Analyst, December, 1979, Vol. 104, pp. 1135-1137 H istochemical ldentif ication of Commercial Wheat Gluten 1135 F. 0. Flint and R. F. P. Johnson Procter Department of Food Science, University of Leeds, Leeds, LS2 9JT Three methods for the microscopical identification of commercial wheat glutens are compared. A periodic acid - Schiff and an iodine - potassium iodide method both indicate gluten by showing the wheat starch present. Toluidine blue contained in an aqueous mountant distinguishes gluten protein from both soya and meat proteins. Each method identified commercial gluten present in a gluten - soya protein - meat mixture. Testing for both starch and protein is recommended for protein products that may contain added starch. Keywords : Commercial wheat gluten identification ; gluten - soya mixture ; gluten - soya - meat mixture In addition to being a significant source of protein for many millions of people, wheat is an important source of industrial starch.l The separated undenatured wheat protein can be dried and forms the so-called “vital gluten’’ of commerce.According to Knight,l the dry commercial product contains 75430% of protein and 5-15% of carbohydrate, which consists largely of wheat starch. The most important use for commercial gluten is in flour protein enrichment for the baking of high-protein or starch-reduced breads, but it is also used in breakfast cereals, pastas and pet foods. Simmonds2 estimates that 10% of Australian gluten is used in processed meats and there are also techniques for the production of texturised vegetable proteins based on g l ~ t e n .~ Methods that distinguish commercial wheat gluten from soya and meat proteins are now needed in order to implement the present meat product regulations. Soya flours and texturised soya products can be identified by the cellular appearance of the carbohydrates that they contain, as shown by the periodic acid - Schiff (PAS) t e c h n i q ~ e . ~ Soya protein can be distinguished from meat proteins by the differential staining given by toluidine blue.5 The various colours (blue, purple and pink) shown by meat tissues and soya material reflect differences in the density of the bound stain and, as wheat proteins are chemically distinct from soya and meat proteins, they could be expected to stain differently. These two techniques and the classical iodine - potassium iodide method for the detection of starch have been compared for their success in identifying “vital gluten” powder and canned wheat glutens and in demonstrating commercial gluten in the presence of soya products and meat tissues.A more recent use for wheat protein is as a meat substitute. Experimental Materials Examined The following materials were examined: (1) laboratory-prepared vital gluten made by mixing bread flour to a stiff dough with distilled water, allowing it to stand for 20 min and then removing starch by washing in running water until the wash water appeared clear; (2) commercial canned gluten sold as a vegetarian food; (3) commercial canned spun soya protein and gluten binder sold as a simulated mince meat; (4) vital gluten powder from RHM Ingredient Supplies Ltd.; (5) vital gluten powder mixed with ground texturised soya protein (TSP); Protena P.M.l from RHM Ingredient Supplies Ltd.was used; and (6) vital gluten powder mixed with TSP [as in (5)] added to finely minced stewing steak. Sample Preparation The laboratory gluten and the com- mercial canned product [(l) and (2)] were sampled by extracting a “core” of material using a 5 mm diameter cork borer. The remaining materials [(3)-(6)] were stirred into a commercial Specimens for sectioning were prepared as follows.1136 FLINT AND JOHNSON HISTOCHEMICAL Analyst, Vol. 104 embedding medium (Tissue Tek OCT Compound from Miles Ames). After rapid freezing, 10-p" sections were cut from each material using a cryostat with a cabinet temperature of -20 "C.Staining Methods Gram's iodine solution (iodine - potassium iodide - water, 1 + 2 + 300) was prepared as a stock solution. Before use, 2 ml of stock solution were diluted to 10 ml with distilled water. The diluted iodine solution was applied directly to the sections, which were then covered with a cover-slip. The PAS method of Coomaraswamy and Flint4 for carbohydrate, with Light Green counterstain for protein, was used exactly as described. Toluidine blue contained in an aqueous mountant, as previously de~cribed,~ was used as a differential staining medium for proteins and other constituents with basic dye binding properties. Results and Discussion The results given by the three staining methods are summarised in Table I and illustrated They show the value of demonstrating both the carbohydrate and the protein in Figs.1-9. components in the identification of commercial wheat protein. TABLE I No. Material STAINING REACTIONS OF GLUTEN AND GLUTEN IN THE PRESENCE OF SOYA AND MEAT PROTEIN Iodine - potassium iodide PA3 and Light Green Toluidine blue A 7- . 