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。     

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.     

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.     

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。     

新疆酸驼乳中细菌素乳酸菌的筛选及其抑菌性

通过双层琼脂扩散打孔法,从新疆哈萨克族传统发酵酸驼乳中分离的23株乳酸菌中,筛选出9株对金黄色葡萄球菌、枯草芽孢杆菌和大肠杆菌具有抑菌活性的菌株(其中茵株MLS5抑菌活性最强).通过有机酸、过氧化氢抑茵的排除试验和蛋白酶敏感性试验,初步确定MLS5产生的抑茵物质为蛋白质类细菌素.温度和pH耐受试验表明,该细菌素具有良好的热稳定性(121℃,20 min),pH 3～6具有抑茵活性.菌株MLS5的发酵曲线表明,菌株在37℃培养条件下具有良好的产细菌素和产酸能力,该细茵素在茵体生长对数中期产生,在生长稳定期抑茵活性达到最大.经抑茵谱试验表明,MLS5所产细菌素不仅能抑制革兰氏阳性细茵,而且对大肠杆菌和毛霉菌也有抑制作用,具有较广的抑茵谱.     

Enhanced antimicrobial activity of nisin in combination with allyl isothiocyanate against Listeria monocytogenes,Staphylococcus aureus,Salmonella Typhimurium and Shigella boydii

This study was designed to evaluate the synergistic antimicrobial effect of nisin and allyl isothiocyanate (AITC) against Listeria monocytogenes, Staphylococcus aureus, Salmonella Typhimurium and Shigella boydii. The synergistic interactions between nisin and AITC were observed against all foodborne pathogens, showing the fractional inhibitory concentrations <1. The populations of L. monocytogenes and S. aureus at the combined treatment of nisin and AITC were decreased to below 1 log CFU mL^?1 after 10‐h incubation at 37 °C. The changes in fatty acid profiles of all strains were substantially influenced by nisin alone and the combined treatment of nisin and AITC. A good agreement was observed among cell viability, membrane permeability and depolarisation activity in response to nisin and AITC. The results suggest that nisin and AITC as synergistic inhibitors could be an effective approach to achieve satisfactory antimicrobial activity against a wide range of foodborne pathogens.     

Characterisation of Argentinean honeys and evaluation of its inhibitory action on Escherichia coli growth

The aim of this work was to evaluate the physicochemical, sensory and Escherichia coli growth inhibitory characteristics of honey of different botanical sources from two geographic origin of Argentina. Honeys were obtained from apiaries located in two zones. The floral identification of honeys allowed to clustered them as monofloral, mixed and polyfloral honeys. The study of the physicochemical parameters such as colour, free acidity, pH and moisture showed that the last one reflected significant differences between honeys. These differences were markedly reflected in the average values of moisture content for each zone, being 18.96% and 14.29% to centre and east zone, respectively. In general, honeys evaluated presented an inhibitory effect on the E. coli growth at different periods of time (bacteriostatic action). Only, two of the samples would show a bactericide action against E. coli at 48 h of incubation. Honeys with higher non‐hydrogen peroxide activity, were collected from a same geographic place at the same season of year, showing a relationship between the antimicrobial activity and the geographic origin, which could be associated with the typical flora of the place.     

Carbon Dioxide Inhibition of Escherichia coli and Staphylococcus aureus on a pH-adjusted Surface in a Model System

Growth of Escherichia coli and Staphylococcus aureuson the surface of Trypticase Soy Agar (TSA) packaged with various CO₂ partial pressures (0, 20, 40, 60, 80, 100%, balance N₂) was compared to the control (N₂ 100%) on TSA in which the pH was adjusted to equal that in CO₂ atmospheres at 15°C and 30°C. At 15°C, the biostatic effect was noted with all CO₂ partial pressures for both species. At 30°C, the biostatic effect of CO₂ was almost completely nullified for E. coli, but that for S. aureus was still effective. S. aureus was more sensitive to the inhibitory effects of CO₂ than E. coli at both the temperatures.     

