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AGNES F. ZAMORA and MARION L. FIELDSDept. of Food Science & Nutrition

University of Missouri, Columbia, MO 65201

PRODUCTION OF CORN AND LEGUME MALTSFOR USE IN HOME

ABSTRACTAqueous extracts of amylases from germinated corn, cowpeas , chick-peas and Great Northern beans were made to determine t he optimumpH, temperature and time of incubation for their activity. Starchhydrolysis, measu red by starch-iodine reaction, and amount of glucoseliberated from soluble starch and from the seedstarcheswere measured.Alpha-amylase from corn had a lower pH and a higher temperatureoptima than cowpeas, chickpeas and Great Northern beans. The amyl-ase (total dextrinizing) activities measured at the optimum pH andtemperature were 18.4, 14.3 and 2.66 for corn, co wpeas and GreatNorthern beans respectively. Chickpeas had a very low alpha-amylaseactivity. Corn and cowpea malts are more practical to use in homefermentation because hey conv erted the starch to dextrin much faster.More reducing sugars were produced from the be an starches by cor nand cowpea malts than by chickpea and Great Northern bean malts.

INTRODUCTIONTODAY, a deficiency of animal proteins prevails in many partsof the world and this will become more serious as the growthin world population rapidly increases. Therefore, mor e plantproteins should be used to meet the required amount of pro-tein as already being don e in many developing countries.Because of availability, low cost and acceptability, corn andlegumes are an important source of protein and calories incountries where animal proteins are limited. However, it is wellrecognized that protein from these sources is of lower qualitythan those from animals.Because of the importance of corn and legumes as foodsources and with increased emphasis on the nutritive value offoods, better use of legumes for home preparations should bemade for the improvement of the diet and nutrition of thepeople (Patwardhan, 1962).Current research is being done to improve the amino acidbalance in corn and legumes (Hackler and Dickson, 1973). Onemethod which is worthwhile nutritionally and economically ismixing different plant protein such that the resulting combina-tion will give a good balance and recommended levels of aminoacid (Bandemer and Evans, 1963).Another possible method of improving the nutritive qualityof corn and legumes is by yeast fermentation of the flour andmeals of these materials. The starch from the corn and legumesshould be hydrolyzed to sugars which the yeast can metabo-lize. This yeast fermentation is a means by which the. proteinquality of corn and some of the legumes can be improved.Industrially, conversion of starch to fermentable sugars isfrequently done by commercially available amylases. But sincethe emphasis on this research was for home preparation, thecorn (Zea mays), cowpeas (Vigna sinensis), chickpeas (Cicerarietinum) and Great Northern beans (Phaseolus vulgaris) weregerminated for possible sour ces of alpha-amylase which can beused in home fermentation. Sprouting can be easily carried-outat home with minimum utensils and materials. Sprouting doesnot need sunlight or soil so it can be done indoors in anyseason.This work was instituted to determine the optimum pH,temperature and time of incubation for degradation of starchwith alpha-amylase produced in germinated corn, cowpeas,

FERMENTATION

chickpeas and Great Northern beans and measure the alpha-amylase (total dextrinizing activity).EXPERIMENTAL

Reduction of maltsCorn, cowpeas, chickpeas and Great Northern beans which we repurchased from the University of Missouri Central Food Stores wereused for this study. They were washed several imes in running waterand soaked overnight in tap water. Th e water was drained and the seedswere put in trays cover ed with wet paper towels. They were incubatedat 30C for 4 days. The s eeds were moistened everyday and pans ofwater were placed in t he incubator to prevent the seeds rom dehydrat-ing. After 4 days, when the sprouts were about 2 in. long, the seedswere dried at 50C and ground through 1 mm mesh screens n a WileyMill.Extraction of enzy me from germinated corn and beans

