Yasushi Morinaga

Structural Features and Properties of Bacterial Cellulose Produced in Agitated Culture

The structure and some properties of bacterial cellulose produced in agitated culture were studied. Scanning electron microscopy revealed that there was almost no difference between reticulated structures of bacterial cellulose fibrils produced in agitated culture and in static culture. Nevertheless, bacterial cellulose produced in agitated culture exhibited microstuctural changes, namely, a low degree of polymerization and a low crystallinity index. A CP/MAS 13C NMR analysis revealed that the cellulose Iα content of the cellulose produced in agitated culture was lower than that of the cellulose produced in static culture. The bacterial cellulose produced in agitated culture had a lower Youngs modulus of sheet, a higher water holding capacity and a higher suspension viscosity in the disintegrated form than that produced in static culture.

Recent Trends in Functional Food Science and the Industry in Japan

International recognition of functional foods has resulted in the recent global development of this field, which originated in Japan. The national policy on functional foods, in terms of “foods for specified health use”, also has been developing and has motivated the food industry to produce a variety of new food items. In Japan as well as in many other countries, academic and industrial scientists have been working in collaboration for the analysis and practical applications of functional food science. Emphasis has been placed on the study of antioxidant and anticarcinogenic food factors as well as pre- and probiotics. This review pinpoints recent trends in the science and industry in this field.

Expression of Escherichia coli promoters in Brevibacterium lactofermentum using the shuttle vector pEB003

Abstract A novel promoter probing vector for examining gene expression in a glutamic acid-producing bacterium Brevibacterium lactofermentum was constructed. This plasmid pEB003, a shuttle vector able to replicate either in B. lactofermentum or Escherichia coli , carries a promoter-less chloramphenicol acetyltransferase (CAT) gene as the index gene for probing promoter strength. By using pEB003, we demonstrated that the E. coli promoters, tac, trp and lac UV5 promoters, worked effectively in B. lactofermentum . As evaluated by the CAT activity, the relative strength of the promoters was the same in B. lactofermentum as in E. coli . Thus, B. lactofermentum seems to have a transcriptional machinery that is similar to that of E. coli .

Sugar fatty acid esters inhibit biofilm formation by food-borne pathogenic bacteria.

Effects of food additives on biofilm formation by food-borne pathogenic bacteria were investigated. Thirty-three potential food additives and 3 related compounds were added to the culture medium at concentrations from 0.001 to 0.1% (w/w), followed by inoculation and cultivation of five biofilm-forming bacterial strains for the evaluation of biofilm formation. Among the tested food additives, 21 showed inhibitory effects of biofilm formation by Staphylococcus aureus and Escherichia coli, and in particular, sugar fatty acid esters showed significant anti-biofilm activity. Sugar fatty acid esters with long chain fatty acid residues (C14-16) exerted their inhibitory effect at the concentration of 0.001% (w/w), but bacterial growth was not affected at this low concentration. Activities of the sugar fatty acid esters positively correlated with the increase of the chain length of the fatty acid residues. Sugar fatty acid esters inhibited the initial attachment of the S. aureus cells to the abiotic surface. Sugar fatty acid esters with long chain fatty acid residues (C14-16) also inhibited biofilm formation by Streptococcus mutans and Listeria monocytogenes at 0.01% (w/w), while the inhibition of biofilm formation by Pseudomonas aeruginosa required the addition of a far higher concentration (0.1% (w/w)) of the sugar fatty acid esters.

Relationship between the physical properties and surface area of cellulose derived from adsorbates of various molecular sizes

An aqueous suspension of bacterial cellulose (BC) has such physical properties as higher viscosity, emulsion-stabilizing effect and filler retention than cellulose of other origins. The specific surface areas of BC, microfibrillated cellulose and wood pulp were evaluated by determining the maximum amounts of adsorption of Congo red, cellobiose dehydrogenase (CDH) and xyloglucan. There was a positive linear correlation between the above-mentioned physical properties of each cellulose sample and the specific surface area derived from the maximum amount of CDH adsorbed. The highest physical property values for BC result from the largest external surface area of the fibrils of BC to which CDH was adsorbed.

Inhibition of Streptococcus mutans Biofilm Formation by Streptococcus salivarius FruA

