【作者】 袁晶；

【导师】 顾青；

【作者基本信息】 浙江工商大学 ， 食品科学与工程， 2011， 硕士

【摘要】 利用乳酸菌发酵食品有着悠久的历史,经乳酸菌发酵加工后的食品不仅可以改善食品风味,提高营养价值,而且还具有特殊营养价值和生理活性。大量研究表明,乳酸菌可以调节机体胃肠道正常菌群,从而抑制肠道内腐败菌生长和腐败产物的产生；其产生的细菌素等代谢产物,还有抑制病菌生长等功效。因其具有高效、无毒、无残留、无抗药性等优势,应用到饲料中可有效改善肉质、提高饲料转化率。本实验室筛选并分离出一株乳酸菌,对其的生理生化特征研究表明它与植物乳杆菌(Lactobacillus plantarum)最为相似,我们将其命名为Lactobacillus plantarum ZJ316。而产生的细菌素则命名为Plantaricin ZJ316。本文主要针对该细菌素的分离纯化及应用做了初步研究,内容包括：植物乳杆菌ZJ316抑菌谱的测定。实验结果表明该菌株具有广谱抑菌性,后经双层平板拮抗法的测定,发现其还具有抗真菌的能力。继而进行植物乳杆菌ZJ316细菌素的分离纯化。首先采用双水相萃取法进行初步分离,PEG/硫酸铵体系的最佳萃取条件是PEG4000浓度为17.5%、(NH4)2SO4浓度为19%、氯化钠浓度是1%,pH值为3.75,经实验验证,在此条件下萃取相的效价为：287.56IU/mL。PEG/硫酸钠双水相体系萃取细菌素的最佳实验条件是PEG分子量为1000,浓度为17%,硫酸钠的浓度为15%,氯化钠的浓度为1.0%,pH为3.5,经试验验证,在此条件下萃取相效价为：256 IU/mL。选择离子柱继续分离纯化。经试验确认,选取pH为5.0的醋酸钠缓冲液,DEAE Sepharose FF填料进行纯化分离,最终获得了较纯的细菌素组分。之后采用Trcine-SDS-PAGE电泳检测,根据电泳结果初步估计植物乳杆菌素ZJ316的分子量大约在6.5 kDa左右,属于小分子多肽类物质。初步探索植物乳杆菌ZJ316作为饲料添加剂应用于仔猪饲喂试验。结果表明：添加乳杆菌ZJ316饲喂的仔猪日增重、饲料转化率均有较明显提高；同时腹泻率和死亡率降低,可以起到替代抗生素的抑菌效果；其对猪肉品质也有一定的改善作用。通过石蜡切片对小肠绒毛形态变化的观察,可看出植物乳杆菌ZJ316对仔猪小肠有一定的影响,从而从生理学角度支持了实验组仔猪日增重、饲料转化率及肉品质均优于对照组的结论。更多还原

【Abstract】 It has a long history of Lactic acid bacteria fermented food, and which could not only improve the flavors and the nutritional values of food, but also have special nutritional values and physiological activities. A large number of studies have showed that lactic acid bacteria can regulate the body normal flora of the gastrointestinal tract, thus inhibiting the growth of intestinal pathogen. Its metabolites which produced by fermentation products also have inhibition of fungal growth and other effects. Because of high efficiency, non-toxic, no residue, no drug resistance and other advantages, they could be added to improve the qualities of meat and the rate of feed conversion.Basic characteristic revealed that the strain had homology to Lactobacillus plantarum. So we named this strain as Lactobacillus plantarum ZJ316 and the bacteriocin as plantaricin ZJ316. In this paper, preliminary studies were done including the purification of bacteriocin ZJ316, its propercities of anti-bacterial and the experiments of feeding piglets.In the anti-fungal experiments, with the determination of double-plate antagonism method, the results showed that Lactobacillus plantarum ZJ316 had anti-fungal activities.Purification of bacteriocin ZJ316. First, aqueous two-phase extraction using a preliminary separation, the best extraction conditions of PEG/ammonium sulfate system were 17.5% PEG4000,19% ammonium sulfate,1% sodium chloride and pH 3.75. Under these conditions the titer was:287.56 IU/mL. The best extraction conditions of PEG/sodium sulfate were 17% PEG 1000,15% sodium sulfate,1% sodium chloride, pH 3.5, the titer was:256 IU/mLThe ion column was selected for purification. The results showed that a more pure-component bacteriocin could be reached by selecting pH 5.0 sodium acetate buffer, DEAE Sepharose FF. After using Trcine-SDS-PAGE electrophoresis, the results showed that the molecular weight of plantaricin ZJ316 was about 6.5 kDa, which was a small molecule peptide substances.In piglets feed tests, it was explored that Lactobacillus plantarum ZJ316 as an feed additive. The results showed that, with the addition of Lactobacillus plantarum ZJ316, the daily gains and the rate of feed conversion had been improved; the rates of diarrhea and mortality were reduced, it had the same anti-fungal effects as mequindox; also it had certain improvements in qualities of meat. With the paraffin sections of small intestine of piglets and observing the morphological changes of small intestine of piglets, it was found that Lactobacillus plantarum ZJ316 had significant effects on small intestine of piglets, which supported the results that the daily gains, the rate of feed conversion and the qualities of meat of experimental group were better than control group from physiological points.更多还原