Aqueous enzyme extracts were prepared from ground malt by theprocedure of Tauber (1949) with t he following modifications: A 10%aqueous enzyme solution was prepared by constant stirring of thewater-ground malt mixture for 30 min at room temperature. The solu-tion was centrifuged at 2SG for 1.5 min in a refrigerated centrifuge(model B 20A, International Equipment Co., MA). The super natant wastaken as the enzyme extract.Reparation of buffered soluble starch solutionsA 2% soluble starch (Fisher Certified Soluble Starch for Iodometry)solution was made by boiling the st arch in each of the 9 buffers (pH 4to 12) prepared. For pH 4 to pH 8, the required amounts of O.lM citricacid and 0.2M Na,HPO, were mixed. To prepare pH 9 buffer, 0.25Mborax and O.lM HCl wer e used. Buffers of pH 10 and 11 were preparedby mixing 0.05M NaHCO, and O.lM N aOH. A 0.2M KC1 and 0.2MNaOH were used n preparing buffer of pg 12.Determination of pH and temperature optimaThe pH, temperature and time of incubation at which t he activity ofthe enz ymes was optimal were determined. Five ml of aqueous enzymeextract fr om each type of malted seeds were added to 10 ml of 2%buffered soluble starch solution at pH 5-12. For corn alpha-amylase,pH 4 was also tested. Incubation temperatures of 40, 50 and 60C for30, 60,90 and 120 min wer e tested.Determination of reducing sugars expressedas glucose)

The concentration of reducing sugars (calculated as glucose) wasdetermined as described by Dubowski (1962).Determination of starch-iodine reaction

Quantitation of the starch-iodine reaction wa s performed as de-scribed by Schwimmer (1947), with the following modifications: 5 mlof a solution of iodine in potassium iodide (O.OOSModine and 0.23Mpotassium iodide) was added to t he starch enzyme solution after incu-bating at different temperatures and times. One ml of this was pipettedinto 10 ml distilled water. The transmittance was read at 660 nm in aBausch and Lomb Spectronic 20 using distilled water to set the instru-ment to 100%.Production of starches rom b eans and corn

The ungerminated seedswere ground through 1 mm mesh screen na Wiley Mill. Water (v/w, 10x) was added to the ground seeds andstirred constantly f or 30 min. The mixture was strained through cheese-cloth to remove large pieces, and the filtrate was centrifuged at 2.5G for15 min in a refrigerated centrifuge (Model B 2OA, International Equip-ment Co., MA). The precipitate was dried at 50C.Hydrolysis of the seed starches

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peas and Great Northern beans were prepared by boiling the seedstarch-buffer mixture. Enzyme hydrolysis of the seed starches wastested at the optimum pH, temperature and time of incubation deter-mined from the pure starch-buffer systems.The starches from the four types of seedswere hydrolyzed by auto-claving at 121C for 60 min in 0.5% H, SO, to determine the maximumamount of reducing sugars which could be formed. This was used as acontrol treatment. The starch-water mixtures were boiled to gelatinizethe starch so that the starch was made available for hydrolysis with thealpha-amylases. The amount of reducing sugars liberated from thestarches n all tr eatments was determined. The reducing sugars presentin the malt extract were measured to correct for the amount deter-mined from the hydrolysis of starch by the enzyme. As another con-trol, the amount of r educing sugars in the nontreated starch wasmeasured.The percent effectiveness of the malt was calculated as follows: theamount of reducing sugar in t he starches was determined. This amountwas subtracted from the acid- and enzyme-treated starch. Since it wasassumed hat the acid-treated starch would yield the maximum amountof reducing sugar, the enzyme-treated starch reducing sugar values weredivided by the acid-treated starch reducing sugar values, multiplied by100 to yield the effectiveness of t he malt at its optimum conditions.Determination of alpha-amylase (total dextrinizing activity)of corn and legume maltsThe reagents and procedure followed were that of Tauber (1949),with the following modifications: the 5% aqueous solution of malt wasfiltered and 5 ml of the filtrate was mixed with.10 ml of 2% solublestarch solution in a test tube. The starch was buffered at pH 5 for corn,pH 7 for chickpeas and pH 8 for cowpeas and Great Northern beans.These were the pH l evels found optimum for the corn and l egumes. Thetubes were incubated at 40C for chickpeas, cowpeas and Great North-ern beans and 50C for corn. At different intervals of time, 1 ml of themixture was added to 5 ml of dilute iodine solution. This procedurewas repeated until the color of the tube matched the standard.Calculation. In this experiment, 1 ml of 2% starch was hydrolyzedby 0.5 ml of the 5% malt extract. If hydrolysis of starch to dextrinoccurred in c min, 1 ml of malt extract will convert in 1 hr 60/c xl/O.5 ml of starch solution.

