The work presented in this study demonstrates the broad applicability of the strategy to isolate strains with enhanced vitamin B2 production characteristics using sub-inhibitory levels of roseoflavin. The resultant strains are made through a non-recombinant method and may therefore be readily applicable in an industrial setting. Various biotechnological processes have been developed for industrial scale riboflavin biosynthesis using the ascomycetes Ashbya gossypii, different yeasts and the bacterium B. subtilis . The latter organism has been successfully employed at a commercial scale to produce riboflavin for feed and food fortification purposes. However, since this production procedure involved a genetically recombinant organism regulatory approval was required where substantial equivalence of the product to non-recombinant riboflavin had to be established, while no DNA from the production strain was allowed to be present in the final product . The purpose of the present study was not to develop strains which would challenge these already available processes, but rather to look at the potential to isolate strains with an improved riboflavin production phenotype that could replace the riboflavin-consuming parent strains in traditional food fermentation processes thus improving the bioavailability of riboflavin.
The Lc. mesenteroides strain, NCDO 2028 used in this study was originally isolated from beetroot silage, and was chosen as a representative of a species, which has many varied industrial applications in the dairy industry and various plant and vegetable fermentation processes [35-37]. Lb. plantarum, used in a large number of fermentation processes was chosen as another candidate. This organism is found in many diverse environments, owing to its metabolic flexibility . In general, descriptions of the strain's nutritional requirements indicate that riboflavin is not required for growth, indicative of a functional riboflavin biosynthetic pathway . Lb. plantarum NCDO 1752, a pickled cabbage isolate, is capable of growth in the absence of the vitamin illustrating its ability to synthesise the vitamin. However, in the course of this work it was revealed that the sequenced strain of Lb. plantarum, WCFS1 does not contain a functional rib operon and consequently is unable to grow in the absence of exogenous riboflavin . Two strains of P. freudenreichii (both isolated from cheese), one subspecies freudenreichii (NIZO B374) and one subspecies shermanii (NIZO B369) were selected from the NIZO culture collection and the resistance strategy was applied also to this strain. All isolated mutants from these two subspecies were shown to be riboflavin overproducers. The successful application of the roseoflavin resistance strategy in three diverse bacterial species shows that it is a readily employable system in an industrial setting to isolate starter strains that produce and secrete vitamin B2. This is particularly appealing when one considers that some yoghurt cultures have been shown to actually decrease the concentration of riboflavin in some products due to their consumption of the vitamin . In a simultaneously performed collaborative study it was demonstrated that a fermented dairy product produced with P. freudenreichii B2336 was able to improve growth and riboflavin status of riboflavin-depleted animals . This proves that the riboflavin produced in yoghurt fermentation is available as a nutrient.
Although not all spontaneous or induced mutations causing riboflavin overproduction in B. subtilis have been analysed in detail, the characterised roseoflavin-induced mutations in this organism have been located either in ribC, the bifunctional flavokinase/FAD synthetase, which converts riboflavin to FMN and FAD [28,30], or in the regulatory region upstream of the rib operon . This knowledge facilitated the identification of the mutations present in the roseoflavin-resistant LAB strains. No mutations were identified in the ribC homologue of any of the roseoflavin-resistant strains, but instead all characterised mutants were found to contain mutations in the regulatory leader region upstream of the rib operon. In both species different point mutations were identified, which were shown to differentially affect the level of riboflavin overproduction. Additionally, for both LAB species spontaneous roseoflavin-resistant mutants were isolated containing various deletions in the regulatory region of the rib operon. It is expected that such mutations are extremely stable. Furthermore, in P. freudenreichii it was shown that after sixty generations in non-selective media the riboflavin-overproducing phenotype does not revert to the wildtype phenotype. In analogy to what is known for the regulation of riboflavin biosynthesis in B. subtilis and L. lactis [24,31,42] it is assumed that also in the species used in this study regulation of the rib operon would be mediated by a termination-antitermination mechanism resulting from two different folding options of the RFN element upstream of the operon in response to riboflavin or FMN. RFN elements have been identified in the rib operon leader region of Lc. mesenteroides (Fig. 2) and Lb. plantarum (Fig. 5) using RFAM . It is likely from the position of the mutations that they affect the stability of the terminator structure making it less energetically favourable for it to form, thus allowing continued transcription of the rib operon.
In order to study the potential usefulness of such strains in an industrial setting a pilot yoghurt trial was set up to compare addition of P. freudenreichii NIZO B374 or its riboflavin-overproducing derivative B2336 either 24 hours prior to the addition of the starter culture or simultaneously with the starter culture. Sequential addition of strain NIZO B2336 to the yoghurt was found to double the concentration of riboflavin in the final product in comparison to yoghurt containing the non-producing wildtype propionibacterium. Furthermore, the mutant showed no differences in comparison to the wildtype strain regarding final cell numbers of the starter culture or final pH of the product. This illustrates that there is a clear benefit of using such riboflavin-overproducing strains in fermentations as it increases the vitamin content of the final product, thus making it more appealing to consumers. Sequential inoculation of the propionibacteria was also found to counteract acidification resulting in a milder product, which could be used as another positive selling point.
The basic concepts of nutrition are changing. The traditional idea of an 'adequate diet', which provides enough nutrients to ensure the individual's survival and meet metabolic needs as well as satisfying hunger is now obsolete. More and more emphasis is being placed on the need for foods to promote health, improve well being and reduce the risk of illness through the adoption of the concept of an "optimum diet" . Selection of strains that have been subjected to uncontrolled genetic alterations has been used in the dairy industry to improve certain intrinsic characteristics of the fermented end product. For example, a spontaneous IS element mediated deletion of the lacZ gene altered lactose metabolism resulting in a decreased fermentation of the sugar. Yoghurt made using this strain is not affected by further acidification . Strains modified by induced mutations are considered non-GMO and are acceptable for deliberate release in the European Union . Such strategies could have important implications for food fermentations as industry constantly strives to increase the marketability of their products to more health conscious consumers in an increasingly competitive market.