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Background Bile inhibits bacterial growth because it is rich in bacteriostatic compounds such as bile acids. Analytical techniques using a high-intensity sequencer recently revealed bacterial flora in the bile of normal gallbladders in brain-dead patients. Therefore, we performed a microbial flora analysis of bile collected from pathologically normal gallbladders surgically removed from patients with hepatobiliary pancreatic diseases and normal liver function. Methods Bacterial DNA was extracted from bile samples and analyzed using 16S rRNA sequencing. Results The culture results of all 12 bile samples were negative. However, the results of the 16S ribosome gene analysis suggested the presence of bacterial flora in all samples. The phyla Firmicutes, Proteobacteria, Actinobacteria, and, more specifically, the genera Anaerobacillus, Delftia, Bacillus, Ralstonia, Ochrobactrum, Acidovorax, and Curvibacter were detected in all 12 samples. The results of the 16S rRNA gene profile analysis revealed that Anaerobacillus and Delftia accounted for 58.62%-87.63% of the bacteria identified in each sample. Conclusion In a bacterial flora analysis targeting the 16S ribosomal gene, a specific bacterial flora was detected in bile collected from the pathologically normal gallbladders of patients with hepatobiliary pancreatic diseases. Although a diverse bacterial flora was previously reported in the bile of brain-dead patients, the present results revealed a simple bacterial flora with no diversity in the bile samples.

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A highly sensitive and convenient method for detecting influenza virus was developed using modified end-point melt curve analysis of a RT-qPCR SYBR Green method and influenza virus-binding sugar chain-immobilized gold-nanoparticles (SGNP). Because SGNPs capture influenza viruses, the virus-SGNP complex was separated easily by centrifugation. Viral RNA was detected at very low concentrations, suggesting that SGNP increased sensitivity compared with standard methods. This method was applied to clinical studies. Influenza viruses were detected in saliva of patients or inpatients who had been considered influenza-free by a rapid diagnostic assay of nasal swabs. Furthermore, the method was applied to a human trial of prophylactic anti-influenza properties of yogurt containing Lactobacillus acidophilus L-92. The incidence of influenza viruses in saliva of the L-92 group was found to be significantly lower compared to the control group. Thus, this method was useful for monitoring the course of anti-influenza treatment or preventive measures against nosocomial infection.

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The antiviral effects of both a live and non-live Lactobacillus acidophilus strain L-92 (L-92) were investigated by oral administration (10 mg/mouse per d) daily for 21 d in a mouse model infected intranasally with influenza virus (H1N1). Virus titres in the lung of mice administered either live or non-live L-92 cells daily for 15 d were repressed 6 d after virus infection compared with the control group. Natural killer (NK) activity in the orally administered non-live L-92 group was higher compared with that of the control group before virus infection and on day 6. In contrast, NK activity in the live L-92 group compared with the control group was not significantly changed on both days, but was significantly higher on day 1. In contrast, live L-92 showed a greater repression of virus proliferation compared with non-live L-92, 6 d after the infection. Live L-92 decreased the number of neutrophils in the lung and suppressed lung weight, leading to the consequent deterioration of consolidation scores of the lung. These results indicated that pretreatment of live or non-live L-92 cells had protective effects against influenza virus infection. Among the measured cytokines and chemokines, eotaxin, macrophage colony-stimulating factor, IL-1b, RANTES (regulated on activation, normal T cell expressed and secreted) and interferon-a were significantly increased in the lung: IL-17 was significantly increased in Peyer's patch of the live L-92 group compared with the control group. A mechanistic study suggested that the enhancement of NK activity in the lung caused by stimulating various antiviral cytokines and chemokines after the oral administration of L-92 cells might be important in protecting against virus infection.

