Synthesis of oligosaccharides with prebiotic potential by crude enzyme preparation from Bifidobacterium.

Oligosaccharides especially prebiotics take high attention in the development of foods because of their physiological properties in human health. They are generally synthetized enzymatically via transferases or hydrolases from mold or bacteria. The fact is that such oligosaccharides synthetized by probiotic bacteria, should be utilized by these microorganisms. This study focused on the production of oligosaccharides with prebiotic potential by crude enzyme preparation from bifidobacteria. Both monosubstrates and bisubstrates systems together with TLC and HPLC techniques, were applied. The crude enzyme preparation has different hydrolase activities such as α-glucosidase (2U/mL), ß-glucosidase (0.3 U/mL), α-galactosidase (1.2 U/mL), ß-galactosidase (0.4 U/mL), ß-fructosidase (11.5 U/mL). Additionally, it also has transglycosylation activities on lactose, lactulose, maltose and sucrose substrates. Two or three types of oligosaccharides were detected. The glycosyltransferase activity peaked at 45 °C, pH 6.6 and 30 g/100 mL substrate concentration. Significant high amount of oligosaccharides were formed in the case of lactose:sucrose combination than others. Both glucooligosaccharides and galactooligosaccharides are detected in the reaction mixtures of bisubstrate. When the lactose is present, the galactosyltransferation is predominated. One-one new types of oligosaccharides were detected in the reaction mixture of bioconversion. Among newly synthetized oligosaccharides, the fraction namely OS4 was utilized by probiotic bifidobacteria only. In conclusion, new types of galacto- and glucooligosaccharides with high prebiotic potentials were synthetized by the crude enzyme from probiotic Bifidobacterium strains.

Probiotic Beverage From Pineapple Juice Fermented With Lactobacillus and Bifidobacterium Strains.

Pineapple is an economically significant plant and the third most important fruit crop in the tropical and subtropical regions of the world. In this study, fermentation of pineapple juice with probiotic bacteria Lactobacillus and Bifidobacterium strains as well as changes of some properties in the beverage during storage were investigated. All tested strains exhibited good growth properties on pineapple juice without supplementation of any nutrient compounds. After 24 h fermentation, the cell counts of lactobacilli passed the level of 5*109 cfu/ml, while the cell number of bifidobacteria reached a level of 109 cfu/ml. The highest volumetric productivity (3.5*108 cfu/ml*h) was observed in L. plantarum 299V. The ratios of lactic acids to acetic acids in the cases of L. plantarum 299V and L. acidophilus La5 were 5.37 and 9.91, respectively. In the case of B. lactis Bb-12, the concentrations of lactic acid and acetic acid were 6 mM and 23 mM in natural juices, and 15 and 21 mM in the case of supplementation with prebiotics at the 16th h of fermentation, respectively. Additionally, supplementation with prebiotics at the initiation of fermentation resulted 7 mM lactic acid and 23 mM acetic acid at the end of fermentation. Fructose was the most preferred sugar for both lactobacilli and bifidobacteria. Both total phenolic content and antioxidant capacity increased slightly during fermentation and dropped during the storage period. The microbial population did not change significantly during the first month of storage. After the storage period (2 months), the probiotic bacteria lost about 0.11 log cfu/ml viability after treatment with 0.3% pepsin for 135 min, and a further 0.1 log cfu/ml after treatment with 0.6% bile salts. These values were 10 times higher than data from the fresh fermented pineapple juice. Our results are very promising and may serve as a good base for developing probiotic pineapple juice.

A novel thermostable alpha-galactosidase from the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b: purification and characterization.

High levels of an extracellular alpha-galactosidase are produced by the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b when grown in submerse culture and induced by sucrose. The enzyme was purified 114-fold from the culture supernatant by (NH(4))(2)SO(4) fractionation, and by chromatographical steps including Sepharose CL-6B gel filtration, DEAE-Sepharose FF anion-exchange, Q-Sepharose FF anion-exchange and Superose 12 gel filtration. The purified enzyme exhibits apparent homogeneity as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and iso-electric focusing (IEF). The native molecular weight of the monomeric alpha-galactosidase is 93 kDa with an isoelectric point of 3.9. The enzyme displays a pH and temperature optimum of 5-5.5 and 65 degrees C, respectively. The purified enzyme retains more than 90% of its activity at 45 degrees C in a pH range from 5.5 to 9.0. The enzyme proves to be a glycoprotein and its carbohydrate content is 5.3%. Kinetic parameters were determined for the substrates p-nitrophenyl-alpha-galactopyranoside, raffinose and stachyose and very similar K(m) values of 1.13 mM, 1.61 mM and 1.17 mM were found. Mn(++) ions activates enzyme activity, whereas inhibitory effects can be observed with Ca(++), Zn(++) and Hg(++). Five min incubation at 65 degrees with 10 mM Ag(+) results in complete inactivation of the purified alpha-galactosidase. Amino acid sequence alignment of N-terminal sequence data allows the alpha-galactosidase from Thermomyces lanuginosus to be classified in glycosyl hydrolase family 36.

