肉桂醛对荧光假单胞菌生物膜形成的抑制作用

本文拟研究肉桂醛在非抑菌浓度条件下对荧光假单胞菌(Pseudomonas fluorescens)FML05-2生物膜形成的抑制作用。首先测定了肉桂醛对模式细菌紫色杆菌(Chromobacterium violaceum)CVO26紫色素产生的影响;再对荧光假单胞菌FML05-2形成的生物膜以及与生物膜形成有关的重要因素胞外多糖进行了测定。结果表明:在非抑菌浓度40、20μg/m L条件下,肉桂醛对紫色杆菌CVO26紫色素的产生具有抑制作用,抑制率分别为31.53%、17.90%;对荧光假单胞菌FML05-2生物膜形成的抑制率分别为44.22%、21.77%;对荧光假单胞菌FML05-2胞外多糖产生的抑制率分别为15.72%、5.34%。因此,肉桂醛在非抑菌浓度条件下对荧光假单胞菌FML05-2生物膜的形成具有抑制作用。     

基于群体感应研究丹皮提取物对嗜水气单胞菌致腐性的抑制作用

首先利用报告菌株紫色杆菌CV026检测模型,确定丹皮提取物具有群体感应抑制活性,然后以从腐败大菱鲆中分离得到的嗜水气单胞菌(Ah-11)为测试对象,以信号分子产生、生物被膜形成、嗜铁素产生、蛋白水解活性以及细菌迁移(群集和泳动)为评价指标,研究丹皮提取物对Ah-11群体感应的抑制作用,并添加外源AHLs分析Ah-11致腐性与其QS系统之间的联系。结果表明,AHLs能够调控Ah-11致腐因子的产生;能够减少Ah-11信号分子的产生,而不影响嗜水气单胞菌的生长速度;能够显著减少其生物被膜的形成量(P0.05),破坏生物被膜的结构,降低其蛋白水解活性(P0.05),抑制其迁移能力(群集和泳动),且抑制作用具有质量浓度依赖性。当丹皮提取物质量浓度为1 mg/m L时,对生物被膜、蛋白水解活性、群集现象和泳动的抑制率分别达到65.47%,43.46%,26.94%和51.68%;嗜铁素试验表明丹皮提取物可能含有能够螯合铁的化合物。     

绿原酸对荧光假单胞菌群体感应现象及其腐败特性的抑制作用

首先以群体感应报告菌株紫色杆菌CV026检测模型,确定绿原酸的群体感应抑制活性,结合HPLC-MS/MS检测其对荧光假单胞菌释放信号分子的影响。以胞外酶活性、嗜铁素、群集与泳动为评价指标,通过添加外源信号分子分析绿原酸对荧光假单胞菌群体感应活性及腐败特性的调控作用。结果表明:在不影响荧光假单胞菌正常生长的剂量下,绿原酸可以减少其信号分子的产生,明显抑制荧光假单胞菌嗜铁素的产生、胞外蛋白酶、胞外脂肪酶活性、群集与泳动等相关腐败特性,且随着绿原酸浓度的升高,抑制效果更趋明显。因此,绿原酸具有较好的群体感应抑制活性,该研究为绿原酸作为群体感应抑制剂的开发提供了理论支持。     

群体感应AHLs对温和气单胞菌体外致腐因子分泌的影响

以大菱鲆腐败菌温和气单胞菌(Aeromonas sobria,AS7)为研究对象,研究了不同处理方式(营养基质、共生培养和添加不同种类的AHLs)对温和气单胞菌的致腐因子(嗜铁素、胞外蛋白酶、生物被膜)产生情况的影响,结果表明,菌株AS7嗜铁素的分泌受到培养基质的影响,在脱脂牛奶等天然基质中可以大量分泌嗜铁素,而市售的合成培养基中不能分泌嗜铁素;AHLs的添加对嗜铁素的产生量有影响,其中,C8-HSL影响显著,随添加的增加,嗜铁素分泌量呈现正相关(P0.05),而C6-HSL影响不显著(P0.05);共生条件下,温和气单胞菌对荧光假单胞菌和腐败希瓦氏菌嗜铁素的分泌有明显促进作用;AHLs的添加可以促进温和气单胞菌胞外蛋白酶和生物被膜的产量,且呈现正相关(P0.05),说明AHLs对温和气单胞菌产生胞外蛋白酶、嗜铁素和生物被膜具有调控作用。     

