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)
D. Lemos et al.rAquaculture 186 (2000 89–105 95
Ten microliters of the sample are added to 1 ml of the substrate solution at 378C and the
change of absorbancy at 410 nm is recorded for 5 min ŽErlanger et al., 1961 .
.
Chymotrypsin activity is evaluated using 0.1 mM N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-
p-nitroanilide ŽSAPFNA in 0.1 M Tris–HCl, pH 7.8, containing 0.01 M CaCl .
.
2
Samples of 10 ml are mixed with 0.69 ml of substrate solution and the absorbancy at
410 nm is recorded over 3 min at 258C ŽDelMar et al., 1979 . Each assay must include
.
blanks in which the changes in absorbancy are recorded without the enzyme extract. The
relative importance of trypsin and chymotrypsin to the total proteinase activity can be
determined by the incubation of enzyme extracts with specific inhibitors ŽTable 1 prior
.
to the incubation with azocasein as described above ŽGarcia-Carreno, 1992b; Garcia-
˜
Carreno et al., 1994 .. Total proteinase, trypsin and chymotrypsin activities can be
˜
expressed as the change in absorbancy per minute per milligram of protein of the
enzyme extract used in the assays or as international units ŽIU of mmol of substrate
.
cleaved per minute, based on the substrate extinction coefficient.
2.5. In Õitro determination of protein digestibility by pH-stat

For an ingredient of suitable essential amino acid composition, the quality of protein
can be evaluated by measuring the digestibility of the ingredient or the feed containing
it. The use of a pH controller as the pH-stat ŽMethohom Ion Analysis, Switzerland , an
.
enzyme extract ŽDimes and Haard, 1994; Ezquerra et al., 1997a, 1998 , and the concept
.
of degree of hydrolysis ŽDH for the in vitro determination of digestibility is based on
.
the fact that the hydrolysis of protein peptide bonds produces a pH drop in the mixture.
The DH of a particular feed can be estimated during the digestion process by the
˜
automatically recorded titration curve ŽDimes and Haard, 1994; Garcia-Carreno et al.,
1997 .. Feeds substrates are homogenized in water and the amount of crude protein
Ž
.
must be adjusted to 8 mg ml y1 ŽEzquerra et al., 1997a . Ten grams of the substrate
.
suspension is added to the hydrolysis vessel and the pH adjusted to 8.0 with 0.1 N
NaOH. The reaction is started with the addition of 1 ml of shrimp enzyme extract
ŽpHs8.0 . The pH is automatically maintained at 8.0 and the volume of 0.1 N NaOH
.
released is recorded. The assay can be conducted at different temperatures from 238Cto
278C ŽEzquerra et al., 1998 . The protein hydrolysis is calculated from the algorithm:
.
x.
DH%s Ž B=N =1.5rM= wS%r100 r8 100
b
where Bsvolume Žml of standard alkali 0.1 N NaOH required to maintain the pH of
.
.
Ž
the reaction mixture at 8.0; N snormality of the titrant; Msmass Ž. g of reaction
b
mixture; Ssprotein concentration in the reaction mixture ŽAdler-Nissen, 1986 .
.
The use of the pH-stat method to monitor protein digestibility offers some advan-
tages: Ž. 1 the rate of hydrolysis can be estimated quickly during the digestion process by
the automatically recorded titration curve; Ž. 2 constant pH during the digestion process
without the use of a physiological buffer ŽPedersen and Eggum, 1983 ; 3 the method is
. Ž .
˜
sensitive to trypsin inhibitors in protein ingredients ŽGarcia-Carreno et al., 1997 ; and
.
Ž. 4 it significantly correlates with in vivo apparent protein digestibility APD Ezquerra
. Ž
Ž
et al., 1998 ..