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Impact of different dietary protein on rat growth, blood serum lipids and protein and liver cholesterol
Nutrition Research 21 (2001) 905–915 www.elsevier.com/locate/nutres
Impact of different dietary protein on rat growth, blood serum lipids and protein and liver cholesterol Helaine B. Jacobucci, Valdemiro C. Sgarbieri*, Na´dia F.G.P. Dias, Patrı´cia Borges, Cristina Tanikawa Institute of Food Technology (ITAL), Av. Brasil, 2880, CEP 13073-001, Campinas, SP, Brazil Received 21 July 2000; received in revised form 15 December 2000; accepted 31 January 2001
Abstract The purpose of this research was to comparatively investigate the effect of whey protein concentrate (WPC), soy protein isolate (SPI) and casein on the growth and lipid concentration on rat liver and blood serum in a 45 days feeding experiment. Hypercholesterolemic diets containing 6% coconut fat plus 1% cholesterol and 20% protein from whey protein concentrate (WPC), soy protein isolate (SPI) and commercial casein (CC) were prepared. The remaining of the diets composition was identical with AIN (AIN-93G) recommendation. After 45 days of feeding CC and SPI diets promoted higher intake and better growth than the WPC diet. Blood serum and liver cholesterol was significantly higher (p ⬍ 0.05) for rats on the casein diet than for the rats on SPI and WPC diets. SPI diet was relatively more hypocholesterolemic than WPC diet. None of the dietary protein was able to maintain blood serum triacylglycerols at or near reference levels, with an increase of nearly two fold for all three dietary treatments. Blood serum total protein increased nearly two and a half fold, and no statistical differences were found among treatments. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Whey protein concentrate; Casein; Soy protein isolate; Hypercholesterolemic diet; Hypocholesterolemia
1. Introduction Milk as food has thousands of years of history. It is considered to be the ideal food for the new born of their species. Isolated milk protein products, as food ingredients, have become
* Corresponding author. Tel.: ⫹55-19-3241-5222 R 213; fax: ⫹55-19-3242-4585. E-mail address: [email protected] (V.C. Sgarbieri). 0271-5317/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 1 ) 0 0 2 9 7 - 4
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commercially available only in this century. In bovine milk 80% of the proteins are caseins and 20% whey proteins. Caseins are traditionally defined as the milk proteins which precipitate from raw skin milk at pH 4.6 and 20°C whereas whey proteins are those proteins which remain in the serum or whey under the same conditions [1]. Caseins are well known for their good nutritive value and excellent functional properties for food formulation [2]. The industrial interest for the milk whey proteins is much newer than for the caseins. Whey proteins are increasingly being used for nutritional purposes because they consistently score high in traditional tests of protein quality. The contents of total essential amino acids and branched-chain amino acids is superior in whey protein than in most dietary proteins. Potter et al [3], considered dietary vegetable protein as an important factor influencing blood serum lipid. According to Carrol [4], Carrol and Kurowska [5], protein of animal origin, such as casein, are generally hypercholesterolemic and atherogenic, when compared with vegetable proteins, as for example soybean protein, in both experimental animals and in humans. On the other hand, Sautier et al [6], and Nagaoka et al [7–8] reported on the hypolipidemic effect of milk whey protein comparatively to casein and soybean protein. Therefore, controversy still exists in the literature regarding the role of vegetable versus animal proteins on blood and liver cholesterol concentrations, particularly with respect to casein and whey protein compared with soybean protein. This was the motivation to investigate a WPC prepared in our pilot plant, with minimum heat-treatment, aiming at preserving the structural and physiological functional properties of the proteins. In this paper, the impact of feeding to Wistar rats, diets containing 20% of casein, whey protein concentrate or soybean protein isolate on growth, lipids and total protein concentration in blood serum, and liver cholesterol, is evaluated.
