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II. The effect of cooking on the digestibility of cereals
II. T H E E F F E C T OF COOKING ON T H E D I G E S T I B I L I T Y OF C E R E A LS JOHN R. Ross, M.D., DORIS MONYPENNu AND S. H. JACKSON, PH.D. TORONTO, ONTARIO
N A previous paper the effect of cooking on the enzymatic digestibility The procedure was to take a measured amount of the cereal to be tested, add a constant amount of water, and cook for varying periods of time, up to four hours. An amount of pancreatic enzyme sufficient to produce the maximum amount of maltose from the starch was added. The digestion was allowed to proceed at 37 ~ C. for one-half hour. The reducing substances were then determined by Willst~tter's iodine titration method3 In a reinvestigation of Wi]lst~tter's method, it was found that certain unsuspected errors were present. In this method an amount of iodine more than required to react with the reducing substances is added, and the excess is titrated with sodium thiosulfate. The maltose is calculated on the basis of the iodine removed by the reducing substances. It was found that the amount of iodine removed in any given digest was influenced by the excess of iodine present. This factor was not recognized in calculating the results in the previous paper. A further investigation of the entire problem was made. The Harding-Downs modification of the Shaffer-Somogyi copper reduction method was used, to avoid the errors encountered with the Willst~tter method. To use the Harding-Downs method it was necessary to treat the digests with West's mercuric sulfate reagent to remove interfering substances. With this method the titration values are directly proportional to the amount of maltose present. It was considered that the rate of digestion of the starch in the cereal after cooking would be a more sensitive index of the digestibility of the cereal than was the previous method of measuring the maximum conversion. For this reason, a smaller concentration of enzyme was used and digestion was allowed to proceed for varying periods of time in successive samples. The percentage of starch converted to maltose in the samples was then plotted against the time of digestion to give a curve expressing the rate of conversion of starch to maltose under these conditions.
I of the starches of various cereals was reported. 1
PROCEDURE
The following procedure was used for the determination of the total starch in the cereals: A 100-rag. sample of cereal was hydrolyzed with 25 ml. N HC1 by boiling for two hours under a reflux condenser. The solution was then neutralized with approximately 1 N NaOH until l~rom the R e s e a r c h L a b o r a t o r i e s of the D e p a r t m e n t of P a e d i a t r i c s , U n i v e r s i t y of Toronto, a n d the I-Iospital for Sick Children, u n d e r t he direction of A l a n Brown, M.D. 395
396
THE JOURNAL OF PEDIATRICS
slightly a d d to litmus, transferred quantitatively to a 500-ml. volumetric flask, and diluted to volume. An aliquot of about 50 ml. of this diluted solution was acidified with one drop of 1:1 H,SO4 and shaken for about four minutes with 0.5 Gin. of Lloyd's reagent. The mixture was then filtered and the acidity adjusted to about pH 5.0 with 40 per cent NaOH and glaeial acetic acid. The total reducing value of this solution was then determined by the Harding-Downs modification of the Shaffer-Somogyi method2 A blank determination was run at the same time. In this the sample of cereal was allowed to stand in water for two hours at room temperature and then diluted to 500 ml. A 50-ml. aliquot was " c l e a r e d " with Lloyd's reagent and the reducing vahle was determined exactly as outlined above. The blank was zero in all cases except with the pablum. When the blank was zero, the total reducing value of the hydrolyzate was calculated as the number of grams of glucose per 100 Gm. of cereal. This value was then converted to the pereentage of stareh in the cereal by multiplying by 0.90 (since 1 Gin. of glucose is produced by 0.90 Gin. of starch). An investigation of the reducing material giving rise to the blank in the pablum was undertaken. One hundred milligrams of cereal plus 50 ml. of water was allowed to stand, cleared by Lloyd's reagent as above, and the filtrate adjusted to pH 6.0 to 6.5. The total reducing value of this solution was determined. Ten milliliter aliquots of the filtrate were fermented with Mar zianus sacchromyces and by M. tropicalls, following the procedure for mycologic analysis given by Harding and Nicholson.