Modern food science deals with the structure, function and interactions between nutrients at the molecular level. Modern methods used in the research give answers to questions concerning the composition and structural, biological and functional properties of food substances, are used.
Owing to the technological resources, the staff of the Chair of Food Biochemistry is able to carry out research on food science and nutrition in broad sense. The methods of separation and determination of the structure and function of food nutrients, which the Chair has at its disposal, are:
Gel electrophoresis, 2D electrophoresis with MALDI -TOF spectrometry, isoelectric focusing, high performance liquid chromatography with ultraviolet spectroscopy and electron scanning microscopy with a fractal characteristics of the microstructure picture of objects under study.
This research is complimented, thanks to a cooperation with foreign and Polish research centres, with a biopolymer analysis with the mass spectrometry method. It enables a molecular weight measurement accurate to 0.02% and a determination of sequences such as peptide sequences. The circular dichroism method is also used for studying the protein secondary structure.
Gel electrophoresis is a classic (having an over 100-year’s tradition) method of the separation of biological material components, using the phenomenon of charged molecules’ dislocation under the influence of an electric field. The technique of polyacrylamide gel electrophoresis with the presence of sodium dodecylsulfonate (SDS-PAGE) is used in the Chair of Food Biochemistry. The possibility of an analysis of proteins that poorly dissolve in solvents other than SDS solutions, is an advantage of this method. Qualitative and quantitative determinations made with this method in the Chair are based on colour reactions between protein/polypeptide and dyes, mainly Coomassie Brilliant blue R-250 and G-250. A quantitative measurement is taken by means of a densitometer.
Examples of the possibilities of using the SDS-PAGE method are the following:
- quantitative determinations and determinations of the molecular weight of plant and animal proteins/polypeptides and enzymes accurate to 5% (in relation to an introduced pattern or on the basis of an electrophoretic mobility calculation);
- an analysis of proteins after modifications; a determination of protein and enzymatic preparation purity;
- a detection of adulterations of milk, powdered milk, milk protein concentrates, cheeses other than cow’s cheeses and meat products by an addition of this milk;
- determinations of locations and the effect of the activity of purposely added enzymes with a characterisics of casein fractions, whey proteins and proteose-peptones in dairy products;
- a study of association processes of e.g. casein-ß; a determination of the effect of protein pasteurization or proteolysis in extracts from ripening and non-ripening cheeses due to differences resulting from an addition of different bacterial cultures.
High performance liquid chromatography
Affinity differences of components separated to two unmixable phases, of which one is mobile and the other stationary, are the basis of the chromatographic division. High performance liquid chromatography (HPLC) is the most frequently used method of separating the components of biological materials. Nearly all bioactive peptides obtained through food protein enzymatic hydrolysis can be isolated by means of reversed phase high performance liquid chromatography (RP-HPLC).
The Chair of Food Biochemistry possesses photodiode and fluorescent equipment for HPLC with UV/VIS detectors for qualitative and quantitative determinations (on the basis of absorbance or fluorescence values) of peptides, proteins, biogenic amines, vitamins and carbohydrates. Moreover, chromatographic separations allow for detecting adulterations of, for instance, soybean protein preparations with cow’s milk proteins, or of meat and dairy products with various types of milk.
Spectral derivatives are an effective tool for identifying and studying changes in, for example, peptides, proteins, carbohydrates, and vitamins. The equipment with appropriate computer software, possessed by the Chair, enables obtaining UV/VIS spectra of biologically active substances under study. The second derivative of UV spectrum can be used for detecting the presence of phenoalanine, tyrosine and tryptophan in a biological material and calculating molar ratios of the content of these amino acids.
The monitoring of the changes in the secondary structure proteins is an example of the application of the second derivative of UV spectrum. On the other hand, the fourth derivative of UV spectrum can be used for quantitative analysis of protein.
Proteomic analysis of proteins
The techniques based on chromatographic and electrophoretic methods together with computer visualisation techniques are mostly applied in proteomic research. Recently many research using techniques of 2D-electrophoresis with MALDI-TOF (matrix-assisted laser desorption/ionisation time-of-flight) have been developed. The mixtures of soluble proteins are initially separated by the use of 2D-electrophoresis. The basis of separation is the difference of pI (isoelectric point) and molecular mass values. Appropriate computer programs applied for gel analyses (e. g. Image Master) allow for initial characteristics of proteins that is based on the comparison of the values of pI and molecular mass of tested proteins and the mixtures of protein standards. Spots which correspond to single proteins are cut from gel. Proteins are digested by proteolytic enzymes (mostly by trypsin). The hydrolysates are analysed by MALDI-TOF mass spectrometr.
Application of peptide mass fingerprinting analysis allows for an idetification of proteins. Appropriate programs such as SONAR PLUS or ProFound convert data concerning the molecular masses of obtained peptide fragments and then compare these data with Internet databases (e. g. NBCI, OWL). Program identifies the proteins by the level of the overlay of amino acid sequence. Additionally the technique PSD or PSD with CAF (chemically assisted fragmentation) allows for the determination of peptide amino acid sequence. Proteomic workroom of the Chair of Food Biochemistry is equipped in 2D-electrophoresis system and the software for gels analysis and documentation (Image Master 2D Platinum) as well as MALDI-TOF Pro mass spectrometr including programs Ettan Maldi Pro, ProFound Search, PSD Sonar Search and own Oracle 9i database which works directly with the National Center for Biotechnology Information (NCBI).