微波加热对三种微生物致死的Weibull模型

探讨不同微波功率加热至不同温度下对沙门氏菌、大肠杆菌和金黄色葡萄球菌等三种对象菌的致死作用,运用Weibull模型描述其致死历程,由Weibull模型计算微波加热使微生物数量减少5-log所需要的时间,并验证其模型的精确度。结果表明,Weibull模型描述沙门氏菌、大肠杆菌和金黄色葡萄球菌的致死历程均有较高的拟合度,通过Weibull模型推算出来的沙门氏菌、大肠杆菌和金黄色葡萄球菌减少5-log的时间与实验结果相近。     

Purification, partial characterisation and mode of action of enterococcin EFS2, an antilisterial bacteriocin produced by a strain of Enterococcus faecalis isolated from a cheese

Enterococcus faecalis strain EFS2, isolated from the surface of a traditional cheese, produced a bacteriocin active against Gram-positive bacteria including Listeria spp. and some Staphylococcus aureus strains. The bacteriocin, named enterococcin EFS2, has been purified to homogeneity by ammonium sulphate precipitation and reversed-phase high performance liquid chromatography (RP-HPLC). The molecular weight was determined by mass spectrometry to be 7149.6. The amino acid composition of enterococcin EFS2 revealed that it contained 67 amino acid residues and had a blocked amino-terminal end. Enterococcin EFS2 induced viability loss, efflux of K⁺ ions and ATP, and cell lysis. Kinetic study of bactericidal activity of enterococcin EFS2 on Listeria innocua strain LIN 11 indicated slower cell destruction than by nisin. At pH 7.0, the activity of enterococcin EFS2 was the highest at 35 °C and was lost at 15 °C. The bacteriocin was more active against L. innocua strain LIN11 in broth adjusted to pH 6.0, 7.0 and 8.0 than to pH 4.5 at 30 °C.     

Aerobic bacterial, coliform, Escherichia coli and Staphylococcus aureus counts of raw and processed milk from selected smallholder dairy farms of Zimbabwe

A cross sectional study was conducted to enumerate total viable bacteria (TBC), coliforms, Escherichia coli and Staphylococcus aureus in raw (n = 120) and processed (n = 20) milk from individual farms from three smallholder dairy schemes of Zimbabwe between October, 2009 and February, 2010. Data on management factors were collected using a structured questionnaire. A standard pour plate technique was used to enumerate total viable bacteria, while for coliforms, E. coli and S. aureus, counts were assessed by the spread plate technique. The association of total viable bacterial counts and management factors was assessed using univariable and a linear regression model. The log₁₀ TBC for raw milk differed significantly (P < 0.05) amongst the schemes with the lowest (5.6 ± 4.7 log₁₀ cfu/ml) and highest (6.7 ± 5.8 log₁₀ cfu/ml) recorded from Marirangwe and Nharira respectively. The mean log₁₀ of TBC of processed milk (6.6 ± 6.0 log₁₀ cfu/ml) were marginally higher than those of raw milk (6.4 ± 5.6 log₁₀ cfu/ml) but not significant (P > 0.05). The coliform, E. coli and S. aureus counts for raw milk significantly differed (P < 0.05) amongst the study areas. The variation in TBC, coliforms, E. coli and S. aureus counts amongst the schemes could be attributed to differences in milking hygiene where farms with more access to training and monitoring of microbiological quality of milk had lower counts. Linear regression analysis revealed dairy scheme, delivery time and season of milking as independently associated with increased TBC of raw milk. The high TBC of raw and processed milk generally indicated low levels of milking hygienic practices, and high level of post-processing contamination, respectively. The high TBC, coliform, E. coli and S. aureus counts of both raw and processed milk may present a public health hazard. Thus, educating the farmers on general hygienic practices, quickening the delivery of milk to collection centres, or availing cooling facilities on-farm will improve the microbiological quality and safety of milk.     

Purification,characterization and action mechanism of plantaricin JY22, a novel bacteriocin against <Emphasis Type="Italic">Bacillus cereus</Emphasis> produced by <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> JY22 from golden carp intestine

A novel bacteriocin-producing strain, Lactobacillus plantarum JY22 isolated from golden carp intestine, was screened and identified by its physiobiochemical characteristics and 16S rRNA gene sequence analysis. This bacteriocin, named plantaricin JY22, was purified using ethyl acetate extraction and gel filtration. Its molecular weight was approximately 4.1 kDa by SDS-PAGE analysis. The partial amino acid sequence of plantaricin JY22 was DFGFDIPDEV. It was highly heat-stable and remained active at pH range from 2.5 to 5.5, but was sensitive to protease. Plantaricin JY22 had a bactericidal mode. Scanning electron microscope analysis indicated that plantaricin JY22 damaged the morphology of cells and spores for Bacillus cereus. Moreover, the plantaricin JY22 destroyed cell membrane integrity as confirmed by the leakage of electrolytes, the losses of Na⁺K⁺-ATP, AKP, nucleic acids (OD_260nm) and proteins. SDS-PAGE of B. cereus proteins further demonstrated that plantaricin JY22 had a remarkable effect on bacterial proteins.     

