Expression and characterisation of neopullulanase from Lactobacillus mucosae

Balolong, M. P.,Chae, J. P.,Kang, D. K. Springer Science + Business Media 2016 Biotechnology letters Vol.38 No.10

<P>To clone and express a neopullulanase gene from Lactobacillus mucosae LM1 in Escherichia coli and characterise the resulting recombinant neopullulanase. An ORF in L. mucosae corresponding to a neopullulanase was cloned and expressed in E. coli. The predicted amino acid sequence of the neopullulanase contained catalytic sites and conserved motifs that are present in members of the neopullulanase subfamily. The resulting recombinant neopullulanase was efficiently purified by Ni-NTA affinity chromatography. The purified enzyme optimally hydrolyses pullulan at 37 A degrees C and pH 6.0, producing panose as the major reaction product. To the best of our knowledge, this is the first report of the cloning, expression and characterisation of a neopullulanase gene from a lactic acid bacterium.</P>

Are They Really Stem Cells? Scrutinizing the Identity of Cells and the Quality of Reporting in the Use of Adipose Tissue-Derived Stem Cells

Balolong, Ernesto,Lee, Soojung,Nemeno, Judee Grace,Lee, Jeong Ik Hindawi Publishing Corporation 2016 Stem cells international Vol.2016 No.-

<P>There is an increasing concern that the term adipose tissue-derived stem cell (ASC) is inappropriately used to refer to the adipose stromal vascular fraction (SVF). To evaluate the accuracy and quality of reporting, 116 manuscripts on the application of ASC in humans and animals were examined based on the 2013 published International Federation for Adipose Therapeutics and Science (IFATS)/ International Society for Cellular Therapy (ISCT) joint statement and in reference to current guidelines for clinical trials and preclinical studies. It is disconcerting that 4 among the 47 papers or 8.51% (CI 2.37–20.38) surveyed after publication of IFATS/ISCT statement reported using ASCs but in fact they used unexpanded cells. 28/47 or 59.57% (CI 44.27–73.63) explicitly reported that adherent cells were used, 35/47 or 74.47% (CI 59.65–86.06) identified expression of surface markers, and 25/47 or 53.19% (CI 14.72–30.65) verified the multilineage potential of the cells. While there are a number of papers examined in this survey that were not able to provide adequate information on the characteristics of ASCs used with some erroneously referring to the SVF as stem cells, there are more room for improvement in the quality of reporting in the application of ASCs in humans and animals.</P>

Stem cells for cartilage repair: what exactly were used for treatment, cultured adipose-derived stem cells or the unexpanded stromal vascular fraction?

Lee, J.I.,Balolong, E.,Han, Y.,Lee, S. Published for the Society by Baillère Tinda 2016 OSTEOARTHRITIS AND CARTILAGE Vol.24 No.7

Isolation and Characterization of a Broad Spectrum Bacteriocin from <i> Bacillus amyloliquefaciens</i> RX7

Lim, Kong Boon,Balolong, Marilen P.,Kim, Sang Hoon,Oh, Ju Kyoung,Lee, Ji Yoon,Kang, Dae-Kyung Hindawi Publishing Corporation 2016 BioMed research international Vol.2016 No.-

<P>We isolated a<I> Bacillus</I> strain, RX7, with inhibitory activity against<I> Listeria monocytogenes</I> from soil and identified it as<I> Bacillus amyloliquefaciens</I> based on 16S rRNA gene sequencing. The inhibitory activity was stable over a wide range of pH and was fully retained after 30 min at 80°C, after which it decreased gradually at higher temperatures. The activity was sensitive to the proteolytic action of <I>α</I>-chymotrypsin, proteinase-K, and trypsin, indicating its proteinaceous nature. This bacteriocin was active against a broad spectrum of bacteria and the fungus<I> Candida albicans</I>. Direct detection of antimicrobial activity on a sodium dodecyl sulfate-polyacrylamide gel suggested an apparent molecular mass of approximately 5 kDa. Ammonium sulfate precipitation and anion-exchange and gel permeation chromatography integrated with reverse phase-high-performance liquid chromatography were used for bacteriocin purification. Automated N-terminal Edman degradation of the purified RX7 bacteriocin recognized the first 15 amino acids as NH<SUB>2</SUB>-X-Ala-Trp-Tyr-Asp-Ile-Arg-Lys-Leu-Gly-Asn-Lys-Gly-Ala, where the letter X in the sequence indicates an unknown or nonstandard amino acid. Based on BLAST similarity search and multiple alignment analysis, the obtained partial sequence showed high homology with the two-peptide lantibiotic haloduracin (HalA1) from<I> Bacillus halodurans</I>, although at least two amino acids differed between the sequences. A time-kill study demonstrated a bactericidal mode of action of RX7 bacteriocin.</P>

