GEHRT: PEPSIN DIGESTIBILITY OF ANIMAL PROTEINS
Pepsin Digestibility Method for Animal Proteins
By ALBERT J. GEHRT (Moorman Mfg. Co., 1000 N. 30th St., Quincy, 111. 62301)
The official method for pepsin digestibility
of animal proteins, 7.040-7.046, has been i m -
proved by replacing the centrifuge isolation
of the indigestible residue with filtration
through glass fiber paper. The modified
method was studied by 25 collaborators. The
study included meat meals, digester tankage,
fish meal, blood meal, poultry by-product
meal, and hydrolyzed feathers. The filtration
method is more rapid, less laborious, and free
from decantation losses. Basic digestion condi-
tions of the official method, pepsin concentra-
tion, acidity, digestion time and temperature,
and continuous agitation, are unchanged. The
study also included grinding the portion of
the samples t h a t was larger t h a n 20 mesh.
Agreement between replicates in individual
laboratories was excellent. Agreement between
laboratories on all samples except poultry by-
product meal and hydrolyzed feathers was
satisfactory; standard deviations ranged from
0.43 to 1.07. Data indicate t h a t part of the
variation was due to slight differences in t h e
samples. The modified method has been
adopted as official first action for all animal
proteins except poultry by-products and hy-
drolyzed feathers.
The AOAC pepsin digestibility method, 7.040-
7.046 (1), was adopted as official in 1959 (2) and
has been widely used since that time for evalu-
ating the quality of animal protein feedstuffs.
The method involves overnight digestion of the
defatted sample with pepsin followed by deter-
mination of the indigestible residue by cen-
trifuging, washing, drying, and weighing. In-
digestible protein can be determined by Kjeldahl
analysis of the indigestible residue or by filtering
the indigestible residue, using a filter aid, and
analyzing the unweighed residue for protein.
Isolation of the indigestible residue by cen-
trifuging has been laborious and fraught with the
possibility of loss of part of the residue during
decantation. For these reasons, several labo-
ratories, including that of the Associate Referee,
have investigated isolation of the indigestible
residue by filtration. It was found that the
indigestible residue can be readily filtered
through glass fiber paper in a California modified
669
Biichner funnel and determined by washing, dry-
ing, and weighing.
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Collaborative Study
Because of the advantages offered by the filtra-
tion method, it was collaboratively studied by 25
collaborators: 8 State feed control laboratories, 5
packers or renderers, 10 feed manufacturers, and
2 commercial testing laboratories.
The official pepsin digestibility method is appli-
cable to meat meal, meat and bone meal, digester
tankage, fish meal, blood meal, whale meal,
poultrjr by-product meal, and hydrolyzed poultry
feathers. To cover this range of feedstuffs the
present study included 3 meat meals of various
levels of indigestible material, 1 digester tankage,
1 blood meal, 1 fish meal, 1 poultry by-product
meal, and 1 hydrolyzed poultry feathers.
In this year's study, the basic digestion condi-
tions of the official method, pepsin concentration,
acidity, digestion time and temperature, and
continuous agitation, were unchanged. The major
improvement in the method is the filtration step
for isolating the residue. Additional changes in-
cluded in the modified method are in the sample
grinding and fat extraction steps. The official
method specifies grinding the sample in the Wiley
mill to pass 2 mm screen. This grind is slightly
coarser than the official grind specified for feeds,
7.002, which calls for the 1 mm screen. The 2 mm
grind was originally chosen for the pepsin method
to permit making the digestion in a state of fine-
ness closer to that actually fed to animals than
the 1 mm grind. Work done in the laboratory of
the Associate Referee has shown that the 1 mm
grind gives results which are practically identical
with the 2 mm grind. The revised method specifies
screening the sample through 20 mesh before
grinding the portion larger than 20 mesh (+20
mesh), followed by re-blending of both portions.
Grinding only the + 2 0 mesh portion is advan-
tageous in minimizing sticking in the mill with
greasy samples. However, it is essential that the
ground portion be thoroughly re-blended with the
portion less than 20 mesh. Elimination of cen-
trifuge extraction of the fat specified in the
670
official m e t h o d is also an improvement because
it is difficult t o avoid loss of p a r t of t h e sample
during transfer to t h e agitator bottle. Also,
extraction on t h e Goldfisch or Soxhlet extractor
is less laborious.
T h e unground samples described above were
sent to t h e collaborators with t h e request t h a t
t h e y be ground a n d analyzed for indigestible
residue a n d indigestible protein, using t h e new
filtration method. T h e y were also asked to ana-
lyze t h e samples for t o t a l protein b y 7.016 a n d to
calculate "protein indigestible" (per cent indi-
gestible protein in sample/per cent total protein
X 100). M o s t of t h e collaborators' results were
received in M a y 1970. All found t h e m e t h o d v e r y
effective for all of t h e samples except t h e p o u l t r y
by-product meal a n d hydrolyzed feathers, which
some found difficult t o filter.
T h e m e t h o d u n d e r s t u d y specified letting t h e
indigestible residue settle in t h e digestion bottle,
filtering t h r o u g h t h e glass fiber paper, washing
the bottle a n d residue with water followed b y
acetone, a n d drying a n d weighing t h e residue.
Because of t h e filtration problem with poultry
by-product meal a n d hydrolyzed feathers this
m a t t e r was studied further b y t h e Associate Ref-
eree. I t was found t h a t settling t h e residue for a t
least 15 min in t h e digestion bottle a t a 45° angle,
filtering carefully to retain as much of t h e residue
as possible in t h e bottle until most of t h e liquid
h a d passed through t h e filter, a n d washing with
acetone alone instead of water followed b y
acetone p e r m i t t e d rapid filtration a n d effective
washing.
Because of t h e success of t h e Associate Ref-
eree's work t h e collaborators were requested t o
re-analyze t h e p o u l t r y by-product meal, t h e
hydrolyzed feathers, a n d Sample 3, t h e m e a t
meal (to include a m e a t meal sample in t h e
second s t u d y ) , using t h e modified filtration m e t h -
od with t h e acetone wash. This method is de-
scribed below.
METHOD
The official final action method for pepsin
digestibility of animal protein feeds, 7.040-7.046,
was revised to employ a filtration isolation step
instead of centrifuging for all products except
poultry by-products and hydrolyzed feathers, and
the revision was adopted official first action. The
revised method is printed below. The official final
action method, 7.040-7.046, applicable to all animal
JOURNAL OF THE AOAC (Vol. 54, N o . 3, 1971)
proteins, is given in Official Methods of Analysis,
11th Ed.
Revised Method—Filtration
7.A01 Principle
Defatted sample is digested 16 hr with warm soln
of pepsin under constant agitation. Insol. residue is
isolated by filtering, washed, dried, and weighed to
