产胶原酶的琥珀葡萄球菌分离鉴定及其培养优化研究

针对胶原蛋白难消化利用的现状,自烟台近海分离筛选产胶原蛋白降解酶的微生物,获得高产菌株SM12,并经形态观察、生理生化试验和16S rDNA鉴定,确定菌株SM12为琥珀葡萄球菌（Staphylococcus succinus）。通过单因素试验确定最佳培养基成分及发酵条件为20 g/L葡萄糖,15 g/L牛肉膏,20 g/L NaCl,10 g/L明胶,初始pH 7.5,培养温度37 ℃,接种量7.5%（V/V）,装液量100 mL/500 mL。在此最佳条件下,筛选的菌株SM12在发酵24 h后获得最大胶原酶活力185.32 U/mL,具备在水产加工下脚料高值化加工领域的应用潜力。     

光皮木瓜绿原酸的提取及抗菌活性测定

为提高木瓜的利用价值,首次利用甲醇研究光皮木瓜中绿原酸的最佳提取工艺,在单因素试验基础上,通过正交试验设计考察浸提溶剂体积分数、浸提温度、浸提料液比和浸提时间对绿原酸提取的影响,并初步考察绿原酸样品液对志贺氏痢疾杆菌及金黄色葡萄球菌的抗菌活性。结果表明从木瓜中提取绿原酸的最佳工艺条件为：以85% 甲醇溶液(85:15,V/V)浸提木瓜粉、浸提料液比1:30(g/mL)、浸提温度40℃、浸提时间14h。在该工艺条件下,绿原酸的最高产率可达到0.142%。绿原酸提取液对志贺氏痢疾杆菌和金黄色葡萄球菌具有明显的抗菌活性,表明民间所用的木瓜白酒浸泡液的主要药理活性成分可能为绿原酸。     

Effect of lactoferrin in combination with penicillin on the morphology and the physiology of Staphylococcus aureus isolated from bovine mastitis

The objective of the present study was to evaluate the therapeutic potential of bovine lactoferrin or lactoferricin in combination with penicillin G against Staphylococcus aureus. Minimal inhibitory concentrations of lactoferrin, lactoferricin, penicillin, and combinations of lactoferrin or lactoferricin with penicillin were determined for 15 S. aureus strains including several strains resistant to beta-lactam antibiotics. The fractional inhibitory concentration index indicated a synergistic effect between lactoferrin and penicillin. Combination of lactoferrin with penicillin increased the inhibitory activity of penicillin by two- to fourfold and reduced the growth rate in S. aureus strains tested, whereas the increase in the inhibitory activity of lactoferrin by penicillin was 16- to 64-fold. The addition of iron to the medium containing a combination of penicillin and lactoferrin had no effect on growth inhibition. Electron microscopy revealed that concentration below the minimal inhibitory concentrations of penicillin induced important ultrastructure alterations, which were further enhanced by the presence of lactoferrin. When S. aureus cells were grown in the presence of a combination of penicillin and lactoferrin, changes in the protein profile of the bacteria, including the disappearance of several protein bands due to the presence of lactoferrin, were observed. These data suggest that bovine lactoferrin or lactoferricin in combination with beta-lactam antibiotics can increase the antibacterial activity of these antibiotics against S. aureus resistant to antibiotics.     

Microbiological study of semi-hard goat's milk cheese (Majorero)

The development of microbial flora in industrially produced semi-hard cheese made from pasteurized goat's milk was studied during manufacture and over a 90-day ripening period.
Estimates of total count, streptococci, lactobacilli, leuconostocs, coliforms, micrococci and staphylococci, Staphylococcus aureus and yeasts and moulds were carried out at various stages of the ripening process; streptococci and lactobacilli were identified by species.
Initially, the total count increased rapidly, primarily as a result of the growth of mesophilic lactic streptococci mainly Streptococcus lactis and Strep, cremoris. Subsequently, both these counts stabilized or decreased. Lactobacilli increased, and by the end of the ripening period were the predominant microorganisms. Most common were Lactobacillus casei var. casei. , especially at the end of storage; L. casei var. rhamnosus , L. casei var. plantarum and L. cellobiosus were also isolated. Leuconostocs were not found in any of the cheeses, and hence no eye formation took place. Coliforms, enterococci, yeasts and moulds remained below 10²–10³ c.f.u. g⁻¹. Maximum levels of micrococci and staphylococci were found after 15–30 days of ripening and decreased gradually towards the end of the ripening period. Neither the milk curd, nor cheese contained Staph. aureus.     

