脆肉鲩鱼在冷藏条件下的特定腐败菌分析

利用16S rDNA克隆文库及限制性内切酶片段长度多态性(Restriction Fragment Length Polymorphism,RFLP)技术分析脆肉鲩鱼在冷藏条件下的特定腐败菌。分别提取新鲜鱼肉及腐败的脆肉鲩鱼肉样品中的细菌总DNA并利用通用引物27F/1492R进行PCR扩增,所得PCR产物用于构建克隆文库,阳性克隆子用RFLP进行分析。结果表明:新鲜鱼肉的克隆文库中细菌种类比较丰富,达15种类型,而腐败鱼肉的细菌种类只有7种,且有2种分型的细菌占优势,共占克隆子总数的88.4%。序列比对分析表明,这2种优势菌的16S rDNA序列与假单孢菌属(Pseudomonas)细菌的核苷酸序列的相似性高达99%,说明脆肉鲩鱼在冷藏条件下的特定腐败菌是假单孢菌(Pseudomonassp.)。     

Behaviour of Brochothrix thermosphacta in presence of other meat spoilage microbial groups

The microbial flora of fresh meat stored aerobically at 5 degrees C up to spoilage was enumerated and collected in order to have mixed spoilage bacterial groups to be used in competition tests against Brochothrix thermosphacta. The bacterial groups collected as bulk colonies were identified by PCR-DGGE followed by partial 16S rDNA sequencing. The predominant bacteria associated with the spoilage of the refrigerated beef were B. thermosphacta, Pseudomonas spp, Enterobacteriaceae and lactic acid bacteria (LAB). The interactions between B. thermosphacta and the other spoilage microbial groups were studied in vitro at 5 degrees C. The results showed that a decrease of the growth of B. thermosphacta was evidenced in presence of LAB at 5 degrees C while the bacterium is the dominant organism when inoculated with mixtures of Pseudomonas spp., LAB and Enterobacteriaceae. A better understanding of bacterial meat spoilage interactions may lead to improved quality of fresh meat stored in refrigerated conditions.     

Predominant enterobacteria on modified-atmosphere packaged meat and poultry

Enterobacteria on modified-atmosphere (MA) packaged meat (n = 54) and poultry (n = 32) products were enumerated, and 899 isolates were picked and ribotyped. For identification, 16S rRNA genes of representative strains were sequenced and analyzed. Altogether 54 (60%) of the samples contained enterobacteria >10⁴ CFU/g. In 34% of the poultry samples, enterobacteria counts were >10⁶ CFU/g suggesting that enterobacteria may contribute to spoilage of MA packaged poultry. The enterobacteria identified were predominantly Hafnia spp. (40%) and Serratia spp. (42%) with Hafnia alvei, Hafnia paralvei, Serratia fonticola, Serratia grimesii, Serratia liquefaciens, Serratia proteamaculans, and Serratia quinivorans being the species identified. In addition, 6% of the isolates were identified as Rahnella spp., 3% as Yersinia spp., and 1% as Buttiauxella spp. Percentage distributions of the predominant genera in different products showed that 89% of the Serratia spp. were from products packaged under a high-O₂ MA containing CO₂ (25–35%), whereas most (76%) isolates of Hafnia originated from anaerobically packaged red meat and poultry. These findings suggest that the gas mixture used for MA packaging influence the selection of enterobacteria growing on meat and poultry.     

