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The quality and safety indicators of commercial dried gluten free (GF) pasta were analyzed to investigate, for the first time, the real nutritional intake through the chemical composition and the heat damage during processing by quantification of furosine. Eight samples of GF spaghetti were compared with wheat spaghetti. Dried and cooked GF pasta had lower protein and ash content than wheat spaghetti. GF samples composed solely by corn flour had higher optimal cooking time. Samples with emulsifier showed lower losses during cooking. Considering their composition, no trend could be established to explain textural behavior. Samples constituted merely by corn showed the highest resilience and elasticity. Spaghetti constituted only from corn and rice showed the highest firmness. The furosine content in dried samples ranged between 19 and 134 mg FUR/100 g proteins and in cooked samples ranged between 48 to 360 mg FUR/100 g proteins. Furosine content of GF pasta was in general lower than in wheat pasta, and those differences were even enlarged when comparing them after cooking. The results of PCA indicated it was possible to discriminate GF pasta regarding their technological and nutritional behavior.

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Corn starch was modified with cyclodextrin glycosyltransferase (CGTase) below the gelatinization temperature. The porous granules with or without CGTase hydrolysis products may be used as an alternative to modified corn starches in foods applications. The amount and type of hydrolysis products were determined, containing mainly ß-cyclodextrin (CD), which will influence pasting behavior and glycemic response in mice. Irregular surface and small holes were observed by microscopic analysis and differences in pasting properties were observed in the presence of hydrolysis products. Postprandial blood glucose in mice fed gelatinized enzymatically modified starch peaked earlier than their ungelatinized counterparts. However, in ungelatinized enzymatically modified starches, the presence of ß- CD may inhibit the orientation of amylases slowing hydrolysis, which may help to maintain lower blood glucose levels. Significant correlations were found between glycemic curves and viscosity pattern of starches.

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Porous starch is attracting very much attention for its absorption and shielding ability in many food applications. The effect of two different enzymes, fungal α-amylase (AM) or amyloglucosidase (AMG), on corn starch at sub-gelatinization temperature was studied as an alternative to obtain porous starch. Biochemical features, thermal and structural analyses of treated starches were studied. Microscopic analysis of the granules confirmed the enzymatic modification of the starches obtaining porous structures with more agglomerates in the case of AMG treated starches. Several changes in thermal properties and hydrolysis kinetics were observed in enzymatically modified starches. Hydration properties were significantly affected by enzymatic modification being greater influenced by AMG activity, and the opposite trend was observed in the pasting properties. Overall, results showed that enzymatic modification at sub-gelatinization temperatures really offer an attractive alternative for obtaining porous starch granules to be used in a variety of foods applications.

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In this work, the wheat flour properties are investigated using ultrasound techniques. Moreover, the flour samples were also characterized by means of well established techniques such as protein content, Alveograph and Mixolab®. A set of 35 dough samples, made of wheat flours with diverse physical and quality properties, were studied. The obtained results shown that ultrasound measurements can detect changes in the dough consistency induced by proteins and also by gelatinization of the starch. Furthermore, ultrasound measurements can be related to parameters indicative of the proteolytic degradation or softening of the dough due to protease activity. Thus, ultrasound can be considered a low cost and rapid tool, complementary to conventional test, for wheat flour characterization.

