Gorse (Ulex europeaus) wastes with 5,6-dimethyl benzimidazole supplementation can support growth of vitamin B12 producing commensal gut microbes.

Many commensal gut microbes are recognized for their potential to synthesize vitamin B12, offering a promising avenue to address deficiencies through probiotic supplementation. While bioinformatics tools aid in predicting B12 biosynthetic potential, empirical validation remains crucial to confirm production, identify cobalamin vitamers, and establish biosynthetic yields. This study investigates vitamin B12 production in three human colonic bacterial species: Anaerobutyricum hallii DSM 3353, Roseburia faecis DSM 16840, and Anaerostipes caccae DSM 14662, along with Propionibacterium freudenreichii DSM 4902 as a positive control. These strains were selected for their potential use as probiotics, based on speculated B12 production from prior bioinformatic analyses. Cultures were grown in M2GSC, chemically defined media (CDM), and Gorse extract medium (GEM). The composition of GEM was similar to CDM, except that the carbon and nitrogen sources were replaced with the protein-depleted liquid waste obtained after subjecting Gorse to a leaf protein extraction process. B12 yields were quantified using liquid chromatography with tandem mass spectrometry. The results suggested that the three butyrate-producing strains could indeed produce B12, although the yields were notably low and were detected only in the cell lysates. Furthermore, B12 production was higher in GEM compared to M2GSC medium. The positive control, P. freudenreichii DSM 4902 produced B12 at concentrations ranging from 7 ng mL-1 to 12 ng mL-1. Univariate-scaled Principal Component Analysis (PCA) of data from previous publications investigating B12 production in P. freudenreichii revealed that B12 yields diminished when the carbon source concentration was ≤30 g L-1. In conclusion, the protein-depleted wastes from the leaf protein extraction process from Gorse can be valorised as a viable substrate for culturing B12-producing colonic gut microbes. Furthermore, this is the first report attesting to the ability of A. hallii, R. faecis, and A. caccae to produce B12. However, these microbes seem unsuitable for industrial applications owing to low B12 yields.

Folate, genomic stability and colon cancer: The use of single cell gel electrophoresis in assessing the impact of folate in vitro, in vivo and in human biomonitoring.

Intake of folate (vitamin B9) is strongly inversely linked with human cancer risk, particularly colon cancer. In general, people with the highest dietary intake of folate or with high blood folate levels are at a reduced risk (approx. 25%) of developing colon cancer. Folate acts in normal cellular metabolism to maintain genomic stability through the provision of nucleotides for DNA replication and DNA repair and by regulating DNA methylation and gene expression. Folate deficiency can accelerate carcinogenesis by inducing misincorporation of uracil into DNA, by increasing DNA strand breakage, by inhibiting DNA base excision repair capacity and by inducing DNA hypomethylation and consequently aberrant gene and protein expression. Conversely, increasing folate intake may improve genomic stability. This review describes key applications of single cell gel electrophoresis (the comet assay) in assessing genomic instability (misincorporated uracil, DNA single strand breakage and DNA repair capacity) in response to folate status (deficient or supplemented) in human cells in vitro, in rodent models and in human case-control and intervention studies. It highlights an adaptation of the SCGE comet assay for measuring genome-wide and gene-specific DNA methylation in human cells and colon tissue.

Effect of increasing fruit and vegetable intake by dietary intervention on nutritional biomarkers and attitudes to dietary change: a randomised trial.

PURPOSE: Low fruit and vegetable consumption is linked with an increased risk of death from vascular disease and cancer. The benefit of eating fruits and vegetables is attributed in part to antioxidants, vitamins and phytochemicals. Whether increasing intake impacts on markers of disease remains to be established. This study investigates whether increasing daily intake of fruits, vegetables and juices from low (approx. 3 portions), to high intakes (approx. 8 portions) impacts on nutritional and clinical biomarkers. Barriers to achieving the recommended fruit and vegetable intakes are also investigated. METHOD: In a randomised clinical trial, the participants [19 men and 26 women (39-58 years)] with low reported fruit, juice and vegetable intake (<3 portions/day) were randomised to consume either their usual diet or a diet supplemented with an additional 480 g of fruit and vegetables and fruit juice (300 ml) daily for 12 weeks. Nutritional biomarkers (vitamin C, carotenoids, B vitamins), antioxidant capacity and genomic stability were measured pre-intervention, at 4-, 8- and 12 weeks throughout the intervention. Samples were also taken post-intervention after a 6-week washout period. Glucose, homocysteine, lipids, blood pressure, weight and arterial stiffness were also measured. Intake of fruit, fruit juice and vegetables was reassessed 12 months after conducting the study and a questionnaire was developed to identify barriers to healthy eating. RESULTS: Intake increased significantly in the intervention group compared to controls, achieving 8.4 portions/day after 12 weeks. Plasma vitamin C (35%), folate (15%) and certain carotenoids [α-carotene (50%) and ß-carotene (70%) and lutein/zeaxanthin (70%)] were significantly increased (P < 0.05) in the intervention group. There were no significant changes in antioxidant capacity, DNA damage and markers of vascular health. Barriers to achieving recommended intakes of fruits and vegetables measured 12 months after the intervention period were amount, inconvenience and cost. CONCLUSION: While increasing fruit, juice and vegetable consumption increases circulating level of beneficial nutrients in healthy subjects, a 12-week intervention was not associated with effects on antioxidant status or lymphocyte DNA damage. TRIAL REGISTRATION: This trial was registered at Controlled-Trials.com; registration ISRCTN71368072.

