双歧杆菌四联活菌片中嗜酸乳杆菌的分离及生长动力学研究

目的以双歧杆菌四联活菌片为实验材料,利用酸化的MRS培养基筛选分离得到嗜酸乳杆菌,对其进行进一步的生长动力学研究,确定嗜酸乳杆菌的生长数学模型。方法通过浓度梯度稀释法利用改良MRS培养基对双歧杆菌四联活菌片中的嗜酸乳杆菌进行分离,利用分光光度仪和平板菌落计数两种不同的方法测定嗜酸乳杆菌在发酵过程中不同发酵时间的细胞浓度的动态变化,经软件处理后拟合出嗜酸乳杆菌细胞生长的Logistic数学模型。结果Logistic方程能很好地拟合嗜酸乳杆菌细胞生长的动态变化,并得到嗜酸乳杆菌在本实验条件下的数学模型,为进一步研究、利用嗜酸乳杆菌生长能力、产酸能力和产香能力等具有重要的理论指导意义。     

胆汁酸盐和低pH值对乳酸菌活性的影响

本文比较了几种乳酸菌对胆汁酸盐的耐受性和ｐＨ值的残活能力。研究结果表明,在０．２％的ＭＲＳ或ＢＭ培养基上,婴儿双歧杆菌的生长速率受到的抑制最小,依次为嗜酸乳杆菌、保加利亚乳杆菌和嗜热链球菌。胆汁酸盐对其生长的完全抑制浓度分别为１．５％、１．２％、０．５％和０．３％。在ｐＨ３时,嗜热链球菌活菌数量降低较为明显,在ｐＨ２时,在３７℃１ｈ过程中,婴儿双歧杆菌活菌数量基本不变;嗜酸乳杆菌在３７℃２０ｍｉｎ后,活菌数量迅速降低;保加利亚乳杆菌数量在３７℃２０ｍｉｎ时几乎为零。     

嗜酸乳杆菌在模拟胃肠环境中抗性的研究

采用MRS培养基,模拟胃肠环境,即低pH值（1.5～4.5）。高胆汁盐（0.1％～0.4％）对嗜酸乳杆菌抗性进行了研究。同时对肠道中致病性大肠杆菌、金黄色葡萄球菌的拮抗特性以及服用抗生素后嗜酸乳杆菌的耐药性进行了研究。结果表明,嗜酸乳杆菌在pH2.5～4.5时具有较强的生存能力,6h活菌数仍达 10⁷cfu/mL以上,pH1.5条件下仍有部分存活。在0.1％～0.3％胆汁盐条件下4h活菌数仍达106cfu/mL以上,且能在0.4％胆汁盐中存活。同时,对致病性大肠杆菌和金黄色葡萄球菌具     

嗜热链球菌培养条件的研究

将嗜热链球菌（Ｓｔｒｅｐｔｏｃｏｃｃｕｓｔｈｅｒｍｏｐｈｉｌｕｓ）在６种固体培养基及７种液体培养基中的生长情况进行了比较,结果表明,改良ＩＲＩＥ固体培养基上的溶钙圈大,活菌数最多;在７种液体培养基中亦以改良ＩＲＩＥ的ＯＤ值最高,活菌最多,达８×１０^８／ｍｌ以上。以此培养基做生长曲线,该菌的平衡期为１２～１６ｈ。     

平菇浸汁促进嗜酸乳杆菌生长的研究

对平菇浸汁可促进嗜酸乳杆菌的生长进行了初步研究。结果表明,平菇浸汁对嗜酸乳杆菌在还原脱脂乳中的生长具有明显的促进作用,添加5％～10％的平菇浸汁,可大大缩短嗜酸乳杆菌生长的世代时间,提高其产酸速率;在37℃下1Oh的培养,其中活菌数的含量较对照组大大提高,可达108/mL。     

