Comparison of bactericidal activity of six lysozymes at atmospheric pressure and under high hydrostatic pressure

The antibacterial working range of six lysozymes was tested under ambient and high pressure, on a panel of five gram-positive (Enterococcus faecalis, Bacillus subtilis, Listeria innocua, Staphylococcus aureus and Micrococcus lysodeikticus) and five gram-negative bacteria (Yersinia enterocolitica, Shigella flexneri, Escherichia coli O157:H7, Pseudomonas aeruginosa and Salmonella typhimurium). The lysozymes included two that are commercially available (hen egg white lysozyme or HEWL, and mutanolysin from Streptomyces globisporus or M1L), and four that were chromatographically purified (bacteriophage lambda lysozyme or LaL, bacteriophage T4 lysozyme or T4L, goose egg white lysozyme or GEWL, and cauliflower lysozyme or CFL). T4L, LaL and GEWL were highly pure as evaluated by silver staining of SDS-PAGE gels and zymogram analysis while CFL was only partially pure. At ambient pressure each gram-positive test organism displayed a specific pattern of sensitivity to the six lysozymes, but none of the gram-negative bacteria was sensitive to any of the lysozymes. High pressure treatment (130-300 MPa, 25 degrees C, 15 min) sensitised several gram-positive and gram-negative bacteria for one or more lysozymes. M. lysodeikticus and P. aeruginosa became sensitive to all lysozymes under high pressure, S. typhimurium remained completely insensitive to all lysozymes, and the other bacteria showed sensitisation to some of the lysozymes. The possible applications of the different lysozymes as biopreservatives, and the possible reasons for the observed differences in bactericidal specificity are discussed.     

The lactoperoxidase system increases efficacy of high-pressure inactivation of foodborne bacteria

The inactivation of eight different bacteria comprising Escherichia coli LMM1010 and MG1655, respectively a pressure-resistant strain and the corresponding wild-type, Salmonella Typhimurium, Pseudomonas fluorescens, Staphylococcus aureus, Enterococcus faecalis, Listeria innocua and Lactobacillus plantarum, by high hydrostatic pressure in skim milk supplemented with the lactoperoxidase-hydrogen peroxide-thiocyanate (LP) system at naturally occurring concentration was studied. In the absence of pressure treatment, the LP system had either no effect, i.e. on S. Typhimurium and E. coli LMM1010, a growth inhibiting effect, i.e. on E. coli MG1655, L. innocua, S. aureus, L. plantarum and E. faecalis, or a bactericidal effect, i.e. on P. fluorescens. The presence of the LP system affected inactivation by high pressure in a cell density-dependent manner. At low cell concentration (10(6) cfu/ml), the LP system strongly increased high-pressure inactivation as measured immediately after pressure treatment of all bacteria except the pressure-resistant E. coli. At high cell density (10(9) cfu/ml), only inactivation of L. innocua, E. faecalis and L. plantarum were enhanced. For both E. coli strains, the fate of the bacteria during 24 h following pressure treatment was also studied. It was found that in the presence of the LP system, considerable further inactivation occurred in the first hours after pressure treatment. The potential of the LP system to improve the bactericidal efficiency of high-pressure treatment for food preservation is discussed.     

Enhancement of nisin, lysozyme, and monolaurin antimicrobial activities by ethylenediaminetetraacetic acid and lactoferrin

A microtiter plate assay was employed to systematically assess the interaction between ethylenediaminetetraacetic acid (EDTA) or lactoferrin and nisin, lysozyme, or monolaurin against strains of Listeria monocytogenes, Escherichia coli, Salmonella enteritidis, and Pseudomonas fluorescens. Low levels of EDTA acted synergistically with nisin and lysozyme against L. monocytogenes but EDTA and monolaurin interacted additively against this microorganism. EDTA synergistically enhanced the activity of nisin, monolaurin, and lysozyme in tryptic soy broth (TSB) against two enterohemorrhagic E. coli strains. In addition, various combinations of nisin, lysozyme, and monolaurin with EDTA were bactericidal to some gram-negative bacteria whereas none of the antimicrobials alone were bactericidal. Lactoferrin alone (2000 microg ml(-1)) did not inhibit any of the bacterial strains, but did enhance nisin activity against both L. monocytogenes strains. Lactoferrin in combination with monolaurin inhibited growth of E. coli O157:H7 but not E. coli O104:H21. While lactoferrin combined with nisin or monolaurin did not completely inhibit growth of the gram-negative bacteria, there was some growth inhibition. All combinations of EDTA or lactoferrin with antimicrobials were less effective in 2% fat UHT milk than in TSB. S. enteritidis and P. fluorescens strains were consistently more resistant to antimicrobial combinations. Resistance may be due to differences in the outer membrane and/or LPS structure.     

