Prolonged Ingestion of Prehydrolyzed Whey Protein Induces Little or No Change in Digestive Enzymes,but Decreases Glutaminase Activity in Exercising Rats

Abstract

Because consumption of whey protein hydrolysates is on the increase, the possibility that prolonged ingestion of whey protein hydrolysates affect the digestive system of mammals has prompted us to evaluate the enzymatic activities of pepsin, leucine-aminopeptidase, chymotrypsin, trypsin, and glutaminase in male Wistar rats fed diets containing either a commercial whey isolate or a whey protein hydrolysate with medium degree of hydrolysis and to compare the results with those produced by physical training (sedentary, sedentary-exhausted, trained, and trained-exhausted) in the treadmill for 4 weeks. The enzymatic activities were determined by classical procedures in all groups. No effect due to the form of the whey protein in the diet was seen in the activities of pepsin, trypsin, chymotrypsin, and leucine-aminopeptidase. Training tended to increase the activity of glutaminase, but exhaustion promoted a decrease in the trained animals, and consumption of the hydrolysate decreased it even further. The results are consistent with the conclusion that chronic consumption of a whey protein hydrolysate brings little or no modification of the proteolytic digestive system and that the lowering of glutaminase activity may be associated with an antistress effect, counteracting the effect induced by training in the rat.

Introduction

T he ingestion of partially hydrolyzed protein has been considered to improve several biological functions. Protein hydrolysates have been reported to improve the digestibility of diets,^1
–3 reduce a specific type of allergenicity,⁴ and stimulate metabolic or physiological advantages in animals^5,6 as well as in humans.⁷ Von Berg et al.⁴ reported that the risk for specific atopic dermatitis can be reduced with certain cow's milk hydrolysates in high-risk infants when breastfeeding was not sufficient. Tassi et al. ⁵ and Pimenta et al.⁶ have observed both better physical resistance of rats to physical exhaustion and a clear improvement of glycogen stores and serum albumin and glycemic levels of rats subject to physical training. Hori et al.,⁷ in turn, have found that the serum cholesterol levels of hypercholesterolemic subjects were significantly reduced after consuming a diet containing soy protein hydrolysate. Feeding a hydrolyzed protein formula with a low degree of hydrolysis to newborn guinea pigs was also reported to protect the animal against the reduced zymogen production caused by artificial feeding.⁸ Other researchers have tested the effect of a hydrolyzed whey protein supplement on the blood pressure of humans in comparison with the intact whey protein as the control.⁹ After completion of the treatment, a follow-up of 4 weeks showed a mean reduction in both systolic and diastolic blood pressure in the experimental group compared to the control. Low-density lipoprotein cholesterol and high-sensitivity C-reactive protein levels were also significantly improved by the treatment.

Both the quantity and quality of dietary protein have been of concern among health workers in the practice of sports, while the use of milk whey proteins continues to gain worldwide acceptance as a valuable supplement for best physical performance. Hayes and Cribb,¹⁰ for instance, have reviewed the nutritional properties and concluded that supplementation with whey protein can be used to augment the beneficial effects of resistance exercise and decelerate the evolution of sarcopenia. Moreover, previous studies have shown that whey protein hydrolysates exhibit broad antioxidant activity, such as in copper-catalyzed liposome emulsions¹¹ or in iron-catalyzed liposome oxidation systems,¹² according to the proteases used.

With time, enzyme-hydrolyzed whey proteins have also found use as bioactive and nutritional ingredients in health and food products.¹³ Antioxidant properties have been generally identified for a growing number of food protein hydrolysates, including those derived from milk whey, and authors continue to propose their use as natural antioxidants that could enhance the antioxidant properties of functional foods,¹⁴ but new methods are being proposed to determine the biological antioxidant activity.¹⁵ With regard to the mechanism of the whey hydrolysates, it has been hypothesized that their antioxidative capacity is related to the amount of glutathione precursor (cysteinylglycine-containing) peptides generated during the hydrolysis process¹⁶ and to the molecular weight of the peptides.¹⁵

In view of the growing application of whey protein hydrolysates in sports and medicine, it was of interest to deepen the current understanding of the body's response to prolonged exposure to hydrolyzed proteins. Because the routine consumption of prehydrolyzed proteins foregoes the normal participation of some endogenous digestive enzymes, we wished to investigate the impact of consuming the enzymatically hydrolyzed milk whey proteins on the enzymatic activities of pepsin, trypsin, chymotrypsin, leucine-aminopeptidase, and intestinal glutaminase in rats subjected to physical activity and fed either the whey protein isolate or a whey protein hydrolysate for 5 weeks. Of special interest was to observe the effect of both diet and exercise on the activity of glutaminase, because of its involvement in stress.

