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.

Bioactive Metabolites of Lactiplantibacillus plantarum K014 Against Methicillin-Resistant Staphylococcus aureus ATCC43300 and In Vitro Evaluation of Its Antibacterial, Antioxidant and Anti-inflammatory Activities.

This study aims to evaluate the effects of bioactive metabolites produced by lactic acid bacteria against methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300. A total of six lactic acid bacteria (LAB) were selected to evaluate the antimicrobial activity against MRSA ATCC 43300, a skin pathogen that is highly resistant to most antibiotics. The K014 isolate from a fermented vegetable recorded the highest inhibition against MRSA ATCC 43300 at 91.93 ± 0.36%. 16S rRNA sequencing revealed the K014 isolate is closely related to L. plantarum and the sequence was subsequently deposited in the GenBank database with an accession number of MW180960, named as Lactiplantibacillus plantarum K014. The cell-free supernatant (CFS) of L. plantarum K014 had tolerance to high temperature as well as acidic pH. The bioactive metabolites, such as hydrogen peroxide, lactic acid and hyaluronic acid, were produced by L. plantarum K014. Result from ABTS assay showed higher antioxidant activity (46.28%) as compared to that obtained by DPPH assay (2.97%). The CFS had showed anti-inflammatory activity for lipoxygenase (LOX) assay at 43.66%. The bioactive metabolites of L. plantarum K014 showed very promising potential to be used topical skin pathogens.

Insight into the pilot-scale fed-batch fermentation for production of Enterococcus faecium CW3801 using molasses-based medium.

Enterococcus sp. has been used as starters in food fermentation due to their probiotic and antimicrobial properties in food biopreservation. The antimicrobial properties were mainly contributed by the bacteriocin called enterocin. Hence, the availability of a cost-effective pilot-scale cultivation conditions is a necessity for the production of probiotic bacteria. This study aims to investigate optimization of medium composition using sugarcane molasses as a carbon source using response surface methodology and the potential use of fed-batch cultivation for improvement of the cell viability of Enterococcus faecium CW3801 for the use as a probiotic starter culture. Two feeding strategies (ramp and constant) were applied in fed-batch cultivation for enhancement of the production of E. faecium in a 2-L stirred tank bioreactor using the optimized medium and scaled up to a 15-L bioreactor. Optimized fermentation medium which comprised of 10% (v/v) of molasses and 10 g/L of yeast extract at pH 7 yielded maximum cell viability of 29.4 × 1011 CFU/mL with 3900 AU/mL of bacteriocin-like inhibitory substances (BLIS) activity. In the fed-batch, the cell viability (8.4 × 1013) and dry cell weight (6.34 g/L) reached the highest in optimized medium when the ramp (stepwise) feeding was applied. In scaling up to 15-L bioreactor, the growth of E. faecium was achieved at 2.3 × 1013 CFU/mL with the dry cell weight of 5.28 g/L under the same condition. The BLIS in 15-L bioreactor was 6% higher than the 2-L bioreactor. This study demonstrated that molasses and yeast extract are good feedstock for the growth of E. faecium. The E. faecium, a non-vancomycin resistant enterococcus (VRE) was successfully produced by a fed-batch cultivation approach and scaled up to a 15-L bioreactor using a ramp feeding strategy. Results from this study revealed that the fed-batch cultivation using molasses-based medium has industrial potential for the production of probiotics.

Recovery of a Bacteriocin-Like Inhibitory Substance from Lactobacillus bulgaricus FTDC 1211 Using Polyethylene-Glycol Impregnated Amberlite XAD-4 Resins System.

Lactobacillus bulgaricus is a LAB strain which is capable of producing bacteriocin substances to inhibit Staphylococcus aureus. The aim of this study was to purify a bacteriocin-like inhibitory substance (BLIS) produced by L. bulgaricus FTDC 1211 using an aqueous impregnated resins system consisting of polyethylene-glycol (PEG) impregnated on Amberlite XAD4. Important parameters influencing on purification of BLIS, such as the molecular weight and concentration of PEG, the concentration and pH of sodium citrate and the concentration of sodium chloride, were optimized using a response surface methodology. Under optimum conditions of 11% (w/w) of PEG 4000 impregnated Amberlite XAD4 resins and 2% (w/w) of sodium citrate at pH 6, the maximum purification factor (3.26) and recovery yield (82.69% ± 0.06) were obtained. These results demonstrate that AIRS could be used as an alternate purification system in the primary recovery step.

Application of bacteriocins in food preservation and infectious disease treatment for humans and livestock: a review.

Infectious diseases caused by bacteria that can be transmitted via food, livestock and humans are always a concern to the public, as majority of them may cause severe illnesses and death. Antibacterial agents have been investigated for the treatment of bacterial infections. Antibiotics are the most successful antibacterial agents that have been used widely for decades to ease human pain caused by bacterial infections. Nevertheless, the emergence of antibiotic-resistant bacteria has raised awareness amongst public about the downside of using antibiotics. The threat of antibiotic resistance to global health, food security and development has been emphasized by the World Health Organization (WHO), and research studies have been focused on alternative antimicrobial agents. Bacteriocin, a natural antimicrobial peptide, has been chosen to replace antibiotics for its application in food preservation and infectious disease treatment for livestock and humans, as it is less toxic.

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.