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Pepsin Digestibility Method For 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 Biichner funnel and determined by washing, dry-
of animal proteins, 7.040-7.046, has been i m - ing, and weighing.
proved by replacing the centrifuge isolation
7.A04 Preparation of Sample H 2 0 . Carry bottle from rack to filter at same angle
Sieve sample, 7.001, thru No. 20 sieve. Grind as settled and pour contents slowly thru filter,
portion retained on sieve to pass No. 20 sieve. Com- retaining as much residue as possible in bottle.
bine both portions and blend by stirring and shaking Filtration proceeds rapidly with moderate suction
in pt jar. Thoro blending is essential. Because of until appreciable amts of residue cover filter surface.
high fat content of many animal products, grinding If filtration rate becomes slow, it may be accelerated
without sieving may cause sticking in mill, loss of with acetone, but only if no significant amt of aq.
moisture or fat, or poorly blended sample. digestion mixt. remains on funnel when acetone is
added. Settling residue at 45° angle followed by
Table 1. Collaborators' results" for total proteins, indigestible residues, indigestible proteins,
and protein indigestibles
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
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
(.Continued)
G E H R T : PEPSIN DIGESTIBILITY OF ANIMAL PROTEINS 673
Table 1. (Continued)
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
S a m p l e 4, T a n k a g e
(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
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,
(Continued)
GEHRT: PEPSIN DIGESTIBILITY OF ANIMAL PROTEINS 675
Table 1. (Continued)
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
Table 2. Collaborators' results'for indigestible residues, indigestible proteins, and protein indigestibles:
modified filtration method
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
(Continued)
residue, indigestible protein, and protein-indi- Coefficients of variation for blood meal (Sample
gestible data for all samples show excellent agree- 6) are high due to the very low level of indigestible
ment between replicates in individual laborato- material present. It is noteworthy that the stan-
ries. Judging from the standard deviations and dard deviations and coefficients of variation for
coefficients of variation the agreement between total crude protein (Kjeldahl method) are some-
laboratories is satisfactor}^ for all samples except what larger than expected. In Samples 1, 2, 4, 5,
poultry by-products and hydrolyzed feathers. 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
"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 according to the above method, the filtration and
indigestible protein, indicating slight non-uni- washing was complete within 5 min and the
formity in the individual collaborators' samples. agreement between replicates was excellent: 13.6,
Since the samples were thoroughly mixed, this 13.0, 14.3, 13.8, 13.6, and 13.7% indigestible
non-uniformity must be due either to variations residue.
inherent in unground samples or to differences At the request of the Associate Referee, the 4
associated with the grinding and reblending of collaborators who reported the highest indi-
the coarse portion of the samples as specified in gestible residue levels in the poultry by-product
the method. meal and hydrolyzed feathers returned these
The modified method (45° angle settling and samples (and also Sample 3, the meat meal with
acetone wash) improved the filtration rate of the highest indigestible residue). The samples were
poultry by-product meal and hydrolyzed feathers analyzed in triplicate in the Associate Referee's
significantly. However, the agreement between laboratory. The indigestible residue results are
laboratories was no better than in the first study. given in Table 3.
The Associate Referee feels that the variations As shown in the table the agreement between
between collaborators with the poultry by-prod- the samples returned by the collaborators and
uct meal and hydrolyzed feathers are due to analyzed by the Associate Referee was excellent.
differences in the filtration step. If these digestion This major improvement demonstrates con-
mixtures are not properly settled or if they are vincingly the effectiveness of the filtration step
poured onto the filter paper too rapidly, the when it is carried out according to directions. The
residues will clog the paper before most of the settled digestion mixture should be carried to the
liquid has passed through, resulting in incomplete Buchner funnel at the same angle as settled and
washing and high results. In our laboratory, poured onto the filter as a continuous small
when the hydrolyzed feather sample was filtered 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
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
H. E. Bagnall, Jr., Farmland Industries, Inc., G. Sakell, American Meat Institute Founda
Kansas City, Mo. tion, Chicago, 111.
R. J. Buswell, Armour and Co., Oakbrook, 111. D. E. Shady, Central Soya Co., Decatur, Ind.
P. L. Cox, Felco, Ft. Dodge, Iowa D. E. Sims, Moorman Mfg. Co., Quincy, 111.
R. H. Durr, Ingman Laboratories, Inc., Minne- K. H. Spitzmueller, Swift & Co., Oak Brook,
apolis, Minn. 111.
Georgia P. French, University of Massachu- R. N. Tulloch, Department of Agriculture and
setts, Amherst, Mass. Commerce, Richmond, Va.