Molecular cloning and sequence analysis of an inulinase gene from an Aspergillus sp

Selected endophytic fungi have been report to be inulin degraders to produce fructose or other oligosaccharides. In this study, the Aspergillus sp. producing inulinase were isolated from selected plant species at Serdang area in Malaysia. Fungal isolates were screened solely based on inulin degrading enzymes production and two isolates named Asf1 and Onf1 were selected as the best inulinase enzyme producers. Genomic DNA of these two isolates were extracted and amplified by polymerase chain reaction (PCR). A 1,341 bp DNA fragment containing inulinase gene was successfully amplified from Asf1 fungal isolate and was named as inu2 gene in this study. Based on the morphological characteristics, rDNA and neighbour-joining phylogenetic analysis, Asf1 fungal isolate could display closely-related to the genus of Aspergillus. The complete sequence designated Asf1 Inu2 gene was successfully obtained via rapid-amplification of cDNA ends-polymerase chain reaction (RACE-PCR). A 2.3 kb DNA fragment encoding endoinulinase, inu2, from Asf1 fungal isolate includes an open reading frame of 1,552 bp with calculated molecular weight of 55,954.1 Da and signal peptide sequence of 23 amino acids. The deduced amino acid sequence of the Asf1 inu2 displayed 97, 96, 69 and 22% identities to that of A. ficuum inu2, A. niger inuB, P. purpurogenum and K. marxianus, respectively. Phylogenetic analysis showed that fungal endo- and exo-inulinases have indepently evolved with the respective hydrolytic activities toward terminal and internal β-(2 → 1)-fructofuranosidic linkages in inulin.     

Cloning and sequence analysis of bile salt hydrolase (bsh) gene from Bifidobacterium longum

A pair of PCR primers for the rapid detection of bile salt hydrolase (bsh) gene from Bifidobacterium longum BB536 has been synthesised and have revealed the bsh gene of approx 970 bp in Bifidobacterium longum BB 536 but not in other species of bacteria tested. The bsh gene was cloned and sequenced showing a high similarity to bsh gene previously published. The resulting nucleotide sequence encodes a predicted protein of 317 amino acids, Mw = 35 kDa.     

Characterization of Salmonella spp. isolated from patients below 3 years old with acute diarrhoea

Fifteen strains of Salmonella were isolated from children with clinically diagnosed diarrhoea aged below 3 years old, who had been admitted to K7 ward, Pediatric Institute, Kuala Lumpur Hospital. The isolates were tested for their susceptibility to a range of antimicrobial agents, and typed by serological tests and randomly amplified polymorphic DNA (RAPD) fingerprinting. All the strains had a similar pattern of antimicrobial susceptibility, where they were susceptible to a wide range of antimicrobial agents. The serological test has typed them into three serovars, which were identified as Salmonella enterica ser. Akanji, Salmonella enterica ser. Hindmarch and Salmonella enterica ser. Richmond. In contrast, the RAPD fingerprinting classed them into two major clusters, cluster 1 consisting of 12 strains of Salmonella and cluster 2 consisting of three strains of Salmonella.     

Growth of Bifidobacterium longum BB536 in medida (fermented cereal porridge) and their survival during refrigerated storage

AIMS: To develop medida, a Sudanese fermented thin porridge as a probiotic dietary adjunct with high total solids. METHODS AND RESULTS: Fifteen per cent brown rice flour of 2-day-old malted paddy and skim milk were used for formulation. Levels of 2.25, 4.5 and 10% of added skim milk were studied. The initial pH was 6.7 and fermentation was run to a final pH of 4.4 using culture of Bifidobacterium longum BB 536. The highest count of 9.9 +/- 0.07 log CFU ml(-1) was obtained with 10% of added skim milk. The total solids at this level was 21%, 11.1 times more compared with the traditionally prepared medida using un-malted brown rice. The viscosity was low and the flowing characteristic was stable. The final productions of lactic and acetic acids were 56.8 +/- 0.80 and 56.3 +/- 2.00 mumol ml(-1) respectively. The high ratio of acetate to lactate decreased as fermentation continues due to the increase in the rate of lactate production. Under refrigerated storage the count of B. longum BB 536 remained relatively stable during the first week (9.7 +/- 0.10 log CFU ml(-1)) then subsequently decreased by 0.9 log CFU ml(-1) in the following week. CONCLUSIONS: The results of this study demonstrated that fermented medida made from malted brown rice is a suitable food system for the delivery of B. longum BB 536 with a relatively stable shelf life. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study is the first attempt to prepare fermented medida from malted flour with bifidobacteria having the highest total solids while still maintaining the flowing characteristics. Previous studies on medida did not go beyond the use of alpha amylase enzyme and pure lactic acid bacteria isolates from spontaneously fermented dough.     

