A BIOCHEMICAL METHOD FOR DETERMINING IN- DIGESTIBLE RESIDUE (CRUDE FIBER) IN FECES:

LIGNIN, CELLULOSE, AND NON-WATER- SOLUBLE HEMICELLULOSES

BY RAY D. WILLIAMS AND W. H. OLMSTED

(From the Department of Internal Medicine, Washington University School of Medicine, St. Louis)

(Received for publication, November 19, 1934)

In a preceding paper one of us (0.) found a 50 per cent crude fiber content in wheat bran free from starch and essentially freed from pentosans by weak acid hydrolysis. This seemed much too low and led us to examine critically the long accepied term “crude fiber.”

For 70 years the method for determining crude fiber has re- mained essentially the same. In 1864 Hennenburg and Stohmann proposed a method with weak acid and alkali digestions, now commonly called the Weende method. A modification of the original method has been adopted by the Association of Official Agricultural Chemists. The original investigators knew that their product did not have a constant chemical composition since it showed varying percentages of C, H, and 0. Many investigators since then have shown the compositional variability of the Weende product (1). Table V indicates that we have confirmed these observations. Many variations of the original procedure have been made and used advantageously (2). Other methods (3) have been described such as: Fellenberg, nitric and acetic acids; Simon and Lohrisch, strong potassium hydroxide and hydrogen peroxide; Konig, glycerol sulfuric acid; Steigler, 10 per cent hy- drochloric acid at 100” and a stream of air; Scharrer and Kursch- ner, acetic acid, nitric acid, and trichloroacetic acid. More re- cently Remy (4) described a biological method with enzymes to isolate cellulose, lignin, and hemicellulose from the starch, protein, and fat; and, after comparing the crude fiber values of certain ma-

653

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654 Indigestible Residue in Feces

terials with the values obtained by the Weende method, concluded that the Weende digestions caused a great loss (his data indicate about 50 per cent) of indigestible residue. His method has dis- tinct advantages over the more drastic treatment by acid and alkali or other chemicals.

It can be said that all methods (except Remy’s) depend upon the incomplete solubility in certain reagents of one or more con- stituents of indigestible residue. That is, the product is defined

TABLE I

Comparison of Fiber (Wee&e Method) of Stools with Indigestible Residue Isolated by Enzyme Method

Material amlyzed

Weende method (fiber)

(2)

25 ml. stool Suspension 1 25 cc 6‘ u 2

25 “ “ “ 3 25 “ I‘ “ 4

25 “ “ “ 5 25 “ “ “ 6

2.5 “ “ “ 7

25 it ‘I 66 g 25 “ “ “ g

25 cc c‘ dc 10

0.5 gm. (80 mesh) wheat bran 0.5 “ Cellu Flour* (80 mesh) 0.5 “ filter papert (80 “ )

ml. m?.

212.5 118.0 249.0 121.0 178.7 88.7 137 4 78.0 167.0 87.0 181.0 117.7 218.0 87.1 308.0 166.6 112.6 43.5 226.3 88.0 273.7 94.6 490.0 276.2 490.0 499.5

per cent

55.5 48.5 49.5 56.7 52.1 64.7 40.0 54.0 38.8 39.0 34.6 56.4

100.0 (d-1.0)

* A commercial product sold by the Chicago Dietetic Supply House, Inc. t Whatman No. 1.

by the process itself. This fact explains why Coleman et al. (5) report that nine collaborators analyzing bran found the results on crude fiber unsatisfactory. Investigators (ourselves and others) of fiber non-digestibility and concomitant laxation have found their work complicated by questionable methods of analysis. For the most part they have realized the shortcomings of the pre- vailing methods but have used time-worn techniques, because most of the digestibility data had been compiled with them. Table I

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R. D. Williams and W. H. Olmsted

shows that by the Weende method, the most universally accepted one, but 50 per cent of the indigestible residue of feces and but 34 per cent of that in bran are determined.