7-- A 1 Laboratory vital 2 Canned gluten gluten 3 Cannedspun soya and gluten mince 4 Gluten powder 6 Gluten powder - TSP 6 Gluten powder - TSP - meat , I , Proteins Carbohydrate Proteins Yellow Starch granules, Green blue - black Yellow Gelled starch, Green dull purple Yellow Gelled starch in Green soya fibres and in gluten binder, similar dull purple shade Yellow Starch granules, Green blue - black Gluten, Starch granules in Green yellow; TSP, paler yellow gluten, ,blue - black Gluten, Starch granules in Green yellow; TSP, paler yellow; muscle fibres, paler yellow gluten, blue - black The distinctive size and shape of gluten and in the commercial gluten and the PAS method (see Figs.1, 2, I , Carbohydrate Proteins Starch granules, Pale blue magenta Gelled starch, Pale blue magenta; bran fragments (sparse), magenta Gelled starch in Fibres (soya), dark fibres (soya) and blue; binder binder (gluten), (gluten), pale blue similar magenta Starch granules, Pale blue magenta Starch granules in Gluten, pale blue; gluten, magenta; soya protein, dark intact and damaged purple - blue cell walls in TSP, niagen t a Starch granules in Gluten, pale blue; gluten, magenta; soya protein, dark soya cell walls in purple - blue; TSP, magenta muscle fibres, pale blue (shade distinct from gluten colour) ; muscle cell nuclei, red - violet Carbohydrate' Unstained Unstained Unstained Unstained Starch in gluten, unstained; cell walls in TSP, deep pink Starch.unstaikd; cell walls in TSP deep pink the wheat starch granules present in the laboratory powder are demonstrated clearly by both the iodine 7 , 8 and 9). Any commercial plant protein derived from a cereal source would hevitably contain starch granules, the morphblogy of which would be diagnostic for that cereal,6 e.g., a commercial plant protein derived from maize would contain corn starch,6 which has a characteristic polyhedral shape. Because wheat starch is widely used as an ingredient, the identification of wheat starch granules in a protein matrix is only a guide to the origin of that protein and there is a need for a confirmatory test that characterises the protein.The spun soya mince examined serves as an example: the fibres contain an amorphous PAS-positive material, which gave a purplish reaction toFig. 1 . Section of laboratory vital gluten stained Section of laboratory vital glutcn stained with iodine reagent. Magnification 90 x . Char- with PAS and light green. Magnification B O X . acteristic wheat starch granules appear deep blue - Characteristic wheat starch granules are magenta, black, wheat protein is yellow. wheat protein is green. Note the similarity of con- trast to Fig. 1. Fig. 2. Fig. 3. Section of canned gluten stained with Section of canned gluten stained with iodine reagent.Magnification 90 x . Gelatinised PAS and light green. Magnification 90 x . Darkcr starch appears dull purple, wheat protein is yellow. areas are of magenta-coloured gelatiniscd starch. Lighter areas are of protein stained green. Note thc similar contrast to iodine staining in Fig. 3. Fig. 4. Fig. 5. Section of canned spun soya and gluten Fig. 6. Section of canncd spun soya and glutcn inince stained with iodine reagent. Magnification mince stained with toluidine blue. Magnification 9 0 ~ . Darker areas are of dull purple staining 90 x . Soya fibres, in transverse and longitudinal gelatiniscd starch; both soya and glutcn protein are section, are de-p blue in contrast to pale blue-stained stained yellow. gluten. Starch fraction is unstained.Note thc differential staining of the two proteins comparctl with uniform staining in Fig. 5. [To fnrc p . 