Investigation of donkey milk bacterial diversity by 16S rDNA high-throughput sequencing on a Cyprus donkey farm.

The interest in milk originating from donkeys is growing worldwide due to its claimed functional and nutritional properties, especially for sensitive population groups, such as infants with cow milk protein allergy. The current study aimed to assess the microbiological quality of donkey milk produced in a donkey farm in Cyprus using culture-based and high-throughput sequencing techniques. The culture-based microbiological analysis showed very low microbial counts, whereas important food-borne pathogens were not detected in any sample. In addition, high-throughput sequencing was applied to characterize the bacterial communities of donkey milk samples. Donkey milk mostly composed of gram-negative Proteobacteria, including Sphingomonas, Pseudomonas, Mesorhizobium, and Acinetobacter; lactic acid bacteria, including Lactobacillus and Streptococcus; the endospores forming Clostridium; and the environmental genera Flavobacterium and Ralstonia, detected in lower relative abundances. The results of the study support existing findings that donkey milk contains mostly gram-negative bacteria. Moreover, it raises questions regarding the contribution of (1) antimicrobial agents (i.e., lysozyme, peptides) in shaping the microbial communities and (2) bacterial microbiota to the functional value of donkey milk.

Bacterial communities and potential spoilage markers of whole blue crab (Callinectes sapidus) stored under commercial simulated conditions.

Bacterial communities composition using 16S Next Generation Sequencing (NGS) and Volatile Organic Compounds (VOCs) profile of whole blue crabs (Callinectes sapidus) stored at 4 and 10 °C (proper and abuse temperature) simulating real storage conditions were performed. Conventional microbiological and chemical analyses (Total Volatile Base-Nitrogen/TVB-N and Trimethylamine-Nitrogen/TMA-N) were also carried out. The rejection time point was 10 and 6 days for the whole crabs stored at 4 and 10 °C, respectively, as determined by development of unpleasant odors, which coincided with crabs death. Initially, the Aerobic Plate Count (APC) was 4.87 log cfu/g and increased by 3 logs at the rejection time. The 16S NGS analysis of DNA extracted directly from the crab tissue (culture-independent method), showed that the initial microbiota of the blue crab mainly consisted of Candidatus Bacilloplasma, while potential pathogens e.g. Listeria monocytogenes, Pseudomonas aeruginosa and Acinetobacter baumannii, were also found. At the rejection point, bacteria of Rhodobacteraceae family (52%) and Vibrio spp. (40.2%) dominated at 4 and 10 °C, respectively. TVB-N and TMA-N also increased, reaching higher values at higher storage temperature. The relative concentrations of some VOCs such as 1-octen-3-ol, trans-2-octenal, trans,trans-2,4-heptadienal, 2-butanone, 3-butanone, 2-heptanone, ethyl isobutyrate, ethyl acetate, ethyl-2-methylbutyrate, ethyl isovalerate, hexanoic acid ethyl ester and indole, exhibited an increasing trend during crab storage, making them promising spoilage markers. The composition of microbial communities at different storage temperatures was examined by 16S amplicon meta-barcoding analysis. This kind of analysis in conjugation with the volatile profile can be used to explore the microbiological quality and further assist towards the application of the appropriate strategies to extend crab shelf-life and protect consumer's health.

Microbial spoilage investigation of thawed common cuttlefish (Sepia officinalis) stored at 2 °C using next generation sequencing and volatilome analysis.

Cephalopods are highly appreciated with increasing demand seafood, but are also very perishable and deteriorate fast mainly due to microbiological spoilage. For this reason exploration of bacterial communities through 16S Next Generation Sequencing (NGS) and Volatile Organic Compounds (VOCs) analysis was performed. Furthermore, sensory evaluation, classical microbiological analysis, Total Volatile Base-Nitrogen/TVB-N and Trimethylamine-Nitrogen/TMA-N determination were also carried out. Shelf-life of thawed cuttlefish (Sepia officinalis) stored at 2°C determined by sensory evaluation was 4 days. Aerobic Plate Counts (APC) reached the levels of 6.6 log cfu/g. The initial and final population of all spoilage microorganisms enumerated with selective media was under detectable levels with the exception of Pseudomonas. Based on 16S NGS analysis, Psychrobacter were the dominants among others, e.g. Pseudomonas, Shewanella, Comamonas, Carnobacterium, Vagococcus, of the initial microbiota. Psychrobacter was also the dominant microorganisms of the spoiled cuttlefish. TVB-N and TMA-N increased considerably only at the late stages of storage. A plethora of VOCs were produced and some exhibited an increasing profile throughout storage, making them promising molecules as freshness indicators in contrast to TVB-N and TMA-N. The application of next generation sequencing revealed the microbiota that escapes the classic microbiological methodologies, showing that other microorganisms different from those determined on selective culture media might be the main cause of microbiological spoilage.