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det. % residue. Residue is examined microscopically
and analyzed for protein. Filtration method is appli-
cable to all animal proteins except hydrolyzed
feather meal and poultry by-product meal. Methods
are not applicable to vegetable proteins or mixed
feeds due to presence of complex carbohydrates and
other compds not digested by pepsin.
7.A02 Apparatus
(a) Agitator.—See Fig. 7:2. Continuous, slow
speed (15 rpm), end-over-end type, to operate inside
incubator at 45 ± 2 ° and carry 8 oz screw-cap pre-
scription bottles, or equiv. Agitator and bottles
available from D . E. Sims, 716 Forrest Ave, Quincy,
IL 62301. Stirring or reciprocating (shaking) type
agitator cannot be used because solid particles
collect on sides of bottle and do not contact pepsin
soln. If heat from agitator motor raises incubator
temp, to >45°, mount motor outside incubator by
drilling hole thru side of incubator and connecting
motor to agitator with extension shaft and coupling
(available from agitator supplier). (Caution: See
46.012.)
(b) Settling rack.—Wood or metal to hold diges-
tion bottles at 45° angle. M a y be made from 2
boards nailed horizontally into " V " cut into vertical
end pieces. Also available from agitator supplier, (a).
(c) Filtering device.—Modified California buchner,
7.055(d), available from Labconco Corp., 8811 S
Prospect Ave, Kansas City, MO 64132, No. 55100.
(If edge of screen is rough, smooth with small-tip
soldering iron.) Use with retainer sleeve, 2 X 2.75"
od stainless steel tube, available from agitator sup-
lier, (a).
(d) Glass fiber filter.—7 cm, Reeve Angel No.
934-AH, or equiv.
(e) Moisture dishes.—Al, 3 X 0.75", with cover
(Matheson Scientific, Inc., 1850 Greenleaf Ave, Elk
Grove Village, I L 60007, No. 19370-30, or equiv.).
7.A03 Reagent
Pepsin soln.—0.2% pepsin (activity 1:10,000) in
0.075^ HC1. Prep, just before use by dilg 6.1 ml HC1
to 1 L and heating to 42-45°. Add pepsin and stir
gently until dissolved. Do not heat pepsin soln on
hot plate or overheat.
GEHRT: PEPSIN DIGESTIBILITY OF ANIMAL PROTEINS
7.A04 Preparation of Sample
Sieve sample, 7.001, thru No. 20 sieve. Grind
portion retained on sieve to pass No. 20 sieve. Com-
bine both portions and blend by stirring and shaking
in pt jar. Thoro blending is essential. Because of
high fat content of many animal products, grinding
without sieving may cause sticking in mill, loss of
moisture or fat, or poorly blended sample.
7.A05 Extraction
(Caution: See 46.011, 46.039, and 46.054.)
Ext ground sample as follows: Prep, extn thimble
from 11 cm Whatman No. 2 paper, or equiv., as
follows: Fold paper in half; straighten paper and
refold at right angles to first fold; turn paper over
and repeat process with folds at 45° to original fold;
while holding creased paper in one hand, place
short test tube (6-8 mm smaller in diam. than
extractor sample holder or cup in which thimble is
to be used) at its center; fold along natural crease
lines to form 4-pointed star around tube; and wrap
points in same direction around tube to complete
thimble.
Weigh 1.000 g sample into thimble and ext 1 hr
with ether at condensation rate of 3-4 drops/sec. (If
Soxhlet is used, top of thimble should extend above
siphon tube to avoid loss of solid particles. If paper
contg sample is totally submerged in siphon cup,
sample must be completely wrapped in paper.) Ob-
serve ether ext to det. that no solid particles were
carried into solv. beaker. If approx. fat content is
desired, evap. ether, and dry and weigh residue. Re-
move paper from sample container or cup and let dry
at room temp. Unfold and quant, brush defatted
sample into digestion bottle, avoiding contamination
by brush bristles or filter paper fibers. Use of
powder funnel is helpful to avoid loss.
7.A06 Pepsin Digestion
To defatted sample in agitator bottle add 150 ml
freshly prepd pepsin soln prewarmed to 42-45°.
Be sure sample is completely wetted by pepsin soln.
Stopper bottle, clamp in agitator, and incubate with
constant agitation 16 hr at 45°.
7.A07 Treatment of Residue
Dry individual sheets of glass fiber filter, (d),
30 min at 110° in moisture dishes with cover open.
Cool in desiccator 30 min with cover closed, and
weigh (Wi).
Remove bottles from agitator. Place in 45° angle
settling rack and loosen caps. Let residue settle
> 15 min. Place weighed filter in California buchner,
(c), apply suction, and moisten with H2O. Place
retainer sleeve on filter and press down gently. Rinse
particles of residue on cap onto filter with small amt
671
H 2 0 . Carry bottle from rack to filter at same angle
as settled and pour contents slowly thru filter,
retaining as much residue as possible in bottle.
Filtration proceeds rapidly with moderate suction
until appreciable amts of residue cover filter surface.
If filtration rate becomes slow, it may be accelerated
with acetone, but only if no significant amt of aq.
digestion mixt. remains on funnel when acetone is
added. Settling residue at 45° angle followed by
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careful pouring of supernatant thru filter in small
stream permits all or most of liq. to pass thru as
fast as it is poured. After supernatant has passed
thru filter, transfer residue quant, onto filter as
follows:
Add 15 ml acetone to bottle. Hold thumb over
bottle neck and shake vigorously. Release pressure,
replace thumb over bottle neck, and shake bottle
in inverted position over filter. Remove thumb,
letting acetone and residue discharge onto filter.
Repeat rinse with second 15 ml portion acetone,
shaking and releasing pressure as above. Inspect
bottle, and rinse further with acetone, using police-
man, if necessary. If >}/%" liq. remains on paper
when acetone washes are started it may be necessary
to use three 15 ml acetone washes instead of 2 to
increase filtration rate.
After all liq. passes thru funnel, wash residue and
inside surface of retainer sleeve with 2 small portions
acetone from wash bottle or hypodermic syringe,
and suck dry. Remove retainer sleeve from funnel.
Transfer filter to original moisture dish. Scrape or
brush any residue particles or filter clinging to
retainer sleeve or funnel onto filter in moisture dish.
D r y in oven, cool, and weigh as before (W2). Calc. %
indigestible residue = (W2 — W\) X 100/g sample.
Det. indigestible protein by transferring filter
contg residue directly to Kjeldahl flask. Proceed
as in 7.016. (Caution: Violent reaction may take
place when NaOH is mixed with dild digestion
mixt., caused by large excess H2SO4 due to small
amt org. material from residue and none from glass
filter. Avoid by thoroly mixing and cooling digestion
mixt. before addn of NaOH or by using 20 ml H2SO4
in Kjeldahl digestion instead of 25 ml.) Make blank
detn on 1 sheet of glass filter and subtract from each
sample detn, if necessary. Calc. % protein based on
original sample wt. Result represents % indigestible
protein in sample. Convert to % crude protein con-
tent of sample not digested, "protein indigestible" =
% indigestible protein in sample X 100/% total
crude protein in sample.
Results and Discussion
Results from t h e first s t u d y are given in T a b l e 1
a n d results from the second s t u d y are given in
T a b l e 2. Statistical analyses of the d a t a are in-
cluded. W i t h a few exceptions t h e indigestible
672 JOURNAL OF THE AOAC (Vol. 54, N o . 3, 1971)

