分光光度法测定黑曲霉孢子浓度的研究

采用分光光度计测定黑曲霉（Aspergillus niger）xj在不同生长期、不同稀释倍数下孢子悬液的OD600 nm值,对比血球板计数法得到的孢子悬液浓度,研究在对数期和稳定期OD600 nm值与孢子浓度之间的关系;采用微电影拍摄法观察黑曲霉xj产孢结构形成过程,并绘制固体发酵条件下的生长曲线。结果表明,在10～20倍稀释区间内,稀释倍数与OD600 nm值之间呈现良好线性关系（对数期R2 =0.984 8、稳定期R2 =0.991 3）,且OD600 nm值与孢子浓度之间也保持良好的线性关系（对数期R2 =0.995 3、稳定期R2 =0.993 6）;通过微电影拍摄法观察到黑曲霉xj产孢结构形成是分阶段进行的过程;在固体发酵过程中,孢子浓度呈现“S”型增长趋势。该研究为测定丝状真菌孢子浓度提供另一种可借鉴的方法。     

微生物吸光值法定量检测实验

通过对铀矿冶生物技术国防重点学科实验室提供的巴氏芽孢杆菌的培养,分别取相同菌量1ml菌悬液(OD600=1.0440),按相应体积倍数稀释后,测定各OD600值。数据的拟合发现,全部稀释倍数乘幂关系拟合线拟合度不高,OD600值大于1或小于0.2时,越靠近坐标轴,数据越失真,误差越大。截取0.2~1.0之间的OD600值,拟合乘幂曲线为y=1.0764x-1.0115(R2=0.9976),应用简单倍数关系y=1.044/x换算,两者误差都维持在5%之内。OD600值在0.2~1.0之间运用简单倍数关系换算准确、快捷。     

食用防腐剂对金黄色葡萄球菌生长参数的影响

为了明确食用防腐剂对金黄色葡萄球菌生长的影响,以乳酸链球菌素、亚硝酸钠、苯甲酸钠、山梨酸钾、焦亚硫酸钠、丙酸钙为实验对象,确定临界生长质量浓度后,分别研究各防腐剂作用下金黄色葡萄球菌的生长规律,并比较部分防腐剂作用于对数期和稳定期菌体时最大比生长速率(μmax)和迟滞期(Lag)的差异。结果表明:乳酸链球菌素具有较强的杀菌作用,培养前6h内菌数持续降低,由6.09(lg(CFU/mL))降至3.68(lg(CFU/mL)),15h左右才恢复到初始值。其余5种防腐剂均表现为抑菌作用,Baranyi方程分析各生长参数,结果显示μmax值均比无防腐剂的对照值低,其中焦亚硫酸钠作用后的μmax值最低为0.45h-1;Lag值则有升有降,其中山梨酸钾作用后的λ值最大为2.87h。将山梨酸钾和焦亚硫酸钠分别作用于对数期和稳定期菌体,发现作用于对数期菌体的μmax值均较稳定期菌体的低,尤其是焦亚硫酸钠作用时,对数期菌体的μmax值(0.45h-1)约为稳定期的(0.88h-1)一半,差异极显著(P<0.01);而两种防腐剂作用于对数期和稳定期菌体时λ值的差异均不显著。     

耐酸耐胆盐乳酸菌的鉴定及筛选

从自然发酵的酸奶中分离出2 株细菌,经16S rDNA分子鉴定为Lactobacillus plantarum SN1和Lactobacillusrhamnosus SN6,并对其生长曲线、产酸速率、耐酸耐胆盐能力进行了研究。L. plantarum SN1和L. rhamnosus SN6在2 h后进入对数期,16 h后达到稳定期,其OD600 nm值分别为8.47、7.43。L. plantarum SN1和L. rhamnosus SN6的产酸速率较快,pH值在8 h后就降到了4.2以下,48 h后降到3.3左右。L. plantarum SN1和L. rhamnosus SN6在pH 4的培养基培养16 h后,其相对OD600 nm值分别为49.29%、47.14%,具有较强的耐酸能力。L. plantarum SN1和L. rhamnosus SN6在0.3 g/L胆盐质量浓度下培养16 h后,相对OD600 nm值分别为57.7%、69.48%;在0.6 g/L胆盐质量浓度下的相对OD600 nm值分别为48.22%、29.56%,具有较强的耐胆盐能力。结果表明：L. plantarum SN1和L. rhamnosus SN6是生长性能好、产酸能力强、耐酸耐胆盐的益生菌株。     

