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- Production of lactan using plain whey, whey permeate and synthetic medium as feedstockPublication . Pintado, Manuela E.; Pintado, Ana I. E.; Malcata, F. XavierWhey (or whey permeate), a by-product of cheese manufacture, has created a worldwide problem of waste disposal owing to its high biological oxygen demand. Production of lactan has been previously described based on a semidefined medium rich in lactose using Rahnella aquatilis. This research was aimed at obtaining lactan directly from whole whey without additional nutrients, as well as and whey permeate obtained after ultrafiltration, using a similar type of strain, and the fermentation process was compared with that using the synthetic medium previously tested. The growth of biomass growth rate, the polysaccharide production rate and the viscosity of the broth were monitored. Organic acids, lactose, peptides and free amino acids were also determined. The growth curves were similar for the three media, showing a maximum specific growth rate of 0.61 h1, 0.65 h-1 and 0.63 h-1 for whey, whey permeate and synthetic medium, respectively. The major increase in polysaccharide production was observed between 12 h (beginning of stationary phase) and 24 h for whey and the synthetic medium; however, the increase in the case of whey permeate is less pronounced and occurs essentially after 24 h. The yield of polysaccharide was 0.59 g/glactose, 0.56 g/glactose and 0.37 g/glactose for synthetic medium, plain whey and whey permeate, respectively. The larger amount of citrate present in whey was used by Rahnella aquatilis with significant formation of acetic acid in the first 12 h and acetoine thereafter; whey permeate and synthetic media did not lead to acetoine formation. The final yields of the various organic acids for the synthetic medium, whey and whey permeate, respectively, were: 0.08, 0.07 and 0.03 (g/glactose) for acetic acid; 0.02, 0.06 and 0,00 (g/glactose) for lactic acid; 0.08, 0.08 and 0.02 (g/glactose) for formic acid; 0.04, 0.01 and 0.00 (g/glactose) for succinic acid; and 0.00, 0.11 and 0.00 (g/glactose) for acetoine. Lactose was almost completely depleted by 48 h of fermentation in the case of whey and synthetic medium, but only part of lactose was consumed in the whey permeate (final yield of 0.43 g/glactose). Small peptides (< 4,000 Da) and most free amino acids were consumed by 24 h in whey and synthetic medium. The whey permeate possessed low amounts of peptides (virtually consumed by 12 h) and very low concentrations of free amino acids, which increased slightly between 12 and 24 h.
- Influence of salt content, degree of proteolysis and aeration on the production of a polymer via fermentation of whey-related media by Rahnella aquatilisPublication . Pintado, Manuela E.; Pintado, Ana I. E.; Malcata, F. XavierUtilization of whey as fermentation feedstock has been attempted widely by the dairy industry. Production of lactan, a polysaccharide composed of mannose, galactose and galacturonic acid (at the molar ratio 5:3:2), starting from a semi-defined medium containing lactose via fermentation under aerobic conditions with Rahnella aquatilis was described previously. In this communication, the effect of salt, previous hydrolysis and aeration were studied during the polysaccharide production from whey in alternative fermentation media: hydrolyzed whey (under (i) aerobic and (ii) anaerobic conditions), hydrolyzed whey with 2.0% NaCl (w/v) (iii) and 0.5% NaCl (w/v) (iv),.and plain whey (v). The growth of biomass and the variation in concentration of organic acids, lactose, peptides and free amino acids were monitored. The polysaccharide production and the variation of viscosity of were also followed throughout 48 h of fermentation. Under the different conditions tested, Rahnella aquatillis showed a maximum specific growth rate of 0.61 h-1, 0.60 h-1, 0.61 h-1, 0.64 h-1, and 0.46 h-1 for hydrolyzed whey under aerobiosis