In vitro Simulation of Gastric and

Pancreatic Phase Digestion of Meat and Bone

Meal: Studies of Autolysis of Pancreatic Enzymes

Y. Qiao and T. van Kempen

Summary

It was demonstrated that the deactivation of pancreatic enzyme cocktail is achieved more by autolysis than simple denaturation. Under more unfavorable conditions (less favorable pH, higher concentration), less autolysis and more denaturation will take place. The autolysis of the pancreatic enzyme cocktail (PT) showed impacts of pH and concentration. The autolysis of PT was faster at more favorable pH (pH 8.0 versus pH 6.0). Without MBM protein, the higher the concentration, the slower the autolysis of trypsin was, and the rate of breakdown of peptide bonds was inversely correlated to the concentration of PT. With the presence of MBM protein, the higher the concentration, the faster the autolysis of trypsin was. MBM was found to greatly extend the half-life of trypsin. The pH and concentration of PT had significant effects on the maximal degree of autolysis (c). At more favorable pH (pH 8.0 versus pH 6.0), the value of c was greater. The c values of PT varied within a narrow range. On average, the maximal degree of autolysis was 22.3%, and the autolysate contained 4.9 amino acid residual.

Introduction

Proteolytic enzymes have a unique feature. Under biological conditions, such an enzyme molecule will digest other enzyme molecules of the same nature as if they are substrate. This hydrolytic process is referred to as autolysis. In the field of in vitro digestion of feed proteins, most researchers have neglected the contamination of enzyme autolysates on the measurement of amino acid. This negligence of autolysis should result in overestimation of digestible amino acids, particularly when a relatively large quantity of enzymes is used.

In our study of in vitro digestion of meat and bone meal (MBM), the hydrolysis of MBM protein was measured by o-pthalaldehyde (OPA). In order to obtain the true hydrolysis of MBM, the hydrolysis of digestive proteases needs to be estimated. We therefore studied the autolysis of pancreatic enzymes (PT).

Materials and Methods

Porcine pancreatic enzymes (PT) were incubated in phosphate buffer solution (PBS, pH 6.0 or pH 8.0). The PT consisted of porcine pancreatin and crystalline trypsin (ratio 5/1, weight basis). Trypsin was added to boost the digestive power of the enzyme mixture. The buffer contained 0.06% sodium azide to prevent microbial fermentation.

All incubation test tubes were fitted into a revolving plate to facilitate the contact between enzyme molecules and enzyme-MBM proteins. The incubation was done at 38 C. For measurement of degree of autolysis, at different time intervals, a small aliquot of the digesta was pipetted out and mixed with at least equal volume of 20% sodium dodecyl sulfate (SDS) to denature the enzyme. After centrifugation at 14,000 g for 5 minutes, the supernatant was used for analysis with the OPA technique. For measurement of enzyme activity decay, benzoyl-L-arginine ethyl ester (BAEE) and benzoyl-L-tyrosine ethyl ester (BTEE) were used as substrates for trypsin and chymotrypsin activity assay, respectively. At time intervals, a small aliquot (usually 5-50 microliters) was taken out from the incubation and the activity was measured immediately. Enzyme activity was expressed in units, defined as an increase of 0.001 units of absorbance per minute per mg of enzyme.

Experiment 1: The autolysis of PT when there was no MBM

PT at three levels was incubated in 0.1 M PBS, pH 6.0 or 8.0 at 38 C. At time intervals, a small aliquot was taken out for measurement of activities of trypsin with 0.5 mM BAEE and chymotrypsin with 1 mM BTEE at 25 C. Data were analyzed with the model [Et]=[Ei] e^{–kd t}, where [Et] is the enzyme activity at time t, [Ei] is the initial enzyme activity, kd is the rate of activity decay, t is time. There were three replicates in each treatment. The rate of decay (kd) and the half life (t _½) are presented in Table 1.

Table 1. The rate of activity decay at different concentration and pH 8.0.

	Trypsin		Chymotrypsin
PT (mg/ml)	kd^1 (1/hr)	t½ (hrs)	kd^1 (1/hr)	t½ (hrs)
0.05	0.41^ae	1.7	1.35^b	0.5
0.20	0.38^e	1.8	0.96^c	0.7
0.40	0.30^e	2.3	0.62^ad	1.1
^1Value was obtained from nonlinear regression of 3 replicates. Different superscripts imply statistical significance based on confidence intervals calculated from the asymptotic standard error (p<0.05).

In the most robust case with PT concentration of 0.40 mg/ml, trypsin lost 95% of initial activity by 10 hours after inoculation, chymotrypsin lost 95% by 5 hours after inoculation. Lower PT resulted in significantly faster decay of chymotrypsin and a tendency of faster decay of trypsin.