Spoilage-related microbiota associated with chilled beef stored in air or vacuum pack

In order to study the spoilage-related microbiota of beef at species level, a combination of culture-independent and culture-dependent methods was used to analyse nine different beef samples stored at 4 °C in air or in vacuum pack. Plate counts on selective agars after 0, 7 and 20 days of storage showed that vacuum packaging reduced the viable counts of Brochothrix thermosphacta, Pseudomonas spp. and Enterobacteriaceae, whereas the growth of lactic acid bacteria (LAB) was unaffected. Storage in vacuum pack mainly affected viable counts and not necessarily the species diversity of microbial populations on meat. Such populations were studied by PCR-DGGE of DNA directly extracted from meat and from bulk cells from culture media, followed by sequencing of DGGE fragments. Pseudomonas spp., Carnobacterium divergens, B. thermosphacta, Rahnella spp. and Serratia grimesii, or close relatives were detected in the meat at time zero. The use of the culture-independent method highlighted the occurrence of species that were not detected by plating. Photobacterium spp. occurred in most meat samples stored in air or in vacuum pack, which indicates this organism probably has a role in spoilage. In contrast, culture-dependent analysis allowed detection of bacterial species that were not found in DNA extracted directly from meat. This was the case for several species of Serratia or Rhanella among the enterobacteria, and Leuconostoc spp. among the LAB. Besides advancing our knowledge of the species involved in the spoilage of vacuum-packaged meat, this study shows the benefits of combining culture-based and direct approaches to enhance understanding of populations of spoilage bacteria.     

Leuconostoc gasicomitatum is the dominating lactic acid bacterium in retail modified-atmosphere-packaged marinated broiler meat strips on sell-by-day

Lactic acid bacteria (LAB) in retail, modified-atmosphere-packaged (MAP), marinated broiler meat strips on sell-by-day were mainly identified as Leuconostoc gasicomitatum. A total of 32 packages, three to five packages of seven differently marinated broiler meat products, were studied at the end of the producer-defined shelf life (at 6 degrees C, 7-9 days depending on the manufacturer). Prior to the microbiological analyses, appearance and smell of the product was checked and pH measured. Bacteria were cultured on MRS and Tomato Juice Agar (TJA), Rogosa SL agar (SLA), Plate Count Agar (PCA) and Streptomycin Thallium Acetate Agar (STAA) for the enumeration of LAB, lactobacilli, total bacterial count and Brochothrix thermosphacta, respectively. The average CFU/g of the 32 packages was 2.3 x 10(8) on PCA. The highest bacterial average, 3.1 x 10(8), was recovered on TJA, the corresponding CFU/g averages on MRS and SLA being 2.3 x 10(8) and 1.3 x 10(8), respectively. Despite the high LAB numbers detected, radical spoilage changes such as unpleasant odor, slime production and formation of gas were not seen. B. thermosphacta did not form a significant part of the bacterial population since none of the levels exceeded the spoilage threshold level of 10(5) CFU/g reported in previous studies for this organism. In order to characterize the dominating LAB population, as many as 85, 85 and 88 colonies from MRS, TJA and SLA, respectively, were randomly picked and cultured pure. LAB were identified to species level using a 16 and 23S rDNA HindIiI RFLP (ribotyping) database. Fifty-six of the 170 isolates picked from the non-selective LAB media (MRS and TJA) were identified as L. gasicomitatum, followed by Carnobacterium divergens (41 isolates), Lactobacillus sakei and Lactobacillus curvatus subsp. melibiosus (31 isolates) and L. curvatus subsp. curvatus (20 isolates) species. SLA proved not to be completely selective for lactobacilli because the growth of Leuconostoc spp. was not inhibited, Carnobacterium spp. were the only species not detected on SLA.     

Characterization and evaluation of the spoilage potential of Lactococcus piscium isolates from modified atmosphere packaged meat

