Summer 2001 Issue — Whey protein Hydrolysate

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Introduction

Davisco recently launched a specialized whey ingredient called BioZate, developed in conjunction with Advitech Solutions, a Canada-based biotechnology company. It brings new hope to sufferers of hypertension, one of the key contributors to heart attacks in industrialized countries and affecting more than 50 million adults in the USA alone.

Astonishingly, almost 30% of adult deaths can be attributed to hypertension or its renal, coronary or neurological complications. Manufactured via a controlled hydrolysis procedure using ion exchange isolated whey proteins as the substrate, BioZate is a pure form of hydrolyzed whey protein. Hydrolysis refers to the chemical process by which an enzyme is used to break the peptide bonds of a protein, resulting in the formation of smaller molecular derivatives called polypeptides and peptides. This new ingredient has an outstanding potential for use as a nutraceutical dairy-based ingredient.

Whey protein hydrolysates aid digestion and absorption, rendering them particularly suitable for infant formulas, dairy beverages and sports nutrition products. Besides its high nutritional value, BioZate has been shown to possess angiotensin converting enzyme (ACE) inhibitory activity and to reduce heart rate and blood pressure in spontaneously hypertensive rats (SHR).

Bioactivity of BioZate

The inhibitory effect of natural peptides has been related to their binding at the active site of ACE. A structure-function study of these various bioactive peptides has revealed that they often possess a Pro-Pro, Ala-pro or Ala-Hyp at their C-terminal end. The occurrence of proline would also contribute to the ACE-inhibitory activity of peptides derived from food proteins.

In Vitro Study

Since ACE plays an important role in contributing to hypertension, it is desirable to use an in vitro screening technique to determine the ACE-inhibitory properties of potential proteins or drugs used to control hypertension. The method used is a spectrophotometric assay that generates IC 50 values. These values indicate the amount of hydrolysate or other material required to inhibit 50% of ACE activity. The in vitro study evaluated the ACE-inhibitory properties of BiPRO (intact whey protein isolate), BioZate (hydrolyzed whey protein isolate) and the synthetic peptide from -lacto globulin f (142-148). BiPROshowed the highest IC 50 value of 0.45mg/ml. To put these values into perspective, the IC 50 value of 376mg/ml indicating that t was least inhibitory of ACE activity. BioZate showed potent ACE-inhibitory activity with a low IC 50 value of 0.45mg/ml. To put these values into perspective, the IC 50 value for the synthetic peptide from -lacto globulin, f (142-148), was 0.04mg/ml. These results clearly demonstrate the effect of enzymatic hydrolysis on the ACE inhibiting activity of whey protein isolate. BioZate was over 800 times more effective than BiPRO at inhibiting ACE activity. Enzymatic hydrolysis leads to a synergistic effect in terms of ACE-inhibitory potential of the hydrolysate obtained. It appears that the synergy could result from the unique compositional characteristics of BiPRO, the unique ion-exchange whey protein isolate used as the substrate to manufacture BioZate , in combination with the use of specific enzymes.

In Vivo Study

Based on the promising results from the in vitro testing, the next step was to conduct an in vivo test, in order to demonstrate the potential for the use of BioZate as an anti-hypertensive ingredient for specialized or clinical nutrition. An in vivo model study was conducted using spontaneously hypertensive rats (SHR). The SHR are considered a genetic model of hypertension and are currently used to understand the development and establishment of hypertension and to determine the blood pressure lowering effect of newly synthesized anti-hypertensive drugs. The study was conducted using male SHR aged 20-24 weeks. There were approximately 10 animals per group. An intravascular catheter was implanted for direct and continuous measurement of heart rate and phasic and mean arterial blood pressure. This method is well developed by researchers and is found to be less stressful and less painful for the animals than using the tail cuff sphygmomanometer technique to measure systolic blood pressure. The animals were randomized into three groups. The control group was administered by a phosphate buffer solution (PBS), one group was administered BiPRO (intact whey protein isolate) and one group was administered BioZate (hydrolyzed whey protein isolate). Isolated doses of 30mg, 75mg and 150mg per kg body weight were administered to the SHR by gastric intubation and the animals were evaluated for a seven-hour period.

Since arterial blood pressure fluctuates up and down with each heartbeat, the fluctuation up being the systolic pressure, and the fluctuation down being the diastolic pressure, the important figure to consider is the mean arterial pressure (MAP). MAP is roughly equal to the diastolic pressure plus one third of the systolic pressure minus the diastolic pressure.

In Vivo Study Results

The in vivo model study generated significant results. At a dose of 75mg/kg body weight, BioZate significantly reduced heart rate at 4, 6 and 7 hours after the administration with a minimum heart rate of 243 beats per minute (bpm) achieved 6 hours after the administration (Figure 1).

Figure 1. Heart rate (HR) of SHR after oral administration of 0.5ml of 0.2mM PBS, BiPRO at a dose of 75mg/kg, or BioZate at a dose of 75mg/kg.

At the dose of 30mg/kg and 150mg/kg BioZate did not significantly affect heart rate. At a dose of 75mg/kg body weight, BioZate elicited a dramatic and significant, long lasting hypertensive effect at 1-7 hours after the administration, with a minimal mean arterial blood pressure of 139mmHg achieved 6 hours after the administration, corresponding to a 17-23% drop in blood pressure (Figure 2).

Figure 2. Mean arterial blood pressure (MAP) of SHR after oral administration of 0.5ml of 0.2mM PBS, BiPRO at a dose of 75mg/kg, or BioZate at a dose of 75mg/kg.

At the dose of 30mg/kg, BioZate did not significantly affect blood pressure. At the highest dose tested of 150mg/kg, BioZate elicited a significant hypertensive effect at 1 and 3-7 hours after the administration with a minimum mean arterial blood pressure of 143mmHg achieved 6 hours after the administration, corresponding to a 10-18% drop in blood pressure. In a second study, similar decreases in blood pressure were observed, and the effect lasted for 24 hours at the BioZate dose of 75mg/kg and 150mg/kg. These results indicate that controlled hydrolysis of a whey protein isolate, BioZate , releases peptides capable of decreasing blood pressure.

Applications

BIOZATE is specially designed for specific nutritional applications. The enzymatic hydrolysis process produces a protein based dairy ingredient which is designed to be applied to nutritional, diabetic and medical foods such as dry mixtures (i.e. milk shakes, soups, puddings, omelettes), drinks, nutritional bars, meal replacers and specialized food for sports nutrition, elderly people, diabetic preparations for weight-loss programs, and meal replacers for people having disorders of the digestive function.

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