Lactic acid bacteria isolated from artisanal dry sausages: characterization of antibacterial compounds and study of the factors affecting bacteriocin production

Lactic acid bacteria (LAB) were isolated from artisanal dry sausages sampled from north-eastern region of Chaco, Argentina. Among 141 isolates, 27 showed antimicrobial activity against Listeria innocua, Staphyloccus aureus or Brochothrix spp. One isolate, identified as Lb. curvatus/sakei, produced bacteriocin like substances (BLIS). These BLIS were heat stable, effective after refrigerated storage and freeze/thaw cycles and even active against pathogens when produced under refrigeration at 3% NaCl concentration. The influence of several factors on production of BLIS was assessed in MRS broth added with: EDTA, ascorbic acid, KCl, potassium sorbate, sodium citrate, 3 and 6% NaCl, Tween 20 or Brij 35. These additives showed different effects towards the effectiveness of the bacteriocin produced by Lb. sakei/curvatus against L. innocua and S. aureus. Conditions that provided high cell density favored high bacteriocin production. BLIS production by this LAB strain was greatly influenced by NaCl concentration and the presence of surfactants.     

溶菌酶功能化金纳米颗粒抑菌性能研究

为了提高溶菌酶的稳定性及对革兰氏阴性菌的抑菌性能,以金纳米颗粒为核,通过表面定向修饰溶菌酶,制备了绿色、高效的溶菌酶功能化金纳米颗粒抑菌材料,研究溶菌酶与金纳米的比例、纳米颗粒质量浓度等对抑菌效果的影响,研究溶菌酶功能化纳米材料对大肠杆菌的抑菌机理及对人神经母细胞瘤(SH-SY5Y)的细胞毒性。结果表明:与未修饰的金纳米及单纯的溶菌酶相比,溶菌酶功能化金纳米颗粒对大肠杆菌和金黄色葡萄球菌的抑菌效果均显著增强,表现出协同抑菌作用,在最优条件下,0.1 g/L溶菌酶功能化金纳米颗粒可以完全杀死2种细菌,并且该溶菌酶功能化金纳米颗粒具有持久抑菌性及良好的生物相容性,对哺乳细胞没有毒性。透射电子显微镜和活菌/死菌荧光染色结果表明:溶菌酶功能化金纳米颗粒主要通过破坏菌体细胞壁和细胞膜结构,从而杀死细菌。     

Influence of bispecific antibodies on the in vitro bactericidal activity of bovine neutrophils against Staphylococcus aureus

We conducted the following study to determine if bispecific antibodies enhance the bactericidal activity of bovine neutrophils. Bispecific antibodies were synthesized by chemically crosslinking bovine neutrophil monoclonal antibodies to Staphylococcus aureus 305 capsule polysaccharide monoclonal antibodies. The efficiency of chemically coupling monoclonal antibody monomers was approximately 50% for each bispecific antibody produced. Monoclonal antibodies against neutrophils enhanced the respiratory burst activity of neutrophils by 2.3- to 2.5-fold. To determine the influence of bispecific antibodies on neutrophil function, S. aureus 305 was preincubated with various concentrations of bispecific antibodies and neutrophils were then added to the opsonized bacteria at different bacteria to neutrophil ratios. The bactericidal activity of neutrophils was expressed as a percentage reduction in colony-forming units in test cultures compared with the number of colony-forming units in control test cultures that did not contain bispecific antibodies or neutrophils. The addition of bispecific antibodies to test cultures increased the bactericidal activity of neutrophils. A reduction in colony-forming units as a function of increasing the S. aureus 305 to neutrophils ratio was observed in both the absence and presence of bispecific antibodies. However, a greater reduction was observed in the presence of bispecific antibodies. Increasing concentrations of bispecific antibodies enhanced the bactericidal activity of neutrophils at a constant S. aureus 305 to neutrophil ratio of 1:500. The results indicate that bispecific antibodies that recognize both S. aureus 305 capsular polysaccharide and neutrophil antigens potentiate the bactericidal activity of neutrophils.     

