Rapid Quantitative Detection of Lactobacillus sakei in Meat and Fermented Sausages by Real ...

Rapid Quantitative Detection of Lactobacillus sakei in Meat and Fermented Sausages by Real ...

9 Pages · 2006 · 174 KB · English

and Fermented Sausages by Real-Time PCR. Belén Martın,1 Anna using both purified DNA and the inoculated sausage model Baboon dental plaque, collection Diagnostic PCR: making internal amplification control man-.

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY , Sept 2006, p 6040–6048Vol 72, No 9 00992240/06/$08000 doi:101128/AEM0285205 Copyright © 2006, American Society for Microbiology All Rights Reserved Rapid Quantitative Detection of Lactobacillus sakeiin Meat and Fermented Sausages by RealTime PCR Bele ´n Mart ´n , 1Anna Jofre ´, 1Margarita Garriga, 1Maria Pla, 2and Teresa Aymerich 1* Institute for Food and Agricultural Research and Technology (IRTA), Meat Technology Center, Granja Camps i Armet, E17121 Monells, Spain, 1and Institute of Food and Agricultural Technology (INTEA), University of Girona, E17071 Girona, Spain 2 Received 5 December 2005/Accepted 20 June 2006 A quick and simple method for quantitative detection of Lactobacillus sakeiin fermented sausages was successfully developed It is based on Chelex100based DNA puri?cation and realtime PCR enumeration using a TaqMan ?uorescence probe Primers and probes were designed in the L sakei16S23S rRNA intergenic transcribed spacer region, and the assay was evaluated using L sakeigenomic DNA and an arti?cially inoculated sausage model The detection limit of this technique was approximately 3 cells per reaction mixture using both puri?ed DNA and the inoculated sausage model The quanti?cation limit was established at 30 cells per reaction mixture in both models The assay was then applied to enumerate L sakeiin real samples, and the results were compared to the MRS agar count method followed by con?rmation of the percentage of L sakei colonies The results obtained by realtime PCR were not statistically signi?cantly different than those obtained by plate count on MRS agar (P > 005), showing a satisfactory agreement between both methods Therefore, the realtime PCR assay developed can be considered a promising rapid alternative method for the quanti? cation of L sakeiand evaluation of the implantation of starter strains of L sakeiin fermented sausages Dry fermented sausages are readytoeat meat products characterized by a bacterial fermentation process followed by a ripening period Indigenous microorganisms have been tradi tionally responsible for fermentation, but starter cultures can also be added to control fermentation and to ensure desired quality (10) Among them, lactic acid bacteria (LAB) play an important role during the fermentation of these products, mainly as a result of competitive growth and the production of inhibitory substances, such as organic acids and bacteriocins (55) The species of LAB most commonly found in meat and meat products, including dry sausages processed with different technologies, are Lactobacillus sakei, Lactobacillus curvatus, and Lactobacillus plantarum (4, 25, 38, 52, 54) LAB have a long safe history of application and consumption in the pro duction of fermented foods and beverages (11, 17, 44) They have been used as a starter culture for the fermentation of meat and meat products to improve microbial safety (21, 26, 27), and L sakei, a facultative heterofermentative LAB, is the most frequently isolated and one of the best choices for further use as a starter culture (5) Studies of the composition of LAB communities in meat and fermented sausages have been conducted using conventional methods (25, 48) or several alternatives methods such as spe ciesspecic hybridization probes (39, 58), conventional PCR (4, 7, 35), and PCRdenaturing gel electrophoresis (14) Con ventional testing methods for the detection of L sakeiin food relying on phenotypical methods have been extensively used (25, 32, 38, 49, 52) They are laborintensive and timeconsum ing, in many instances requiring from 8 to 10 days to be com pleted One of the current limitations of rapid processing in the laboratory is the requirement to subculture isolates to perform biochemical or other tests needed for bacterial identication: Gram staining, catalase and oxidase testing plus biochemical identication by carbohydrate fermentation proles, absence of diaminopimelic acid in the cell, production of DLlactic acid, and hydrolysis of arginine Moreover, physiological or bio chemical criteria are sometimes ambiguous (8, 29) L sakeiis biochemically different from L curvatusby melibiose con sumption and arginine degradation (52), which sometimes may be confusing Reliable and fast identication methods are of great impor tance to control and monitor either endogenous or inoculated starter cultures Development of a molecular cultureindepen dent enumeration method appears to be particularly valuable in the case of

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