Pyrosequencing-based Analysis of Fecal Microbial Communities in Three Purebred Pig Lines

Edward Alain B. Pajarillo,채종표,Marilen P. Balolong,김현범,서강석,강대경 한국미생물학회 2014 The journal of microbiology Vol.52 No.8

This study examined the fecal bacterial diversity of 15-weekoldpigs from three purebred lines: Duroc, Landrace, andYorkshire. Taxon-dependent and -independent analyses wereperformed to evaluate differences in the fecal bacterial communitiesand to identify bacterial genera that can be used todiscriminate breeds, following high-throughput pyrosequencingof 16S rRNA genes. Among the breeds evaluated, Landracehad the most diverse bacterial community composition. Prevotella, Blautia, Oscillibacter, and Clostridium were detectedin all samples regardless of breed. On the other hand,Catenibacterium, Blautia, Dialister, and Sphaerochaeta weredifferentially detected among breeds, as demonstrated bythe canonical loading plot. The discriminant analysis of principalcomponents plot also showed clear separation of thethree purebred pig lines, with a certain degree of similaritybetween Landrace and Yorkshire pigs and a distinct separationbetween Duroc pigs and the other two breeds. Otherfactors not related to breed, such as season or time of samplingand pen effects, may contribute to shaping the gutmicrobiota of pigs.

Carbohydrate-binding specificities of potential probiotic Lactobacillus strains in porcine jejunal (IPEC-J2) cells and porcine mucin

Valerie Diane Valeriano,BAGON BERNADETTE,Marilen P. Balolong,Dae-Kyung Kang 한국미생물학회 2016 The journal of microbiology Vol.54 No.7

Bacterial lectins are carbohydrate-binding adhesins that recognize glycoreceptors in the gut mucus and epithelium of hosts. In this study, the contribution of lectin-like activities to adhesion of Lactobacillus mucosae LM1 and Lactobacillus johnsonii PF01, which were isolated from swine intestine, were compared to those of the commercial probiotic Lactobacillus rhamnosus GG. Both LM1 and PF01 strains have been reported to have good adhesion ability to crude intestinal mucus of pigs. To confirm this, we quantified their adhesion to porcine gastric mucin and intestinal porcine enterocytes isolated from the jejunum of piglets (IPEC-J2). In addition, we examined their carbohydrate-binding specificities by suspending bacterial cells in carbohydrate solutions prior to adhesion assays. We found that the selected carbohydrates affected the adherences of LM1 to IPEC-J2 cells and of LGG to mucin. In addition, compared to adhesion to IPEC-J2 cells, adhesion to mucin by both LM1 and LGG was characterized by enhanced specific recognition of glycoreceptor components such as galactose, mannose, and N-acetylglucosamine. Hydrophobic interactions might make a greater contribution to adhesion of PF01. A similar adhesin profile between a probiotic and a pathogen, suggest a correlation between shared pathogen–probiotic glycoreceptor recognition and the ability to exclude enteropathogens such as Escherichia coli K88 and Salmonella Typhimurium KCCM 40253. These findings extend our understanding of the mechanisms of the intestinal adhesion and pathogen-inhibition abilities of probiotic Lactobacillus strains.

