A novel bacteriocin against Staphylococcus aureus from Lactobacillus paracasei isolated from Yunnan traditional fermented yogurt: Purification,antibacterial characterization,and antibiofilm activity

Staphylococcus aureus and its biofilm have emerged as a significant threat to the safety of dairy products. In recent years, lactic acid bacteria (LAB) bacteriocins have been widely acknowledged as the potential natural antibacterial substance in food biopreservation due to their excellent antibacterial effects. However, few LAB bacteriocins with antibacterial and antibiofilm activity against S. aureus have been reported in dairy products. In the present study, a novel bacteriocin LSX01 of Lactobacillus paracasei LS-6 isolated from a traditional fermented yogurt produced in Yunnan, China, was purified and characterized extensively. The LSX01 possessed a molecular weight of 967.49 Da and an AA sequence of LDQAGISYT. The minimum inhibitory concentration of LSX01 against S. aureus_45 was 16.90 μg/mL, which was close to or lower than the previously reported bacteriocins. The LSX01 exhibited an extensive antimicrobial spectrum against both gram-positive and gram-negative bacteria. Moreover, LSX01 exhibited excellent tolerance to heat and acid-base treatments, and sensitivity to the proteolytic enzymes, such as pepsin and proteinase K. Furthermore, the treatment of S. aureus_45 planktonic cells with LSX01 significantly reduced their metabolic activity and disrupted the cell membrane integrity. Scan electron microscopy results demonstrated that LSX01 induced cytoplasmic content leakage and cell deformation. Additionally, biofilm formation of S. aureus_45 was also significantly inhibited by LSX01. Overall, the results suggested that the novel LAB bacteriocin LSX01 possessed antibacterial activity and antibiofilm activity against S. aureus and, hence, could have potential for improving safety of dairy products.     

Antibacterial activity and mechanism of a novel bacteriocin produced by Lactiplantibacillus plantarum against Escherichia coli and Staphylococcus aureus

The purpose of the study was to explore the antibacterial effect and mechanism of a novel bacteriocin of Lactiplantibacillus plantarum (L. plantarum) against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The antibacterial activity was determined by the Oxford cup method, and the dynamic growth curves were conducted with continuous shaking incubation. The result showed that the bacteriocin was a protein or protein-like antibacterial substance susceptible to pepsin and trypsin. And it had good antibacterial activity, pH stability, thermostability and enzyme treatment stability against E. coli and S. aureus. The SEM, flow cytometry and nucleic acid leakage showed that the bacteriocin disrupted the cell structures of the two bacteria by damaging cell walls and cell membranes. An agarose gel electrophoresis and SDS-PAGE analysis showed that the bacteriocin inhibited DNA replication and interfered with the protein formation, which resulted in the inhibition of the two bacteria's growth. Therefore, the use of the L. plantarum bacteriocin might be a promising biocontrol strategy to inhibit the pollution of E. coli and S. aureus simultaneously in foods.     

肉桂醛对大肠杆菌和金黄色葡萄球菌的抑菌作用及抑菌机理研究

研究了肉桂醛对大肠杆菌和金黄色葡萄球菌的抑菌作用及其抑菌机理。本文采用滤纸片扩散法测定抑菌圈大小,双倍稀释法测定最低抑菌浓度、最低杀菌浓度,以此评价肉桂醛对大肠杆菌和金黄色葡萄球菌的抑菌性,通过扫描电镜观察、胞膜通透性、胞膜完整性、膜电位实验,阐述肉桂醛抑菌的机理。结果表明:肉桂醛对大肠杆菌和金黄色葡萄球菌的抑菌圈分别为21.75 mm、29.37mm,最低抑菌浓度均为0.25μL/m L,最低杀菌浓度均为0.5μL/m L;肉桂醛破坏了菌体细胞的形态,出现形变;随着肉桂醛浓度的增大,悬液中相对电导率迅速升高,表明肉桂醛影响细菌的膜通透性,而成倍数增长的核酸、蛋白质含量表示胞膜的完整性遭到破坏,肉桂醛降低菌体膜电位,影响其代谢活性,从而抑制细菌生长。肉桂醛主要作用于细胞膜,适宜作为天然防腐剂,抑制食品中腐败菌和致病菌的生长,延长食品货架期。     

