17
Sugar Formation by Diastatic bnzymes of Flour -
H. C. GOREAKD S. J ~ Z S AFleischmann , Laboratories, Standard Brands, Inc., New York, N . Y. PREVIOUS INVESTIGATIONS C O M M E R C I A L FLOC RS were found to
L
liquefying and s a c c h a r i f y i l l g p o w e r that permit the rapid and exact estimation of t h e s e components of diastase, and to relate, if possible, these enzyme activities to the sugar-forming power of flour as determined by the Rumsey method. It w a s n e c e s s a r v to s t a t e the diastatic activitik of flour in terms of grams of the substrates, starch paste, and soluble starch, used in the two methods, converted by 1 gram of the enzyme preparation. By comparing the values found with the sugar formed during saccharification in the flour by the Rumsey method, the three activities could be compared. I n the liquefying method the activity of the enzyme is measured directly in terms of substrate. The liquefying power is there defined as the weight of dry starch, in the form of a special starch paste, liquefied a t 21" C. in 1 hour a t 4.8 pH by 1 part of active substance. For example, the liquefying power of a distiller's malt was found to be 128. I n making the measurement, the starch paste containing 4.211 grams of dry starch per 100 grams lost 64.9 per cent of its viscosity when 150 grams of the paste were digested for 1 hour a t 21" C. a t 4.8 p H with 15 cc. of malt infusion, each cc. of which represented 1 mg. of malt. This loss in viscosity, represented 1.28 grams of dry starch liquefied in 100 grams of paste per hour by 10 mg. of malt infusion. Thus,
contain both liquefying and saccharifying enzymes. The addition of s n z d quantities of salt Or Papain to %floursusPensior@ greatly increased the amounts of soluble liquefying and saccharifying diastase. The presence of neither salt nor papain appreciably changed the rate of sacchari$cation in $our.
INTNER m e t h o d s plied t o f l o u r d o n o t give very significant results, a c c o r d i n g t o R u m s e y (21). Rumsey thinks that flour showing greater Sugar-fOrming p o w e r s h o u l d s h o w greater itrength and consequently greater baking value, proyiding the quality and quantity of gluten are relatively the same. He proposed a method in which the diastatic power was expressed in terms of the reducing sugar, calculated as maltose, formed when all the flour in water suspension was used to autolyze under gil en conditions. -illthough this niethod has been of great value in studying saccharification in flours, the relationships between the diastatic activity, as determined by Lintner methods, and saccharification remain unknown. The earlier literature leads to the belief that the liquefying power of diastase is responsible for the direct action on the raw starch of the flour. Brown and Morris (3) state, "We have always found the power to liquefy starch paste and to erode the starch granule to go hand in hand. A diastase which will liquefy starch paste will, under favorable conditions, also dirintegrate the starch granule and vice versa. It is always safe to predict that a non-liquefying diastase will not attack the starch granule by pitting and disintegration, and that a diastase which will not so act on the starch granule will not liquefy starch paste." Baker and Hulton ( 1 ) found a n abundance of the saccharifying enzyme of diastase in flour, but they were unable to obtain the expected correlation between the diastjitic power and sugar formation in doughs. The value of malt, however, in causing increased sugar for gas formation JTas clearly recognized. They found that as little as 0.25 per cent of malt (50 mg. for 20 grams of flour) produced a marked effect. They believed that many flours have an inadequate supply of the liquefying enzyme of diastase, and that such flours should be supplemented with malt. Olson (20) held that low gas production in a weak flour is caused by an inadequate supply of starch liquefying enzyme. Ford and Guthrie (6)observed the effect of tht: duration of extraction on the diastatic activity of flour as measured by the action of the filtered extract on soluble starch solutions. When the period of standing a t 18" C. was increased from 10 minutes to 2 hours, the activity of the filtered 4 per cent suspensions decreased markedly. These investigations found that while their results were somewhat inconsistent, not surprising as the p H relations of diastase (12) were not known until 1915, various substances (glycine, gelatin, certain proteases, primary and secondary phosphates, and potassium chloride) greatly increased the diastatic power when added to the water in vhich the flour mas digested. Similar effects mere obtained n-ith barley flour ( 7 ) . LIQUEFYING AND s ICCHARIFYING P O W E R The primary object of the studies reported in this paper was to apply to flour two new methods (8, 9) for measuring DETERMIIN I T I O N O F
