Succession of bacterial microbiota in tilapia fillets at 4 °C and in situ investigation of spoilers

  • Shan Duan
  • Xingzhi Zhou
  • Jianyin Miao
  • Xingxing Duan
Original Paper


The succession of bacterial microbiota in tilapia fillets during cold storage at 4 °C was investigated employing PCR-DGGE method. Results showed that Pseudomonas was the most dominant genus during entire storage period. Shewanella and Psychrobacter were also always present, but became dominant only after 3 days of storage. Acinetobacter, Brevibacterium, Flavobacterium, Dietzia and Janthinobacterium were always the minor genera, among which Acinetobacter and Brevibacterium disappeared 6 days later, and Dietzia and Janthinobacterium only appeared at the end of storage. Further, the potential spoiler(s) of tilapia fillets at 4 °C were investigated in situ. The spoilage ability of a specific group of bacteria was evaluated as follows: Certain preservatives were selectively added to fillets to inhibit a specific group of bacteria, and then the changes in spoilage degree of fillets were determined. In this way the spoilage ability of the inhibited bacteria was evaluated. Our experiments showed that protamine strongly inhibited Pseudomonas but rarely inhibited Psychrobacter, Acinetobacter and Brevibacterium, but garlic juice, on the contrary, strongly inhibited the latter three but rarely inhibited the former. The mixed preservative, which consisted of protamine and garlic juice, didn’t play better than protamine alone in preventing the spoilage of fillets. This indicated that Psychrobacter, Acinetobacter and Brevibacterium contribute little to the spoilage of tilapia fillets.

Graphical abstract


Fish Spoilage Pseudomonas Psychrobacter Acinetobacter Brevibacterium In situ Tilapia 



The work was supported by the Administration of Science and Technology of Guangdong Province (Project Numbers: 2013B090600111 and 2010A020104004). The authors express their sincere thanks to the Administration.

Supplementary material

11274_2018_2452_MOESM1_ESM.docx (13 kb)
Supplementary material 1 (DOCX 13 KB)


