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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.
- Dominant microflora of Picante cheese: effects on proteolysis and lipolysisPublication . Freitas, A. Cristina; Pintado, Ana E.; Pintado, Manuela E.; Malcata, F. XavierFour species of bacteria (Enterococcus faecium and E. faecalis, Lactobacillus plantarum and L. paracasein and three species of yeasts (Debaryomyces hansenii, Yarrowia lipolytica and Cryptococcus laurentiU previously isolated from Picante cheese, were assayed for proteolysis and lipolysis. Milk type (caprine or ovine), ripening time (0 to 65 d) and concentration of NaCl (0 to 14 %(w/v)) have been assessed in terms of their effects upon in vitro curdled milk. Good evidence of proteolytic and peptidolytic activities was provided for Y. lipolytica, and at much lower levels for the other strains. Milk type, ripening time and content of NaCl appeared to be statistically significant processing factors in terms of proteolysis. Clear lipolytic activity was detected for Y. lipolytica, but release of free fatty acids to lesser extents was also observed for the other strains under study. Ripening time was statistically significant with regard to lipolysis but milk type was not. Lipolytic activities were strongly affected by presence of NaCl. According to experimental results, it is suggested that a mixed-strain starter for Picante cheese including L. plantarum, E. faecium (or E. faecalis,) and D. hansenii (and/or Y. lipolyticaj is of potential interest.
- Acidifying and aromatic properties of enterococcus strains in ovine and bovine milksPublication . Pimentel, L. L.; Soares, J. C.; Pintado, M. M. E.; Pintado, A. I. E.; Gomes, A. M. P.; Ferreira, A. C.; Malcata, F. X.Enterococci are lactic acid bacteria present in a wide variety of dairy and other food products. Although their presence in the former has been considered an indicator of poor hygienic conditions, nowadays a number of biotechnological and probiotic useful characteristics are already known. A few compounds resulting from the bacterium metabolism contribute to the aroma of cheese. Knowledge of those components, as well as of the acidifying capacity is very important to the dairy industry, including application toward selection of strains as better starters. The aim of this research effort was to study the role of some enterococci in ovine and bovine milks, in terms of acidifying properties and aroma potential. A total of seven strains, previously isolated from traditional Portuguese cheeses, were assayed for: Enterococcus faecium 28A, E. durans 13 and E. faecalis 6 (from Serra da Estrela cheese); and E. faecium 42, E. durans 15, E. faecalis 3 and E. casseliflavus 40 (from Terrincho cheese). Sterilized ovine and bovine milks were inoculated with 2% (v/v) inoculum and incubated at 37 °C. Microbiological viable counts on M17 agar, pH and titrable acidity were determined in duplicate at 0, 1, 4 and 7 d of incubation. Free fatty acids, recognized as strong odorants in Serra da Estrela and other traditional Portuguese cheeses, were quantified in each sample using SPME – GC/MS. All strains showed similar growth patterns, either in bovine or ovine milks, and reached levels of 108 –109 cfu/ml by 1 d of incubation (except E. durans 13, which exhibited a better growth in bovine than ovine milk). An important decrease in pH was observed in both types of milk by 1 d, for all strains. The highest reduction was observed for E. casseliflavus 40 and E. durans 15 in bovine milk, and for E. faecium 42 and E. durans 13 in ovine milk. Enterococcus faecalis 6 revealed the poorest acidification power.