L, P. = 1.28 - = 128 0.01
A substance, then, is defined as having a liquefying p mer of 100 when 1 part will liquefy starch paste under standard conditions a t the rate of 100 times its weight of dry starch per hour. Statement, in terms of substrate, of the activity of the wccharifying enzyme was much more difficult. The starch present in the special solution of soluble starch used in the niethod, in all probability, is not split quantitatively into maltose. The nearest approach to the correct solution of the problem is believed to be, to define the saccharifying power in terms of rate of formation of reducing sugar, calculated as maltose, formed when the diastase acts on the special soluble starch under the standard conditions specified in the polarimetric Lintner method (9). Here the following expression was used in calculating th(. activity from the fall in polarization observed: L =
IT-here L
=
I1 =
t I
= =
4.6 =
100 D t
x I x
4.6
degrees Lintner fall in polarization measured on sugar scale at 20" C. time in hours length of tube in decimeters constant determined experimentally
The rate of reducing sugar formation in the polarimetric niethod was determined by the following procedure: Add Fehling solution, or Fehling solution and water, to a mixture of 50 cc. soluble starch solution and 5 cc. malt infusion a t the end of different digestion periods. Determine reducing
99
100
INDUSTRIAL AND ENGINEERING CHEMISTRY
substances, reckoned as maltose, by the Munson and Walker method. Determine the polarization declines in a parallel experiment, using the same procedure. Mix 50-cc portions of standard starch solution at 21' C. with 5-cc. portions of malt infusion also at 21' C., counting time from the moment when the first of the starch solution reaches the malt infusion. After incubating for the desired period at 21' C., stop enzyme action by adding Fehling solution. Calculate the quantities of Fehling solution and of water added so that the 100-cc. portion later removed and boiled represents a known aliquot containing 50 cc. of Fehling solution. The dilutions used are given in Table I. TABLEI. DILUTIONS USED STARCH SOLUTION MALTIN- INCUBATICYN FEHLING TIME SOLUTIOX WATER MALT FUSION CC. Cc. Minutes cc* cc
.
TOTAL VOLUME
cc*
The quantities of reducing sugars, as maltose, and the polarizations, are given in Table 11. SUGARS AND POLARIZATIONS TABLE11. REDUCING niaLT08E
FORMED
Vol. 24. No. 1
power is expressed in terms of enzyme and apparent sugar formed from substrate. Under the conditions of the two methods a malt having a liquefying power (L. P.) of 100 will liquefy starch paste a t the rate of 100 times its weight per hour and 1 gram of a malt having a Lintner value of 100 will produce sugar from soluble starch a t the rate of 7.029 grams of reducing substance, calculated as maltose, per hour. I n using the liquefying power method, 10 grams of flour were mixed with 100 cc. of water for 1 minute by a highspeed laboratory stirrer. The flour suspension was allowed to stand for 1 hour a t laboratory temperature, with occasional mixing, and poured on a folded filter. (This method of preparing the infusion differs from that used in preparing the infusion in case of malt, which is about 100 times more active.) Then 15-cc. portions of the filtrate were mixed with 150 grams of the standard starch paste and the viscosity was determined. I n the application of the polarimetric Lintner method the infusions of flour and water were made up in substantially the same manner, except that the high-speed stirrer was not used. Twenty-five grams of flour were shaken with 250 cc. of water in Erlenmeyer flasks, let stand for an hour, and filtered. I n each determination 5-cc. portions of the filtrate were used.
PER
10
v.
(4-DCM.