Use of a ground beef model to assess the effect of the lactoperoxidase system on the growth of Escherichia coli O157:H7, Listeria monocytogenes and Staphylococcus aureus in red meat

The ability to preserve food in a state that is both appetising and nutritious is a basic requirement for health. Food poisoning represents a major source of illness and loss of productivity in many developed countries. Of particular concern in recent years are outbreaks of food poisoning associated with Escherichia coli O157:H7 or Listeria monocytogenes, many of which have been associated with the consumption of ground meat. Many of the chemicals presently licensed for use as food preservatives are increasingly being questioned with regard to their effects on humans, creating pressure on food suppliers to consider the use of 'natural' alternatives to these chemical agents. The potential use of one such agent, the lactoperoxidase system (LPS), for use in ground meat preparations is examined in this study. The degree of inhibition of growth of E. coli O157:H7, L. monocytogenes L45 and S. aureus R37 by LPS was examined in a broth system at 37 degrees C and in a ground beef system at 0, 6 and 12 degrees C. The degree of inhibition by LPS of natural populations of microorganisms present in ground beef obtained from eight retail outlets and incubated at room temperature was also examined. For each of the strains examined, sensitivity from most to least sensitive followed the order L. monocytogenes L45, S. aureus R37 and E. coli O157:H7. In each case the ability of LPS to inhibit growth was highly temperature dependent and maximal at a temperature permissive but not optimal for growth of the test strain. The numbers of bacteria detected in ground beef obtained from retail outlets varied considerably between the eight samples. In all cases, numbers of bacteria increased markedly in the uninhibited control over the 4 h incubation time and, with the exception of one faecal coliform count, growth of the microbial populations was strongly inhibited by the presence of LPS. It was concluded that LPS could potentially be applied to a considerably wider range of food products than those to which it is presently restricted.     

Purification, characterization, and primary structure of a novel N-acyl-d-amino acid amidohydrolase from Microbacterium natoriense TNJL143-2

A novel N-acyl-d-amino acid amidohydrolase (DAA) was purified from the cells of a novel species of the genus Microbacterium. The purified enzyme, termed AcyM, was a monomeric protein with an apparent molecular weight of 56,000. It acted on N-acylated hydrophobic d-amino acids with the highest preference for N-acetyl-d-phenylalanine (NADF). Optimum temperature and pH for the hydrolysis of NADF were 45°C and pH 8.5, respectively. The k(cat) and K(m) values for NADF were 41?s(-1) and 2.5?mM at 37°C and pH 8.0, although the enzyme activity was inhibited by high concentrations of NADF. Although many known DAAs are inhibited by 1?mM EDTA, AcyM displayed a 65% level of its full activity even in the presence of 20?mM EDTA. Based on partial amino acid sequences of the purified enzyme, the full-length AcyM gene was cloned and sequenced. It encoded a protein of 495 amino acids with a relatively low sequence similarity to a DAA from Alcaligenes faecalis DA1 (termed AFD), a binuclear zinc enzyme of the α/β-barrel amidohydrolase superfamily. The unique cysteine residue that serves as a ligand to the active-site zinc ions in AFD and other DAAs was not conserved in AcyM and was replaced by alanine. AcyM was the most closely related to a DAA of Gluconobacter oxydans (termed Gox1177) and phylogenetically distant from AFD and all other DAAs that have been biochemically characterized thus far. AcyM, along with Gox1177, appears to represent a new phylogenetic subcluster of DAAs.     

Inhibition of Escherichia coli O157:H7, Listeria monocytogenes and Staphylococcus aureus on sliced roast beef by cetylpyridinium chloride and acidified sodium chlorite

The effects of 0.5% cetylpyridinium chloride (CPC), 0.12% acidified sodium chlorite (ASC) and a mix of equal volume of the two (0.25% CPC-0.06% ASC) on Escherichia coli O157:H7, Listeria monocytogenes and Staphylococcus aureus were evaluated on inoculated sliced roast beef. The antimicrobial agents were, respectively, sprayed on the beef surfaces and tray absorbent pads, and samples were stored at 4 degrees C for 10 days (d). At 0 d, L. monocytogenes and S. aureus were reduced to undetectable levels in 2 h after spraying with CPC. CPC-ASC treatment reduced E. coli O157:H7, L. monocytogenes and S. aureus by 4.07, 6.37 and 4.32 log cfu/cm2, respectively, at 0 d. ASC treatment reduced the population of E. coli O157:H7 by 6.09 log cfu/cm2 at 10 d. CPC treatment caused a slight discoloration and ASC-treated beef surfaces demonstrated the lowest redness and highest lightness. The grey colour and off-odour were significant in the ASC-treated beef samples, while CPC-treated samples demonstrated less off-odor and brown colour from 0 to 4 d. Based on our results, it appears that the application of CPC on sliced roast beef can extend the shelf-life of the product without impairing its quality.     