Materials and Methods

Animals

One hundred forty-four male Wistar rats were purchased from the Multidisciplinary Center for Biological Investigations (Cemib, University of Campinas, Campinas, SP, Brazil) and housed in controlled ambient conditions (23 ± 1°C, 50–60% relative humidity, 12-hour inverted light-dark cycle, and free access to water.

Diet composition and nitrogen balance

After an adaptation period of 1 week on commercial chow (Nuvital, Curitiba, PR, Brazil), the animals were fed the experimental diets formulated on the basis of the AIN-93-G diet (American Institute of Nutrition),¹⁷ except that the experimental proteins took the place of casein and its content was reduced to about 14%, which was below the 17% recommended for maximal growth. The experimental protein sources were the commercial whey (intact) protein isolate (Alacen™ 895), its hydrolysate (Alacen™ 817) (11% degree of hydrolysis, monitored by the trinitrobenzenesulfonic acid method of Adler-Nissen¹⁸), and casein as the control, all from New Zealand Milk Products (Wellington, New Zealand).

The proximal composition of the diets (Table 1) was determined by standard procedures: protein, lipids, fiber, ash, and moisture were by the AOAC International procedures,¹⁹ using the 6.38 protein conversion factor for the diets and 6.25 for the feces. A nitrogen balance evaluation was carried out by standard procedures in order to maintain the vegetative effect of the diets under control.

Table 1.

Proximal Composition of the Experimental Diets (Dry Basis)

Diet	kcal/100 g	Protein (%)	Lipid (%)	Carbohydrate (%)	Fiber (%)	Moisture (%)	Ash (%)
Casein	444.1 ± 0.3	139.2 ± 0.1	88.1 ± 0.1	685.2 ± 0.2	29.9 ± 0.1	90 ± 0.4	28.8 ± 0.1
Isolate	446.05 ± 0.1	136.6 ± 0.2	92.1 ± 0.2	683.6 ± 0.2	32.1 ± 0.1	77 ± 0.1	27.8 ± 0.0
Hydrolysate	447.65 ± 0.9	139.5 ± 0.3	95.3 ± 0.2	671.9 ± 0.3	30.7 ± 0.0	76 ± 0.6	31.3 ± 0.1

Data are mean ± SD values of determinations in triplicate.

Training protocol and exhaustion test

After 1 week of adaptation to the experimental diet, the animals started a 4-week feeding-training protocol, adapted as described by Smolka et al. ²⁰ The experiment started with a total of 120 rats (10 rats in each of 12 groups), but a screening physical activity test of 5 minutes, at the speed of 10 m/minute, was run in an attempt to exclude from the study those animals that did not adapt to exercising.

The procedure for training and exhaustion was the same used by Pimenta et al.,⁶ except that our present protocol was 5 days shorter because the nitrogen balance test was run during the 2 weeks of adaptation. Ten animals of each group were removed from their cages and subjected to physical exhaustion, running at 32.5 m/minute. The exhaustion point was defined as the time elapsed until the animal remained standing, receiving the shock for more than 5 seconds. After exhaustion the animals were allowed to recover in their cages, with free access to diet and water, and thereafter were sacrificed by decapitation to start collecting the tissues for determining the 24-hour responses. The research protocol was approved by the institutional Ethics Committee for Animal Experimentation (Institute of Biology, University of Campinas).

Determination of enzymatic activities

The pepsin activity was determined according to the method of Anson.²¹ The procedure of Curthoys and Lowry²² was followed for the glutaminase activity, quantifying the generated glutamate by the method of Bergmeyer.²³ For the determination of the pancreatic enzymes and leucine-aminopeptidase, the luminal contents were prepared according to the recommendations of Ohtani et al.²⁴ Chymotrypsin and trypsin activities were measured by the methods of Rick,^25,26 whereas the leucine-aminopeptidase assay followed the procedure of Appel.²⁷ All colorimetric determinations were carried out using a spectrophotometer (model DU-640, Beckman-Coulter, Fullerton, CA, USA).