A total of 222 psychrotrophic lactococci isolated from use-by day, modified atmosphere packaged (MAP) meat were identified to the species level by numerical analyses of EcoRI and ClaI ribopatterns and phylogenetic sequence analyses of 16S, rpoA and pheS genes. In addition, their meat spoilage potential was studied. The majority of the isolates (n=215) were identified as Lactococcus piscium, while seven isolates belonged to Lactococcus raffinolactis. L. piscium was shown to be adapted to growing in a variety of MAP meat products including broiler, turkey, pork, and minced meat from beef and pork, where they belonged to the predominating microbiota at the end of the storage. Numerical analyses of EcoRI and ClaI ribopatterns, and phylogenetic sequence analyses of rpoA and pheS genes were shown to be reliable tools in species level identification of meat lactococci. The spoilage potential of L. piscium was evaluated by inoculating representative isolates to MAP pork stored at 6 °C for 22 days. Development of spoilage population was monitored using a culture-independent T-RFLP approach. The sensory shelf life of pork inoculated with L. piscium was shortened compared to the uninoculated control. Alongside with the inoculated L. piscium isolates, Leuconostoc spp. present as initial contaminants in the samples thrived. This shows that even though lactococci were inoculated at higher levels compared to the natural microbiota, they did not occupy the niche and prevent the growth of other lactic acid bacteria.     

Molecular identification of dominant microflora associated with 'Hawaijar' - a traditional fermented soybean (Glycine max (L.)) food of Manipur, India

The dominant microorganisms in 'Hawaijar', a traditional non-salted fermented soybean (Glycine max (L.)) food of Manipur, India, were isolated and identified by molecular techniques. Bacillus spp. were the predominant microorganisms in 'Hawaijar'. A total of 274 isolates were obtained from forty-one 'Hawaijar' samples collected from household preparations and markets of valley districts of Manipur. Phenotypic grouping followed by Amplified Ribosomal DNA Restriction Digestion Analysis (ARDRA), PCR amplification of 16S-23S rDNA region, RFLP by Hae III and Hind III double digest (by comparing MTCC type strains) and sequencing of 9-1514 region of 16S rDNA resulted in three major phylogenic groups. Bacillus subtilis group comprising B. subtilis and B. licheniformis representing 112 isolates, B. cereus group representing of 128 isolates and Staphylococcus spp. group comprising S. aureus and S. sciuri representing 23 isolates. A few bacterial isolates belongs to Alkaligenes spp. and Providencia rettgeri were also found. Genetic diversity of B. subtilis phylogenic group was investigated by RAPD-PCR. Eighty-two strains of B. subtilis phylogenic group were identified using RAPD-PCR using OPA-18 and OPA-20 primers. These strains will be investigated for potential starter culture selection.     

Identification of extracellular DNase-producing bacterial populations on catfish fillets during refrigerated storage

Food spoilage is a major problem faced by consumers across the globe. As an enzyme that degrades DNA, DNase production on fish tissue seemed likely to aid in fish spoilage. Based on physical characteristics, bacteria producing extracellular DNase were isolated on selective media. 16S rDNA sequences were obtained identifying isolates as bacteria belonging to Aeromonas spp., Serratia spp., Shewanella spp., and Rahnella spp. Aeromonas spp. were the predominant bacteria isolated in this study; this statistically suggests that Aeromonas spp. are dominant in DNase-producing bacterial populations on catfish tissue. Results obtained in this study suggest that extracellular DNase-producing bacteria play a large role in catfish spoilage and support the need for further research on the role of Aeromonas spp. in fish spoilage. Rahnella spp. was isolated from catfish fillets in this study and identified, for the first time, as DNase producing bacteria.     

新疆椒麻鸡中腐败细菌的分离与鉴定

为研究新疆椒麻鸡中腐败细菌的种类和特性,采用平板划线法分离腐败菌。利用生理生化实验和16S rDNA序列分析方法,对从椒麻鸡中分离的31 株腐败菌进行鉴定。结果表明：所筛选的31 株菌中,肠杆菌20 株、葡萄球菌3 株、气单胞菌2 株、乳酸菌4 株、溶酪大球菌2 株;新鲜椒麻鸡中的主要腐败菌为溶酪大球菌、葡萄球菌和肠杆菌;低温5 ℃贮藏条件下腐败样品中的主要腐败菌为乳酸菌、葡萄球菌和肠杆菌;常温25 ℃贮藏条件下腐败椒麻鸡中的腐败菌主要为肠杆菌、气单胞菌、葡萄球菌和乳杆菌。     