亚硝酸钠对金黄色葡萄球菌和副溶血性弧菌生物被膜形成的抑制作用

本文以金黄色葡萄球菌和副溶血性弧菌为研究对象,采用微孔板检测法,分别以OD630值与生物被膜清除率反映两种菌的生物被膜生成量及不同培养时间添加不同浓度亚硝酸钠对两种菌形成生物被膜的抑制效果,并通过细胞表面反应和细胞间聚集实验初步研究其抑制机理。结果表明:亚硝酸钠对两种致病菌生物被膜的形成均有一定的抑制作用,600 mg/L、培养48 h对金黄色葡萄球菌生物被膜清除率达37.31%,抑制效果最佳;而对副溶血性弧菌而言,添加200 mg/L亚硝酸钠培养12 h时清除率达36.67%,效果最佳。由细胞表面反应与细胞间聚集实验结果表明,亚硝酸钠能有效抑制两种菌的表面粘附和菌体聚集,其主要通过影响细胞聚合与表面粘附,从而抑制金黄色葡萄球菌和副溶血性弧菌生物被膜的形成与生长。      

戊糖乳杆菌素pentocin LPEM818的初步纯化及特性研究

对戊糖乳杆菌(Lactobacillus pentosus)LPEM818所产戊糖乳杆菌素(pentocin)LPEM818进行了初步纯化,并对其生物学特性进行了研究。结果表明,采用80%硫酸铵沉淀和Sephadex G-25凝胶层析分离纯化后,细菌素的纯化倍数为11.09倍,回收率为6.4%;该细菌素在pH 2.0～8.0条件下稳定,121℃加热15 min保留84.52%的抑菌活性;对胰蛋白酶和蛋白酶K敏感;该细菌素的作用方式为杀菌;对供试的部分革兰氏阳性菌(G+)和革兰氏阴性菌(G-)具有较强抑制作用,因其抑菌谱较广,对多数致病菌和食品腐败菌有较好抑菌作用,所以具有作为食品生物防腐剂的潜在应用价值。     

培养条件对金黄色葡萄球菌-大肠杆菌混合生物被膜的影响

以常见食源性致病菌金黄色葡萄球菌和大肠杆菌为研究对象,采用微孔板法对混合生物被膜形成过程中的不同影响因素进行了研究。结果表明：在25 ℃或30 ℃条件下,采用质量分数0.8%的胰酶大豆肉汤（TSB）或0.8%的脑心浸出液（BHI）培养基培养时,生物被膜形成量较高;添加适量的葡萄糖、乳糖、麦芽糖和蔗糖可提高混合生物被膜形成能力;培养基中NaCl质量分数＞8%时,混合生物被膜的生长明显受到抑制。     

Short communication: Antioxidative and antibacterial activities on Staphylococcus aureus and Escherichia coli O157:H4 in milk with added ginseng marc extract fermented by Lactobacillus plantarum KCCM 11613P

Ginseng marc, a by-product of the extraction of fresh ginseng, is known to have bioactive compounds, but is frequently discarded as agriculture waste. The objectives of our study were to assess the antioxidative activity of fermented ginseng marc extract using Lactobacillus plantarum KCCM 11613P and to evaluate antibacterial activity of fermented milk with added ginseng marc extract during fermentation. After 24 h of fermentation of ginseng marc extract, the viable cell number was increased to 7.7 ± 0.1 log cfu/mL, and the pH and total titratable acidity were 4.2 ± 0.4 and 0.6% lactic acid, respectively. The total phenolic and flavonoid contents of fermented ginseng marc extract increased by 32.4 and 23.3%, respectively. Higher antioxidative activity of fermented ginseng marc extract was obtained in the β-carotene bleaching, ferric-reducing ability of plasma, and ferric thiocyanate assays than the 1,1-diphenyl-2-picrylhydrazy assay. However, the 1,1-diphenyl-2-picrylhydrazy scavenging effect decreased due to lowered pH. During production of fermented milk with ginseng, inhibition rate of Staphylococcus aureus and Escherichia coli were 9.7 and 2.3%, respectively. The present study shows the possibilities of Lactobacillus plantarum KCCM 11613P used as a fermentation strain and ginseng marc used as a functional supplement in milk.     

