SMITH, R. J., and CARUSO, J. L. 1964. Determination of phosphorus. TUSCHHOFF, J. V., KESSINGER, G. L., and HANSON, C. E. 1969. Page 42 in: Methods in Carbohydrate Chemistry. Vol. 4. R. L. Whistler, Phosphorus oxyhalide cross-linked hydroxypropyl starch derivative. ed. Academic Press: New York. U.S. patent no. 3,422,088. SRIVASTAVA, H. C., and PATEL, M. M. 1973. Viscosity stabilization of WETZTEIN, H. L., and LYON, P. 1956. Manufacture of modified tapioca starch. Starch/ Staerke 25:17. starch. U.S. patent no. 2,754,232. SWINKELS, J. J. M. 1985. Composition and properties of commercial WHISTLER, R. L. 1953. Starch retrogradation. Page 213 in: Starch and native starches. Starch/ Staerke 37:1. its derivatives. Vol. 1. J. A. Radley, ed. Chapman and Hall: London. TAKEDA, Y., SHIRASAKA, K., and HIZUKURI, S. 1984. Examination WITNAUER, L. P., SENTI, F. R., and STERN, M. D. 1955. Light of the purity and structure of amylose by gel permeation scattering investigation of potato amylopectin. J. Polym. Sci. 16:1. chromatography. Carbohydr. Res. 132:83. WOLF, M. J., RUGGLES, V. J., and MacMASTERS, M. M. 1962. TAKEDA, Y., TOKUNAGA, N., TAKEDA, C., and HIZUKURI, S. Refractive indices of wheat starch granules at various moisture levels 1986. Physicochemical properties of sweet potato starch. Starch/ Staerke determined with an interface microscope. Biochim. Biophys. Acta. 38:345. 57: 135. TAN, S. L., and MORRISON, W. R. 1979. The distribution of lipids in the WURZBURG, 0. B. 1964. Acetylation. Page 286 in: Methods in germ, , pericarp, and tip cap of amylomaize, LG-l 1 hybrid Carbohydrate Chemistry. Vol. 4. R. L. Whistler, ed. Academic Press: maize and waxy maize. J. Am. Oil Chem. Soc. 56:531. New York.

[Received April 4, 1988. Revision received October 20, 1988. Accepted December 30, 1988.]

Rheological Changes in Cracker Sponges During an 18-Hour Fermentation1

J. Y. WU2 and R. C. HOSENEY 3

ABSTRACT Chem. 66(3):182-185

Of variables tested, the pH of cracker sponges was the most effective in determined from the slope of the linear relationship between resistance to reducing their resistance to extension. The effect was attributed to extension and time. The slopes showed that sponges at pH 4.1 and 3.5 had proteolytic enzymes, because the optimum pH of 4.1 coincides with the much greater enzyme activities than sponges at pH 4.7 and 2.7. reported optimum pH of indigenous protease. A mixture of Reconstituting an enzyme-free composited residue with enzyme extracts commercial and wheat starch was used to study the effect of from several cracker showed that different flours differed in enzyme proteolytic enzymes. Their optimum pH for decreasing the resistance to activity. extension ranged from 3.8 to 4.1. Relative enzyme activities were

Conventionally, the saltine cracker making process requires about 24 hr of fermentation. One purpose of this long fermentation moisture 13.0%, ash 0.45%), flour D (protein 9.1%, moisture is to obtain an optimum modification of the flour gluten. That 12.6%, ash 0.46%), flour E (protein 9.6%, moisture 12.1%, ash modification is believed to be closely related to rheological changes 0.52%), flour F (protein 7.8%, moisture 13.1%, ash 0.42%), and in the cracker sponge that are important in determining flour G (protein 10.1%, moisture 13.8%, ash 0.48%). The handling properties and final product qualities (Rogers and commercial cracker flours (flours C-G) were supplied by Nabisco Hoseney 1989). Physical testing methods have been used to Brands, Peavey, Lance, and Dixie-Portland. In addition, two soft measure the rheological changes in cracker sponges during , a soft white wheat (SWW, A) and a soft red winter (SRW, fermentation (Faridi 1975; Pizzinatto and Hoseney 1980; B) were milled on the Kansas State University pilot mill. A Doescher and Hoseney 1985a,b). The influences of the various commercial flour (protein 12.2%, moisture 12.6%, ash formula ingredients of cracker sponges on dough rheology also 0.47%) was also used in certain studies. were studied by Pizzinatto and Hoseney (1980). In this study, the major inherent factors in flours that caused Yeast rheological changes in cracker sponges were investigated. Of Red Star commercial compressed baker's yeast (70% moisture) special interest was the importance of flour proteolytic enzymes on from Universal Food Corporation was used. the rheological properties of cracker sponges. Gluten and Wheat Starch MATERIALS AND METHODS Gluten and wheat starch samples were provided by Midwest Solvent Co., Atchinson, KS. The protein and moisture contents Flour were 80 and 11.1%, respectively, for gluten and 0.3 and 7.9% for A series of soft wheat cracker flours with different wheat starch. properties was used: soft white A (protein 9.5%, moisture 12.7%, ash 0.46%) and soft red wheat flour B (protein Chemicals 10.1%, moisture 13.0%, ash 0.49%), flour C (protein 9.1%, All chemicals were reagent grade.

