Comparative study on microwave and conventional methods for protein hydrolysis in food

Summary A rapid microwave hydrolysis procedure was developed for amino acid determination in food. The hydrolysis was performed with 6M HCl in sealed vessels using a microwave digestion system.Bovine Serum Albumin was chosen as a model protein to compare its theoric amino acid sequence with the experimental results obtained after hydrolysis by both the traditional oven heating and the microwave methods. Furthermore the selected microwave methods were carried out on different food matrices (cheese and durum wheat) and the obtained data were compared with the traditional method results.This comparative study shows that the rapid microwave hydrolysis is as accurate and precise as the traditional hydrolysis for determining amino acids in food.These results were presented at the Third International Congress on Amino Acids, Vienna, August 23–27th, 1993.     

Characterization of leucine amino peptidase from Streptomyces gedanensis and its applications for protein hydrolysis

The aim of this work was to purify and characterize the extra-cellular leucine amino peptidase (LAP) from Streptomyces gedanensis and also study its applications for protein hydrolysis. The enzyme was purified to homogeneity by ammonium sulfate fractionation and sequential chromatography steps. LAP appeared to be a monomeric enzyme with a molecular weight of ～75 kDa determined by sodium dodecyl sulfate poly acryl amide gel electrophoresis (SDS-PAGE). The enzyme preferentially hydrolyzed leucine p-nitroanilide followed by Met, Phe, Lys and Arg derivatives. Kinetic studies on the purified enzyme confirmed that it can hydrolyze peptide as well as ester substrates at comparable rates. This amino peptidase was highly resistant to different concentrations of various organic solvents. The characteristics of this amino peptidase, including thermo stability, organic solvent resistance, its activity against various substrates, and also it showed esterase and peptidase activity at comparable rates; identified this amino peptidase as a novel one. The specificity towards aromatic and hydrophobic amino acid residues, the solvent-resistance and thermo stability make this amino peptidase could offer interesting possibilities for various industrial applications including debittering of protein hydrolysates, peptide and ester synthesis.     

Determination of methionine sulfoxide in protein and food by hydrolysis with p-toluenesulfonic acid

Methionine sulfoxide in peptides and proteins was determined by use of 3 N p-toluenesulfonic acid as a hydrolyzing agent. Samples were hydrolyzed at 110 degrees C for 22 h in an evacuated sealed tube and analyzed for amino acid content. Amino acid analysis showed that the recovery of methionine sulfoxide from a synthetic peptide and its mixture with proteins was consistently better than 90%. The recovery of all other amino acids except tryptophan was complete, and was similar to that observed after hydrolysis with 6 N HCl. The presence of carbohydrates had no effect on the yield. Thus, the present procedure can be used for general and simultaneous determination of methionine sulfoxide as well as other amino acids in proteins.     

Process development for the enzymatic hydrolysis of food protein: Effects of pre-treatment and post-treatments on degree of hydrolysis and other product characteristics

An enzymatic process was developed to produce protein hydrolysate from defatted soya protein. Various unit operations were tried, and the effects of pre- and post-treatments on the product characteristics such as degree of hydrolysis (DH), free amino acid content (%FAA) and average molecular weight (MW) were investigated. The use of acid washes showed no difference in %DH. Increasing pH during pre-cooking gave lower %DH. Alkaline cooking made too much insoluble protein, thus the protein yield was too small. A better hydrolysis with more acceptable taste was obtained when the combination of Neutrase/Alcalase/Flavourzyme was used in place of Alcalase/Flavourzyme combination. Untoasted defatted soya was more effective on the proteolysis than toasted one. The MW of the evaporated and spray dried product was higher than that of undried product, due to precipitation of low-solubility components. When the product separation was carried out by ultrafiltration and the product concentration by reverse osmosis, the solubility and the taste of the product were improved. The difference between enzyme hydrolysate and acid hydrolysate was significant in free amino acid composition, especially in tyrosine, phenylalanine, glutamine and asparagine.     