RESULTS & DISCUSSIONEffect of pH, temperature and time of incubationon amylase activitiesAlpha-amylase from corn showed maximum activity (bothby color of iodine-starch and amount of reducing sugar) at pH5 when incubated at 50C after 90 min incubation (Fig. 1).This closely agrees with the results of Wahl (197 1) who con-cluded that the maximum activity was at pH 5.2. Greenwoodand Milne (1968) also said that the optimum activity of alpha-amylases from higher plants is between 5 and 6. It was ob-

Fig. 1 Starch hydrolysis (blue value-reductio n of starch-iodinereaction measured as percent transmittance) and amount of reduc-ing sugars (expressed as glucose) produced by corn alpha-amylaseafter 90 min incubation at different pH and temperature combina-tions.

served in this study that the optimum pH for the corn amylaseactivity was near the pH of the ungerminated corn which was5.2 and corn malts which was 5.5. There was a slight decreasein activity at pH 4. At 40 and 50C, t he lowest activity AS Ooccurred at pH 7-l 1. The activity at 60C appeared t o belowest at all pH levels.The effect of other incubation time was not shown in thegraph because it did not seem to affect the enzyme activity.The readings were almost the same at different incubationtimes at the particular pH and temperature.Figure 1 also shows the amount of reducing sugar producedat different pHs and temperatures of incubation. The amountof reducing sugar was highest at pH 5. Production of reducingsugar reflected the hydrolyzing power of the amylase at pHvalues and temperatures tested. The starch-iodine and reducingsugar tests complemented each other.The activity of cowpea alpha-amylase gradually i ncreasedfrom pH 5 to pH 8 with optimum at pH 8-9 as measured bystarch-iodine reaction (Fig. 2). This optimum is above thenormal pH of the ungerminated cowpeas and malt which was6.2. As the temperature was increased from 40 to 60C, moreinactivation of amylase probably occurred. This was reflectedmore at pH values between 5-9 than at higher pH levels withthe starch-iodine reaction. The amount of reducing sugarproduced at 40C, however, was not the maximum. More re-ducing sugar was detected at 50C at pH 8 than at other pHvalues.The optimum pH for the activity of chickpea al pha-amylasewas at pH 7 at 40C, although there was only a slight decreasein activity at pH 8-12 as measured by the starch-iodine reac-tion. In contrast, there was a rapid increase in activity from pH5 to 7 (Fig. 3).Much more reducing sugar was produced at 40C regardlessof pH than at 50 or 6OC. At 40C more reducing sugar wasproduced at pH 7 than at other pH units; the least reducingsugar was produced at pH 5. Quantities of reducing sugar liber-ated were nearly constant at 50 and 6OC (Fig. 3).The behavior of alpha-amylase from Great Northern beanswas similar to that of chickpeas and cowpeas (being leastactive at pH 5 and more active at pH above 7). The alpha-amylase from these beans was the only one that was affectedby time of incubation. At the temperatures tested, 120 minincubation was found suitable for this enzyme. At 120 minincubation and 40C, regardless of pH, there was much more

Fig. l-Starch hydrolysis (blue value-reducti on of starch-iodine reac-tion measured as percent transmittance) and amount of reducingsugars (expressed as glucose) produced by cowpea alph asmylaseafter 90 min incubation at different pH and temperature combina-tions.