~ M. saochromyces removes only glucose, whereas M. tropicalis removes maltose as well as glucose. As a result of this, it was found that the reducing blank of pablum was due entirely to maltose, since fermentation by M. sacc~romyccs had no effect on this reducing value, whereas fermentation by M. tropicalis removed it completely. This information was used in the calculation of the amount of starch in the precooked cereal. Th~ total reducing value after hydrolysis was calculated as glucose. From this total glucose value was subtraeted the amount of glueose that would have resulted from the hydrolysis of the free maltose found in the eereal. The residual glucose value was then multiplied by 0.90 to convert it to the percentage of starch. The aetual estimation of the enzymatic digestibility of the stareh was made by the following procedure: An amount, of cereal calculated to contain 51.7 rag. of starch was accurately weighed out into each of five test tubes and 2.5 ml. of water was added to each. The tubes were tightly stoppered with nonabsorbent cotton and placed in a wire basket , with the bottoms as dose together as possible. The basket was then placed over one opening in a steam bath, and the basket and tubes were wrapped in cloths so that the tubes were entirely surrounded b y the
ROSS E T AL. :
D I G E S T I B I L I T Y OF CEREALS
397
steam. They were cooked in this way for one hour and then cooled, and the water loss was replaced to make up the 2.5 ml. vohmle. The mixture was stirred with a stirring rod so as not to splash the cereal on the sides of the tube. One milliliter of phosphate buffer (0.2 M, pH 6.8) and 0.2 ml. of 0.2N NaC1 were added to each tube. The tubes were then placed in a water bath at 37 ~ C. for about five minutes to bring them to the temperature of the bath. One-tenth milliliter of 100 rag. per cent pangestin (1:75 Difco) in 1 per cent NaHCO, was accurately added to four of the tubes. The contents were then mixed and incubated for exactly ten, twenty, forty, and eighty minutes from the time of addition of the enzyme. The action was stopped at the end of the incubation period by adding 0.25 ml. of West's reagent (30 per cent HgSQ in 10 per cent H 2 S Q ) . The mixture was neutralized with freely ground Ba.CQ, transferred quantitatively to a 100-ml. volumetric flask, and diluted to the mark. A blank was prepared in the same manner except that the order of addition of the enzyme and the West's reagent was reversed and it was not incubated. 60 ~/TqBLUm
40
MZ-,9~ ~- CERE,e ~.
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Chart L - - S t a r c h
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digestion of pabIum and cereals cooked one hour.
The solutions were then filtered, acidified with one drop of 1:1 tI2SO~ and about 100 rag. of lead acetate was added. H~S was passed in to complete precipitation of the lead and mercury sulfides and the excess H~S removed with a stream of nitrogen (or air). The solutions were filtered and the acidity adjusted with 18N NaOtI and 1:1 H~SO~ to about p tI 6.0. The reducing value was determined on 2-ml. aliquots, using the Harding-Downs method, and this reducing value was calculated as maltose. The procedure with pablum was the same except that the cooking process was dispensed with, since this cereal is precooked. The results obtained on a number of different cereals are shown in Chart 1. It is seen there is a significant difference in the rate of
398
THE JOURNAL OF PEDIATRICS
starch digestion under constant conditions in the different cereals, cracked wheat having the lowest and pablum the highest rate of starch digestion. 6o 30 Z~
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~o OROIJv,~I7 ~ Gk'IIVD
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Chart 2.--Effect of the physical condition of c.ereal on starch digestion.
In Chart 2 is shown the rate of digestion of starch in cracked wheat as ordinarily purchased and also in the same cracked wheat finely ground. F r o m this it is evident that the ease of digestibility of the starch is dependent, to some degree, on the physical state of the cereal. In this regard, it is of interest to note the difference in the rate of starch digestion between Mead's cereal and pablum. Both products have the same composition, but in the process of manufacture pablum is so treated that the final product is extremely porous. This no doubt accounts in no small measure for its case of digestibility. CONCLUSIONS
There is a significant difference in the digestibility of starch under constant conditions in different cereals. Changing the physical state of a cereal alters the ease of digestibility of its starch. REFERENCES
1. l%ss~ J. 1% and 2. Willst~tter~ 1%.: 1232. 3. H a r d i n g , V. J , 4. Handing, V. J.,
BurriH, L. M. : J. PEDIAT. 4: 654, 1934. U n t e r s u e h u n g e n fiber Enzymes, Berlin, 1928, Julius Springer, p. and Downs, C . E . : J. Biol. Chem. 101: 487, 1933. and Nicholson, T. 1~. : Biochem. J. 27: 1082, 1933.
