The term "nutraceutical" was first coined in 1989 as a contraction of the words "nutrition" and "pharmaceutical", and refers to a food compound that not only supplements the diet but also aids in the prevention and/or treatment of disease and/or disorder [1]. Similarly, functional foods contain at least one component, whether it be a nutrient or not, that affects a target function of the organism in a specific, positive way, thereby generating a physiological effect beyond its nutritional value [2]. Functional food ingredients include probiotics, prebiotics, vitamins and minerals, and can be found in such diverse products as fermented dairy products, sports drinks and chewing gum [3,4]. Interest in and acceptance of functional foods is gaining impetus, for a number of reasons that include changing consumer demands and social attitudes, scientific evidence of the health benefits of particular ingredients and commercially driven interests to add value to existing products. Consumer awareness on nutrition raises the demand for healthy food options, ideally delivered in a convenient form [5]. Functional foods have a significant and growing global market with recent estimates indicating up to a $50 billion annual share [4].
Since Metchnikoff first theorised that fermented milk products provided health benefits, including longer life expectancy, these products have been viewed as "healthy" by consumers [6]. In many modern societies fermented dairy products make up a substantial proportion of the total daily food consumption. Lactic acid bacteria (LAB) are an industrially important group of micro-organisms used all over the world for centuries in a large variety of food fermentations, such as those of meat and vegetables, but undoubtedly with the major application in the dairy industry. LAB have been shown to be ideal cell factories because the biosynthetic capacity and metabolic versatility of these bacteria is generally quite limited, their physiology is relatively simple, while their energy metabolism and biosynthesis processes are almost completely separated [7]. It has therefore been possible to exploit LAB for the production of many nutraceuticals [8]. The dairy propionibacteria, especially P. freudenreichii ssp. shermanii, are the main ripening flora in Swiss-type cheeses, where they play a critical role in the development of the characteristic flavour and "eye" production [9]. Propionibacteria are also used for the production of vitamin B12 [10] and there is increasing interest in their potential use as probiotics [11]. Considering the current widespread use of LAB and propionibacteria in the food industry, coupled with consumer demand for healthier foods, the potential to use these food grade microorganisms as "vitamin factories" was investigated.
Riboflavin is a water-soluble vitamin produced by plants and many micro-organisms. However, this biosynthetic capability is lacking in higher animals and they must therefore obtain this essential nutrient from their diet. Riboflavin is the precursor of the enzyme cofactors FMN and FAD, which are vital in many of the body's enzymatic functions for the transfer of electrons in oxidation-reduction reactions. Riboflavin deficiency is most commonly seen in developing countries [12], among the elderly [13], and in chronic alcoholics [14]. Clinical symptoms of riboflavin deficiency are rarely seen in developed countries but the subclinical stage of deficiency, characterised by a change in biochemical indices, is seen in a significant portion of the population of these nations as exemplified by Ireland [15]. Riboflavin deficiency mainly manifests itself clinically in the mucocutaneous surfaces of the mouth, through the occurrence of cracks at the corners, and inflammation of the lips and tongue [16], but deficiency is also associated with vision deterioration and growth failure. In recent years the vitamin has been found to be effective in the treatment of migraine [17], malaria [18] and Parkinson's disease [19]. Riboflavin is commonly obtained in the diet from meat, eggs, fortified cereals and green leafy vegetables, in addition to dairy products, which contribute most significantly to riboflavin intake [20].
Riboflavin has been traditionally synthesised for food and feed fortification by chemical means but in more recent years biotechnological processes employing various bacteria, yeast and fungi have been found to be commercially competitive and are replacing chemical synthesis [21]. One of these biotechnological processes employs Bacillus (B.) subtilis as the riboflavin cell factory and much work has been carried out in characterising the vitamin's biosynthetic pathway in this organism (For a review see [22]). For B. subtilis and Lactococcus (L.) lactis it has been shown that mutants that are isolated on the basis of their resistance to the toxic riboflavin analogue roseoflavin also exhibit a riboflavin-overproduction phenotype [23,24]. Recently, it was demonstrated that fermented dairy products produced either with a roseoflavin-resistant strain of P. freudenreichii or L. lactis, was able to improve growth and riboflavin status of riboflavin-depleted animals [25,26].
The current study reports on the isolation of roseoflavin-resistant mutants of various strains used in the food industry and analysis of their resulting riboflavin-overproducing phenotype. In the case of Lb. plantarum and Lc. mesenteroides the mutations responsible for riboflavin overproduction were identified and the possible effects of these mutations on transcription of the rib operon are discussed. In the case of P. freudenreichii the riboflavin-overproducing strains were examined in comparison to a control strain in a yoghurt production model.
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