Purification of lipase from Burkholderia metallica fermentation broth in a column chromatography using polymer impregnated resins.

In this work, porous glass beads grafted with polyethylene glycol (PEG) were used as an adsorbent to purify lipase from Burkholderia metallica in column chromatography. The purification parameters viz. salt stability, types and concentrations of PEG and salt, pH of the binding solution, and flow rate were studied to determine the performance of the purification system in an XK16/20 column. The crude lipase was mixed with different types and concentrations of salts 1-5% (w/w) (sodium citrate, potassium citrate, and sodium acetate) and subjected to the column containing the polymeric glass bead. One-variable-at-a-time experimentation revealed that 20% (w/w) PEG 6000 g/mol impregnated glass beads with a binding solution of 5% sodium citrate at pH 7.7, a flow rate of 1.0 mL/min and extraction time of 10 min resulted in the highest purification factor and recovery yield at 3.67 and 88%, respectively. The purified lipase has 55 ∼ 60 kDa molecular mass. The outcome of the study showed PEG could be applied to modify the inert glass beads into polymeric form, providing a biocompatible and mild separation condition for lipase. Thus, PEG could be successfully applied for the purification of lipase from B. metallica fermentation broth using column chromatography.

Safety and Effects of Lactobacillus paracasei TISTR 2593 Supplementation on Improving Cholesterol Metabolism and Atherosclerosis-Related Parameters in Subjects with Hypercholesterolemia: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial.

Probiotics have the potential as a multi-target approach to modulate hypercholesterolemia associated with premature atherosclerosis. Various strains of Lactobacillus paracasei have been reported to affect hypercholesterolemia positively. This study aimed to investigate the effects of L. paracasei TISTR 2593 on lipid profile, cholesterol metabolism, and atherosclerosis according to the registration of Thai Clinical Trial Registry as identification number TCTR 20220917002. A total of 50 participants with hypercholesterolemia were randomly and equally assigned to consume L. paracasei TISTR 2593 or a placebo in maltodextrin capsules daily. Biomarkers of lipid profiles, oxidative stress state, inflammatory state, and other biological indicators were examined on days 0, 45, and 90. The results showed that subjects taking the L. paracasei TISTR 2593 could significantly reduce the level of serum low-density lipoprotein-cholesterol (p < 0.05), malondialdehyde (p < 0.001), and tumor necrosis factor-α (p < 0.01). Moreover, L. paracasei TISTR 2593 increased the level of serum apolipoprotein E (p < 0.01) and adiponectin (p < 0.001) significantly. No changes in serum total cholesterol, high-density lipoprotein-cholesterol, triglyceride, total bile acids, and monocyte chemoattractant protein-1 were observed during L. paracasei TISTR 2593 supplementation. Therefore, L. paracasei TISTR 2593 could be an adjuvant probiotic supplement to ameliorate hypercholesterolemia and prevent or delay the development of atherosclerosis.

Influence of dietary supplementation with new Lactobacillus strains on hematology, serum biochemistry, nutritional status, digestibility, enzyme activities, and immunity in dogs.