传统发酵蔬菜中抑制嗜水气单胞菌群体感应及生物膜形成乳酸菌的筛选与鉴定

目的:从来源于传统发酵蔬菜的乳酸菌中筛选对嗜水气单胞菌群体感应及生物膜形成有抑制作用的菌株。方法:采用牛津杯打孔法筛选乳酸菌菌株,测定其对N酰基高丝氨酸内酯(AHLs)信号分子的降解率。利用96孔板法测定其对生物膜的抑制率,应用光镜和扫描电镜观察其对生物膜形成的影响。最终通过生理生化试验和16S rRNA序列分析鉴定乳酸菌菌株。结果:分离自安徽绩溪酸豆角的菌株AJS2-4对嗜水气单胞菌群体感应有抑制作用,当其代谢产物粗提物质量浓度为4 mg/mL时,对嗜水气单胞菌AHLs的降解率为49.36%,对其生物膜抑制率为32.25%;当其质量浓度达到8 mg/mL时,可使嗜水气单胞菌的AHLs完全降解。该粗提物经121℃处理30 min活性基本不变,蛋白酶处理使其活性完全丧失,可初步判定菌株AJS2-4粗提物中的抑制群体感应活性成分为蛋白类物质,且具有热稳定性。光镜结果显示:菌株AJS2-4粗提物可有效抑制嗜水气单胞菌生物膜的合成。扫描电镜结果显示:菌株AJS2-4粗提物不仅能有效降低其生物膜的生成量,还可使其结构破裂变得疏松。经生理生化和16S r RNA鉴定菌株AJS2-4为植物乳杆菌(Lactobacillus plantarum)。结论:从安徽绩溪酸豆角中获得1株抑制嗜水气单胞菌群体感应和生物膜形成的植物乳杆菌AJS2-4。     

滤纸片法筛选抑制嗜水气单胞菌群体感应的乳酸菌及抑制作用分析

采用滤纸片法从传统发酵蔬菜中筛选抑制嗜水气单胞菌群体感应的乳酸菌菌株,应用96?孔板法测定其对生物膜的抑制率,光学显微镜和扫描电镜观察其对嗜水气单胞菌生物膜形成的影响,同时以蛋白酶、嗜铁素、群集及泳动为指标研究其对嗜水气单胞菌毒力作用的影响。结果表明：来源于辽宁锦州酸菜的乳酸菌菌株SCT-2对嗜水气单胞菌群体感应有明显抑制作用,当其代谢产物粗提物质量浓度为8?mg/mL时,对嗜水气单胞菌生物膜抑制率为45.16%,光学显微镜下显示菌株SCT-2粗提物可有效抑制嗜水气单胞菌生物膜的形成,扫描电镜结果进一步表明菌株SCT-2粗提物不仅降低了嗜水气单胞菌生物膜的生成量,而且使其生物膜断裂。8?mg/mL的SCT-2粗提物可使嗜水气单胞菌蛋白酶和嗜铁素的分泌量分别减少27.18%和22.11%,对信号分子的降解率为32.27%,且对嗜水气单胞菌的群集和泳动现象抑制明显。经生理生化和16S?rRNA鉴定菌株SCT-2为植物乳杆菌（Lactobacillus plantarum）。从传统东北酸菜中获得对嗜水气单胞菌群体感应有抑制作用的植物乳杆菌SCT-2,为开发一种抑制嗜水气单胞菌群体感应的乳酸菌生物制剂提供了理论支持。     

