In vitro Simulation of Gastric

and Pancreatic Phase Digestion of

Meat and Bone Meal: Maximizing the

Hydrolysis with Minimal Enzyme Usage

Summary

An in vitro digestion system was established to mimic the gastric and pancreatic phase digestion of MBM. It was shown that the hydrolysis of MBM was maximized at minimal costs of enzymes. The cost was about 5% MBM protein. The average peptide length of the digest was 3.6 amino acids.

Introduction

The in vitro technique as an alternative for evaluation of protein quality and available amino acids in feedstuffs has been widely used. However, the conditions for in vitro digestion varied 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. As a consequence of these subjective treatments, feed proteins are hydrolyzed to various degrees. In most cases, they are not maximally hydrolyzed.

We have defined the conditions to optimize the efficiency of the digestive proteolytic enzymes in our model. We also found that pepsin was inefficient to increase the hydrolysis of meat and bone meal (MBM), and it was dispensable for maximal hydrolysis. To simulate the gastric and pancreatic phase digestion, we chose a low level of pepsin for subsequent work and concentrated on the pancreatic enzymes to reach maximal degree of hydrolysis. This paper will report the final estimation of the amount of enzymes needed to achieve maximal hydrolysis based on the test of the hypothesis that upon sufficient action of enzymes, the hydrolysis of MBM would reach a plateau in our 2-stage digestion model.

Materials and Methods

MBM was used as it was obtained from the mills. Nine MBM were randomly chosen from over 150 MBM samples to estimate the average amount of enzymes required for maximal hydrolysis of MBM proteins. Crystalline pepsin (4100 hemoglobin units/mg), trypsin (12,000 BAEE units/mg) and pancreatin (8 x) were used. All enzymes were of porcine origin. Enzyme solutions were made fresh. Pepsin was made at 10 mg/ml in 0.1 M citrate buffer solution (CBS, pH 4.0), and the pancreatic enzymes were made at 10 mg/ml in 0.2 M phosphate buffer solution (PBS, pH 8.0).

A two-stage in vitro digestion system was adopted. In the first stage, pepsin (PP) was used for incubation of MBM in 20 ml of 0.1 M citrate buffer solutions (CBS, pH 2.0) for 24 hours. In the 2^nd stage, 30 ml of 0.2 M phosphate buffer solution (PBS pH 8.0, then raised to pH 12 by adding NaOH) was added to adjust pH to around 7 followed by inoculation of pancreatic enzyme cocktail (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 (500 mg per test tube) were hosted in 50 ml Corning screw capped test tubes. 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 ml) 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 the degree hydrolysis using the o-pthalaldehyde (OPA) method.

The two-stage digestion model

We established a model to describe the processes of digestion of MBM protein by pepsin (PP) and pancreatic enzymes (PT).

The 1^st stage digestion

MBM protein (Si) is first digested by pepsin (E1) to release the free amino group (product P1). Meanwhile, pepsin (E1) digests itself to release free amino group (Pe1).

Si + E1 à P1 + E1

E1 à Pe1

[Si]=initial MBM, [E1]=initial pepsin, [P1]=product from MBM, [Pe1]=product from pepsin.

The 2^nd stage digestion

After the 1^st stage digestion, those MBM peptide bonds that have not been digested (Sr) are further hydrolyzed by PT (E2) to release free amino group (product P2). Those pepsin peptide bonds that are not autolyzed in the first stage are further hydrolyzed by PT (E2) to give free amino group (Pe1r). In addition, PT autolyzes itself to yield free amino group (Pe2).

Sr + E2 à P2 + E2

E1r + E2 à Pe1r + E2

E2 à Pe2

[Sr]=[Si]-[P1], [E2]=initial PT, [E1r]=[E1]-[Pe1], [P2]=product from Sr, [Pe1r]=product from [E1r], [Pe2]=product from PT.

At the end of the 2^nd stage digestion, the sum of free amino group released by digestive enzymes is [Pmbm]=[P1]+[P2]. The sum of free amino group released from enzymes is [Penzyme]=[Pe1] + [Pe1r] + [Pe2].

Because the hydrolysates of MBM protein and the autolysates of the enzymes are not separable using OPA method, it is the apparent degree of hydrolysis (ADH) that is measured. ADH is defined as:

Where [Pmbm] is the hydrolysate from MBM, [Penzyme] is the autolysate from enzyme; [Si] is the initial substrate. The term [Pmbm]/[Si] is defined as the true degree of hydrolysis (TDH):

DEC is estimated by measuring the autolysate of enzymes (Penzyme) from incubation of enzymes without substrates. In estimating DEC, the [Si] is pseudo and it is the MBM concentration used for calculating ADH.