In vitro peripheral blood mononuclear cell proliferation in a crossbred cattle population

Immune function measured by Staphylococcus aureus- and phytohemagglutinin- (PHA-) induced cell proliferation was assessed in a population of 445 genetically defined, F2 and backcross Charolais-Holstein crossbred cattle when the animals were approximately 5 mo of age. Variation in Staph. aureus-induced, PHA-induced, and control proliferation [peripheral blood mononuclear cell (PBMC) and media only] was observed at d 2, 3, 4, 5, 9, and 10 of in vitro culture. The levels of Staph. aureus-induced, PHA-induced, and control proliferation were strongly positively correlated between days of culture within-assay (e.g., between d 2 and d 3 or between d 4 and d 5). Responses were also positively correlated when the same individuals were resampled and the assay repeated within 3 mo. Analyses fitting linear mixed models using REML showed that Staph. aureus-induced and PHA-induced proliferation was significantly associated with control proliferation and the year of birth. The age of the animal at sampling influenced only Staph. aureus-induced proliferation, with Staph. aureus-induced proliferation increasing with the age of the animal. Control proliferation was influenced by a sex × cross interaction, although in this study, sex was confounded by management, as female cattle were housed and reared differently from male cattle. All 3 measures of immune function were influenced by sire, demonstrating that these traits are partially under genetic control, and indicating that it may ultimately be possible to identify quantitative trait loci for these measures of immunity.     

Generation of volatile fatty acids by axillary bacteria

It is generally accepted that short-chain (C(2)-C(5)) volatile fatty acids (VFAs) are among the causal molecules of axillary malodour. It is also widely acknowledged that malodour generation is attributable to the biotransformation of odourless natural secretions, into volatile odorous products, by axillary bacteria. However, little information is available on the biochemical origins of VFAs on axillary skin. In these studies, assay systems were developed to investigate the generation of VFAs from substrates readily available to the bacteria resident on axillary skin. Propionibacteria and staphylococci were shown to ferment glycerol and lactic acid to the short-chain (C(2)-C(3)) VFAs, acetic and propionic acid. Furthermore, staphylococci are capable of converting branched aliphatic amino acids, such as leucine, to highly odorous short-chain (C(4)-C(5)) methyl-branched VFAs, such as isovaleric acid, which are traditionally associated with the acidic note of axillary malodour. However, in vitro kinetic data indicates that these pathways contribute less to axillary VFA levels, than fatty acid biotransformations by a recently defined sub-group of the Corynebacterium genus, corynebacteria (A). The results of these studies provide new understanding on the biochemical origins of VFA-based axillary malodour which, in turn, should lead to the development of novel deodorant systems.     

中草药祛痘复方对痤疮致病菌和外泌酶的影响

通过药敏试验和抑菌试验考察中草药祛痘复方对痤疮丙酸杆菌(Propionibacterium acnes,PA)、金黄色葡萄球菌(Staphylococcus aureus,SA)和表皮葡萄球菌(Staphylococcus epidermidis,SE)的抑菌功效,同时考察其对3种菌的抑菌时间动力学的影响。采用脂肪酶和蛋白酶水解底物形成的透明圈,研究中草药祛痘复方对PA释放脂肪酶和蛋白酶的影响。结果表明,中草药祛痘复方明显抑制了PA、SA和SE,并呈时间和剂量依赖性关系;中草药祛痘复方抑制脂肪酶和蛋白酶的作用主要是通过抑制PA而发挥作用。     