N A previous paper the effect of cooking on the enzymatic digestibility The procedure was to take a measured amount of the cereal to be tested, add a constant amount of water, and cook for varying periods of time, up to four hours. An amount of pancreatic enzyme sufficient to produce the maximum amount of maltose from the starch was added. The digestion was allowed to proceed at 37 ~ C. for one-half hour. The reducing substances were then determined by Willst~tter's iodine titration method3 In a reinvestigation of Wi]lst~tter's method, it was found that certain unsuspected errors were present. In this method an amount of iodine more than required to react with the reducing substances is added, and the excess is titrated with sodium thiosulfate. The maltose is calculated on the basis of the iodine removed by the reducing substances. It was found that the amount of iodine removed in any given digest was influenced by the excess of iodine present. This factor was not recognized in calculating the results in the previous paper. A further investigation of the entire problem was made. The Harding-Downs modification of the Shaffer-Somogyi copper reduction method was used, to avoid the errors encountered with the Willst~tter method. To use the Harding-Downs method it was necessary to treat the digests with West's mercuric sulfate reagent to remove interfering substances. With this method the titration values are directly proportional to the amount of maltose present. It was considered that the rate of digestion of the starch in the cereal after cooking would be a more sensitive index of the digestibility of the cereal than was the previous method of measuring the maximum conversion. For this reason, a smaller concentration of enzyme was used and digestion was allowed to proceed for varying periods of time in successive samples. The percentage of starch converted to maltose in the samples was then plotted against the time of digestion to give a curve expressing the rate of conversion of starch to maltose under these conditions.
I of the starches of various cereals was reported. 1
PROCEDURE
The following procedure was used for the determination of the total starch in the cereals: A 100-rag. sample of cereal was hydrolyzed with 25 ml. N HC1 by boiling for two hours under a reflux condenser. The solution was then neutralized with approximately 1 N NaOH until l~rom the R e s e a r c h L a b o r a t o r i e s of the D e p a r t m e n t of P a e d i a t r i c s , U n i v e r s i t y of Toronto, a n d the I-Iospital for Sick Children, u n d e r t he direction of A l a n Brown, M.D. 395
396
THE JOURNAL OF PEDIATRICS
slightly a d d to litmus, transferred quantitatively to a 500-ml. volumetric flask, and diluted to volume. An aliquot of about 50 ml. of this diluted solution was acidified with one drop of 1:1 H,SO4 and shaken for about four minutes with 0.5 Gin. of Lloyd's reagent. The mixture was then filtered and the acidity adjusted to about pH 5.0 with 40 per cent NaOH and glaeial acetic acid. The total reducing value of this solution was then determined by the Harding-Downs modification of the Shaffer-Somogyi method2 A blank determination was run at the same time. In this the sample of cereal was allowed to stand in water for two hours at room temperature and then diluted to 500 ml. A 50-ml. aliquot was " c l e a r e d " with Lloyd's reagent and the reducing vahle was determined exactly as outlined above. The blank was zero in all cases except with the pablum. When the blank was zero, the total reducing value of the hydrolyzate was calculated as the number of grams of glucose per 100 Gm. of cereal. This value was then converted to the pereentage of stareh in the cereal by multiplying by 0.90 (since 1 Gin. of glucose is produced by 0.90 Gin. of starch). An investigation of the reducing material giving rise to the blank in the pablum was undertaken. One hundred milligrams of cereal plus 50 ml. of water was allowed to stand, cleared by Lloyd's reagent as above, and the filtrate adjusted to pH 6.0 to 6.5. The total reducing value of this solution was determined. Ten milliliter aliquots of the filtrate were fermented with Mar zianus sacchromyces and by M. tropicalls, following the procedure for mycologic analysis given by Harding and Nicholson.