乳酸、乙酸及pH对嗜酸乳杆菌和 阴道加德纳菌的生长及交互作用影响

目的探讨乳酸、乙酸及pH值变化对嗜酸乳杆菌和阴道加德纳菌的生长及交互作用影响,为探究细菌性阴道病患者治疗失败的可能原因提供依据。方法 (1)两菌经不同浓度乳酸、乙酸作用后,检测其细菌浓度变化。(2)两菌经不同pH的SBHIG肉汤单独及混合培养,检测其细菌浓度变化并涂片观察生长情况。结果乳酸、乙酸浓度1.0%~5.0%时,均可抑制两菌的生长。0.1%乳酸对阴道加德纳菌生长的抑制能力较弱,但可促进嗜酸乳杆菌的生长。肉汤培养基在pH 3.5时,几乎只有嗜酸乳杆菌能生长;pH 8.5时,生长的细菌中阴道加德纳菌占绝大多数;pH 5.5和7.0时,两菌虽然共同生长,但嗜酸乳杆菌生长明显受抑制。结论嗜酸乳杆菌与阴道加德纳菌存在交互作用,但受细菌浓度、乳酸和乙酸浓度及pH的影响。细菌性阴道病患者需多疗程疗治疗或治疗失败可能与乳杆菌累积浓度未能达到抑制阴道加德纳菌生长的要求有关。     

pH值对萼花臂尾轮虫种群增长及繁殖的影响

采用种群积累培养法,实验观察了 ｐＨ在３．５～１１．５之间（间隔 １）萼花臂尾轮虫 （Ｂｒａｃｈｉｏｎｕｓ ｃａｌｙｃｉｆｌｏｒｕｓ）种群的增长及繁殖．结果表明,该轮虫种群在ＰＨ６．５～８．５之间增 长较快, ８． ５时增长最快,即瞬时增长率 ｒ和种群密度较大和最大; ｐＨ在 ３． ５～４． ５和 ９． ５ ～１０． ５之间,种群为负增长,即 ｒ为负值; ｐＨ在 ５． ５～９． ５之间种群为正增长,即正为正 值．该轮虫存活的ｐＨ上限为１１．５,下限为３．５．在种群增长最适ｐＨ（８．５）条件下,该轮虫 的繁殖最快,即绝对带卵量最高（１３２个·ｍｌ－１）;ｐＨ在９．５时,其相对带卵量最高．为其它 ｐＨ值条件下的２～４倍．本研究结果可为淡水轮虫的大批量培养提供可靠的ｐＨ值技术指 标．     

液氮保存疫霉属菌种存活期检测

对Ｐｈｙｔｏｐｈｔｈｏｒａ和Ｐｅｒｏｎｏｐｈｙｔｈｏｒａ所属１２个种２９株菌在液氮中保存５年零８个月至６年零３个月后检测证明有６８．９％的菌种存活下来。但有些未能存活,这些种有Ｐｈｙｔｏｐｈｔｈｏｒａ ｃｏｌｏｃａｓｉａｅ（ＸＨ３０）,Ｐ．ｄｒｅｃｈｓｌｅｒｉ（ＡＴＣＣ１５４２８）,Ｐ．ｅｒｙｔｈｒｏｓｅｐｔｉｃａ（ＡＴＣＣ３６３０２）与Ｐｅｒｏｎｏｐｈｙｔｈｏｒａ ｌｉｔｃｈｉｉ（ＡＴＣＣ３５９     

多菌种酸奶中活性乳酸菌的计数方法研究

利用4种选择性培养基MRS、MRS-山梨醇、MRS-5．2、Elliker,采用平板涂布法和倾注接种法。在蜡烛缸法（缺氧）、封口膜法（微氧）和供氧条件下对4种市售多菌种酸奶中保加利亚乳杆菌、嗜热链球菌、嗜酸乳杆菌、双歧杆菌进行选择性计数方法研究。结果表明：蜡烛缸法较能反映乳酸菌活菌数量的实际情况,封口膜法次之;在不同培养条件下4种选择性培养基对于乳酸菌活菌的计数都是灵敏的;而涂布法和倾注法接种活菌数有显著差异,倾注法要优于涂布法。     

化感物质对枯草芽孢杆菌（Bacillus subtilis）在厌氧条件下和生长？…

研究由秸秆腐解产生的化感物质：阿魏酸（ｔ－ＦＡ）、对羟基苯甲酸（ｐ－ＨＡ）和苯甲酸（ＢＡ）在不同浓度下对厌氧培养的枯草芽孢杆菌（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）的生长及其反硝化活性的影响。结果表明,３种浓度的阿魏酸（５．１５、２．５８、０．２６ｍｍｏｌ／Ｌ）均表现出对枯草芽孢杆菌的生长有抑制作用。对羟基苯甲酸（０;．３６、３．６２、７．２４ｍｍｏｌ／Ｌ）对生长影响不明显。８．１９ｍｍｏｌ／Ｌ和     