Table 1. Collaborators' results" for total proteins, indigestible residues, indigestible proteins,
and protein indigestibles

Coll. % Protein % In d. Res d u e % In d. Protein % Protein Indigest.

S a m p l e 1, Meat Mea

1 55.3, 55.2 9.4, 9.7, 9 6 4.3, 4.3, 4.3 7.8, 7.8, 7.8
2 54.4 54.4 8.5, 8.6 3.5, 3.9 6.4, 7.2
3 54.4 54.2 8.6, 8.6 4.4, 3.9 8.1, 7.2
4 51.6 52.9 8.6, 8.9 4.7, 4.2 9.1, 7.9
5 54.4 54.5 8.8, 8.4 3.9, 3.4 7.3, 6.3

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6 53.6 53.6 7.0, 7.5 5.7, 6.1 10.6, 11.4
7 55.0 55.0 9.8, 10.0 4.9, 4.9 8.9, 8.9
8 55.9 55.4 9.0, 9.0 3.5, 3.6 6.3, 6.5
9 55.6 55.1 9.3, 9.3 4.0, 4.0 7.2, 7.3
10 53.0 52.9 10.0, 9.7 4.5, 4.4 8.5, 8.3
11 54.7 54.4 9.1, 8.3 3.6, 2.2'' 6.6, 4.0 b
12 54.7 9.8, 9.3, 8 9 4.3, 3.7 7.9, 6.8
13 54.0 54.1 8.8, 9.1 4.0, 4.2 7.4, 7.8
14 54.6 54.6 8.7, 9.2 3.7, 3.8 6.8, 7.0
15 56.0 55.1, 54.6 10.3, 9.8, 9 3 4.7, 3.8, 4.0 8.4, 6.9, 7.3
16 54.0 54.6 8.8, 9.2 3.5, 3.9 6.5, 7.1
17 55.3 55.2 8.6, 9.2 4.2, 4.4 7.6, 8.0
18 54.7 54.7 8.6, 8.7 3.3, 3.5 6.0, 6.4
19 53.8 54.2 9.4, 7.7 3.8, 3.8 7.1, 7.0
20 54.2 53.1 8.9, 8.8 4.0, 3.8 7.4, 7.2
21 52.4 52.2 8.4 4.4 8.4
22 54.2 54.0 9.3, 9.0 3.4, 3.3 6.3, 6.1
23 55.2 54.9 9.5, 9.9 4.5, 4.4 8.2, 8.0
24 54.4 54.8 9.1, 9.1 4.1, 3.6 7.5, 6.6
25 55.0 54.8 8.6, 8.9 4.1, 3.9 7.5, 7.1