Obviously then indigestible residue must be determined by methods other than physical; and, once defined, by these methods of determination all of it must be estimated. No one has conclusively demonstrated that the mammalian digestive tract se- cretes enzymes which digest cellulose, lignin, and hemicellulose. There is evidence that one or all three of these may disappear from the mammalian gut as a result of bacterial action. Thus we de- fine non-digestible residue as those vegetable materials not at- tacked by digestive enzymes in the mammalian gut and consist- ing of lignin, cellulose, and non-water-soluble hemicelluloses.

Hemicelluloses have been defined by Schorger ((6) p. 141) as ‘I . . . a polysaccharide soluble in dilute alkalis and convertible into simple sugars by heating with dilute acids at atmospheric pressure . . . in the natural state should be insoluble in boil- ing water.” This group includes everything of a carbohy- drate nature except cellulose, starch, lignin, and soluble sugars. Some are true carbohydrates such as pentosans, hexosans, galac- toarabans and some are not true carbohydrates, such as uranic 6-carbon sugar acids (galacturonic and glucuronic acids, the latter of which occurs as an anhydrous form in straw and wood (7)). Galacturonic acid is found in pectins (7). McCance has classified this group as (1) pentosans and other furfural precursors, (2) in- ulin and fructosans (levulosans), (3) other hexosans such as man- nans and galactans. Group 1, according to McCance, has a more or less universal distribution in the plant kingdom and is com- posed chiefly of (a) pentosans, (b) true hemicelluloses and pectins, which contain some xylose and arabinose but are mostly composed of galacturonic and glucuronic acids, (c) nucleic acids from animal tissues, and (d) precursors of methyl furfural such as methyl pen- toses and hexoses. Group 2 is an unimportant human dietary constituent in this country. Group 3 is more extensive. Man- nans occur in yeast, woods, and common seeds such as beans and peas. Galactans occur in gums, leguminous seeds, and in higher plants (7). This group represents a variable and considerable part of the human dietary.

It will be evident that estimation of the hemicellulose group by

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Indigestible Residue in Feces

the furfural method may lead to very erroneous results. Groups 1 and 3 (except mannan), however, possess two properties in com- mon. When hydrolyzed to their constituents, they reduce alkaline copper reagents and are essentially non-fermentable by Fleischmann’s yeast. Mannose, a reducing sugar, appears with the fermentable fraction (glucose from cellulose). This method of determining the hemicellulose group as a whole is an admit- ted approximation but a very close one; and, until the relative amounts of these constituents in foods are known, a closer ap- proximation is not practical. The relative reduction values of the principal non-fermentable sugars involved in our work with the re- agent as used are arabinose as 100, xylose 109, galactose 92. Uranic acid reductions are not given, for under the conditions of our method they would be converted into pentoses (8). The rela- tive reduction values of the principal fermentable sugars involved are glucose as 100, mannose 96. The furfural precursors (xylose, arabinose, and uranic acids) constitute more than 70 per cent of the non-fermentable fraction in the materials which we analyzed. McCance’s data (7) indicate a percentage of the same order. In this paper we assume that xylose and arabinose are present in equal amounts; and the non-fermentable fraction is estimated as such a mixture. Such a mixture is improbable but the results would be substantially the same with one or the other predominat- ing. The furfural method for pentosans coupled with Link’s method for uranic acid determination (9) would further differen- tiate the constituents of the hemicellulose group.

The second portion of indigestible residue is lignin. Schorger ((6) p. 70) has defined it as “. . . the non-carbohydrate por- tion of lignified tissue after it has been freed from tannins, resins, fats, and similar secondary constituents. . . It is easily oxi- dized by nitric acid. Halogens produce oxidation and substitution, the halogenated product being readily soluble in alkalis. It is soluble in acetyl bromide in the cold and in alkalis, sulphurous acid, and sulphite solutions at high temperatures.” One reliable and accepted method for the actual separation and estimation of lignin is that of Ost and Wilkemng (10). This method was re- cently investigated and improved by Sherrard and his coworkers (11). It is based on the principle that 72 per cent sulfuric acid under controlled conditions dissolves the carbohydrate fraction

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R. D. Williams and W. H. Olmsted 657

and retains it in solution when diluted but the lignin is precipi- tated quantitatively. We have used the principle of their method with modifications necessitated by our determination of cellulose and hemicellulose.

