样品采集地区,对该地区猕猴桃生产线从果园到生产车间再到成品进行了全面采样。样品富集培养后分离嗜酸耐热菌,提取其基因组DNA,通过16S rDNA测序进行鉴定。分离菌株通过构建系统发育树和随机引物扩增多态性分析,以及代表菌株的API糖醇利用实验及基因组DNA杂交实验考察其生物多样性。然后通过代表菌株的生长曲线实验、D90℃值测定和产污染物分析考察其污染能力。最后研究了nisin对其增殖能力和抗热性的抑制情况,并研究了射频联合nisin对其芽孢的杀灭效果。论文的主要研究结果如下:(1)自陕西猕猴桃主产区的生产线采集了共401个样品,76个样品检出嗜酸耐热菌(阳性),检出率(阳性率)19.0%。从中分离出85株嗜酸耐热菌,其中1株来自猕猴桃产品,16株分离自加工工厂,其余68株分离自果园。85株分离菌中76株为Alicyclobacillus,包括4个种:A.acidoterrestris(46株)、A.contaminans(10株)、A.herbarius(19株)和A.cycloheptanicus(1株)。其中A.contaminans和A.herbarius,在猕猴桃果园内的分离国内属首次报道。另外还分离到9株Bacillus(分属3个种)。(2)所有85株分离菌以及4株标准菌的基于16S rDNA的N-J系统发育树图分为7个类群,与随机引物扩增多态性分析结果基本吻合,显示出分离菌具有一定的生物多样性。代表菌株的API实验49项糖醇利用反应中,分离菌与本次实验使用的标准菌株的不同反应结果数目为3项至19项,与文献报道中标准菌株差异为7项至22项,从代谢方面显示出分离菌株间的多样性。基因组杂交实验显示10株代表菌株与相应标准菌株基因组的杂交率为74.75~104.79%,从基因组同源性方面显示出分离菌株之间的多样性。(3)代表菌株的生长曲线显示,在BAM中所有菌株均可生长,生长量最高的2周增加了2.61对数单位。而在猕猴桃汁(pH 4.2)中,只有7株菌可以生长,生长量最高的2周增加了3.23个对数单位。在BAM中,D90℃值在不同菌株之间为16.95 min到45.45 min,在猕猴桃汁中,D90℃值在不同菌株之间为12.50 min到38.46 min。在所有10株受试菌株的GC-MS分析中没有发现2,6-DBP和2,6-DCP,只有愈创木酚。愈创木酚产量在2.56 ppb和42.74 ppb之间(静置21天后)。(4)Nisin对受试菌株的生长和抗热性均有一定的抑制作用。在BAM及猕猴桃汁中,当nisin浓度超过20 IU/mL时所有菌株均不能生长,最快的在第4天就检测不到具有活力的细胞。当nisin浓度从0增至100 IU/m L时,BAM在各个菌株的D90℃值的削减量为0.56 min(3.30%)至6.21 min(25.46%),在猕猴桃汁中的范围则是0.45min(3.60%)到17.11 min(53.04%)。以上结果表明nisin可以抑制Alicyclobacillus营养细胞的增殖,也能削弱其芽孢的抗热性,但是在菌株间有差异。RF与nisin在杀灭Alicyclobacillus芽孢中有复合作用,90℃处理15 min最高可以使芽孢浓度下降4.17对数单位,添加100 IU/mL nisin时达到5.19对数单位。RF结合nisin比相同条件下普通加热结合nisin效果更好,同样90℃处理15 min,在20 IU/mL nisin情况下最大差距2.08对数单位,这表明RF结合nisin是一种较传统加热方法更有效的杀灭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.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：TS207.4
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本文编号：1996512