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AIMS: To screen for phosphatase and phytase activities in Lactobacillus isolated from diverse ecosystems and to determine the biochemical properties and the factors that regulate the synthesis of the enzyme responsible for these activities in the selected strain, Lactobacillus pentosus CECT 4023. METHODS AND RESULTS: These activities were determined spectrophotometrically by using p-nitrophenyl phosphate and sodium phytate as substrates. They were maximal at the onset of the stationary phase of growth and repressed in the presence of high glucose concentration and inorganic phosphate. The enzyme responsible for these activities was an acid phosphatase (E.C.3.1.3.2.), with a molecular mass of 69 kDa. The activity was optimum at pH 5.0 and 50 degrees C. It hydrolysed mono-phosphorylated substrates and phytate, albeit at lower rates. It was inhibited by iodoacetic acid, phenyl-methylsulphonyl fluoride, di-sodium pyrophosphate and Ca+2 while activated by Co+2 and low concentrations of L-ascorbic acid and EDTA. CONCLUSIONS: Lactobacillus pentosus CECT 4023 produces a nonspecific acid phosphatase that hydrolyses a number of mono-phosphorylated substrates and phytate. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that the phosphatase from L. pentosus CECT 4023 could partly contribute to reduce the phosphorylation degree of phytate and its derivatives and, thereby, their anti-nutrient properties during fermentation processes.

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The possible use of phytase as a breadmaking improver has been tested in whole wheat breads by adding different amounts of fungal phytase. The effect of phytase addition on the fermentation stage and the final bread quality was analyzed. The phytase addition shortened the fermentation period, without affecting the bread dough pH. Regarding the whole wheat bread, a considerable increase of the specific bread volume, an improvement of the crumb texture, and the width/height ratio of the bread slice were obtained. An in vitro assay revealed that the improving effect of phytase on breadmaking might be associated with the activation of alpha-amylase, due to the release of calcium ions from calcium-phytate complexes promoted by phytase activity. As a conclusion, phytase offers excellent possibilities as a breadmaking improver, with two main advantages: first, the nutritional improvement produced by decreasing phytate content, and second, all the benefits produced by alpha-amylase addition can be obtained by adding phytase, which promotes the activation of endogenous alpha-amylase.

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alpha-Amylases from different origins (wheat, malted barley, fungi, and bacteria) are used extensively to improve breadmaking. However, the enzyme activities, in addition to the differences associated with their origins, are strongly affected by the process conditions and the presence of other compounds in the medium. The activity of different alpha-amylases was tested under different conditions (pH and temperature), and in the presence of some bread ingredients (salt and sugar), some breadmaking additives (ascorbic acid and sodium propionate), and some metabolites (organic acids and saccharides) generated during the fermentation step, to envisage the behavior of these alpha-amylases during the breadmaking process. The alpha-amylase activities were affected to a different extent by the addition of these compounds depending on the enzyme origin. In general, the alpha-amylases from cereals (wheat and malted barley) were less sensitive to the presence of some ingredients, additives, and metabolites. These results show the great variation of the alpha-amylase activity with the process conditions and the importance of its knowledge in the selection of the appropriate alpha-amylase for a specific breadmaking process.

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Parameters relevant to the thermodynamically controlled synthesis of cephalothin utilizing highly active stabilized penicillin G acylase derivatives were studied. These included solubility/stability of substrates, enzyme derivative activity/stability, reaction course and synthetic yields. These parameters were altered by varying the pH, dimethylformamide concentration and temperature. Simultaneous optimization of the selected parameters could not be achieved with a single set of conditions. However, continuous adjustment of conditions throughout the reaction course allowed each parameter to be optimized (dynamic reaction design). This strategy works by optimizing those parameters that are critical to the overall reaction at a given point, whilst leaving others sub-optimal when their contribution to the total is minimal. This strategy has achieved a 90% transformation of antibiotic nucleus to cephalothin at a final concentration of 20 g/l, high enzyme and reactant stability, with a reaction period of 3 h (using 1 ml of derivative/40 ml of reaction solution).

