Fermentation profile, cholesterol-reducing properties and chemopreventive potential of ß-glucans from Levilactobacillus brevis and Pediococcus claussenii - a comparative study with ß-glucans from different sources.

The aim of the present study was to investigate whether ß-glucans obtained from the lactic acid bacteria (LAB) Levilactobacillus (L.) brevis and Pediococcus (P.) claussenii exhibit similar physiological effects such as cholesterol-binding capacity (CBC) as the structurally different ß-glucans from oat, barley, and yeast as well as curdlan. After in vitro fermentation, fermentation supernatants (FSs) and/or -pellets (FPs) were analyzed regarding the concentrations of short-chain fatty acids (SCFAs), ammonia, bile acids, the relative abundance of bacterial taxa and chemopreventive effects (growth inhibition, apoptosis, genotoxicity) in LT97 colon adenoma cells. Compared to other glucans, the highest CBC was determined for oat ß-glucan (65.9 ± 8.8 mg g-1, p < 0.05). Concentrations of SCFA were increased in FSs of all ß-glucans (up to 2.7-fold). The lowest concentrations of ammonia (down to 0.8 ± 0.3 mmol L-1) and bile acids (2.5-5.2 µg mL-1) were detected in FSs of the ß-glucans from oat, barley, yeast, and curdlan. The various ß-glucans differentially modulated the relative abundance of bacteria families and reduced the Firmicutes/Bacteroidetes ratio. Treatment of LT97 cells with the FSs led to a significant dose-dependent growth reduction and increase in caspase-3 activity without exhibiting genotoxic effects. Though the different ß-glucans show different fermentation profiles as well as cholesterol- and bile acid-reducing properties, they exhibit comparable chemopreventive effects.

Study on chemopreventive effects of raw and roasted ß-glucan-rich waxy winter barley using an in vitro human colon digestion model.

Due to their unique dietary fibre composition, in particular ß-glucan, the consumption of barley may contribute to a healthy diet and the prevention of colon cancer. The aim of the present study was to analyse chemopreventive effects of barley flakes obtained from a ß-glucan-rich barley cultivar. In order to address the impact of heat treatment on potential chemopreventive effects, barley flakes were roasted (160 °C-180 °C, approx. 20 min). The flakes were subjected to in vitro digestion and fermentation. Fermentation supernatants (FS) were analysed for the concentrations of short-chain fatty acids (SCFA) and ammonia. Chemopreventive endpoints (growth inhibition, apoptosis, DNA integrity, gene expression of detoxifying enzymes) were analysed in LT97 colon adenoma cells. Concentrations of SCFA were increased in barley FS (2.5-fold, on average) with a shift of molar ratios towards butyrate production, while ammonia levels were significantly decreased (0.7-fold, on average) compared to the fermentation control. The growth of LT97 cells was significantly reduced by barley FS in a time- and dose-dependent manner, and caspase-3 activity of treated cells was significantly enhanced (up to 6.3-fold, on average). On average, treatment of cells resulted in increased mRNA levels of CAT (2.1-fold), SOD2 (2.2-fold) and GSTP1 (3.9-fold), while expression of GPx1 (0.3-fold) was significantly decreased in some cases. The roasting process did not cause genotoxic effects of barley FS and had no impact on chemopreventive properties. Our results indicate chemopreventive potential of in vitro fermented waxy winter barley, mediated primarily by growth inhibitory and apoptotic effects, which is largely unaffected by roasting.

Impact of different roasting conditions on sensory properties and health-related compounds of oat products.

Due to their high content of ß-glucan, the consumption of oat products can contribute to a healthy diet. Roasting may improve sensory properties but could also affect the nutritional value of oat products. Therefore, the aim of the present study was to analyse the impact of different roasting conditions (140-180 °C, approx. 20 min) on sensory quality, health-related compounds (e.g. acrylamide, ß-glucan) and viscosities of oat kernels and flakes. Roasting resulted in oat flakes with improved sensory properties. Acrylamide formation increased with higher roasting temperatures in kernels, thin and thick flakes. Contents of fat, protein, starch and ß-glucan were not affected by roasting, whereas dietary fibre fractions were marginally modulated. Viscosities were significantly reduced with increasing roasting temperatures. The results indicate that roasting up to 160 °C is a processing technique with potential to generate oat products with improved sensory quality and favourable nutritional composition.