A multiplex RTi-PCR reaction for simultaneous detection of Escherichia coli O157:H7, Salmonella spp. and Staphylococcus aureus on fresh, minimally processed vegetables

In this work, a new multiplex single-tube real-time PCR approach is presented for the detection of Escherichia coli O157:H7, Salmonella spp. and Staphylococcus aureus, three of the more frequent food-borne bacterial pathogens that are usually investigated in a variety of food matrices. The study includes the design and specificity testing, of a new primer and probe specific for Salmonella spp. Reaction conditions were adjusted for the simultaneous amplification and detection of specific fragments in the beta-glucuronidase (uidA, E. coli) and Thermonulease (nuc, Sta. aureus) genes, and in the replication origin sequence (oriC, Salmonella spp.). Melting-curve analysis using a SYBR Green I RTi-PCR approach showed characteristic T(m) values demonstrating the specific and efficient amplification of the three fragments. Subsequently, a TaqMan RTi-PCR approach was settled, using FAM, NED and VIC fluorescently labelled specific probes for an automated detection. It was equally sensitive than uniplex RTi-PCR reactions in Sta. aureus and E. coli O157:H7, using same amounts of purified DNA, and allowed detection of 10 genome equivalents in the presence of 10(2) or 10(4) genome equivalents of the other two pathogens. Finally, it was tested in artificially inoculated fresh, minimally processed vegetables, revealing a sensitivity of 10(3)CFUg(-1) each of these pathogens in direct detection, following DNA extraction with DNeasy Tissue Kit (Qiagen). The multiplex RTi-PCR developed scored the sensitivity recognised for PCR in food and it allows a high-throughput and automation, thus it is promising as a rapid and cost-effective test for the food industry.     

Purification and characterization of a novel fungi Se-containing protein from Se-enriched <Emphasis Type="Italic">Ganoderma Lucidum</Emphasis> mushroom and its Se-dependent radical scavenging activity

Our previous studies have established that Ganoderma lucidum (GL) can biotransform inorganic selenium into organic selenium, which was stored preferentially in its water-soluble protein component. In the present study, a novel water-soluble fungi Se-containing protein, named as Se–GL–P, has been purified for the first time from the Se-enriched GL to establish the relationship between the antioxidant activity of protein and its Se content. This protein was isolated and purified to homogeneity using ammonium sulfate precipitation followed by two consecutive anion exchange chromatography and size exclusion chromatography. This protein in its native state was identified as a monomer of 36,600 Da estimated by 19.8% weight carbohydrate. The protein is acidic (pI=4.0) and rich in Asp, Glu, Gly, and Ala. The N-terminal shows a sequence of DINGGGATLPQKLYLTPDVL, suggesting that this protein belongs to a family of D I N G protein. The Se-content of this protein (4.87 mg Se/g protein) is ∼3-fold larger than that of the water-soluble protein extract (1.91 mg Se per protein). Perfectly consistent with its higher Se-content, the protein exhibits approximately three times stronger activity of scavenging superoxide and hydroxyl radicals as compared to the water-soluble protein extract, a finding demonstrating that the increasing antioxidant property of this protein depends quantitatively on its Se content.

Evaluation of a novel antimicrobial (lauric arginate ester) substance against biofilm of Escherichia coli O157:H7, Listeria monocytogenes,and Salmonella spp.

The purpose of this study was to evaluate the activity of a novel antimicrobial substance lauric arginate ester (LAE) against selected foodborne pathogens (Escherichia coli O157:H7, Listeria monocytogenes and Salmonella spp.) in biofilm. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined and showed that LAE exhibits a strong antimicrobial activity. Biofilms were grown on abiotic stainless steel, rubber, MBEC biofilm device) and biotic (lettuce) surfaces. The efficacy of LAE (50, 100 and 200 ppm) at reducing the biofilm cells on these surfaces was examined by applying LAE for 2 h. Results revealed that LAE exhibited the reduction in biofilm bacteria up to 7 log CFU cm^?2, 3.5 log CFU cm^?2, 4.0 log CFU peg^?1 and 1.5 log CFU cm^?2 on stainless steel, rubber, MBEC and lettuce surfaces, respectively. Overall, these results suggest that LAE has been shown to be a potential alternative to control bacteria in biofilm mode in food industry.