Isolation and characterization of promising probiotic lactobacilli and bifidobacteria from stools of breast-fed infants and honeybees’ gut

Members of the Bifidobacterium and Lactobacillus genera are widely recognized as health-promoting since they are able to exert nutritional or therapeutic benefits to the host. Recently, there is a growing interest in the isolation and identification of new potential probiotics to be used as feed supplements or to setup functional foods. Honeybees’ gut and fresh fecal samples of breast-fed Algerian infants, aged 3-6 months, were collected, homogenized and inoculated in a reducing medium containing Brain Heart Infusion (BHI) broth (Oxoid, Italy), 0.5% glucose, 0.5% yeast extract (Oxoid, Italy), 0.25% cysteine (Merck), 10 μg/L vitamin K1 (Sigma Aldrich, Italy), and 0.02 g/L hemin (Sigma Aldrich, Italy). Ten-fold serial dilutions were made and plated in duplicate on De Man, Rogosa and Sharpe agar (MRS, Oxoid, Italy) supplemented with 0.25 % (w/v) L-cysteine hydrochloride (MRSc) and on Bifidobacterium Selective Medium agar (BSM, Sigma Aldrich, Italy) and incubated under anaerobic conditions at 37 °C for 24-72h. Forty-eight isolates were presumptively identified as Bifidobacterium sp. and Lactobacillus sp. based on mmorphological characteristics, physiological and biochemical properties such as catalase, oxidase, spore formation, gelatinase activities, production of indole, NH3 from arginine, and CO2 from glucose. Molecular tools (genus-specific PCR and 16S rRNA gene sequencing) were applied for confirmation. The identified stains were screened for safety features (DNAse, gelatinase, haemolytic activity, antibiotic susceptibility, genes encoding for virulence factors and antibiotic resistance) and functional properties (resistance to low pH and bile salts, lysozyme tolerance, gastrointestinal survival, antagonistic activity against pathogens, hydrophobicity, auto-aggregation, and co-aggregation abilities). The strains, fulfilling the criteria described above, were evaluated for growth in camel milk and their viability, under refrigerated conditions, was monitored till 15 days. Seven stains ascribed to Lactobacillus paracasei and Bifidobacterium asteroides species beyond satisfying the safety requirements, were able to tolerate the harsh environmental condition occurring during the GIT passage and to antagonize both foodborne and intestinal pathogens. The selected stains were able to growth in camel milk and their viability was unaffected during refrigerated storage for 15 days. The seven promising probiotic strains were able to grow and survive in camel milk suggesting their possible use for the formulation of new functional foods.