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Characterization of microbial population of ‘Alheira’ (a traditional Portuguese fermented sausage) by PCR-DGGE and traditional cultural microbiological methods

2008; Oxford University Press; Volume: 105; Issue: 6 Linguagem: Inglês

10.1111/j.1365-2672.2008.03947.x

1365-2672

Helena Albano, Isabel Henriques, António Correia, Tim Hogg, Paula Teixeira,

Identification and Quantification in Food

Journal of Applied MicrobiologyVolume 105, Issue 6 p. 2187-2194 Free Access Characterization of microbial population of ‘Alheira’ (a traditional Portuguese fermented sausage) by PCR-DGGE and traditional cultural microbiological methods H. Albano, H. Albano Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, PortugalSearch for more papers by this authorI. Henriques, I. Henriques CESAM and Departamento de Biologia, Universidade de Aveiro, 3810-193 Aveiro, PortugalSearch for more papers by this authorA. Correia, A. Correia CESAM and Departamento de Biologia, Universidade de Aveiro, 3810-193 Aveiro, PortugalSearch for more papers by this authorT. Hogg, T. Hogg Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, PortugalSearch for more papers by this authorP. Teixeira, P. Teixeira Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, PortugalSearch for more papers by this author H. Albano, H. Albano Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, PortugalSearch for more papers by this authorI. Henriques, I. Henriques CESAM and Departamento de Biologia, Universidade de Aveiro, 3810-193 Aveiro, PortugalSearch for more papers by this authorA. Correia, A. Correia CESAM and Departamento de Biologia, Universidade de Aveiro, 3810-193 Aveiro, PortugalSearch for more papers by this authorT. Hogg, T. Hogg Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, PortugalSearch for more papers by this authorP. Teixeira, P. Teixeira Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, PortugalSearch for more papers by this author First published: 21 November 2008 https://doi.org/10.1111/j.1365-2672.2008.03947.xCitations: 31 Paula Teixeira, Rua Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal. E-mail: pcteixeira@esb.ucp.pt AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Aims: This study evaluates the microbial ecology of ‘Alheira’ by traditional microbiological analysis and a PCR-denaturing gradient gel electrophoresis (DGGE) protocol. Methods and Results: Total microbial DNA from ‘Alheiras’ was extracted directly from the products and subjected to PCR using Eubacterial primers for 16S rDNA. The amplicons were separated by DGGE. The results demonstrated that different products of the same batch display identical profiles, whereas products from different batches of the same producer could display different DGGE profiles. ‘Alheiras’ from different producers were distinguishable based on the respective DGGE profiles. The obtained sequences from prevalent phylotypes affiliated with order Lactobacillales and order Bacillales and class Gammaproteobacteria. The same samples were subjected to traditional microbiological analysis. In both methods, lactic acid bacteria were dominant and were present together with other organisms, mainly members of the family Micrococcaceae. Conclusions: The approach explored in this study allowed the description of the microbial community present in ‘Alheira’ in particular the diversity of lactic acid bacteria. Significance and Impact of the Study: This can be useful for the microbiological characterization of traditional products in order to develop new methods of quality control capable of supporting a standardization of the processes, while preserving their typical traits. Introduction The microbiology of fermented sausages is complex. The type of microflora that develops is often closely related to the ripening technique utilized. The manufacture of fermented sausages has a long history in Portugal where there is a wide variety of typical preparations. ‘Alheira’ is a traditional fermented meat sausage typical of the North of Portugal (Trás-os-Montes). ‘Alheira de Mirandela’ is a much appreciated product and represents an important resource for this region where more than 500 tons are produced annually. ‘Alheira de Mirandela’ is in the process of name registration as ‘Traditional Guaranteed Speciality’. The specific characteristics of the final product mainly arise from the raw materials employed, the agro-ecosystem of the area of production and the traditional technology of manufacture. ‘Alheira de Mirandela’ is produced from pork and poultry meat, lard and pork fat, wheat bread and olive oil. Ingredients are mixed with salt, garlic and spices until they form a paste which is then stuffed into natural or artificial casings (horseshoe-shaped sausages approximately 15 ± 60 mm long) and submitted to a smoking process, at low but uncontrolled temperature (generally 7·5 log CFU g−1, with the exception of one sample from producer Gr (Table 1). In addition, enterococci counts were >6·5 log CFU g−1 in most samples. With the exception of one of the samples from producers Gr, Ef and Tx, counts on mannitol salt agar were >log 4·44 log CFU g−1, although values varied between 4·4 log CFU g−1 and >6·20 log CFU