2. Materials and methods 2.1. Material of study Commercial casein was acquired from the local market (M. Cassab Company), soy protein isolate–Samprosoy 90 NB, was furnished by the company “Ceval Alimentos”, and the whey protein concentrate (WPC) was produced in our pilot plant. 2.2. Preparation of WPC WPC (80 – 85% protein) was produced in our pilot plant applying the following operations: defatted and pasteurized milk (72°C, 15 sec) was obtained in a milk farmer’s cooperative near our laboratory. On arriving at the pilot plant, the milk was immediately coagulated (Chymosin, 34°C, 45 to 60 min), following the separation of whey by filtration, after breaking the casein coagulum. The whey was concentrated by ultrafiltration (Kock Membrane Systems Inc., MW cut-off 10 kDa), to a concentration factor of 10. The operation was completed by diafiltration of the retentate, by using 15 times the retentate volume of deionized water (15 cycles). The retentate was then frozen and freeze-dried. All operations were performed at around or below 40°C to prevent protein denaturation.
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2.3. Analytical determinations Total solids, ashes and protein (N x F) were determined by the procedures described in the A.O.A.C. [9]. The appropriate factor (F) was applied to each protein source. Total lipids were determined by the method of Bligh and Dyer [10], and carbohydrate calculated by difference to 100%. Amino acids were determined after acid hydrolysis (6N HCl, 110°C, 22h), under vacuum, by cation exchange chromatography (Dionex-300 Amino Acid Analyser), and the amino acids quantified by post-column reaction with ninhydrin. Tryptophan was determined in a pronase hydrolysate by the colorimetric method of Spies [11]. 2.4. Rat assays, animals and diets A 45-days rat assay was performed using 64 weanling male rats of the Wistar strain, (30 to 35 g) initial body weight. The animals were furnished by the Central Animals Breeding Facilities of the University of Campinas, Sa˜o Paulo, Brazil, and were specific pathogen-free (SPF). Ten rats were sacrificed at the start of the experiment (T0) for determination of initial reference values. The remaining rats were randomly distributed in three groups to receive diets containing 20% protein diets from commercial casein (CC), soy protein isolate (SPI) or whey protein concentrate (WPC). Other dietary components were according with AIN-93G recommendation, reported by Reeves et al [12], except for the 7% vegetable oil in the AIN-93G which was replaced by 6% coconut fat plus 1% cholesterol, to make the diets more cholesterolemic. The animals were kept individually in stainless steel screen cages and received diets and water ad libitum. The temperature of the assay room was kept at 22 ⫾ 2°C with periods of light-dark alternating every 12 hours. Samples of blood and liver were collected at 15, 30 and 45 days of feeding, after anesthesia with ethyl ether. 2.5. Blood collection and analysis Blood collection was performed after 16 h fasting by total bleeding. The animals were anesthetized with ethyl ether, the axillary veined plexus was opened and the axillary vein cut. Blood was collected by using a Pasteur pipette. After collection the animals were immediately killed by cervical dislocation. The coagulated blood was maintained in water-bath (37°C, 15 min) followed by ice-bath (4°C, 15 min). Blood serum was obtained by centrifugation (1500 g, 15 min). Total cholesterol was determined by the Liebermann Burchard reaction using a Laborlab kit [13]. Triacylglycerols were determined by the enzymatic method of Fossati and Prencipe [14], by specific Laborlab kit. Total protein in the blood serum was determined by the Coomassie Brilliant Blue complexation method of Bradford [15]. 2.6. Liver collection and analysis After blood collection and the rats killed, the livers were collected, weighed and kept into saline solution prior to analysis. Liver extracts for analysis were prepared on isopropyl alcohol from 10% w/v suspensions of liver tissue. The cells suspensions were transferred to
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Table 1 Proximate percent composition (dry basis) of three dietary protein sources Component (%)
WPC1
SPI2
CC3
Protein (N ⫻ F) Total lipids Ash Carbohydrate (difference)
83.8 4.5 2.8 8.9
92.0 0.5 4.0 3.5
84.3 1.4 3.5 10.8
1
Milk whey protein concentrate (F ⫽ 6.38); 2 Soy protein isolate (F ⫽ 6.25); 3 Commercial casein (F ⫽ 6.38).
centrifuge tubes and let them stand for 48 h and then centrifuged (3000 g, 15 min). The supernatant was used for cholesterol [13] and triacylglycerol [14] determinations. 2.7. Statistical analysis The experimental results were submitted to analysis of variance and to the Tukey test of means at a confidence level of 95% (p ⬍ 0.05), according to Gomes [16]. The Statistica: Basic Statistics and Tables Program was used.