Amino acid determination by liquid chromatography

The amino acid profiles of the protein sources were determined for the acid hydrolysate of each source, using the phenylisothiocyanate derivatives, according to the method of White et al.²⁸ as modified by Hangen et al.²⁹ The chromatographic procedure included the use of an RP C-18 Luna column (catalog number 00G-4252-EQ; 100 Å; particle size, 5 μm; 250 × 4.6 mm; Phenomenex, Torrance, CA, USA) and a binary eluant system basically consisting of (A) 60 mM sodium acetate buffer and triethylamine and (B) acetonitrile in water with EDTA.

Statistical analysis

The dependent variables were tested for normality; only chymotrypsin did not pass the test and was therefore subjected to the nonparametric Kruskal-Wallis analysis. To the remaining enzymes a general linear monovariate model was applied, followed by the test of homogeneity of the variance. The data for pepsin and leucine-aminopeptidase and times of exhaustion were shown to have normality and homogeneity of variance and were thus subjected to the significance tests of Tukey's HSD and Bonferroni. The variance of both trypsin and glutaminase, however, showed heterogeneity of variance and were therefore subjected to the Tamhane analysis, which compares multiple means among treatments. For comparison of the amino acid profiles, Tukey's test was applied. All analyses were performed using Statistical Package for Social Sciences version 17 software (SPSS, Chicago, IL, USA) adopting the standard criterion of P ≤ .05.

Results and Discussion

Growth and physical performance

We have examined the influence of both the protein source in the diet and physical activity, with and without exhaustion, on the activity of stomach and intestinal proteolytic enzymes and glutaminase of rats trained in the treadmill. The first biological evaluation showed that a 3-day nitrogen balance was positive for all diets, with no significant differences in body mass accretion (0.87 ± 0.15, 0.81 ± 0.17, and 0.83 ± 0.17 g of N/day for casein, the isolate, and the hydrolysate, correspondingly), indicating that the diets were indistinguishable considering the overall nitrogen balance. The animals had a smooth, steady growth, and the mean daily body weight gains and food intakes during week 5 can be seen in Table 2.

Table 2.

Mean Individual Daily Body Weight Gain and Average Individual Food Intake During the Last Week of the Experiment

	Activity group
	S	T	SX	TX
Body weight gain (g)
Casein	5.50 ± 0.61	4.98 ± 0.79	6.30 ± 0.86	6.41 ± 0.85
Isolate	4.95 ± 0.57	5.13 ± 0.77	6.98 ± 0.78	5.05 ± 0.80
Hydrolysate	5.13 ± 0.68	4.38 ± 0.59	7.34 ± 0.96	6.16 ± 0.79
Food intake (g)
Casein	24.90	24.31	18.33	18.10
Isolate	17.75	18.22	17.80	22.95
Hydrolysate	19.06	16.98	17.93	19.52

For the last 4 weeks of the experiment, the animals were housed in collective cages (n = 6).

S, sedentary; T, trained; SX, sedentary-exhausted; TX, trained-exhausted.

As seen in Table 3, the amino acids supplied by the protein sources of the experimental diets varied somewhat in comparison with that from casein and the recommendations set forth by the Institute of Laboratory Animal requirements.³⁰ Both leucine and lysine in the isolate and the hydrolysate were, respectively, 100% and 33% above the standard profile, whereas the sulfur amino acids were 50% above and 12% below, respectively. In spite of the fact that tryptophan is at least twofold more abundant in the whey proteins than in casein, this amino acid was still below the recommended level, and the minimal differences in amino acid contents between the whey protein diets did not cause any significant difference in growth (data not shown).

Table 3.

Essential Amino Acids Supplied by the Experimental Diets Compared with the Recommended Profile for Growing Rats

	Level (g/kg of diet) for protein sources
Amino acid	Casein	Isolate	Hydrolysate	Nutrient requirements ^*
Arg	4.1 ± 0.02^a	3.5 ± 0.19^a	3.7 ± 0.37^a	6.0
His	1.4 ± 0.01^a	1.4 ± 0.05^a	1.5 ± 0.15^a	3.0
Ile	6.1 ± 0.03^a	7.8 ± 0.18^a	6.5 ± 0.66^a	5.5
Leu	10.4 ± 0.10^bc	16.8 ± 0.12^a	14.2 ± 1.25^ab	7.5
Lys	10.8 ± 1.89^ab	14.2 ± 0.14^a	12.1 ± 0.88^ab	9.0
Met + Cys^†	4.1 ± 0.37^b	9.3 ± 5.8^a	5.3 ± 2.19^ab	6.0
Phe + Tyr	12.8 ± 0.23^a	10.2 ± 0.42^ab	10.7 ± 0.8^ab	8.0
Thr	4.9 ± 0.0^a	6.3 ± 0.01^a	5.6 ± 0.84^a	5.0
Trp	0.21 ± 0.01^a	0.5 ± 0.01^a	0.5 ± 0.01^a	1.5
Val	7.3 ± 0.02^a	7.0 ± 0.03^a	6.5 ± 0.55^a	6.0

