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TNO’s intestinal screening model (TNO i-screen) helps to quickly identify food ingredients that modulate the intestinal microbiota composition. For manufacturers, searching for health-promoting ingredients is a complex and time-consuming process. Large numbers of substances have to be screened, while for some components almost no proper identification methods are available. When a functional ingredient has finally been selected, extensive in vitro and human volunteer studies are required to demonstrate its safety and to validate its efficacy prior to marketing it as beneficial to health.

Single dose live oral vaccines are most urgently needed to meet the prerequisites as phrased in the `Children's Vaccine Initiative'. The use of a Generally Regarded as Safe (GRAS) micro-organism as Lactobacillus as oral vaccine carrier has the advantage a.o. that these bacteria have probiotic, health beneficial properties and can be used as `food-grade' delivery vehicles. In mice, using model antigens we have investigated the properties of various Lactobacillus strains as a carrier and as an adjuvant. It was shown that some Lactobacillus-strains do possess adjuvant properties to a level as observed with well established water-in-oil adjuvants whereas other strains do not provide immuno-enhancing effects. Haptenated Lactobacillus demonstrated the carrier function which is provided in serum antibody as well as DTH responses. Synthetic peptides with at least one B-cell and one T-cell epitope coupled to the surface of the bacteriae indicated that surface expression best supports immune response induction (antibodies, DTH). Expression vectors for Lactobacillus with surface anchored viral- and bacterial peptide-antigens as well as vectors allowing the intracellular expression and secretion of antigens have been developed and are being tested in vaccine feasibility studies.

The DNA sequences of a 2.4 kb plasmid (p353-2) from Lactobacillus pentosus MD353 and a 1.9 kb plasmid (p8014-2) from Lactobacillus plantarum ATCC 8014 show 81.5% overall similarity. Both plasmids carry elements (replication protein gene, plus-origin and minus-origin of replication), which are typical of plasmids that replicate via a rolling-circle mechanism of replication (RCR). Direct evidence for an RCR mechanism was obtained by showing the accumulation of single-stranded plasmid intermediates in the presence of rifampicin. A minus-origin of replication was defined for plasmids p353-2 and p8014-2 based on DNA sequence analysis and on its ability to convert single-stranded into double-stranded plasmid DNA. Plasmids pLPE323, pLPE350 and pLPC37 that are derived from the p353-2 or p8014-2 replicon are structurally and segregationally stable in L. pentosus MD353, L. plantarum ATCC 8014 and in Lactobacillus casei ATCC 393. The presence of Escherichia coli or λ DNA fragments in vectors derived from p353-2 or p8013-2 does not affect the structural stability but results in segregational instability of the vectors. The instability increases with increasing size of the inserted DNA fragment. Since vectors based on these replicons can be effciently propagated in a wide variety of Lactobacillus species, they are highly suitable for cloning and expression of foreign DNA in Lactobacillus, provided that selective pressure is applied.