Exopolysaccharides from milk fermented by lactic acid bacteria enhance dietary carotenoid bioavailability in humans in a randomized crossover trial and in rats.

BACKGROUND: Dietary supplementation with carotenoids can have beneficial health effects, but carotenoids are poorly absorbed. OBJECTIVES: We aimed to evaluate how milk fermented by lactic acid bacteria affects dietary carotenoid bioavailability in humans and rats and to investigate mechanisms by which active components in milk fermented by Lactobacilli enhance dietary carotenoid absorption. METHODS: Male rats (n = 8/group) were administered ß-carotene or ß-carotene + fermented milk. Rats (n = 6/group) were also pretreated with ezetimibe, a cholesterol absorption inhibitor, to investigate ß-carotene transport mechanisms. In humans, 3 studies were conducted using a randomized crossover method. Subjects (n = 16/study) consumed a vegetable (carrot, tomato, or spinach) drink alone or with a fermented milk drink. Blood samples were collected at various time points after consumption. RESULTS: In rats, the serum ß-carotene area under the concentration-time curve (AUC) was significantly higher for the ß-carotene + fermented milk than for ß-carotene only. A significant correlation (r = 0.83, P < 0.001) between the exopolysaccharide (EPS) content of fermented milk and serum ß-carotene AUC was observed. Ezetimibe treatment did not suppress elevations in serum ß-carotene concentrations induced by fermented milk ingestion. In humans, the incremental area under the concentration-time curve (iAUC) for ß-carotene in the plasma triacylglycerol-rich lipoprotein (TRL) fraction was significantly (1.8-fold, range: 0.6-3.9) higher when carrot + fermented milk was consumed compared with carrot drink alone. A significantly (6.5-fold, range: 0.04-7.7) higher iAUC for lycopene in the plasma TRL fraction was observed for subjects who consumed tomato + fermented milk compared with tomato drink alone. A significant increase in plasma lutein in all fractions was observed after consumption of spinach + fermented milk, but not with spinach drink alone. CONCLUSIONS: Co-ingestion of ß-carotene and fermented milk significantly increased dietary ß-carotene bioavailability in humans and rats. EPSs could affect the physical properties of fermented milk to enhance dietary ß-carotene absorption mediated by simple diffusion mechanisms. These findings may be relevant for methods to increase dietary carotenoid bioavailability.This trial was registered at umin.ac.jp/ctr as UMIN000034838, UMIN000034839, and UMIN000034840.

Age-related and seasonal changes in covalently bound ceramide content in forearm stratum corneum of Japanese subjects: determination of molecular species of ceramides.

The stratum corneum (SC) consists of corneocytes surrounded by a neutral lipid-enriched intercellular matrix. Ceramides represent approximately 50% of intercellular lipids, and play important roles in retaining epidermal water. The SC also contains covalently bound ceramides, which are thought to play a crucial role in the formation of lamellar structures, and are involved in maintaining skin barrier function. A previous report showed that levels of free ceramides in human SC changed with the seasons and age, although whether the content of different species of covalently bound ceramides also underwent such temporal changes was unclear. Here, SC samples were taken from 99 healthy individuals of different ages (24-64 years) and during different seasons. The content of different molecular species of covalently bound ceramides in the samples was quantified using HPLC-MS/MS. The levels of total covalently bound ceramides (Total-Cers) significantly decreased approximately 50% in autumn and winter, compared with that of spring and summer. The levels of covalently bound ceramides containing saturated fatty acids (SFA-Cers) in the spring and summer were approximately 2.3-fold higher than that seen in autumn and winter, whereas the level of covalently bound ceramides containing unsaturated fatty acids (USFA-Cers) in spring and summer were approximately 1.6-fold higher than that in autumn and winter. Furthermore, the ratio between SFA-Cers and USFA-Cers was significantly lower in spring and summer than in autumn and winter. The levels of SFA-Cers, but not USFA-Cers, were significantly lower in individuals ≥ 50 years old compared to those who are 30- and 40-years old in the spring. Our study showed for the first time that, similar to free ceramides, the level of covalently bound ceramides changed with the seasons. However, age-related changes in covalently bound ceramide content were limited in that only the amount of SFA-Cers in the spring was lower in older individuals.