The fate of indigenous microbiota, starter cultures, Escherichia coli, Listeria innocua and Staphylococcus aureus in Danish raw milk and cheeses determined by pyrosequencing and quantitative real time (qRT)-PCR

The purpose of this work was to study the bacterial communities in raw milk and in Danish raw milk cheeses using pyrosequencing of tagged amplicons of the V3 and V4 regions of the 16S rDNA and cDNA. Furthermore, the effects of acidification and ripening starter cultures, cooking temperatures and rate of acidification on survival of added Escherichia coli, Listeria innocua and Staphylococcus aureus in cheeses at different stages of ripening were studied by pyrosequencing and quantitative real time (qRT)-PCR. A high diversity of bacterial species was detected in raw milk. Lactococcus lactis, Streptococcus thermophilus, Lactobacillus casei and Lactobacillus rhamnosus were the main bacteria detected in raw milk and cheeses. Bacteria belonging to the genera Brevibacterium, Staphylococcus, Escherichia, Weissella, Leuconostoc, Pediococcus were also detected in both 16S rDNA and cDNA obtained from raw milk and cheeses. E. coli, which was added to milk used for production of some cheeses, was detected in both DNA and RNA extracted from cheeses at different stages of ripening showing the highest percentage of the total sequence reads at 7 days of ripening and decreased again in the later ripening stages. Growth of E. coli in cheeses appeared to be affected by the cooking temperature and the rate of acidification but not by the ripening starter cultures applied or the indigenous microbiota of raw milk. Growth of L. innocua and S. aureus added to milks was inhibited in all cheeses at different stages of ripening. The use of 16S rRNA gene pyrosequencing and qRT-PCR allows a deeper understanding of the behavior of indigenous microbiota, starter cultures and pathogenic bacteria in raw milk and cheeses.     

甘肃陇南大红袍花椒芳香油成分分析及其抑菌活性

目的:确定甘肃陇南大红袍花椒芳香油的化学成分及对于大肠杆菌、金黄色葡萄球菌、破伤风杆菌、荧光假单胞杆菌四种细菌的抑菌效果。方法:采用水蒸气蒸馏法提取陇南大红袍花椒芳香油,运用气相色谱-质谱联用(GC-MS)对其成分进行分析鉴定,并研究了陇南大红袍花椒芳香油的抑菌活性。结果:陇南大红袍花椒芳香油鉴定出50种化学成分,占总量的(92.49%),主要的化学成分包括D-柠檬烯(32.08%)、右旋香芹酮(20.02%)、石竹烯(12.26%)等;通过抑菌实验发现陇南大红袍花椒芳香油对于4种细菌均具有抑菌活性,其中对金黄色葡萄杆菌和荧光假单胞杆菌抑菌效果较好,最低抑菌浓度(MIC)和最低杀菌浓度(MBC)分别为2.5和5.0 mL/L、2.5和5.0 mL/L。结论:本文确定了甘肃省陇南大红袍花椒芳香油的化学成分,并得出了陇南大红袍花椒挥发油对于大肠杆菌、金黄色葡萄球菌、破伤风杆菌、荧光假单胞杆菌的MIC和MBC,为花椒的进一步应用奠定一定的理论基础。     