Av.c 54.4 9.0 4.1 7.5

Std dev. c 0.89 0.56 0.53 1.02
Coeff. of var. %c 1.6 6.2 13.0 13.6

S a m p l e 2, Meat Mea

1 55.3, 55.6 8.9, 9.2, 8 6 4.4, 4.4, 4.3 8.0, 7.9, 7.7
2 55.3 55.1 7.8, 7.6 3.7, 3.9 6.7, 7.1
3 55.0 54.0 7.5, 7.5 3.5, 3.5 6.4, 6.5
4 55.6 54.3 7.9, 7.7 4.4, 5.1 7.9, 9.4
5 51.6 51.1 7.6, 7.6 3.9, 3.5 7.6, 6.9
6 54.6 54.6 7.3, 7.0 3.5, 3.5 6.4, 6.4
7 53.8 53.8 8.8, 9.1 4.6, 4.7 8.6, 8.7
8 55.3 55.4 8.3, 8.6 3.8, 4.0 6.9, 7.2
9 55.0 54.0 7.7, 8.0 3.7, 3.7 6.7, 6.9
10 54.5 54.6 9.8, 9.5 5.5, 5.0 10.1, 9.2
11 55.5 55.2 7.7, 7.4 2.5, 3.1 4.5, 5.6
12 54.1 8.2, 8.0, 8 2 3.6, 3.5 6.7, 6.5
13 55.0 55.1 9.0, 8.2 4.6, 4.5 8.4, 8.2
14 54.9 56.1 7.8, 8.4 3.5, 4.1 6.4, 7.3
15 55.3 56.0, 55.0 9.1, 9.2, 8 7 4.3, 4.1, 4.3 7.8, 7.3, 7.8
16 54.7 55.2 7.9, 8.1 3.5, 3.7 6.4, 6.7
17 54.6 55.2 8.2, 7.9 4.3, 4.5 7.9, 8.2
18 55.4 55.6 7.6, 7.4 3.2, 3.2 5.8, 5.8
19 55.0 55.0 8.2, 9.1 3.8, 3.8 6.9, 6.9
20 55.0 54.5 8.4, 8.3 4.2, 4.0 7.6, 7.3
21 54.2 53.8 8.8 5.4 10.0
22 54.7 55.0 8.1, 7.5 3.3, 3.4 6.0, 6.2
23 56.1 56.4 8.9, 9.3 4.6, 4.8 8.2, 8.5
24 54.6 54.8 7.5, 7.5 3.5, 3.6 6.4, 6.6
25 55.3 54.1 8.3, 6.9 3.7, 5.5 6.7, 10.2

Av.c 54.8 8.2 4.0 7.4

Std dev. r 0.90 0.65 0.65 1.19
Coeff. of var. % c 1.6 7.9 16.0 16.1

(.Continued)
G E H R T : PEPSIN DIGESTIBILITY OF ANIMAL PROTEINS 673

Table 1. (Continued)

Coll. % Protein % I n d . Residue % I n d . Protein % Protein Indigest.

S a m p l e 3, M e a t M e a l

53.3 53.4 14.7 14.3, 14.7 5.7, 5.3, 5.3 10.7, 9.9, 9.9
52.8 53.1 13.7 14.0 4.9, 5.2 9.3, 9.8
49.8 51.7 13.2 13.9 4.9, 5.3 9.8, 10.3
53.0 53.4 13.7 14.0 6.2, 4.9 11.7, 9.2
53.2 53.0 13.8 13.9 5.3, 5.5 9.9, 10.3
53.4 53.4 13.0 5.0, 4.7 9.4, 8.8

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53.0 53.0 15.4 6.1, 6.4 11.5, 12.1
53.3 53.5 14.4 5.3, 5.1 9.9, 9.5
54.1 53.9 14.5 5.0, 5.1 9.2, 9.5
52.4 52.5 16.3 6.3, 6.6 11.9, 12.6
53.7 54.0 14.0 13.9 4.9, 5.0 9.1, 9.3
51.3 14 14.6, 14.6 5.2, 4.9 10.1, 9.6
52.6 52.6 14 14.7 4.0, 5.8 7.6, 11.0
52.1 52.2 13 14.0 5.1, 5.0 9.8, 9.6
54.0 53.7, 53.6 15 14.7, 16.3 5.7, 5.3, 5.5 10.6, 9.9, 10.3
52.5 53.0 13 14.1 5.0, 5.2 9.5, 9.8
53.5 53.5 14.0 14.6 5.8, 5.8 10.8, 10.8
53.3 53.1 13.5 13.2 4.4, 4.4 8.3, 8.3
52.2 53.0 14 14.5 5.2, 5.2 10.0, 9.8
53.4 51.8 14 15.2 6.1, 5.8 11.4, 11.2
51.6 51.6 14 6.0 11.6
52.6 52.5 14 13.8 4.6, 4.7 8.7, 9.0
52.9 53.3 15 16.0 6.0, 6.3 11.3, 11.8
53.1 53.5 11.0 11.0 4.3, 4.7 8.8
8.1,
54.1 53.0 15.2 13.7 5.7, 7.3 10.5, 13.8

Av. c 52.9 14.3 5.4 10.2

Std dev. c 0.79 1.07 0.59 1.11
Coeff. of v a r . , % c 1.5 7.5 11.0 10.9

S a m p l e 4, T a n k a g e

63.9 63.8 8.9, 9.4 2.1, 2.1, 2.1 3.3, 3.3

64.5 64.6 7.9, 1.7, 1.8 2.6,
59.7 59.1 7.6, 2.0, 2.1 3.4,
63.9 63.0 8.2, 2.5, 1.8 3.9,
64.2 64.2 8.2, 2.4, 2.4 3.8,
63.4 63.1 8.7, 2.2, 1.9 3.
63.3 63.3 9.2, 2.8, 2.6 4.
64.0 64.0 9.2, 1.7, 1.7 2.
65.0 64.0 8.5, 2.1, 2.4 3.
63.7 62.8 9.4, 2.3, 2.3 3.
63.0 63.8 8.5, 8.0 1.7, 2.2 2.
63.3 8.4, 8.5, 2.0, 2.3 3.
63 63.6 8.4, 8.0 2.0, 2.4 3.
63 63.1 8.5, 8.2 1.9, 1.9 3.
63 63.8, 64.4 9.7, 8.8, 2.3, 2.0, 2.1 3. 3.3
63 64.1 8.5, 8.9 2.1, 2.1 3.
64 64.7 8.5, 8.6 2.6, 2.7 4.
63 64.1 8.3, 8.1 1.5, 1.3 2.
64 64.6 8.7, 8.7 1.8, 2.0 2.
63 64.5 7.9, 8.1 2.0, 2.0 3.
62.0 61.8 7.7 2.4 3.
64.0 63.8 8.5, 8.7 1.9, 2.1 3.
64.4 64.6 9.3, 8.9 1.2, 2.3 1.
64.1 64.5 8.1, 7.5 2.3, 2.0 3.
63.7 62.7 7.1, 7.0 2.2, 2.9 3,

Av.c 63.6 8.4 2.1 3.4

Std dev. c 1.07 0.54 0.30 0.48
C o e f f . o f var., %c 1.7 6.4 14.2 14.4

(Continued)
674 JOURNAL OF THE AOAC ( V o l . 5 4 , N o . 3 , 1 9 7 1 )

