Validation of the AnticFast® Beta-Lactams Rapid Test Kit for Detection of Beta-Lactams (Penicillins and Cephalosporins) in Raw Cow's Milk: AOAC Performance Tested MethodSM 032303.

BACKGROUND: AnticFast® Beta-Lactams Rapid Test Kit is a qualitative two-step (2 min + 5 min) rapid lateral flow assay to detect ß-lactam (penicillins and cephalosporins) antibiotic residues in raw commingled cow's milk. OBJECTIVE: The method performance was evaluated according to Commission Decision 2002/657/EC, Commission Implementing Regulation 2021/808, and Community Reference Laboratories Residues Guidelines for the Validation of Screening Methods for Residues of Veterinary Medicines. METHODS: The AnticFast Beta-Lactams Rapid Test Kit was evaluated for detection capability, selectivity, false-positive results, repeatability, robustness, suitability for various milk types and milk compositions, milks from various species, and test kit consistency and stability. Samples included milks spiked at concentrations bracketing the EU maximum residue limits (MRLs) for ß-lactams as well as bulk farm and tanker milks. RESULTS: The AnticFast Beta-Lactams Rapid Test Kit is specific for the detection of ß-lactams in milk and does not detect compounds from other antibiotic families. Interference was seen with clavulanic acid, a ß-lactamase inhibitor, which was expected. The test can detect all residues of ß-lactams (penicillins and cephalosporins) present on the EU-MRL list for milk at their respective MRL except for desfuroylceftiofur and cephalexin, which were above the MRL. No false positives were detected in the 602 (300 blank farm and 302 tanker load) samples tested. Robustness testing indicated that the detection in heat-treated milk types may be slightly hampered. For substances with a detection capability well below the MRL, this interference does not cause problems since detection at MRL remains guaranteed, but care should be taken for substances with a CCß at or near their MRL. Diminished sample flow was seen with reconstituted milk powder and blank ewes' milk, so sample flow should always be verified for these milk types. CONCLUSIONS: Results of this validation show that the AnticFast Beta-Lactams Rapid Test Kit is a reliable test for rapid screening of raw cows' milk for residues of ß-lactam antibiotics. HIGHLIGHTS: AnticFast Beta-Lactams Rapid Test Kit is an easy, realiable, robust and highly specific test for screening of raw cows' milk for residues of penicillins and cephalosporins.

Microbial ecology of Vietnamese Tra fish (Pangasius hypophthalmus) fillets during processing.

There are numerous factors that can have an impact on the microbial ecology and quality of frozen Pangasius hypophthalmus fillets during processing in Vietnam. The presence of spoilage bacteria along the processing line can shorten the shelf-life of thawed frozen fish products. Therefore, the spoilage microbiota throughout the processing chain of two companies (BC: large scale factory, chlorine-based process, BW: large scale factory, water-based process and SC: small scale factory, chlorine-based process) was identified by culture-dependent techniques and 16S rRNA gene sequencing. The microbiological counts were observed to be insignificantly different (p>0.05) between BC and BW. Surprisingly, chlorine treated fillets from the SC line were revealed to have significantly higher microbial counts than potable water treated fillets at BW line. This was determined to be a result of temperature abuse during processing at SC, with temperatures even greater than 10 °C being recorded from skinning onwards. On the contrary, the microbiota related to spoilage for BC and BW lines was determined by 16S rRNA gene sequencing to be more diverse than that on the SC line. A total of 174 isolates, 20 genera and 38 species were identified along the processing chains. The genera Aeromonas, Acinetobacter, Lactococcus and Enterococcus were prevalent at various processing steps on all the processing lines evaluated. A diverse range of isolates belonging to the Enterobacteriaceae such as Providencia, Shigella, Klebsiella, Enterobacter and Wautersiella were isolated from fillets sampled on the SC line whereas Serratia was only observed on fillets sampled on the BC and BW lines. The results can be used to improve Good Manufacturing Practices for processed Pangasius fillets and to select effective measures to prolong the shelf-life of thawed Vietnamese Pangasius fillets products.

Volatile compounds associated with Psychrobacter spp. and Pseudoalteromonas spp., the dominant microbiota of brown shrimp (Crangon crangon) during aerobic storage.

The spoilage potential of several Psychrobacter and Pseudoalteromonas species (Psychrobacter cibarius, Psychrobacter maritimus, Pseudoalteromonas elyakovii, Pseudoalteromonas paragorgicola and Pseudoalteromonas nigrifaciens) was determined and quantified based on the presence of volatile organic compounds (VOCs). Psychrobacter and Pseudoalteromonas species dominate the microbiota of cooked brown shrimp (Crangon crangon). Additionally, API ZYM analyses determined the species' enzymatic capacity to contribute to spoilage by degrading lipids, amino acids and proteins. The bacterial species used in this study were isolated from cooked brown shrimp during storage under different storage and processing conditions and were selected for analysis of their spoilage potential based on their difference in the (GTG)5-rep profile, 16S rRNA and gyrB sequences and API ZYM profile. The isolates were inoculated as pure cultures on heat-sterilised shrimp. The inoculated samples were stored at 4 °C and the production of VOCs by the pure strains on the shrimp matrix was identified via gas chromatography coupled to mass spectrometry (GC-MS). VOC production was quantified daily by selected ion flow tube mass spectrometry (SIFT-MS) until the bacterial count exceeded 108-109 cfu/g. The sensory profile of Psychrobacter species revealed very low spoilage potential as measured by the production of VOCs, but these species may nevertheless contribute to spoilage. Based on the API ZYM results, Pseudoalteromonas as well as Psychrobacter species might enhance spoilage by breaking down lipids and hydrolysing amino acids and proteins. Pseudoalteromonas species, especially Psa. elyakovii and Psa. nigrifaciens, have a high spoilage potential and might be responsible for the off-odours produced during spoilage of brown shrimp. These isolates produced significant amounts of volatile compounds such as sulphides, acetone, ammonia, and ethanol, which are all involved in seafood spoilage.

Molecular identification of the microbiota of peeled and unpeeled brown shrimp (Crangon crangon) during storage on ice and at 7.5 °C.

The dominant microbiota of brown shrimp (Crangon crangon) were systematically identified during storage under different conditions. Freshly caught shrimp were processed on board the fishing vessel under the best possible hygienic conditions (IDEAL), unpeeled and manually (sterile) peeled, then stored on ice and at 7.5 °C until microbiologically spoiled. Results were compared with industrially processed (INDUSTRIAL) shrimp. Isolates grown on various media were identified by 16S rRNA and gyrB gene sequencing. We examined the total microbiota and microbial population shifts of shrimp under various storage conditions using denaturant gradient gel electrophoresis (DGGE). The microbiota differed somewhat during storage and among the various storage conditions; however, members of the genera Psychrobacter and Pseudoalteromonas were found to dominate the microbiota of all shrimp samples regardless of processing procedures or storage conditions. Most isolates could be identified by gyrB gene sequencing as Psychrobacter immobilis or Psychrobacter cibarius. Also Pseudoalteromonas nigrifaciens, Pseudoalteromonas elyakovii or Pseudoalteromonas paragorgicola dominated the microbiota of brown shrimp during storage. Also species from the genera Planocuccus, Exiguobacterium, Carnobacterium, Pseudomonas, Chryseobacterium and Staphylococcus were detected during storage of brown shrimp. Culture-dependent and culture-independent DGGE analysis produced different results in band patterns. Both methods are therefore required to accurately identify the microbiota and bacterial population shifts on seafood during storage.