- Exopolysaccharide production by lactobacillus acidophilus for potential applications in fresh cheesePublication . Pintado, A. I. E.; Truszkowska, K.; Pintado, M. M. E.; Gomes, A. M. P.; Malcata, F. X.Many strains belonging to the group of lactic acid bacteria (LAB) produce exopolysaccharides (EPS). The ability of EPS to act as viscosifying, stabilizing, and/or water-binding agents in various foods makes it an effective natural alternative to commercial synthetic stabilizers, so it may play an important role in the dairy industry. In fact, EPS-producing LAB are not only important in manufacture of yoghurt and fermented milks, but also in cheese production. Recent research efforts have shown that EPS cultures are useful to increase moisture retention and improve functional properties of low-fat Mozzarella cheese and whey cheese. In this work, the ability of Lb. acidophilus to produce EPS, and the performance of two different methods for EPS isolation was investigated. Fermentations were carried out in a Braun Biostat B 2-L fermentor, filled with 1.5 L of MRS broth containing 20 g/L lactose. The yield of the EPS isolation method was tested by adding xanthan gum to the medium. The experiments were carried out at 37 °C, 150 rpm, pH 5.5 under an N2 atmosphere. A 50 mL/L standard inoculum was prepared from a subculture of Lb. acidophilus, previously grown in the corresponding medium for 20 h at 37 °C, and was used to start-up every fermentation batch. Fermentation batches took 48 h, and samples were taken periodically. Growth was monitored spectrophotometrically and by plate enumeration. To optimise EPS isolation, cells and residual polypeptides were removed by centrifugation (4000 rpm for 20 min) upon precipitation via two different processes: addition of pronase E solution and one volume of 20 % trichloroacetic acid, or precipitation with 20 g/kg 5-sulfosalysilic acid. The EPS was precipitated by three volumes of cold ethanol and was collected by centrifugation. The weight of isolated and dried polymers was measured, and the total amount of carbohydrates was determined by the phenol-sulphuric method. Exponential growth took place for ca. 12 h, and EPS was produced mainly during the stationary phase.
- Characterization of the microstructure of Terrincho ewe cheesePublication . Gomes, A. M. P.; Braga, T. M.; Pintado, Ana I. E.; Pintado, Maria Manuela; Malcata, F. XavierThe microstructure of Terrincho cheese – a Portuguese traditional cheese bearing a Protected Denomination of Origin, was assessed by scanning electron microscopy (SEM). Terrincho cheese was manufactured according to the traditional protocol using pre-filtered, plain raw ewe’s milk of Churra da Terra Quente breed, without addition of starter of any other type of cultures, and coagulated with animal rennet. The cheeses were salted upon unmoulding, and placed in ripening chambers held at 10–12 °C and 88–89 % relative humidity for a period of 30 days. Cheeses were sampled at 0, 15 and 30 days. Cheese specimens were prepared for SEM by fixing small blocks of cheese (cut from freshly sampled cheeses) in formaldehyde, at room temperature for 1 month, cutting it into 1–2 mm thick slices using sharp razor blades and dehydrating in a graded ethanol series. Slices in absolute ethanol were critical point dried from carbon dioxide, mounted on SEM stubs using adhesive tabs, sputter-coated with gold, and examined using a JEOL scanning electron microscope. The microstructure of the (semi-soft) curd of Terrincho cheese appeared as a continuous casein network possessing a spongy appearance. The various microbial populations present within the cheese were found to grow in the form of colonies, embedded in the casein network and close to the void spaces originally occupied by fat. Microbial colonies were roughly spherical, with bacteria tightly packed in the centre. A similar pattern of microbial growth was observed throughout ripening. Other inclusions were also pinpointed, namely salt crystals.
- Food, Beverages and Agricultural ApplicationsPublication . Tóth, Ildikó V.; Segundo, Marcela A.; Rangel, António O. S. S.This chapter provides an overview of the flow methods described for the analysis of food, beverage, and agricultural samples. A beverage is a drink specifically prepared for human consumption, other than water, such as alcoholic drinks, coffee, fruit juices, and tea. The evolution of flow-injection application to food analysis is described along with distribution by commodity. Some of the analytical features of flow methods for alcoholic beverages, meat, and fish products are tabulated. The analytes illustrate the dual role that flow-based methods have in food analysis. These include flow systems devised for routine analysis based on well-established methods. However, flow systems are also applied to novel analytical tasks, such as the determination of analytes related to sensory properties or to characteristics that contribute to a value-added product. Modern agricultural economies are highly dependent on the use of pesticides. A group of key analytes of interest in monitoring food safety is the antimicrobial agents. The evaluation of freshness and the quality of meat and fish products is based on sensorial evaluation. The application of flow systems to the determination of parameters can be correlated to astringency, bitterness, body, and smoothness.
