Valutazione della vitalità dei probiotici negli integratori commerciali per animali domestici

Abstract: The gut microbiome is considered as an organ, harbours bacteria, virus, archaea and fungi. Bacteria constitute most microorganisms in the gastrointestinal tract, supplies the digestion of certain nutrients such as fibre, and the synthesis of vitamins. Microbiome influence the host metabolism, the immune system and protect from pathogens. Diet and age are factors that greatly influence the microbiome in dogs and cats. Various diseases alter the gut microbiota in dogs and cats such as inflammatory bowel disease (IBD), obesity, diabetes, kidney disease, allergies, and constipation. Nutritional interventions help restore gut flora, such as the intake of prebiotics (fructo-oligosaccharides, lactitol, mannan-oligosaccharides, beta-glucans) and probiotics. Microorganisms authorized in Europe in pet foods include Enterococcus faecium, Lactobacillus acidophilus and Bacillus velezensis. In this study, market research was conducted that identified 23 probiotic supplements for pets with microorganisms in the additives. Specifically, 11 tablets, 2 chewable tablets, 1 capsule, 4 pastes, and 5 powders were examined. Each supplement was compared with EC regulations to assess compliance. Then, of these 23, four supplements in different pharmaceutical forms were identified: one tablet, one paste, one powder, and one capsule. For each of the four probiotics, analysis by Maldi-Tof mass spectrometry was performed to identify the microorganisms. Sample preparation was done by plate seeding with the stay of 72 hours in incubator. Subsequently, 12 samples were taken for each supplement. By means of the Maldi-Tof, the results of the tablet probiotic show that Enterococcus faecium (present on the label) was detected in 8 % of the samples and Sphingomonas molluscorum was detected in 25 %. Regarding probiotics in powder and capsule form, the colonies grown on the plates had a yeast-like appearance; in fact, although the spectrum obtained was of good quality, no corresponding spectrum was found in the database to allow unambiguous identification of the microorganism. Enterococcus faecium could be detected in the probiotic paste in 58 % of the samples. Further analysis was performed to detect the total bacterial load of each probiotic supplement. Generic Plate Count Agar (PCA) medium and MRS agar were used to detect lactic acid bacteria. The plates were seeded and placed in an incubator at 30°C for 48 hours. The concentrations detected within the probiotic supplements complied in the tablet, paste, and powder. As for the sample consisting of the capsule, the label charge was found to be overestimated. In all the supplements examined, the strain identification numbers of the microorganisms, the expiration date and the instructions for use are indicated on the label, which comply with EC Regulation No. 1831/2003 (consolidated version) (Annex III). In conclusion, therefore, it could be seen that in three out of four probiotic samples the bacterial concentration, thus the viability of bacteria, in most of the cases, remains stable. Regarding the Maldi-Tof analysis, given the failure to grow bacteria in two different samples (powder and capsule), it can be inferred that the generic culture media were not ideal for growing bacilli. Another reason for the incomplete success of bacterial identification, is that the strains present in the probiotics are different from the strains included in the Bruker company database used for the above study.