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Packed bed hollow fiber membrane reactors were used to carry out organic phase biocatalysis at constant water activity. The performance of the device was tested by carrying out the esterification of dodecanol and decanoic acid in hexane. Lipase from Candida rugosa, immobilized on microporous polypropylene and packed in the shell space of the reactor, was used to catalyze the reaction. In situ water activity control was accomplished by pumping appropriate saturated salt solutions through the microporous hollow fiber polypropylene membranes. Water generated by reaction in the organic phase, pumped continuously through the shell of the reactor, was transferred into the bulk of the aqueous phase under the water activity gradient. The reactor performance was found to be strongly dependent on the controlling water activity. By carefully selecting this control activity it was found possible to obtain complete esterification. The water activity of the organic phase could be maintained very close to that of the saturated salt solution used. The reactor could be operated in the continuous mode for 100 h without any degradation in its performance. (c) 1996 John Wiley & Sons, Inc.

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Addition of miscible organic solvents to water increases the solubility of naphthalene. The logarithm of the solubility is linearly dependent on the co-solvent concentration, in an intermediate range. The relative solubilising effects of different solvents correlate well with their known tendency to denature proteins (using literature data for trypsin, cytochrome c, chymotrypsinogen, chymotrypsin, laccase and myoglobin). This is expected if denaturation occurs when the hydrophobic effect has been reduced by a characteristic extent for a given protein. Naphthalene solubility predicts denaturation as well as does the denaturation capacity model.

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We have developed integrated studies of enzyme reaction engineering for the hydrolysis of penicillin G catalysed by very active penicillin G acylase (PGA) derivatives. We have studied the distinct effect of a key variable (pH) on different industrial parameters (e.g. activity/stability parameters). In this way we have demonstrated, in contrast with that proposed by other authors, that the generation of gradients of pH inside the porous structure of very active enzyme derivatives may be not a problem but a 'very profitable tool' to improve the whole set of industrial parameters. In this way we can establish two distinct 'optimal pH values': (i) the one inside the particle of the biocatalyst and (ii) the one in the bulk solution. The use of an external pH of 8.0 associated with the promotion of a controlled decrease in internal pH (e.g. around a mean value of 5.5) was very useful to simultaneously obtain interesting values of all industrial parameters: (i) very high hydrolytic yields (higher than 97%); (ii) a very important increase on the stability of PGA derivatives (higher than a 50-fold factor); and (iii) a very small decrease in operational activity (approximately 15%) as compared with the one of soluble enzyme at pH 8.0 with no diffusional hindrances.

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We have developed a strategy for immobilization-stabilization of penicillin G acylase (PGA) from Kluyvera citrophila by controlled multipoint covalent attachment to agarose-aldehyde gels. This enzyme is composed by two dissimilar subunits noncovalently bound. Thus, in this article we establish clear correlations between enzyme stabilization and the multipoint immobilization and/or between enzyme stabilization and the involvement of the two subunits in the attachment of them to the support. We have demonstrated that important thermal stabilizations of derivatives were only obtained through a very intense enzyme-support multipoint attachment involving the whole enzyme molecule. In this way, we have prepared derivatives preserving more than 90% of catalytic activity and being more than 1000-fold more stable than soluble and one-point attached enzyme. In addition, the involvement of the two subunits in the covalent attachment to the support has proved to be essential to develop interesting strategies for reactivation of inactivated enzyme molecules [e.g., by refolding of immobilized PGA after previous unfolding with urea and sodium dodecyl sulfate (SDS)].

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A method for the preparation of new aminated agarose gels containing monoaminoethyl-N-aminoethyl structures, MANA-agarose gels, has been developed. These gels contain primary amino groups with a very low pK value (6.8). In addition to that, we have been able to prepare very highly activated gels (e.g., 10% agarose gels containing up to 200 mu Eq of primary amines per milliliter). These two properties make these activated supports suitable for performing novel and interesting methods for protein immobilizations via very mild carbodiimide activation of carboxy groups. For example, very effective coupling reactions can be performed at pH 5.0-6.0 in the presence of low concentrations of activating agent, e.g., 1 mM. By using a model industrial enzyme, beta-galactosidase from Aspergillus oryzae, we have been able to demonstrate the excellent prospects of these novel activated supports.