Data are mean ± SD values of triplicate determinations for comparisons made on a dry weight basis.

abc

Different lowercase letters represent significant differences between any two sources (Tukey's HSD, P ≤ .05).

According to the Institute of Laboratory Animal requirements.³⁰

†

Cysteine was added to the casein diet as recommended by the AIN 93.

Arg, arginine; His, histidine; Ile, isoleucine; Leu, leucine; Lys, lysine; Met + Cys, methionine + cysteine; Phe + Tyr, phenylalanine + tyrosine; Thr, threonine; Trp, tryptophan; Val, valine.

The influence of each protein source on the physical performance of the animals as measured by the exhaustion times on a treadmill was variable as shown in Figure 1. A statistical difference between the isolate and the hydrolysate was detected in their performance test only in the animals that underwent training (isolate-consuming trained to exhaustion and hydrolysate-consuming trained to exhaustion: P = .017 [Tukey's test] and P = .021 [Bonferroni's test]), but no differences were observed between the casein and the isolate diets, independent of the animals having been sedentary or trained. It should be noted, however, that the trained animals consuming the hydrolysate ran on average for 100% longer times than those fed the casein, whereas those consuming the isolate endured for 63% longer times before reaching exhaustion.

FIG. 1.

Mean ± SD exhaustion times of the groups consuming one of the three protein sources (casein, whey protein isolate, or whey protein hydrolysate) and comparison of performances of the trained versus the sedentary regimens for every diet group. TX, trained-exhausted; SX, sedentary-exhausted. Capital letters qualify the difference between the two levels of activity for each diet group; lowercase letters compare the diets within the same level of activity. By Tukey's and Bonferroni's tests, P ≤ .05.

In general, these data are in accord with those of Pimenta et al.,⁶ suggesting that the enzymatically hydrolyzed milk whey proteins may improve physical performance in the rat brought to exhaustion.

In another experiment, however, Ramos³¹ found that an enzymatic hydrolysate with 30% degree of hydrolysis did not produce such an advantage in rats undergoing swimming bouts in comparison to the intact protein. Such a result was attributed to differences in the nature and abundance of peptides that were made available to the animal, in contrast to the findings of Tassi et al.⁵ and Pimenta et al.,⁶ which were obtained with hydrolysates with about 10% degree of hydrolysis. It seems likely, therefore, that the physiological effect could depend on the degree of hydrolysis.

Effect of hydrolysate on enzymatic activities

This work has evaluated the possible effect that either the type of protein alone, physical activity alone, or a combination of both could have on the activity of some stomach and intestinal proteases and glutaminase.

Preliminary evaluation of the data showed no statistical interaction between diet and physical exercise if the two were to be considered simultaneously. The values reported in Table 4 for pepsin were somewhat higher than those encountered by Coppi et al.³² (27.5 ± 1.7 mg of tyrosine/g of N) for adult Sprague-Dawley rats. Our data in Table 4 show that although no effect was detected from the diet, physical activity did have an influence on the enzymatic activity of pepsin; a significant decrease in peptic activity was noticed when the sedentary animals were brought to exhaustion.

Table 4.

Protease Activities in Stomach and Jejunum of Rats Fed Diets with Casein, Whey Protein Isolate, or Whey Protein Hydrolysate and Either Sedentary, Trained, Sedentary and Eventually Brought to Exhaustion, or Trained and Eventually Brought to Exhaustion

