Detection of pathogenic and spoilage micro-organisms in food with the polymerase chain reaction

Traditional biochemical and immunochemical methods for the detection of micro-organisms in food have been supplemented by a number of DNA-based methods during the last decade. Besides the development of direct hybridization techniques, emphasis has been laid onin vitroamplification methods.In vitromethods such as the self-sustained sequence replication (3SR) or the nucleic acid sequence based amplification (NASBA), the Q-beta replicase amplification and the ligase chain reaction (LCR) have had, until now, only limited practical relevance for food monitoring and control. The most developedin vitroamplification method is the polymerase chain reaction (PCR) that allows rapid and selective identification of micro-organisms. Because PCR is increasingly raising interest as a detection method in food hygiene and control, an overview of PCR-systems for the detection of bacteria and viruses in food is provided with this paper. Although the PCR method has advantages (particular specificity, sensitivity) it also has limitations, one of which is its inability to allow differentiation between viable and non-viable micro-organisms. Furthermore, the inhibition of PCR by food-derived inhibitors can result in false negative results. Possible reasons for PCR inhibition and ways to differentiate between viable and dead micro-organisms are discussed in this review.     

七种单离食用香料对食品腐败菌抑菌活性的研究

通过测定抑菌圈直径和最低抑菌浓度,研究百里香酚、水杨醛、肉桂醛、丁香酚、茴香脑、柠檬醛和香兰素七种单离食用香料对引起食品腐败的供试菌种的抑菌活性.结果表明:七种香料均对细菌、霉菌和酵母具有一定的抑制能力,且表现出对霉菌抑菌较强的特点;其综合抑菌能力强弱顺序为:百里香酚＞水杨醛＞肉桂醛＞丁香酚＞茴香脑＞柠檬醛、香兰素.实验进一步选取百里香酚、丁香酚、肉桂醛、水杨醛四种香料进行优化重组,重组配比为5:5:5:1时,0.05μL/mL重组香料就可以完全抑制所有供试菌的生长.     

Antimicrobial activity of Microgard against food spoilage and pathogenic microorganisms

Microgard, a commercially available fermented milk product containing antimicrobial metabolites, was a potent inhibitor for Gram-negative bacteria such as Pseudomonas, Salmonella, and Yersinia when 1% concentration was incorporated into agar media. Gram-positive Bacillus cereus, Staphylococcus aureus, and Listeria monocytogenes were insensitive to Microgard. Kluyveromyces marxianus, an unidentified black yeast, and Penicillium expansum were partially suppressed, whereas Aspergillus niger and a yogurt spoilage yeast were tolerant to 5% Microgard. Optimum activity of Microgard was at pH 5.3 and below; the concentration that gave complete inhibition depended upon the number of bacteria present as well as the genus tested. Blood agar base reversed the antagonistic activity of Microgard against Pseudomonas putida compared with plate count agar.     

Antagonistic action of lactic cultures toward spoilage and pathogenic microorganisms in food

The antibacterial properties of cell-free filtrate from lactic cultures were assessed against 10 bacterial cultures. All the five species of lactic culture examined showed antimicrobial activity against tested bacteria. S. aureus was least sensitive of the tested bacteria, followed by E. coli and S. typhosa. E. aerogenes was the most sensitive one. L. casei had the greatest antimicrobial activity. Leu. mesenteroides weakly inhibited the growth of the tested bacteria. In general, inoculum density had little effect on inhibition.     

Antibiotic susceptibility of bacteria isolated from pasteurized milk and characterization of macrolide-lincosamide-streptogramin resistance genes

The presence of antibiotic-resistant bacteria in pasteurized milk was detected by plating 18 milk samples on selective media containing beta-lactams, macrolides, or a glycopeptide. Most samples contained gram-positive bacteria that grew on agar plates containing oxacillin, erythromycin, and/or spiramycin. The disk-diffusion method confirmed resistance to erythromycin and/or spiramycin in 86 and 65% of the coryneform bacteria and Micrococcaceae tested, respectively. PCR and sequence analysis revealed the presence of an ermC gene in 2 of the 25 Micrococcaceae strains investigated for their resistance to erythromycin and/or spiramycin. None of the 14 corynebacteria strains resistant to erythromycin and/or spiramycin harbored the erm(X) gene. No gene transfer could be demonstrated between the two erm(C) staphylococcal isolates and recipient strains of Enterococcus faecalis JH2-2 or Staphylococcus aureus 80CR5.     

