Periodic Report Summary - COLORSPORE (New sources of natural, gastric stable, food additives, colourants and novel functional foods)
Carotenoids are lipophilic antioxidants recognised widely as giving many plants, fruits, flowers and vegetables their red, orange and yellow colours and it is through diet that many animals such as salmon, trout and flamingos obtain their flesh and feather colourations. Carotenoids in animals including humans are widely recognised as having important anti-oxidant activities and some act as a precursor source of vitamin A. There is also increasing evidence that some of these play an important role in benefiting human health.
The COLOSPORE project has originated from the discovery that a certain class of bacterium that is able to form spores carries species that are rich in carotenoids. These spore-forming bacteria are mostly of the Bacillus genus and have been largely unnoticed. Interestingly, and of significant industrial interest is the finding that Bacillus carotenoids have been shown to be gastric stable, that is, they can survive passage through the stomach which is an environment of low pH. This is the central problem with existing carotenoids that are used as dietary supplements. Here carotenoids are rapidly destroyed in the stomach requiring large doses to be consumed. The ability of Bacillus carotenoids to survive conditions of low pH is probably linked to the unique resistance properties of these dormant life forms.
The COLOSPORE consortium in year 1 devoted its efforts to identifying and characterising the bacteria and the carotenoids produced by them. This has now been achieved. In year 2 our efforts have focused on short-listing key Bacillus strains that produce carotenoids in workpackage 1 (WP1). These represent well-characterised strains for which we know the identity at the species level. For these strains we have determined their carotenoid profiles, their safety (i.e. ensuring they carry no virulence or pathogenic traits) and production efficiency (i.e. can we prepare large quantities easily). For two isolates (HU36 and GB1) we have identified media with which to grow high yields using industrial conditions (WP3). We have also determined the complete genome sequences of both organisms and with these we have identified all genes (crt) involved in carotenoid biosynthesis (WP2). The crt genes enable us to define the biosynthetic pathways and this has been complemented by structural analysis of individual carotenoids in each pathway (WP2). Thus, at the end of year 2 we know the biosynthetic pathways from two Bacillus type-strains, one producing a yellow/orange end product (HU36), the other a pink/red carotenoid (GB1). The individual compounds are now known and will be named shortly.
Regarding safety these strains have been evaluated in animal (in vivo) and using in vitro laboratory models for safety (WP5). We have shown that none produce any cause for concern and this has been supported by metabolomic studies. In WP3 and WP5 a GC-MS platform has been set up and validated and used to evaluate all compounds produced by HU36 and GB1 during cell growth. This has been conducted in parallel to a food-grade strain of Bacillus subtilis (Natto). GB1 appears to be equivalent to Natto while HU36 does show some differences. The compounds that are new to HU36 are being evaluated but are expected to be innocuous.
In WP4 extensive evaluation of the stability of purified carotenoids has been made. In the presence of iron the carotenoids are shown to be significantly more stable than either b-carotene or lycopene. Moreover, antioxidant activity is far superior in the presence of iron. These results are encouraging and provide a potential avenue for further exploitation.
Finally, in WP6 a preliminary yet exhaustive analysis of the potential of using pigmented Bacilli as probiotic food ingredients has been assessed. Here, spores of HU36 have been incorporated into baked food products and assessed for taste, odour, colour and stability. Results suggest that this approach has potential with industry and should be considered.
The COLOSPORE project has originated from the discovery that a certain class of bacterium that is able to form spores carries species that are rich in carotenoids. These spore-forming bacteria are mostly of the Bacillus genus and have been largely unnoticed. Interestingly, and of significant industrial interest is the finding that Bacillus carotenoids have been shown to be gastric stable, that is, they can survive passage through the stomach which is an environment of low pH. This is the central problem with existing carotenoids that are used as dietary supplements. Here carotenoids are rapidly destroyed in the stomach requiring large doses to be consumed. The ability of Bacillus carotenoids to survive conditions of low pH is probably linked to the unique resistance properties of these dormant life forms.
The COLOSPORE consortium in year 1 devoted its efforts to identifying and characterising the bacteria and the carotenoids produced by them. This has now been achieved. In year 2 our efforts have focused on short-listing key Bacillus strains that produce carotenoids in workpackage 1 (WP1). These represent well-characterised strains for which we know the identity at the species level. For these strains we have determined their carotenoid profiles, their safety (i.e. ensuring they carry no virulence or pathogenic traits) and production efficiency (i.e. can we prepare large quantities easily). For two isolates (HU36 and GB1) we have identified media with which to grow high yields using industrial conditions (WP3). We have also determined the complete genome sequences of both organisms and with these we have identified all genes (crt) involved in carotenoid biosynthesis (WP2). The crt genes enable us to define the biosynthetic pathways and this has been complemented by structural analysis of individual carotenoids in each pathway (WP2). Thus, at the end of year 2 we know the biosynthetic pathways from two Bacillus type-strains, one producing a yellow/orange end product (HU36), the other a pink/red carotenoid (GB1). The individual compounds are now known and will be named shortly.
Regarding safety these strains have been evaluated in animal (in vivo) and using in vitro laboratory models for safety (WP5). We have shown that none produce any cause for concern and this has been supported by metabolomic studies. In WP3 and WP5 a GC-MS platform has been set up and validated and used to evaluate all compounds produced by HU36 and GB1 during cell growth. This has been conducted in parallel to a food-grade strain of Bacillus subtilis (Natto). GB1 appears to be equivalent to Natto while HU36 does show some differences. The compounds that are new to HU36 are being evaluated but are expected to be innocuous.
In WP4 extensive evaluation of the stability of purified carotenoids has been made. In the presence of iron the carotenoids are shown to be significantly more stable than either b-carotene or lycopene. Moreover, antioxidant activity is far superior in the presence of iron. These results are encouraging and provide a potential avenue for further exploitation.
Finally, in WP6 a preliminary yet exhaustive analysis of the potential of using pigmented Bacilli as probiotic food ingredients has been assessed. Here, spores of HU36 have been incorporated into baked food products and assessed for taste, odour, colour and stability. Results suggest that this approach has potential with industry and should be considered.