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Probiotic microbes may confer a variety of positive health effects on the host. Until now, development of probiotic products has mainly focused on Lactobacillus and Bifidobacterium species, which generally tolerate the stresses encountered during processing, storage and delivery well. In recent years, newly-discovered gut microbes have gained attention due to their association with healthy host conditions. However, these microbes, designated next generation probiotics, are often oxygen-sensitive, do not tolerate the established product processing techniques, and need protection during delivery and gastric transit. Here, we review the challenges related to development of next generation probiotic products. The applications of current microbial processing and delivery techniques for the oxygen-sensitive next generation probiotics are assessed, and putative process optimizations are discussed. Current microbial product processing techniques are not suited for next generation probiotics, thus optimizations or entirely novel processing approaches are needed. Freeze-drying is currently the only method that keeps cells viable during processing and storage, but optimization of the process for individual strains is required, e.g. by adding antioxidants to the drying solution.Oral delivery of live next generation probiotics is poorly investigated. The strains in question are often known to colonize primarily in the colon, and carriers, such as microparticles and microdevices, which have been verified for colon-targeted delivery of drugs, may represent a novel choice as delivery vehicle for next generation probiotics. • Antioxidants increases viability of oxygen-sensitive microbes during freeze-drying. • Desiccation of oxygen-sensitive microbes is required for prolonged storage. • Freeze-drying is the best method for processing of oxygen-sensitive microbes. [ABSTRACT FROM AUTHOR]

Encapsulation of bacteria into a polymer matrix can potentially enhance the delivery of probiotics. In this study, we present a multilayer electrospun construct to facilitate enhanced delivery of probiotics, where the internal layer is loaded with bacterial cells and the external layers sandwich the internal layer for increased protection and potential mucoadhesion properties. In this proof of concept study, Lactobacillus rhamnosus GG (LGG), which is a robust and well-studied probiotic strain, was encapsulated into pullulan nanofibers, with two electrospun PLGA (Poly-lactic-co-glycolic acid) layers covering it. According to our in vitro study, there was a large decrease in the viability of LGG released from the monolayer sample (LGG:pullulan). However, the multilayer construct maintained excellent viability and an acceptable storage potential. Our in vivo competition study showed that LGG, delivered by multilayer construct, were able to survive intestinal transit and were recovered from all segments of the intestine. Not only did the multilayer construct perform as well as non-encapsulated spray-dried cells in terms of viability and establishment of LGG, but it also showed some cases of increased LGG colonization from fibers in jejunum and cecum compared to spray-dried LGG three days after dosage. We believe, the multilayer construct has the potential to enhance the delivery of the strains that require additional protection, and can benefit from mucus embedment with the help of the covering layers. Particularly, the use of electrospun fibers for the protection of next-generation probiotics sensitive to oxygen and/or gastric conditions could have major commercial interest. Schematic representation of the procedure performed to load LGG into nanofibers and to fill the capsules either with Strep-resistant spray-dried LGG (StrepR) or Rif-resistant LGG (RifR) loaded into fiber (or the opposite composition) to perform a competition study between the two forms of LGG dosed into an individual rat. [Display omitted] • Multilayer electrospun construct for enhanced encapsulation/delivery of probiotics. • Higher viability of LGG from multilayer construct compared to monolayer construct. • Multilayer construct as good as spray-dried cells in establishment of LGG in vivo. • We recommend layered structure for encapsulation of next generation probiotics. [ABSTRACT FROM AUTHOR]