【作者】 高莉莉；

【导师】 张柏林；

【作者基本信息】 河北农业大学 ， 微生物学， 2005， 硕士

【摘要】 明串珠菌是一类进行异型乳酸发酵的革兰氏阳性细菌,可以代谢柠檬酸形成风味成分双乙酰,在乳制品、食品防腐和医药行业有重要用途,是乳品加工中占有重要地位的发酵剂菌种之一。本文从不同来源的基质中,分离、筛选明串珠菌,研究其代谢和生理特性,旨在提供新的适应于商业性发酵剂规模化生产的优良菌株和资源,为开发新的发酵乳产品和满足人们的消费需求提供技术支持。 采用HP、LUSM和LMM三种选择性培养基,从来源不同的基质材料中分离到45株疑似明串珠菌;经万古霉素抗性试验、柠檬酸代谢试验和葡聚糖生成试验,获得柠檬酸代谢和葡聚糖生成均为阳性的菌株6株,柠檬酸代谢阳性菌株6株,能够将蔗糖转化为葡聚糖的菌株8株。经过革兰氏染色、细胞形态学特征观察、37℃下生长试验、pH 4.8生长试验、6.5%和3%NaCl生长试验、过氧化氢酶试验、水解精氨酸试验、从葡萄糖产酸产气试验、水解七叶灵试验以及碳水化合物发酵试验,并以肠膜明串珠菌肠膜亚种6055作为鉴定试验模式菌株,对所获得的20株疑似菌株分别进行生理和生化特征测试,参照《伯杰细菌鉴定手册》（第八版）确定20株参选菌株均属于明串珠菌属,其中10株被鉴定为肠膜明串珠菌肠膜亚种,4株被鉴定为肠膜明串珠菌右旋葡聚糖亚种、1株被鉴定为肠膜明串珠菌乳脂亚种、1株被鉴定为乳明串珠菌、4株被鉴定为假肠膜明串珠菌。 基于柠檬酸代谢试验以及双乙酰产量初步测定结果,确定了柠檬酸代谢能力较强的3个菌株,即肠膜明串珠菌肠膜亚种DM1-2、肠膜明串珠菌右旋葡聚糖亚种WZ4-3和假肠膜明串珠菌WZ-9-2作为进一步的研究菌株。 以模式菌株肠膜明串珠菌6055作为对照,研究了葡萄糖、蔗糖和乳糖等碳源对菌株DM1-2、WZ4-3和WZ-9-2细胞生长和发酵活性的影响。结果表明,蔗糖和葡萄糖均可促进菌株6055、DM1-2和WZ-9-2良好生长,且效果相近,而菌株WZ4-3的最佳碳源是蔗糖。 研究了蛋白胨、胰蛋白胨和胰酶水解酪蛋白等氮源对菌株6055、DM1-2、WZ4-3和WZ-9-2的生长和发酵活性的影响。结果表明,三种氮源在刺激明串珠菌的生长和酸化活性方面并没有显著的差别,都可以作为氮源使用。 研究了柠檬酸对菌株6055、DM1-2,WZ4-3和WZ-9-2的细胞生长和发酵活性的影响。结果表明,添加柠檬酸均不同程度的降低了四菌株的酸化活力;在刺激细胞生长方面,除明显刺激WZ4-3的细胞增长之外,对其它3个菌株并无明显效果。 研究了Mn²⁺和Mg²⁺的不同添加方式对菌株6055、DM1-2、WZ4-3和WZ-9-2的细胞生长和发酵活性的影响。结果表明,Mn²⁺对4株菌的细胞生长和发酵活性均有促进效果,而Mg²⁺并无明显效果。更多还原