3. Results The proximate composition of the three protein sources under study is shown in Table 1. WPC and commercial casein (CC) showed greater similarity in terms of gross composition, except for total lipids which was considerably lower in CC. Soy protein isolate (SPI) showed very low total lipids, a lower carbohydrate and a higher protein concentration, compared with WPC and CC. The essential amino acid profiles of the three proteins are shown in Table 2, in comparison with the FAO/WHO [17] recommendation for children 2 to 5 years old. The amino acid Table 2 Essential amino acid profiles of three dietary protein sources and amino acid scores based on the FAO/WHO theoretical profile Amino acid (g/100gP)
WPC1
SPI2
CC3
FAO/WHO
Threonine Valine Methionine⫹Cystine Isoleucine Leucine Phenylalanine⫹tyrosine Lysine Histidine Tryptophan AAS4
6.40 5.04 3.43 5.29 9.81 6.15* 9.27 5.15 1.12 0.98
3.60 4.50 2.10* 4.30 7.80 9.50 6.50 2.70 1.00 0.84
4.15 6.16 2.17* 4.62 8.87 9.92 7.67 2.84 1.96 0.87
3.4 3.5 2.5 2.8 6.6 6.3 5.8 1.9 1.1
1
2
3
4
—
Milk whey protein concentrate; Soy protein isolate; Commercial casein; Amino acid score, FAO/WHO [17]; * limiting essential amino acids.
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Table 3 Average diet consumption (DC), protein consumption (PC), body weight gain (BWG) and BWG/PC ratio, for rats fed three different sources of dietary protein at 20% concentration, for a 45 days period Determined Index1
Diet consumption (g) Protein consumption (g) Body weight gain (g) Body weight gain/protein consumption 1
Dietary protein (20% w/w) WPC2
SPI3
CC4
822.2 ⫾ 26.2b 164.4 ⫾ 5.2b 159.2 ⫾ 17.2b 0.96
940.0 ⫾ 40.3a 188.0 ⫾ 8.0a 188.0 ⫾ 8.0a 1.00
972.0 ⫾ 61.8a 194.4 ⫾ 12.4a 199.9 ⫾ 11.5a 1.02
Means ⫾ SD of four rats; 2 Whey protein Concentrate; 3 Soy protein isolate; 4 Commercial casein. Different superscript letters (lines) indicate statistical different results (p ⬍ 0.05).
a,b
composition of SPI and CC are quite similar, except for higher concentrations of valine and tryptophan in CC, compared to SPI. WPC presents higher concentrations of threonine, sulfur-containing amino acids (methionine ⫹ cystine), branched-chain amino acids (valine, leucine, isoleucine), lysine and histidine, and lower concentrations of the aromatic chain amino acids (phenylalanine ⫹ tyrosine), in comparison with SPI and CC. The essential amino acid scores, compared with FAO/WHO [17] is higher (essentially 1.0) for WPC and lower (0.84 and 0.87) respectively, for SPI and CC. Table 3 illustrates the values for some indices of growth promotion and dietary protein utilization for the three protein sources under study. SPI and CC diets had a better performance than WPC diet for all the indices determined. Although no statistical treatment was given to the ratio of body weight gain/protein consumption it is believed that no difference should exist between these ratios for the three dietary proteins. Therefore it is suggested that the lower body weight gain for the rats on WPC should be attributed to lower food intake, rather than difference in protein quality. Results of total serum cholesterol are shown in Fig. 1. Statistical differences for means (p ⬍ 0.05) were found among the dietary treatments for each time interval. Total serum cholesterol was consistently higher for the rats on CC diet and consistently lower (p ⬍ 0.05) for the rats on WPC and SPI diets. After 30 and 45 days on the diets (T30 and T45) serum cholesterol was lower for the rats on SPI than for the rats on WPC diet. Changes in the liver total cholesterol are illustrated in the Fig. 2. A net increase in liver cholesterol, compared with the initial value (T0) can be noticed for all three dietary treatments. After the first 15 days, liver cholesterol concentration was identical for the three dietary treatments. After 30 days (T30), cholesterol was higher and identical for the groups on CC and WPC and significantly lower (p ⬍ 0.05) for SPI diet. At the end of the experiment (T45), cholesterol was higher for the rats on CC diet but lower for the rats on SPI and WPC, which did not differ statistically among themselves. Changes in rat serum triacylglycerols, as a function of time and dietary protein type are shown in Fig. 3. Contrary to serum cholesterol, triacylglycerols did not follow a definite pattern for any one of the dietary treatment, alternating statistical differences at the three time intervals studied. Compared with the starting value (T0) all three dietary treatments promoted a pronounced rise in the blood serum triacylglycerols. At the end of the experiment (T45) the
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Fig. 1. Blood serum cholesterol of rats fed on hypercholesterolemic diets containing 20% (w/w) protein of the following sources: WPC, whey protein concentrate; SPI, soy protein isolate; CC, commercial casein. a,b,cDifferent letters indicate statistical differences among treatments at each time interval (p ⬍ 0.05).