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Table 3-Main effects of storage time, storage temperature, flow rateand blanch methods on sensory quality of canned snap bean@

Main effectsAbsense of General

Color browning appearance Flavor

a. Storage time ihr)0 8.5a 8.0a24 7.6b 7.8a

48 5.8~ 6.7bb. Storage temperature (Cl

13 8.3a 8.2a21 8.0a 7.8a29 7.2b 7.2b38 5.6c 6.7~

c. Flow rateslow 7.8a 7.9afast 6.7b 7 .Ob

d. Blanch methodwater 7.4a 7.4asteam (rotary) 7.2a 7.5a

8.6a 7.3a7.5b 7.2a5.2~ 6.8b

8.4a7.8b7 .oc5.3d

7.6a7.6a7 .Ob6.3~

7.7a6.5b

6.3b7.8a

7.2a7 .Oa 7.la7.la

aSensory ratings made on a scale of 1 (poor) to 10 (best) by atrained panel, comparing each sample against a standard reference.

b Means separated within main effect and columns followed by thesame letter or letters not significantly different at the 5% level by

Duncans multiple range test.

respiration. High CO z in beans stored at slow flow rates is anindication of anaerobic conditions (Fig. la).In conclusion, holding snap beans after harvest in loadedtrucks for 24 hr or longer is detrimental to quality, especiallyat temperatures above 21C. Snap beans held at the highertemperatures under fast flow rates of air had higher respirationrates, shear p ress values and weight losses as well as higher %seed, fiber, hemicellulose and cellulose. Fast air flow acceler-ated browning although color was not affected at lower tem-peratures. Extending the storage time of beans magnified thechanges in quality. Beans stored under low air flow, which wascomparable to conditions found in loaded trucks, lost lessweight during storage and were lower in % fiber, seed, hemi-

celluIose and cellulose but the canned beans were off-flavored.Therefore, movement of air through loaded trucks during longperiods of holding is necessary to prevent anaerobi c condi-tions. High COa atmospheres are beneficial in the inhibition ofbrowning when adequate air circulation is provided (Reitmeierand Buescher, 1975). Holding the beans at temperatures below21C was less detrimental to quality than those held at highertemperatures.REFERENCES

Freeman, D. and Sistrunk. W.A. 1973. Comparison of steam and waterin the blanching of snap beans. Ark. Farm. Res. 22(5): 11.Groeschel. E., Nelson. A.I. and Steinberg, M.P. 1966. Changes in colorand other characteristics of green beans stored in controlled refrig-erated atmospheres. J. Food Sci. 31: 488.Guyer, R.B. and Kramer, A. 1950. Factors affecting the qualitymeasurements of raw and canned green and wax beans, a pre-liminary report. Proc. Amer. Sot. Hort. Sci. 56: 303.Henderson, J.R. 1977. Studies on the control of broken-end discolora-tion in snap bean pods during post-harvest handling. Masters thesis,University of Arkansas, Fayetteville.James. W.O. 1953. Plant Respiration. Oxford at the Clarendon Press.Great Britain.Kidd. F. 1917. The controlline influence of carbon dioxide. 3. Theretarding effect of carbon djoxide on respiration. Proc. ROY. Sot.B89: 136.Kramer, A., Haut, I.C. and Scott, L.E. 1949. Objective method formeasuring quality factors of raw, canned, and frozen asparagus.Proc. Amer. Sot. Hort. Sci. 53: 411.Lieberman. M.H., Schemer, A. and Ezell, B.D. 1950. Effect of storagetemperature and type of film on weight loss, package atmosphere,decay, ascorbic acid, content and salability of prepackaged greenbeans. Pre-Pack-Age 4(4): 24.Lutz. J.M. 1938. Evaluating quality changes in certain vegetables afterharvesting. Proc. Amer. Sot. Hort. Sci. 36: 754.Parker, M.W. and Stuart, N.W. 1935. Chang es in the chamical composi-tion of green snap beans after storage. Md. Agri. Expt. Sta. Bull.383.Reitmeier, C.A. 1975. Physiological studies of brown end discolorationin snap bean pods. Masters thesis, University of Arkansas, Fayette-tie.Reitmeier, C.A. and Buescher, R.W. 1975. Control of brown-end dis-coloration of snap beans. Ark. Farm Res. 24(l): 12.Sistrunk, W.A. 1965a. Influence of post-harvest storage of snap beanson chemical and physical changes during canning. J. Food Sci. 30:34nIS .Sistrunk. W.A. 1965b. Effect of storage time and temperature of freshsnap beans on chemical composition of the canned product. Proc.Amer. Sot. Hort. Sci. 86: 380.USDA. 1972. United States Standards for grades of canned green beansand canned wax beans. FDA title 21, part 51.11.MS received 4111177; revised 7118177; accepted 7125177.Snap beans for the study were graciously supplied by Steele CanningCo. and Allen Canning Co.Use of commercial brand names does not signify endorsement bythe University of Arkansas.

