A novel selective medium for isolation of Limosilactobacillus reuteri from dietary supplements

Limosilactobacillus reuteri is a probiotic bacterium known for its numerous beneficial effects on human health and is commonly utilized in various dietary supplements. Previously, we encountered difficulties in isolating L. reuteri from retail dietary supplements containing complex probiotic compositions by using non-selective media such as de Man, Rogosa, and Sharpe (MRS) agar. Our findings reveal that MRS agar with d-gluconic acid as the carbon source and peptone from soymeal as the nitrogen source provides a growth advantage for L. reuteri. Furthermore, all the tested L. reuteri strains exhibit higher resistance to oxacillin compared with non-L. reuteri strains, and the recovery of L. reuteri is significantly higher than that of non-L. reuteri strains on modified MRS agar (MRS-GSOT agar) supplemented with either 4 or 10 μg/mL oxacillin. Results of spiking tests indicate that MRS-GSOT agar with 10 μg/mL oxacillin can selectively inhibit the growth of species other than L. reuteri in single culture or mixed bacterial broth within food matrices. However, the recovery of L. reuteri is relatively low when subjected to the spiking tests with various ratios of non- L. reuteri. Testing results of 15 retail dietary supplements also show that MRS-GSOT agar could efficiently isolate L. reuteri from retail dietary supplements with complex compositions of probiotic bacteria. In addition, we observe that L. reuteri exhibits two different colony morphologies on MRS-GSOT agar with 10 μg/mL oxacillin, yet they shared a common feature: a noticeable metallic (golden) sheen on the colony surface when the plate is slightly tilted, which can be used to distinguish them from non-L. reuteri species, such as Lactiplantibacillus plantarum subsp. plantarum, Levilactobacillus brevis, and Bifidobacterium longum subsp. longum. In conclusion, we have developed MRS-GSOT agar containing d-gluconic acid, peptone from soymeal, oxacillin, and 2,3,5-triphenyltetrazolium chloride for efficient isolation of L. reuteri from dietary supplements.