豆浆作为脱位牙保存液对牙周膜成纤维细胞活性的影响研究

目的:通过探讨牙周膜成纤维细胞保存在豆浆中对其细胞活性的影响,评价豆浆作为脱位牙保存液的可行性。方法:将因口腔正畸而拔除12-18岁青少年的前磨牙进行细胞分离,选用分离出来的牙周膜成纤维细胞进行培养,将其分别置于ViaSpan、DMEM高糖培养基、豆浆、自来水、牛奶、Hank平衡盐溶液这6种液体中进行培养,采用cck-8法测定其在不同保存液中1h、2h、4h、8h、24h五个时间点的细胞活性。结果:豆浆组1h、2h、4h、8h、24h细胞残存率都显著高于自来水组(P0.01);豆浆组1h、2h、4h、8h细胞残存率与DMEM组、ViaSpan组比较均无统计学差异(P0.05),在24h时的细胞残存率显著高于DMEM组和ViaSpan组(P0.05);豆浆组2h、8h细胞残存率和牛奶组比较无统计学差异(P0.05),但牛奶组1h、4h、24h细胞残存率显著高于豆浆组(P0.05);豆浆组1h、2h、4h、8h、24h细胞残存率和HBSS组比较均无统计学差异(P0.05)。结论:豆浆在保存牙周膜成纤维细胞活性效果等效于Hank平衡盐溶液,是一种有效的脱位牙保存液。     

Influence of Storage at Freezing and Subsequent Refrigeration Temperatures on beta-Galactosidase Activity of Lactobacillus acidophilus

The ability of three strains of Lactobacillus acidophilus to survive and retain beta-galactosidase activity during storage in liquid nitrogen at -196 degrees C and during subsequent storage in milk at 5 degrees C was tested. The level of beta-galactosidase activity varied among the three strains (0.048 to 0.177 U/10 organisms). Freezing and storage at -196 degrees C had much less adverse influence on viability and activity of the enzyme than did storage in milk at 5 degrees C. The strains varied in the extent of the losses of viability and beta-galactosidase activity during both types of storage. There was not a significant interaction between storage at -196 degrees C and subsequent storage at 5 degrees C. The strains that exhibited the greatest losses of beta-galactosidase activity during storage in milk at 5 degrees C also exhibited the greatest losses in viability at 5 degrees C. However, the losses in viability were of much greater magnitude than were the losses of enzymatic activity. This indicates that some cells of L. acidophilus which failed to form colonies on the enumeration medium still possessed beta-galactosidase activity. Cultures of L. acidophilus to be used as dietary adjuncts to improve lactose utilization in humans should be carefully selected to ensure that adequate beta-galactosidase activity is provided.     

Flow Cytometry Approach to Quantify the Viability of Milk Somatic Cell Counts after Various Physico-Chemical Treatments

Flow cytometry has been used as a routine method to count somatic cells in milk, and to ascertain udder health and milk quality. However, few studies investigate the viability of somatic cells and even fewer at a subpopulation level to follow up how the cells can resist to various stresses that can be encountered during technological processes. To address this issue, a flow cytometry approach was used to simultaneously identify cell types of bovine milk using cell-specific antibodies and to measure the cell viability among the identified subpopulations by using a live/dead cell viability kit. Confirmation of the cell viability was performed by using conventional microscopy. Different physico-chemical treatments were carried out on standardized cell samples, such as heat treatment, various centrifugation rates and storage in milk or in PBS pH 7.4 for three days. Cytometry gating strategy was developed by using blood cell samples stored at 4°C in PBS and milk cell samples heat-treated at 80°C for 30 min as a control for the maximum (95.9%) and minimum (0.7%) values of cell viability respectively. Cell viability in the initial samples was 39.5% for all cells and varied for each cell population from 26.7% for PMNs, to 32.6% for macrophages, and 58.3% for lymphocytes. Regarding the physico-chemical treatments applied, somatic cells did not sustain heat treatment at 60°C and 80°C in contrast to changes in centrifugation rates, for which only the higher level, i.e. 5000×g led to a cell viability decrease, down to 9.4%, but no significant changes within the cell subpopulation distribution were observed. Finally, the somatic cells were better preserved in milk after 72h storage, in particular PMNs, that maintained a viability of 34.0 ± 2.9% compared to 4.9±1.9% in PBS, while there was almost no changes for macrophages (41.7 ± 5.7% in milk vs 31.2 ± 2.4% in PBS) and lymphocytes (25.3 ± 3.0% in milk vs 11.4 ± 3.1% in PBS). This study provides a new array to better understand milk cell biology and to establish the relationship between the cell viability and the release of their endogenous enzymes in dairy matrix.     