辉光放电低温等离子体技术对微生物的杀菌动力学及杀菌机制

应用研制的辉光放电低温等离子体杀菌设备,以大肠杆菌、志贺氏菌、金黄色葡萄球菌、单核细胞增生李斯特氏菌、黄曲霉菌、白色念珠菌6 种微生物为实验菌株,探讨辉光放电低温等离子体杀菌技术对不同微生物的杀菌动力学;应用扫描电镜、透射电镜、DNA电泳、光谱分析等技术,探讨辉光放电低温等离子体的杀菌机制。结果表明：辉光放电低温等离子体杀菌技术对大肠杆菌、志贺氏菌、金黄色葡萄球菌、单核细胞增生李斯特氏菌、黄曲霉菌、白色念珠菌6 种微生物的有效杀灭时间分别为2、1.5、3、4、10、10 min。依据杀菌动力学曲线,可以将6 种微生物准确分为革兰氏阴性细菌、革兰氏阳性细菌和真菌3 类,这可能与微生物的细胞壁结构有关;辉光放电低温等离子体可能是通过带电的高能粒子对细菌的细胞壁造成破坏,进而杀死细菌。该研究为辉光放电低温等离子体技术广泛用于微生物杀菌提供了重要的方法依据。     

阿魏酸化学修饰溶菌酶及扩展抑菌谱的研究

为了使溶菌酶扩展抑菌谱,同时作用于革兰氏阳性菌和革兰氏阴性菌,本文采用了阿魏酸修饰溶茼酶.采用EDAC作为缩合剂,使溶菌酶赖氡酸残基上的ε-氨基共价结合阿魏酸的羧基,形成一定程度的阿魏酸修饰酶结果显示:与天然溶菌酶相比,修饰酶的酶活力略有下降,但是修饰酶扩展了抑菌谱,对革兰氏阴性菌的抑菌作用增强.修饰酶对大肠杆菌和沙门氏菌的最小抑菌浓度均为0.5mg/mL,而对金黄色葡萄球菌的抑菌作用略有下降.     

溶菌酶酶解物的抗菌活性研究

目的:考察溶菌酶酶解物的抑菌能力,为扩展溶菌酶的应用提供理论依据。方法:采用胃蛋白酶对溶菌酶进行酶解,得到溶菌酶酶解物(Lysozyme hydrolysate,LH);采用二倍稀释法检测LH对几种常见食品腐败菌的抑菌能力;以金黄色葡萄球菌为受试菌种,通过抑菌圈实验探讨LH的抑菌活性,并与溶菌酶和乳酸链球菌素作比较。结果:LH对几种常见阳性菌都有很强的抑制作用,其中对金黄色葡萄球菌的抑制作用最强;未经热处理时,LH的抑菌活性略低于溶菌酶,而高于乳酸链球菌素;经热处理后,LH的抑菌活性高于溶菌酶和乳酸链球菌素。菌酶酶解物有望被开发成一种天然的食品防腐剂。     

Evaluation of cell wall binding domain of Staphylococcus aureus autolysin as affinity reagent for bacteria and its application to bacterial detection

We evaluated the cell wall binding (CWB) domain of Staphylococcus aureus autolysin as an affinity reagent for bacteria. A fusion of CWB domain and green fluorescent protein (CWB-GFP) bound to S. aureus with a dissociation constant of 15 nM. CWB-GFP bound to a wide range of gram-positive bacteria, but not to most gram-negative bacteria. We suspected that the outer membrane of gram-negative bacteria inhibits the access of CWB-GFP to peptidoglycan layer. Indeed, CWB-GFP bound to gram-negative bacteria when they were treated with benzalkonium chloride. Because CWB-GFP bound to the bacterial peptidoglycan layer, it appeared to be an effective affinity reagent for bacteria and CWB fusion with reporter proteins could be applied to detect bacteria. We also constructed a fusion of CWB and luciferase, which can be used for the rapid detection of bacteria.     