and under anaerobiosis, hydrolyzed whey with 2.0% NaCl (w/v) and 0.5% NaCl, and plain whey, respectively; the final yields of the various organic acids were: 0.07, 0.18, 0.07, 0.04 and 0.05 (g/glactose) for acetic acid; 0.06, 0.07, 0.00, 0.04 and 0.02 (g/glactose) for lactic acid; 0.08, 0.09, 0.03, 0.04 and 0.04 (g/glactose) for formic acid; 0.01, 0.04, 0.01, 0.01 and 0.02 (g/glactose) for succinic acid; and 0.11, 0.09, 0.14, 0.19 and 0.00 (g/glactose) for acetoine. Lactose was almost completely depleted during the 48 h of fermentation for hydrolyzed whey; however, lactose was only partly consumed in plain whey (final yield of 0.48 g/glactose).Small peptides (< 2,000 Da) and most free amino acids were consumed by 24 h in hydrolyzed whey fermented under anaerobiosis and plain whey, but these peptides were present until the end of fermentation in the remaining media. R. aquatilis showed similar behavior in free amino acid consumption in hydrolyzed whey with NaCl and hydrolyzed whey fermented under aerobiosis. Plain whey yielded very low concentrations of free amino acids throughout the whole fermentation. The yield of polysaccharide was 0.56, 0.26, 0.39, 0.40 and 0.44 g/glactose for hydrolyzed whey fermented under aerobiosis and under anaerobiosis, hydrolyzed whey with 2.0% NaCl (w/v) and 0.5% NaCl, and plain whey, respectively.
- Dominant microflora of picante cheese: Independent role upon proteolysis and lipolysis in model systemsPublication . Freitas, A. Cristina; Pintado, Ana E.; Pintado, Manuela E.; Malcata, F. XavierFour species of bacteria (two species of enterococci, Enterococcus faecium and Enterococcus faecalis, and two species of lactobacilli, Lactobacillus plantarum and Lactobacillus paracasei) and three species of yeasts (Debaryomyces hansenii, Yarrowia lipolytica and Cryptococcus laurentii), previously isolated from Picante cheese were assayed for biochemical performance in proteolysis and lipolysis. In addition to the difference of the microbiological strains, the milk type (caprine or ovine), the ripening time (0 to 65 days) and the concentration of NaCl (0 to 14%(w/v)) have been deliberatly fixed in vitro curdled milk (previously prepared from heat-sterilized milk, coagulated with animal rennet and inoculated with each strain) and subject to 12 ºC. High proteolytic activity was demonstrated by Y. lipolytica and by all the other strains to a lesser extent; Y. lipolytica produced ca. 85% of WSN by 65 days of ripening whereas E. faecium, D. hansenii and C. laurentii produced levels of WSN ranging in 40-50%, and E. faecalis, L. plantarum and L. paracasei in 30- 40%. In terms of peptidolytic activity, measured by NPN contents and by release of free amino acids, once again Y. lipolytica presented the highest activity, followed by L. plantarum, L. paracasei, E. faecium and E. faecalis. Milk type, ripening time, and content of NaCl revealed to be statistically significant processing factors in terms of proteolysis; caprine milk, 65 days of ripening and lower contents of NaCl led to the highest values. The lipolytic activity, assessed by the release of butyric acid from tributyrin, was strong for Y. lipolytica and C. laurentii, whereas release of free fatty acids was observed at different rates for all strains under study. Ripening time proved to be a statistically significant factor for lipolysis, whereas milk type was not; lipolytic activities, measured as fat acidity index, were strongly affected by NaCl content and, as happened with release of free amino acids, the extent of fat hydrolysis was much more affected by the increase of NaCl from 0 to 7% than by its increase from 7% to 14%. Although it is not possible to directly compare results obtained in vitro using pure, single cultures with those obtained in loco using actual cheese, our results suggest that a mixed-strain starter for Picante cheese including L. plantarum, E. faecium (or E. faecalis) and D. hansenii (and/or Y. lipolytica) would be of potential interest.