Bacteriophage endolysins: applications for food safety

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Improving starch for food and industrial applications

Progress in understanding starch biosynthesis, and the isolation of many of the genes involved in this process, has enabled the genetic modification of crops in a rational manner to produce novel starches with improved functionality. For example, potato starches have been created that contain unprecedented levels of amylose and phosphate. Amylose-free short-chain amylopectin starches have also been developed; these starches have excellent freeze-thaw stability without the need for chemical modification. These developments highlight the potential to create even more modified starches in the future.     

Fluorometric assay for intestinal peptidases

A fluorometric assay for intestinal peptidases has been developed. Amino acids liberated by hydrolysis are estimated by use of l-amino-acid oxidase, peroxidase, and the fluorogenic reagent p-hydroxyphenylacetic acid, which yields a highly fluorescent compound on oxidation. During development of fluorescence, continued hydrolysis of peptides by peptidases which contaminate available preparations of l-amino-acid oxidase is prevented by the use of two inhibitors, 1,10-phenanthroline and p-hydroxymercuribenzoate. The assay is at least 10 times more sensitive than comparable spectrophotometric methods which employ the potentially carcinogenic chromogen o-dianisidine for detection of amino acids.     

Nomenclature for enkephalin degrading peptidases

The use of trivial names for enkephalin degrading peptidases such as "aminoenkephalinase" and "carboxyenkephalinase" imply a specificity and cellular localization which is not inherent in any of the peptidases implicated in the degradation of endogenous enkephalins. Rather than name these enzymes on the basis of one of their many substrates, it is proposed that they be named according to their general reaction type. Such a nomenclature has already been proposed for the enkephalin degrading endopeptidase 24.11 given the trivial name "enkephalinase".     

Equine peptidases: Correspondence with human peptidases and polymorphism for erythrocyte peptidase a

Equine erythrocyte peptidases were compared to the six human erythrocyte peptidases, A, B, C, D, E, and F, regarding substrate specificity, relative activity, and electrophoretic mobility. Five equine erythrocyte peptidases appeared homologous to human peptidases A, B, D, E, and F. In contrast to human, equine peptidase C was absent in red cells, although it was weakly active in white cells. On the other hand, an equine peptidase, probably homologous to human peptidase S, was weakly active in red cells as well as present in white cells. Polymorphism for equine erythrocyte peptidase A is reported.     

Debittering effect of Actinomucor elegans peptidases on soybean protein hydrolysates

Effects of the enzymes in Actinomucor elegans extract and the enzyme Alcalase 2.4L on debittering the soybean protein hydrolysates were investigated. When the protein was treated only with the latter, a strong bitterness formed; but it decreased if the protein was treated with both the enzymes. The more the enzymes were used, weaker was the bitterness tasted. SDS-PAGE profile and ESI-MS spectrum of the hydrolysates evidenced that the Alcalase could convert the protein into peptides rapidly, while the enzymes in the A. elegans extract were able to further degrade some peptides which were difficult or unable to be hydrolyzed by the Alcalase. Further systematic analysis of the peptidases showed that the Alcalase exhibited a significant endopeptidase activity towards NBZ-Phe-pNA substrate (p < 0.01), whereas many exopeptidases in the A. elegans extract had the carboxypeptidase activity towards N-CBZ-Ile-Leu (p < 0.01). It is concluded that those exopeptidases presented in the A. elegans extract can benefit by decreasing the bitterness of the soybean protein hydroysate. They are also capable of being used with the Alcalase in a single-step enzymatic reaction to prepare the bitterless protein hydrolysate, which may be an efficient application for food industry.     

