Agata Fabiszewska

Carbon source impact on Yarrowia lipolytica KKP 379 lipase production

The production of lipases by microorganisms is strongly influenced by the culture conditions. The optimum culture conditions for enzyme production are strain- and species-dependent. The aim of this study was to evaluate the impact of the carbon source used in the culture medium on the profile of lipases produced by Yarrowia lipolytica KKP 379. We observed a different pattern of extracellular and cell-bound lipase production, which was the highest in the early exponential phase. The extracellular lipase activity increased in the late exponential phase due to the lower accumulation of lipase molecules in cell walls. The best carbon source for extracellular lipase production by Y. lipolytica KKP 379 was olive oil. Glucose, dodecane and olive oil had a positive effect on biomass yield. Dodecane and/or glycerol utilization in microbiological lipase production was possible, but this process could not proceed without the addition of some activators such as olive oil in the cultivation medium.

Synthetic and Natural Lipase Inhibitors

Lipases are enzymes that catalyse the hydrolysis of ester bonds of triglycerides ranging among biocatalysts of considerable physiological significance and industrial potential. Better understanding of the catalytic functions and achieving the possibility to control the biocatalysis process, in particular exploring some activators and inhibitors of lipases, seems to be crucial in the context of novel applications. The lipase activity is a function of interfacial composition: the enzyme can be there activated as well as denaturated or deactivated and the interface is an appropriate site for modulating lipolysis. Lipase inhibitor, interacts directly with the enzyme and inhibits lipase action. Alternatively, some compounds can postpone the lipolytic reaction via adsorption to the interphase or to the substrate molecules. The aim of this review is to summarise the current knowledge concerning human, animal and microbial lipase inhibitors, which were grouped into two categories: synthetic lipase inhibitors (including phosphonates, boronic acids and fats analogues) and natural compounds (including β-lactones and some botanical foodstuffs - plant extracts and plant metabolites, mainly polyphenols and saponins as well as peptides and some dietary fibers). The topics discussed include also inhibition issues from the viewpoint of obesity treatment. Among natural compounds able to inhibit lipase activity are β- lactones including orlistat. Orlistat is the only registered drug for obesity treatment in many countries and lipases are essential enzymes for lipid absorption - thus fat absorption or obesity can be controlled by lipase inhibition, especially pancreatic lipase which is responsible for the hydrolysis of over 80% of total dietary fats. Its effectiveness in obesity treatment was also described.

Plant and microbial sources of antioxidants

In recent years there has been growing interest in substances with antioxidative properties, which reduce or prevent harmful effects of free radicals on living tissues, and inhibit aging processes and the development of certain diseases. The objective of this paper is to review new methods of obtaining antioxidants of plant origin and new trends in research aiming to improve their quality and profitability on an industrial scale. Among the issues discussed, there are the methods that use techniques of plant and microbial genetic engineering. A brief description of antioxidants and natural sources of their occurrence is also presented in this paper. In view of the fact that the biosynthesis of flavonoids and isoflavonoids is probably the best-known metabolic pathway of natural plant products, the review of achievements of recent years in the field of metabolic engineering was shown with the example of flavonoids. The modifications of flavonoid biosynthetic pathways were related to changes in the expression level of structural or regulatory genes, silencing of competitive genes or modifying catalytic properties of enzymes using techniques of protein engineering. The paper also presents the achievements of microorganism engineering in the field of application of fermentation processes as a source of specific flavonoid compounds, which was possible by designing the phenylpropanoid biosynthetic pathway in cells of microorganisms such as the bacterium E. coli or S. cerevisiae, bakers yeast. Both approaches can be used in the production of flavonoids attractive in terms of application.

Different aspects of Lactobacillus inoculants on the improvement of quality and safety of alfalfa silage

There is a significant range of bacterial inoculants for forage ensiling, but there is still a need for formulations to improve the safety of feed. The objective of the study was to evaluate the usefulness of three lactobacilli strains in alfalfa (Medicago sativa L.) The following bacterial inoculants were used: Lactocacillus plantarum K KKP 593p (LPK), L. plantarum C KKP 788p (LPC), L. buchneri KKP 907p (LB), and mix of all three strains (LPK+LPC+LB). The application of bacterial inoculants in alfalfa ensiling resulted in a reduction of the total number of molds, Clostridium perfringens and Listeria sp. (up to 5, 7, and 5 times respectively for LB inoculant in comparison to untreated silage). Total inhibition of Salmonella sp. and Escherichia coli growth was achieved in silages treated with all inoculants except for LPC. Aerobic stability in the control silage was the lowest (77 h) and doubled under the influence of bacterial inoculants. The most stable according to aerobic stability was silage treated with LB inoculant (175 h), where the highest concentrations of acetic acid (4.8 g kg-1), propionic acid (0.7 g kg-1) and 1,2-propanediol (526 mg kg-1) were reported. The study discussed that it is important to evaluate not only the effect of bacterial inoculants on physicochemical and microbiological silage properties, as the presence and expression of antibiotic resistance genes in lactic acid bacteria have been reported. The results of antimicrobial susceptibility testing of the strains showed that almost all minimum inhibitory concentrations values for eight antibiotics were equal to or below the corresponding breakpoints proposed by the European Food Safety Authority, Additives and Products or Substances used in Animal Feed Panel.

Study on the Properties of Immobilized Biocatalysts with Lipase Activity Produced by Yarrowia lipolytica in Batch Culture

Three kinds of matrices (calcium alginate, gelatin, and PVA) were employed as supports to immobilize lipases from Y. lipolytica KKP 379 via physical adsorption. The stability of biocatalysts (free and immobilized) was evaluated by measuring the enzyme activity before and after treatment with the method based on the hydrolysis of p-nitrophenyl laurate. Two fractions of enzymes were immobilized: cell-bound (yeast biomass) and extracellular (supernatant). The yield of immobilization and catalytic properties of immobilized lipases were investigated. Satisfactory results for lipolytic activity and biocatalyst stability were obtained for cell-bound enzymes immobilized in alginate (0.38 U g–1 d.m.) and crosslinked gelatin (0.18 U g–1 d.m.). Immobilization of the supernatant was successful only on the alginate (0.026 U g–1 d.m.). After lyophilization, no significant difference was noticed between treated and untreated biocatalysts. Lyophilized catalysts were successfully immobilized in all three matrices, but the process reduced their lipolytic activity probably due to an insufficient amount of water in the reaction solution.