~ M. saochromyces removes only glucose, whereas M. tropicalis removes maltose as well as glucose. As a result of this, it was found that the reducing blank of pablum was due entirely to maltose, since fermentation by M. sacc~romyccs had no effect on this reducing value, whereas fermentation by M. tropicalis removed it completely. This information was used in the calculation of the amount of starch in the precooked cereal. Th~ total reducing value after hydrolysis was calculated as glucose. From this total glucose value was subtraeted the amount of glueose that would have resulted from the hydrolysis of the free maltose found in the eereal. The residual glucose value was then multiplied by 0.90 to convert it to the percentage of starch. The aetual estimation of the enzymatic digestibility of the stareh was made by the following procedure: An amount, of cereal calculated to contain 51.7 rag. of starch was accurately weighed out into each of five test tubes and 2.5 ml. of water was added to each. The tubes were tightly stoppered with nonabsorbent cotton and placed in a wire basket , with the bottoms as dose together as possible. The basket was then placed over one opening in a steam bath, and the basket and tubes were wrapped in cloths so that the tubes were entirely surrounded b y the
ROSS E T AL. :
D I G E S T I B I L I T Y OF CEREALS
397
steam. They were cooked in this way for one hour and then cooled, and the water loss was replaced to make up the 2.5 ml. vohmle. The mixture was stirred with a stirring rod so as not to splash the cereal on the sides of the tube. One milliliter of phosphate buffer (0.2 M, pH 6.8) and 0.2 ml. of 0.2N NaC1 were added to each tube. The tubes were then placed in a water bath at 37 ~ C. for about five minutes to bring them to the temperature of the bath. One-tenth milliliter of 100 rag. per cent pangestin (1:75 Difco) in 1 per cent NaHCO, was accurately added to four of the tubes. The contents were then mixed and incubated for exactly ten, twenty, forty, and eighty minutes from the time of addition of the enzyme. The action was stopped at the end of the incubation period by adding 0.25 ml. of West's reagent (30 per cent HgSQ in 10 per cent H 2 S Q ) . The mixture was neutralized with freely ground Ba.CQ, transferred quantitatively to a 100-ml. volumetric flask, and diluted to the mark. A blank was prepared in the same manner except that the order of addition of the enzyme and the West's reagent was reversed and it was not incubated. 60 ~/TqBLUm
40
MZ-,9~ ~- CERE,e ~.
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/o
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7"2/',1E O F /-/YDJP O L Y # / #
Chart L - - S t a r c h
o
-
./V//N U ' / 1 E ~
digestion of pabIum and cereals cooked one hour.
The solutions were then filtered, acidified with one drop of 1:1 tI2SO~ and about 100 rag. of lead acetate was added. H~S was passed in to complete precipitation of the lead and mercury sulfides and the excess H~S removed with a stream of nitrogen (or air). The solutions were filtered and the acidity adjusted with 18N NaOtI and 1:1 H~SO~ to about p tI 6.0. The reducing value was determined on 2-ml. aliquots, using the Harding-Downs method, and this reducing value was calculated as maltose. The procedure with pablum was the same except that the cooking process was dispensed with, since this cereal is precooked. The results obtained on a number of different cereals are shown in Chart 1. It is seen there is a significant difference in the rate of
398
THE JOURNAL OF PEDIATRICS
starch digestion under constant conditions in the different cereals, cracked wheat having the lowest and pablum the highest rate of starch digestion. 6o 30 Z~
CZ~C~ZD PFJc~I~-/NE ~,4mIND
~o OROIJv,~I7 ~ Gk'IIVD
2~ ~3 ZO
T//~C
Of"
94 0 /C/VDRO.I.y.3"I0"
,gO -
/v,I/NUTE~"
Chart 2.--Effect of the physical condition of c.ereal on starch digestion.
In Chart 2 is shown the rate of digestion of starch in cracked wheat as ordinarily purchased and also in the same cracked wheat finely ground. F r o m this it is evident that the ease of digestibility of the starch is dependent, to some degree, on the physical state of the cereal. In this regard, it is of interest to note the difference in the rate of starch digestion between Mead's cereal and pablum. Both products have the same composition, but in the process of manufacture pablum is so treated that the final product is extremely porous. This no doubt accounts in no small measure for its case of digestibility. CONCLUSIONS
There is a significant difference in the digestibility of starch under constant conditions in different cereals. Changing the physical state of a cereal alters the ease of digestibility of its starch. REFERENCES
1. l%ss~ J. 1% and 2. Willst~tter~ 1%.: 1232. 3. H a r d i n g , V. J , 4. Handing, V. J.,
BurriH, L. M. : J. PEDIAT. 4: 654, 1934. U n t e r s u e h u n g e n fiber Enzymes, Berlin, 1928, Julius Springer, p. and Downs, C . E . : J. Biol. Chem. 101: 487, 1933. and Nicholson, T. 1~. : Biochem. J. 27: 1082, 1933.