The third constituent of indigestible residue is cellulose. Since the work of Irvine and his coworker (12) on cellulose chemistry, there has been little doubt that cellulose is made up of glucose re- mainders and that by suitable methods cellulose could be quanti- tatively converted into glucose. To make this conversion we used a modification of the sulfuric acid method originally pre- sented by Ost and recently improved by Ritter, Seborg, and Mitchell (13).

Thus, methods have been presented for determining separately hemicelluloses, lignin, and cellulose. But the best application of these methods depends upon the preliminary removal of protein, fats, resins, gums, and starch.

EXPERIMENTAL

Pretreatment of Fecal Material

We first used Remy’s enzymatic (pepsin-hydrochloric acid, neutral malt diastase, pancreatin-sodium carbonate) digestion and found the starch completely removed, the proteins, fats, and resins adequately removed, but a substantial loss (12 per cent) in the hemicellulose fraction. Successive analyses of the steps indicated that the loss was essentially in the malt diastase treatment. Pringsheim ((14) p. 318) mentions the investigations of Luers on the malt enzyme, zytase, which splits hemicellulose. We then tried another plant diastase, taka-diastase, but found a much greater loss (31 per cent). Finally we found that the animal diastase, pancreatin, in neutral solution removed starch without the concurrent loss of hemicellulose. The proteolytic enzymes of pancreatin are quite efhcient and the amylolytic power is not greatly reduced at pH 8. To obtain simultaneous digestion of protein and starch the pH of the digest was adjusted to 8 and the hemicellulose recovery determined. None of the hemicellulose fraction was lost in feces and wheat bran; but, when the treatment was applied to air-dried vegetables, as much as 40 per cent loss occurred. Successive analyses of the steps indicated that the loss

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Indigestible Residue in Feces

was due to the alkalinity of the digest and not to the pancreatic enzymes. It was concluded that the hemicelluloses which pass the digestive tract and those found in wheat bran are sufficiently resistant to encounter negligible loss by this pretreatment. How- ever, the labile hemicelluloses may suffer so much loss that an additional check must be made.’

Table II indicates the efficiency of our final method of pretreat- ment for removing starch, protein, and other primary interfering substances. Separately it was found that the pretreatment was adequate for removing and rendering achromatic to iodine 4 gm. of potato starch (wet weight) and for digesting 4 gm. of lean beef

TABLE II Ejkiency of Pretreatment

25 ml. stool suspension.. . . . . 25 “ “ ‘I + 0.25 gm. starch 25 “ ‘I “ + 0.25 gm. air-

dried meat.. . . . . . . . . . 25 ml. stool suspension + 0.25 gm. cellu-

lose (subtracted).................... 25 ml. stool suspension from non-residue

diet. . . . . . . . . .

Fer~lYta- reduction

‘0.006 N thio

ml

1.82 1.80

1.80 3.85 194.6

1.85

0.0

NW- fermentable

reduction Lignin

0.005 N thio

ml. ma.

3.86 190.7 3.82

3.77

0.0 0.0

meat (wet weight) or 1 gm. of casein (dry weight). Hence if all of the residue from the feces suspension were starch and protein, or either one, the treatment is adequate for removing it.

As a further control a man was kept on a milk-cream diet for 8 days. At the end of 3 days a cathartic was taken to insure com- plete removal of residue. Stools were collected on the 6th, 7th,

1 As a check for the labile hemicelluloses of food we suggest that to a sample (0.25 pm.) treated to remove the resins and fats only (no enzyme digestion) the 21.4 N sulfuric acid be added directly, the subsequent pro- cedure being the same, and the non-fermentable reduction determined. The strong-weak acid hydrolysis is, to a great extent, independent of the size of the particles and the results are more reliable than the usual 2.5 per cent hydrochloric acid hydrolysis.