Milk Fermented by Lactic Acid Bacteria Enhances the Absorption of Dietary Sphingomyelin in Rats.

Supplementation with sphingomyelin has been reported to prevent disease and maintain good health. However, intact sphingomyelin and ceramides are poorly absorbed compared with glycerolipids. Therefore, if the bioavailability of dietary sphingomyelin can be increased, supplementation would be more effective at lower doses. The aim of this study in rats was to evaluate the effect of fermented milk on the bioavailability of dietary sphingomyelin in rats. After the rats had fasted for 15 h, test solutions were administrated orally. Blood samples were collected from the tail vein before and 90, 180, 270, and 360 min after administration. Compared with sphingomyelin/milk phospholipids concentrate (MPL) alone, co-ingestion of sphingomyelin/MPL with fermented milk caused an approximate twofold significant increase in serum ceramides containing d16:1 sphingosine with 16:0, 22:0, 23:0 and 24:0 fatty acids, which was derived from the ingested sphingomyelin. While nonfat milk also increased the serum levels of these ceramides, fermented milk was more effective. Co-ingestion of the upper layer of fermented milk or exopolysaccharide concentrate prepared from fermented milk significantly increased serum ceramide levels. X-ray diffraction analysis also showed addition of fermented milk or EPS concentrate to sphingomyelin eliminated the characteristic peak of sphingomyelin. This study demonstrated for the first time that co-ingestion of dietary sphingomyelin and fermented milk, compared with ingestion of dietary sphingomyelin alone, caused a significant increase in the absorption of sphingomyelin. Our results indicate exopolysaccharides in fermented milk may contribute to inhibition of sphingomyelin crystallization, resulting in enhanced absorption of dietary sphingomyelin in rats.

Exopolysaccharides Isolated from Milk Fermented with Lactic Acid Bacteria Prevent Ultraviolet-Induced Skin Damage in Hairless Mice.

BACKGROUND: We studied the mechanism by which fermented milk ameliorates UV-B-induced skin damage and determined the active components in milk fermented with lactic acid bacteria by evaluating erythema formation, dryness, epidermal proliferation, DNA damage and cytokine mRNA levels in hairless mice exposed to acute UV-B irradiation. METHODS: Nine week-old hairless mice were given fermented milk (1.3 g/kg BW/day) or exopolysaccharide (EPS) concentrate (70 mg/kg BW/day) orally for ten days. Seven days after fermented milk or EPS administration began, the dorsal skin of the mice was exposed to a single dose of UV-B (20 mJ/cm²). RESULTS: Ingestion of either fermented milk or EPS significantly attenuated UV-B-induced erythema formation, dryness and epidermal proliferation in mouse skin. Both fermented milk and EPS were associated with a significant decrease in cyclobutane pyrimidine dimers and upregulated mRNA levels of xeroderma pigmentosum complementation group A (XPA), which is involved in DNA repair. Furthermore, administration of either fermented milk or EPS significantly suppressed increases in the ratio of interleukin (IL)-10/IL-12a and IL-10/interferon-gamma mRNA levels. CONCLUSION: Together, these results indicate that EPS isolated from milk fermented with lactic acid bacteria enhanced DNA repair mechanisms and modulated skin immunity to protect skin against UV damage.

Post-exercise impact of ingested whey protein hydrolysate on gene expression profiles in rat skeletal muscle: activation of extracellular signal-regulated kinase 1/2 and hypoxia-inducible factor-1α.

We have previously shown that whey protein hydrolysate (WPH) causes a greater increase in muscle protein synthesis than does a mixture of amino acids that is identical in amino acid composition. The present study was conducted to investigate the effect of WPH on gene expression. Male Sprague-Dawley rats subjected to a 2 h swimming exercise were administered either a carbohydrate-amino acid diet or a carbohydrate-WPH diet immediately after exercise. At 1 h after exercise, epitrochlearis muscle mRNA was sampled and subjected to DNA microarray analysis. We found that ingestion of WPH altered 189 genes after considering the false discovery rate. Among the up-regulated genes, eight Gene Ontology (GO) terms were enriched, which included key elements such as Cd24, Ccl2, Ccl7 and Cxcl1 involved in muscle repair after exercise. In contrast, nine GO terms were enriched in gene sets that were down-regulated by the ingestion of WPH, and these GO terms fell into two clusters, 'regulation of ATPase activity' and 'immune response'. Furthermore, we found that WPH activated two upstream proteins, extracellular signal-regulated kinase 1/2 (ERK1/2) and hypoxia-inducible factor-1α (HIF-1α), which might act as key factors for regulating gene expression. These results suggest that ingestion of WPH, compared with ingestion of a mixture of amino acids with an identical amino acid composition, induces greater changes in the post-exercise gene expression profile via activation of the proteins ERK1/2 and HIF-1α.