The database of proteins and bioactive peptides (BIOPEP)
Computer methods of analyzing sequences of biologically active compounds are well-applied in biochemistry and biotechnology. They are used most frequently for modelling the secondary structure of proteins and for creating databases.
BIOPEP database designed in Chair of Food Biochemistry contains information on proteins (about 1000) which are precursors of bioactive peptides. Peptides (about 2000) of which information is gathered in the database, show different (total 44) types of activities including antihypertensive, immunomodulating, antithrombotic, opioid and others. An additionally created computer application – the BIOPEP, enables an evaluation of food proteins according to the following factors/criteria: the frequency of the occurrence of fragments with the given activity in a protein chain, a potential activity of protein fragments, and profiles of a potential biological protein activity (i. e. the type and location of a bioactive fragment in the protein chain).
Using BIOPEP as the one of in silico tools of protein evluation, there is also possible to design the processes of the release of bioactive peptides from protein. The above-mentioned methods can be applied for designing food with the special designed and desired properties (functional food) as well as production of nutraceuticals i. e. food with therapeutic properties.
A method for detecting and determining unknown proteins in protein preparations
This offer concerns a method enabling to detect, identify and determine quantitatively, in the 1-99% range, the “alien” protein in mixtures with a protein preparations. Various methods for detecting and determining proteins (including milk proteins and soybean proteins in mixtures), such as electrophoretic, immunological and chromatographic methods, have limited capabilities, which made the authors of this offer search for a standard, routine procedure that would allow to detect and determine milk proteins in soybean protein preparations.
According to the method proposed, samples of protein preparations suspected to contain unknown proteins are subjected to enzymatic hydrolysis by specific proteolytic enyzme, and then analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC).
The chromatographic analysis provides chromatographic images of specific products of enzymatic hydrolysis of particular protein preparations, i.e. the so called peptide profiles. On the basis of statistical analysis of results obtained from chromatograms, peptide fractions with specified retention times, unique to each type of milk protein preparations are selected. They are called “markers”. The markers were correlated with the percentage of a given protein preparation to the highest degree, which allow to distinguish between a pure protein preparation and a protein preparation containing an unknown protein. Chromatographic profiles of a pure protein preparation and 15 various concentrations of unknown proteins in mixtures with a protein preparation enable to create a data matrix (correlation between areas of chromatographic peaks and content of protein in a preparation), in the form of a spreadsheet. In the spreadsheet the profiles obtained were considered reference patterns (matrices) for particular types of detected protein preparations, to be used in a commercial method for identifying and determining unknown proteins in the protein preparations.
The main advantage of this strategy is that it enables both to detect very low concentrations of uknown protein in a protein preparations (at a level of at least 1%), and to perform a qualitative analysis of protein preparations (i.e. to determine the type of preparation). This makes it possible to confirm the authenticity of protein products. The use of this method for commercial purposes allowed to detect “alien” proteins in food preparations and products, and exclude the proteins that cause allergies. In addition, this strategy combines several modern analytical methods, supported by computer programs and a spreadsheet enabling routine determination of the qualitative and quantitative composition of a given protein preparation. Having obtained results from a chromatogram, the user has to enter peak areas into the appropriate column of the spreadsheet. All other computations are made automatically.
The libraries of UV spectra of proteins and peptides
The derivatives of ultraviolet/visible spectra appear to be a sufficient tool of studies on changes occurring e.g. in peptides, proteins and dyes. The UV/Vis spectra of substances separated via high-performance liquid chromatography (HPLC) were acquired using diode-array detector (DAD). The chair possesses HPLC-DAD assembly with the program enabling acquisition and processing of UV/Vis spectra derivatives. The second derivatives of UV spectra may serve e.g. for detection of presence phenylalanine, tyrosine or tryptophan in biological material and for calculation of molar ratios of these amino acids. Determination of molar ratios of aromatic amino acids was applied e.g. for discrimination of casein and whey protein fractions of bovine milk, products of bovine beta-casein hydrolysis by plasmin as well as wheat alpha/beta- and gamma-gliadins. Chemometrical analysis of UV spectra involving calculation of so called similarity indices between reference spectra derivatives and spectra derivatives of analytes became a suitable tool for substance identification. Identification on the basis of third derivatives of UV spectra appeared to be most reliable. The libraries of derivatives (from first to fourth) of spectra of major bovine milk proteins (6 proteins), bovine beta-casein fragments originating form hydrolysis by plasmin (8 fractions); wheat alpha/beta-gliadins (2 fractions) as well as wheat gamma-gliadins (2 fractions) were used. Enabling access to the libraries of protein UV spectra and their derivatives at the website of the Chair of Food Biochemistry is planned in 2007