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R. D. Williams and W. H. Olmsted 659

and 8th days and analyzed for indigestible residue. The findings of the non-residue diet are given in Table II. The fatty character of these stools suggested the use of added bile salts as an important step in pretreatment.

Methods for Analyzing Prepared Material

Concentration of Sulfuric Acid-We have already indicated that cellulose is known to be dissolved by very strong sulfuric acid solu- tions (60 to 72 per cent) and lignin is dissociated from it. It would appear, therefore, that the first step in the quantitative analysis of cellulose, lignin, and hemicellulose is the treatment with strong sulfuric acid.

It was observed that with the 72 per cent acid at 6-10’ there was a tendency to charring, the pentosan recovery was low, and

TABLE III

Relation of Pentose Recovery to Concentration of Sulfuric Acid

Sulfuric acid

pm cent by volume

50 60 (21.4 N)

72

w.

194.0 200.0 140.0

the lignin value was high. This apparently confirmed the ob- servation of Jenkens (15) that a lignin-like material was formed from pentose by 72 per cent sulfuric acid. We further determined that completely carbonized material (charred toast, etc.) appears quantitatively in the lignin fraction, and this emphasized the necessity of having diets free from charred foods. The furfural method and weak acid hydrolysis showed that the hemicellulose lost by the 72 per cent acid solution was not lost when the concen- tration of acid was reduced to 60 per cent. Thus 0.5 gm. of water- washed wheat bran gave 196.0 mg. of pentose by weak acid hydrol- ysis or 200.0 mg. of pentose by the furfural method. Table III presents our findings and indicates that the optimum for pentosans would be less than 50 per cent acid; but in our method lignin and cellulose must be determined. With 50 per cent acid, cellulose precipitated on being diluted to 4 per cent acid. 60 per cent acid does not appreciably decrease the yield of pentose and converts

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cellulose (Whatman No. 1) between 96 and 98 per cent of the theo- retical. Therefore, 60 per cent acid was adopted for our method, because the pentose recovery is equal to that by other methods and lignin values were constant on several different determinations and presented a good, light brown appearance. With 72 per cent sulfuric acid the lignin was very dark.

Time Necessary for Weak Acid Hydrolysis-Since the introduc- tion of the sulfuric acid method for hydrolysis of cellulose, it has been known that a weak acid hydrolysis is necessary to convert the dissolved cellulose into glucose. With 4 per cent sulfuric acid we found that a maximum reduction value was attained after 2.5 hours and that this value was maintained through 6 hours. We selected 3 hours as an optimum time.

Temperature-The temperature control of the strong acid (60 per cent) is known to be very important. Within certain limits, the higher the temperature, the shorter the time necessary. We found that with 60 per cent acid and 6-10’ an optimum was reached between 16 and 34 hours. With this concentration of acid and at this temperature the glucose recovery is excellent, no charring occurs, and considerable variations in time do not affect the yield. This fact adds to the flexibility of the method.

To summarize, the principle of the analysis is this: 21.4 N (60 per cent by volume) sulfuric acid under controlled conditions dissolves the cellulose and hemicellulose completely and disso- ciates them from the lignin. The 21.4 N acid is then diluted to 1.426 N (4 per cent by volume) and boiled for 3 hours. The lignin is precipitated quantitatively; the cellulose and hemicellulose are converted into their constituent simple sugars which are soluble. The lignin is filtered off and weighed; the cellulose and hemicellu- lose, now converted into simple sugars, are estimated by copper reduction; the non-fermentable reduction represents the hemi- cellulose, and the fermentable, the cellulose (and mannanj.

Reduplication of results depends primarily on familiarity with copper reduction technique. We stress the importance of adjust- ing the sugar solutions to neutrality before analysis.