  1. Akarsubasi AT, Ince O, Kirdar B, Oz NA, Orhon D, Curtis TP, Head IM, Ince BK (2005) Effect of wastewater composition on archaeal population diversity. Water Res 39:1576–1584CrossRefPubMedGoogle Scholar
  2. Ampe F, Omar NB, Moizan C, Wacher C, Guyot JP (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 65:5464–5473PubMedPubMedCentralGoogle Scholar
  3. Bandara MK, Masoudi S, Zhu H, Bandara R, Willcox MDP (2016) Evaluation of protamine as a disinfectant for contact lenses. Optometry Vision Sci 93:1349–1355CrossRefGoogle Scholar
  4. Boon N, Windt W, Verstraete W, Top EM (2002) Evaluation of nested PCR-DGGE (denaturing gradient gel electrophoresis) with group-specific 16S rRNA primers for the analysis of bacterial communities from different wastewater treatment plants. FEMS Microbiol Ecol 39(2):101–112. PubMedCrossRefGoogle Scholar
  5. Boziaris IS, Parlapani FF (2016) Specific Spoilage Organisms (SSOs) in Fish. In: Bevilacqua A, Corbo MR, Sinigaglia M (eds) The microbiological quality of food, 1st edn. Woodhead Publishing, Cambridge, pp 61–98Google Scholar
  6. Broekaert K, Heyndrickx M, Herman L, Devlieghere F, Vlaemynck G (2011) Seafood quality analysis: Molecular identification of dominant microbiota after ice storage on several general growth media. Food Microbiol 28:1162–1169CrossRefPubMedGoogle Scholar
  7. Broekaert K, Noseda B, Heyndrickx M, Vlaemynck G, Devlieghere F (2013) Volatile compounds associated with Psychrobacter spp. and Pseudoalteromonas spp., the dominant microbiota of brown shrimp (Crangon crangon) during aerobic storage. Int J Food Microbiol 166:487–493CrossRefPubMedGoogle Scholar
  8. Cao R, Liu Q, Yin BZ, Wu B (2012) Chitosan extends the shelf-life of filleted tilapia (Oreochromis niloticus) during refrigerated storage. J Ocean Univ China 11:408–412CrossRefGoogle Scholar
  9. Dalgaard P (1995) Qualitative and quantitative characterization of spoilage bacteria from packed fish. Int J Food Microbiol 26:319–333CrossRefPubMedGoogle Scholar
  10. Díez B, Pedrós-Alió C, Marsh TL, Massana R (2001) Application of denaturing gradient gel electrophoresis (DGGE) to study the diversity of marine picoeukaryotic assemblages and comparison of DGGE with other molecular techniques. Appl Environ Microbiol 67:2942–2951. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Gafan GP, Lucas VS, Roberts GJ, Petrie A, Wilson M, Spratt DA (2005) Statistical analyses of complex denaturing gradient gel electrophoresis profiles. J Clin Microbiol 43:3971–3978. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Gram L, Dalgaard P (2002) Fish spoilage bacteria – problems and solutions. Curr Opin Biotechnol 13:262–266CrossRefPubMedGoogle Scholar
  13. Gram L, Huss HH (1996) Microbiological spoilage of fish and fish products. Int J Food Microbiol 33:121–137CrossRefPubMedGoogle Scholar
  14. Gram L, Wedell-Neergaard C, Huss HH (1990) The bacteriology of fresh and spoiling Lake Victorian Nile perch (Lates niloticus). Int J Food Microbiol 10:303–316CrossRefPubMedGoogle Scholar
  15. Heuer H, Krsek M, Baker P, Smalla K, Wellington EMH (1997) Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients. Appl Environ Microbiol 63:3233–3241PubMedPubMedCentralGoogle Scholar
  16. Johansen C, Gill T, Gram L (1995) Antibacterial effect of protamine assayed by impedimetry. J Appl Microbiol 78:297–303. CrossRefGoogle Scholar
  17. Koziñska A, Pekala A (2004) First isolation of Shewanella putrefaciens from freshwater fish—a potential new pathogen of fish. Bull Eur Assoc Fish Pathol 24:189–193Google Scholar
  18. Li B, Yu RR, Yu SH, Qiu W, Fang Y, Xie GL (2009) First report on bacterial heart rot of garlic caused by Pseudomonas fluorescens in China. Plant Pathol J 25:91–94CrossRefGoogle Scholar
  19. Meng XH, Hu CT, Xu QQ, Chen XW, Duan S (2012) Antiseptic effects of several natural biological preservatives on fresh tilapia fillets. Acad Period Farm Prod Process 274:51–54Google Scholar
  20. Mounier J, Rea MC, O’Connor PM, Fitzgerald GF, Cogan TM (2007) Growth characteristics of Brevibacterium, Corynebacterium, Microbacterium. and Staphylococcus spp. isolated from surface-ripened cheese. Appl Environ Microbiol 73:7732–7739CrossRefPubMedPubMedCentralGoogle Scholar
  21. Muyzer G, De Wall EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 59:695–700PubMedPubMedCentralGoogle Scholar
  22. Nikolcheva LG, Cockshutt AM, Bärlocher F (2003) Determining diversity of freshwater fungi on decomposing leaves: comparison of traditional and molecular approaches. Appl Environ Microbiol 69:2548–2554CrossRefPubMedPubMedCentralGoogle Scholar
  23. Nubel U, Engelen B, Felske A, Snaidr J, Wieshuber A, Amann RI, Ludwig W, Backhaus H (1996) Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis. J Bacteriol 178:5636–5643CrossRefPubMedPubMedCentralGoogle Scholar
  24. Pakingking R Jr, Palma P, Usero R (2015) Quantitative and qualitative analyses of the bacterial microbiota of tilapia (Oreochromis niloticus) cultured in earthen ponds in the Philippines. World J Microbiol Biotechnol 31:265–275CrossRefPubMedGoogle Scholar
  25. Parlapani FF, Meziti A, Kormas KA, Boziaris IS (2013) Indigenous and spoilage microbiota of farmed sea bream stored in ice identified by phenotypic and 16S rRNA gene analysis. Food Microbiol 33:85–89CrossRefPubMedGoogle Scholar
  26. Pękala A, Kozińska A, Paździor E, Głowacka H (2015) Phenotypical and genotypical characterization of Shewanella putrefaciens strains isolated from diseased freshwater fish. J Fish Dis 38:283–293CrossRefPubMedGoogle Scholar
  27. Shabir A, Dar J, Gijs K, Gerard M (2005) Nested PCR-denaturing gradient gel electrophoresis approach to determine the diversity of sulfate-reducing bacteria in complex microbial communities. Appl Environ Microbiol 71:2325–2330CrossRefGoogle Scholar
  28. Slusarenko AJ, Patel A, Portz D (2008) Control of plant diseases by natural products: Allicin from garlic as a case study. Eur J Plant Pathol 121:313–322CrossRefGoogle Scholar
  29. The national health and family planning commission of the People’s Republic of China (2016a) Food microbiological examination: detection of aerobic bacterial count GB4789.2-2016. In: Food safety national standards. Beijing, pp. 1–5Google Scholar
  30. The national health and family planning commission of the People’s Republic of China (2016b) The determination of volatile base nitrogen in foods GB5009.228-2016. In: Food safety national standards. Beijing, pp. 1–8Google Scholar
  31. Thi ANT, Noseda B, Samapundo S, Nguyen BL, Broekaert K, Rasschaert G, Heyndrickx M, Devlieghere F (2013) Microbial ecology of Vietnamese Tra fish (Pangasius hypophthalmus) fillets during processing. Int J Food Microbiol 167:144–152CrossRefGoogle Scholar
  32. Xing SH, Zhang XH, Liu WJ, Tian D, Hu JY (2012) Modeling growth of specific spoilage organisms in tilapia: comparison Baranyi with chi-square automatic interaction detection (CHAID) model. Afr J Biotechnol 11:6910–6917Google Scholar
  33. Zhang YM, Li Q, Li DP, Liu XC, Luo YK (2015) Changes in the microbial communities of air-packaged and vacuum packaged common carp (Cyprinus carpio) stored at 4 C. Food Microbiol 52:197–204CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.College of Food Science and TechnologySouth China Agricultural UniversityGuangzhouPeople’s Republic of China
  2. 2.Department of Food ScienceUniversity of MassachusettsAmherstUSA
  3. 3.CapitalBio Genomics Co., Ltd.DongguanPeople’s Republic of China

Personalised recommendations