Post-exercise whey protein hydrolysate supplementation induces a greater increase in muscle protein synthesis than its constituent amino acid content.

It is well known that ingestion of a protein source is effective in stimulating muscle protein synthesis after exercise. In addition, there are numerous reports on the impact of leucine and leucine-rich whey protein on muscle protein synthesis and mammalian target of rapamycin (mTOR) signalling. However, there is only limited information on the effects of whey protein hydrolysates (WPH) on muscle protein synthesis and mTOR signalling. The aim of the present study was to compare the effects of WPH and amino acids on muscle protein synthesis and the initiation of translation in skeletal muscle during the post-exercise phase. Male Sprague­Dawley rats swam for 2 h to depress muscle protein synthesis. Immediately after exercise, the animals were administered either carbohydrate (CHO), CHO plus an amino acid mixture (AA) or CHO plus WPH. At 1 h after exercise, the supplements containing whey-based protein (AA and WPH) caused a significant increase in the fractional rate of protein synthesis (FSR) compared with CHO. WPH also caused a significant increase in FSR compared with AA. Post-exercise ingestion of WPH caused a significant increase in the phosphorylation of mTOR levels compared with AA or CHO. In addition, WPH caused greater phosphorylation of ribosomal protein S6 kinase and eukaryotic initiation factor 4E-binding protein 1 than AA and CHO. In contrast, there was no difference in plasma amino acid levels following supplementation with either AA or WPH. These results indicate that WPH may include active components that are superior to amino acids for stimulating muscle protein synthesis and initiating translation.

Dietary whey protein hydrolysates increase skeletal muscle glycogen levels via activation of glycogen synthase in mice.

Previously, we have shown that consuming carbohydrate plus whey protein hydrolysates (WPHs) replenished muscle glycogen after exercise more effectively than consuming intact whey protein or branched-chain amino acids (BCAAs). The mechanism leading to superior glycogen replenishment after consuming WPH is unclear. In this 5 week intervention, ddY mice were fed experimental diets containing WPH, a mixture of whey amino acids (WAAs), or casein (control). After the intervention, gastrocnemius muscle glycogen levels were significantly higher in the WPH group (4.35 mg/g) than in the WAA (3.15 mg/g) or control (2.51 mg/g) groups. In addition, total glycogen synthase (GS) protein levels were significantly higher in the WPH group (153%) than in the WAA (89.2%) or control groups, and phosphorylated GS levels were significantly decreased in the WPH group (51.4%). These results indicate that dietary WPH may increase the muscle glycogen content through increased GS activity.

Comparison of fructooligosaccharide utilization by Lactobacillus and Bacteroides species.

The utilization of 1-kestose (GF(2)) and nystose (GF(3)), the main components of fructooligosaccharides (FOS), by Lactobacillus and Bacteroides species was examined. Of seven Lactobacillus and five Bacteroides strains that utilized FOS, L. salivarius, L. rhamnosus, L. casei, and L. gasseri utilized only GF(2), whereas L. acidophilus and all the Bacteroides strains utilized both GF(2) and GF(3). Only the strains able to utilize both GF(2) and GF(3) had ß-fructosidase activity in the culture supernatants. The culture supernatants of the Lactobacillus strains had higher ß-fructosidase activity for GF(2) than for GF(3), whereas those of the Bacteroides strains had higher activity for GF(3) than for GF(2). Furthermore, ß-fructosidase activity of the culture supernatants of the Lactobacillus cells grown in the GF(3) medium was much higher than that of the cells grown in the GF(2) medium, whereas the activity of the culture supernatants of the Bacteroides cells grown in the GF(3) medium was almost the same as that of the cells grown in the GF(2) medium. These results indicate that Lactobacillus species metabolize FOS in a different way from that of Bacteroides species.