Table 1. (Continued)
Coll % Protein % Ind. Residue % Ind. Protein % Protein Indigest.
Sample 5, Fish Meal

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Av.c
Std dev.c
Coeff. of var., %'

Sample 6, Blood Meal

1 90.3, 90.3 1.2, 2.2, 2.2 1.1, 1.2, 1.3 1.2, 1.3, 1.4
2 89.9, 90.0 1.1, 1.2 0.7, 0.9 0.8, 1.0
3 89.5, 89.7 1.4, 1.4 0.8, 1.3 0.9, 1.4
4 89.7, 90.1 1.8, 1.7 1.6, 1.2 1.8, 1.3
5 90.6, 90.7 1.8, 1.5 1.3, 1.3 1.5, 1.5
6 88.8, 88.8 1.8, 1.6 1.2, 1.1 1.4, 1.2
7 90.2, 90.2 2.4, 2.6 1.9, 1.8 2.1, 2.0
8 88.3, 88.7 2.2, 2.1 1.2, 1.0 1.4,
9 89.8, 90.4 2.6, 2.1 1.6, 1.5 1.8,
10 89.4, 87.3 1.9, 2.0 1.2, 1.3 1.3,
11 90.1, 89.7 2.2, 1.8 0.4b 1.3 0.4b,
12 89.3 1.7, 1.8, 1.7 1.4, 1.3 1.6,
13 89.7, 89.2 89.0, 89.0 1.2, 1.5, 2.3, 1. } 1.4, 1.3, 1.6, 1.4 1.6, 1.5, 1.8. 1.6
14 87.8, 88.9 2.7, 2.5 1.5, 1.4 1.7, 1.6
15 91.3, 91.0 89.6 2.9, 2.5, 2.1 1.7, 1.1, 1.1 1.9, 1.2, 1.2
16 88.6, 88.9 2.3, 2.5 1.1, 1.2 1.2, 1.3
17 90.2, 90.2 2.3, 2.3 2.0, 2.0 2.2, 2.2
18 90.1, 90.2 1.9, 2.1 0.9, 1.1 1.0, 1.2
19 90.2, 89.0 2.0, 2.1 1.6, 1.0 1.8,
20 89.5, 88.8 1.3, 1.5 1.1, 0.9 1.2,
21 88.0, 88.0 1.5 1.2 1.4
22 90.0, 89.6 2.6, 2.9 1.1, 1.4 1.2,
23 91.1, 90.9 1.8, 2.0 1.3, 1.2 1.4,
24 90.7, 90.5 2.1, 2.1 1.2, 1.2 1.3,
25 90.2, 89.7 1.0, 1.7 1.4, 1.6 1.5,

Av.c 89.7 2.0 1.3 1.5

Std dev. c 0.83 0.43 0.26 0.29
Coeff. of var., %c 0.9 21.6 19.7 20.0

(Continued)
GEHRT: PEPSIN DIGESTIBILITY OF ANIMAL PROTEINS 675

Table 1. (Continued)

Coll. % Protein % Ind. Residue % Ind. Protein % Protein Indigest.

d
Sample 7, Poultry By-Product

1 64.8, 64.9 16.1, 16.2, 16.2 12.7, 12.6, 13.0 19.6 19.4, 20.0
2 64.0, 64.5 14.5, 15.2 11.4, 11.8 17.8 18.3
3 64.8, 64.4 15.9, 16.0 12.8, 12. 19.8 19.4
4 64.8, 64.3 14.8, 15.4 11.7, 11. 18.1 18.2
5 63.3, 63.7 19.0, 18.0 15.5, 14. 24.5 22.0
6 63.4, 63.5 16.8, 17.2 12.3, 12. 19.4 19.1

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7 63.4, 63.4 18.4, 18.4 14.9, 14. 23.5 23.2
8 62.8, 63.1 18.2, 18.3 13.7, 13. 21.8 20.8
9 64.6, 65.3 16.6, 15.8 12.5, 12. 19.3 18.8
10 62.7, 63.9 24.3, 20.0 19.7, 20. 31.4 31.9
11 64.6, 64.6 18.3, 17.5 14.1, 14.0 21.8 21.7
12 63.0 15.7, 15.8, 16.0 12.1, 12.0 19.2 19.1
13 63.6, 63.8 20.4, 19.6 17.0, 17.8 26.7 27.9
14 62.4, 63.2 23.0, 22.7 17.8, 17.7 28.5 28.0
15 65.1, 64.9, 65.1 18.7, 21.5, 19.5 14.7, 15.9 22.6 24.4
17 64.4, 64.7 16.6, 15.9 13.2, 13.2 20.5 20.4
18 64.6, 64.4 16.6, 17.4 13.1, 13.8 20.3 21.4
19 64.2, 64.8 15.6, 15.9 11.6, 12.0 18.1 18.5
20 63.7, 63.0 21.5, 20.0 17.4, 16.5 27.3 26.2
21 61.8, 62.0 21.1 24.0 6 38.7b
22 64.4, 64.5 14.2, 13.0 10.5, 9.7 16.3 15.0
23 64.7, 64.4 22.9, 23.0 19.6, 19.3 30.3 30.0
24 64.0, 64.0 13.2, 13.8 12.2, 12.4 19.1 19.4
25 64.3, 63.2 24.9, 25.3 22.6, 26.3 35.2 41.7
Av.c 64.0 18.2 14.6 22.8
Std dev. c 0.81 3.13 3.41 5.42
Coeff. of var. c 1.3 17.2 23.8
% 23.4

