In vitro Simulation of Gastric and
Pancreatic Phase Digestion of Meat and
Bone Meal: Defining Optimal Conditions
for Maximizing the Efficiency of Enzymes

Y. Qiao and T. van Kempen

Summary

The conditions to maximize the effects of digestive protease were defined for the two-stage digestion model. For the 1^st stage, the incubation time, the pH of the buffer, the concentration of the MBM protein were 24 hours, 0.1 M CBS pH 2.0 and 12.5 mg/ml, respectively. For the 2^nd stage, they were 72 hours, pH 7.0 and 5.0 mg/ml, respectively. Theoretically, at each pepsin level, there is a minimal level of PT for achieving maximal hydrolysis of MBM proteins. It was found that pepsin was not indispensable to achieve maximal ADH in the digestion model, and pepsin was inefficient to maximize the hydrolysis. In order to maintain the model, pepsin usage of 0.25% was subjectively chosen. Once the usage of pepsin was selected, subsequent work would focus on minimizing the usage of PT to maximize the hydrolysis of MBM proteins.

Introduction

Due to concerns of time and cost required for in vivo assays of feedstuffs, researchers have approached in vitro techniques as an alternative for evaluation of protein quality and available amino acids. However, conditions for in vitro digestion vary considerably. The pH could change from 2 to 4 for pepsin incubation, from 6 to 8 for pancreatic enzyme incubation, and from fixed to variable during the incubation. The incubation time differed from as short as 10 minutes to 72 hours. The amount of enzyme used ranged from 0.025% to 20% of the feed proteins. Given this information, it is extremely difficult to compare results obtained from different laboratories.

Our goal is to set up an in vitro experimental system that bypasses the in vivo assay for amino acid analysis. This system should be able to maximize the hydrolysis of meat and bone meal (MBM) proteins with minimal enzyme level. To do this, it is necessary to define the incubation conditions and the relationship of digestive enzymes in term of increasing the hydrolysis of substrate proteins.

Materials and Methods

Meat and bone meal (MBM) was obtained from several mills. One MBM was randomly chosen from over 150 MBM samples to study the conditions for optimal digestion by PP and PT.

A two-stage in vitro digestion system was adopted. Briefly, porcine pepsin (PP) was used for incubation of MBM in acidic citrate buffer solutions (CBS), followed by adding phosphate buffer solution (PBS) to adjust pH that is more favorable for the digestion by porcine pancreatic enzymes (PT). The PT contained pancreatin and crystalline trypsin (5/1 ratio, pancreatin to trypsin) to boost the digestive power of the enzyme mixture. The buffer contained 0.06% sodium azide to prevent microbial fermentation. Enzymes and MBM were hosted in 50ml Corning screw capped test tubes. For the first stage (PP), 20 ml of 0.1 M CBS (pH 2 or pH 4) was used. For the second stage (PT), 30 ml of phosphate buffer solution (PBS, 0.2 M, pH 8.0, then raised to pH 12 by adding NaOH) was added, and the final pH was around 7. Usually 500 mg of MBM was weighed into each test tube. Test tubes were fitted into a revolving plate to facilitate enzyme-MBM contact and incubated at 38 C. At different time intervals, a small aliquot of the digesta (usually 25-100 microliters) was pipetted out and mixed with equal volume of 20% sodium dodecyl sulfate (SDS) to denature the enzyme. After centrifugation at 14,000 g for 5 minutes, the supernatant was pipetted out for analysis of hydrolysis of MBM proteins using the o-pthalaldehyde (OPA) method.

Because the products of MBM and enzyme were not separable using OPA method, it was the apparent degree of hydrolysis (ADH) that was measured. ADH was defined as

Where [Pmbm] was the product from MBM, [Penzyme] was the product from enzyme autolysis; [Si] was the initial substrate.

Results

Experiment 1: Determination of Incubation time for the 1^st stage

MBM (12.5mg protein/ml) was incubated with pepsin (0.25%, pepsin to MBM protein ratio) at 38 C. At different hours after inoculation, an aliquot was taken for measurement of ADH. The results are shown in Figure 1.

Figure 1. Time course of pepsin digestion of MBM in 0.1 M CBS pH 4.0 at 38 C. PP/MBM protein ratio was 0.25%. Results of 5 replicates.

It was demonstrated that 24 hours of incubation was sufficient. This agreed with the study of pepsin activity decay in the incubation with MBM (Qiao and Van Kempen, unpublished data). In that case, the half-life of pepsin was about 3.3 hours, and the time needed to deactivate 95% of pepsin was 14.3 hours.

Experiment 2: Determination of Incubation time for the 2^nd stage

MBM (5.0mg protein/ml) was incubated with 1.0% PT at pH 7.0 after digestion by 0.25% of pepsin for 24 hours. The ADH was measured at different time after inoculation. The results are shown below in Figure 2.

Figure 2. Time course of digestion of MBM by PT in PBS pH 7.0 following the digestion by 0.25% PP for 24 hours. PT/MBM protein was 1.0%. Results of 3 replicates.

The results showed that at least 72 hours of incubation should be used in order to allow the enzyme to work maximally. Therefore, at least 72 hours was used for the 2nd stage incubation.