	Protease activity
Protease	S	T	SX	TX
Pepsin
C^*	46.0 ± 8.7^a	34.5 ± 3.8	33.3 ± 10.0^b	40.4 ± 3.9
I^*	34.9 ± 4.1^a	42.6 ± 4.5	32.8 ± 2.5^b	30.5 ± 5.8
H^*	48.4 ± 10.5^a	47.4 ± 10.1	29.7 ± 6.3^b	34.5 ± 3.1
Leu-AP
C^†	291.9 ± 14^b	315.1 ± 8^a	282.6 ± 21^b	310.4 ± 21.2^a
I^†	296.5 ± 29^b	356.8 ± 8^a	268.7 ± 21^b	333.6 ± 13.9^a
H^†	301.2 ± 8^b	338.2 ± 8^a	282.6 ± 8^b	324.3 ± 16.0^a
Chymotrypsin
C^†	214.6 ± 25.4	221.8 ± 20.7	185.6 ± 12.6	210.4 ± 7.8
I^†	236.4 ± 6.2	414.7 ± 3.6	198.0 ± 1.8	363.9 ± 8.2
H^†	256.0 ± 16.4	241.5 ± 7.2	216.7 ± 20.7	240.5 ± 6.5
Trypsin
C^†	1,955.6 ± 81^b	1,990.0 ± 59^b	1,952.4 ± 105^b	1,970.7 ± 71^b
I^†	2,190.7 ± 77^a	2,442.9 ± 29^a	2,292.6 ± 21^a	2,395.6 ± 13^a
H^†	2,220.7 ± 29^a	2,416.7 ± 22^a	2,066.2 ± 154^a	2,295.9 ± 32^a
Glutaminase
C^†	47.8 ± 2.2^ABb	63.2 ± 4.38^ABa	32.7 ± 7.52^Abc	48.4 ± 3.9^ABb
I^†	52.2 ± 4.5^Ab	66.4 ± 1.39^Aa	37.6 ± 2.68^Ac	49.5 ± 6.7^Ab
H^†	38.4 ± 4.5^Bab	49.7 ± 1.01^Ba	28.5 ± 1.31^Bc	35.4 ± 3.4^Bb

For each enzyme, ^ABdifferent capital letters within the same column indicate significant statistical differences due to the dietary protein; ^abcdifferent lowercase letters within the same line indicate that statistically significant differences exist because of exercise (Tukey's HSD, Bonferroni's, and Tamhane's tests, P ≤ .05).

In mg of tyrosine/g of N.

†

In nmol/minute/mg of protein.

Leu-AP, leucine-aminopeptidase; C, diet with casein; I, diet with whey protein isolate; H, diet with whey protein hydroysate.

Insofar as the activity of leucine-aminopeptidase, training produced an increase (10–20%) in activity, whereas diet again had a null effect. Exhaustion, however, did not show any alteration of this effect on the animals, regardless of being sedentary or trained.

The activity of trypsin, in turn, was nearly 20% higher in the animals consuming both the isolate and the hydrolysate, compared to casein (P = .000), but no effect could be attributed to either training or exhaustion, whereas the difference between the isolate and hydrolysate was not significant (P = .37).

On the other hand, application of the Kruskal-Wallis test to the chymotrypsin data showed that, in spite of the substantially higher (75%; Table 4) activity exhibited by the trained animals consuming the isolate, no significant effect on this enzyme could be attributed to either physical activity or diet.

The apparent immutability of the activity levels of either trypsin or chymotrypsin in the casein group could be partly understood if we took into account the independent observations of Lindberg et al.³³ and Ohtani et al.,²⁴ who reported that casein alone exerts a partial inhibition of both trypsin and chymotrypsin.

With regard to glutaminase, it can be seen (Table 4) that both diet and physical activity had an independent influence on this enzyme's activity, although no interaction was observed between the two variables. Training alone was found to produce a general increase of the glutaminase activity. Surprisingly, however, it was also noted that physical exhaustion was responsible for a mean reduction of nearly 29% in glutaminase activity in the animals of the three diet groups.

Alterations in glutaminase activity have been explained by several authors as resulting from modifications in either the levels of the glucocorticoid or glucagon hormones, or even from manipulations of various dietary components. Among the latter factors, a study by Pinkus and Windmueller³⁴ showed that phosphate, ammonium, sulfate, malate, bicarbonate, citrate, pyrophosphate, and oxalate ions activated glutaminase. In another study, the release of large amounts of bicarbonate ion by the pancreas in the duodenum was proven to elicit the activation of glutaminase in enterocytes, in phase with the process of stomach emptying and neutralization of the acid products generated during stomach digestion.³⁵