g−1. Enterobacteriaceae counts were >4 log CFU g−1 in most samples, with the exception of producer Gr. Listeria spp. was isolated in countable quantities in some samples, the highest values coming from two samples from the producer Tx (3·8 CFU g−1 and 4·3 CFU g−1). Table 1. Microbiological characterization of ‘Alheira de Mirandela’ from six different producers (Ag, Am, Ef, Gr, Tp, Tx;): important microbial parameters in fermented products Producer Total counts 30°C (log CFU g−1) Counts on MRS (log CFU g−1) Counts on M17 (log CFU g−1) Counts on MSA (log CFU g−1) Enterococci (log CFU g−1) Enterobacteria (log CFU g−1) Listeria spp. (log CFU g−1) Gr1 6·9 ± 0·6 6·0 ± 0·0 6·0 ± 0·3 5·4 ± 0·2 7·7 ± 0·3 2·3 ± 0·1 <1 Gr2 9·7 ± 0·2 8·8 ± 0·6 8·8 ± 0·2 6·2 ± 0·3 9·3 ± 0·2 7·1 ± 0·0 <1 Am2 1·0 ± 0·2 8·8 ± 0·2 8·5 ± 0·2 4·4 ± 0·6 7·8 ± 0·5 6·3 ± 0·0 <1 Am3 9·7 ± 0·1 8·5 ± 0·1 7·9 ± 0·1 4·2 ± 0·0 7·2 ± 0·1 6·8 ± 0·2 2·3 ± 0·3 Ef1 9·6 ± 0·2 7·6 ± 0·8 7·7 ± 0·1 <1 4·6 ± 0·2 3·3 ± 0·2 <1 Ef2 8·7 ± 0·4 7·7 ± 0·1 7·9 ± 0·4 5·9 ± 0·1 7·0 ± 0·0 5·6 ± 0·3 6·2 >7·2 4·3 ± 0·8 <1 Tx3 9·3 ± 0·2 8·0 ± 0·2 9·1 ± 0·2 7·2 >7·5 10·5 9·8 ± 0·5 >10·5 >6·2 >7·2 6·9 ± 0·4 10·5 5·9 ± 0·1 6·8 ± 0·3 4·9 ± 0·2 6·2 >7·2 >7·5 4·2 log CFU g−1 in most samples. Intriguingly, although the separation of the V1 and V3 rRNA gene amplicons by PCR-DGGE has been described as a reliable tool to investigate the LAB and CNC diversity in fermented sausages (Cocolin et al. 2001a,b), only one band was identified as being part of this latter microbial group (Macrococcus caseolyticus, Band 5) in the denaturing gels generated in this present study. This finding might be due to the selective amplification of the most abundant templates, a bias previously recognized when performing PCR-DGGE analysis of heterogeneous DNA template mixtures (Felske et al. 1998; Ampe et al. 1999; Ercolini et al. 2001b). According to the guidelines for the microbiological quality of fermented meats published by Gilbert et al. (2000), most of the samples tested (80%) would be considered unsatisfactory, with Enterobacteriaceae counts greater than 4 log CFU g−1. The detection of Listeria spp. in some samples points to the possibility of product contamination with the pathogenic specie L. monocytogenes. Sequences closely related to Enterobacteriaceae were retrieved from DGGE bands. Brochothrix-related phylotypes were also detected in almost all the samples, as were close relatives of Pseudomonas; in both cases suggesting the presence of potential spoilage strains. Quantitative approaches, such as those using real-time PCR would be needed in order to quantify the presence of specific potentially problematic bacteria. As shown by Rantsiou and Cocolin (2006), the combination of cultivation strategies and DNA-based techniques allows the identification of the dominant microbial species in fermented sausages, since it permits the generation and comparison of characteristic patterns from cultivated isolates and complex microbial communities. However, Ercolini (2004) described possible pitfalls of the whole procedure when applied to food products, like sampling, DNA extraction, DNA purity, PCR conditions, formation of heteroduplex and chimeric molecules, small fragments, between others. PCR-DGGE applied to template DNA, directly extracted from a food matrix, generates a specific profile of that product in that moment, given the conditions used. DGGE analysis and sequencing of the resultant bands allows the identification of the dominant phylotypes present in all products. The approach explored in this study allowed the description of the microbial community present in ‘Alheira’ in particular the diversity of LAB. DGGE could be used in monitoring the growth kinetics during the ripening process and the effects of these on the hygienic quality, allowing tracking of the microbial ‘typicity’ of the ‘Alheira de Mirandela’, and collecting of important information for the designing of autochthonous starter cultures. Acknowledgements This work received financial support via project POCTI/AGG/39587/2001, Fundação para a Ciência e Tecnologia (FCT)/Fundo Europeu Desenvolvimento Regional (FEDER). Financial support for author H. Albano was provided by PhD fellowship, SFRH/BD/13161/2003 (FCT) and for I. Henriques by PhD fellowship (SFRH/BPD/21384/2005). References Albano, H., Van Reenen, C.A., Todorov, S.D., Hogg, T., Dicks, L.M.T. and Teixeira, P. (2007) Ecology of lactic acid bacteria in “Alheira”, a fermented sausage traditionally produced in Portugal: isolation, identification and molecular characterization. Personal communication in MICRO’07 – BIOTEC’07-XXXIII, Lisbon, Portugal, 30 November, 2007–2 December, 2007. Google Scholar Ampe, F., Ben Omar, N., Moizan, C., Wacher, C. and Guyot, J.-P. (1999) Polyphasic study of the spatial distribution of microorganisms in Mexican pozol, a fermented maize dough, demonstrates the need for cultivation independent methods to investigate traditional fermentations. Appl Environ Microbiol 65, 5464– 5473. CASPubMedWeb of Science®Google Scholar Aquilanti, L., Santarelli, S., Silvestri, G., Osimani, A., Petruzzelli, A. and Clementi, F. (2007) The microbial ecology of a typical Italian salami during its natural fermentation. Int J Food Microbiol 120, 136– 145.