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To understand high amount of production and detailed processing of antihypertensive peptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), in Lactobacillus helveticus CM4 fermented milk, whole genome sequence of the CM4 strain was completed and compared to previously reported whole genome sequence of L. helveticus DPC4571. It revealed 2,028,493 bp of DNA sequence and encoding of 2174 open reading frames in the whole genome sequence with the highest homology to the genome sequence of L. helveticus DPC 4571. Comparative analysis focused on proteolytic enzymes between CM4 and DPC4571 strains revealed existence of 23 kinds of identical intracellular peptidase genes in both strains but no prtY type proteinase gene in DPC4571. Immunoblotting analysis with an antibody raised against the PrtY proteinase showed existence of the 45 kDa PrtY protein in CM4 but not in DPC4571 in the cell extracts. The cell wall-associated proteinase activity was higher in the CM4 than that in the DPC4571 throughout all fermentation period, and the amounts of VPP and IPP in CM4 and DPC4571 fermented milk were correlated with the proteinase activity on the cell wall. Moreover, slight difference of the ß-casein hydrolysates by cell wall-associated extracellular proteinases between CM4 and DPC4571 cells was detected by a MALDI-TOF/TOF analysis. These results suggest that the extracellular proteinase activity might affect on the productivity of VPP and IPP in L. helveticus fermented milk and some peptidases might play important role in following precise processing to release VPP and IPP.

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The adhesive activities of eight Lactobacillus acidophilus strains toward intestinal epithelial Caco-2 cells were studied to understand the probiotic characteristics of the L. acidophilus L-92 strain. Most of the strains, including L-92, showed high adhesive activity; CP23 showed the lowest adhesive activity. CP23 was selected for comparative analysis of cell wall-associated proteins versus the L-92 strain. Cell wall-associated proteins extracted from L-92 and CP23 were subjected to two-dimensional electrophoresis, and major spots observed in the former were compared to the corresponding spots in the latter. To understand the effects of key components of L-92 on its adhesion to Caco-2 cells, 18 spots with stronger signals in L-92 than those in CP23 were identified by a MALDI-TOF/TOF of Ultraflex analysis. Among the identified proteins of L-92, surface-layer protein A (SlpA) was considered strongly involved in adhesion in the eight L. acidophilus strains. To study the importance of SlpA in the adhesion of L. acidophilus, the amounts of SlpA proteins in LiCl extracts of the eight strains were compared by SDSpolyacrylamide gel electrophoresis. As a result, the adhesive abilities of L. acidophilus strains to Caco-2 cells correlated closely to the amount of SlpA in the cells and the productivity of IL-12, an inflammatory cytokine, in all eight strains. These results strongly suggested that SlpA in L. acidophilus might play a key role in its attachment to Caco-2 cells and in the release of IL-12 from dendritic cells.

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Two proteolytic enzymes capable of releasing the angiotensin I-converting enzyme (ACE) inhibitor Ile-Pro-Pro from casein were identified by purification of an Aspergillus oryzae extract by three-step column chromatography. First, proteins capable of producing Ile-Pro-Pro from beta-casein were eluted using a DEAE-sepharose FF column with a linear sodium chloride gradient. An endopeptidase capable of releasing Pro-Ile-Pro-Gln-Ser-Leu-Pro-Gln-Asn-Ile-Pro-Pro from Pro-Ile-Pro-Gln-Ser-Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu-Thr-Gln and an aminopeptidase producing Ile-Pro-Pro from Gln-Asn-Ile-Pro-Pro were separated from the resultant fraction using a hydroxyapatite column. Each active enzyme was then loaded onto a Develosil 300Diol gel filtration column for high performance liquid chromatography (HPLC) and purified to homogeneity. The endopeptidase had a molecular mass of approximately 46,000 Da and exhibited an N-terminal amino acid sequence identical to that of neutral protease I (NP I) of A. oryzae. Meanwhile, the aminopeptidase had a molecular mass of 36,000 Da and an N-terminal amino acid sequence similar to that of Leucine aminopeptidase (LAP), as reported in Aspergillus sojae and A. oryzae. The eluted endopeptidase and aminopeptidase were thus identified as NP I and LAP, respectively. Analysis of peptide production using synthetic proteins containing an Ile-Pro-Pro sequence showed that NP I processed the C-terminal end and LAP processed the N terminus to produce Ile-Pro-Pro. While Ile-Pro-Pro was successfully produced from casein by the addition of these two purified enzymes, it was not generated with the addition of only a single enzyme. Based on our experimental findings, we suggest that NP I and LAP are key proteolytic enzymes in the release of Ile-Pro-Pro from casein in A. oryzae.

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