菊粉低聚果糖与蔗糖低聚果糖对双歧杆菌体外增殖的研究

目的:研究两种来源的低聚果糖对双歧杆菌的体外增殖效果。方法:配制以两种低聚果糖为碳源的培养基,对两种双歧杆菌进行体外培养,以培养时间为横坐标,吸光度(OD值)为纵坐标,绘制生长曲线,通过对比吸光度(OD值)反馈双歧杆菌的增殖情况,进而评价两种低聚果糖对双歧杆菌的增殖效果。结果:从双歧杆菌Ⅰ(BI-07)的生长曲线看出,两种低聚果糖均能促进双歧杆菌Ⅰ(BI-07)的生长,从对数期到稳定期,菊粉低聚果糖的吸光度(OD值)值始终高于蔗糖低聚果糖;从双歧杆菌Ⅱ(BB-12)的生长曲线看出,两种低聚果糖均能促进双歧杆菌Ⅱ(BB-12)的生长,当双歧杆菌处于对数期时,初期蔗糖低聚果糖的吸光度(OD值)高于菊粉低聚果糖,但在24h后,菊粉低聚果糖的吸光度(OD值)逐渐高于蔗糖低聚果糖,最终是菊粉低聚果糖的吸光度(OD值)高于蔗糖低聚果糖。结论:两种低聚果糖均能促进双歧杆菌的体外增殖,但菊粉来源的低聚果糖对双歧杆菌的体外促生长作用优于蔗糖来源的低聚果糖。     

双麦麦精对嗜热链球菌生长影响的研究

主要研究双麦麦精对嗜热链球菌生长过程的影响。通过在营养成分缺省的培养基中添加双麦麦精,采用分光光度法测定菌液吸光值(OD600),根据OD值确定对应的嗜热链球菌的活菌数,从而分析双麦麦精对嗜热链球菌生长情况的影响。利用正交实验,通过直接计数法确定双麦麦精与其他营养成分的最佳比例。结果表明,双麦麦精对嗜热链球菌的生长有明显的促进作用,可为嗜热链球菌提供充足的碳源和生长因子。双麦麦精的添加量为16g/L时效果最佳。分析比较吸光值法和直接计数法测得的生长曲线,结果表明两种方法具有良好的相似性,其中在对数期有强相关性。     

不同生长阶段保加利亚乳杆菌关键蛋白酶基因表达变化规律

乳酸菌自身不能直接利用外源蛋白质,必须通过蛋白水解体系水解外源蛋白质,生成供菌体正常生长需要的短肽和游离氨基酸。该研究选择6株保加利亚乳杆菌(KLDS1.0207、1.0501、1.1007、1.9201、1.9203、1.0208)进行脱脂乳发酵,在mRNA转录水平上,应用实时荧光定量PCR技术,探讨保加利亚乳杆菌的蛋白水解体系中关键蛋白酶基因(prtB、oppD、pepC、pepF、pepQ、pepX、pepT)在菌体不同生长阶段表达的动态变化。结果表明,所有菌株生长曲线均呈S型。随着菌体在脱脂乳中的不断生长,蛋白水解活力极显著增强(P<0.01),7个目的基因相对表达量呈现总体上调。6株菌的prtB和oppD基因在对数期的相对表达量极显著高于对照组(4h)(P<0.01)。对数期的胞内肽酶基因(pepC、pepF、pepQ、pepX、pepT)的表达量与对照组(4 h)相比,呈极显著上调(P<0.01),但菌株KLDS 1.0207的pepQ基因在对数期下调,而稳定期上调。由此可见,蛋白水解活力较高的菌株,其生长和产酸特性也较高;发现在脱脂乳中培养保加利亚乳杆菌,其关键蛋白酶基因的表达量呈时间依赖性,且菌株间各蛋白酶基因表达量有较大差异。     

pH值对聚唾液酸分批发酵的影响及补料发酵

研究了不同pH值对聚唾液酸发酵过程中菌体生长和产聚唾液酸的影响.发酵初期pH自然下降时有利于菌体生长,菌体生长对数期较长,最大菌体干重可达6.9g/L;发酵中后期pH控制在6.4时有利于聚唾液酸的延续合成,合成对数期比其它pH条件下的合成对数期延长了11h.动力学特性表现为部分相关模式,而在其它pH条件下,动力学特性表现为相关模式.对聚唾液酸的流加补料发酵进行了初步研究,最终使菌体干重达到11.16g/L,聚唾液酸产量达到2.606g/L.     