Impact of different roasting conditions on chemical composition, sensory quality and physicochemical properties of waxy-barley products.

The consumption of ß-glucan-rich barley can contribute to a healthy diet. Sensory properties may be improved by roasting whereby the nutritional value should be preserved. The aim of the present study was to investigate the impact of different roasting conditions (160-200 °C, 20 minutes) on sensory quality, health-related compounds and viscosity in ß-glucan-rich barley kernels, thin and thick flakes. Sensory quality was improved by roasting. Acrylamide levels increased due to roasting to maximum values of 322 µg kg-1 (kernels), 586 µg kg-1 (thin flakes) and 804 µg kg-1 (thick flakes). No relevant impact of roasting on the contents of fat, protein, starch and ß-glucan was observed, whereas dietary fibre fractions were marginally modulated. Roasting significantly decreased viscosity 1.9-fold (kernels), 2.4-fold (thin flakes) and 2.7-fold (thick flakes), on average. In conclusion, improved sensory quality along with a favourable healthy composition of barley products may be achieved by roasting over a low to medium temperature range.

In vitro-fermented raw and roasted walnuts induce expression of CAT and GSTT2 genes, growth inhibition, and apoptosis in LT97 colon adenoma cells.

Walnuts are rich in bioactive compounds such as polyunsaturated fatty acids, polyphenols, and dietary fiber. Therefore, the consumption of walnuts can contribute to a healthy diet and may reduce the risk for colon cancer. Heat treatment like roasting may change the chemical composition of walnuts and therefore their chemopreventive properties. Therefore, the hypothesis of the present study is that different roasting conditions (RCs) alter the chemopreventive effects of walnuts. Thus, the aim of the present study was to investigate whether different RCs (RC1=139.7°C/25 min, RC2=154.5°C/20 min, and RC3=185.5°C/25 min) alter the chemopreventive effects of walnuts. Raw and roasted walnuts were subjected to in vitro digestion and fermentation. After treatment of LT97 colon adenoma cells with fermentation supernatants (FSs), expression of CAT, SOD2, GPx1, GSTP1, and GSTT2 genes as well as cell growth and apoptosis was examined. In comparison to the fermentation blank control, walnut FS particularly increased mRNA levels of CAT 1.7-fold and GSTT2 3.1-fold, whereas GPx1 levels were significantly decreased 0.6-fold. Walnut FS decreased growth of adenoma cells in a time- and dose-dependent manner. In particular, higher concentrations of walnut FS (5%) significantly increased the number of early apoptotic cells 2.0-fold and induced caspase-3 activity 6.8-fold compared with the blank control. The roasting process had no direct impact on the observed effects. In sum, our results indicate that walnuts exhibit chemopreventive effects regarding the risk for colon cancer development by inducing expression of genes involved in detoxification (CAT, GSTT2) and by inducing growth inhibition and apoptosis in colon adenoma cells unaffected by moderate roasting.

Comet assay: an essential tool in toxicological research.

The comet assay is a versatile, reliable, cost-efficient, and fast technique for detecting DNA damage and repair in any tissue. It is useable in almost any cell type and applicable to both eukaryotic and prokaryotic organisms. Instead of highlighting one of the numerous specific aspects of the comet assay, the present review aims at giving an overview about the evolution of this widely applicable method from the first description by Ostling and Johanson to the OECD Guideline 489 for the in vivo mammalian comet assay. In addition, methodical aspects and the influence of critical steps of the assay as well as the evaluation of results and improvements of the method are reviewed. Methodical aspects regarding oxidative DNA damage and repair are also addressed. An overview about the most recent works and relevant cutting-edge reviews based on the comet assay with special regard to, e.g., clinical applications, nanoparticles or environmental risk assessment concludes this review. Taken together, the presented overview raises expectations to further decades of successful applications and enhancements of this excellent method.

In vitro fermentation of nuts results in the formation of butyrate and c9,t11 conjugated linoleic acid as chemopreventive metabolites.