The effect of UV-C irradiation on lipids and selected biologically active compounds in human milk

The effect of UV-C irradiation of human milk on lipid oxidation, content of antioxidants (vitamin C and catalase, CAT) and bactericidal compounds (lysozyme), as well as the total antioxidant capacity (TAC), of the breast milk was investigated. In parallel, the extent of inactivation of some bacteria was also determined. UV-C at doses from 85 to 740 J L⁻¹ caused total inactivation of Escherichia coli and Staphylococcus aureus, but bacteria of the genus Enterococcus were reduced only partially. There was a significant increase in content of primary (lipid peroxides, LP) and secondary (thiobarbituric acid reactive substances, TBARS) oxidation products of lipids (by 33% and 36%, respectively) but decreased vitamin C and lysozyme content (by 35% and 41%). UV-C had no effect on the value of the TAC and caused a smaller decrease in CAT activity (by 14%) than conventional pasteurisation (by 60%).     

Detection and preliminary characterization of a bacteriocin (plantaricin 35d) produced by a Lactobacillus plantarum strain

Lactic acid bacteria (134) from Italian sausages were tested for the production of antimicrobial substances (bacteriocins). Six percent of these showed antibacterial activity against one or several closely related microorganisms used as indicators. Lactobacillus plantarum 35d in particular produced a bacteriocin of high activity (320 AU ml(-1)) and a wide range of antimicrobial activity including S. aureus, L. monocytogenes, and A. hydrophila. The bacteriocin withstood heating at 80 degrees C for 120 min and storage at 4 degrees C for 6 months. The mode of action was identified as bactericidal. The apparent molecular weight of the bacteriocin extracted with n-butanol was estimated to be 4.5 kDa.     

INHIBITION OF LISTERIA MONOCYTOGENES BY LACTOCOCCUS LACTIS SUBSP. LACTIS ISOLATED FROM ITALIAN RAW HAM

A total of 168 strains of lactic acid bacteria were isolated from Italian raw ham and screened for antagonistic activity against Listeria monocytogenes by using an agar spot assay. Only one strain of Lactococcus lactis subsp . lactis produced antagonistic effects other than inhibition by low pH and hydrogen peroxide. The proteinaceous nature of the compound produced by L. lactis B10 was demonstrated by its inactivation by proteolytic enzymes. The cell-free culture super-natants (filtered and heat-treated) showed a bactericidal mode of action. Bacteriocin produced by L. lactis B10 was inhibitory to other lactic acid bacteria and one strain of Staphylococcus aureus, but not to the gram-negative bacteria tested .     

Impedance measurements to study the antimicrobial activity of essential oils from Lamiaceae and Compositae

A wide range of essential oils from sage, mint, hyssop, camomile and oregano were tested for their inhibitory effects against nine strains of gram-negative bacteria and six strains of gram-positive bacteria. Three principles were used in describing the antimicrobial effects of the essential oils: the overall antimicrobial activity determined by use of an impedometric method, the bactericidal effect determined as colony forming units after exposure to the essential oils, and the number of apparent dead cells determined after further enrichment. The data obtained indicate that while the essential oils of sage, mint, hyssop and camomile had generally a bacteriostatic activity, the essential oil from oregano appeared to be bactericidal at concentrations above 400 ppm, probably because of high contents in phenolic compounds. For the other essential oils, the chemical analysis was unable to explain the antimicrobial effect. The bacteriostatic activity was more marked against gram-positive bacteria; in contrast, the bactericidal activity was greatest against gram-negative bacteria. The most sensitive strain was Escherichia coli O157:H7 and, of the gram-positive species even at the lowest oil concentrations, Listeria innocua was the most sensitive. The data obtained from the study of the bactericidal effect of oregano essential oil indicated that the major part of the species was irreversibly inactivated, i.e. they could not be revived by enrichment.     

异硫氰酸苄酯及其类似物抑菌活性的初步探究

本文对异硫氰酸苄酯及其类似物(异氰酸苄酯、异硫氰酸苯酯、异硫氰酸苯乙酯、异硫氰酸乙酯)对大肠杆菌及金黄色葡萄球菌的抑菌活性进行初步探究。通过对抑菌圈、最低抑菌浓度、最低杀菌浓度、生长曲线、能量代谢及构效关系研究,初步探究了异硫氰酸苄酯及其类似物的抑菌基团及抑菌机理。结果表明:5种物质对两种受试菌种均具有抑菌作用,尤其对金黄色葡萄球菌最为敏感;其抑菌活性是由异硫氰酸基团、亚甲基以及苯环三者共同作用的结果,并且异硫氰酸基团对其抑菌活性的决定性作用最大、亚甲基次之、苯环最弱。结构不同物质对细菌的代谢活性及相关酶活性的影响也不同。其中,异硫氰酸基团对能量代谢影响最大,氧原子被硫原子取代后进一步增强了这种作用,苯环的存在对能量代谢的影响产生反向作用。但对能量代谢影响最为明显的抑菌物质,其抑菌活性并不是最强的,这说明可能还存在其他抑菌机理。     

