Comparative Analysis of Bacterial Diversity for Cutlassfish (Trichiurus haumela) during Cold Storage by the Culture-Dependent and Culture-Independent Methods

Abstract:

The methods of culture-dependent and denaturing gradient gel electrophoresis (DGGE) based on the sequence of 16S rRNA V3 region gene were described to comparatively characterize the microbial population and community structure of cutlassfish (Trichiurus haumela) under the cold storage. The results showed that 13 kinds of bacteria were identified by the traditional culture-dependent methods, the dominant bacteria belonged to Shewanella putrefaciens and Pseudomonas fluorescens. To determine the community profiles of the samples on variable V3 region, the bacteria of 16S rRNA gene were amplified by PCR and 11 distinct PCR products were separated by DGGE fingerprinting technology. From the sequence analysis, Psychrobacter sp. was found to be the predominant bacteria in the initial stage of the storage. The proportion of Shewanella sp., Pseudomonas sp. increased gradually with the extension of storage time, and they took the place of Psychrobacter sp. to be the dominant bacteria. Thereinto, both Pseudomonas fluorescens and Vibrio sp. took high proportions in the process of storage due to the deterioration of cutlassfish (Trichiurus haumela).

• Periodical: Advanced Materials Research (Volumes 535-537)
• Pages: 1046-1053
• DOI: https://doi.org/10.4028/www.scientific.net/AMR.535-537.1046
• Online since: June 2012
• Authors: Wei Qing Lan, Jing Xie
• Keywords: 16S rRNA Gene, Cold Storage, Culture-Dependent, Culture-Independent, Denaturing Gradient Gel Electrophoresis (DGGE), Microbial Diversity, PCR, Trichiurus haumela

References

[1] Bi J.Z., Shao C.W., Miao G.D., Ma H.Y., Chen S.L., Isolation and characterization of 12 microsatellite loci from cutlassfish (Trichiurus haumela). Conserv Genet. Vol.10 (2009), pp.1171-1173.

DOI: 10.1007/s10592-008-9736-5

[2] Jackson, T.C., Acuff, G.R., & Dickson, J.S., Meat, poultry and seafood. In M. P.Doyle, & T. J. Beuchat (Eds.), Food microbiology: fundamentals and frontiers Washington, D.C: ASM Press, 1997, p.83−100.

[3] Hilario, E., Buckley, T. R., & Young, J.M.. Improved resolution of the phylogenetic relationships among Pseudomonas by the combined analysis of atpD, carA, recA and 16S rDNA. Antonie van Leeuwenhoek, Vol.86 (2004), p.51−64.

DOI: 10.1023/b:anto.0000024910.57117.16

[4] Gennari M, Tomaselli S, Cotrona V. The microflora of fresh and spoiled sardines (Sardina pilchadus) caught in Adriatic (Mediterranean) sea and stored in ice. Food Microbiology, Vol.16 (1999), pp.15-28.

DOI: 10.1006/fmic.1998.0210

[5] Taoukis P S, Koutsoumanis K, Nychas G J E., Use of time temperature integrators and predictive modeling for shelf life control of chilled fish under dynamic storage conditions. International Journal of Food Microbiology, Vol.53 (1999), pp.1-31.

DOI: 10.1016/s0168-1605(99)00142-7

[6] Gram L, Huss H H., Microbiological spoilage of fish and fish product. International Journal of Food Microbiology, Vol. 33(1996), pp.121-137.

DOI: 10.1016/0168-1605(96)01134-8

[7] Gillespie N C, Maerae I C., The bacterial flora of some Queensland fish and its ability to cause spoilage. Journal of Applied Bacteriology, Vol. 39(1975), pp.91-100.

DOI: 10.1111/j.1365-2672.1975.tb00549.x

[8] Amann R.I,Ludwig W,Schlefer K.H., Phylogenetic identification in situ detection of individual microbial cells without cultivation. Microbial Reviews, Vol. 59(1995), pp.143-169.

DOI: 10.1128/mr.59.1.143-169.1995

[9] Pace N.R., A Molecular View of Microbial Diversity and the Biosphere. Science, Vol. 276(1997), pp.734-740.