酱香型窖泥古菌群落结构的研究

采用ARDRA免培养手段研究酱香型窖泥中的古菌群落结构,通过对窖泥总DNA的提取,扩增古菌16SrDNA序列,构建古菌16S rDNA文库。随机挑选39个阳性克隆子,通过HhaI限制性内切酶酶切,选择酶切图谱不同的25个克隆子用于后续测序,测序结果与NCBI数据库比对分析,获得其分类信息。克隆文库分析结果表明,酱香型窖泥中古菌主要分布于广古菌门中的甲烷袋状菌属（Methanoculleus）、甲烷八叠球菌属（Methanosarcina）、甲烷鬃毛菌（Methanosaeta）和甲烷杆菌属（Methanobacterium）,分别占44%、41%、3%、9%。     

Characterization of psychrotrophic bacterial communities in modified atmosphere-packed meat with terminal restriction fragment length polymorphism

Characterization of psychrotrophic lactic acid bacteria (LAB) and Brochothrix thermosphacta communities is needed to understand the microbial ecology of spoilage of modified atmosphere-packed (MAP) meats. To overcome the limitations of the currently used methods for the characterization of psychrotrophic bacterial communities in meat, we developed a culture-independent, 16S rRNA gene-targeted terminal restriction fragment length polymorphism (T-RFLP) method. An identification library consisting of 100 Gram-positive and 30 Gram-negative meat-associated bacterial strains was set up to identify the terminal restriction fragments derived from the communities. The taxonomic resolution level of the T-RFLP method was in between genus and species within the investigated LAB strains and within family and genus within the investigated Gram-negative strains. The established library was applied to identify the members of bacterial communities in MAP minced meat at the end of the shelf life. The T-RFLP results and plate counts on Man-Rogosa-Sharpe, Violet Red Bile Glucose, and Streptomycin sulfate thallium acetate actidione agars indicated that LAB and B. thermosphacta predominated in meat. The bacterial taxa associated with the T-RFLP results were compared to those identified among plate-grown LAB isolates by numerical ribopattern analysis. Both methods agreed that Leuconostoc spp. and Carnobacterium spp. prevailed in the LAB community in minced meat followed by Lactobacillus algidus, Lactococcus spp. and Weissella spp. Colony identification revealed that Leuconostoc gasicomitatum, L. gelidum, Carnobacterium divergens and C. maltaromaticum were the predominant LAB species. The T-RFLP results were shown to correlate with viable counts of Leuconostoc spp. and B. thermosphacta. The T-RFLP method was found to be a useful tool enabling rapid and high-throughput characterization of psychrotrophic bacteria prevailing in MAP meat.     

羊肉屠宰加工场主要污染菌的分布及其对熟制羊肉的致腐能力

对江苏省苏州市具有代表性的某羊屠宰场的屠宰环境及熟制品加工车间进行微生物检测,重点对熟制品加工过程进行检测,包括煮制车间的接触面、各加工车间的空气以及清洗前后的生肉与煮制后的熟肉,以确定熟制品加工过程中污染菌群的分布。结果表明,熟制品加工过程中各加工车间空气细菌数均低于10 CFU/皿,表明空气质量合格。而煮制车间的接触面污染较严重致使交叉污染造成熟制羊肉的菌落总数达到3.23(lg(CFU/cm~2))。结合形态观察和16S rDNA菌种鉴定,所污染的菌主要为芽孢杆菌、腐生葡萄球菌、变形杆菌、金黄杆菌和微杆菌等。随后选取3个污染菌芽孢杆菌(Bacillus sp.M1、Bacillus sp.M9)和腐生葡萄球菌(Staphylococcus saprophyticus M7),通过对熟制羊肉的感官评分、pH值、菌落总数、挥发性盐基氮值和腐败代谢产物产量因子的测定评定它们对熟制羊肉的致腐能力。结果显示,Bacillus sp.M1的致腐能力最强且明显高于S.saprophyticus M7和Bacillus sp.M9,为有效预防和控制熟制羊肉的微生物污染提供依据。     