传统腌渍蔬菜中抑制嗜水气单胞菌群体感应及生物膜形成乳酸菌的筛选

从传统腌渍蔬菜分离的乳酸菌中筛选对嗜水气单胞菌群体感应及生物膜形成具有抑制作用的菌株。采用牛津杯打孔法从21株乳酸菌中筛选出具有抑制活性的6株乳酸菌,其中分离自辽宁大连酸黄瓜的菌株DLH513效果较好。当菌株DLH513粗提物质量浓度为16.0 mg/mL时,对嗜水气单胞菌AHLs信号分子降解率为51.1%,对其生物膜抑制率为40.9%;当粗提物质量浓度为20.0 mg/mL时,可使其AHLs信号分子完全降解。应用不同蛋白酶处理菌株DLH513粗提物后,对嗜水气单胞菌群体感应无抑制作用,表明其粗提物具有蛋白特性;在40～121℃处理30 min,其粗提物仍具有抑制群体感应活性,表明其粗提物具有耐热特征;在pH 3.0～4.5范围,菌株DLH513粗提物表现出抑制群体感应活性,在酸性条件下较稳定。光镜和扫描电镜结果表明,菌株DLH513粗提物对嗜水气单胞菌生物膜形成有显著的破坏作用。经生理生化反应和16S r RNA鉴定菌株DLH513为植物乳杆菌。研究表明植物乳杆菌DLH513可作为革兰氏阴性菌群体感应的抑制剂,以期为研发一种新的用于控制引起食品腐败和食源性疾病的革兰氏阴性菌群体感应的抑制剂提供理论基础。     

一株产抗真菌活性物质海洋细菌的鉴定和系统发育分析

从中国南海海域海底淤泥中筛选得到一株产抗真菌活性物质的菌株JIV-49,该菌产生的活性物质对白色念珠菌、新型隐球菌、烟曲霉、黑曲霉、红色毛癣菌和须癣毛癣菌等多种真菌有明显抑制作用。对JIV-49菌株进行了形态、生理生化鉴定及16S rRNA基因序列分析。菌株JIV-49为短杆状、革兰氏染色阴性、不形成芽孢、有单生极鞭毛、无荚膜、不积累PHB作为胞内贮存物、菌体大小为0.4-0.6μm×1.0-1.4μm,无精氨酸双水解酶系统、生长需要海水。根据生理生化特征及16S rRNA基因序列分析结果,JIV-49被鉴定为假交替单胞菌,其生理生化特征与Pseudoalteromonas ruthenica的性状较相似却又不尽相同,因此可能是P.ruthenica的新菌株或变种。     

luxR基因对希瓦氏菌群体感应特性的影响研究

希瓦氏菌(Shewanella baltica)是冷藏水产品常见的优势腐败菌,可通过LuxR受体系统感受酰基高丝氨酸内酯(AHLs)和二酮哌嗪类化合物(DKPs)信号分子,从而调控水产品的腐败进程。通过构建希瓦氏菌LuxR基因不同片段缺陷菌株,对比研究希瓦氏菌野生型与LuxR基因缺陷型菌株生长、生物膜形成、蛋白酶产生、产硫能力等群体感应特性的差异,揭示希瓦氏菌的致腐机制。结果表明,希瓦氏菌LuxR基因缺陷菌株生长、产蛋白酶能力及生物膜形成能力均降低,部分缺陷菌株群集运动能力和产硫能力也同步降低。希瓦氏菌群体感应相关的大部分性状受LuxR基因影响,且不同LuxR基因片的片段所控制的群体感性状不同。研究结果为LuxR系统作为希瓦氏菌控制的新靶点提供了理论依据与技术支撑。     

鱼源荧光假单胞菌生物被膜形成和致腐表型的研究

荧光假单胞菌是养殖鱼类低温贮藏中的优势腐败菌。本研究比较分析5种荧光假单胞菌的生物被膜形成和腐败表型。采用结晶紫法、苯酚硫酸法、珠涡流法和荧光显微镜观察荧光假单胞菌的生物被膜形成和粘附能力,并测定细菌泳动性、蛋白酶活性、嗜铁素等致腐表型。结果表明,5株荧光假单胞菌在28℃LB肉汤中生长良好,经24 h培养后气-液界面上出现较厚的膜,在微孔板中生物被膜形成较快,其中鱼源PF01、PF06、PF07和PF10分离株在12 h含量最高,而标准菌株PFuk4在18 h最高。随着培养时间延长,细菌胞外多糖的含量逐步积累,在18～24 h达到最高,并较快粘附到不锈钢片表面,其中PF07的粘附量最高。5株荧光假单胞菌还具有较强的泳动性和蛋白酶活性,且均产嗜铁素。在PF07和PFuk4中还检测出短链高丝氨酸内酯(AHLs)活性,可能与其较高的被膜、粘附能力、泳动性及蛋白酶活性有关。本研究结果为从AHLs角度探究荧光假单胞菌的致腐机理打下良好的基础。     