食源性金黄色葡萄球菌生物膜形成调控与生物防控策略研究进展

生物膜是很多食源性致病菌应对各种极端环境、杀菌理化因子以及维持菌体内环境稳定的重要基质屏障。数据显示,有超过80%的细菌感染由生物膜引起,尤以金黄色葡萄球菌感染多见。食源性金黄色葡萄球菌是引发细菌性食物中毒和食品安全事故的重要风险源, 特别是多重耐药菌株。金黄色葡萄球菌的耐药性、致病性和免疫逃逸与生物膜复杂的三维结构有重大关系。由于生物膜结构基因和调控因子结构相对保守, 现已成为金黄色葡萄球菌生物防控新的重要的效应靶点。本文从多糖细胞间黏附素、胞外DNA和生物膜形成相关蛋白等角度阐明了生物膜形成机制,从群体感应系统（如Agr系统和LuxS/AI-2群体感应系统）、全局性调控因子（如附属调节因子Sar和转录因子SigB）以及双组分信号转导系统（如SrrAB系统、SaeRS系统、ArlSR系统、LytRS系统和WalKR系统）等角度系统阐述了生物膜形成调控机制。最后,从抗菌肽（蛋白）、植物源化合物、生物酶、抗菌药物等角度提出新的防控策略, 以期为食源性金黄色葡萄球菌的生物防控提供指导。     

Identification and molecular mechanism of action of antibacterial peptides from Flavourzyme-hydrolyzed yak casein against Staphylococcus aureus

Antibacterial peptides can be released from yak milk casein. To date, the amino acid sequences and mechanism of action of yak casein–derived antibacterial peptides remain unknown. The current study identified antibacterial peptides from yak casein and their molecular mechanism of action. Our results showed that yak α-casein, β-casein, and κ-casein could be effectively hydrolyzed by Flavourzyme (Solarbio Science and Technology Co. Ltd.), and the 2-h hydrolysate showed the highest antibacterial rate of 43.07 ± 2.59% against Staphylococcus aureus. The 1,000 to 3,000 Da fraction accounted for 23.61% of the 2-h hydrolysate and had an antibacterial rate of 62.64 ± 4.40%. Three novel peptides with antibacterial activity were identified from this fraction, and the β-casein–derived peptide APKHKEMPFPKYP showed the strongest antibacterial effect (half-maximal inhibitory concentration = 0.397 mg/mL). Molecular docking predicted that APKHKEMPFPKYP interacted with 2 important enzymes of Staph. aureus, dihydrofolate reductase and DNA gyrase, through hydrophobic, hydrogen bonding, salt bridge, and π-π stacking interactions. Our findings suggest that the yak casein–derived peptides may serve as a potential source of natural preservatives to inhibit Staph. aureus.     

Short communication: Activity of nisin,lipid bilayer fragments and cationic nisin-lipid nanoparticles against multidrug-resistant Staphylococcus spp. isolated from bovine mastitis

Staphylococci are the main etiological agents of bovine mastitis. Bacteriocins and nanoparticles have emerged as promising alternatives for the future development of antimicrobial agents. This study evaluated the activity of the bacteriocin nisin and bicelles of the synthetic cationic lipid dioctadecyldimethylammonium bromide, alone and in combination, against multidrug-resistant Staphylococcus spp. strains isolated from bovine mastitis. In summary, cationic nisin/dioctadecyldimethylammonium bromide nanoparticles are shown to be a promising alternative for the control of mastitis caused by multidrug-resistant Staphylococcus spp.     