Background and Aim: The use of antibiotics is associated with many side effects, with the development of bacterial resistance being particularly important. It has been found that dogs and their owners host similar resistant bacteria. This contributes to increased concurrent bacterial resistance and a possible trend of increased bacterial resistance in humans. Thus, using probiotics in dogs is an alternative option for preventing and reducing the transmission of bacterial resistance from dogs to humans. Probiotics are characterized by their potential to endure low pH levels and high concentrations of bile acids in the gastrointestinal tract. Lactobacilli are more acid-tolerant and resistant to bile acid, so they are ideal probiotics to be added to the canine diet. According to the previous studies, the benefits of Lactobacillus are a stable nutritional status and greater digestibility, along with improved fecal scores and reduced ammonia in dogs. However, no studies have been conducted with Lactobacillus plantarum CM20-8 (TISTR 2676), Lactobacillus acidophilus Im10 (TISTR 2734), Lactobacillus rhamnosus L12-2 (TISTR 2716), Lactobacillus paracasei KT-5 (TISTR 2688), and Lactobacillus fermentum CM14-8 (TISTR 2720), or their use in combination. Hence, the aim of this study was to examine the possible effects of the aforementioned Lactobacillus on hematological indices, nutritional status, digestibility, enzyme activities, and immunity in dogs. From the results, a new and safe strain of Lactobacillus may emerge for use as a probiotic in the future. Materials and Methods: In this study, 35 dogs were allocated equally into seven groups: Group 1 received a basal diet (control), while Groups 2-7 received the same diet further supplemented with L. plantarum CM20-8 (TISTR 2676), L. acidophilus Im10 (TISTR 2734), L. rhamnosus L12-2 (TISTR 2716), L. paracasei KT-5 (TISTR 2688), L. fermentum CM14-8 (TISTR 2720), or a mixture of probiotics (L. plantarum, L. acidophilus, L. rhamnosus, L. paracasei, and L. fermentum), respectively. All probiotics were administered at a dose of 109 colony-forming unit/dog for 28 days. Nutritional status, hematology, serum biochemistry, digestibility, enzyme activities, and immunity parameters were assessed. Results: There were no differences among the groups in body weight, feed intake, body condition score, fecal score, and fecal dry matter on the different sampling days. The hematology and serum biochemical analyses showed a difference only in creatinine activity (p < 0.001), with higher values in group L. fermentum CM14-8 (TISTR 2720) and lower values in group L. paracasei KT-5 (TISTR 2688) than in controls. However, all measurements were within the normal laboratory reference ranges. Fecal characteristics (fecal ammonia and fecal pH), fecal digestive enzyme activities, serum immunoglobulin (IgG), and fecal IgA did not differ significantly among the groups (p > 0.05). Conclusion: Lactobacillus plantarum CM20-8 (TISTR 2676), L. acidophilus Im10 (TISTR 2734), L. rhamnosus L12-2 (TISTR 2716), L. paracasei KT-5 (TISTR 2688), and L. fermentum CM14-8 (TISTR 2720), along with their mixture are safe and non-pathogenic additives for use as new probiotic strains of Lactobacillus in dogs. Although the new Lactobacillus strains had no effect on hematology, serum biochemistry, nutritional status, digestive enzyme activities, immunity, body weight, feed intake, or body condition scores in dogs, further studies should investigate the intestinal microbiota and the development of clinical treatments.

Adjuvant-free immunization with hemagglutinin-Fc fusion proteins as an approach to influenza vaccines.

The hemagglutinins (HAs) of human H1 and H3 influenza viruses and avian H5 influenza virus were produced as recombinant fusion proteins with the human immunoglobulin Fc domain. Recombinant HA-human immunoglobulin Fc domain (HA-HuFc) proteins were secreted from baculovirus-infected insect cells as glycosylated oligomer HAs of the anticipated molecular mass, agglutinated red blood cells, were purified on protein A, and were used to immunize mice in the absence of adjuvant. Immunogenicity was demonstrated for all subtypes, with the serum samples demonstrating subtype-specific hemagglutination inhibition, epitope specificity similar to that seen with virus infection, and neutralization. HuFc-tagged HAs are potential candidates for gene-to-vaccine approaches to influenza vaccination.

Synthesis of uniform submicron poly(lactic acid)-based particles/capsules by radical precipitation polymerization.

Poly(l-lactic acid) (PLLA) is a well-known biopolymer, usually synthesized via step-growth or ring-opening polymerization from lactic acid or a lactide monomer, respectively. PLLA microspherical particles are produced by dispersion polymerization with a ring-opening lactide monomer using a particular copolymer chain as a stabilizer. This is not easy to achieve when dehydration is needed. Here, a robust and simple synthesis of a nearly monodisperse, submicron PLLA-based particle/capsule was proposed via radical precipitation polymerization without the use of surfactant. A commercial PLLA was first glycolyzed with ethylene glycol to obtain a low molecular weight glycolyzed PLLA (GPLLA). Then, the GPLLA was copolymerized with methacrylic acid and ethylene glycol dimethacrylate monomers using a benzoyl peroxide initiator. Active sites on the GPLLA backbone were generated by hydrogen abstraction of benzoyloxy radicals that further copolymerized before self-assembly to form the polymer particles. Uniform particle size of about 580 nm with a low polydispersity index (PDI) of 0.012 was obtained. This method was also implemented to produce nearly monodisperse capsules containing linalool. The particle size of PLLA-based capsules was about 280 nm with narrow particle size distribution (PDI of 0.120). The PLLA-based capsules effectively inhibited microbial growth of Staphylococcus aureus, Escherichia coli and Candida albicans and were not toxic to human cells.

Development of qualitative and quantitative immunochromatographic strip test assay for rapid and simple detection of leucomalachite green residual in aquatic animals.

