全脂乳中2种乳酸菌的HPLC研究

研究了可应用于高效液相离子交换色谱分析的嗜热链球菌及保加利亚乳杆菌的培养方法,同时探讨了全脂乳中培养的乳酸菌HPLC上样菌悬液的制备方法。对全脂乳中培养不同时间的嗜热链球菌及保加利亚乳杆菌进行色谱表征,并首次发现保加利亚乳杆菌在全脂乳培养过程中菌体形态变化与色谱表征的一致性。     

Streptococcus thermophilus strains of plant origin as dairy starters: Isolation and characterisation

Streptococcus thermophilus strains have been isolated mainly from dairy environments. To prospect for new strains of S. thermophilus, isolation was made from different plant sources. In this study, 74 plant isolates were characterised as S. thermophilus by a polyphasic approach and by 16S rRNA sequencing. The isolates were further evaluated for their physiological and biochemical properties. Plant isolates exhibited good acid production and varying proteolytic activity. All the isolates showed acetaldehyde and capsular exopolysaccharide (EPS) production and few revealed diacetyl production. In contrast to industrial strains, six isolates were able to ferment galactose and 25 were found to have no urease activity. Both the plant isolates and reference dairy cultures were found to possess similar physiological and biochemical properties and can be considered for developing new starters.     

青花椒精油对致龋菌的体外抑菌活性

为研究青花椒精油（Zanthoxylum schinifolium Siebold & Zucc. essential oils,ZSEOs）对变形链球菌、血链球菌和远缘链球菌的体外抑菌活性,采用水蒸气蒸馏法从重庆、贵州、四川、云南4 个产地的青花椒果皮中提取ZSEOs,分别记为ZS1EO、ZS2EO、ZS3EO、ZS4EO。通过全二维气相色谱-飞行时间质谱分析不同ZSEOs的化学组成,同时测定抑菌圈直径、最小抑菌质量浓度（minimum inhibitory concentration,MIC）、最小杀菌质量浓度（minimum bactericidal concentration,MBC）、致龋菌生长曲线评估ZSEOs的抑菌活性,并通过扫描电子显微镜观察ZSEOs处理后细菌的形态变化。结果表明,芳樟醇（18.86%～22.00%）、柠檬烯（9.56%～14.84%）、β-月桂烯（5.79%～12.70%）、桧烯（5.49%～8.93%）和β-蒎烯（3.33%～6.41%）在4 个产地ZSEOs中的含量均普遍较高。ZSEOs对3 种致龋菌均有一定的抑制效果,其中ZS1EO抑菌活性最强,对3 种致龋菌的MIC和MBC范围分别为0.5～1.0 mg/mL和1～2 mg/mL。在致龋菌生长的延迟期和对数生长期加入MIC ZS1EO会导致细菌停止增殖。ZS1EO的抑菌活性与细菌链状结构的破坏和细菌细胞膜的损伤有关。综上,ZSEOs可作为植物来源的潜在龋病抗菌剂。     

乳酸菌微囊化的初步研究

运用喷雾干燥方法对保加利亚乳杆菌，嗜热链球菌的微囊化进行了初步研究，结果表明，微胶囊化对菌体活性的保护具有良好的效果，其中各组壁材所得的最佳结果，其存活率均达５０％以上，与未微囊化样品相比，微囊铧对菌全活性的保持具有明显的效果。     

嗜热链球菌胞外多糖的结构生物合成与遗传调控

介绍了嗜热链球菌胞外多糖的组成结构、生物合成、代谢途径和针对途径中关键酶的遗传调控,为胞外多糖研究提供参考.     

Strain-specific pathogenicity of putative host-adapted and nonadapted strains of Streptococcus uberis in dairy cattle

Streptococcus uberis is an important cause of intramammary infection in dairy cattle. Strains of Strep. uberis appear to differ in their ability to cause disease based on previous epidemiological studies. We explored the pathogenicity of 2 strains of Strep. uberis, where one strain represented a putatively host-adapted type based on its ability to cause persistent infection and to spread from cow to cow in a lactating herd. This type was part of a clonal complex that is commonly associated with bovine mastitis. The other strain, which was isolated from a transient infection in a single animal in the same herd and did not belong to any known clonal complex, was selected as putatively nonadapted type. Cows (6 per strain) were experimentally challenged in a single hind quarter and the adjacent hind quarter was used as mock challenged control quarter. Both strains showed an equal ability to grow in the milk of challenge animals in vitro. All cows that were challenged with the putatively host-adapted strain developed clinical signs of mastitis, including fever and milk yield depression as well as elevated somatic cell count due to influx of polymorphonuclear leucocytes and lymphocytes. The cytokine response followed a specific order, with an increase in IL-1β, IL-6, and IL-8 levels at the time of first SCC elevation, followed by an increase in IL-10, IL-12p40, and tumor necrosis factor-α levels approximately 6 h later. In 4 of 6 animals, IL-17A was detected in milk between 57 and 168 h postchallenge. The increase in IL-17A levels coincided with inversion of the prechallenge CD4⁺-to-CD8⁺ T lymphocyte ratio, which was observed from 96 h postchallenge. This was followed by normalization of the CD4⁺-to-CD8⁺ ratio due to continued increase of the CD8⁺ concentration up to 312 h postchallenge. Spontaneous resolution of infection was observed in 5 animals and coincided with a measurable IL-17A response in 4 animals, suggesting that IL-17 may be involved in the resolution of intramammary infection. With the exception of minor elevation of IL-8 levels, no clinical, cytological, or immunological response was detected in quarters challenged with the nonadapted strain. The observed strain-specific pathogenicity was consistent across animals, implying that it is determined by pathogen factors rather than host factors.     

