Mª Concepción García

Analysis of Soyabean Proteins in Meat Products: A Review

Dr. Lourdes Amigo, Instituto de Fermentaciones, Industriales (CSIC), Juan de la Cierva, 3, 28006 Madrid, Spain The use of soyabean proteins as meat extenders has spread significantly due to the interesting nutritional and functional properties that are present in soyabean proteins. Together with these, health and economical reasons are the major causes for the addition of soyabean proteins to meat products. Nevertheless, despite the good properties associated to soyabean proteins, there are many countries in which the addition of these proteins is forbidden or in which the addition of soyabean proteins is allowed up to a certain extent. Thus, the need of analytical methods enabling the detection of added soyabean proteins in meat products is obvious. Microscopic, electrophoretic, immunologic, and chromatographic methods are the most widely used for this purpose. However, the detection of soyabean proteins in meat products presents difficulties related to the composition (meat species, meat quality, soyabean protein source, presence of other non-meat proteins, etc.) and the processing of the meat products, and, although these analytical methods have tried to overcome all these difficulties, there is still not a method enabling quantitative assessment of soyabean proteins in all kinds of meat products.

Effect of the mobile phase composition on the separation and detection of intact proteins by reversed-phase liquid chromatography-electrospray mass spectrometry.

Various buffers (ammonium acetate, ammonium formate, and ammonium hydrogencarbonate), acids (formic acid, acetic acid, heptafluorobutyric acid, and trifluoroacetic acid), and bases (ammonium hydroxide and morpholine) covering the range from 2 to 11.5 have been investigated for their performance in the separation of proteins by reversed-phase liquid chromatography (RPLC) and in their detection by electrospray mass spectrometry (ESI-MS). These additives were first tested for the detection of standard proteins by ESI-MS by flow-injection analysis (FIA). Those additives yielding the highest signals were employed for the separation of standard proteins by using three different reversed-phase columns: two C18 columns (4.6 mm I.D. and 2.1 mm I.D.) and one perfusion column (2 mm I.D.). The sensitivity of the LC-MS system was evaluated with the column giving the best results and with those LC eluents enabling the LC separation of the proteins and also yielding the highest MS signals. For that purpose, calibration curves were compared for both LC-MS and FIA-MS. Formic acid was the additive yielding the highest responses in FIA-MS and trifluoroacetic acid (TFA) gave the best separation and recovery of the proteins. However, problems related to poor recovery of the proteins in the column when formic acid was used and the significant signal suppression observed in MS when TFA was employed, made neither of them suitable for the sensitive detection of the proteins in LC-MS.

Perfusion chromatography: an emergent technique for the analysis of food proteins

Perfusion chromatography is a technique arised to overcome the problem associated with mass transfer in the separation of large molecules such as proteins by high-performance liquid chromatography (HPLC). Perfusion media are constituted by two set of pores: throughpores (6000-8000 A) and diffusive pores (800-1500 A) which enable better access of macromolecules to the inner of the particle by the combination of convective and diffusive flow. As a consequence, times required for a chromatographic separation are reduced. Perfusion media are available in different chromatographic modes: reversed-phase, ion-exchange, hydrophobic interaction, and affinity. From the theoretical models developed to explain the dynamic of retention of solutes in perfusive supports, it was derived that efficiency of a separation was independent of the flow-rate and only depended slightly on the particle diameter. Furthermore, loading capacity was also independent of the superficial velocity. All these advantages have promoted the use of this chromatographic technique for the separation of biomolecules both in analytical and preparative chromatography. Characteristics of perfusion chromatography make this technique very interesting for the analysis of food proteins. Perfusion chromatography enables the assessment of protein composition of a foodstuff at sufficient speed and low cost to be suitable in routine analysis.

Rapid separation of soybean globulins by reversed-phase high-performance liquid chromatography

A rapid separation of the main soybean proteins (7S and 11S globulins) was carried out by reversed-phase high-performance liquid chromatography. For this purpose, a linear binary gradient acetonitrile--water--0.1% trifluoroacetic acid, at a flow-rate of 1 ml/min and 50 degrees C temperature was designed. Under the experimental conditions of this work, it was possible to separate five peaks corresponding to the globulins from a soybean protein isolate in 9 min. The characterization of soybean proteins was accomplished by analyzing the 7S and 11S purified fractions obtained from a soybean protein isolate. The method was applied to the separation of soybean proteins from commercial foodstuffs: soybean flour, textured soybean and soybean milks.

