Lorenzo Pastrana

Nutritional factors affecting the production of two bacteriocins from lactic acid bacteria on whey

The ability of Lactococcus lactis subsp. lactis CECT 539 and Pediococcus acidilactici NRRL B-5627 to produce bacteriocins on both diluted and concentrated whey was investigated in batch fermentations. Both strains produced the higher amounts of biomass and bacteriocin titres on diluted whey. Luedeking and Piret expression was able to model the production of nisin, which was produced as a primary metabolite on both culture media. However, the pediocin production could not be typified in any case due to the negligible growth of P. acidilactici. Although the whey supported the growth and bacteriocin production by the two strains, both biomass and bacteriocin productions were lower than those obtained on MRS broth. The effect of total sugar, nitrogen, phosphorous and buffer concentrations on the production of nisin and pediocin was studied in diluted whey using factorial experiments and empirical modelling. The production of nisin was greatly inhibited by the increase in nitrogen, buffer, and to a lesser extent, sugar concentration in the medium, nevertheless, the used phosphorous source produced a light stimulatory effect on bacteriocin synthesis. In addition, the growth of Lc 1.04 was mainly affected by the nitrogen source used. On the other hand, pediocin was inhibited by the increase in buffer, phosphorous, and to a lesser degree, by the sugar and nitrogen concentration. The inhibitory activity of pediocin disappeared almost totally after 15 min of treatment with trypsin, papain, subtilisin and pepsin. The activity of nisin was drastically reduced by treatment with trypsin, subtilisin and pepsin. Nevertheless, 50% of the initial activity was retained when nisin was treated with papain. Both bacteriocins showed the highest heat stability at acidic pH and short incubation times.

Reactivity of Pure Candida rugosa Lipase Isoenzymes (Lip1, Lip2, and Lip3) in Aqueous and Organic Media. Influence of the Isoenzymatic Profile on the Lipase Performance in Organic Media

Three pure isoenzymes from Candida rugosa lipase (CRL: Lip1, Lip2, and Lip3) were compared in terms of their stability and reactivity in both aqueous and organic media. The combined effect of temperature and pH on their stability was studied applying a factorial design. The analysis of the response surfaces indicated that Lip1 and Lip3 have a similar stability, lower than that of Lip2. In aqueous media, Lip3 was the most active enzyme on the hydrolysis of p‐nitrophenyl esters, whereas Lip1 showed the highest activity on the hydrolysis of most assayed triacylglycerides. The highest differences among isoenzymes were found in the hydrolysis of triacylglycerides. Thus, a short, medium, and long acyl chain triacylglyceride was the preferred substrate for Lip3, Lip1, and Lip2, respectively. In organic medium, Lip3 and Lip1 provided excellent results in terms of enantioselectivity in the resolution of ibuprofen (EF value over 0.90) and conversion, whereas initial esterification rate was higher for Lip3. However, the use of Lip2 resulted in lower values of conversion, enantiomeric excess, and enantioselectivity. In the case of trans‐2‐phenyl‐1‐cyclohexanol (TPCH) resolution, initial esterification rates were high except for Lip3, which also produced poor results in conversion and enantiomeric excess. The performance of the pure isoenzymes in the enantioselectivity esterification of these substrates was compared with different CRL crude preparations with known isoenzymatic content and the different results could not be explained by their isoenzymatic profile. Therefore, it can be concluded that other factors can also affect the catalysis of CRL and only the reproducibility between powders can ensure the reproducibility in synthesis reactions.

Modelling the influence of pH on the kinetics of both nisin and pediocin production and characterization of their functional properties

Abstract A kinetic model of the production of bacteriocins on MRS medium by Lactococcus lactis subsp. lactis CECT 539 and Pediococcus acidilactici NRRL B-5627 has been developed. The model is a modification of the Luedeking and Piret expression, including a term for the influence of pH on both nisin and pediocin production. With this model it was demonstrated that these bacteriocins are produced as primary metabolites depending on the pH. By modeling the effects of temperature and pH on their activities, the high heat stability of both bacteriocins was demonstrated, with optimum values at acidic pH and short incubation times. The initial activity of both nisin and nisaplin was drastically reduced by treatment with trypsin, subtilisin and pepsin, but 50% activity was retained when both antibacterial substances were treated with papain. The inhibitory activity of pediocin almost totally disappeared after 15 min of treatment with these four proteases.

Influence of pH drop on both nisin and pediocin production by Lactococcus lactis and Pediococcus acidilactici.

Aims: To develop a kinetic model for describing the specific effect of pH drop on nisin and pediocin production in whey.