Short-chain fructooligosaccharide regulates hepatic peroxisome proliferator-activated receptor alpha and farnesoid X receptor target gene expression in rats.

Prebiotic short-chain fructooligosaccharide (scFOS) is known to have various beneficial effects in humans and animals. Using a nutrigenomic approach, we have previously identified marker genes for the intestinal immunomodulatory and lipid-lowering effects of scFOS. The present study aimed to predict novel physiological effects of scFOS through nutrigenomic analyses. DNA microarray analysis revealed that administration of scFOS changed the expression of the nuclear receptors peroxisome proliferator-activated receptor alpha (PPARalpha) and farnesoid X receptor (FXR) target genes in the rat liver. Gene expression analysis provided some new interesting hypotheses, for instance, the possible improvement of bile secretion via FXR target genes, and regulation of amino acid metabolism and the urea cycle via PPARalpha and/or FXR target genes. Our findings clearly indicated that nutrigenomics may make it possible to screen for novel physiological effects of dietary ingredients.

Identification of marker genes for lipid-lowering effect of a short-chain fructooligosaccharide by DNA microarray analysis.

Administration of short-chain fructooligosaccharide (scFOS) is known to lower serum triglyceride levels in rats fed a high-fat diet, but the molecular mechanisms remain unclear. This study aimed to identify marker genes for lipid-lowering effect of scFOS administration. The changes in hepatic gene expressions in rats fed scFOS were investigated using DNA microarray and quantitative RT-PCR analysis. The DNA microarray showed that phytanoyl-CoA 2-hydroxylase 2 (Phyh2), lipoprotein lipase (Lpl) and tyrosine aminotransferase (Tat) were significantly affected by scFOS administration (p < .05). Since Lpl is involved in lipid metabolism, the up-regulation of Lpl in the liver can be a potential marker of the lipid-lowering effect of scFOS.

Identification of marker genes for intestinal immunomodulating effect of a fructooligosaccharide by DNA microarray analysis.

Prebiotic fructooligosaccharides are noted for their intestinal immunodulating effects, and the identification of markers for the effects is a matter of great concern. This study aimed to identify marker genes for physiological effects of a particular fructooligosaccharide (FOS) on a host animal and also to define the target of its function in the small intestine. DNA microarray technology was used to screen candidate marker genes, and comprehensive changes in gene expressions in the ileum of mice fed with FOS were investigated. One of the major physiological effects of FOS was intestinal immunomodulation. Marker genes were then identified for major histocompatibility complex classes I and II, interferon, and phosphatidylinositol metabolites. Also, the ileum was segmented into Peyer's patch (PP) and the other ileal organ (DeltaPP), and these were analyzed by quantitative RT-PCR method, with the result that the site for recognizing the FOS function was the DeltaPP rather than the PP. This is the first paper showing the markers for the physiological effects of FOS in the small intestine at gene expression level. Applying these marker genes would make it possible to clarify the mechanisms of how the administration of dietary FOS and associated changes in the intestinal environment are recognized by host organisms as well as how its immunomodulating effects are expressed in the body.

Increase in terminal restriction fragments of Bacteroidetes-derived 16S rRNA genes after administration of short-chain fructooligosaccharides.

It is well known that short chain fructooligosaccharides (scFOS) modify intestinal microbiota in animals as well as in humans. Since most murine intestinal bacteria are still uncultured, it is difficult for a culturing method to detect changes in intestinal microbiota after scFOS administration in a mouse model. In this study, we sought markers of positive change in murine intestinal microbiota after scFOS administration using terminal restriction fragment length polymorphism (T-RFLP) analysis, which is a culture-independent method. The T-RFLP profiles showed that six terminal restriction fragments (T-RFs) were significantly increased after scFOS administration. Phylogenetic analysis of the 16S rRNA partial gene sequences of murine fecal bacteria suggested that four of six T-RFs that increased after scFOS administration were derived from the 16S rRNA genes of the class Bacteroidetes. Preliminary quantification of Bacteroidetes by real-time PCR suggests that the 16S rRNA genes derived from Bacteroidetes were increased by scFOS administration. Therefore, the T-RFs derived from Bacteroidetes are good markers of change of murine intestinal microbiota after scFOS administration.