'Contribution no. 88-417J. Kansas Agricultural Experiment Station, Manhattan Preparation of Slurry 66506. A slurry was prepared as described by Doescher and Hoseney 2 Graduate research assistant and professor, respectively, Department of Grain (1985b). The mixture contained flour and water (1:2 ratio), 0.62% Science and Industry, Kansas State University, Manhattan. yeast, and 3% sucrose based on flour weight. It was fermented for 'Present Address: Food Industry Research and Development Institute, Hsinchu, 18 hr at 30°C and 90% relative humidity. A feed of 20% flour Taiwan. (based on sponge flour weight) was then added, and the slurry was

© 1989 American Association of Cereal Chemists, Inc. given a second 18-hr fermentation.

182 CEREAL CHEMISTRY Rheological Studies by Extensigraph RESULTS AND DISCUSSION To study the rheological changes occurring during fermentation of cracker sponges an extensigraph procedure (Pizzinatto and Proteolytic Enzyme Effects in a Commercial Gluten Hoseney 1980) was used. Sponges that contained yeast (0.36% and Starch System based on flour weight), slurry (4.52% based on flour weight), and To study the rheological effects of proteolytic enzymes we water (22% based on flour weight) were mixed for 3 min in a pin needed an enzyme-free system. We chose a system of commercial mixer (TMCO-National Mfg. Company, Lincoln, NE) modified to gluten and starch. However, it was found that the resistance to 32 rpm and allowed to ferment for 18 hr (30'C, 90% rh). After extension of this system decreased during 12 hr of rest at pH 4. 1. fermentation, the sponge was mixed for 5 min with shortening This indicated that at least part of the proteolytic enzymes (11%), (1.8%), and soda (0.45%) but no additional flour, and remained associated with the gluten or that the low p H changed the divided into three pieces of 150 g each. Each piece was rounded, sponge rheology. molded, allowed to rest for 45 min, and then measured with an To examine the possible effects of acid, two series of sponges extensigraph. Each treatment was duplicated. were adjusted with lactic acid to pH values between 2.7 and 4.7 and To study proteolytic enzyme activity, a constant (or control) allowed to stand for 0 hr and 12 hr. Resistance to extension was gluten and starch system was used. No shortening was added at the dough-up stage to aid in handling of the sponges. In all the rheological studies, soda was added at the dough-up stage, unless specifically mentioned otherwise.

Extraction of Proteolytic Enzymes One kilogram of flour was extracted with 6 L of ammonium sulfate solution (18% saturation), by stirring for 1.5 hr at 40 C. The slurry was then centrifuged at 1,000 X g for 15 min. The residue was wm lyophilized and prepared for reconstitution studies. The super- natant was adjusted to 40% saturation by adding solid ammonium z sulfate, allowed to stand for 24 hr at 4° C, then centrifuged. The supernatant was then adjusted to 80% saturation and allowed to set for another 24 hr at 40 C. The precipitate was collected by Cl, centrifugation and lyophilized. This preparation was used as the w crude proteolytic enzyme in reconstitution studies. The isolation procedure is essentially that reported by Salgo (1981). LL. C.)0 Reconstitution Studies w In studying the Theological effects, different amounts of the crude proteolytic enzyme extract were added back: zero, one third, two thirds, and all. w a:0

11001- 0 2.0 4.0 4.5 5.0

1 0001 pH Fig. 2. Effect of pH on the activity of the proteolytic enzymes. 9001 Ohr