Profiling of recovery efficiencies for three standard protocols (FDA-BAM, ISO-11290, and modified USDA) on temperature-injured Listeria monocytogenes

There have been a number of studies conducted in order to compare the efficiencies of recovery rates, utilizing different protocols, for the isolation of L. monocytogenes. However, the severity of multiple cell injury has not been included in these studies. In the current study, L. monocytogenes ATCC 19112 was injured by exposure to extreme temperatures (60°C and -20°C) for a one-step injury, and for a two-step injury the cells were transferred directly from a heat treatment to frozen state to induce a severe cell injury (up to 100% injury). The injured cells were then subjected to the US Food and Drug Administration (FDA), the ISO-11290, and the modified United States Department of Agriculture (mUSDA) protocols, and plated on TSAyeast (0.6% yeast), PALCAM agar, and CHROMAgar Listeria for 24 h or 48 h. The evaluation of the total recovery of injured cells was also calculated based on the costs involved in the preparation of media for each protocol. Results indicate that the mUSDA method is best able to aid the recovery of heat-injured, freeze-injured, and heat-freeze-injured cells and was shown to be the most cost effective for heat-freeze-injured cells.     

Cell viability of microencapsulated Bifidobacterium animalis subsp. lactis under freeze-drying, storage and gastrointestinal tract simulation conditions

The purpose of this study was to improve the survival of Bifidobacterium animalis subsp. lactis 10140 during freeze-drying process by microencapsulation, using a special pediatric prebiotics mixture (galactooligosaccharides and fructooligosaccharides). Probiotic microorganisms were encapsulated with a coat combination of prebiotics–calcium-alginate prior to freeze-drying. Both encapsulated and free cells were then freeze-dried in their optimized combinations of skim milk and prebiotics. Response surface methodology (RSM) was used to produce a coating combination as well as drying medium with the highest cell viability during freeze-drying. The optimum encapsulation composition was found to be 2.1 % Na-alginate, 2.9 % prebiotic, and 21.7 % glycerol. Maximum survival predicted by the model was 81.2 %. No significant (p?>?0.05) difference between the predicted and experimental values verified the adequacy of final reduced models. The protection ability of encapsulation was then examined over 120 days of storage at 4 and 25 °C and exposure to a sequential model of infantile GIT conditions including both gastric conditions (pH 3.0 and 4.0, 90 min, 37 °C) and intestinal conditions (pH 7.5, 5 h, 37 °C). Significantly improved cell viability showed that microencapsulation of B. lactis 10140 with the prebiotics was successful in producing a stable symbiotic powdery nutraceutical.     

Utilisation of enterobacterial repetitive intergenic consensus (ERIC) sequence-based PCR to fingerprint the genomes of Bifidobacterium isolates and other probiotic bacteria

Enterobacterial repetitive intergenic consensus based on PCR (ERIC-PCR) was used to generate DNA fingerprints for bifidobacteria and other probiotic bacteria. Two primers (ERIC 1R and ERIC 2) used in ERIC-PCR revealed that all of the probiotic bacteria tested possess enterobacterial repetitive intergenic consensus sequences with the PCR products ranging from 250 bp to 5000 bp. The bacterial strains can be differentiated by comparing fingerprint patterns. The dendrogram of the fingerprints revealed that most of the bifidobacterial wild type strains fell into one cluster at similarity level of approximately 79%.     

Safety evaluation of Bifidobacterium pseudocatenulatum G4 as assessed in BALB/c mice

AIMS: To assess the safety of Bifidobacterium pseudocatenulatum G4 in BALB/c mice that involves examination of bacterial translocation, changes in the internal organs and histology of the intestinal lining. METHODS AND RESULTS: Forty male BALB/c mice were randomly assigned into five groups (n = 8). Three groups were orally fed with 50 microl of three different concentrations of B. pseudocatenulatum G4 (2 x 10(4), 1 x 10(8) and 1 x 10(11) CFU day(-1)) for 4 weeks. One group was orally administered with 50 microl of 1 x 10(8) CFU B. longum BB536 per day for 4 weeks and last group was used as a nonbifidobacterial treatment control, which received 50 microl of skim milk. The administered strains did not affect the general health of mice and incapable of carrying out translocation to blood or liver. There were no significant differences in the internal organ (liver, heart, kidney and spleen) indices, serum enzymes of liver (aspartate aminotransferase, alkaline phosphate, alanine aminotransferase) and kidney (urea and creatinine) and histology (villi height, crypts height, mucosa thickness and epithelial cell height) of caecum, ileum and colon. CONCLUSION: Administration of high dose of up to 1 x 10(11) CFU B. pseudocatenulatum G4 per day to mice did not show any health threatening symptoms. SIGNIFICANCE AND IMPACT OF THE STUDY: Bifidobacterium pseudocatenulatum G4 is none pathogenic to BALB/c mice and could be safe probiotic for human consumption.     

An integrated bioreactor-expanded bed adsorption system for the removal of acetate to enhance the production of alpha-interferon-2b by Escherichia coli

A stirred tank bioreactor (STB) integrated with an expanded bed adsorption (EBA) system containing anion-exchange resin (Diaion WA30) was developed for in situ removal of acetate to increase the production of α-interferon-2b (α-PrIFN-2b) by Escherichia coli (E. coli). Although the total acetate (9.79 g/L) secreted by E. coli in the integrated STB/EBA system was higher than that in a bioreactor with dispersed resin or a conventional batch bioreactor, cell growth (14.97 g/L) and α-PrIFN-2b production (867.4 μg/L) were significantly improved owing to the high efficiency of acetate removal from the culture. The production of α-PrIFN-2b in the integrated STB/EBA system was improved by 3-fold and 1.4-fold over that obtained in a conventional batch bioreactor and a bioreactor containing dispersed resins, respectively.