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We have tested the effect of chemical modifications with formaldehyde on the activity/stability of immobilized derivatives of the enzyme penicillin G acylase (PGA). These derivatives were previously stabilized through enzyme-support multipoint covalent attachment. We carried out very different chemical treatments of our derivatives by testing the effect of different variables which control the intensity and the nature of these amine-formaldehyde reactions. The variables tested were: formaldehyde concentration, pH, time, and temperature. We also developed a colorimetric titration of the free amine groups on immobilized PGA in order to evaluate the extension of the reaction between formaldehyde and the amine groups of the enzyme. As a consequence of these studies, we have been able to get additional stabilizations of our previously stabilized-immobilized derivatives: e.g. a factor of 24-fold was achieved in terms of stabilization against irreversible thermal inactivation. The integrated effect of additional chemical modification plus previous multipoint covalent attachment has allowed us to prepare PGA derivatives which are 50,000 more thermostable than native PGA as well as most of the commercial PGA derivatives.

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We have developed a strategy for immobilization-stabilization of alpha-chymotrypsin by multipoint covalent attachment of the enzyme, through its amino groups, to agarosealdehyde gels. We have studied the role of the main variables that control the intensity of these enzyme-support multi-interaction processes (surface density of aldehyde groups in the activated gel, contact time between the immobilized enzyme and the activated support prior to borohydride reduction of the derivatives, etc.). In this way, we have prepared a number of very different chymotrypsinagarose derivatives. Our best derivatives, with the most intense multipoint attachment, were more stable than one-point attached derivatives and were more than 60,000-fold more stable than soluble enzyme in the absence of autolysis phenomena. In spite of the dramatic stabilization, the catalytic activity of these derivatives is little changed (they only lose 35% of intrinsic activity after this intense enzyme-support multi-interaction process). In addition, we have also demonstrated the very high capacity of 6% aldehyde-agarose gels to immobilize pure chymotrypsin (40 mg enzyme/mL catalyst). Furthermore, we have been able to establish a clear correlation between enzyme-support multipoint covalent attachment, stabilization against very different denaturing agents (heat, urea, organic cosolvents), and insensitivity of those immobilized chymotrypsin molecules to some activating agents.

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By using very active and very stable penicillin G acylase (PGA)--agarose derivatives we have studied the industrial design of equilibrium-controlled synthesis of lactamic antibiotics. In the presence of high concentrations of organic cosolvents we have carried out the direct enzymatic condensation of phenylacetic acid and 6-aminopenicillanic acid to yield the model antibiotic penicillin G. We have mainly studied the integrated effect of different variables that define the reaction medium on a number of parameters of industrial interest:time course of antibiotic synthesis, highest synthetic yields, stability of the catalyst, and solubility and stability of substrates and products. The main variables tested were the nature and concentration of the organic cosolvent, pH, and temperature. The effects of the variables tested on different parameters were quite different and sometimes opposite. Hence, the optimal experimental conditions for antibiotic synthesis catalysed by PGA were established, as a compromise solution, in order to obtain good values for every parameter of industrial interest. These conditions seem to be important parameters for scale-up (e.g. we have been able to reach more than 95% of synthetic yields with productivities around 0.5 tons of model antibiotic per year per liter of catalyst).

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Synthesis of dipeptides benzoyl Arginine leucinamide and kyotorphin catalyzed by highly stabilized derivatives of trypsin and chymotrypsin have been performed. Extreme experimental conditions could be tested and parameters of industrial interest could be improved provided the high activity and stability of the derivatives in these unfavourable environments. Thermodynamically controlled synthesis catalyzed by trypsin could be optimized and 97% conversion was obtained in 90% organic cosolvents. 100% yields were achieved in kinetically controlled synthesis catalyzed by trypsin in aqueous medium in the presence of IM Ammonium Sulphate. Higher starting concentrations of poorly soluble substrates of chymotrypsin could be used in a reaction medium containing 50% DMF and 95% yield were obtained.

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