Glucocorticoids in turn are capable of modifying the activity of intestinal glutaminase as a result of the alteration of the hormone levels caused by physical exercise. Increases of more than 100% in cortisol levels were reported by França et al.³⁶ in male marathon runners after a competition, while similar changes also have been observed in male runners by Acevedo et al.³⁷ and Minetto et al.³⁸

Because glutamine is the principal source of energy for the enterocyte and glucocorticoids play a role as mediators of the metabolic rate, these hormones are likely to regulate the levels of glutamine in tissues. Salleh et al. ³⁹ showed that when glucocorticoids were injected in rats, the utilization of glucose by the enterocyte diminished, whereas that of glutamine increased as a source of energy. It should be noted in our experiment (Table 4) that although training alone was followed by a glutaminase increase in all diet groups, the increase in the group that consumed the hydrolysate was significantly (P = .01) and substantially lower from that seen in the group that consumed the isolate.

Because all animals in our experiment, including those of the sedentary group, were similarly exposed to the stress produced by the handling and the electric shock, any changes in glutaminase activity were therefore attributed to only diet, training, and exhaustion. As shown in Table 4, training alone raised the enzymatic activity by about 30%, whereas exhaustion caused a decrease of 29% below the sedentary level, for both the casein and the isolate diets. It should be also noted that the sedentary animals fed the hydrolyzed whey protein exhibited levels at least 10% lower than those that received the whole proteins and that neither training nor exhaustion altered such low levels. This advantage was particularly evident when comparing the hydrolysate with the isolate.

Two features of these results should be kept in mind: one being the fact that all groups were allowed to have a 24-hour recovery period before the measurements were made, and second that when the animals fed either the isolate or the hydrolysate were brought to exhaustion, these worked significantly more than did the casein-fed or the sedentary cohorts. When forced to exhaustion, therefore, the animals consuming the whey proteins performed significantly better and, therefore, withstood greater activity-derived stress than either the sedentary or the casein-fed groups. It could be understood, therefore, that the hydrolysate, or some of its constituting peptides, exerted a compensating effect on the accelerated metabolism caused by either training or exhaustion, thus suggesting the existence of an antistress effect.

It is still noteworthy in Table 4 that besides the lowering effect on the glutaminase by the hydrolysate alone, evident in all physical activity groups, there was an additional effect that was common to all the proteins, seemingly caused by subjecting the trained animals to the resistance exercise (TX column). Therefore, the glutaminase values observed in column TX for each of the three proteins appear to be the sum of the two effects, even 24 hours after exhaustion. Again, when comparing groups SX and TX, account should be taken of the fact that animals of the former reached exhaustion with substantially less work accomplished than the TX cohorts.

The possibility that an antistress effect was the result of consuming the whey hydrolysate in exercising animals would be consistent with recent claims made by workers about the antioxidant properties of three whey protein hydrolysates as measured by the thiobarbituric acid test^12,40 and could be a plausible explanation of the higher resistance to exhaustion that has been observed in our study.

Comparing the effects caused by a dietary milk whey protein fed in both the whole and the partially hydrolyzed forms to the rat supports the conclusion that few physiological differences can be observed in response to the form in which the protein was supplied to the animal. Pepsin activity did not undergo any apparent modification due to the diet, but the activity of leucine-aminopeptidase was also moderately (∼13%) increased as a result of training. Additionally, whereas chymotrypsin did not vary as a result of the proteins fed, it was possible to detect minor yet significant differences in the activities of the pancreatic protease trypsin against the whey proteins, but they were probably related more to the inhibiting effect of casein than to the form of the protein in the diet. Significantly lower values were observed, however, in the activity of glutaminase when the partly hydrolyzed protein was fed, suggesting that the apparently mandatory increase in activity caused by physical training, combined with the consumption of the intact proteins, could be ameliorated by substituting the hydrolysate for the intact isolated protein in the diet.

It is tempting to consider the elevation in glutaminase activity during exercise as being the result of a glucocorticoid-dependent demand for higher intestinal energy consumption, in turn, as being a result of the accelerated metabolism caused by the higher level of physical activity. If this were the case, the enzymatic activity-lowering effect could be considered as an extra beneficial effect of the hydrolyzed whey protein, in addition to the health properties already assigned to the intact whey proteins.

Footnotes

Acknowledgments

The authors thank CAPES and the Brazilian Research Council (CNPq) for the scholarships and New Zealand Milk Products for the gift of the whey derivatives.

Author Disclosure Statement

No competing financial interests exist.

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