Sample 8, Hydrolyzed Feathers^

1 83.3 83.5 16.1 14.0, 15.4 14.2 11.2 17.0, 13.4

2 83.7 83.9 12.4 12.8 10.2 10.7 12.2, 12.8
3 80.3 81.9 14.3 14.9 12.3 13.1 15.3, 16.0
4 83.6 82.3 11.6 12.0 10.8 9.8 12.9, 11.9
5 83.5 84.5 9.8 6.6 9.2 5.9 11.0, 7.1
6 82.8 83.1 17.2 17.4 14.0 14.5 16.9, 17.4
7 82.3 82.3 16.8 17.0 14.3 14.3 17.4, 17.4
8 83.3 83.3 19.2 19.0 13.4 13.3 16.1, 16.0
9 83.1 82.6 17.2 14.1 14.0 11.0 16.8, 13.3
10 83.0 81.6 32.0 33.0 27.8 28.1 33.5, 34.4
11 83.2 83.8 15.4 16.1 13.9 13.6 16.6, 16.2
12 83.2 15.3 14.5, 14.2 12.6 12.5 15.2, 15.0
13 83.2 82.9 24.8 25.0 22.4 22.0 26.9, 26.5
14 82.7 82.6 25.7 24.6 21.6 20.4 26.1, 24.7
15 85.3 85.0, 83.6 19.0 24.0, 21.8 14.9 21.7, 17.3 17.5, 25.5, 20.7
17 84.0 84.2 15.7 15.6 13.9 14.8 16.5, 17.6
18 84.4 84.0 18.9 18.4 14.7 15.5 17.4, 18.5
19 83.4 83.2 13.2 13.5 10.4 10.0 12.5, 12.0
20 83.0 82.7 22.4 21.4 20.6 18.6 24.8, 22.5
21 81.0 81.2 25.1 13.6 16.7
22 83.5 83.3 12.2 11.8 9.0 9.0 10.8, 10.8
23 84.3 84.6 30.3 31.0 27.7 28.2 32.9, 33.3
24 83.2 83.0 12.8 13.4 13.2 , 13.5 6
l
16.5 b 16.3 b
25 85.0 83.8 32.8 33.7 16.4 17.9 19.3, 21.4

Av.c 83.2 18.8 15.3 18.3

Std dev. c 0.91 6.80 5.42 6.49
Coeff. of var. c 1.1 36.3 35.5 35.4
%
° Values shown in table are individual determinations made on different days.
6
Results inconsistent, not used in statistical analysis.
c
Calculated from average of each collaborator's results.
d
Collaborator 16 did not analyze Samples 7 and 8.
676 JOURNAL OF THE AOAC ( V o l . 5 4 , N o . 3 , 1971)

Table 2. Collaborators' results'for indigestible residues, indigestible proteins, and protein indigestibles:
modified filtration method

Coll % Ind. Residue % Ind. Protein % Protein Indigest.'

Sample 3, Meat Meal

1 14.0 14.2, 14.3 5.2, 9.8

2 13.8 15.3 5.2, 10.4
3 13.2 13.5 6.0, 11.6
4 14.7 13. 6.0, 10.5
5 14.5 14. 10.0

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6 14.6 13. 9.9
7 23.8' 24.
8 14.3 14. 10.1
9 14.6 14. 9.4
10 18.2 17.1 13.3
11 14.9 14.7 10.2
13 15.8 14.5 6.4, 11.8
14 13.6 13.8 4.9, 10.0
15 14.3 14.8 5.6, 10.2
18 13.6 13.4 4.8, 9.0
19 14.5 14.6 5 4, 10.5
20 14.7 15.0 5 5, 10.6
21 15.7 15.7 7. 6, 13.8
23 15.4 14.8 6, 4, 11.7
24 11.0 11.0 4. 3, 8.4
25 13.6 15.1, 15.0 6.1, 5.8 11.4

Av.rf 14.5 5.6 10.6

Std dev. d 1.22 0.65 1.32
Coeff. of var. 8.4 11.5 12.4

Sample 7, Poultry By-Product

1 18.3
2 18.4
3 22.3
4 17.3
5 21.4
6 21.1
7 18.6
8 23.5
9 20.3
10 29.4
11 22.3
13 27.2
14 24.7
15 23.2
18 20.5
19 18.9
20 23.2
21 33.9
23 29.4
24 19.2
25 30.1

Av. d 18.2 14.7 23.0

Std dev. d 3.21 2.81 4.58
Coeff. of var., % d 17.6 19.1 19.9

(Continued)

residue, indigestible protein, and protein-indi-
gestible data for all samples show excellent agree-
ment between replicates in individual laborato-
ries. Judging from the standard deviations and
coefficients of variation the agreement between
laboratories is satisfactor}^ for all samples except
poultry by-products and hydrolyzed feathers.
Coefficients of variation for blood meal (Sample
6) are high due to the very low level of indigestible
material present. It is noteworthy that the stan-
dard deviations and coefficients of variation for
total crude protein (Kjeldahl method) are some-
what larger than expected. In Samples 1, 2, 4, 5,
and 6 the standard deviations are larger than the
GEHRT: PEPSIN DIGESTIBILITY OF ANIMAL PROTEINS 677

Table 2. (Continued)
Coll. % In d. Residue % Ind. Protein % Protein Indigest. 6

S a m p l e 8, Hydrolyzed Feathers

1 13.6, 13.0, 14.3 11.6, 11.4 13.8

2 13.3, 14.0 11.6, 11.6 13.8
3 16.8, 16.5 13.7, 15.1 17.8
4 12.9, 12.0 11.2, 10.8 13.3
5 15.3, 14.9 12.3, 12.3 14.6
6 15.7, 16.2 12.4, 13.6 15.7

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7 14.0, 14.3 11.4, 11.4 13.9
8 18.9, 19.1 15.8, 16.3 19.3
9 17.7, 17.1 14.6, 13.8 17.1
10 29.7, 30.0 25.2, 25.7 31.0
11 17.8, 17.9 14.1, 14.1 16.9
13 25.2, 24.6 21.6, 21.4 25.9
14 20.8, 20.6 18.5, 17.7 21.9
15 20.6, 21.0 17.9, 17.9 21.2
18 16.8, 17.8 13.4, 14.6 16.6
19 14.0, 14.4 11.6, 11.6 13.9
20 21.7, 22.3 19.0, 19.4 23.2
21 32.2, 32.5 29.2, 28.4 35.5
23 28.4, 27.8 25.8, 24.9 30.1
24 12.8, 13.4 13.7C, 13.5C
25 30.7, 26.4, 27.0 28.2, 23.8, 24.2 30.1
d
Av. 19.4 16.9 20.3
Std dev. d 6.02 5.65 6.85
Coeff. of var. %d 31.0 33.5 33.8