The oral microbial flora consists of many beneficial species of bacteria that are associated with the healthy condition and control the progression of oral disease. Cooperative interactions between oral streptococci and the pathogens play important roles in the development of dental biofilms in the oral cavity. To determine the roles of oral streptococci in multi-species biofilm development and the effects of the streptococci in biofilm formation, the active substances inhibiting S. mutans biofilm formation were purified from Streptococcus salivarius ATCC 9759 and HT9R culture supernatants using ion-exchange and gel filtration chromatography. MALDI-TOF mass spectrometry analysis was performed and the results were compared to data bases. The S. salivarius HT9R genome sequence was determined; and used to indentify candidate proteins for inhibition. The candidates inhibiting biofilms were identified as S. salivarius fructosyltransferase (FTF) and exo-beta-D-fructosidase (FruA). The activity of the inhibitors was elevated in the presence of sucrose; and the inhibitory effects were dependent on the sucrose concentration in the biofilm formation assay medium. Purified and commercial FruA from Aspergillus niger (31.6% identity and 59.6% similarity to the amino acid sequence of FruA from S. salivarius HT9R) completely inhibited S. mutans GS-5 biofilm formation on saliva-coated polystyrene and hydroxyapatite surfaces. The inhibition was induced by decreasing polysaccharide production dependent on sucrose digestion rather than fructan digestion. The data indicate S. salivarius produces large quantities of FruA; and FruA alone may play an important role in multi-species microbial interactions for sucrose-dependent biofilm formation in the oral cavity.ABSTRACT The oral microbial flora consists of many beneficial species of bacteria that are associated with a healthy condition and control the progression of oral disease. Cooperative interactions between oral streptococci and the pathogens play important roles in the development of dental biofilms in the oral cavity. To determine the roles of oral streptococci in multispecies biofilm development and the effects of the streptococci in biofilm formation, the active substances inhibiting Streptococcus mutans biofilm formation were purified from Streptococcus salivarius ATCC 9759 and HT9R culture supernatants using ion exchange and gel filtration chromatography. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis was performed, and the results were compared to databases. The S. salivarius HT9R genome sequence was determined and used to indentify candidate proteins for inhibition. The candidates inhibiting biofilms were identified as S. salivarius fructosyltransferase (FTF) and exo-beta-d-fructosidase (FruA). The activity of the inhibitors was elevated in the presence of sucrose, and the inhibitory effects were dependent on the sucrose concentration in the biofilm formation assay medium. Purified and commercial FruA from Aspergillus niger (31.6% identity and 59.6% similarity to the amino acid sequence of FruA from S. salivarius HT9R) completely inhibited S. mutans GS-5 biofilm formation on saliva-coated polystyrene and hydroxyapatite surfaces. Inhibition was induced by decreasing polysaccharide production, which is dependent on sucrose digestion rather than fructan digestion. The data indicate that S. salivarius produces large quantities of FruA and that FruA alone may play an important role in multispecies microbial interactions for sucrose-dependent biofilm formation in the oral cavity.

Acute Effect of Poly-γ-Glutamic Acid on Calcium Absorption in Post-Menopausal Women

Objective: Poly-γ-glutamic acid (PGA) increases calcium (Ca) solubility in vitro and in vivo, and is associated with reduced bone loss in post-menopausal Japanese women. This study is the first to examine the effect of PGA on Ca absorption in humans. Methods: A single-blind, randomized, crossover study with a 3–4 week wash-out was performed to determine the effect of PGA (80.6% glutamic acids) on Ca absorption measured by the double stable isotope method. Twenty-four healthy, non-smoking, postmenopausal women (mean age: 56.4 ± SE 0.9) were given 200 g of orange juice containing 200 mg Ca as Ca-44 enriched CaCO3, with or without 60 mg of PGA, after an overnight fast. The two tests were separated by 3–4 weeks. An intravenous injection of Ca-42 (CaCl2 solution) was given 30 min after consuming the drink and a complete urine collection carried out from 24–48 h post-dosing. Ca absorption was calculated from the Ca isotope ratios measured by thermal ionization quadrupole mass spectrometry (TIQMS). Results: Mean Ca absorption with PGA was significantly higher (P < 0.01) than without PGA, 39.1 (SE 1.6) % and 34.6 (SE 1.9) %, respectively. The effect of PGA on increasing Ca absorption was more marked in a sub-group of subjects whose baseline Ca absorption (without PGA) was lower than the population mean value. Conclusion: Postmenopausal women who received a single dose of PGA increased their intestinal Ca absorption particularly those individuals with lower basal absorptive capacity.

Degree of Polymerization of Cellulose from Acetobacter xylinum BPR2001 Decreased by Cellulase Produced by the Strain

Acetobacter xylinum produces both cellulase and bacterial cellulose, but some report believed that this cellulase activity does not decrease the degree of polymerization (DP) of bacterial cellulose during cultivation. A. xylinum subsp. sucrofermentans BPR2001 produces two enzymes that hydrolyze CM-cellulose and cellotriose, respectively. We examined the effect of the two cellulase activities on the DP of bacterial cellulose when bacterial cells were cultured with agitation at pH 4, where little cellulase is produced, and at pH 5, where much cellulase is produced. The weight-average degree of polymerization (DPw) of bacterial cellulose remained in the range of 14,000 of 16,000 during cultivation at pH 4, but at pH 5, the DPw decreased from 16,800 to 11,000. The mechanical strength of a sheet prepared from the bacterial cellulose produced at pH 4 was higher than those of BC produced at pH 5. These results suggest that the two cellulase activities cause the decrease in DP and deterioration of physical properties of bacterial cellulose seen during cultivation.

Significance of microbial symbiotic coexistence in traditional fermentation

Symbiosis has long been a central theme in microbiology. There have been many studies on the symbioses between microorganisms and higher organisms such as plants and animals. There also have been some studies on the symbiosis or coexistence of microorganisms, such as yeasts, lactic acid bacteria (LAB), acetic acid bacteria (AAB) and koji molds, in traditional fermentation (brewing). These microorganisms are considered to interact and cooperate with each other in various natural environments, such as dropped cereal crops or ripe fruits. Human beings have taken advantage of these microbial interactions for producing various fermented foods.

The Importance of Inter-Species Cell-Cell Co-Aggregation between Lactobacillus plantarum ML11-11 and Saccharomyces cerevisiae BY4741 in Mixed-Species Biofilm Formation

Cells of Lactobacillus plantarum ML11-11, an isolate from Fukuyama pot vinegar, and the yeast Saccharomyces cerevisiae formed significant mixed-species biofilms with concurrent inter-species co-aggregation. The co-aggregation did not occur with heated or proteinase K-treated ML11-11 cells, or in the presence of D-mannose, suggesting that surface proteins of ML11-11 and mannose-containing surface substance(s) of yeast were the predominant contributing factors. Sugar fatty acid ester inhibited mixed-species biofilm formation, but did not inhibit co-aggregation, suggesting that the cell-cell adhesion and cell-polystylene adhesion are controlled by different mechanisms. Microscopic observation and microflora analysis revealed that inter-species co-aggregation plays an important role in the formation of the mixed-species biofilm.