Characterization of the Fecal Microbial Communities of Duroc Pigs Using 16S rRNA Gene Pyrosequencing

Edward Alain B. Pajarillo,채종표,Marilen P. Balolong,김현범,서강석,강대경 아세아·태평양축산학회 2015 Animal Bioscience Vol.28 No.4

This study characterized the fecal bacterial community structure and inter-individual variation in 30-week-old Duroc pigs, which are known for their excellent meat quality. Pyrosequencing of the V1–V3 hypervariable regions of the 16S rRNA genes generated 108,254 valid reads and 508 operational taxonomic units at a 95% identity cut-off (genus level). Bacterial diversity and species richness as measured by the Shannon diversity index were significantly greater than those reported previously using denaturation gradient gel electrophoresis; thus, this study provides substantial information related to both known bacteria and the untapped portion of unclassified bacteria in the population. The bacterial composition of Duroc pig fecal samples was investigated at the phylum, class, family, and genus levels. Firmicutes and Bacteroidetes predominated at the phylum level, while Clostridia and Bacteroidia were most abundant at the class level. This study also detected prominent inter-individual variation starting at the family level. Among the core microbiome, which was observed at the genus level, Prevotella was consistently dominant, as well as a bacterial phylotype related to Oscillibacter valericigenes, a valerate producer. This study found high bacterial diversity and compositional variation among individuals of the same breed line, as well as high abundance of unclassified bacterial phylotypes that may have important functions in the growth performance of Duroc pigs.

Labeling Cells Correctly as Stromal Vascular Fraction Matters

Lee, J.I.,Lee, S.,Han, Y.,Balolong, E. Raven Press 2017 Arthroscopy Vol.33 No.8

Characterization of the Fecal Microbial Communities of Duroc Pigs Using 16S rRNA Gene Pyrosequencing

Pajarillo, Edward Alain B.,Chae, Jong Pyo,Balolong, Marilen P.,Kim, Hyeun Bum,Seo, Kang-Seok,Kang, Dae-Kyung Asian Australasian Association of Animal Productio 2015 Animal Bioscience Vol.28 No.4

This study characterized the fecal bacterial community structure and inter-individual variation in 30-week-old Duroc pigs, which are known for their excellent meat quality. Pyrosequencing of the V1-V3 hypervariable regions of the 16S rRNA genes generated 108,254 valid reads and 508 operational taxonomic units at a 95% identity cut-off (genus level). Bacterial diversity and species richness as measured by the Shannon diversity index were significantly greater than those reported previously using denaturation gradient gel electrophoresis; thus, this study provides substantial information related to both known bacteria and the untapped portion of unclassified bacteria in the population. The bacterial composition of Duroc pig fecal samples was investigated at the phylum, class, family, and genus levels. Firmicutes and Bacteroidetes predominated at the phylum level, while Clostridia and Bacteroidia were most abundant at the class level. This study also detected prominent inter-individual variation starting at the family level. Among the core microbiome, which was observed at the genus level, Prevotella was consistently dominant, as well as a bacterial phylotype related to Oscillibacter valericigenes, a valerate producer. This study found high bacterial diversity and compositional variation among individuals of the same breed line, as well as high abundance of unclassified bacterial phylotypes that may have important functions in the growth performance of Duroc pigs.

Assessment of fecal bacterial diversity among healthy piglets during the weaning transition.

Alain B Pajarillo, Edward,Chae, Jong-Pyo,Balolong, Marilen P,Bum Kim, Hyeun,Kang, Dae-Kyung Institute of Applied Microbiology, University of T 2014 The Journal of general and applied microbiology Vol.60 No.4

<P>The high level of genetic diversity in the microflora of the gastrointestinal tract has the potential to provide numerous beneficial functions to the host. Thus it is now acknowledged that the complexity in animal functioning is linked to the interacting microbiome in the gut. Despite the importance of gut microbiome, there is a lack of information concerning the microbial communities in the pig gut during the weaning transition. This study describes the fecal microbial shifts of healthy piglets during the weaning transition using barcoded pyrosequencing of the prokaryotic 16S rRNA gene. Fecal samples were obtained from 15 piglets during the pre-weaning period (fourth week after birth) and post-weaning (sixth week after birth) and were subjected to community genomic DNA extraction for pyrosequencing analysis. As the piglets underwent the weaning transition a trend toward increased bacterial diversity was observed, based on species abundance as measured by the Shannon-Weaver index. Firmicutes (54.0%) and Bacteroidetes (59.6%) were the most dominant phyla during pre-weaning and post-weaning, respectively. During the weaning transition a distinct shift from Bacteroides to Prevotella as the most abundant genus was observed. Additionally, we detected a number of abundant gut bacterial species that have not been reported previously. Clostridium rectum, C. clostridioforme, C. lactatifermentans and Butyricimonas virosa were uniquely detected prior to weaning while Roseburia cecicola and Blautia wexlerae were detected during the post-weaning period only.</P>