Possibility of breast cancer prevention: use of soy isoflavones and fermented soy beverage produced using probiotics.

The various beneficial effects of soybeans, which are rich in phytochemicals, have received much attention because of increasing health awareness. Soy milk that has been fermented using lactic acid bacteria has been used to prepare cheese-like products, tofu (bean-curd), and yogurt-type products. However, the distinct odor of soybeans has limited the acceptance of such foods, particularly in Western countries. In Japan, while tofu and soy milk have long been habitually consumed, the development of novel, palatable food products has not been easy. The unpleasant odor of soy milk and the absorption efficiency for isoflavones can be improved using a recently developed fermented soy milk beverage. Cancer has been the leading cause of death, and breast cancer is the most common malignancy among women. The most common type of breast cancer is estrogen-dependent, and the anti-estrogenic effects of isoflavones are known. The present review focuses on the characteristics of soy milk fermented using probiotics, an epidemiological study examining the incidence of breast cancer and soy isoflavone consumption, and a non-clinical study examining breast cancer prevention using fermented soy milk beverage.

Continuous intake of galacto-oligosaccharides containing syrup contributes to maintaining the health of household dogs by modulating their gut microbiota.

Interest is growing in the relationship of the microbiota and intestinal environment with health in companion animals. Galacto-oligosaccharides (GOS), typical prebiotics, are expected to provide benefits in dogs. Previous studies of GOS in dogs have involved dogs with similar rearing conditions and diets, which may have biased the results. We conducted an open study of 26 healthy dogs kept in households with diverse rearing environments in order to evaluate how the intake of a GOS-containing syrup affects the intestinal microbiota and its metabolites. Each dog was fed 1.2-4.8 g of the GOS-containing syrup (GOS 0.5-2.0 g equivalent) for 8 weeks. Fecal microbiota, fecal concentrations of organic acids and putrefactive products, fecal odor, and serum uremic toxin concentrations were evaluated before intake (0 weeks), during the 8-week intake period (4 and 8 weeks), and 4 weeks after intake (12 weeks). The activity of N-benzoyl-DL-arginine peptidase in dental plaque, which may be associated with periodontal disease, was evaluated at 0 and 8 weeks. Continuous intake of GOS resulted in changes in fecal microbiota, with a particularly marked increase in the abundance of Megamonas, which produces propionic acid. Other findings included a significant increase in the fecal acetic, propionic, and n-butyric acid concentrations. Additionally, significant decreases in fecal odor, fecal phenol concentration, and serum indoxyl sulfate concentration. Intake of GOS was also associated with a significant decrease in N-benzoyl-DL-arginine peptidase activity in dental plaques. These results suggest that continuous intake of GOS may contribute to canine health.

Lactobacillus casei Shirota enhances the preventive efficacy of soymilk in chemically induced breast cancer.

Soy foods are known to be effective for breast cancer prevention. The habitual consumption of soy isoflavones in combination with the probiotic Lactobacillus casei Shirota (LcS) was shown to decrease the risk of breast cancer occurrence in our previous population-based case-controlled study among Japanese women. The present study aimed to elucidate the cooperative prevention mechanism of soymilk and LcS using an animal carcinogenic model. Female Sprague-Dawley rats received a high-fat, AIN-76A diet containing soymilk, LcS, both soymilk and LcS, or none and were orally exposed to 2-amino-1-methyl-6-penylimidazo[4,5-b]pyridine at a dose of 85 mg/kg bodyweight eight times for 2 weeks. The development of palpable mammary tumors was monitored for 17 weeks. Tumor tissues were immunohistochemically examined for estrogen receptor (ER)-α, Ki-67 and CD34. Compared with the control group, the incidence and multiplicity of mammary tumors were reduced by soymilk alone and soymilk in combination with LcS, while tumor volume was decreased by LcS alone and LcS in combination with soymilk. An immunohistochemical analysis revealed that soymilk in combination with LcS more effectively reduced the numbers of ER-α-positive and Ki-67-positive cells in tumors than soymilk alone and that both soymilk and LcS inhibited tumor angiogenesis. These results demonstrated that soymilk prevents the development of mammary tumors and that LcS suppresses tumor growth, potentially enhancing the preventive efficacy of soymilk. The habitual consumption of LcS in combination with soymilk might be a beneficial dietary style for breast cancer prevention.