市售即食食品中非致病菌的耐药性及耐药基因转移的研究

目的 了解西安市即食食品中的非致病菌的耐药性分布、耐药程度及耐药基因的转移情况,分析潜在的食品安全问题.方法 对西安市的几家大型超市零售的熟肉、果盘、凉拌菜3大类即食食品样品进行食源性非致病菌的红霉素和氨苄青霉素耐药性分析、耐药基因转移、菌株16 S rRNA序列测序鉴定等.结果 42.5％的分离株具有不同程度的红霉素耐药性,47.5％的分离株具有不同程度的氨苄青霉素耐药性,21.2％的分离株同时具有2种抗生素耐药性;11.3％的红霉素耐药株耐药性高达160 mg/L,6.3％的氨苄青霉素耐药株耐药性高达500 mg/L.随机挑选的部分耐药株在无抗生素培养基中连续传代1000代以后,60％的氨苄青霉素耐药株和45％的红霉素耐药株的耐药性消失.部分耐药株的耐药基因可水平转移至非耐药株中.这些耐药株多为肠球菌、葡萄球菌、芽孢杆菌、假单胞菌等.结论 即食食品中的非致病菌有潜在的风险,耐药基因在细菌间的转移可能对人体敏感肠道菌群和致病菌有一定影响.     

Development of active plastic crate and evaluation of phytopathogenic and pathogenic food spoilage micro‐organism inhibition

BACKGROUND: The plastic crates used in fruit and vegetable shipping can be vehicles of disease dissemination among production fields, since there is a chance of phytopathogenic micro‐organism adhesion on the crate surfaces when in contact with soil, contaminated produce or handling. The aim of this study was to develop an active plastic crate incorporated with a triclosan‐based antimicrobial agent and to evaluate its efficiency of micro‐organism inhibition. RESULTS: Staphylococcus aureus (a human pathogen), Clavibacter michiganensis ssp. michiganensis and Erwinia carotovora ssp. carotovora (phytopathogens) were the most sensitive micro‐organisms when in contact with samples of plastic crate incorporated with 30 g kg^?1 of antimicrobial agent. They presented diameters of approximately 5.0, 3.5 and 3.5 cm respectively in the halo test. Mean specific growth rates decreased in samples with 30 g kg^?1 of antimicrobial agent, compared with control samples, from 1.13 to 0 h^?1 for S. aureus, from 1.26 to 0.47 h^?1 for Escherichia coli and from 1.75 to 0.18 h^?1 for Listeria monocytogenes. The antimicrobial agent did not influence the mechanical properties of the crates. CONCLUSION: The active plastic crate has great potential to contribute to the safety of horticultural produce by restraining the proliferation of micro‐organisms among production fields. Copyright © 2008 Society of Chemical Industry     

The distribution of Enterobacteriaceae and some pathogenic micro-organisms in nonbranded white cheese samples sold in Ankara city

In this research, 75 samples of nonbranded white cheese, presented for sale in small markets from various regions and bazaars in Ankara, were studied. Eighty-six isolates, 71 of which are Gram-negative isolates and 15 of which are Gram-positive isolates, were obtained. These isolates were identified by using bioMérieux API20E and classical methods. Total mesophilic bacteria counts and total coliform bacteria counts were carried out for each white cheese sample. The total average mesophilic bacteria of 75 white cheese samples was 15.5 × 10⁵ cfu/g and the total average coliform bacteria were 13.6 × 10⁵ cfu/g.     