PRODUCTI ON OF CORN AND LEGUME MALTS. . .From page 207Higher pH optima were observed for cowpeas, chickpeas andGreat Northern bean alpha-amylases. These enzymes weremore active at 40C than at 50 or 60C. Except for GreatNorthern bean amylase, the time of incubation did not seemto affect t he total activity of the enzyme.The total dextrinizing activities for corn and cowpeas weremuch higher than f or Great Northern beans and chickpeas,which would make them more suitable in hydrolyzing starchesfrom various sources for use in home fermentation.

REFERENCESAllen, W.G. and Spradlin, J.E. 1974. Amylases and their properties.Brewers Digest. 49(7): 48.Bandemer, S.L. and Evans. R.J. 1963. The amino acid composition ofsome seeds. Ag. Food Chem. ll(2): 134.Dubowski. K.M. 1962. An o-toluidine method for body-fluid glucosedetermination. Clinical Chem. 8: 215.Greenwood, C.T. and Milne, E.A. 1968. Starch-degrading and synthesiz-ing enzymes: A discussion of their properties and action pattern. In

Advances in Carbohydrate Chemistry, Vol 23, p. 281. AcademicPress, New York.

Hackler. L.R. and Dickson, M.H. 1973. A comparison of the amino acidand nitrogen content of pods and seeds of beans (Phaseolus vulgarisL). Search Agriculture. 3(5): 1.Patwardhan, V.N. 1962. Pulses and beans in human nutrition. Am. J.Clio. Nut. 11: 12.Pfisterer. E. 1974. Enzymes in fermentation. MBAA Technical Quar-terlY:ll: 9.PAG Bulletin. 1973. PAG Statement (No. 22) on upgrading humannutrition through the improvement of food legumes. 3(2): 1.Schivimmer, S. 1947. Purification of barley malt alpha-amylase. CerealChem. 24: 35.Tauber. H. 1949. Chemistry and Technology of Enzymes. J. Wiley,New York.Thoma. J. and Spradlin, J. 1971. Plant and animal amylases. In TheEnzymes, Ed. Bayer. P. Vol 5. p. 115. Academic Press. New York.Wahl, G. 1971. Biochemical-technological studies on wet-processing ofmaize. 5. Amylolytic enzyme system of maize. (in German).Staerke. 23: 85. [In Chem. Abstr. (1971) 75(l)].Whitaker, J.R. 1972. Enzyme cofactors. In Principles of Enzymologyfor the Food Sciences. Vol 2, p. 369. Marcel Dekker, Inc., NewYork.Ms received 3/19/l?; revised 5126177: accepted 613171.

Contribution from the Missouri Agricultural Experiment Station.Journal Series No. 7629.

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