Systematic review and critical reflection on the isolation and identification methods for spoilage associated bacteria in fresh marine fish

Consumers demand more fresh, safe, and high-quality food. As this is partiallycorrelated to the microbial profile, several microbiological examination tools are available. Incontrast to meat, no microbiological normalized methods to assess the microbiological quality of fresh marine fish have been agreed on. As a result, studies on the detection and diversity of spoilage associated organisms (SAOs) in fish often apply various detection, isolation, and identification techniques. This complicates the comparison and interpretation of data reported, and often results in different or inconclusive results. Therefore, the present review aimed to present a critical overview of the isolation/cultivation and detection techniques currently applied in fish microbiology. After a comprehensive search in the PubMed, Web of Science and Scopus databases, a total of 111 studies fulfilled the review selection criteria. Results revealed that when relying on culture media for the isolation of SAOs in fish, it is essential to include a salt-containing medium next to plate count agar that is currently used as the reference medium for the enumeration of bacteria on fish. In terms of identification, MALDI-TOF MS and 16S rRNA gene sequencing are currently the most promising tools, though other housekeeping genes should be targeted as well, and, the biggest challenge at this point is still the lack of comprehensive proteomic and sequence databases for SAOs. A full replacement of cultivation by next generation sequencing is difficult to recommend due to the absence of a standardized experimental methodology, especially for fish, and the relatively high sequencing costs. Additionally, a discrepancy between culture-dependent and independent methods in revealing the bacterial diversity, and abundancy, from marine fish was demonstrated by several authors. It is therefore recommended to consider both approaches as complements of one another, rather than substitutes, and to include them simultaneously to yield more complete results regarding the SAOs in fresh marine fish. As such, a thorough understanding of the biology of spoilage organisms and process will be obtained to prolong the shelf-life and deliver a high-quality product.