Analysis of fecal microbial profiles in dogs with intestinal lymphangiectasia.

Intestinal lymphangiectasia (IL) is a common complication in dogs. This study analyzed intestinal microbiota using 16S rRNA amplicon analysis as candidate factors that strongly influence the small intestinal lymphatic vessels in dogs with and without IL. Twelve dogs were included, of which six were diagnosed with lymphoplasmacytic enteritis, four with small-cell lymphoma, and two with large-cell lymphoma. Seven of these dogs had IL, whereas five did not. First, the microbial diversity analyzed by Faith pd index was significantly decreased in dogs with IL compared to dogs without IL. Then, the relative amounts of each bacterial taxa were compared between dogs with and without IL using Linear discriminant analysis effect size analysis. At the genus level, the Ruminococcus gnavus group significantly increased in dogs with IL compared to dogs without IL. A total of four genera, including Ruminococcus torques group and Faecalibacterium, which produce butyrate, significantly decreased in dogs with IL. This study showed decreased intestinal bacterial diversity and several alterations of intestinal microbiota, including a decrease in butyrate-producing bacteria in dogs with IL, compared to dogs without IL.

Impacts of Habitual Diets Intake on Gut Microbial Counts in Healthy Japanese Adults.

Although diet is an important factor influencing gut microbiota, there are very few studies regarding that relationship in Japanese people. Here, we analyzed the relationship between habitual dietary intake surveyed by food frequency questionnaire and the quantitative features of gut bacteria by quantitative PCR and next generation sequencer in 354 healthy Japanese adults. The α-diversity of gut microbiota was positively correlated with the intake of mushrooms and beans and negatively correlated with the intake of grains. The ß-diversity was significantly associated with the intake of fruits, mushrooms, seaweeds, seafoods, and alcoholic beverages. Multiple linear regression analysis of the relationship between food groups associated with the diversity of gut microbiota and the number of gut bacteria at the genus level found 24 significant associations, including a positive association between alcoholic beverages and the number of Fusobacterium. These results support that habitual dietary intake influenced the diversity of gut microbiota and was strongly associated with the number of specific gut bacteria. These results will help us to understand the complex relationship between habitual diet and gut microbiota of the Japanese.

Peptide array-based screening of human mesenchymal stem cell-adhesive peptides derived from fibronectin type III domain.

Human mesenchymal stem cell-adhesive peptides were screened based on the amino acid sequence of fibronectin type III domain 8-11 (FN-III(8-11)) using a peptide array synthesized by the Fmoc-chemistry. Using hexameric peptide library of FN-III(8-11) scan, we identified the ALNGR (Ala-Leu-Asn-Gly-Arg) peptide that induced cell adhesion as well as RGDS (Arg-Gly-Asp-Ser) peptide. After incubation for 2h, approximately 68% of inoculated cells adhere to the ALNGR peptide disk. Adhesion inhibition assay with integrin antibodies showed that the ALNGR peptide interacts with integrin beta1 but not with alphavbeta3, indicating that the receptors for ALNGR are different from RGDS. Additionally, the ALNGR peptide expressed cell specificities for adhesion: cell adhesion was promoted for fibroblasts but not for keratinocytes or endotherial cells. The ALNGR peptide induced cell adhesion and promoted cell proliferation without changing its property. It is therefore useful for the construction of functional biomaterials.

Computationally assisted screening and design of cell-interactive peptides by a cell-based assay using peptide arrays and a fuzzy neural network algorithm.

We developed a method of effective peptide screening that combines experiments and computational analysis. The method is based on the concept that screening efficiency can be enhanced from even limited data by use of a model derived from computational analysis that serves as a guide to screening and combining the model with subsequent repeated experiments. Here we focus on cell-adhesion peptides as a model application of this peptide-screening strategy. Cell-adhesion peptides were screened by use of a cell-based assay of a peptide array. Starting with the screening data obtained from a limited, random 5-mer library (643 sequences), a rule regarding structural characteristics of cell-adhesion peptides was extracted by fuzzy neural network (FNN) analysis. According to this rule, peptides with unfavored residues in certain positions that led to inefficient binding were eliminated from the random sequences. In the restricted, second random library (273 sequences), the yield of cell-adhesion peptides having an adhesion rate more than 1.5-fold to that of the basal array support was significantly high (31%) compared with the unrestricted random library (20%). In the restricted third library (50 sequences), the yield of cell-adhesion peptides increased to 84%. We conclude that a repeated cycle of experiments screening limited numbers of peptides can be assisted by the rule-extracting feature of FNN.