12hr w 8001 z C- M 10001- 7001 Q--, - pH4.7 w 900 - 6001 z 0 0 800- l_ Co w z 0 500k w 7001- x 600 1 400k w z 0 500p w 3001 I- CO w 4001- : pH27 CO3 2001 z 3001- LiL a: CO, 1001 2001 7pH 3.5 w pH 4.1 Al I I I I I II 100 U. 2Z5 3D 404 5.0 n v - 4 8 12 pH Fig. 1. Rheological changes of sponges made from gluten and starch set at TIME (Hr.) different pH levels for 0 or 12 hr. Fig. 3. Activity of indigenous proteolytic enzymes at different pH levels.

Vol. 66, No. 3,1989 183 measured after the sponges were neutralized to pH 7.0. At zero largest Theological changes were found to be in the range from pH time, the acid greatly changed sponge rheology (Fig. 1). 3.8 to 4.1 (Fig. 2), which was the optimum range reported for the The effect of the acid was accounted for by subtracting the proteolytic enzymes (Salgo 1981). This evidence further supported effects at 0 hr from the results at 12 hr. With this technique, the the theory that proteolytic enzymes were playing important roles in changing the sponge rheology. Sponges were adjusted to pH 2.7, 3.5, 4.1, and 4.7, and then allowed to set for different times. As shown in Figure 3, good linear 1200 relationships were found for decreases in resistance to extension at all pH values. At pH 3.5 and 4.1, much steeper slopes were 1100 obtained, which indicated greater enzyme activities in that pH ' ( no enzesww range. - (no enzyme) 1000 The slope at pH 3.5 was slightly greater than the slope at pH 4. 1; however, z this changed after subtracting the effect of the acid. 0 9001 Sponges at pH 4.1 showed a more rapid Theological change than the others. z 8001 Rheological Effects of the Proteolytic Enzymes xl 7001 Extracted from Soft White Wheat Flour Sponges made from the soft white wheat residue after enzyme LU 0 6001 extraction showed no Theological change (Fig. 4) during 18-hr "fermentation" at pH 4.1. This indicated that the proteolytic I- 500[ enzymes, presumably completely extracted from the residue, did have a very important effect on sponge rheology. z 400F NIb A reconstitution study, combining the enzymes and the residue to give a reconstituted sponge, gave resistance to extension curves (0) 300[ measured after 18 hr of fermentation similar to those of the rn unextracted control flour. This showed that the enzymes were n sww (0 200F active when added back to the residue. The resistance to extension of this sponge was 432 Brabender units, so about 82% of the enzyme activity was recovered. 100 As shown in Figure 5, the rheology of the sponge was closely

A I I related to the amount of the extract added. Therefore, the 2 4 6 8 10 12 14 16 18 rheological change caused by fermentation can be materially TIME (Hr.) influenced by increasing the proteolytic enzyme in the cracker Fig. 4. Changes in the Theological properties of cracker sponges at pH 4.1 sponge. during an 18-hr setting time. Soft white wheat (SWW) = complete flour; SWW no enzyme = extracted with ammonium sulfate. Rheological Effects of the Proteolytic Enzymes Extracted from Different Cracker Flours The proteolytic enzymes were extracted from seven cracker 1200 flours with different baking properties. The residues from six flours were combined to study the relative enzyme activities. As 1100 shown in Table I, reconstituted flours made from the combined z residue and the enzyme extract from each flour were made into mo 10001_ sponges and allowed to stand for 18 hr. The proteolytic enzyme 9 extracts from the different flour sources showed different abilities 9001 to change sponge rheology. z- 8001- Comparison of Rheological Changes in Cracker Flours After 9 hr z Wu and Hoseney (1989) showed that the rapid change in rheology that occurs during the first 5 hr of fermentation x 7001_ (Doescher and Hoseney 1985b) has essentially no influence on the LU cracker dough. In addition, they showed that sponges made with 6001- yeast plus slurry have a pH that favors proteolytic enzyme action I- after about 9 hr of fermentation. Therefore, the actual period of 5001F time for proteolytic enzyme activity would be from about 9 hr until

z 400I_ TABLE I Rheological Effects of the Proteolytic Enzymes Extracted w 3001- Co) from Different Cracker Floursa LUnl Extracted Resistance to Extension 200j_ from (BU)b Control (no extract) 1,150 100 Flour D 280 Flour C 336 urn Flour G 370 0 33 66 100 Soft white wheat flour A 453 Soft red wheat flour B 462 ENZYME AMOUNT Flour E 474 ( % OF EXTRACT) Flour F 493 Fig. 5. Rheological effects after setting 18 hr at pH 4.1 with different aEnzymes were added to a constant residue and fermented for 18 hr. amounts of enzyme extract expressed as percent of total extract. bLSDo.os = 32.