- Recuperación y revalorización del lactosuero para la producción de nuevos produtos con propriedades funcionalesPublication . Madureira, A. R.; Pintado, Ana I. E.; Gomes, Ana M. P.; Malcata, F. X.; Pintado, M. E.En los últimos años han surgido gran cantidad de alimentos funcionales a los que se les atribuyen diversas propiedades beneficiosas. Entre estos alimentos se encuentran aquellos que contienen proteínas del lactosuero así como aquellos que incorporan bacterias probióticas en su composición. En la actualidad existen matrices de proteínas de suero que contienen bacterias probióticas que se mantienen viables durante 28 días a temperaturas de refrigeración. Además, también se han realizado diferentes estudios relacionados con la actividad metabólica, las propiedades antimicrobianas y la resistencia a las enzimas del tracto gastrointestinal. Por último, se ha evaluado el uso de cepas de bacterias del ácido láctico con propiedades probióticas en la producción de exopolisacáridos en lactosuero.
- Angiotensin-converting enzyme-inhibitory activity of extracts of hydrolysed k-casein glycomacropeptide: stability under simulated gastrointestinal digestionPublication . Tavares, Tânia; Amorim, Manuela; Pintado, Manuela; Récio, Isidra; Malcata, F. XavierDietary proteins usually possess a wide range of nutritional, functional and biological properties; many of such biological properties have been attributed to physiologically active peptides, which are encrypted within the protein sequence. k‐Casein glycomacropeptide (CMP) – one of the main components of whey, is released in the first step of (enzymatic) cheese making – and has been claimed to act as mediator in important biological pathways. Peptides released from CMP via enzymatic or microbial activity have indeed been proven to posses inhibitory activity against the angiotensin‐converting enzyme (ACE). However, they will not act properly in the human body unless they can resist gastrointestinal digestion, as well as be absorbed and reach the cardiovascular system in active form. On the other hand, several studies have already demonstrated the important role of gastrointestinal digestion upon ACE‐inhibitory peptide formation. Therefore, the aim of this study was to evaluate the ACE‐inhibitory activity of peptide extracts obtained via hydrolysis of CMP, using an extract of Cynara cardunculus as enzymatic vector. Moreover, such peptides were subjected to simulated gastrointestinal digestion, in order to assess their stability and the evolution of their ACE‐inhibitory activity. A 40 g/L solution of CMP, hydrolysed with 8.5 %(w/w) commercial crude extract of C. cardunculus, exhibitedACE‐inhibitory activity corresponding to IC50 values of 296.0 mg/mL (total fraction), 63.0 mg/mL (3000 Da fraction). The identification of peptides proceeded by LC‐MS, and permitted identification of the following peptides: MAIPPKKNDQD (k‐CN f106‐115), as potentially responsible for antihypertensive activity; and TVQVTSTAV (k‐CN f161‐169) and MAIPPKKNDQD (k‐CN f106‐115), as potentially responsible for antithrombotic activity, because both encompass sequences analogous to the bioactive fragment f400‐411 of fibrinogen g‐chain. A new peptide, KTEIPIN (k‐CN f116‐123), was also identified to relatively high concentrations, with a promising antihypertensive activity. Studies concerning the in vitro simulation of gastrointestinal digestion were conducted; the gastrointestinal stability of the total extract and of the 3000 Da peptide fraction, as well as of the plain CMP (used as control), following incorporation in water and fruit juice, was tested in said gastrointestinal model, but none of said fractions was significantly affected.