Peptide array-based interaction assay of solid-bound peptides and anchorage-dependant cells and its effectiveness in cell-adhesive peptide design.

Peptide array, the designable peptide library covalently synthesized on cellulose support, was applied to assay peptide-cell interaction, between solid-bound peptides and anchorage-dependant cells, to study objective peptide design. As a model case, cell-adhesive peptides that could enhance cell growth as tissue engineering scaffold material, was studied. On the peptide array, the relative cell-adhesion ratio of NIH/3T3 cells was 2.5-fold higher on the RGDS (Arg-Gly-Asp-Ser) peptide spot as compared to the spot with no peptide, thus indicating integrin-mediated peptide-cell interaction. Such strong cell adhesion mediated by the RGDS peptide was easily disrupted by single residue substitution on the peptide array, thus indicating that the sequence recognition accuracy of cells was strictly conserved in our optimized scheme. The observed cellular morphological extension with active actin stress-fiber on the RGD motif-containing peptide supported our strategy that peptide array-based interaction assay of solid-bound peptide and anchorage-dependant cells (PIASPAC) could provide quantitative data on biological peptide-cell interaction. The analysis of 180 peptides obtained from fibronectin type III domain (no. 1447-1629) yielded 18 novel cell-adhesive peptides without the RGD motif. Taken together with the novel candidates, representative rules of ineffective amino acid usage were obtained from non-effective candidate sequences for the effective designing of cell-adhesive peptides. On comparing the amino acid usage of the top 20 and last 20 peptides from the 180 peptides, the following four brief design rules were indicated: (i) Arg or Lys of positively charged amino acids (except His) could enhance cell adhesion, (ii) small hydrophilic amino acids are favored in cell-adhesion peptides, (iii) negatively charged amino acids and small amino acids (except Gly) could reduce cell adhesion, and (iv) Cys and Met could be excluded from the sequence combination since they have less influence on the peptide design. Such rules that are indicative of the nature of the functional peptide sequence can be obtained only by the mass comparison analysis of PIASPAC using peptide array. By following such indicative rules, numerous amino acid combinations can be effectively screened for further examination of novel peptide design.

Intake of a fermented soymilk beverage containing moderate levels of isoflavone aglycones enhances bioavailability of isoflavones in healthy premenopausal Japanese women: a double-blind, placebo-controlled, single-dose, crossover trial.

This study aimed to investigate the bioavailability of serum isoflavones after the intake of soymilk fermented by Lactobacillus casei strain Shirota containing 32.5% isoflavone aglycones (FSM) or placebo soymilk containing no isoflavone aglycones (SM). In a double-blind, placebo-controlled, single-dose, crossover trial, 7 healthy premenopausal Japanese women (mean age: 35.3 ± 11.0) consumed FSM or SM on day 1 and crossed over to the other soymilk after a 6-day washout period. Serum isoflavones in blood samples collected at 0, 1, 2, 3, 4, and 5 hr after intake were analyzed by liquid chromatography coupled with tandem mass spectrometry. The area under the curve (AUC) values for the serum concentrations of genistein and total isoflavones were significantly higher, by about 1.4-fold, up to 5 hr after FSM intake compared with SM intake (each p<0.05), and that of daidzein tended to be higher after FSM intake. In addition, AUC analysis of total isoflavones for individual subjects revealed that 5 out of 7 subjects had higher AUC values after FSM intake compared with SM intake and that the 2 remaining subjects had similar AUC values. These 2 subjects had higher AUC values after SM intake (mean, 2,502 ± 348) than those of the other subjects (mean, 1,158 ± 269). These results indicate that the bioavailability of isoflavones, especially genistein, is enhanced after the intake of FSM containing 32.5% isoflavone aglycones compared with intake of SM containing no isoflavone aglycones and that the enhancement is observed in healthy premenopausal Japanese women whose isoflavone absorption capacity is low after SM intake.

Consecutive Intake of Fermented Milk Containing Bifidobacterium breve Strain Yakult and Galacto-oligosaccharides Benefits Skin Condition in Healthy Adult Women.