Microbial analysis of raw cows’ milk used for cheese-making: influence of storage treatments on microbial composition and other technological traits

Raw milk used to produce Grana cheese was subjected to several treatment regimes, including varying temperatures and storage times. Milk from morning and evening milking were transferred to a dairy factory separately (double delivery) or together (single delivery), after storage at the farm for 12 h; in the former case, milk was stored at 12 or 8°C, whereas, in the latter, it was kept at ambient temperature or 18°C. Values of pH of the vat milk were lower for milk samples kept at room temperature, while other physico-chemical parameters and rheological characteristics tested did not show significant differences linked to the different storage temperatures of milk used for “Grana Trentino” cheese production. Total microorganisms and several microbial groups (psychrotrophic bacteria, coliforms, mesophilic and termophilic lactic acid bacteria, including enterococci, pseudomonads and clostridia) were detected and quantified by classical (plate count and most probable number) techniques, after each technological treatment for a total of 212 milk and cream samples. The application of a culture-independent microbiological strategy, consisting of denaturing gradient gel electrophoresis, allowed the recognition of several bacterial genera and species.     

Survival and growth of Cronobacter species (Enterobacter sakazakii) in wheat-based infant follow-on formulas

Aims:  To determine the survival and growth characteristics of Cronobacter species ( Enterobacter sakazakii ) in infant wheat-based formulas reconstituted with water, milk, grape juice or apple juice during storage.
Methods and Results:  Infant wheat-based formulas were reconstituted with water, ultra high temperature milk, pasteurized grape or apple juices. The reconstituted formulas were inoculated with Cronobacter sakazakii and Cronobacter muytjensii and stored at 4, 25 or 37°C for up to 24 h. At 25 and 37°C, Cronobacter grew more (>5 log₁₀) in formulas reconstituted with water or milk than those prepared with grape or apple juices ( c. 2–3 log₁₀). The organism persisted, but did not grow in any formulas stored at 4°C. Formulas reconstituted with water and milk decreased from pH 6·0 to 4·8–5·0 after 24 h, whereas the pH of the formulas reconstituted with fruit juices remained at their initial pH values, c. pH 4·8–5·0.
Conclusions:  Cronobacter sakazakii and C. muytjensii can grow in reconstituted wheat-based formulas. If not immediately consumed, these formulas should be stored at refrigeration temperatures to reduce the risk of infant infection.
Significance and Impact of the Study:  The results of this study will be of use to regulatory agencies and infant formula producers to recommend storage conditions that reduce the growth of Cronobacter in infant wheat-based formulas.     

Influence of Storage at Freezing and Subsequent Refrigeration Temperatures on β-Galactosidase Activity of Lactobacillus acidophilus

The ability of three strains of Lactobacillus acidophilus to survive and retain β-galactosidase activity during storage in liquid nitrogen at −196°C and during subsequent storage in milk at 5°C was tested. The level of β-galactosidase activity varied among the three strains (0.048 to 0.177 U/10⁷ organisms). Freezing and storage at −196°C had much less adverse influence on viability and activity of the enzyme than did storage in milk at 5°C. The strains varied in the extent of the losses of viability and β-galactosidase activity during both types of storage. There was not a significant interaction between storage at −196°C and subsequent storage at 5°C. The strains that exhibited the greatest losses of β-galactosidase activity during storage in milk at 5°C also exhibited the greatest losses in viability at 5°C. However, the losses in viability were of much greater magnitude than were the losses of enzymatic activity. This indicates that some cells of L. acidophilus which failed to form colonies on the enumeration medium still possessed β-galactosidase activity. Cultures of L. acidophilus to be used as dietary adjuncts to improve lactose utilization in humans should be carefully selected to ensure that adequate β-galactosidase activity is provided.     