1136Fig. 7. Section of gluten powder stained with Fig. 8. Section of gluten powder stained with iodine reagent. Magnification 90 x . Starch PAS and light green. Magnification 9Ox. granules stained deep blue - black, wheat protein Starch granules stained magenta, wheat protein stained yellow. counterstained green. Note the similar contrast to iodine staining in Fig. 7 . Fig. 9. Section of gluten powder niixed with tcxturised soya protein and meat. Magnification 90 x . Top: particles of green - blue stained gluten (some coalesced), starch granules unstained. Bottom left: muscle fibres stained pale blue with red - violet nuclei. Bottom right: soya protein stained dark purple - blue, associated carbohydrate deep pink. Stain : toluidine blue.December, 1979 IDENTIFICATION O F COMMERCIAL WHEAT GLUTEN 1137 iodine, suggesting that it is gelatinised starch and the less well defined binder contains a similar material (see Fig.5). The toluidine blue serves to distinguish the two proteins present (see Fig. 6, which shows the deep staining of the fibrous soya set against the much paler colour of the declared gluten binder, the staining of which matches that of the other glutens examined). Spun soya protein (Fig. 6) gives a dark blue colour with toluidine blue, whereas extruded soya gives a more purple colour. In both instances the colour is much deeper than the pale shades given by commercial gluten and meat tissues.Both gluten and muscle fibres stain pale blue, but the gluten colour is a distinctly more greenish shade of blue (see Fig. 9). In addition to muscle fibres, meat contains connective tissue, which stains characteristically with toluidine blue. Collagenous connective tissue is coloured pale pink and elastinous connective tissue blue - green.5 The green - blue of the toluidine blue-stained glutens examined is similar to that shown by stained elastic tissue fibres, but there is little possibility of confusion of these proteins because of the characteristic fibrous form in which elastic tissue occurs and the amorphous “granular” appearance shown by raw and heat-processed gluten (see Figs. 9 and 6). The only other differential staining of protein that was observed was that given by iodine with the gluten powder-TSP-meat mixture [(S)].Here the gluten powder stained a full yellow and the TSP and raw meat a paler yellow, but the difference was not sufficiently well marked for this to form a reliable distinguishing test. The advantage of the iodine method is that by demonstrating the presence of wheat starch (even when gelatinised), an important fraction of present commercial glutens may be demonstrated, and this makes it a useful rapid first test although one which should be followed by subsequent protein staining. The PAS technique also demonstrates intact and gelati- nised starch but additionally it reveals plant cell walls and their debris so that TSP can be positively identified. An advantage of the PAS method is that the slide preparation can form a permanent record whereas the iodine and toluidine blue methods yield only temporary slides.Conclusions Each of the three micro methods can aid in the identification of fresh and processed com- mercial gluten. In the detection of commercial glutens, the toluidine blue technique is the preferred method but either the iodine or the PAS technique should be used to confirm that there is a starch fraction present and, with uncooked products, that it is wheat starch. Because it is a one-stage method of staining that clearly distinguishes commercial wheat gluten from meat tissues and soya protein, the toluidine blue method is the most suitable for the quantitative microscopy of processed meats containing added commercial gluten. The method previously described for the quantitative determination of texturised soya protein in comminuted meat5 could be used for determining added commercial gluten without further modification. References 1. Knight, J . W., “The Chemistry of Wheat Starch and Gluten and Their Conversion Products,” 2. 3. 4. 5. 6. Leonard Hill, London, 1965. Simmonds, D. H., Food Technol., Aust., 1976, 28, 84 and 116. Gutcho, M., “Artificial Meat: Textured Foods and Allied Products,” Noyes Data Corp., New Jersey Coomaraswamy, M., and Flint, F. O., A n a l ~ ~ s t , 1973, 98, 542. Flint, F. O., and Meech, M. V., Analyst, 1978, 103, 252. Kent- Jones, D. W., and Amos, A. J . , “Modern Cereal Chemistry,” Sixth Edition, Food Trade Press, and London, 1973. London, 1967. Received February 12th, 1979 Accepted July 9fh, 1979