Cell surface hydrophobicity and attachment of pathogenic and spoilage bacteria to meat surfaces

Cell surface hydrophobicity of several pathogenic and spoilage bacteria was determined by hydrocarbon adherence, adhesion to nitrocellulose filter, salt aggregation and adherence to phenyl-sepharose beads. Hydrophobicity of each bacterium was dependent on the method of measurement. Hydrophobicity was compared with the strength of attachment (Sr) of the bacteria to beef muscle surfaces. Bacterial cell surface hydrophobicity, as determined by the bacterial adherence to xylene correlated well with attachment strength (r = 0.800, P < 0.05). Staphylococcus aureus, Clostridium perfringens and Yersinia enterocolitica showed the highest values of attachment strength.     

香辛料提取物对葡萄主要致病菌抑菌效果的研究

采用11种香辛料提取物对葡萄的5种主要致病菌进行抑菌实验,并对5种抑菌效果较好的提取物最低抑菌浓度(MIC)值进行了测定,又比较了5种香辛料提取物的任意3种1∶1∶1比例配伍对参试菌的抑制作用。结果表明:丁香、百里香、牛至、八角茴香、肉桂提取物的抑菌效果较好,尤以丁香提取物对5种主要致病菌的抑菌作用最明显,其抑菌圈直径分别为:大肠杆菌17.45mm,金黄色葡萄球菌31.45mm,黑曲霉38.38mm,灰葡萄孢霉54.70mm,青霉25.18mm;这5种提取物的MIC也以丁香最低,分别为:大肠杆菌6.25mg/m L,金黄色葡萄球菌12.5mg/m L,黑曲霉3.13mg/m L,灰葡萄孢霉1.56mg/m L,青霉12.5mg/m L。配伍提取液的抑制效果以百里香、丁香、八角茴香的配伍方案最优,但低于丁香单一提取液的抑菌效果。表明丁香提取液可以应用于葡萄的保鲜。     

食品中腐败酵母的实时荧光PCR鉴定

为研究食品中腐败酵母实时荧光PCR快速鉴定方法,设计和筛选出了可用于腐败酵母鉴定的多条探针和引物,并建立了针对酿酒酵母、鲁氏接合酵母、斯巴达克毕赤酵母和布鲁塞尔德克酵母等4种腐败酵母菌的实时荧光PCR鉴定方法。用文中建立的方法对从糕点、蜂蜜、饮料等市售食品中分离出的60余株酵母菌进行了鉴定,发现其中4株为酿酒酵母,21株为鲁氏酵母,其他为与上述4种不同的酵母。以实时荧光PCR方法鉴定酵母,全过程仅需约3 h,与常用的生化鉴定方法相比,简化了鉴定步骤,提高了鉴定准确性,缩短了鉴定时间。     

几种香辛料对鱼油抗氧化及消臭作用的研究

用乙醇、石油醚分别从辣椒、生姜和香菜中提取抗氧化物质 ,比较了不同溶剂的提取率和不同提取物对鱼油的抗氧化效果。结果表明 ,乙醇对三者均有较高的提取率 ,抗氧化作用以辣椒的乙醇提取物为最强。此外 ,还初步研究了上述几种香辛料提取物对鱼油的消臭效果     