"Values shown in table are individual determinations made on different days. Collaborators 12, 16, 17, and 22
did not re-analyze the samples.
b
Calculated by dividing average indigestible protein values by average total protein values from Table 1.
c
Results inconsistent, not used in statistical analysis.
d
Calculated from average of each collaborator's results.

standard deviations for indigestible residue and
indigestible protein, indicating slight non-uni-
formity in the individual collaborators' samples.
Since the samples were thoroughly mixed, this
non-uniformity must be due either to variations
inherent in unground samples or to differences
associated with the grinding and reblending of
the coarse portion of the samples as specified in
the method.
The modified method (45° angle settling and
acetone wash) improved the filtration rate of the
poultry by-product meal and hydrolyzed feathers
significantly. However, the agreement between
laboratories was no better than in the first study.
The Associate Referee feels that the variations
between collaborators with the poultry by-prod-
uct meal and hydrolyzed feathers are due to
differences in the filtration step. If these digestion
mixtures are not properly settled or if they are
poured onto the filter paper too rapidly, the
residues will clog the paper before most of the
liquid has passed through, resulting in incomplete
washing and high results. In our laboratory,
when the hydrolyzed feather sample was filtered
according to the above method, the filtration and
washing was complete within 5 min and the
agreement between replicates was excellent: 13.6,
13.0, 14.3, 13.8, 13.6, and 13.7% indigestible
residue.
At the request of the Associate Referee, the 4
collaborators who reported the highest indi-
gestible residue levels in the poultry by-product
meal and hydrolyzed feathers returned these
samples (and also Sample 3, the meat meal with
highest indigestible residue). The samples were
analyzed in triplicate in the Associate Referee's
laboratory. The indigestible residue results are
given in Table 3.
As shown in the table the agreement between
the samples returned by the collaborators and
analyzed by the Associate Referee was excellent.
This major improvement demonstrates con-
vincingly the effectiveness of the filtration step
when it is carried out according to directions. The
settled digestion mixture should be carried to the
Buchner funnel at the same angle as settled and
poured onto the filter as a continuous small
stream. The liquid passes through the paper as
678 JOURNAL OF THE AOAC (Vol. 54, N o . 3, 1971)

Table 3. Comparison of individual collaborators' results for per cent indigestible residues with
Associate Referee (AR) on same samples
Sample 7, Sample 8,
Sample 3, Poultry Hydrolyzed
Coll. Meat Mea 3y-Product Feathers

AR 14.0 14.2 14.3 15.8 15.5 15.5 13.6 13.0 14.3

10 18.2 17.1 23.8 25.1 29.7 30.0

AR 13.7 14.2 14.6 13.5 15.7 14.6 14.3 14.0 15.5

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21 15.7 15.7 24.6 25.3 32.2 32.5
AR 14.1 14.2 14.3 14.6 15.0 14.1 14.2 15.8 15.1

23 15.4 14.8 22.7 22.0 28.4 27.8

AR 13.8 14.2 13.9 14.7 14.5 16.0 15.8 15.5 14.9

25 13.6 15.1 15.0 23.5 21.7 21.5 30.7 27.0

AR 14.1 14.5 14.3 14.3 14.2 14.2 16.2 15.6 16.1

Std dev. a 1.22 3.21 6.02

Std dev.& 0.34 0.74 0.98
Coeff. of var." 8.4 17.6 31.0
Coeff. of var. 6 2.4 5.0 6.5

" All collaborators, Table 2.

6
Associate Referee's samples and samples from 4 collaborators; analyses made by Associate Referee.

fast as poured, with the residue gradually Table 4. Indigestible residues in collaborative
samples by official method vs. proposed
spreading over the surface of the filter but not filtration method
covering it completely until all or practically all
Sample Products Off. Prop."
of the liquid has passed through. Any unnecessary
agitation of the mixture in the bottle while it is 1 meat meal 8.7 9.0
2 meat meal 8.0 8.2
being poured should be avoided. 3 meat meal 13.9 14.3
Strict adherence to the technique described 4 tankage 7.7 8.4
above permits rapid filtration and complete 5 fish meal 4.5 5.3
6 blood meal 0.9 2.0
washing of the residues from all of the animal 7 poultry by-produ cts 15.3 18.2
products studied, including poultry by-product 8 hydrolyzed feath ers 15.3 19.4
meal and hydrolyzed feathers, with excellent a
Collaborators' average.
reproducibility of results.
Many of the collaborators expressed the feeling and acid-insoluble ash (sand) were determined
that the new method is a major improvement chemically on the samples. The Associate Ref-
over the official method, which isolates the indi- eree's laboratory also determined fat. These data
gestible residue by centrifuging, because the new are given in Table 5. The agreement between the
method is more rapid, less laborious, and less microscopically estimated indigestible residues
liable to residue losses by decantation. The levels and the collaborators' averages (from Tables 1
of indigestible residue as found by the official and 2) is noteworthy. Inclusion of ash (bone),
method and the new method were compared in blood meal, and fat in the table is not meant to
the laboratory of the Associate Referee. Results imply that these ingredients are indigestible, but
are given in Table 4 and show good agreement rather to give more complete information regard-
between the methods. The filtration method ing the composition of the samples.
gives very slightly higher results due undoubtedly
Conclusions and Recommendations
to slight losses of colloidal material when the
residues are isolated by centrifuging. The results of this year's study show that the
Although not related directly to the collabo- filtration method represents a major improve-
rative study, a microscopic identification and ment over the centrifuge method for all of the
estimation of the indigestible matter in the sam-
The recommendations of the Associate Referee were approved
ples was made in the laboratory of one of the by the General Referee and by Subcommittee A and were
collaborators. The blood meal content of the meat adopted by the Association, except that the pepsin digestibility
method was adopted as an alternative to 7,040-7.046. See
meals was also estimated and ash (mostly bone) J AOAC 54, 3§3 (1971).
G E H R T : PEPSIN DIGESTIBILITY OF ANIMAL PROTEINS 679

products studied except poultry by-product meal Table 5. (Continued)

and hydrolyzed feathers. This conclusion is based % How
on the rapidity and simplicity of the filtration Ingredient Present Measured™
step; the statistical data which are equal to that S a m Pl e 3 , Meat M eal
obtained in the 1958 and 1959 studies on which
adoption of the method as official was based; the Hoof, hair, hide trimmings 7.0 A
satisfactory agreement between laboratories in Stomach contents 5.2 A
Acid-insoluble ash 1.7 B
this year's study which involved unground sam-
ples whereas ground samples were sent to the Total indigestible 13.9