冷鲜牛肉贮藏中菌群结构及优势菌致腐性的分析

为探究牛肉在0?℃冷藏下微生物菌群变化和优势腐败菌,采用培养依赖的16S rRNA结合高通量测序技术分析牛肉样品的微生物多样性变化。通过定期测定接种牛肉汁的生长、感官、蛋白酶活性、pH值和挥发性盐基氮（total volatile basic nitrogen,TVB-N）值,分析分离株的致腐特征。结果表明,牛肉冷藏中感官品质保持良好,15?d出现异味,18?d有腐臭味。而样品中菌落总数第3天快速上升,15?d菌落总数为8.73（lg（CFU/g））,之后呈现稳定,假单胞菌（Pseudomonas spp.）、热死环丝菌（Brochothrix thermosphacta）、肠杆菌（Enterobacter）和乳酸菌4 种分离培养基中细菌与菌落总数增长趋势相似,其中假单胞菌增长最快,肠杆菌数和乳酸菌数生长最慢。两种菌群鉴定结果显示,冷鲜牛肉初始微生物构成复杂,包括热死环丝菌、不动杆菌属（Acinetobacter spp.）、气单胞菌属（Aeromonas spp.）和假单胞菌等多种菌属构成,而腐败末期菌群构成趋于单一,假单胞菌和热死环丝菌为优势腐败菌,特别是莓实假单胞菌（P. fragi）。将腐败分离株10?株假单胞菌、4?株热死环丝菌和1?株蜂房哈夫尼菌（Hafnia alve）接种于冷藏的牛肉汁,发现假单胞菌和蜂房哈夫尼菌的接种组感官评分、pH值和TVB-N值高于热死环丝菌,且假单胞菌有较强的蛋白酶活性。研究表明,结合感官和微生物评价冷鲜牛肉贮藏期为15?d,且菌群多样性下降,假单胞菌和热死环丝菌是优势腐败菌,其中假单胞菌致腐性较强。     

Comparison of culture-dependent and independent techniques for characterisation of the microflora of peroxyacetic acid treated,vacuum-packaged beef

The diversity of microflora associated with peroxyacetic acid (POAA) treated and untreated beef was investigated by 16S rDNA gene cloning, DGGE analysis and conventional bacterial cultivation. Following vacuum packaging, POAA treated and untreated meat samples were stored for up to 18 weeks at −1.5 °C. Each culture independent method showed Carnobacterium spp. to predominate on both POAA treated and untreated meat. However, 16S rDNA gene analysis also detected the presence of psychrotolerant Clostridium spp. in the POAA-treated beef. Culture-dependent analysis did not distinguish Carnobacterium spp. from Lactobacilli. Although culture-dependent analysis showed an increase in the ratio of Enterobacteriaceae to lactic acid bacteria from weeks 6–18 in the POAA treated compared with the untreated meat, the numbers of Enterobacteriaceae were significantly less on POAA treated than on untreated meat. The combination of data collected by culture-dependent and independent techniques provided the most robust approach for elucidating the efficacy of chemical sanitization of chilled vacuum-packaged beef. If conventional cultivation is used for monitoring bacterial spoilage of vacuum-packaged chilled meats it is recommended that culture methods specific for Carnobacterium and Clostridium spp. should be included in order to provide a more complete indication of microbial diversity.     