2012年中国食源性单核细胞增生李斯特菌耐药特征及多位点序列分型研究

研究2012年中国23个省市自治区10类食品中分离的635株单核细胞增生李斯特菌的耐药特征及多位点序列分型(multilocus sequence typing,MLST)测定。方法 采用CLSI推荐的微量肉汤稀释法,选择氨苄西林、复方新诺明、四环素、庆大霉素、红霉素、环丙沙星、氯霉素和万古霉素8种抗生素,对耐药株进行MLST分型。结果 635株单增李斯特菌检测出66株耐药菌,平均耐药率为10.39%。耐四环素菌株最多为49株,其次为耐环丙沙星20株、红霉素10株、氯霉素7株、复方新诺明3株、氨苄青霉素1株、庆大霉素1株、万古霉素1株。耐受2种抗生素有8株,耐受3种及以上抗生素有7株。77株菌耐药性介于中介度,其中75株菌对环丙沙星耐药性介于中介度。耐药株MLST分型表明,ST155、ST9、ST705和ST87为我国单增李斯特菌耐药株常见型别。四环素和四环素-红霉素-氯霉素耐药谱在MLST聚类分析中有集中趋势,耐药菌株主要来源于熟肉制品和中式凉拌荤菜。结论 我国目前食品来源的单增李斯特菌耐药率普遍较低,但与往年相比呈现逐渐增加的趋势。耐药株的主要MLST型别分布与相关耐药谱在聚类分析中相互关联。     

香辣蟹中特征性腐败菌的分离鉴定以及香料的抑制作用研究

目的分析香辣蟹产品的特征性腐败菌,用于指导筛选特定的香辛料配方,以延长产品保质期。方法从自然腐败的市售香辣蟹产品中分离纯化出特征性腐败菌,通过生理生化实验和16SrRNA序列比对法对腐败细菌进行鉴定,选取不同属的腐败菌进行常用香辛料提取物的抑菌效果评价。通过进一步测定香辣蟹在储藏过程中菌落总数和挥发性盐基氮值,评估不同香辛料提取物对香辣蟹的保鲜效果。结果分离所得的15株腐败菌中10株为革兰氏阳性细菌,其余均为革兰氏阴性细菌, 3株属于葡萄球菌属(Staphylococcus. sp), 6株属于白色杆菌属(Leucobacter.sp),3株属于变形杆菌属(Proteus.sp),2株属于普罗威登斯菌(Providencia vermicola),1株属于节杆菌属(Arthrobacter.sp)。不同品种的香辛料对腐败菌生长的抑制作用差异较大,公丁香、八角、香砂、千里香、栀子、草寇对5种指示腐败菌均有体外抑菌作用,研究采用的香辛料配方降低香辣蟹储藏过程中菌落总数和挥发性盐基氮值的增加速度。结论合适的香辛料能延长香辣蟹储藏时间,可以作为香辣蟹天然保鲜剂。     

食品防腐剂的概况与发展

食品的腐败变质会引起巨大的经济损失，如何防止腐败是食品科学工作者最为关注的一个问题。目前，常用的防腐措施是添加食品防腐剂，食品防腐剂分为化学防腐剂、天然防腐剂和复合型防腐剂，其中使用最为广泛的是化学防腐剂。但随着人们对健康的重视，天然防腐剂和复合型防腐剂的应用会越来越广泛。本文将对食品防腐剂分类进行阐释。     

High Pdr12 levels in spoilage yeast (Saccharomyces cerevisiae) correlate directly with sorbic acid levels in the culture medium but are not sufficient to provide cells with acquired resistance to the food preservative

Sorbic acid is a commonly used food preservative against yeast and fungal food spoilage. Understanding its effect on the molecular physiology of yeast cells will allow the food industry to develop knowledge-based strategies to make more optimal use of its preservative action. Here we show that the yeast membrane protein Pdr12, previously shown to be prominently involved in sorbic acid resistance development in laboratory strains, was strongly induced by the presence of sorbic acid in the culture medium in Saccharomyces strains isolated from spoiled foods. Induction of Pdr12 expression was seen both under laboratory conditions and upon growth in a commercial soft drink. Induction was rapid and maintained for the duration of the stress. No Pdr12-like protein induction was seen in Zygosaccharomyces bailii or Zygosaccharomyces lentus, two well-known beverages spoilage organisms. Finally, unexpectedly, our studies showed for the first time that pre-inducing Pdr12p to maximal levels by subjecting cells to a mild sorbic acid stress did not lead to cells with an acquired resistance. Neither more rapid growth in the presence of the acid nor growth at higher sorbic acid concentrations at a given environmental pH was observed. Thus we have shown that while important in resistance development against sorbic acid, by itself induction of the pump is not sufficient to acquire resistance to the preservative.     