TUBE)
INCOBATION
TIME Cur0 Minuter Gram 0 0.1526 16 0.3566 30 0.3851 45 0.3850 60 0,3407
hfhLTOSE
Gram 0.1189 0.2803 0,3027 0,3027 0.2678
TOTAL MALTOSE
Grams 0.1308 0.7008 1.2108 1.5135 1.6068
POLARIDEZATION POLARICLIXF, MAL- (20° C. ZATIOK IN TOSE 4-DCM. DE- POLARIZAFORMEDTUBE)CLINES TION Grams * V. V. Gram 90.0 6:O 0:096 0:6700 8 4 . 0 11.2 0.096 1.0800 78.7 14.1 0.098 1.3827 75.9 15.1 0.098 1.4760 74.9
The results in Table I1 show that the development of reducing power, calculated as maltose, during the early stages of digestion of soluble starch by malt diastase closely parallels the declines in polarization. The percentage of starch solids was determined by drying 10-cc. samples of the starch solution used in this work in vacuum a t 70" C. before the buffer solution was added. The dry matter was 4.737 grams per 100 cc. After the buffer (2 cc. per 100 cc.) was added the dry starch present, calculated from the foregoing figures, was 4.427 grams per 100 cc. The results are shown graphically in Figure 1. The quantities of reducing substance calculated as maltose formed per degree of polarization decline during the first 15- and 30-minute intervals, when these declines were nearly linear, were 0.095 and 0.096 gram. If the average value, 0.0955, be accepted as correct, the saccharifying power determined by the polarimetric method can be stated in terms of rate of formation of reducing sugars. When 5 cc. of a n infusion of diastase equivalent to 250 mg. of a malt of 100" Lintner acts on 50 cc. of a soluble starch containing 2.213 grams of starch solids, under standard conditions (21" C. and 4.8 pH), the time being so chosen that the fall in polarization with respect to time is linear, the rate of fall of polarization measured in a 4-dcm. tube is 18.4" V. per hour. This corresponds t o the formation rate of 1.757 grams (18.4 X 0.0955) of reducing carbohydrate calculated as maltose. Thus, 1 gram of malt, or other substance, of 100" L. acting on soluble starch under the conditions of the polarimetric method produces reducing substances a t the rate of 7.029 grams of reducing sugar as maltose per hour. When determined by the polarimetric method, then, saccharifying
lb
45
30
T I N E I h WINUTES OF jIGESTlffi A? 2:'
60
C.
FIGURE1. RELATION BETWEEN FALLIN POLYMERIZATION AXD REDUCING SUGARFORMEDDURING DIGESTION OF STARCH BY MALTDIASTASE (POLARIMETRIC LINTNER METHOD)
These two methods m r e applied to the flours with and without the addition of salt. The salt (2.5 per cent and 2.0 per cent of the flour, in case of the liquefying and saccharifying powers) was dissolved in the water used in mixing. Sugar formation in the flours was determined by a slight modification of the Rumsey (11) method, the details of which follow: Mix 25 grams of flour with 241.5 cc. of water a t 27" C. and let stand for 1 hour, with occasional mixing, at 27" C. Add 7.5 cc. of a solution of sodium tungstate contaling 15 grams per 100 cc. and 1 cc. of concentrated sulfuric acid. After thorough mixing filter the suspension and determine the reducing sugar on 50-cc. portions of the filtrate by the Munson and Walker method. Following Rumsey's suggestion, add to the Fehling
January, 1932
IRDUSTRIAL AND ENGINEERING
CHEMISTRY
101
AND SICCH.UUFYING POWER OF 11 FLOURS TABLE111. LIQUEFYING
LIQUBFYINQ POWER
SACCHhRIFYINQ P O W E R
2.5%
2t
N o salt present
TYPEOF FLOUR
salt present
In- No salt orease present present
% Patent from choice hard spring wheat Short patent from hard winter Kansas wheat Same as No.2, unbleached Northwestern flour from spring Montana wheat First patent from blend of sprlng and Kansas wheat Patent from northern s ring wheat First clear flour from %ansa8 hard winter wheat (the dear flour from the same wheat from which No. 2 was made,' Very short patent soft winter wheat flour Patent from mixture of high-protein Kansas wheat Cake flour from soft winter wheat 11. Whole wheat flour from Northern hard apring wheat Average of flours 1 t o 10 a Determined by Rumsey Method ( 1 1 ) .
1. 2. 3. 4 5: 6 7: 8 9: 10.