1 株水开菲尔粒中植物乳杆菌的鉴定及产细菌素基因分析

目的：对从水开菲尔粒中分离得到1 株能产生抑菌物质的菌株QF01进行菌种鉴定,并对其产细菌素进行实验、鉴定和基因序列分析。方法：通过牛津杯法测定抑菌能力,采用聚合酶链式反应（polymerase chain reaction,PCR）扩增16S rDNA和细菌素相关基因并测序,利用ProParam tool、TMHMM 2.0、InterProScan、SOPM和SWISS-MODEL在线软件对基因编码产物进行分析。结果：该菌株产的细菌素对大肠杆菌（Escherichia coli JM109）有明显的抑制作用,通过16S rDNA序列分析初步确定该菌株属于植物乳杆菌（Lactobacillus plantarum）。该菌株含有plnD、plnEF、plnV、plnR四种基因,其中plnV基因编码产物有跨膜螺旋结构,其结构存在信号肽特征。此外,从三级结构的模型预测得到4?种基因的编码产物均存在α-螺旋、β-转角、伸展链和无规则卷曲。结论：从水开菲尔粒分离得到的L. plantarum QF01菌株,含有plnD、plnEF、plnV、plnR细菌素基因,对大肠杆菌有很好的抑制作用。     

Antibacterial activity and action target of phenyllactic acid against Staphylococcus aureus and its application in skim milk and cheese

Phenyllactic acid (PLA) has been demonstrated to possess antibacterial activity and capacity to prolong food shelf life. However, studies on the performance of PLA in inhibiting Staphylococcus aureus and its effectiveness when applied to dairy products are largely lacking. Here, antibacterial activity (planktonic and biofilm states) of PLA against S. aureus CICC10145 (S. aureus_45) were investigated. The results showed that PLA inhibited growth of S. aureus_45 and formation of S. aureus_45 biofilm. Next, the antibacterial action target of PLA was uncovered from both physiological and phenotypic perspectives. The results showed that PLA decreased cell metabolic activity and cell viability, damaged cell membrane integrity, triggered leakage of intracellular contents (DNA, proteins, and ATP), and caused oxidative stress damage and morphological deformation of S. aureus_45. In practical application, the antibacterial activity of PLA against S. aureus_45 cells was further confirmed in skim milk and cheese as dairy food models, and the antibacterial effects can be adequately maintained during storage for 21 d, at least at 4°C. These findings suggested that PLA could be a potential candidate for controlling S. aureus outgrowth in dairy foods.     

广东地区46株食源性金黄色葡萄球菌耐药性、生物被膜形成能力及IcaAB基因的特征分析

目的 分析广东地区食源性金黄色葡萄球菌耐药现状、生物被膜形成能力及生物被膜相关基因IcaAB的特征。方法 对46株食源性金黄色葡萄球菌采用微量肉汤稀释法进行药敏实验,采用结晶紫法检测生物被膜生成能力,运用聚合酶链式反应(polymerase chain reaction, PCR)检测生物被膜相关基因。结果 97.83%的菌株对至少1种抗生素耐药, 30.43%为多重耐药菌株,对青霉素(97.83%)、氨苄西林(97.83%)耐药性最高; 65.22%菌株能够形成生物被膜,其中强、中和弱粘附生物被膜能力的菌株分别占17.39%、30.43%和17.39%,且产生物被膜能力强的菌株对特定抗生素(头孢西丁、环丙沙星)的耐药性更高(P<0.05); PCR结果显示, icaA、icaB的检出率分别为56.67%、36.67%,基因携带率与产膜能力无显著关联(P>0.05)。结论 广东地区食源性金黄色葡萄球菌普遍表现出耐药性和生物被膜形成能力,对公共卫生带来挑战,需采用措施以确保公共安全。     

大蒜汁对耐甲氧西林金黄色葡萄球菌食品分离菌株的生长和生物被膜形成的影响

研究紫皮蒜和白皮蒜汁对耐甲氧西林金黄色葡萄球菌(MRSA)的生长和生物被膜形成的影响。以紫皮蒜和白皮蒜为原料制备大蒜汁;采用平板抑菌实验研究两种大蒜汁对7株MRSA食品分离菌株的抑菌效果;同时采用96孔板法研究两种大蒜汁对MRSA细菌生物被膜形成影响。结果表明:白皮蒜与紫皮蒜汁对于7株MRSA食品分离菌株均具有明显抑菌效果,抑菌圈大于19 mm,紫皮蒜汁效果优于白皮蒜汁,紫皮蒜汁的最低抑菌浓度为6.25 mL/L,白皮蒜汁的最低抑菌浓度为12.5 mL/L;两种大蒜汁在不同亚抑菌浓度下对MRSA的生物被膜形成均具有明显抑制作用。本研究结果为MRSA菌株的消除和防控措施提供参考。     