Thus TDH was obtained by:

Results

Experiment 1: Determination of the DEC

In the measurement of the amino group released from MBM proteins, the blank control used for optical absorbance was MBM in the absence of enzymes. Thus the measurement also contained the free -NH₂ of the amino acid residuals of the intact enzymes. Therefore, in correction of the ADH, the DEC should be measured using the buffer as the control. In estimation of the DEC, it is assumed that the liberated peptide bonds from enzymes remain unchanged regardless of the presence of substrates.

Suppose the pseudo MBM protein concentration was 12.5 mg/ml and 5.0 mg/ml for the 1^st stage incubation and 2^nd stage incubation, respectively, the concentrations of pepsin and PT for the enzyme only incubations were calculated with varying ratios of enzyme to MBM protein. Pepsin was incubated in 20 ml of 0.1 M CBS pH 2.0 for 24 hours at 38 C. Then 30 ml of 0.2 M PBS pH 8.0 was added followed by inoculation of PT. The experiment procedure is illustrated below. At each PP level, there were 10 replicates. Each two of them were used for one level of PT incubation in the 2^nd stage. The experiment had 35 treatment combinations and 2 replicates for each treatment combination.

1^st stage	Pepsin (mg/ml)
PP/MBM protein (%)	0, 0.25, 0.5, 1.0, 1.5, 2.0, 3.0
2^nd stage	PT (mg/ml)
PT/MBM protein (%)	0, 0.25, 0.5, 1.0, 1.5

At 48 hours after PT inoculations when the auto-digestion ceased, an aliquot was taken out and mixed with equal volume of 20% SDS. After centrifugation, the absorbance was measured against corresponding buffer mixture with the OPA method.

The experiment was a split plot design. The main effects of pepsin and PT were tested with the 2-way interaction term. The effects of replication and interaction were insignificant. The effects of pepsin and PT levels were significant. The linear regression that gave predicted DEC is shown in Figure 1.

Figure 1. The degree of enzyme contamination (DEC) measured using the buffer as the blank control. The predicted DEC was calculated using [Si]=5.0 mg/ml, the chosen concentration of the 2^nd stage digestion. The DEC could be predicted from the levels of pepsin and PT used in the 2 stages of incubation.

The predicted DEC was: DEC=0.19*PPR + 0.60*PTR (R²=0.98)

Where DEC is in percent (%), PPR stands for pepsin/MBM ratio (%), PTR stands for PT/MBM ratio (%).

Experiment 2: Estimation of minimal enzyme for complete digestion.

Nine MBM samples were digested with a fixed level of pepsin at 0.25% for 24 hours in 20 ml CBS pH 2.0. Then, after adding 30 ml of PBS to adjust the pH to 7.0, PT was added at graded levels from 0 to 9% (except 1% PT level, a few to 11%) and incubated for 96 hours. The ADH was measured as usual. The TDH was calculated from ADH subtracted by the DEC. The DEC was calculated using the regression derived from Experiment 1. Each level of PT had 3 replicates.

From the two-stage digestion model, a formula was derived as the model of the nonlinear regression. When time was not limiting, at timeà¥, the true degree of hydrolysis (TDH) was

Where c was the predicted maximal TDH, c ´ b0 was the TDH exhibited by pepsin, k was the digestion rate constant of the PT, PT was the levels of PT (%) used in the digestion.

The minimal PT (PTmin) to reach 95% of c was calculated with:

Estimation of the parameters was performed with SAS NLIN (version 7.0, SAS Institute Inc, 1998). An example is given in Figure 2 to illustrate the estimation of c, b0, k and PTmin from TDH.

Figure 2. Nonlinear Regression of TDH with PT level as the independent variable. Data of MBM No.43. MBM was first incubated with 0.25% pepsin for 24 hours. After adding PBS buffer, PT was added from 0 to 9% (except the 1% level, PT to MBM protein ratio) and another 96 hour incubation was allowed. Then a small aliquot was taken out for measurement of ADH. Values are means of 3 replicates.

Nine different MBM samples were treated using the procedure. All MBM samples showed the convergence to maximal degree of hydrolysis. It was estimated that on average, 5.7% PT was needed to achieve maximum TDH of MBM. The 95% upper confidence limit of this mean value was 6.30%.

Since the protein content of pancreatin was 71% (assayed by Kjeldahl method), therefore, 6.30% of PT contained 4.78% protein. The usage of pepsin was 0.25%. Therefore, the average minimal enzyme protein level needed to reach the maximal degree of hydrolysis of MBM was 5.03%.

The mean degree of hydrolysis of MBM at plateau was 27.4%. This was equivalent to the average peptide length of 3.6 amino acid residuals. This figure did not seem to contradict the conclusion that the digestion was complete.