Identification and characterisation of lactic acid bacterial secondary metabolites with functional and health benefits

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Hill, Daragh
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University College Cork
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During fermentation lactic acid bacteria (LAB) produce and secrete a range of secondary metabolites. These metabolites have a range of potential applications in both medical and food sectors. They can be used to enhance a food product through the production of desirable metabolites, they can synthesise exopolysaccharides (EPS) and polyols, and LAB can impact both food and medicine through the production of potent antimicrobial compounds. In the research described in this thesis LAB were isolated from ovine, bovine, and caprine raw milks, raw milk cheeses, honey, sheep faeces and other environmental sources to create a biobank of 799 putative LAB strains. This biobank was screened for exopolysaccharide (EPS) and polyol producing LAB for applications in dairy fermentations to produce foods with enhanced functionality and potential health benefits. EPS production has been used to improve the functional properties of food products; a property that is increasingly important given the rising demand for clean label food ingredients. In Chapter Three the entire biobank was tested for EPS production using three phenotypic screening methods in order to identify both ropy and mucoid EPS producers. In total 466 (58%) positive strains were identified, five of which were utilised as adjunct cultures in skim milk fermentations. Following assessment of both safety and acidification characteristics, three strains were selected for inclusion as adjunct cultures in a goat milk fermentation. All three strains significantly improved the texture of the yogurt produced in comparison to the control containing only the starter culture. This work highlights the prevalence of EPS production among LAB and the advantage of using multiple screening methods for identification of EPS producing isolates. Mannitol is a six-carbon polyol that is 50-70% as sweet as sucrose but with only half the calories. With the rising demand for low calorific dairy products there is a need for alternative sweetening methods. In Chapter Four we included a mannitol producing LAB in dairy fermentations to produce a low-calorific, sweetened fermented product. Two hundred and sixty-one isolates of the LAB biobank were screened for their ability to produce mannitol using a high throughput HPLC-based screening method. Twenty-five strains produced mannitol, with five high mannitol producers identified that had yields of over 1.5g/L in fermentation broth. These five strains were applied in skim milk fermentations to investigate if mannitol could also be produced in milk. Small quantities of mannitol were detected in the fermented product, highlighting the potential for in situ production in a dairy product, albeit that production would require further optimisation for commercial application. In Chapter Five the properties of a bacteriocin produced by Streptococcus gallolyticus LL009 were examined. LL009 was one of the high EPS producing strains identified in an earlier chapter. Gallocin D is a narrow spectrum two component bacteriocin with potent activity against vancomycin-resistant enterococci (VRE). It can be readily synthesized and has potential in the treatment of VRE infections. In silico analysis revealed that gallocin D is distinct from a previously identified S. gallolyticus bacteriocin, gallocin A, although the gene clusters encoding both bacteriocins share a high degree of gene synteny. The structural genes are highly variable and appear to have undergone gene shuffling with other streptococcal species. S. gallolyticus is part of the Streptococcus bovis/Streptococccus equinus complex (SBSEC), which contains seven subgroups. Members of the SBSEC show high genome plasticity and in Chapter Six the bacteriocin capabilities of the complex were further examined by including all sequenced strains from each subgroup. The genomes of 105 strains were downloaded and analysed using Bagel4 to identify potential bacteriocin clusters. Forty-four strains showed at least one area of interest (AOI) with a total of 67 AOI identified, and 166 predicted structural genes. Each bacteriocin cluster was aligned using MAUVE and the 67 clusters were assigned to six subgroups, with ten remaining unassigned. The predicted structural genes within each group were compared using MUSCLE to determine whether these structural peptides are identical, variants or unrelated within and/or between species. The source of each isolate was also examined but no obvious pattern was evident. The high prevalence of structural genes among the strains led us to speculate why one operon in a strain would harbour multiple diverse bacteriocin structural genes. Whether or not these strains are capable of producing each of these bacteriocins, there is a fitness cost associated with the presence of extra genes. If these are all transcribed at once, or production is changed depending on the niche the strain is growing in, remains unknown. In depth study on these strains involving growth in a range of conditions and mass spectrometry could help to answer some of these questions.
Microbiology , Bacteriocins , Exopolysaccharide , Polyol
Hill, D. M. 2020. Identification and characterisation of lactic acid bacterial secondary metabolites with functional and health benefits. PhD Thesis, University College Cork.