0.950 1.220 1.240 1,000 1,100 1.097
26 56 46 39 47 29
26 23 29 27 35 26
0.855 0.840 0.745 0.915 1.550 0.804
1.125 1,100 1.050 1.240 1.700 1.11
31 31 41 35 9 38
56 35 24
Rumsey used a 1: 10 ratio of flour to water :md centrifugalized instead of filtering the suspensions. Table I11 shows the liquefying power of 11 commercial flours, the saccharifying power of the flours, and the sugar present in 1: 10 flour suspensions before and after incubation by the Rumsey method. I n all cases filtrates of the flour suspensions were used. The results in Table I11 show that all the flours possessed high liquefying and saccharifying power. Omitting the whole wheat flour, the average liquefying power of the flour filtrates was 0.804. In the presence of 2.5 per cent of salt this increased to 1.11. On the average, enough power to liquefy 80.4 grams of starch paste, or 111 grams of starch paste in the presence of salt, under the standard conditions of the method, was present in each 100 grams of flour. Similarly, the average saccharifying power of the 10 flour filtrates was 33.1" L. without salt, and 88.4" L. with salt (2 per cent of the flour). This activity corresponds to the power of forming 240 and 621 parts of sugar as maltose, under the conditions of the method, from every 100 parts of flour. The average formation of reducing sugar as maltose a t 27" C. in the 10 flours was only 1.20 per cent. S o correlation was apparent between either the liquefying or saccharifying power and saccharification. For example, flour KO. 4 (L. P., 0.716) developed more sugar, 1.496 per cent, than flour KO.10 (L. P., 0.915), which formed 1.005 per cent of sugar. Again, flour No. 7 (56" L.) formed less sugar than flour No. 4 (27" L.). The effect of the salt on the liquefying and saccharifying powers of the flour filtrates was striking. Increases of 26 to 56 per cent (average, 38 per cent) in liquefying power were observed. The saccharifying powers were affected to a much greater extent, the increases ranging from 60 t o 273 per cent (average, 160 per cent). I n case of the whole wheat flour, the increases in liquefying and saccharifying powers of the filtrates due to salt were much less (9 and 18 per cent). EFFECTOF PAPAIN AND SALTON LIQUEFYING ASD FYING
OL.
0.750 0.780 0.845 0.715 0.747 0,850
solution enough concentrated soda solution to neutralize the 0.2 cc. of sulfuric acid present in 50 cc. of filtrate. In case of the checks (determinations in which the reducing power of the original flours is measured) mix each 25-gram sample with a solution consisting of 241.5 cc. of water, 7.5 cc. of tungstate solution, and 1 cc. of sulfuric acid.
QACCHARI-
POWER
The remarkable effect of malt in increasing sugar formation in doughs shown by Baker and Hulton ( I ) has been repeatedly observed by others (4, 5 ) . Ford and Guthrie (G,7 ) showed the influence of papain in increasing the solubility of the diastases of wheat and barley flour. Baker and Hulton ( 2 ) reported a n extended investigation of the attack of precipitated malt diastase on raw barley starch. To determine whether or not the rate of attack of the
Increase
62
77 34.1
97 73 75 80
96 81 89 80
87
116 91 88.4
REDUCINQSUB3TANCB MALTOSE"
AS
Maltose Inou- apparently bated formed
Check
%
%
%
273 218 159 196 191 211
1.083 0.881 0.844 1.092 1.155 1.154
1.835 1.989 2.397 2.588 2.744 2.678
% 0.752 1.108 1.553 1.496 1.589 1.524
60 129 262 87 18 160
1.020 0.997 1.413 0.907 1.496 1.055
2.018 1.945 2.437 1.912 2.693 2.254
0.998 0.948 1.024 1.005 1.197 1.200
enzymes of malt on the starch of flour could be increased by the presence of papain or salt, sugar formation in flour suspensions containing added diastatically active malt sirup was measured. Hard flour KO. 6 and soft flour No. 8 were used. The amount of malt sirup (Lintner) used was 4 per cent of the flour; of papain, 0.25 per cent; and of salt, 2 per cent. I n each experiment malt sirup alone, nialt sirup and papain, and malt sirup and salt were dissolved in water to a total volume of 241.5 cc., warmed to 27" C., and mixed with 25 grams of flour. The reducing substances formed upon incubating for 1 hour at 27" C. were determined by the modified Rumsey method. The results are given in Table IY.
--
TABLEIv. REDWING SUBST4NCES AS REDUCING SUGAR3
Checks (not SIRUP PAPAIN SALT incubated) Gram Gram Gram Grams ,MALT
Incubated Crams
AMMALTOSE
AS
Increase Grams
MALTOSEIncrease due t o papain and to papain and mlt Gram
HARD FLOUH NO, 8
0 1 1
1 1
0 0 0,0625 0 0.0625
0 0 0
0.5 0.5
0.34 3.08
..