大菱鲆荧光假单胞菌的群体感应现象及不同碳源培养下的腐败特性研究

本文以大菱鲆分离的荧光假单胞菌作为研究对象,紫色杆菌CVO26平行划线法检测信号分子。不同碳源(葡萄糖、蔗糖、果糖、木糖、乳糖、麦芽糖)的AB培养基培养并检测其生物被膜、嗜铁素和胞外蛋白酶的产生量,同时添加外源的信号分子标准品,研究AHLs与腐败因子之间的相关性。研究表明:大菱鲆分离的荧光假单胞菌能够发生群体感应现象,经不同碳源培养,其腐败因子产生情况有明显差异,碳源的添加对生物被膜的产生有促进作用,对胞外蛋白酶的产生没有显著影响;以葡萄糖、麦芽糖为碳源,生物被膜的产生量较高;以蔗糖和乳糖为碳源,嗜铁素的产量较高。添加外源信号分子标准品,生物被膜、嗜铁素和胞外蛋白酶的产生量有显著提高。因此,生物被膜、嗜铁素和胞外蛋白酶的产生与AHLs有关,群体感应现象可调控腐败特性的表达,并在水产品腐败过程中发挥作用。     

Molecular Characterization and Identification of Bioactive Peptides Producing Lactobacillus sps. Based on 16S rRNA Gene Sequencing

In the present study, 3 bacterial cultures were isolated from faecal samples of human infant. The biochemical traits showed similarity with Lactobacillus sps and 16S rRNA sequence analyses, confirmed as Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus rhamnosus. The cultures were screened for their proteolytic activity and good ability to release peptides from milk proteins was found. Hence, these bacteria were used as a proteolytic starter culture for the fermentation of skim milk and whey for the liberation of small peptides. Bioactive nature of the peptides released from whey and skim milk was tested, and results demonstrated that peptides obtained after fermentation of whey and skim milk by Lactobacillus strains showed antimicrobial activity against all the pathogens causing food borne infections in humans. These peptides also indicated antioxidant as well as ACE (angiotensin-converting enzymes) inhibitory activity.     

Spoilage patterns of skim and whole milks

The reason for the reported difference in spoilage behaviour of skim and whole pasteurised milks was investigated. The rates of growth of psychrotrophic bacteria were not significantly different in the two milks and the bacterial types, all pseudomonads, present at spoilage were also similar. However, when representative spoilage organisms were cultured into freshly pasteurised skim and whole milks, skim milks exhibited predominantly bitter flavours while whole milk showed mostly sour flavours. The different spoilage behaviours can be largely explained by greater proteolvsis in skim milk than in whole milk, caused by higher production of protease and greater susceptibility of the protein to protease attack. In addition, lipolysis in whole milk, caused by the substantial quantities of lipase produced by spoilage pseudomonads in this milk, also contributes to the different flavours produced during cold storage of these milk types.     

Biofilm Formation Characteristics of Pseudomonas lundensis Isolated from Meat

Biofilms formations of spoilage and pathogenic bacteria on food or food contact surfaces have attracted increasing attention. These events may lead to a higher risk of food spoilage and foodborne disease transmission. While Pseudomonas lundensis is one of the most important bacteria that cause spoilage in chilled meat, its capability for biofilm formation has been seldom reported. Here, we investigated biofilm formation characteristics of P. lundensis mainly by using crystal violet staining, and confocal laser scanning microscopy (CLSM). The swarming and swimming motility, biofilm formation in different temperatures (30, 10, and 4 °C) and the protease activity of the target strain were also assessed. The results showed that P. lundensis showed a typical surface‐associated motility and was quite capable of forming biofilms in different temperatures (30, 10, and 4 °C). The strain began to adhere to the contact surfaces and form biofilms early in the 4 to 6 h. The biofilms began to be formed in massive amounts after 12 h at 30 °C, and the extracellular polysaccharides increased as the biofilm structure developed. Compared with at 30 °C, more biofilms were formed at 4 and 10 °C even by a low bacterial density. The protease activity in the biofilm was significantly correlated with the biofilm formation. Moreover, the protease activity in biofilm was significantly higher than that of the corresponding planktonic cultures after cultured 12 h at 30 °C.     