传统风吹肉加工过程中的微生物演替及优势菌群分析

研究传统风吹肉在整个生产过程的不同阶段中微生物的种类、优势菌群及其数量变化情况。选用7 种培养基对不同加工阶段风吹肉样品中的不同微生物进行分离纯化,并对其进行16S或18S rDNA分子生物学鉴定,鉴定得到加工过程存在于样品肉中的优势微生物9 种,其中细菌7 种和真菌2 种。结果表明：在传统风吹肉生产过程中,料泡引起微生物大量死亡后,风吹肉样品中的各类微生物总数在风吹前10 d持续上升,在中后期达到稳定。以乳酸乳球菌（Lactococcus lactis）为主的乳酸菌是川味风吹肉风吹过程中的主要优势菌群,其次是以巴氏葡萄球菌（Staphylococcus pasteuri）为主的葡萄球菌,汉逊德巴利酵母（Debaryomyces hansenii）和解脂耶氏酵母（Yarrowialipolytica）次之。此外,芽孢杆菌（Bacillus spp.）和水生拉恩菌（Rahnella aquatilis）为川味风吹肉制作过程中的主要腐败菌,但随着风吹过程显著减少。     

小龙虾(Procambarus clarkii)加工前后优势腐败菌的分离与鉴定

以原料小龙虾和卤制加工后的即食小龙虾为对象研究其优势腐败菌种类,为后续研发适宜的杀菌工艺提供理论依据。采用传统平板分离法,从原料小龙虾与开始腐败的即食小龙虾中分别分离出优势腐败菌,通过菌落形态观察、革兰氏染色镜检对微生物种类进行初步分类。采用16S rDNA PCR扩增、限制性内切酶酶切分析以及测序的方法,进行了菌种鉴定试验。结果表明,原料小龙虾优势腐败菌为阴沟肠杆菌(Enterobacter cloacae)、溶酪巨球菌(Macrococcus caseolyticus)、嗜水气单胞菌(Aeromonas hydrophilia)、微小杆菌(Exiguobacterium indicum)和芽胞杆菌属(Bacillus spp.);即食小龙虾优势腐败菌仅检测到以蜡样芽孢杆菌(Bacillus cereus)与苏云金芽孢杆菌(Bacillus thuringiensis)为代表的芽孢杆菌,而其他类型的细菌在卤制加工过程中已被灭活。小龙虾加工前后优势腐败菌鉴定结果对比分析可知,高温卤制工艺难以除灭原料小龙虾中的芽孢杆菌,芽孢杆菌仍存活导致即食小龙虾腐败。     

Study of the bacterial ecosystem in tropical cooked and peeled shrimps using a polyphasic approach

The characterization of the microbial ecosystem of cooked tropical shrimps was carried out using a polyphasic approach. First, culture-dependent methods were used for bacterial enumeration and the phenotypic and molecular identification of bacterial isolates. Then, culture-independent methods, including PCR-TTGE (V3 region of the 16S rRNA gene), provided a fingerprinting of bacterial DNA directly extracted from shrimps. Two batches of cooked and peeled tropical shrimps were stored at 5 and 15 degrees C for 5 and 3 weeks, respectively. Trained panelists carried out a sensory evaluation and microbiological enumerations were performed. When spoilage of samples was perceived, several colonies were isolated from the total viable count media. Thus, 137 bacterial strains were identified by phenotypic and molecular tests. Lactic acid bacteria (LAB) constituted the major group with the most represented genera being Carnobacterium (C. divergens, C. maltaromaticum and indiscernible C. alterfunditum/pleistocenium), Vagococcus (indiscernible V. carniphilus/fluvialis) and Enterococcus (E. faecalis and E. faecium). The other groups corresponded to Brochothrix thermosphacta and Enterobacteriaceae (Serratia liquefaciens). In PCR-TTGE profiles some of DNA fragments were assigned to those of standard strains (S. liquefaciens, B. thermosphacta, E. faecalis, C. divergens and C. maltaromaticum) or identified isolates from culture-dependent analysis (E. faecium). Other additional informations were provided by fragment cloning (Psychrobacter sp, Citrobacter gillenii and Firmicute). In conclusion, TTGE is an excellent tool to monitor the evolution of the microbial ecosystem in seafood products.     