A rapid and simple immunochromatographic strip test assay based on competitive format was developed for leucomalachite green (LMG) detection. LMG-bovine serum albumin and rabbit anti-sheep IgG were immobilized on nitrocellulose membrane for the test line and control line, respectively. Anti-LMG-colloidal gold conjugate was immobilized onto the conjugate pad. For qualitative detection, the cut-off limit of the strip test was determined at 2 µg/L by the naked eye. For quantitative analysis, the working range of the LMG detection was 0.7-2 µg/L with LOD at 0.28 µg/L. A one-step immunochromatographic strip test for LMG detection can be completed within 5 min without any incubation, washing and blocking steps. Analysis results of LMG in aquatic animals obtained from the immunochromatographic strip test were in good agreement with those realized from enzyme-link immunosorbent assay. The developed the immunochromatographic strip test offered rapid detection as a simple (one-step), cost-effective, instrument-free assay and no need for handling reagents.

Isolation and characterization of Enterococcus faecium DSM 20477 with ability to secrete antimicrobial substance for the inhibition of oral pathogen Streptococcus mutans UKMCC 1019.

Streptococcus mutans and Candida albicans are the main oral pathogens which contribute to dental caries that affects all ages of human being. OBJECTIVES: This study focuses on the potential of crude cell free supernatant (CCFS) from lactic acid bacteria (LAB) to inhibit of the growth of S. mutans UKMCC 1019. DESIGN: A total of 61 CCFS from LAB strains were screened for their inhibitory ability against S. mutans UKMCC 1019 by broth microdilution method. The selected LAB with highest antimicrobial activity was identified and its CCFS was characterized for pH stability, temperature tolerance, enzyme sensitivity, metabolism of carbohydrates, enzymatic activities and antimicrobial activity against S. mutans UKMCC 1019 and C. albicans UKMCC 3001 by well diffusion assay. The effect of CCFS on cell structure of S. mutans UKMCC 1019 was observed under transmission electron microscopy (TEM). RESULTS: The CCFS from isolate CC2 from Kimchi showed the highest inhibition against S. mutans UKMCC 1019, which was 76.46 % or 4406.08 mm2/mL and it was identified to be most closely related to Enterococcus faecium DSM 20477 based on 16 s rRNA sequencing. The CCFS of E. faecium DSM 20477 had high tolerance to acidic and alkaline environment as well as high temperature. It also shows high antifungal activities against C. albicans UKMCC 3001 with 2362.56 mm2/mL. Under TEM, the cell walls and the cytoplasm membrane of S. mutans UKMCC 1019 were disrupted by the antimicrobial substance, causing cell lysis. CONCLUSIONS: Hence, the CCFS from E. faecium DSM 20477 is a potential bacteriocin in future for the treatment of dental caries.

Purification of ß-mannanase derived from Bacillus subtilis ATCC 11774 using ionic liquid as adjuvant in aqueous two-phase system.

The partitioning of ß-mannanase derived from Bacillus subtilis ATCC 11774 in aqueous two-phase system (ATPS) was studied. The ATPS containing different molecular weight of polyethylene glycol (PEG) and types of salt were employed in this study. The PEG/salt composition for the partitioning of ß-mannanase was optimized using response surface methodology. The study demonstrated that ATPS consists of 25% (w/w) of PEG 6000 and 12.52% (w/w) of potassium citrate is the optimum composition for the purification of ß-mannanase with a purification fold (PF) of 2.28 and partition coefficient (K) of 1.14. The study on influences of pH and crude loading showed that ATPS with pH 8.0 and 1.5% (w/w) of crude loading gave highest PF of 3.1. To enhance the partitioning of ß-mannanase, four ionic liquids namely 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF4), 1-ethyl-3-methylimidazolium tetrafluoroborate ([Emim]BF4), 1-butyl-3-methylimidazolium bromide ([Bmim]Br), 1-ethyl-3-methylimidazolium bromide ([Emim]Br) was added into the system as an adjuvant. The highest recovery yield (89.65%) was obtained with addition of 3% (w/w) of [Bmim]BF4. The SDS-PAGE analysis revealed that the ß-mannanase was successfully recovered in the top phase of ATPS with the molecular size of 36.7kDa. Therefore, ATPS demonstrated a simple and efficient approach for recovery and purification of ß-mannanase from fermentation broth in one single-step strategy.