Toxicity of unsaturated fatty acids to the biohydrogenating ruminal bacterium, Butyrivibrio fibrisolvens.

BACKGROUND: Health-promoting polyunsaturated fatty acids (PUFA) are abundant in forages grazed by ruminants and in vegetable and fish oils used as dietary supplements, but only a small proportion of PUFA finds its way into meat and milk, because of biohydrogenation in the rumen. Butyrivibrio fibrisolvens plays a major role in this activity. The aim of this study was to investigate the mechanisms by which PUFA affect the growth of B. fibrisolvens, how PUFA are metabolized and the metabolic response to growth in the presence of PUFA. RESULTS: Linoleic acid (LA; cis-9, cis-12-18:2) and alpha-linolenic acid (LNA; cis-9, cis-12, cis-15-18:3) increased the lag phase of B. fibrisolvens JW11, LNA having the greater effect. Growth was initiated only when the PUFA had been converted to vaccenic acid (VA; trans-11-18:1). The major fish oil fatty acids, eicosapentaenoic acid (EPA; 20:5(n-3)) and docosahexaenoic acid (DHA; 22:6(n-3)), were not metabolized and prevented growth. Cellular integrity, as determined fluorimetrically by propidium iodide (PI) ingression, was affected as much by 18:1 fatty acids, including VA, as 18:2 fatty acids. The methyl esters of LNA, LA, EPA and DHA had no effect on growth or other measurements. The ATP pool decreased by 2/3 when LA was added to growing bacteria, whereas most acyl CoA pools decreased by >96%. CONCLUSIONS: It was concluded that biohydrogenation occurs to enable B. fibrisolvens to survive the bacteriostatic effects of PUFA, and that the toxicity of PUFA is probably mediated via a metabolic effect rather than disruption of membrane integrity.

Biphasic effect of falcarinol on caco-2 cell proliferation, DNA damage, and apoptosis.

The polyacetylene falcarinol, isolated from carrots, has been shown to be protective against chemically induced colon cancer development in rats, but the mechanisms are not fully understood. In this study CaCo-2 cells were exposed to falcarinol (0.5-100 microM) and the effects on proliferation, DNA damage, and apoptosis investigated. Low-dose falcarinol exposure (0.5-10 microM) decreased expression of the apoptosis indicator caspase-3 concomitantly with decreased basal DNA strand breakage. Cell proliferation was increased (1-10 microM), whereas cellular attachment was unaffected by <10 microM falcarinol. At concentrations above 20 microM falcarinol, proliferation of CaCo-2 cells decreased and the number of cells expressing active caspase-3 increased simultaneously with increased cell detachment. Furthermore, DNA single-strand breakage was significantly increased at concentrations above 10 microM falcarinol. Thus, the effects of falcarinol on CaCo-2 cells appear to be biphasic, inducing pro-proliferative and apoptotic characteristics at low and high concentrations of falcarinol, respectively.

Influence of galangin on HL-60 cell proliferation and survival.

The effect of galangin, a flavonol component of India root spice and the 'herbal' medicine propolis, on HL-60 human leukaemia cell survival is characterised. Galangin (1-100 microM) exerted an antiproliferative effect that, with dose and exposure longevity, was progressively associated with an elevated hypodiploid DNA content and expression of the active form of caspase-3, principally prior to membrane damage. At >or=50 microM, plasmamembrane phosphatidylserine exposure was observed. There was no evidence for intracellular oxidative stress as an orchestrator of cytotoxicity and significant phagocyte-like differentiation was not detected. We discuss whether such cytotoxicity will be therapeutically exploitable or contribute to cancer prevention within a pharmacological or dietary context.

The effect of short-term kaempferol exposure on reactive oxygen levels and integrity of human (HL-60) leukaemic cells.

Flavonoids may be a principal contributor to the cancer preventative activity of fruit- and vegetable-rich diets and there is interest in their use as dietary supplements. However, there is potential conflict between the cytoprotective and cytotoxic activities of flavonoids, and their efficacy as anti-cancer agents is unresolved. Here, the integrity and survival of HL-60 promyelocytic leukaemia cells following short-term (90 min) exposure to the dietary abundant flavonoid kaempferol (1-100 microM) is reported. Supplementation initially decreased reactive oxygen levels but, paradoxically, a dose-dependent increase in single-strand DNA breakage occurred. However, there was no increase in oxidised DNA purines or membrane damage. Following a 24-h recovery period in non-kaempferol supplemented media, DNA single-strand breakage had declined and kaempferol exposed and control cultures possessed similar reactive oxygen levels. A reduction in (3)H-thymidine incorporation occurred with > or =10 microM kaempferol. One hundred micromolar kaempefrol increased the proportion of cells in G(2)-M phase, the proportion of cells with a sub-G(1) DNA content and enhanced 'active' caspase-3 expression but only induced a loss of mitochondrial membrane potential within a minority of cells. The relevance of induced DNA damage within a non-overtly oxidatively stressed environment to the disease preventative and therapeutic use of kaempferol is discussed.