Reagents Procedure

Buffer-bile salt solution. 50 ml. of 0.2 M potassium acid phos- phate, 23.4 ml. of 0.4 N sodium hydroxide, 6.6 ml. of water, and

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R. D. Williams and W. H. Olmsted 661

2.0 gm. of sodium taurocholate (exclusive of the sodium tauro- cholate, pH 8 on final dilution; see @lark and Lubs).

Pancreatin-sodium chloride is made fresh each day. To 100 ml. of 8.5 per cent sodium chloride add 10 gm. of pancreatin (Merck’s U.S.P. or 5 gm. of Merck’s absolute). Shake frequently for 30 minutes and then filter through a medium paper.

Strong sulfuric acid. 21.4 N (600 ml. of C.P. 95 per cent con- centrated sulfuric acid per liter).

Stools are collected and weighed. The wet weight is multi- plied by 4 and this amount of water added and further diluted to the nearest 25 ml. mark. The total volume is then put into a suitably sized ball mill and ground for 20 minutes or until the suspension will pass a 20 mesh sieve. Owing to the character of the suspension a brisk rubbing may be necessary to sieve it. 25 ml. of this suspension2 are transferred to a 50 ml. glass-stoppered container, stoppered loosely, and steam-sterilized (15 pounds steam pressure, 30 minutes, to kill the spores and gelatinize the starch). The material is allowed to cool below 50” and these reagents added. 20 ml. of buffer-bile salt solution, 5 ml. of pancreatin-sodium chlo- ride solution, and a few drops of toluene. The materials are well mixed and incubated for 3 days at 45” with occasional shaking. Filter the digest through a 125 mesh silk cloth.3 This is easily done if the digest is added slowly and with a stream of distilled water from a wash bottle. Wash the residue with at least 200 ml. of water, then with 50 ml. of hot ethyl alcohol, followed by 25 ml. of hot benzene, and finally with 25 ml. of ethyl ether. Transfer the residue carefully to a 50 ml. glass-stoppered container. This transfer is facilitated if done just after the residue becomes crumbly and before the ether is completely evaporated. Place the con-

2 0.25 gm. of air-dried food (20 mesh), to which have been added 25 ml. of water, can be pretreated as the feces suspension; but an additional check must be made as indicated in foot-note 1. If the material is finely pow- dered, the precaution of foot-note 3 should be observed.

3 This cloth was obtained through Essmueller Mill Furnishing Company of St. Louis. The holes are approximately 0.075 mm. square. Where the material will permit (foods) we recommend the 150 to 200 mesh cloth. If the material is powdered, we suggest putting the sample in a 50 ml. glass- stoppered centrifuge tube. The enzyme digestion, all washings by centrif- ugation, and acid treatment (described later) can be done without remov- ing the residue from that vessel. This minimizes mechanical losses.

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662 Indigestible Residue in Feces

tamer with the residue in the oven at 70’ for 2 hours or until the residue is dry.

To the prepared material (in a glass-stoppered container) add 20 ml. of chilled (6-10”) 21.4 N sulfuric acid, shake briskly, and put in the ice box (S-10’). Shake again at hour intervals (par- ticularly during the first 5 hours) and keep at this temperature for 24 hours. Dilute rapidly to 300 ml. (1.426 N acid) with dis- tilled water. Keep at the boiling temperature (gentle ebulition) under a reflux for 3 hours, cool to room temperature, and filter through a loose layer of ignited asbestos (Gooch crucible), and wash thoroughly with distilled water, collecting the first 50 ml. of the washings and adding it to the filtrate. Then wash carefully with at least 100 ml. of distilled water and follow with adequate alcohol, benzene, and ether washings. Dry the residue at llO“, weigh, ignite, reweigh, and calculate the loss as the lignin frac- tion. The filtrate is neutralized with 50 per cent sodium hydrox- ide (about 40 ml.) to phenol red, and further diluted to 500 ml. The total reduction is determined by the Shaffer-Somogyi copper reagent4 (16). A portion (40 ml.) is fermented by the Somogyi washed yeast procedure (17), and the non-fermentable reduction interpreted on the xylose-arabinose curve and multiplied by the factor 0.88 to convert pentose to pentosan (hemicellulose).’ The fermentable reduction is interpreted on the glucose curve and multiplied by the factor 0.90 to convert glucose to cellulose.