Physiology of Batrachochytrium dendrobatidis, a chytrid pathogen of amphibians

Batrachochytrium dendrobatidis is a pathogen of amphibians that has been implicated in severe population declines on several continents. We investigated the zoospore activity, physiology and protease production of B. dendrobatidis to help understand the epidemiology of this pathogen. More than 95% of zoospores stopped moving within 24 h and swam less than 2 cm before encysting. Isolates of B. dendrobatidis grew and reproduced at temperatures of 4-25 C and at pH 4-8. Growth was maximal at 17-25 C and at pH 6-7. Exposure of cultures to 30 C for 8 d killed 50% of the replicates. B. dendrobatidis cultures grew on autoclaved snakeskin and 1% keratin agar, but they grew best in tryptone or peptonized milk and did not require additional sugars when grown in tryptone. B. dendrobatidis produced extracellular proteases that degraded casein and gelatin but had no measurable activity against keratin azure. The proteases were active against azocasein at temperatures of 6-37 C and in a pH range of 6-8, with the highest activity at temperatures of 23-30 C and at pH 8. The implications of these observations on disease transmission and development are discussed.     

Survival of Enterobacter cloacae and Pseudomonas paucimobilis in yoghurts manufactured from cow's milk and soymilk during storage at different temperatures.

The survival of two microbial contaminants, Enterobacter cloacae and Pseudomonas paucimobilis, in yoghurts manufactured from cow's milk and soymilk was investigated during storage for 45 days at 4 and 12 degrees C. Sensory panel tests performed before microbiological investigation, showed that the flavor of soy-yoghurts made with cocoa powder or malt added did not have the beany taste of soy beans. Both contaminants were significantly resistant to low pH values during storage for 32 days at 4 degrees C. The survival at 4 degrees C was remarkable in both plain and flavored yoghurts and a population close to 10(2) C.F.U./ml was observed after 38 days of storage. Experiments performed with soymilk yoghurts showed an enhanced survival of P. paucimobilis at 4 degrees C compared to the storage in cow's milk yoghurts; microbial values were close to 7-8 x 10(6) C.F.U./ml after 16 days. Soymilk exhibited a protective effect on L. delbrueckii subsp. bulgaricus and S. thermophilus at 12 degrees C and, compared to the survival in cow's milk yoghurts, a larger number of viable cells of both probiotic microorganisms (10(6) and 10(8) C.F.U./ml, respectively) were observed after 36 days of storage.     

Preservation by the Lactoperoxidase system (LP-S) of a contaminated infant milk formula

Enterococcus, Pseudomonas spp. and Enterobacteriaceae did not grow in an infant milk formula inoculated with the mixed flora contained in lake water and supplemented with an activated lactoperoxidase system (LP-S). Yeasts did grow and, with the exhaustion of thiocyanate (SCN^-) after 48 h, the one which had attained numerical dominance, Rhodotorula glutinis , was displaced by a non-pigmented yeast. The growth of the bacteria mentioned above occurred along with that of the latter yeast. Absence of the LP-S from the infant milk was associated with the unrestricted proliferation of Enterobacteriaceae in general and Escherichia coli in particular. These organisms dominated the microbial population after 24 h storage. The LP-S also prevented significant glucose utilization and stabilized the pH.     

A comparative study of preservation and storage of Haemophilus influenzae

The aim of this study was to compare the efficacy of conservation by freezing the strains of Haemophilus influenzae at -20 degrees C and -70 degrees C. Skim milk supplemented with glucose, yeast extract and glycerol allowed highest viability of H. influenzae both at -20 degrees C and -70 degrees C from the media analyzed. Trypticase soy broth and brain heart infusion broth supplemented with glycerol, allowed excellent recovery. Use of cotton swaps as supporting material, with or without addition of cryoprotective agents, did not modify H. influenzae viability after six months of storage. Concentration of the initial inoculum positively affected viability when stored at -20 degrees C. Initial concentration did not influence survival after storage at -70 degrees C. Thawing at room temperature should not exceed 3 h as to get highest survival percentage.