【作者】 李红娟；

【导师】 吕加平；

【作者基本信息】 中国农业科学院 ， 食品科学， 2011， 硕士

【摘要】 天然防腐及生物防腐已成为现代食品科学和生物科学的前沿及热点,筛选具有抗菌活性的天然资源成为生物、食品研究的新趋势。乳酸菌用在食品和饲料中有着悠久的历史,它可以降低食品pH值并产生抑菌的代谢物质。近几年,许多研究发现乳酸菌对霉菌和酵母有一定的抑菌功能,将其应用到食品中具有重要的实际应用价值。本论文首先,采用琼脂扩散法对东北泡菜及传统乳制品中分离出的60株乳酸菌进行抗真菌活性初筛,筛选得到一株具有良好抗真菌效果的乳酸菌菌株;其次,对该乳酸菌的发酵特性及抑菌作用条件进行了研究;第三,对抑菌物质进行了初步的分离纯化;随后,将该菌株AST18作为辅助发酵剂添加到酸奶制作中,考察其防霉效果及对酸奶理化指标和感官指标的影响。主要研究结果如下:(1)通过牛津杯—琼脂扩散法对本实验室保藏及分离出的60株乳酸菌进行了抗真菌初筛。其中的7株显示出了良好的抗真菌性能。对显示出最强抑菌活性的乳酸菌菌株AST18进行16S r DNA鉴定,鉴定结果为Lactobacillus casei(干酪乳杆菌)。本实验所筛出的7株乳酸菌经HPLC检测,均可产生苯乳酸(PLA)。发酵液最高PLA产量为16.24 mg/L,最低为6.86 mg /L,但分析发现PLA产量和抑菌活性无相关关系。(2)研究不同培养时间、培养温度及通气状况对AST18生长及抑菌性能的影响,确定AST18最适生长温度37℃,在培养基初始pH6.0-7.0,摇床条件下菌株生长状况及抑菌活性较好。考察AST18发酵上清液对pH、热处理及蛋白酶处理的敏感度。研究表明,Lactobacillus caseiAST18发酵上清液在低pH范围内(2.0-4.0)抑菌活性高,耐热性较差,100℃以上加热失活,对胰蛋白酶、胃蛋白酶不敏感。本实验中分别用乙酸、乳酸和盐酸将MRS肉汤调节pH至3.88,未能观测到抑菌圈。(3)AST18上清发酵液中抑菌物质为小分子,分子量小于3000Da,在发酵液中,第2部分(4.5-7.5min)和第4部分(11.4-13.4min)出现相对较大抑菌圈。对第2部分进行初步分析,可推测其抑菌作用主要是由于高浓度的有机酸(主要是乳酸),由于其本身抑菌作用或对pH值的降低起到了抑菌效果。对第4部分进行GC-MS分析,分离出三种小分子有机化合物,包括环二肽ctclo-(Leu-Pro)、2,6-二苯基哌啶及小分子吡嗪类物质。(4)Lactobacillus casei AST18作为辅助发酵剂添加到酸奶中可提高酸奶中的乳杆菌数,降低酸奶中的乳链球菌数,2%AST18添加量中乳链球菌数同对照组相比降低1.0 Log(cfu/mL)。这可能是由于乳杆菌和乳链球菌的拮抗作用造成的。AST18的添加对酸奶黏度、持水力、及感官品质无显著影响且AST18后酸化水平较低,对酸奶pH值、滴定酸度无显著影响。因此,AST18作为辅助发酵剂在酸奶中的添加对其理化指标及感官品质影响较小,可作为辅助发酵剂在应用于酸奶产品的防腐剂。(5)4℃下,添加2%Lactobacillus caseiAST18的酸奶在21天货架期内可完全抑制霉菌和酵母的生长。28℃条件下,Lactobacillus caseiAST18的添加可延迟霉菌的生长,并抑制霉菌孢子生成。更多还原

【Abstract】 The lactic acid bacteria that can reduce pH value and produce Antibacterial material had a long history in food and fodder field. The lactic bacteria that were applied in food had important pratical value, such as many studies have found that mould and yeast can be inhibited by it in recent years. In this paper , Agar-well diffusion method were used to select the antifungal LAB which came from 60 stains screened form Northeast kimchi and traditional dairy product . Then the antifungal properties of the screened LAB AST18 was studied, that including fermentation properties、physical and chemical properties of the antifungal material、the isolation and purification of the antifungal material and the applications in the yogurt production process as adjunct culture . when the AST18 was used as adjunct culture ,the Anti-fungal properties、physical and chemical and sensory evaluation of the yogurt were also studied. The main results are as follows:(1) Agar-well diffusion method was used to select the antifungal LAB which from 60 strains preserved in laboratory. There are 7 strains had better antifungal properties among 60 stain LAB. Of which the LAB of best antifungal activities was identified as Lactobacillus casei by 16S r DNA method. The 7 screened LABS all can produce PLA through HPLC detecting. Although the PLA from fermentation broth can reach highest content 16.24 mg l/L and the minimum content 6.86 mg l/L , the test result showed PLA content have no relations with antifungal activities. The MRS broth pH was adjusted to 3.88 by acetic acid、lactic acid and hydrochloric acid separately, but there is no inhibition zone. The further research about antibacterial material needs to be done to further identify antibacterial material.(2)The effects of different incubation time、incubation temperature and aeration condition on the growth and antibacterial properties of AST18 were studied, The optimum growth conditions and antibacterial activities was at 37°C and at the initial pH of 6.5-7.0 and by shaking culture . by studying the different sensitivity of culture supernatants of Lactobacillus casei AST18 with pH、heat treatment and protease treatment, the results showed the antibacterial activities of the culture supernatants of Lactobacillus casei AST18 was high at the pH of 2.0-4.0, it was vulnerable to heat treatment and even lost bioactivity by above 100℃heat treatment ,but it was not affected after treatment of trypsin and pepsin.(3)The part 2(4.5-7.5min)and part 4(11.4-13.4min)showed the biggest antifungal activities. Through the primary research, the high level of lactic acid was the main antifungal materials in part 2. Part 4 was analysed by GC-MS. There are three organic compounds departed: cyclic peptidesctclo-(Leu-Pro),2,6-diphenyl-piperidine and small molecular substances of pyrazine.(4)The physical and chemical properties and sensory properties were evaluated when the Lactobacillus casei AST18 as Secondary fermentation agent was used in yogurt. The result showed the addition of Lactobacillus caseiAST18 had little effection on viscosity、water-retaining capability and sensory evaluation of yogurt, and the weak acid yield ability, and little effection on pH value and titratable acidity of yogurt. The AST18 can be used as secondary fermentation and its addition had little effection on physical and chemical properities and sensory evaluation. At the same time, AST18 addition can increase the number of Lactobacillus in yogurt and reduce the number of Lactococcus, such as, Compared with the control group the 2% addition AST18 can reduce by 1.0 Log (cfu/mL) and other test groups reduce by 0.5 Log (cfu/mL). This may because of the Antagonism of Lactobacillus and Lactococcus.(5) The shelf life experiment and mouldproof accelerated testing were done at 4℃and 28℃. The test result showed, the mould growth situation can be totally inhibited by addition 2% Lactobacillus caseiAST18 in yogurt at shelf life experiment and mouldproof accelerated testing at 4℃; the mould growth situation can be delayed by addition 2% Lactobacillus caseiAST18 in yogurt at shelf life experiment and mouldproof accelerated testing at 28℃更多还原