1.68 2.82
1.14 7.72 7.90
1.33 4.74 4.07 4.64 4.82
0 0
1.03 3.48 3.85 3.08 3.65
0:37
0.08
S O F T F L O U R NO. 8
0
0
0
1 1 1 1
0 0.0625 0 0.0625
0 0
0.5 0.5
0.27 3.05
..
.. ..
1.30 6.52 6.89 6.13 6.69
.. 0
0.17
The remarkable effect of diastatic malt sirup on sugar formation in flours is clearly brought out by the results in Table IV. The hard flour formed 4.74 grams and the soft flour 3.48 grams of reducing substances reckoned as maltose. Seither papain nor salt, alone or together, appreciably increased the rate of attack of the diastatic enzymes present on the raw starch of the flours.
CONCLUSION^ Neither the liquefying power nor the saccharifying power of diastase, using starch paste or a solution of soluble starch, has any direct bearing on the effective diastatic properties of flour. The idea that the liquefying power is concerned directly with the attack of diastase on the raw starch of flour therefore must be abandoned. The work here reported shows that commercial flours have both types of diastatic power in excess. It shows also that the presence of salt or papain in flour suspensions effects no substantial changes in the rate of the attack on the raw starch of flour. The effect of malt extracts on sugar formation found by previous investigators was confirmed. kKNOWLEDGMENTS
All sugar determinations were made by G. E. Miller, H. J. Steele, and Q. Landis of the Fleischmann Laboratories. The writers also wish t o express their gratitude to C. N. Frey for his suggestions.
INDUSTRIAL AND ENGINEERING CHEMISTRY
102
LITERaTURE C I T E D
(1) Baker and Hulton, J. SOC.Chem. ind., 27, 368 (1908). (2) Baker and Hulton, J . Chem. SOC.,105, 1529 (1911). Brown and Morris, I b i d . , 57, 508 (1890). Collatz, A m . inst. Baking, Bull. 9 (1922). Collatz and Racke, Cereal Chem., 2, 213 (1925). Ford and Guthrie, J. SOC.Chem. Ind., 27, 389 (1908). (7) Ford and Guthrie, J . 1 m t . Brewing, 14, 61 (1908). (8) Gore, IND. ESG.CHEM.,20, 865 (1928). (3) (4) (5) (6)
(9) (10) (11) (12)
Vol. 24, No. 1
J6zsa and Gore, ibid.. Anal. Ed.. 2. 26 (1930). Olson, Wash. Agr. Expt. Sta., &U.' 114.' Rumsey, Am. Inst. Baking,Bull. 8 (1922). Sherman, Thomas, and Baldwin, J . A m . Chem. Soc., 41, 231 (1919'.
RECEIVED July 13, 1931. Presented before the Division of Agricultural and Food Chemistry a t the 79th Meeting of the American Chemical Society, Atlanta, Ga , April 7 to 11 1930.
Diastatic Enzymes in Certain Foods H. C. GORE. ~ N DS. JO/zs.&,Fleischmann Laboralories, Standard Brands, Znc., ;Vew York, N . Y
A
LTHOUGH the power to liquefy starch paste is usually considered a specific function of diastase, only recently has it been possible readily to measure this property with a satisfactory degree of exactness. The new method (5) is rapid and exact, and its results are expressed directly in terms of substrate (anhydrous starch) and enzyme. Thus, a liquefying power of 100 means that 100 parts of dry starch in the form of standard starch paste are liquefied by 1 part of enzyme sample under the prescribed conditions. The object of the work here reported was to obtain an idea of the distribution of the diastatic enzymes, especially the starch-liquefying enzyme, in various food crops, a t the same time measuring the influence of salt. I n determining the liquefying power of the samples, 20 grams of fine-ground seeds or 50 grams of fine-ground vegetables were digested with 100 cc. of water alone and with 100 cc. of water containing 5 grams of sodium chloride per liter for 1 hour at room temperature. The solutions were then filtered and the liquefying power was determined in the usual manner. TABLE
I.