Formation of novel hydrogel bio-anode by immobilization of biocatalyst in alginate/polyaniline/titanium-dioxide/graphite composites and its electrical performance.

A new bio-anode containing gel-entrapped bacteria in alginate/polyaniline/TiO2/graphite composites was constructed and electrically investigated. Alginate as dopant and template as well as entrapped gel was used for immobilization of microorganism cells. Increase of polyaniline concentration resulted an increase in the conductivity in gels. Addition of 0.01 and 0.02 g/mL polyaniline caused 6-fold and 10-fold higher conductivity, respectively. Furthermore, addition of 0.05 g/mL graphite powder caused 10-fold higher conductivity and 4-fold higher power density, respectively. The combination of polyaniline and graphite resulted 105-fold higher conductivity and 7-fold higher power-density output. Optimized concentrations of polyaniline and graphite powder were determined to be 0.02 g/mL and 0.05 g/mL, respectively. Modified hydrogel anode was successfully used in microbial fuel cell systems both in semi- and continuous operations modes. In semi-continuous mode, about 7.88 W/m3 power density was obtained after 13 h of fermentation. The glucose consumption rate was calculated to be about 7 mg glucose/h/1.2·107 CFU immobilized cells. Similar power density was observed in the continuous operation mode of the microbial fuel cell, and it was operated stably for more than 7 days. Our results are very promising for development of an improved microbial fuel cell with new type of bio-anode that have higher power density and can operate for long term.

Novel method for screening microbes for application in microbial fuel cell.

The ability to produce and to transport exo-electrons by microbes either to external acceptors or to electrodes are reported in our study. All investigated microorganisms (exception of Lactobacillus plantarum) exhibited strong iron-reducing capabilities in the absence of mediator meaning production and secretion of exo-electrons to the growth medium. L.plantarum, Saccharomyces cerevisiae and Escherichia coli need an electron shuttle molecule to reduce Fe(3+) ion. Significant correlation was observed between growth and iron-reducing capacity, as well as between initial cell counts and iron-reducing capacity. Changes of bio-current generated in MFC and iron-reduction were experimentally monitored, and a mathematical model was established by regression analysis. Based on these results, a novel and rapid screening method was developed for the selection of microorganisms for potential application in MFC. The method is based on the measurement of absorbance of bacterial and yeast cultures at 460 nm, providing a robust and high sample throughput approach.

Performances of new isolates of Bifidobacterium on fermentation of soymilk.

Growth and metabolic activity of several new, human origin isolates of Bifidobacterium strains were investigated. All tested bifidobacteria strains were grown well on the native soymilk medium without any additional nutrients. The fermentation processes cultured with initial cell concentrations in 105 -107 cfu/ml resulted in 108 cfu/ml after 8-12 h of incubation in soymilk, and were kept viable up to the end of fermentation (48 h). Volumetric productivities of B. bifidum B3.2, B. bifidum B7.1 and B. breve B9.14 were 1.6 × 10¹° cfu/L.h, 4.5 × 10¹° cfu/L.h and 7.6 × 109 cfu/L.h, respectively, whereas these values of B. lactis Bb-12 and B. longum Bb-46 probiotic strains were 2.7 × 109 cfu/L.h and 1.0 x 10¹° cfu/L.h. The α-galactosidase activities were also detected in the intracellular fraction of the disrupted cells. Productions of lactic and acetic acids were in the range of 23-60 mmol/L and 2.4-5.6 mmol/L, respectively. Molar ratios of acetate to lactate in all tested strains varied from 0.05-0.1 that are very promising for further technological development of probiotic fermented soy-based food products.