核桃青皮中胡桃醌对大肠杆菌的抗菌作用及机制

为研究胡桃醌对大肠杆菌的抗菌活性及其作用机理,用不同质量浓度胡桃醌处理大肠杆菌,分别对最小抑菌质量浓度（minimal inhibitory concentration,MIC）、生长曲线、细胞膜相对电导率、荧光发射光谱等进行测定,并进行生物膜形成和细胞活性分析、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulphate-polyacrylamide gel electrophoresis,SDS-PAGE）以及大肠杆菌基因组合成分析。结果表明,胡桃醌对大肠杆菌具有有效的抗菌活性,MIC为0.062 5 mg/mL。经不同质量浓度胡桃醌处理后大肠杆菌细胞膜相对电导率升高,表明胡桃醌破坏了大肠杆菌膜的完整性,增加了细胞膜的通透性。荧光发射光谱分析结果表明,胡桃醌能够与膜蛋白相互作用从而改变大肠杆菌细胞膜结构。结晶紫和刃天青染色实验结果表明胡桃醌能够通过抑制大肠杆菌生物膜的形成减弱大肠杆菌的呼吸作用,进而抑制其活性。SDS-PAGE和大肠杆菌基因组合成分析结果显示胡桃醌可抑制大肠杆菌中蛋白质、DNA和RNA的表达。通过分子对接实验可知,胡桃醌可以结合到基因组DNA的...     

微波杀菌动力学及其影响因素的研究

本实验研究了不同温度条件下微波杀菌动力学D值;并且就食品体系中相应的pH和盐浓度对微波杀菌效果的影响作了探讨;同时对微波杀菌的机理进行了初步的研究。结果表明,相同温度下,微波功率越大,细菌的致死率越高;相同功率下,设定微波温度越高,其D值越小;当pH低于最适生长温度时,细菌对微波的敏感性增加,致死率提高;金黄色葡萄球菌(60℃)在20g/L的NaCl溶液中的致死率最大,而大肠杆菌(55℃)致死率随NaCl浓度增加而降低。微波处理后的金黄色葡萄球菌,经Flou-3/AM作用,其荧光值随温度的升高而增加,说明微波致金黄色葡萄球菌死亡的原因主要是改变了细胞膜的通透性。     

脱镁叶绿酸a对金黄色葡萄球菌、大肠杆菌的抑制作用

以叶绿素为提取物，进行化学修饰得到光敏活性剂-脱镁叶绿酸α，其对微生物生长影响的研究表明：在光照条件下，脱镁叶绿酸α对金黄色葡萄球菌的生长和大肠杆菌原生质体的再生有明显的抑制作用；在暗培养条件下，脱镁叶绿酸α的抑制作用减弱。     

山苍子精油对MRSA生物被膜及其调控基因ica A的抑制机理研究

耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)是一种常见的人畜共患病原菌,极易在食品加工设备表面生成生物膜,增加食物污染的风险。山苍子精油作为植物源天然提取物,对S.aureus有良好的抗菌效果,但其抗MRSA生物膜能力及作用机制还知之甚少。本实验研究了山苍子精油对MRSA生物被膜及其多糖细胞间黏附素(PIA)的调控基ica A的抗菌机制。结果表明:山苍子精油对MRSA生物膜的MBIC是2.0mg/mL,MBEC是4.0mg/mL;由菌落计数法可知,山苍子精油的抗MRSA生物膜能力与精油浓度和处理时间呈正相关。抗生物膜机制的初步探索结果表明,MRSA生物膜的代谢水平和细胞外聚合物(胞外多糖,胞外蛋白和胞外DNA)的形成受到山苍子精油的抑制。此外,在分子水平上的实验结果表明,山苍子精油可以显著抑制MRSA生物膜中ica A基因的转录水平。