Results

Table IV presents analyses of (a) a prepared bran which con- tains lignin, hemicellulose, and cellulose, (b) Cellu Flour which contains hemicellulose and cellulose, and (c) a filter paper which is pure cellulose. Analytical literature is replete with failures to account for 100 per cent of such a complex mixture as wheat bran.

Residues from aliquots of feces suspensions and other materials were isolated by the Weende method (crude fiber) and by the enzy- matic pretreatment (indigestible residue). Table V compares the gross values and the analyses of the two products.

4 The Shaffer-Somogyi Reagent 50 (with 5 gm. of potassium iodide) is calibrated for 30 minute heating periods for glucose and for xylose-arabinose (l:l). The sugar solutions for calibration are made up with 0.85 N sodium sulfate solution.

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R. D. Williams and W. H. Olmsted 663

The data of Table V permit calculation of the percentages of the original lignin, cellulose, and hemicellulose remaining after the Weende digestions; and the results are given in Table VI. From these results it would appear that in the Weende digestions hemi- cellulose suffers the greatest lost, lignin less, and cellulose least. It has been suggested ((14) p. 206) that lignin protects native cellulose and hemicellulose against bacterial and chemical de- struction. However, native cellulose and hemicellulose in the absence of lignin vary greatly in their resistance to chemical treat- ment. The resistance of the hemicellulose particularly may be so strong that concentrated alkali is required to dissolve it or so

TABLE IV

Composite Analysis of Materials by New Method

Moisture. ....... Fats, resins, etc. Lignin .......... Cellulose. ....... Pentosan. ....... Protein. ........ Ash. ............

.

.

Total. . . 95.52

Acid-treated waShed bran

per cent 3.27

20.51 21.44 32.22

8.60 8.06 1.42

Cellu Flour Filter pwm

per cent per cent 2.50 4.41 0.0 0.0 0.0 0.0

78.84 94.0 17.20 0.0 0.0 0.0 0.0 0.0

98.54 98.41

weak that a phosphate buffer (pH 8) will remove as much as 40 per cent. Therefore, with the solubility of these three fractions (lignin, cellulose, and hemicellulose) varying widely, it is reason- able to expect the Weende product to have a variable composition and, even though the materials have the same crude fiber content, the chemical composition to vary. Table VII gives an example of two materials (Table V, stool Suspension 3, and water-washed bran) with essentially the same indigestible residue values and crude fiber contents, but without a single instance of similar pro- portions of lignin, cellulose, and hemicellulose. Whereas con- trolled Weende digestion of the same material will give a product of similar composition, the value obtained is meaningless, for it

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TABL

E V

Anal

ysis

of

Resid

ues

afte

r W

eend

e Tr

eatm

ent

and

afte

r En

zym

atic

Dige

stio

n

Enzy

mat

ic m

etho

d (in

dige

stib

le

resid

ue)

Mat

erial

Li

gnin

WY.

25

ml.

fece

s Su

spen

sion

1..

......

. 17

7.8

25

“ “

“ 2.

. ...

....

91.4

25

‘I

‘I “

3..

......

. 11

5.7

25

“ “

“ 4.

. ...

....

67.6

25

‘t

6‘

6‘

5..

......

. 51

.2

25

6‘

6‘

~6

6..

......

. 78

.0

25

‘6

ic

66

7..

......

. 91

.7

25 ‘I

“

“ 8.

. ...

....

58.6

25

66

‘6

66

9.

. ...

....

113.

4 0.