【Abstract】 Leuconostoc spp. are gram-positive and heterofermentative lactic acid bacteria found in raw foodstuffs, dairy products and vegetable fermentations. The microorganisms are able to convert citric acid from media into diacetyl, one important compound contributing to the aroma of dairy products like butter, cheeses or buttermilk. Moreover, Leuconostoc strains have found commercial applications as one of the most important starter cultures in dairy and vegetable processing, food preservation and even medical industry. The objectives of the present study were: (1) to isolate and screen leuconostoc strains from various sources like raw milks and fermented sauerkrauts; (2) to investigate their physiological and citrate-metabolizing properties under controlled conditions; (3) to obtain new leuconostoc strains available for the commercial preparation of starters used in dairy products. The following are main results.The three selective media HP, LUSM and LMM were used to isolate 45 strains of Leuconostoc spp. from raw cow milk, goat milk and fermented vegetables obtained form Inner Mongolia and Baoding City of Hebei Province based on vancomycin-resistant, citrate-fermenting and dextran-producing from saccharose. Among 45 isolates, 20 isolates were identified as Leuconostoc species based on the morphological, gram-staining, physiology and carbohydrate fermentations with reference to the type strain Leuconostoc mesenteriodies subsp. mesenteriodies 6055. Out of 20 leuconostoc species, 10 strains were identified as Leuconostoc mesenteriodies subsp. mesenteriodies, 4 strains as Leuconostoc mesenteriodies subsp. dextranicum, 1 strain as Leuconostoc mesenteriodies subsp. cremoris, 1 strain as Leuconostoc lactic, and 4 strains as Leuconostoc prarmesenteriodies.L. mesenteriodies subsp. mesenteriodies DM 1-2, L .mesenteriodies subsp. dextranicum WZ4-3 and L.prarmesenteriodies WZ-9-2, along with L.mesenteriodies subsp. mesenteriodies 6055, were tested for further studies according to their citrate-metabolizing and the amount of formed diacetyl. Compared to strain 6055 used as the control, the effects of glucose, saccharose and lactose on the cell growth and acidifying ability of strains DM 1-2, WZ4-3 and WZ-9-2 were carried out. The results showed that glucose and saccharose stimulated the growth and acidifying activity of strains 6055, DM 1-2 and WZ-9-2 very well, whereas saccharose was the optimal sugar supporting the cell growth of strain WZ4-3.Peptone, tryptone and casein-hydrolysate were utilized well as nitrogen sources by the strains6055. DM1-2, WZ4-3 and WZ-9-2. The cell growth and acidifying activity of 4 strains studied showed that no significant differences were found among the utilization of 3 nitrogen resources used as growth-promoting factors.Inclusion of citrate into MRS media incubated at 37°C, to some extent, reduced the acidifying activity of Leuconostoc strains 6055, DM 1-2, WZ4-3 and WZ-9-2, Moreover, addition of 0.15% citric acid to MRS media had not give a clear stimulation to the cell growth of 6055, DM1-2 and WZ-9-2 except strain WZ4-3.The effects of metal irons like Mn2+, Mg2* or Mn2+ plus Mg2+ on the cell growth and acidifying ability of Leuconostoc strains DM 1-2, WZ4-3 and WZ-9-2 were carried out. The results indicated that Mn2+ improved clearly the growth and acidifying activity of 4 strains tested, whereas Mg~" produced no clear stimulation to the growth and activity of leuconostoc strainsFour strains of Leuconostoc tested grew at a wide temperature range from 21°C to 32°C. Cell growth and acidifying activity showed that strains 6055 and DM 1-2 grew very well at 32°C. Strains WZ4-3 and WZ-9-2 had the largest growth and acidifying activity at 25°C. It was clear that the optimal cell growth and activity were strain-dependent.The activities of diacetyl reductase and lactic dehydrogenase (LDH) of the strains 6055, DM 1-2, WZ4-3 and WZ-9-2 were investigated based on the changes of the two enzymes in pH from 4.6 to 6.6. It was seen that the diacetyl reductase of strain 6055 showed a change in the shape of "v" (i.e. firstly declining, then reaching the lowest, and finally rising) as pH increased. When pH was 5.5, the level of diacetyl reductase from strain 6055 achieved the minimum having 0.290U. Its LDH activity decreased from 0.188U to 0.083 U when pH was raised. The levels of diacetyl reductase and LDH from strain DM 1-2 decreased gradually with increase in pH. When pH changed between 6.6 and 4.6, the enzyme’s activity of strain DM 1-2 declined from 0.291U to 0.195U in diacetyl reductase or from 0.173U to 0.062U in LDH. Data from the present study showed that the activities of diacetyl reductase and LDH of strains WZ4-3 varied in the shape of "a" as pH rose from 4.6 to 6.6. The activity of its diacetyl reductase had a maximal value of 0.231U at pH 4.8; and its LDH reached maximum with 0.165 at pH 5.8. The diacetyl reductase of.strain WZ-9-2 increased slowly between 0.059U and 0.149U as pH declined from 6.6 to 4.6. Its LDH activity showed a decline from 0.278U to 0.114U when pH varied between 6.6 and 4.6.The colorimetric estimation of ONPG was used to determine [i-galactosidase activity of the four ieuconostoc strains 6055, DM 1-2, WZ4-3 and WZ-9-2. The levels of (3-galactosidase was 6.8x 10~’U for strain 6055, 8.4x 10"3U for strain DM1-2,2.9x I0~3U for strain WZ4-3 and 9.3x 10~2U for strain WZ-9-2, respectively. It was clear that the levels of |3-galactosidase formed by leuconostoc depended upon strains used.Steam-distillation-spectrophotometric method was used to deal with citrate utilization and subsequent diacetyl formation by the Leuconostoc strains DM 1-2 and WZ4-3 under different fermentation conditions. The results showed that addition of 0.15% citric acid to milk before inoculation increased the level of diacetyi from I.571ug/mL to 2.186|ag/mL produced by strain更多还原