胡桃醌抑制细菌生长作用的研究

目的:以大肠杆菌和金黄色葡萄球菌为研究对象,探讨胡桃醌对微生物不同阶段生长及形态特征的影响,为胡桃醌抑菌机理的研究奠定基础。方法:采用紫外分光光度计和平板计数法绘制生长曲线,通过透射电镜观察菌体形态特征的变化。结果:大肠杆菌的迟缓期和对数期受到胡桃醌的抑制作用明显;处于对数期的金黄色葡萄球菌对胡桃醌的抑制作用更为敏感。透射电镜的结果表明,最低抑菌浓度下的胡桃醌作用大肠杆菌2h,菌体即出现细胞质壁分离现象,细胞质分布不均匀;8h后,细胞壁破坏严重,内溶物流出。胡桃醌作用金黄色葡萄球菌4h,菌体细胞吸水膨胀,部分细胞隔膜已被破坏。8h后,菌体互相粘连,细胞与细胞间的界限变得模糊。结论:胡桃醌的抑菌效果主要表现在细菌生长的对数期,并可能通过破坏菌体的细胞壁或细胞膜结构来抑制细菌的生长。     

鸡源乳酸杆菌C最佳培养条件的确定

以鸡源乳酸杆菌C为实验材料,确定其最佳的培养条件.通过将乳酸杆菌接种于MRS培养基中扩大培养,然后取适当菌株分别置于不同温度、不同pH、不同接种密度的条件下,通过测定600nm下的OD值,测定其最佳生长条件.在pH为6.0左右的时候,OD值达到最大,证明pH为6.0时微生物数量最多,生长最好,为其最佳培养pH条件.在37℃左右OD值达到最大值,因此其最佳培养温度为37℃.在50mL培养基中接种0.2mL时,OD值为最大,证明其最佳接种量为0.2mL.     

Effect of starvation on expression of the ribosomal RNA rrnB P2 promoter during the lag phase of Pseudomonas fluorescens

Mathematical modelling of food-borne pathogen survival and growth is an important and expanding area of food microbiology. Effective models have been developed for growth rate as influenced by the environment; however, reliable models which describe the lag phase prior to exponential growth are more difficult to obtain. In order to improve our understanding of the physiological changes that take place in the microbial cell during this adaptation period, the effect of starvation on the expression of a gene for ribosomal RNA (rRNA) synthesis-an important step in preparing the cells for growth-was examined. A strain of Pseudomonas fluorescens containing the Tn7-luxCDABE gene cassette regulated by the rRNA promoter rrnB P(2) was used as a model system. Growth was measured as optical density at 600 nm (OD(600)), and fitting was achieved with a two-phase linear model to obtain the parameters growth rate (R(OD)) and lag phase duration (LPD(OD)). The increase in bioluminescence (measured as natural log [ln] relative light units per unit OD(600)) after inoculation of stationary phase cells into fresh tryptic soy broth (TSB) followed an exponential association model, with lag (LPD(Exp)) and rate (R(Exp)) parameters. Starvation of cells in either spent TSB or in MOPS buffer resulted in time-dependent linear increases in both lag parameters and, in the case of TSB, a decrease in the R(Exp) parameter. The results show that models can be developed for expression of genes during the lag phase, which will improve our ability to make accurate predictions of food-borne pathogen growth.     

乳酸链球菌素对乳酸菌抑菌作用的研究

目的：为了观察Nisin 对乳酸菌的抑菌效果,探讨Nisin 作为食品级载体筛选方法的最佳抑菌浓度和持续时间。方法：采用试管稀释法和纸片扩散法检测Nisin 对乳酸菌的抑菌浓度,并根据乳酸菌的OD600nm 作出生长曲线,以研究Nisin 对乳酸菌的抑菌效果。结果：Nisin 对乳品工业常用乳酸菌具有较强的抑菌作用,仅某些乳酸乳球菌对Nisin 表现出抗性;Nisin 对乳酸菌有较持久的抑菌效果,可达30～48h 以上。结论：该实验为Nisin 作为食品级载体筛选方法的最佳抑菌浓度和持续时间提供了依据。     

Growth curve prediction from optical density data

A fundamental aspect of predictive microbiology is the shape of the microbial growth curve and many models are used to fit microbial count data, the modified Gompertz and Baranyi equation being two of the most widely used. Rapid, automated methods such as turbidimetry have been widely used to obtain growth parameters, but do not directly give the microbial growth curve. Optical density (OD) data can be used to obtain the specific growth rate and if used in conjunction with the known initial inocula, the maximum population data and knowledge of the microbial number at a predefined OD at a known time then all the information required for the reconstruction of a standard growth curve can be obtained. Using multiple initial inocula the times to detection (TTD) at a given standard OD were obtained from which the specific growth rate was calculated. The modified logistic, modified Gompertz, 3-phase linear, Baranyi and the classical logistic model (with or without lag) were fitted to the TTD data. In all cases the modified logistic and modified Gompertz failed to reproduce the observed linear plots of the log initial inocula against TTD using the known parameters (initial inoculum, MPD and growth rate). The 3 phase linear model (3PLM), Baranyi and classical logistic models fitted the observed data and were able to reproduce elements of the OD incubation-time curves. Using a calibration curve relating OD and microbial numbers, the Baranyi equation was able to reproduce OD data obtained for Listeria monocytogenes at 37 and 30°C as well as data on the effect of pH (range 7.05 to 3.46) at 30°C. The Baranyi model was found to be the most capable primary model of those examined (in the absence of lag it defaults to the classic logistic model). The results suggested that the modified logistic and the modified Gompertz models should not be used as Primary models for TTD data as they cannot reproduce the observed data.     