Occurrence of proteolytic activity and N-acyl-homoserine lactone signals in the spoilage of aerobically chill-stored proteinaceous raw foods

Proteolytic pseudomonads dominate the spoilage flora of aerobically chill-stored proteinaceous raw foods. Proteolysis during spoilage of these food systems affects both food quality and the dynamics of the bacterial community because it increases the availability of nutrients to the community as a whole. Quorum sensing, or cell-cell signaling, is associated closely with ecological interactions among bacteria in mixed communities. The potential role of quorum sensing in proteolytic food spoilage was examined, based on the evaluation of N-acyl-homoserine lactone (AHL) signal molecules. The occurrence of proteolytic activity and AHL signals was studied during spoilage of aerobically chill-stored ground beef, fish, chicken, and raw milk. Pseudomonads dominated the psychrotrophic flora, followed distantly by members of the Enterobacteriaceae. The growth of pseudomonads was correlated with the occurrence of proteolytic activity in all food systems. AHL concentration began increasing significantly only after the onset of proteolytic activity. Widely divergent AHL profiles were revealed by thin-layer chromatography analysis of the different food samples, and these profiles were likely determined by the undefined bacterial flora in these systems and by the characterized pseudomonads and Enterobacteriaceae. Although Hafnia alvei was a major component of the Enterobacteriaceae flora in all foods tested and a strong AHL producer, the signal molecules produced by H. alvei strain EB1 did not influence protease production by Pseudomonas fluorescens strain 395 in vitro. These results do not indicate any clear correlation between the overall detectable AHL signal molecules accumulated in the food samples and proteolytic activity.     

Productions and monomer compositions of exopolysaccharides by Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains isolated from traditional home-made yoghurts and raw milk

In this study, a total of forty‐five strains of lactobacilli and streptococci were determined exopolysaccharide (EPS) production in skim milk and Man Rogosa and Sharpe (MRS)/M17 medium, viscosity and proteolytic activity. The exopolysaccharide production by lactobacilli strains during growth in MRS medium was twenty‐one to 211 mg L^?1, while in skim milk was to thirty‐six to 315 mg L^?1. The EPS production by streptococci strains during growth in M17 medium was sixteen to 114 mg L^?1, while in skim milk was to twenty‐four to 140 mg L^?1. The EPS production of strains was lower in MRS/M17 medium than skim milk. Results showed that it was not clear correlation between the viscosity and EPS production of some strains. All strains were shown proteolytic activity. Positive correlations between exopolysaccharide production and proteolytic activity in skim milk were found some strains of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. These results indicated that the high exocellular protease‐producing strains can produce high EPS in skim milk. The monomer compositions of the EPSs formed by selected five strains were analysed. Mannose dominated (99–100%) on the EPS produced by L. delbrueckii subsp. bulgaricus and S. thermophilusstrains (except L. delbrueckii subsp. bulgaricus 22) in skim milk and MRS/M17 medium. Besides, the EPSs of strains in skim milk contained small amount of lactose.     

Quorum Sensing Inhibitory and Anti-Biofilm Activity of Essential Oils and Their in vivo Efficacy in Food Systems