Novel applications for microbial transglutaminase beyond food processing

Transglutaminase (EC 2.3.2.13) initially attracted interest because of its ability to reconstitute small pieces of meat into a 'steak'. The extremely high cost of transglutaminase of animal origin has hampered its wider application and has initiated efforts to find an enzyme of microbial origin. Since the early 1990s, many microbial transglutaminase-producing strains have been found, and production processes have been optimized. This has resulted in a rapidly increasing number of applications of transglutaminase in the food sector. However, applications of microbial transglutaminase in other sectors have been explored to a much lesser extent. Here, we will present the wider potential of transglutaminases and discuss recent efforts that could contribute to the realization of their potential.     

An apparatus for rapidly preparing protein samples for hydrolysis

A device is described which allows 12 samples to be prepared for acid hydrolysis in approximately 1 h. Screw-cap test tubes containing the weighed samples are placed into the apparatus along with 6 n HCl reagent. Air is removed rapidly and simultaneously from samples and reagent by alternate evacuation and nitrogen flushing. Reagent is measured into the sample tubes, and caps are threaded into place under a nitrogen atmosphere.     

Complete amino acid analysis of peptides and proteins after hydrolysis by a mixture of Sepharose-bound peptidases

Incubation with a mixture of Sepharose-bound peptidases was shown to result in the quantitative release of amino acids from certain peptides and S-aminoethylated proteins. Subtraction of the low background values of amino acids generated by the enzymes enables amino acid ratios of corticotrophin-(1-24)-tetracosapeptide to be determined with a standard deviation on repeat digestions of 3-5%. Good values were obtained for amino acids that are completely or partially destroyed on acid hydrolysis, i.e. tryptophan, tyrosine, serine, asparagine and glutamine. Experiments with peptides containing d-amino acids showed that the enzyme mixture is stereospecific and could therefore be used to detect the presence of d-residues in peptides. The enzyme mixture completely hydrolyses peptide fragments obtained after Edman degradation and should therefore be useful for determining sequences of peptides containing acid-labile amino acid residues. The activities of the bound enzymes were unaltered over a period of 7 months and they provide a simple, reproducible procedure for the quantitative determination of amino acids in peptides and proteins containing l-amino acids.     

Flow cytometry applications in the food industry

Flow cytometry has become a valuable tool in food microbiology. By analysing large numbers of cells individually using light-scattering and fluorescence measurements, this technique reveals both cellular characteristics and the levels of cellular components. Flow cytometry has been developed to rapidly enumerate microorganisms; to distinguish between viable, metabolically active and dead cells, which is of great importance in food development and food spoilage; and to detect specific pathogenic microorganisms by conjugating antibodies with fluorochromes, which is of great use in the food industry. In addition, high-speed multiparametric data acquisition, analysis and cell sorting, which allow other characteristics of individual cells to be studied, have increased the interest of food microbiologists in this technique. This mini-review gives an overview of the principles of flow cytometry and examples of the application of this technique in the food industry.     

Nanoliposomes and their applications in food nanotechnology

Food nanotechnology involves the utilization of nanocarrier systems to stabilize the bioactive materials against a range of environmental and chemical changes as well as to improve their bioavailability. Nanoliposome technology presents exciting opportunities for food technologists in areas such as encapsulation and controlled release of food materials, as well as the enhanced bioavailability, stability, and shelf-life of sensitive ingredients. Liposomes and nanoliposomes have been used in the food industry to deliver flavors and nutrients and, more recently, have been investigated for their abilityto incorporate antimicrobials that could aid in the protection of food products against microbial contamination. In this paper, the main physicochemical properties of liposomes and nanoliposomes are described and some of the industrially applicable methods for their manufacture are reviewed. A summary of the application of nanoliposomes as carrier vehicles of nutrients, nutraceuticals, enzymes, food additives, and food antimicrobials is also presented.     

Recombinant protein expression for therapeutic applications

In recent years, the number of recombinant proteins used for therapeutic applications has increased dramatically. Many of these applications involve complex glycoproteins and antibodies with relatively high production needs. These demands have driven the development of a variety of improvements in protein expression technology, particularly involving mammalian and microbial culture systems.