Phylogenetic and spectroscopic analysis of Alicyclobacillus isolates by 16S rDNA sequencing and mid-infrared spectroscopy

Alicyclobacillus spp. are a group of thermophilic, acidophilic, spore-forming bacteria, some of which cause spoilage in commercially pasteurized fruit and vegetable juice products including apple, orange, tomato and carrot juice. In this study, we characterized seven isolates of Alicyclobacillus (14-2, KF, A-Gala 2-1, C-Fuji 6, Gala 9-2, 1016, 18-1) and a reference strain A. acidiphilus DSM 14558 by a 16S rDNA sequencing technique and Fourier transform infrared (FT-IR) spectral features. The degree of similarity between Alicyclobacillus isolates based upon phylogenetic analysis of sequence data and multivariate statistical analysis of FT-IR spectral data was determined. Results from both methods were consistent, suggesting that a combination of methods targeting both 16S rDNA and mid-infrared spectroscopic signatures may be a suitable and more accurate approach to either identify or discriminate Alicyclobacillus isolates from fruit and vegetable juice products.     

Detection of cold-tolerant clostridia other than Clostridium estertheticum in raw vacuum-packed chill-stored meat

Samples from raw chill-stored vacuum-packed beef, lamb and venison or the meat processing environment, associated with a spoilage problem, but negative for Clostridium estertheticum using a specific real-time PCR test, were examined for other Clostridium spp. using direct 16S rDNA PCR-RFLP and sequencing. Of 291 samples tested by PCR, presence of clostridia was indicated in 123 and there was sufficient PCR product in 35 to be further investigated. Presence of Clostridium spp. was confirmed by RFLP and sequencing in 25/35 samples (11 of 14 incidents). Species detected in spoiled meat were (incidents): Clostridium tagluense-like (4), Clostridium putrefaciens (2), Clostridium algidicarnis (3), Clostridium frigoris/estertheticum-like (3) and Clostridium. gasigenes (2). More than one species was detected in some incidents. All of the above species have previously been associated with spoiled meat apart from the Cl. tagluense-like species. Clostridia were also confirmed in 4/7 samples from the environment, with two Cl. frigoris/estertheticum-like and two mesophilic species of Clostridium. Our study showed that, cold-tolerant Clostridium species other than Cl. estertheticum are occasionally associated with spoiled vacuum-packed meat, particularly lamb. Further studies are required to confirm the exact identity of the Cl. tagluense-like species and its role in meat spoilage.     

Characterization of molds isolated from smoked paprika by PCR-RFLP and micellar electrokinetic capillary electrophoresis

Molds are common contaminants of paprika meat products. The drying and storage stages of paprika processing are critical because they can provide molds with the conditions particularly appropriate for their growth and proliferation. Thus, an efficient and accurate characterization of the toxigenic molds of paprika is necessary. An RFLP analysis of the rRNA genes was performed by using a TaqI restriction enzyme. In addition, a micellar electrokinetic capillary chromatography (MECC) method was tested to analyze secondary metabolites produced by mold strains commonly found in paprika. This study was confirmed with a 5.8S-ITS region sequence analysis. A total of 31 isolates were identified by RFLP and MECC analysis. These showed stable RFLP profiles that were clearly different for the different genera and species, and were grouped into clusters together with the profiles of the 16 reference strains. MECC analysis provided additional characteristic peak patterns for the characterization of the mold species present. The characterized isolates were species of the genera Fusarium spp., Aspergillus spp., Penicillium spp., Cladosporium spp., Mucor spp. and Phlebia spp. The identifications were confirmed by the 5.8S-ITS region sequence analysis and by a BLAST search of the GenBank database. RFLP patterns with TaqI restriction enzyme and MECC profiles, either singly or combined, could be of great interest to distinguish molds in paprika.