Experiment 2: The degree of autolysis of PT

PT at different concentrations was incubated at either pH 6.0 or pH 8.0 at 38 C. At time intervals, the peptide bond release [Penzyme] was measured using the OPA method. Data were analyzed using [Penzyme]=[Ei](1-e^{-ka t}). The degree of autolysis (DA) was calculated from DA=[Penzyme]/[Ei]. [Ei] is the initial peptide bond concentration, ka is the rate of release of peptide bonds, t is time. Figure 1 presents the data obtained for PT at different concentrations incubated in PBS pH 8.0 at 38 C.

Figure 1. Pancreatic enzyme cocktail (PT) at different concentration was incubated in 0.1 M PBS pH 8.0. Results of 2 replicates. The degree of autolysis (DA) was calculated from absorbance. The results demonstrated that the DA converged to a plateau.

The rate of autolysis (ka) and the time needed (t_½) to reach 50% of the maximal degree of autolysis (c) are presented in Table 2.

Table 2. The rate of autolysis (ka) of PT at different concentration and pH measured by the release of free amino group from PT without MBM. The half-life, t _½ (hrs), stands for the time needed to reach 50% of maximal degree of autolysis (c).

	pH 6.0		pH 8.0
PT (mg/ml)	ka^1 (1/hr)	t½ (hrs)	ka^1 (1/hr)	t½ (hrs)
0.05	0.2253^ad	3.1	0.5933^c	1.2
0.20	0.2142^a	3.2	0.4119^bc	1.7
0.40	0.1490^a	4.7	0.3458^bd	2.0
^1Value was obtained from nonlinear regression of 2 replicates. Different superscripts imply statistical significance based on confidence intervals calculated from the asymptotic standard error (p<0.05).

Statistics show that there was a significant effect of pH and the concentration of PT on the rate of autolysis. Like pepsin, the rate of release of peptide bonds from the enzyme cocktail was inversely correlated to the concentration. More optimal pH facilitated the breakdown of the enzymic peptide bonds.

The maximal degree of autolysis (c), predicted from [DA]=c (1-e^{-ka t}) when the t à ¥, is shown in Table 3.

Table 3. The maximal degree of autolysis (c) of PT at different concentration and pH measured by the release of free amino group from PT without MBM.

	c^1 (%)
PT (mg/ml)	pH 6.0	pH 8.0
0.05	21.5^a	22.8^a
0.20	15.4^b	22.7^a
0.40	14.9^b	24.3^a
^1Value was obtained from nonlinear regression of 2 replicates. Different superscripts imply statistical significance based on confidence intervals calculated from the asymptotic standard error (p<0.05).

Statistics shows that there were effects of pH and concentration of PT on the value of c. Nonetheless, the range of variation was not large. The average of degree of autolysis was 20.3%. This implied that the average peptide length was about 4.9 amino acid residual. Compared to 9.5 amino acid residual for pepsin autolytic products, this figure suggested that PT digested itself to a greater extent than pepsin did.

The degree of autolysis was significantly different from 0. This implied breakdown of the enzymatic peptide bonds during loss of activity. This supported the assumption that under in vitro conditions the inactivation of enzymes was mainly achieved by autolysis rather than pure denaturation.

Experiment 3: The decay of trypsin in the presence of MBM proteins

The activity of trypsin was measured after inoculation of PT following the digestion of MBM with 1% pepsin. Five mM BAEE was used as the substrate for the trypsin activity assay. MBM protein concentration was 5 mg/ml. PT levels were 0.05, 0.30 and 0.55 mg/ml, respectively. The results are presented in Table 4.

Table 4. Rate of decay (kd) of trypsin when PT was incubated with MBM. The rate of decay (kd) was obtained by nonlinear regression using the model [Et]=[Ei] e^{-kd t}. The half life was calculated using the formula t _½=ln2/kd.

PT (mg/ml)	kd^1 (1/hr)	t½ (hrs)
0.05	0.009^a	77
0.30	0.021^b	33
0.55	0.033^c	21
^1Values were obtained from nonlinear regression of 3 replicates. Different superscripts imply statistical significance based on confidence intervals calculated from the asymptotic standard error (p<0.05).

The half-life of trypsin in the presence of MBM was significantly longer than the half life of trypsin without MBM (Experiment 1). This provided evidence for the protective effects of substrate for enzymes.

Furthermore, when there was MBM, the half-life of trypsin became longer when the enzyme concentration decreased. In Experiment 1, when there was no MBM, the half- life of trypsin tended to become shorter when the enzyme concentration decreased. The presence of MBM reversed the tendency of changes in the half-life of trypsin.

It is proposed that the enzyme/substrate ratio played a role. When the enzyme/substrate ratio was lower, the chance of enzymes to meet and digest each other decreased. Therefore, trypsin activity lasted longer in PT of 0.05 mg/ml than in PT of 0.30 mg/ml, and PT of 0.30 mg/ml longer than PT of 0.55 mg/ml.