A double-blind, placebo-controlled, randomized trial was conducted to investigate the beneficial effect of probiotic and prebiotic fermented milk on the skin of healthy adult women. Forty healthy Japanese adult female volunteers with healthy skin randomly received either a bottle of probiotic and prebiotic fermented milk containing Bifidobacterium breve strain Yakult and galacto-oligosaccharides (GOS) (active group) or a non-fermented placebo milk containing neither probiotics nor GOS (placebo group) daily for 4 weeks. Before and after intake, hydration levels and cathepsin L-like activity in the stratum corneum and phenol levels in the serum and urine were determined. After intake, the hydration level of the stratum corneum decreased significantly in only the placebo group and was significantly lower than in the active group (p=0.031). Cathepsin L-like activity, an indicator of keratinocyte differentiation, was significantly increased in the active group (p=0.027). Serum and urine phenol levels decreased significantly in the active group (p=0.014, p=0.002, respectively), and serum phenol levels were significantly lower in the active group compared with the placebo group (p=0.006). The consecutive intake of probiotic and prebiotic fermented milk can benefit skin condition without dryness and decrease the levels of phenol production by gut bacteria in healthy adult women.

Screening of peptides with a high affinity to bile acids using peptide arrays and a computational analysis.

Bile acid binding peptides have attracted attention for the improvement and prevention of hypercholesterolemia. In this study, screening of bile acid high affinity peptides was investigated using computationally-assisted peptide array analysis. Starting with the screening data obtained from a limited, random 6-mer library (2212 sequences), the peptides with a high affinity to bile acid were characterized by comparison of high- and low-affinity peptides using fuzzy neural network (FNN) analysis. The physical properties of amino acids at specific positions that contribute to bile acid binding activity were extracted as the structural rule; optimization was carried out using three repeated screening cycles of the rule extraction. The extracted structural rule indicates that Trp, Tyr, Phe, Leu, Ile and Val are enriched in bile acid binding peptides. The yields of bile acid binding peptides with an affinity of above the VAWWMY peptide (soystatin, control sequence) were significantly higher in the optimized structural rule (32.5%) compared to that of the random library (3.1%), and 6 peptides were obtained with above 2.0-fold increased binding activity.

Screening of peptides with a high affinity for ZnO using spot-synthesized peptide arrays and computational analysis.

Metal-binding peptides have attracted attention for their usefulness in biomineralization processes. In this work screening of high affinity peptides against ZnO was performed using a combinatorial library approach with a peptide array and computational analysis. The computationally assisted peptide screening and design enabled identification of linear peptides with affinity for ZnO with limited experimentation. Starting with the screening data obtained from a random 6-mer library of 420 sequences, the characteristics of the peptides with high ZnO affinity were analyzed using a fuzzy neural network algorithm by comparison of high and low affinity peptides. Three physical properties of amino acids (hydrophobicity, isoelectric point and size) and positional information of each residue were analyzed and a peptide rule with restricted amino acids at certain positions was extracted. The average affinity for ZnO increased 2.0-fold when 300 sequences were synthesized according to the restricted random library, compared with the non-restricted library. In addition, for a peptide library with the amino acids at the restricted sites exchanged the average binding capacity decreased to 0.7-fold. Interestingly, the peptides with high ZnO affinity obtained exhibited binding specificity for ZnO. Computationally assisted screening is an invaluable means for finding peptides with limited experimentation.

Screening of a novel octamer peptide, CNSCWSKD, that induces caspase-dependent cell death.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is known to induce apoptosis to various tumor cells but not in normal cells. We have screened cell death-inducing peptides from the extracellular domain sequence of TRAIL, using a peptide array. Peptides of higher activity were found through amino acid substitution, and the CNSCWSKD peptide induced >90% cell death in treated Jurkat cells. Features of apoptosis, such as DNA fragmentation, activation of caspase, phosphatidylserine externalization, chromatin condensation, and competition with TRAIL for binding to the death receptor (DR) 4 or DR5 were observed, suggesting that this peptide is a TRAIL mimic. Caspase-3 activation was observed in various tumor cells treated with this peptide as well as with TRAIL, while no activation was observed in human normal fibroblasts. The CNSCWSKD peptide is a potential candidate for use in cancer therapy.