羊奶粉生产中金黄色葡萄球菌分离鉴定及其分子特性和耐药性检测

为探究羊奶粉在加工过程中金黄色葡萄球菌污染的关键环节及菌株的分子特征和耐药性,对某羊奶粉加工厂不同生产环节进行样本收集,通过采用选择培养和聚合酶链式反应扩增nuc基因对金黄色葡萄球菌进行分离鉴定,然后对分离的菌株进行毒素基因（21 种）、耐药性（16 种常用抗生素）及葡萄球菌A蛋白（staphylococcal protein A,SPA）、多位点序列分型（multilocus sequence typing,MLST）和脉冲场凝胶电泳（pulsed field gel electrophpresis,PFGE）分型研究。结果表明,收集的112 份样本中有6 份样本被污染（5.4%,6/112）,其中包括加工设备（2 份）、加工人员（2 份）、罐奶（1 份）和车间落地粉（1 份）。所有的分离株至少携带1 种毒素基因,其中以pvl基因的携带率最高（100.0%,6/6）,其次为sea、sec、see、seh、sek和seq（50.0%,3/6）,seg、sej和ser（33.3%,2/6）,sed、sei、sem和seo（16.7%,1/6）。所有分离株至少耐受4 种抗生素,其中对氨苄西林、头孢哌酮、甲氧苄啶/磺胺甲恶唑和青霉素的耐药性最为普遍（100.0%,6/6）,其次为红霉素和阿莫西林/克拉维酸（83.3%,5/6）、四环素（50.0%,3/6）和庆大霉素（16.7%,1/6）。此外,所有菌株对苯唑西林、头孢西丁、万古霉素、利奈唑胺等抗生素敏感。所有分离株共有4 种spa分型和3 种ST分型,其中以ST1-t127（50.0%,3/6）为主,其次为ST5-t002、ST5-t548和ST188-t189（16.7%,1/6）。对于PFGE分型,可分为3 个大簇（I、II和III簇）和4 个基因型（A、B、C和D）,其中加工设备和落地粉存在相同的PFGE分型。研究结果表明在羊奶粉加工过程中存在金黄色葡萄球菌污染现象,罐奶、加工设备、加工人员和落地粉是受污染的关键环节,且在不同的加工环节中存在一定的交叉污染。虽然污染率较低,有必要对奶厂不同加工环节的污染情况进行长期调查,确认关键污染环节,可以有效控制金黄色葡萄球菌在奶粉制品中的扩散。     

工业大麻叶对金黄色葡萄球菌的抑菌机制

探索工业大麻叶的抑菌组成及对金黄色葡萄球菌的抑菌机制,为以工业大麻叶成分为先导化合物开发新型天然防腐剂提供理论依据。工业大麻叶乙醇提取物经乙酸乙酯萃取、硅胶柱分离,采用微量二倍稀释法确定最低抑菌浓度(MIC),获得高活性组分,GC-MS分析抑菌组成;研究作用前后菌体细胞壁完整性、细胞膜通透性、能量代谢和氧化损伤变化,探讨抑菌机制。结果表明,工业大麻叶Fr5(石油醚和乙酸乙酯的体积比为1∶1)组分抑菌效果最佳,对金黄色葡萄球菌的MIC为31.25μg/mL,GC-MS共鉴定出24种化合物;经Fr5处理后,细菌表面明显凹陷,胞外碱性磷酸酶(AKP)活力升高,培养液电导率升高,核酸相对浓度增加,可溶性蛋白质质量浓度增大,菌体三磷酸腺苷(ATP)浓度和超氧化物歧化酶(SOD)活性均先升后降。工业大麻叶Fr5可破坏金黄色葡萄球菌细胞壁和细胞膜结构,导致细胞膜通透性增加,内容物外泄,进而影响菌体能量代谢并造成质膜氧化损伤,最终抑制细胞生长。