Testing nano-silver food packaging to evaluate silver migration and food spoilage bacteria on chicken meat

Migration of nanomaterials from food containers into food is a matter of concern because of the potential risk for exposed consumers. The aims of this study were to evaluate silver migration from a commercially available food packaging containing silver nanoparticles into a real food matrix (chicken meat) under plausible domestic storage conditions and to test the contribution of such packaging to limit food spoilage bacteria proliferation. Chemical analysis revealed the absence of silver in chicken meatballs under the experimental conditions in compliance with current European Union legislation, which establishes a maximum level of 0.010 mg kg^–1 for the migration of non-authorised substances through a functional barrier (Commission Regulation (EU) No. 10/2011). On the other hand, microbiological tests (total microbial count, Pseudomonas spp. and Enterobacteriaceae) showed no relevant difference in the tested bacteria levels between meatballs stored in silver-nanoparticle plastic bags or control bags. This study shows the importance of testing food packaging not only to verify potential silver migration as an indicator of potential nanoparticle migration, but also to evaluate the benefits in terms of food preservation so as to avoid unjustified usage of silver nanoparticles and possible negative impacts on the environment.     

Antibiotic resistance of coagulase-negative staphylococci associated with food and used in starter cultures

The resistance of 330 coagulase-negative staphylococci (CNS) associated with food or used in starter cultures and belonging to the species Staphylococcus carnosus, Staphylococcus condimenti, Staphylococcus piscifermentans, Staphylococcus equorum, Staphylococcus succinus and Staphylococcus xylosus, against 21 antibiotics was determined using the disk diffusion method. The incidence and number of resistances was found to be species and source of isolation dependent. Most strains of S. equorum (63%), S. succinus (90%) and S. xylosus (95%) exhibited resistances against up to seven antibiotics, whereas only few strains of S. carnosus (12%) and S. piscifermentans (27%) were antibiotic resistant. Resistances to lincomycin, penicillin, fusidic acid, oxacillin, ampicillin and tetracycline were predominant. Among strains of S. xylosus, the incidence of resistance ranged from 22% for tetracycline up to 69% for penicillin. Concerning the source of isolation, resistances were often determined in strains of S. equorum, S. succinus and S. xylosus isolated from cheese (87%) and sausage (83%), and strains of S. xylosus obtained from meat starter cultures (93%). Remarkably, all CNS were sensitive to the clinically important antibiotics chloramphenicol, clindamycin, cotrimoxazol, gentamicin, kanamycin, linezolid, neomycin, streptomycin, synercid and vancomycin. The phenotypic resistances to ss-lactam antibiotics, lincomycin and tetracycline were verified by PCR amplification and could be traced back to the genes blaZ, lnuA and tetK, respectively. This study permitted a comprehensive insight into the incidence of antibiotic resistances in food-associated CNS.     

Chemometric approach to evaluate trace metal concentrations in some spices and herbs

Herbs (mint, thyme and rosemary) and spices (black pepper, chili pepper, cinnamon, cumin, sweet red pepper and turmeric) were analysed using atomic spectrometry and then subjected to chemometric evaluation in an attempt to classify them using their trace metallic analyte concentrations (As, Ba, Ca, Cd, Co, Cr, Cu, Fe, Mg, Mn, Ni, Sr and Zn). Trace metals in acid digests of these materials were determined using both inductively coupled plasma-atomic emission spectrometry and inductively coupled plasma-mass spectrometry. The chemometric techniques of principal component analysis (PCA), linear discriminant analysis (LDA) and cluster analysis (CA) were used for the classification studies. These herbs and spices were classified into five groups by PCA and CA. When the results of these techniques were compared with those from LDA, it was found that all group members determined by PCA and CA are in the predicted group that 100.0% of original grouped cases correctly classified by LDA.