PURPOSE: The consumption of foods rich in dietary fiber and polyunsaturated fatty acids such as nuts can contribute to a healthy diet. Therefore, the formation of fermentation end-products which might exert chemopreventive effects regarding colon cancer was investigated after an in vitro simulated digestion and fermentation of nuts using human fecal microbiota. METHODS: Fermentation supernatants (FS) and pellets (FP) were obtained after an in vitro fermentation of hazelnuts, almonds, macadamia, pistachios and walnuts. Short-chain fatty acids (SCFA) and bile acids (BA) in FS as well as fatty acids in FP were analyzed via gas chromatography. Malondialdehyde (MDA) levels in FS were determined photometrically. RESULTS: Fermentation of nuts resulted in 1.9- to 2.8-fold higher concentrations of SCFA compared to the control and a shift of molar ratios toward butyrate production. In vitro fermentation resulted in the formation of vaccenic acid (C18:1t11, 32.1 ± 3.2 % FAME; fatty acid methyl ester) and conjugated linoleic acid (c9,t11 CLA, 2.4 ± 0.7 % FAME) exclusively in fermented walnut samples. Concentrations of secondary BA deoxycholic-/iso-deoxycholic acid (6.8-24.1-fold/4.9-10.9-fold, respectively) and levels of MDA (1.3-fold) were significantly reduced in fermented nut samples compared to the control. CONCLUSION: This is the first study that demonstrates the ability of the human fecal microbiota to convert polyunsaturated fatty acids from walnuts to c9,t11 CLA as a potential chemopreventive metabolite. In addition, the production of butyrate and reduction in potential carcinogens such as secondary BA and lipid peroxidation products might contribute to the protective effects of nuts regarding colon cancer development.

Influence of miRNA-106b and miRNA-135a on butyrate-regulated expression of p21 and Cyclin D2 in human colon adenoma cells.

Epigenetic and posttranslational modifications of the expression of cell cycle-relevant genes or proteins like p21, e.g., by miRNAs are crucial mechanisms in the development or prevention of colon cancer. The present study investigated the influence of butyrate and trichostatin A (TSA) as histone deacetylase inhibitors on the expression of colon cancer-relevant miRNA (miR-135a, miR-135b, miR-24, miR-106b, miR-let-7a) in LT97 colon adenoma cells as a model of an early stage of colon carcinogenesis. The impact of distinct miRNAs (miR-106b, miR-135a) on butyrate-mediated regulation of p21 and Cyclin D2 gene and protein expression as well as the effect on LT97 cell proliferation (non-transfected, miR-106b and miR-135a mimic transfected) was analyzed. Butyrate and partial TSA reduced the expression of miR-135a, miR-135b, miR-24 and miR-let-7a (~0.5-fold, 24 h) and miR-24, miR-106b and miR-let-7a (~0.5-0.7-fold, 48 h) in LT97 cells. Levels of p21 mRNA and protein were significantly increased by butyrate and TSA (~threefold and 4.5-fold, respectively, 24 h) in non-transfected but not in miR-106b transfected LT97 cells. Levels of Cyclin D2 mRNA were significantly reduced by butyrate and TSA (~0.3-fold, 24 h) in non-transfected and miR-135a-transfected LT97 cells, whereas protein levels were predominantly not influenced. MiR-106b and miR-135a significantly reduced butyrate-/TSA-mediated inhibition of LT97 cell proliferation (72 h). These results indicate that butyrate is able to modify colon cancer-relevant miRNAs like miR-106b and miR-135a which are involved in the regulation of cell cycle-relevant genes like p21 and might influence inhibition of adenoma cell proliferation.

Influence of roasting conditions on health-related compounds in different nuts.

Due to their health-beneficial ingredients the consumption of nuts can contribute to a healthy diet. The composition of hazelnuts, almonds, macadamia nuts, pistachios and walnuts regarding health-promoting and potentially harmful compounds was examined before and after roasting under different time and temperature conditions. Fatty acid compositions were not affected by roasting. Malondialdehyde increased with higher roasting temperatures (17-fold in walnuts). Levels of tocopherol isomers were reduced after roasting (α-T: 38%, ß-T: 40%, γ-T: 70%) and hydrophilic antioxidant capacity decreased significantly in hazelnuts (1.4-fold), macadamia nuts (1.7-fold) and walnuts (3.7-fold). Increasing roasting temperatures supported the formation of significant amounts of acrylamide only in almonds (1220 µg kg(-1)). In general, nuts roasted at low/middle temperatures (120-160°C) exhibited best sensory properties. Therefore, desired sensory quality along with a favourable healthy nut composition may be achieved by roasting over a low to medium temperature range.