Ultrarapid detection of bovine whey proteins in powdered soybean milk by perfusion reversed-phase high-performance liquid chromatography

A perfusion reversed-phase high-performance liquid chromatographic method has been developed to simultaneously separate soybean and bovine whey proteins (alpha-lactalbumin and beta-lactoglobulins (A + B)) in a very short analysis time (approximately 5 min). The method consisted of a linear binary gradient water-acetonitrile-0.10% trifluoroacetic acid at a flow-rate of 3 ml/min, with the column thermostated at 60 degrees C, and ultraviolet detection at 254 nm. This method enables the rapid detection of adulterations of powdered soybean milks by addition of bovine whey proteins. When bovine whey proteins were too low to be detected by direct injection of the sample, a previous acidic precipitation step was required in order to concentrate these proteins. Quantitatve analysis of bovine whey proteins was also successfully performed. In fact, it was possible to detect about 1% and 1.3% of alpha-lactalbumin and beta-lactoglobulins, respectively, in a commercial powdered soybean milk in which these proteins were included in its formulation. Results were compared with those obtained by conventional reversed-phase high-performance liquid chromatography.

Characterization of commercial soybean products by conventional and perfusion reversed-phase high-performance liquid chromatography and multivariate analysis

Conventional and perfusion reversed-phase high-performance liquid chromatography are used to characterize commercial soybean products for human consumption. For this purpose, previously optimized methods of conventional and perfusion chromatography applied to the separation of soybean proteins are employed. Sixty different samples corresponding to 26 different trademarks of soybean products [soybean protein isolate, soybean flour, textured soybean, soybean milks (liquid and powdered), and soybean infant formulas] are analyzed. Characterization of soybean products is carried out on the basis of their protein profiles obtained by both chromatographic methods. Data obtained are processed using multivariate methods such as principal components and discriminant analysis. Perfusion chromatography enables a further and faster characterization of commercial soybean products than conventional chromatography, of great value in the quality control of this kind of product.

First approach based on direct ultrasonic assisted enzymatic digestion and capillary-high performance liquid chromatography for the peptide mapping of soybean proteins.

This work proposes, for the first time, the use of a high intensity ultrasonic probe to accelerate the tryptic digestion of soybean proteins. Different digestion parameters were optimized: protein extracting solution, reduction, and alkylation conditions (time, concentration, and temperature), trypsin:protein ratio, and ultrasonic conditions (sonication amplitude and time). Separation of peptide profiles was carried out by capillary-HPLC. The effect of the variation of chromatographic conditions (elution gradient, column temperature, and injection volume) on peptide separation was also studied using two capillary-HPLC columns with different column diameters and particle sizes. Moreover, samples were focused at the top of the column in order to obtain an increasing sensitivity without loss of efficiency. This method was successfully applied to the profiling of soybean peptides from transgenic and non-transgenic soybeans and from different pigmented beans commercialized as soybeans.

Determination by perfusion reversed-phase high-performance liquid chromatography of the soybean protein content of commercial soybean products prepared directly from whole soybeans.

The use of soybean flour as external standard for the determination of soybean proteins in soybean products directly prepared from whole soybeans is investigated. For that purpose a perfusion reversed-phase high-performance liquid chromatography method consisting of a linear binary gradient acetonitrile-water (both with 0.1% trifluoroacetic acid) in 3 min at a flow-rate of 3 ml/min, and a temperature of 60 degrees C is used. Samples dissolved in water are directly injected in the chromatographic system. The method is validated by evaluating detection limits, precision, and accuracy and applied to the quantitation of soybean proteins in soybean products directly prepared from whole soybeans.

DETERMINATION OF THE SOYBEAN PROTEIN CONTENT IN SOYBEAN LIQUID MILKS BY REVERSED-PHASE HPLC

A reversed-phase high performance liquid chromatography method, previously optimized, was applied to determine the soybean protein content in commercial soybean liquid milks. Quantitation of soybean proteins was first performed by using soybean protein isolate as external standard. Although this standard seemed suitable for the quantitation of soybean proteins in some soybean liquid milks, for those soybean milks directly prepared from whole soybeans, a best estimation of the soybean protein content was obtained by using soybean flour as external standard.

USE OF PHASTGEL SODIUM DODECYL SULPHATE POLYACRYLAMIDE GEL ELECTROPHORESIS FOR RAPID CHARACTERIZATION OF SOYBEAN PROTEINS IN COMMERCIAL SOYBEAN PRODUCTS

Commercial soybean products for human consumption (soybean protein isolate, soybean flour, textured soybean, and soybean powdered milks) are characterized for the first time by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) using the PhastSystem®, a semi-automatic electrophoretic system. It makes SDS-PAGE very fast and gives very sharp bands that simplify the identification. The time from loading the gel to characterization is less than 1 hour and the resolution is similar to that of large conventional gels. Proteic fractions obtained from these commercial soybean products by using a fractionation procedure described in literature are also analyzed. The electrophoretic patterns of these globulin fractions exhibited some differences which were attributed to the different technological process used for the preparation of each product investigated. Furthermore, the electrophoretic patterns obtained for commercial soybean products enabled the differentiation between products derived from soybean protein isolates and those prepared directly from whole soybeans.