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collaborators in 1958 and 1959. Ash (mostly bone) 23.1 B
It is therefore recommended— Fat 12.5 C

(1) That the official pepsin digestibility

Sample 4, Digester Ta nkage
method, 7.040-7.046, be repealed for all animal
proteins except poultry by-product meal and hy- Stomach contents 6.0 A
drolyzed feathers and retained for these 2 Hoof, hair 0.7 A
Acid-insoluble ash 0.6 B
products.
(2) That the method described in this report Total indigestible 7.3
be adopted as official first action for all animal Ash (mostly bone) 10.9 B
proteins except poultry by-product meal and hy- Fat 12.0 C
drolyzed feathers.
Sarr Pi e 5 , Fish Meal
(3) That the study be continued to fully re-
solve the filtration problem with poultry by- Only traces of contaminants found—
product meal and hydrolyzed feathers. such as iron filings; small amount
of material might be overcooked
Acknowledgments Acid-insoluble ash (sand) 1.0
Ash (mostly bone) 21.0
The Associate Referee wishes to thank the Fat 8.8
following collaborators for their excellent co-
operation: Sample 6, Blood Meal
L. A. Baggenstoss, W. 11. Grace & Co.. At- Stomach contents (indigestible) 1.0 A
lantic, Iowa Ash (mostly bone) 2.2 B
Fat 0.3 C

Table 5. Microscopic evaluation of collaborative

samples Sample 7, Poultry By-Product Meal

% How Sample appears to be poultry

Ingredient Present Measured™ hatchery by-product. Many tiny
Sample 1, Meat Meal feathers; egg albumen, egg shell
present
Hoof, hair, trace of feathers 3.9 Feathers 16.3 A
Stomach contents 3.6 Vegetable fiber 1.0 A
Acid-insoluble ash (sand) 1.0 Acid-insoluble ash (sand) 0.3 B

Total indigestible 8.5 Total indigestible 17.6

Ash (mostly bone) 26.1 Ash (mostly bone) 7.2 B

Blood meal 5.5 Fat 18.9 C
Fat 10.8
Sample 8, Hydrolyzed Feathers
Sample 2, Meat Meal

Hoof, hair 4.4 Unhydrolyzed and/or partially

Stomach contents 2.0 unhydrolyzed feathers 11.8 A
Acid-insoluble ash 0.8 Acid-insoluble ash (sand) 0.9 B

Total indigestible 7.2 Total indigestible 12.7

Ash (mostly bone) 24.1 Ash (mostly bone) 5.7 B

Blood meal 1.3 Fat 3.8 C
Fat 12.9
™A = microscope; B = chemical analysis; C = ex-
(Continued) traction.
680
H. E. Bagnall, Jr., Farmland Industries, Inc.,
Kansas City, Mo.
R. J. Buswell, Armour and Co., Oakbrook, 111.
P. L. Cox, Felco, Ft. Dodge, Iowa
R. H. Durr, Ingman Laboratories, Inc., Minne-
apolis, Minn.
Georgia P. French, University of Massachu-
setts, Amherst, Mass.
Mary Heckman, Ralston Purina Co., St.
Louis, Mo.
Norma H. Holm, Board of Agriculture Labo-
ratory, Topeka, Kan.
R. H. Jones, Darling & Co., Chicago, 111.
H. Kuechenmeister, Geo. A. Hormel & Co.,
Austin, Minn.
J. R. Ledin, Woodson-Tenerit Laboratories,
Des Moines, Iowa
G. W. Lenser, Nebraska Consolidated Mills
Co., Omaha, Neb.
L. Jann, Department of Agriculture, Spring-
field, 111.
V. C. Midkiff, Agricultural Experiment Sta-
tion, Lexington, Ky.
J. P. Minyard, Jr., Mississippi State Chemical
Laboratory, State College, Miss.
R. J. Noel, Agricultural Experiment Station,
Lafayette, Ind.
H. Olson, Hubbard Milling Co., Mankato,
Minn.
G. Knorr, Agway, Inc., Buffalo, N.Y.
JOURNAL OF THE AOAC ( V o l . 5 4 , N o . 3 , 1 9 7 1 )
G. Sakell, American Meat Institute Founda
tion, Chicago, 111.
D. E. Shady, Central Soya Co., Decatur, Ind.
D. E. Sims, Moorman Mfg. Co., Quincy, 111.
K. H. Spitzmueller, Swift & Co., Oak Brook,
111.
R. N. Tulloch, Department of Agriculture and
Commerce, Richmond, Va.
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D. Whitson, Department of Agriculture, Des
Moines, Iowa
Janet B. Windsor, Cargill, Inc., Minneapolis,
Minn.
Special appreciation is directed to the follow-
ing: Merle A. Altemeier, Iowa Department of
Agriculture, Des Moines, for his work in develop-
ing the filtration technique using the glass fiber
paper; Marvel Herrera, Iowa Department of
Agriculture, Des Moines, for the microscopic
estimations and ash and acid-insoluble ash
analyses; Burks Oakley, Moorman Mfg. Co.,
Quincy, 111., for the statistical analysis.
REFERENCES
(1) Official Methods of Analysis, 11th Ed., AOAC,
Washington, D.C., 1970.
(2) Elmslie, W. P., JAOAC 43, 320-328 (1960);
"Changes in Methods," JAOAC 43, 156 (I960).
This report of the Associate Referee was presented at the
84th Annual Meeting of the AOAC, Oct. 12-15, 1970, at
Washington, D.C.