184 CEREAL CHEMISTRY TABLE II from different flours showed different decreases in resistance to Comparison of Rheological Changes in Different Cracker Flours extension. This may be a useful indicator to determine flour quality After Setting for 9 hr at pH 4.1 and predict baking results. Resistance to Extension In studying the rate of the Theological changes with standing 2 Samples (BU) time (Fig. 6), it was found that although the Theological properties . Flour B 970 were the same for several sponges after 9 hr of standing, they had Flour D 864 different rates of change in the reduction of resistance to extension. Flour C 811 A particularly high rate was found for the sponge mixed from the Flour E 476 soft red winter flour. Flour F 425 The sponge mixed from bread flour is also shown in Figure 6. Flour G 252 The high resistance to extension infers either a low proteolytic enzyme activity or a very strong residue that was not affected by aLSDo.o 5 z= 33. enzyme activity. It would be difficult to bring the resistance to extension of that sponge to a reasonable (low) value for cracker production.

Oreaa CONCLUSIONS

Sponge pH 4.1 was found to be the most effective in reducing the resistance to extension. The effect was attributed to proteolytic 0:M enzymes. A constant system using commercial gluten and wheat z starch was made for studying the effect of proteolytic enzymes. The C,,z optimum pH was in the range 3.8-4.1. Relative enzyme activities determined from the slope of the linear relationship between wIn resistance to extension versus time showed that the sponges at pH z 4.1 and 3.5 had much greater enzyme activities than at pH 4.7 and 2.7. Rheological properties of sponges made with flours from x w which enzymes had been extracted did not change during fermenta- Flour C tion. The Theological changes returned when enzyme extracts were 0 added back to the sponges. Thus, proteolytic enzymes have I- important effects on sponge rheology. w Reconstituting a constant residue with enzyme extracts from SWW different flours showed that enzyme activity differed in different z flours. The proteolytic enzymes also exhibited different abilities to change sponge rheology when acting on their own flour residue. en Flour D This may be a useful indicator to determine flour quality and to Fn predict baking performance, as Rogers and Hoseney (1989) have \Flour D shown that dough rheology and baking performance are related. cic IW ~ % LITERATURE CITED

DOESCHER, L. C., and HOSENEY, R. C. 1985a. Saltine crackers: Changes in cracker sponge rheology and modification of a cracker 7 8 9 baking procedure. Cereal Chem. 62:158. 1 2 3 4 5 6 DOESCHER, L. C., and HOSENEY, R. C. 1985b. Evaluation of commer- TIME (Hr.) cial cracker flours by test baking and study of changes occurring during Fig. 6. Comparison of the change in Theological prop)erties of different sponge fermentation. J. Cereal Sci. 3:261. flours with short standing times at pH 4.1. SWW= soft white wheat, FARIDI, H. 1975. Physical and chemical studies of saltine cracker lengthy SRW= soft red wheat; flours B-D= commercial crackcer flours. fermentation. Ph.D. dissertation, Kansas State University, Manhattan. PIZZINATTO, A., and HOSENEY, R. C. 1980. Rheological changes in cracker sponges during fermentation. Cereal Chem. 57:185. the end of 18 hr of fermentation. Thus, 9 hr of reaiction time could ROGERS, D. E., and HOSENEY, R. C. 1989. Effects of fermentation on saltine cracker production. Cereal Chem. 66:6-10. be used to simulate cracker fermentation. SALGO, A. 1981. Study of wheat proteases. Ph.D. dissertation, Technical The differing activities of enzymes on a composeted residue flour University of Budapest. extracted from different cracker flours raised the question of how WU, J. Y., and HOSENEY, R. C. 1989. Rheological changes in cracker those enzymes would function on each flour's own* residue. As sponges during the first five hours of fermentation. J. Food Process. shown in Table II, after setting for 9 hr at pH 4. 1, sponges made Preserv. In press.

[Received July 15, 1988. Accepted January 6, 1989.]

Vol. 66, No. 3, 1989 185