5 gm

. wa

ter-w

ashe

d br

an

(80

mes

h)

......

......

......

......

. 34

.2

0.5

gm.

Cellu

Fl

our

(80

mes

h).

...

0

-

( -

-

:~llU

lO%

3

w.

mg.

51.3

79

.2

57.6

62

.5

60.3

73

.0

48.6

62

.5

36.0

50

.2

45.0

44

.0

35.1

54

.5

17.1

36

.9

49.5

63

.4

81.0

15

8.4

394.

2 86

.0

Hem

i- ce

llulo

se

-

1

-

Tota

l in

dige

stib

le

resid

ue

w.

w.

208.

3 85

.3

212.

5 33

.0

249.

0 32

.7

178.

7 39

.0

137.

4 33

.5

167.

0 25

.3

181.

0 40

.0

112.

6 28

.2

226.

3 36

.4

273.

7 12

.7

490.

0 0

Lign

in

-

(

-- -

Wee

nde

met

hod

(cru

de

fiber

)

ml.

39.6

37

.8

36.0

29

.7

21.6

21

.6

22.5

13

.5

27.0

58.0

27

1.8

- . _

-

Hem

i- ce

llulo

se

Tota

l re

cove

ry (li

gnin

+

eel-

l&se

+

hem

i- ce

llulG

se)

Tota

l cr

ude

fiber

mg.

m

g.

“8.

16.7

14

1.6

146.

9 34

.3

105.

1 11

0.0

26.4

95

.1

100.

0 17

.6

86.3

90

.0

11.1

66

.2

70.0

16

.7

63.6

65

.0

15.8

78

.3

80.0

6.

2 47

.9

49.0

15

.8

79.2

82

.0

20.2

90

.9

94.6

0

271.

8 27

6.2

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R. D. Williams and W. H. Olmsted 665

cannot be ascertained by the method whether the fractions dis- appearing are lost in the digestive tract or by the chemical degra- dation of the analytical method.

TABLE VI

Indicating Percentages of Indigestible Fractions Remaining after Weende Treatment

Material

Feces Suspension 1.. .............. “ “ 2 ................ “ “ 3 ................ “ ‘I 4. ............... I‘ “ 5 ................ ‘I ‘I 6 ................ “ “ 7. ............... “ “ 8. ............... “ “ 9. ...............

Water-washed bran ................ Cellu Flour. ......................

Lignin. ....................... Cellulose. .................... Hemieelluloses ................

Total.........................

Lignin remaining

per cent 48.0 36.2 28.4 57.5 65.7 32.1 43.7 48.2 32.1 37.2

CellUlOSe remaining

per cent

77.1 65.7 59.7 61.1 60.0 48.0 64.0 79.0 54.6 71.7 69.1

lemicelluloses remaining

per cent

42.6 55.0 36.2 28.2 22.1 38.0 29.0 16.8 24.9 12.7 0

TABLE VII

Stool Suspension 3 Water-washed bran

lnf$;;;~le Crude fiber ln;z;;tle Crude fiber

m7. WT. w7. ml. 116 33 34 13 60 36 81 58 73 26 158 20

249 95 273 91

SUMMARY

The Weede method is inadequate, ,because the product has an inconstant chemical composition; i.e., it determines a variable fraction of the cellulose, hemicellulose, and lignin.

Indigestible residue is defined and a biochemical method for its determination is described. The method is advantageous, be- cause by it are determined quantitatively the fractions of indi- gestible residue.

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Indigestible Residue in Feces

The biochemical method has been successfully applied to fecal material and certain other substances; and precautions are recom- mended for its successful use in food analysis.

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Ray D. Williams and W. H. OlmstedHEMICELLULOSES

NON-WATERSOLUBLELIGNIN, CELLULOSE, AND

RESIDUE (CRUDE FIBER) IN FECES:DETERMINING INDIGESTIBLE

A BIOCHEMICAL METHOD FOR

1935, 108:653-666.J. Biol. Chem. 

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