Evaluation of a monoclonal antibody able to detect live Listeria monocytogenes and Listeria innocua

A monoclonal Listeria antibody, designated B4, was evaluated. The ability of the antibody to bind to viable bacteria belonging to Listeria spp. compared to bacteria of the same species killed by heat treatment, acid or base treatment, sanitizers, and irradiation was examined. The antibody was found to react with viable L. monocytogenes and L. innocua, but not with heat-killed (72 degrees C, 5 min) strains of these organisms. When L. monocytogenes and L. innocua were killed by methods other than heat treatment, it was ambiguous whether the antibody detected the organism or not. It was concluded that the B4 antibody has potential to be used in an immuno capture step to capture live L. monocytogenes and L. innocua from foods prior to identification of L. monocytogenes by polymerase chain reaction (PCR).     

Antimicrobial effects of chitosans and chitooligosaccharides, upon Staphylococcus aureus and Escherichia coli, in food model systems

The objective of this study was to elucidate the controversial relationship between the molecular weight (MW) of chitosans and their antibacterial activity (upon different inoculum levels, at several concentrations). The influence of food components on the activity was also ascertained, as well as acceptance by a sensory panel. All the compounds tested exhibited antibacterial activity against Staphylococcus aureus and Escherichia coli. This activity was shown to be closely dependent on the inoculum level, MW and concentration used. Within 4h at 10(3) cells/mL, all five compounds, at every concentration (0.5%, 0.25% and 0.1%, w/v), proved to be bactericidal; for higher inocula, 0.1% (w/v) was only bacteriostatic; at 10(7) or 10(5) cells/mL, and independently of the inoculum level, 0.25% (w/v) of any chitooligosaccharide (COS) mixture was sufficient to reduce the E. coli initial population by at least 3 log cycles; COS never exhibited bactericidal action over S. aureus, unlike high and medium MW chitosans-which, at 0.5% (w/v), presented a bactericidal effect even against 10(7) cells/mL. When incorporated in liquid food matrices, medium and high MW chitosans maintained their activity, for both matrices and bacteria, although a slower activity was noticeable in milk; however, COS lost their activity upon both bacteria in milk after 4-8h. Furthermore, addition of chitosans to apple juice led to several unpleasant off-flavors, such as astringency and after taste--which increased in magnitude with MW.     

Evaluation of liquid smoke treated ready-to-eat (RTE) meat products for control of Listeria innocua M1

ABSTRACT:  Liquid smoke fractions (S1, S2, S3, and S4) were applied on ready-to-eat (RTE) meat products to control the growth of inoculated Listeria innocua M1. Turkey rolls and roast beef products were dipped in liquid smoke, surface inoculated with L. innocua M1 (10² CFU/25 cm² RTE meat surface), vacuum packaged, and stored at 4 °C. Section 8.5 of USDA's detection and isolation procedure for L. monocytogenes was employed in conjunction with a Micro-ID™ system for L. innocua M1 identification (ID). Products treated with smoke fractions S1, S2, and S3 were negative for L. innocua M1 at 2 and 4 wk during incubation at 4 °C. Products treated with S4 were positive for L. innocua M1 immediately following inoculation and after storage for 2 and 4 wk. Smoke fractions S1, S2, and S3 exhibited pH values lower than 4.6, acidity values higher than 1.5%, and carbonyl concentrations higher than 110 mg/mL. All liquid smoke fractions contained similar phenol concentrations (0.3 to 0.6 mg/mL), suggesting that phenols may have a limited role in the bactericidal effects of liquid smoke fractions against specific microorganisms.