STARCH
LIQUEFYIVG Ahiu S 4 C C H A R I F Y I V C SEEDS
PORER OF
S i C C H i S I t YISG
SEED^
LIQUEFYING Pon E R Wlthout Wlth NaCl NaCl
POWER Without With NaCl NaCl 0 L. L. 121, 115 Inactive Inactive Inactive Inactive Inactive Inactive Inactive Inactive Inactive Inactive Inactive Inactive 76 6 !1 2 67 64
4.36 4.00 Soy beans, Mammoth Yellow 0.90 1.15 Cow peas black-eye 0.30 1.00 Peas Hedderson's New Jubilant 1.25 0.10 Bean's Scarlet Runner, pole 2.10 1.25 White'lupines 3.20 3.60 Lentils 1.35 0.90 Japanese buckwheat 4.35 4.60 Kafir corn 3.20 2.60 Rioe Blue Rose" 2.76 2.10 Field corn, New Eureka. Dent 2.10 1.65 Field corn, Flint Longfellow 2.35 1.40 Sweet corn Golden Bantam 0.25 0.65 Sweet corn: Stowell's Everareen 5.75 7 80 Spring rye 2 30 0.90 Oats, Storm King 5,40 3.45 Barley ... 160 90.0 164.0 Malted barley 96 5 15 78 3.40 Wheat, spring, Marquis a Seed rice, supplied by Chambllss, U 9 Department of .Igriculture
In determining the saccharifying power, 5 grains of fineground sample were digested for 1 hour a t room temperature with 100 cc. of water alone, and, if the sample showed diastatic activity, with 100 cc. of water containing 0.5 gram of sodium chloride, the procedure already outlined by the writers (-?) was followed. The products used were not previously sterilized, nor Tvac toluene or other preservative used during these digestions. It is possible, therefore, that some of the activities measured were due to microiirganisms. The results are given in Tables I and 11.
All the seeds and vegetables showed marked liquefying action of starch paste, varying from 0.10 for Scarlet Runner beans t o 5.75 for rye. The high liquefying activity of unmalted rye is interesting because malted rye is known ( I ) to have the highest liquefying power recorded for malted grains. I n all products tested, except cow peas, the presence of salt increased the liquefying power. With potatoes this increase was only slight (0.26 to 0.42). With other materials it was very great, the liquefying power of Scarlet Runner beans, for example, increasing from 0.1 to 1.25. Of the legumes listed, soy beans alone exhibited any saccharifying poaer. Here, however, the activity found, 121' L., was of the same order as that of barley and malt. Buckwheat, kafir corn, field and sweet corn, and rice were substantially lacking in saccharifying power, and oats showed only 1" L. Salt increased the saccharifying power in all the active materials except that from soy beans. The results as a whole reveal a n ability to liquefy starch paste widely distributed in food plants. Whether this liquefaction is the action of a phosphatase (6) or a necessary step in the digestion of starch by the saccharifying enzymes of plants and animals is nat known. This function of diastase is of great importance in the digestion of starch because of the enormous quantities of starch metabolized by plants and animals. The cause of the influence of salt is unknown. LIQUEFYIXG AND SACCHARIFYING POWER OF TABLE 11. STARCH VEGETABLES TEGET~BLES Jersey potatoes Turnip rutabaga Parsnips New potatoes Carrots a S o activity.
LIQUEFYINQ POWER S4CCHARIFYINQ POWER Without With Without With NaCl NaCl NaCl NaCl L. L. 1.48 2.14 36 !l .. 2.64 2.84 1.10 1.60 .. 0.26 0.42 .. 0.40 0.78
From the data here given the saccharifying power seems to be far less widely distributed in plant products than the liquefying power of diastase.
LITERATURE CITED (1) Chrzaazcz, T., Wochschr. Brau., 30, 538 (1913). (2) Effront, "The Enzymes and Their Application," 1901. (3) Gore, ISD.E x G . C H m f . , 20, 865 (1928). (4) Hesse, "Enzyrn. Tech. Gahrungs Ind.," IS (1929). ($5) J6zsa and Gore, ISD. ESG. CHEM.,Anal. Ed., 2, 26 (1930). (6) Ohlsson, 2. phi/siol. ChenL., 119 (1922). ( i )Pawlowski-Doermens, "Brau. Tech. Untera. Methoden," Munich and Berlin, 1927. RECEIVED July 13, 1931. Presented before the Division of Agricdtural a n d Food Chemistry a t the 79tn Meeting of the American Chemical Society, Atlanta, Ga.. April 7 to 11. 1930.