Prevalence and comparison of Streptococcus infantarius subsp. infantarius and Streptococcus gallolyticus subsp. macedonicus in raw and fermented dairy products from East and West Africa

Streptococcus infantarius subsp. infantarius (Sii) and Streptococcus gallolyticus subsp. macedonicus are members of the Streptococcus bovis/Streptococcus equinus complex (SBSEC) associated with human infections. SBSEC-related endocarditis was furthermore associated with rural residency in Southern Europe. SBSEC members are increasingly isolated as predominant species from fermented dairy products in Europe, Asia and Africa. African variants of Sii displayed dairy adaptations to lactose metabolism paralleling those of Streptococcus thermophilus including genome decay. In this study, the aim was to assess the prevalence of Sii and possibly other SBSEC members in dairy products of East and West Africa in order to identify their habitat, estimate their importance in dairy fermentation processes and determine geographic areas affected by this potential health risk. Presumptive SBSEC members were isolated on semi-selective M17 and SM agar media. Subsequent genotypic identification of isolates was based on rep-PCR fingerprinting and SBSEC-specific16S rRNA gene PCR assay. Detailed identification was achieved through application of novel primers enhancing the binding stringency in partial groES/groEL gene amplification and subsequent DNA sequencing. The presence of S. thermophilus-like lacS and lacZ genes in the SBSEC isolates was determined to elucidate the prevalence of this dairy adaptation. Isolates (n = 754) were obtained from 72 raw and 95 fermented milk samples from Côte d'Ivoire and Kenya on semi-selective agar media. Colonies of Sii were not detected from raw milk despite high microbial titers of approximately 10⁶ CFU/mL on M17 agar medium. However, after spontaneous milk fermentation Sii was genotypically identified in 94.1% of Kenyan samples and 60.8% of Kenyan isolates. Sii prevalence in Côte d'Ivoire displayed seasonal variations in samples from 32.3% (June) to 40.0% (Dec/Jan) and isolates from 20.5% (June) to 27.7% (Dec/Jan) present at titers of 10⁶–10⁸ CFU/mL. lacS and lacZ genes were detected in all Kenyan and 25.8% (June) to 65.4% (Dec/Jan) of Ivorian Sii isolates. Regional differences in prevalence of Sii and dairy adaptations were observed, but no clear effect of dairy animal, fermentation procedure and climate was revealed. Conclusively, the high prevalence of Sii in Kenya, Côte d'Ivoire in addition to Somalia, Sudan and Mali strongly indicates a pivotal role of Sii in traditional African dairy fermentations potentially paralleling that of typical western dairy species S. thermophilus. Putative health risks associated with the consumption of high amounts of live Sii and potential different degrees of evolutionary adaptation or ecological colonization require further epidemiologic and genomic investigations, particularly in Africa.     

溶血性链球菌LAMP检测方法的建立

目的：研究溶血性链球菌的环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测方法,实现对溶血性链球菌的快速检测。方法：针对溶血性链球菌的scpA 基因设计4 条特异性LAMP 内外引物进行LAMP扩增;优化扩增反应条件;采用包括溶血性链球菌在内的6 种不同菌株进行LAMP 的特异性检测,并酶切鉴定;对溶血性链球菌菌液以无菌水进行10 倍系列梯度稀释后,进行LAMP 扩增检测其灵敏度;将菌掺入牛奶样本并进行LAMP 检测。结果：本法可快速灵敏地检测出溶血性链球菌,反应特异性高,检出限为16.7CFU/mL,而牛奶样本的检出限则达10CFU/mL。结论：LAMP 法可用于食品溶血性链球菌的快速检测。     

Simultaneous detection of mastitis pathogens, Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae by multiplex real-time polymerase chain reaction

The objective of this study was to develop a multiplex real-time polymerase chain reaction (PCR) method for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus uberis directly from milk. A genetic marker specific for Staph. aureus was used for primers and dual-labeled probe design. The target for Strep. agalactiae primers and dual-labeled probe was selected from the cfb gene encoding the Christie-Atkins-Munch-Petersen factor. The plasminogen activator gene was the target for primers and dual-labeled probe design for Strep. uberis. Quarter milk samples (n = 192) were analyzed by the multiplex real-time PCR assay and conventional microbiological methods. An additional 57 quarter milk samples were analyzed in a separate real-time PCR assay for Strep. agalactiae only. Using an overnight enrichment step, the real-time PCR technique correctly identified 96.4% of all quarter milk samples; 91.7% of Staph. aureus, 98.2% of Strep. agalactiae, and 100% of Strep. uberis. Results of conventional microbiological methods were used to determine the sensitivity and specificity of the multiplex real-time PCR procedure. The sensitivity of the procedure to correctly identify Staph. aureus, Strep. agalactiae, and Strep. uberis directly from milk was 95.5%, and the specificity was 99.6%. Results of this study indicate that the multiplex real-time PCR procedure has the potential to be a valuable diagnostic technique for simultaneous identification of Staph. aureus, Strep. agalactiae, and Strep. uberis directly from quarter milk samples.     

The influence of metallic surface wettability on bacterial adhesion

The wettability of AISI 304 stainless steel with 2B and 2RB surface finishes expressed in terms of the solid surface free energy was investigated with respect to the cleaning process. It was shown that cleaning affects the wettability of a solid surface. Depending on the cleaning method, ranged from 43.4 to 277.8 mJ m^-2 for the 2RB surface and from 34.4 to 122.8 mJ m ² for the 2B surface. There was no direct relationship between the number of adhering bacteria and or the wettability of solids. However, it was found that the adhesion of Streptococcus thermophilus was driven by a balance between and The experimental results are as expected based on thermodynamic predictions when the spreading pressure is accounted for in the surface free energy of bacteria, determination.