DOI: 10.1126/science.276.5313.734

[10] Blixt, Y., & Borch, E., Comparison of shelf life of vacuum-packed pork and beef. Meat Science, Vol. 60(2002), p.371−378.

DOI: 10.1016/s0309-1740(01)00145-0

[11] Ercolini, D., PCR–DGGE fingerprinting: novel strategies for detection of microbes in food. Journal of Microbiological Methods, Vol. 56(2004), p.297−314.

DOI: 10.1016/j.mimet.2003.11.006

[12] Maria B.H., Morten S., Bjorn T.L., et.al, Characteriasation of the dominant bacterial population in modified atmosphere packaged farmed halibut (Hippoglossus hippoglossus) based on 16S rDNA-DGGE. Food Microbiology, Vol.24 (2007), pp.362-371.

DOI: 10.1016/j.fm.2006.07.018

[13] Maria B.H., Bjorn T.L., Morten S., Jan T.R., Characterisation of the bacterial flora of modified atmosphere packaged farmed Atlantic cod (Gadus morhua) by PCR-DGGE of conserved 16S rRNA gene regions. International Journal of Food Microbiology, Vol.117 (2007), pp.68-75.

DOI: 10.1016/j.ijfoodmicro.2007.02.022

[14] Jiang Y., Gao F., Xu X.L., Su Y., Ye K.P., Zhou G.H., Changes in the bacterial communities of vacuum-packaged pork during chilled storage analyzed by PCR-DGGE. Meat Science, Vol. 86(2010), pp.889-895.

DOI: 10.1016/j.meatsci.2010.05.021

[15] Satokari, R. M., Vaughan, E. E., Akkermans, A. D. L., Saarela, M., & de Vos, W. M., Bifidobacterial diversity in human feces detected by genus-specific PCR and denaturing gradient gel eletrophoresis. Applied and Environmental Microbiology, Vol.67 (2001), p.504−513.

DOI: 10.1128/aem.67.2.504-513.2001

[16] Randazzo, C. L., Torriani, S., Akkermans, A. D. L., de Vos,W. M., & Vaughan, E. E., Diversity, dynamics, and activity of bacterial communities during production of an artisanal Sicilian cheese as evaluated by 16S rRNA analysis. Applied and Environmental Microbiology, Vol. 68(2002), p.1882−1892.

DOI: 10.1128/aem.68.4.1882-1892.2002

[17] Sambrook.J., DAVID W.RUSSELL, Molecular Cloning a laboratory Manual, USA. (2002)

[18] Muyzer G., DGGE/TGGE a method for identifying genes from natural ecosystems, Current of Opinion Microbiology, Vol.2(1999), pp.317-322.

DOI: 10.1016/s1369-5274(99)80055-1

[19] Muyzer, G. de Weal, E.C., & Uitterlinden, A., Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Applied of Environment Microbiology, Vol. 59(1993), pp.695-700.

DOI: 10.1128/aem.59.3.695-700.1993

[20] Muyzer G., Smalla, K., Application of denaturing gradient gel electrophoresis（DGGE）and temperature gradient gel electrophoresis (TGGE) in microbial ecology, Antonie van Leeuwenhoek, Vol. 73(1998), pp.127-141.

DOI: 10.1007/springerreference_76317

[21] Nübel, U., Engelen, B., et al., Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel elelectrophoresis, Journal of Bacteriology, Vol.178(1996), pp.5636-5643.

DOI: 10.1128/jb.178.19.5636-5643.1996

[22] Theelen, B., Silvestri, M., Gueho, E., van Belkum, A., & Boekhout, T., Identification and typing of Malassezia yeasts using amplified fragment length polymorphisms (AFLP), random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE). FEMS Yeast Research, Vol.1 (2001), pp.79-86.

DOI: 10.1111/j.1567-1364.2001.tb00018.x

[23] Gill C.O., Extending the storage life of raw. Meat Science, Vol.43 (1996), pp.99-109.

DOI: 10.1016/0309-1740(96)00058-7

[24] Gurtler, V., Garrie, H.D., & Mayall, B.C., Denaturing gradient gel electrophoretic multilocus sequence typing of Staphylococcus aureus isolates. Electrophoresis, Vol.23 (2002), pp.3310-3320.