Behaviour of log-phase Escherichia coli at temperatures near the minimum for growth

The behaviour of cold-adapted, log-phase Escherichia coli in broth cultures incubated at temperatures between 7 and 15 degrees C was examined by determinations of numbers of colonies recovered on plate count agar (PCA); absorbance at 600 nm (A600); cell lengths from photomicrographs; and cell size distributions by flow cytometry. Cultures incubated between 7 and 10 degrees C were evaluated for 8 days or until A600 values approached 1.0. Cultures incubated at > or =12 degrees C were subcultured to maintain them in the log phase for up to 8 days. Numbers of colonies recovered declined when cultures were incubated at 7 degrees C, but increased when cultures were incubated at higher temperatures. However, A600 values increased during incubation at all temperatures. The mean lengths of cells doubled during incubation at 7 degrees C for 8 days, but remained constant during incubation at 10 degrees C for 1.25 days. Forward angle light scatter (FALS) measurements obtained by flow cytometry indicated that the mean length of cells increased at < or = 8 degrees C, but not at 10 degrees C. A reference value at the 90th percentile of FALS measurements on day 0 was used to determine changes in the distribution of the lengths of cells. About 80% or 17% of the cells were above the reference value after 5 days of incubation at 7 degrees C or 1.25 days of incubation at 10 degrees C, respectively. Cultures that were maintained in the log phase at 12 degrees C became increasingly heterogeneous in cell size after 2 days, but cultures that were maintained at 13 degrees C remained constant in cell size for 8 days. The observations have implications for the prediction of mesophile proliferation at temperatures that approach their minima for growth.     

枯草芽孢杆菌发酵提取物对大肠杆菌的抑制作用

为探索枯草芽孢杆菌发酵提取物对大肠杆菌的抑菌效果及作用机理,利用倍半稀释法确定其对大肠杆菌的最小抑菌浓度,通过研究枯草芽孢杆菌发酵提取物对大肠杆菌生长曲线、细胞膜通透性以及细胞超微结构的影响,探讨提取物对大肠杆菌的抑制作用机理,同时对该提取物的化合物类型进行了初步分析。结果表明,该提取物对大肠杆菌具有明显的抑制作用,其最小抑菌浓度为0.614 mg·mL-1,在大肠杆菌生长的延滞期和对数期加入该提取物,比在稳定期加入能够显现出更好的抑菌效果。经提取物作用后的细胞表面粗糙,边缘模糊,细胞膜破裂,表明该提取物能够增加大肠杆菌细胞膜的通透性。化合物分析显示,提取物中抑菌成分主要是多烯类化合物和脂肽类化合物。     

辣椒素对辣椒发酵中乳酸菌的影响

针对辣椒乳酸发酵中的技术问题,采用植物乳杆菌、短乳杆菌、罗伊氏乳杆菌、瑞士乳杆菌、鼠李糖乳杆菌这5种乳酸菌为研究对象,探讨辣椒中主要成分辣椒素和发酵过程中食盐质量浓度对乳酸菌纯培养物生长的影响,同时结合电镜实验讨论辣椒素对乳酸菌的作用效果。结果表明：辣椒素对乳酸菌的生长均有一定的延迟作用,辣椒素对乳酸菌抑制的最低质量浓度为0.133mg/mL,使其对数生长期延后2h,并且随着其质量浓度的增大,这种延迟效果更加明显。同时还降低了培养液中达到稳定期的OD600nm值,降低了0.2左右。通过电镜实验发现,一定质量浓度的辣椒素对乳酸菌细胞具有一定的伤害作用,会破坏其细胞形态及结构,但不会严重影响发酵效果。     

酸奶发酵过程中物理性质和乳酸菌生长规律研究

本文研究了酸奶发酵过程中乳酸菌双菌的生长规律，酸奶电导率变化和pH值下降曲线以及二者间的关系。同时研究了酸奶凝乳与pH值的相关性。研究表明：发酵过程中双菌存在相同的迟滞期，二者同时进入对数生长期，对数期球菌(ST)的生长较杆菌(LB)快得多，球菌比杆菌提前进入稳定期；体系的电导率随pH值下降逐渐增高，当pH值到达4.5以后，其电导率保持不变，存在一个最终恒定值：pH值处于6．3～4.5范围时，电导率与pH值存在很好的线性关系；pH值下降曲线和电导率上升曲线都呈现五次多项式的性质．具有很好的拟合性；酸奶开始发生凝乳与体系pH之间存在显著相关，当pH值下降至5．3～5．4范围时发酵液即出现凝乳。