The present study investigated the effect of the essential oils of Cuminium cymium, Murraya koenigii, Curcuma longa, Zingiber officinale, Myristica fragrans (mace and nutmeg), Trigonella foenum graceum, and Elettaria cardamomum on AHL-mediated quorum sensing and biofilm formation. Inhibition of quorum sensing (QS) regulated phenotypes by the essential oils at subinhibitory concentrations was investigated using the biosensor bacteria Chromobacterium violaceum (C. violaceum 12472 and C. violaceum CV026) and Pseudomonas aeruginosa PA01. Sub-MIC of essential oils was screened for inhibition of quorum-sensing regulated pigment production, motility and biofilm formation in the biosensors. The essential oil of M. koenigii showed the most promising QS inhibitory and anti-biofilm activity at a concentration of 0.02% v/v and hence was used for further studies. Anti-biofilm activity against food spoilage Pseudomonas species was studied by crystal-violet MTP method and fluorescence microscopy. The essential oil of M. koenigii at sub-MICs inhibited biofilm formation in the pseudomonads by inhibiting cell attachment, reducing metabolic activity and EPS production, and preventing biofilm maturation. Further, it was capable of delaying spoilage by psychrotrophic P. psychrophila PSPF19 in refrigerated milk. The major components present in M. koenigii essential oil were caryophyllene, caryophyllene oxide, cinnamaldehyde, α-, and β-phellandrene and eugenol. Application of M. koenigii essential oil and its constituents based on the quorum-sensing inhibitory and anti-biofilm activity would be a novel intervention strategy to enhance shelf life and food safety.     

Risk evaluation for staphylococcal food poisoning in processed milk produced with skim milk powder

The growth of S. aureus and the production of staphylococcal enterotoxin A (SEA) in skim milk concentrates stored at inappropriate temperatures in a recovery milk tank (tank for excess concentrated skim milk) used in the manufacture of skimmed milk powder were investigated. Also, it was estimated if a possible outbreak of food poisoning would occur if the contaminated skimmed milk powder was used in the manufacture of processed milk. Skim milk concentrates with milk solid content of 15, 25, and 35% were inoculated with S. aureus at 1-2 log CFU/ml and incubated at 15, 25, or 35 degrees C for 0 to 24 h with or without shaking. Bacterial growth and the level of SEA production were measured. At 35 degrees C with shaking, there was a significant difference (p<0.05) in one way layout analysis of variance, and it was demonstrated that the growth of S. aureus and SEA production could be milk solid content-dependent. Shaking accelerated the growth of S. aureus and SEA production at 35 degrees C. Generally, skim milk powder is produced by mixing a set percentage of skim milk concentrates (recovery milk) from the recovery milk tank into raw milk. If recovery milk contaminated with S. aureus at levels of 1-2 log CFU/ml is kept at 15 to 35 degrees C due to a power failure, it was estimated that processed milk consumption of 670-1200 ml, 420-1500 ml and 18-83 ml would trigger the onset of food poisoning symptoms when skim milk concentrates (recovery milk) are stored at 25 degrees C for 24 h, 35 degrees C for 10 h, and 35 degrees C for 24 h, respectively, during the production of the skim milk powder. Based on these consumption levels, it was concluded that, if recovery milk cannot be refrigerated and is stored at room temperature (25 to 35 degrees C), it must be used within 8 h and preferably within 6 h.     

Shelf life and quality of skim milk processed by cold microfiltration with a 1.4-μm pore size membrane,with or without heat treatment

The objective of this study was to evaluate the effectiveness of cold microfiltration (MF), alone or in combination with heat treatment, in extending the shelf life of skim milk. Raw skim milk underwent MF at 6 ± 1°C with a ceramic membrane of 1.4-μm pore size, at a transmembrane pressure of 75.8 kPa and a crossflow velocity of 7 m/s. Samples of raw skim milk; MF skim milk; high-temperature, short-time (HTST)-pasteurized milk; and MF+HTST-pasteurized skim milk were stored at 6°C for 92 d. During the shelf-life study, the total bacterial count and degree of proteolysis were evaluated weekly. The study was replicated 3 times. Cold MF was very effective in reducing the microbial load in skim milk, and an average of 3.4 log reduction in vegetative bacteria was obtained. Although HTST pasteurization reduced the bacterial load by ～2 log, the MF+HTST process resulted in near complete elimination of vegetative microflora, with a total of ～4 log reduction. A 9-member sensory panel found no significant differences between skim milk samples processed with or without MF. The MF+HTST skim milk had only minor microbial growth after 92 d at 6°C, but its proteolytic shelf life was limited by plasmin activity. A reduction of plasmin activity and a slower rate of proteolysis were obtained by increasing the heat treatment temperature to 85°C. The results of this study can be used to make decisions regarding processing strategies that lead to increased skim milk shelf life.