Production of red pigments by Monascus ruber in synthetic media with a strictly controlled nitrogen source

Abstract A complete factorial design was used to determine the composition of a synthetic culture medium with glucose as carbon source and monosodium glutamate as sole nitrogen source for Monascus ruber to maximize simultaneously strict exocellular pigment production and their red-to-yellow proportion. When culture conditions improved pigment production, the red/yellow ratio decreased. The compromise conditions for both objectives were obtained when the concentrations of glucose, monosodium glutamate, KH 2 PO 4 and K 2 HPO 4 in the culture medium were respectively 26, 5, 5 and 5 g/litre. In Erlenmeyer flask cultures, high ethanol production and CO 2 as coproducts were found associated with large pellet size, while for smaller pellet size a decrease in ethanol production and an increase in pigment synthesis were detected. Fermenter analysis of kinetic data showed a growth limitation that could be overcome by aeration with CO 2 -air mixtures. In all cases the maximum specific rate of pigment production occurred unexpectedly before the maximum specific growth rate was reached. Mycelial morphological changes are the precursor to decreasing specific rates of pigment production. Neither a complete sexual cycle nor pigment accumulation structures into mycelium were detected.

Strategies for improving extracellular lipolytic enzyme production by Thermus thermophilus HB27

In Thermus thermophilus HB27 cultures the localisation of lipolytic activity is extracellular, intracellular and membrane bound, with low percentage for the former. Therefore, the extracellular secretion must be increased in order to simplify the downstream process and to reduce the economic cost. This study focuses on the design of an innovative operational strategy to increase extracellular lipolytic enzyme production by T. thermophilus HB27 at bioreactor scale. In order to favour its secretion, the effect of several operational variables was evaluated. Among them, the presence of oils in the culture medium leads to improvements in growth and lipolytic enzyme activity. Sunflower oil is the most efficient inducer showing better results when added after 10h of growth. On the other hand, although surfactants lead to an almost complete inhibition of growth and lipolytic enzyme production, their addition along the culture could affect the location of the enzyme. Thus, by addition of surfactants at the stationary phase, a release of intracellular and membrane enzyme which increases the extracellular enzyme proportion is detected. Based on these results, strategies with successive addition of oil and surfactant in several culture phases in shake flask are developed and verified in a laboratory scale stirred tank bioreactor.

Amylase production by solid state culture of Aspergillus oryzae on polyurethane foams. Some mechanistic approaches from an empirical model

Abstract Using systems formed by particles of polyurethane foam of different densities soaked in an amylaceous effluent, the production of amylases by Aspergillus oryzae in solid state culture was studied by a complete factorial plan with four variables — support density, particle diameter, volume of liquid phase and substrate concentration. The empirical model obtained is consistent and allows a simple control of the response, superior in several respects to that of the corresponding submerged cultures. Moreover, it enables a mechanistic approach to the behaviour of the system, whose principal kinetic characteristics can be interpreted in terms of intra- and interparticular diffusional effects, which have a determining influence on basic state variables, such as water activity and gas transfer.

Characterization of microbial biomasses and amylolytic preparations obtained from mussel processing waste treatment

This work describes a treatment of glycogen-rich wastes from industrial mussel processing, involving the production of a protein fraction and a medium suitable for the culture of amylolytic microfungi. A strain of Aspergillus oryzae was chosen which allows the simultaneous production of single cell protein and a highly stable amylolytic preparation. The characteristics of the biomass obtained (proportion and in vitro digestibility of the proteins, levels of essential amino acids and nucleic acids, types of fatty acids present) were shown to be suitable, in principle, for animal feeds. The amylolytic preparation, obtained by ultrafiltration (with cut-off at 30 kD) of the cell-free medium, was very similar to commercial α-amylase preparations, containing mainly α-amylase, together with a small proportion of glucoamylase.

Nisin and pediocin production on mussel-processing waste supplemented with glucose and five nitrogen sources

Aims: Optimization of bacteriocin production by L. lactis subsp. lactis CECT 539 and Ped. acidilactici NRRL B‐5627 on mussel‐processing wastes.

Purification and characterization of Lip2 and Lip3 isoenzymes from a Candida rugosa pilot-plant scale fed-batch fermentation

Previous purification of a crude extracellular enzyme preparation from Candida rugosa ATCC 14830 pilot-plant fed-batch fermentations showed the presence of two lipase isoenzymes, Lip2 and Lip3, differing in their molecular masses (58 and 62 kDa, respectively). These enzymes were purified but the lipases were forming active aggregates with a molecular mass higher than 200 kDa. In this work we developed a purification method following three steps: ammonium sulfate precipitation, sodium cholate treatment and ethanol/ether precipitation, and anion exchange chromatography which allowed the sequential disaggregation of the isoenzymes. Pure and monomeric Lip2 and Lip3 were characterized according to pI, glycosylation and activity for p-nitrophenol esters and triacylglycerols of varying acyl chain. Lip3 was the best catalyst for the hydrolysis of the simple esters and triacylglycerols with short and medium acyl chains.