At 72 hours, the trypsin activity was lost slight over 50% (Qiao and van Kempen, unpublished data). The fact that the ADH virtually stopped to improve at 72 hours post inoculation even if the trypsin activity was still about half indicated that other enzymes than trypsin was limiting for the digestion.

Experiment 3: Determination of initial pH for the 1^st and 2^nd stage incubation.

Initially we used CBS pH 4.0 for the first incubation. To optimize the conditions for the 1^st stage incubation, we examined the effects of CBS pH 2.0 and pH 4.0. MBM protein was incubated with 1.0 % of pepsin (pepsin to MBM protein ratio) in different pH buffer. The results are presented in Figure 3 below.

Figure 3. The effects of initial pH on ADH (%). Replicates of 3. MBM was incubated in 0.1 M CBS with 1% pepsin for 24 hours.

More acid pH (2.0) significantly increased the ADH. Therefore, CBS pH 2.0 was chosen for future assays.

In the 2nd stage, the effects of pH 6.5 and pH 7.0 were not significantly different. We selected pH 7.0 as the ambient pH for the 2^nd stage digestion.

Experiment 4: The effects of initial MBM protein concentration on ADH in the 1^st stage. In order to find the suitable substrate concentration for incubation, three levels of MBM protein concentration (6.25, 12.5, 25 mg/ml) were used for incubation with pepsin (1%, pepsin to MBM protein ratio). ADH was measured at different hours after inoculation. The results are presented in Figure 4.

Figure 4. The effects of pepsin on the kinetics of MBM digestion. Replicates of 3. MBM was incubated with CBS pH 2.0.

The difference between 6.25 mg/ml and 12.5 mg/ml was not significant. At higher concentrations of 25mg protein/ml, both the rate of digestion and the endpoint of the ADH of MBM were negatively effected. Therefore, 25.0 mg/ml was excluded.

Experiment 5: The effects of initial MBM protein concentration on ADH in the 2^nd stage.

After digestion of MBM protein of 6.25, 12.5, 25 mg/ml with 1% pepsin for 24 hours, the MBM was diluted to 2.5, 5.0, 10.0 mg/ml, respectively by adding PBS buffer. They were further incubated with 1.0% PT. ADH was measured at 72 hours after inoculation. The results are shown in Figure 5 below.

Figure 5. The effects of MBM protein concentration on ADH in the 2^nd stage of digestion by 1% PT. Results of 3 replicates. ADH was measured 72 hours after inoculation of PT.

At higher concentration (10 mg/ml), MBM ADH was greater than lower concentration (2.5 mg/ml). The 5 mg/ml was neither different from the two sides. Therefore, from the results of Experiment 4 and 5, MBM protein concentration of 12.5 mg/ml and 5.0 mg/ml were chosen for the 1^st and 2^nd stage digestion, respectively.

Experiment 6: Determination of pepsin usage in the 1^st stage

MBM (protein 12.5 mg/ml) was incubated with different pepsin levels (0.5%, 1.0%, 4.0%, 10%, pepsin to MBM protein ratios) in 0.1 M CBS pH 4.0. ADH was measured at 24 hours after inoculation. The results are presented in Table 1.

Table 1. ADH of MBM proteins after incubation with different pepsin levels for 24 hours. Values are means with at least 5 replicates. Significant difference (p<0.05) is shown by the different superscripts.

PP/MBM protein (%)	ADH (%)
0.5	2.2^c
1.0	3.4^bc
4.0	4.0^b
10.0	7.8^a

Increasing the pepsin/MBM ratio did not result in desired increase in ADH. In contrast, 1% PT could increase the ADH from 3% to over 20% (Experiment 2). From the perspective of minimizing enzyme proteins, it is undesirable to use pepsin to increase the digestion of MBM.

Experiment 7: The relationship between pepsin and PT usage

To test the importance of pepsin to reach maximal hydrolysis of MBM proteins in this 2 stage model, MBM protein (12.5 mg/ml) was incubated in 0.05 M CBS buffer with 7 levels (0, 0.25, 0.50, 1.0, 1.5, 2.0 and 3.0%) of pepsin for 24 hours. Then, in the 2^nd stage, PT of 5 levels (0, 0.25, 0.50, 1.0, 1.5, 2.0, 3.0, 4.0 and 5.0%) was inoculated and MBM protein at 5.0 mg/ml was further incubated at pH 7.0 for 48 hours. The ADH, measured at 48 hours in the 2^nd stage, was presented in the 3-D graph below.

Figure 6. The effects of combinations of pepsin and PT usage on ADH. MBM was first digested with pepsin, then with PT. All units are in percent (%).

Although the relationship between ADH, pepsin and PT usage is nonlinear and complex, this graph shows that the effects of pepsin were minimal in achieving maximal hydrolysis of MBM proteins. Statistics show that at PT levels of 4.0% and 5.0%, the ADH reached maximum regardless of the action of pepsin. At PT levels of 3.0%, 4.0% and 5.0%, the effects of pepsin became insignificant. The results suggested that pepsin was not indispensable for the ADH to reach plateau. It was concluded that in the subsequent studies, PT rather than pepsin should be focused in order to improve the degree of hydrolysis of MBM. A level of 0.25% pepsin was thus chosen subjectively for the 1^st stage digestion.