concentration was lower (p ⬍ 0.05) for the CC diet and significantly higher, although not different among themselves, for the SPI and WPC diets. Net total blood serum protein raised around three fold during the 45 days experiment, as
Fig. 2. Liver cholesterol of rats fed on hypercholesterolemic diets containing 20% (w/w) protein of the following sources: WPC, whey protein concentrate; SPI, soy protein isolate; CC, commercial casein. a,bDifferent letters indicate statistical differences among treatments at each time interval (p ⬍ 0.05).
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Fig. 3. Blood serum triacylglycerols of rats fed on hypercholesterolemic diets containing 20% (w/w) protein of the following sources: WPC, whey protein concentrate; SPI, soy protein isolate; CC, commercial casein. a, b, c Different letters indicate statistical differences among treatments at each time interval (p ⬍ 0.05).
illustrated in Fig. 4. There was no statistical differences for blood serum protein, among the dietary treatments.
4. Discussion The essential amino acid composition and the amino acid scores [17], for the three protein sources, permitted to anticipate a higher growth for the WPC diet, in comparison with the CC and SPI diets. However, food intake and growth (Table 3) was lower for WPC diet compared with the SPI and CC diets. It is not apparent what could have caused lower food intake which resulted in lower growth for the rats on WPC diet. Work reported by Boirie et al [18] and Fru¨hbeck [19] demonstrated that the speed of amino acid absorption after protein ingestion has a major impact on the postprandial metabolic response to a single protein meal. They showed that casein was absorbed slowly and promoted postprandial protein deposition by inhibition of protein breakdown without excessive increase in blood amino acid concentration. On the other hand, whey protein was absorbed very fast and rapidly stimulated protein synthesis but also amino acids oxidation. These investigators classified casein and whey protein, respectively as slow and fast metabolizing proteins. There is a possibility that the rapid and high influx of amino acids into the blood from ingestion of whey protein may have triggered two simultaneous physiological processes, i.e.:
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Fig. 4. Blood serum total protein of rats fed on hypercholesterolemic diets containing 20% (w/w) protein of the following sources: WPC, whey protein concentrate; SPI, soy protein isolate; CC, commercial casein. a, b Different letters indicate statistical differences among treatments at each time interval (p ⬍ 0.05).
inhibition of appetite and food intake by a momentary accumulation of free amino acids in the blood; secondly the fast stimulation of protein synthesis and breakdown, all above the dietary casein, might be responsible for an energy deficit relative to casein diet that might be responsible for the lower body weight gain of rats on whey protein diets. The inhibition of food intake by a fast and high influx of free amino acids in the rat blood could be compared to what happened when carbohydrates were given to the rat as a single meal [20]. The voluntary food intake suppression was proportional to the dose and dependent on the type of carbohydrate. The results described in this paper differed from published data [6 – 8], which reported no differences in food intake and growth of rats fed on diets containing similar but not identical concentrations of the proteins under study, in the present paper. Differences not only in protein concentrations but also in the nature and proportions of other dietary components might have influenced diet intake by the rats. With regard to blood serum total cholesterol, our results also show some differences with reported data. Sautier et al [6] using diets with 23% protein from casein, whey protein and soy protein, in experiment of 49 days of duration, demonstrated a cholesterol lowering effect of whey protein, compared with casein and soy protein. Compared with casein they could not demonstrate a cholesterol lowering effect of soy protein in the rat serum. Nagaoka et al [7,8] arrived essentially to the same conclusions as Sautier et al [6]. These authors also demonstrated a cholesterol lowering effect for whey protein and a cholesterol raising effect of both casein and soy protein, but no statistical significant difference for casein and soy protein. These authors, particularly Nagaoka et al [8] questioned the suggested hypocholesterolemic property of soy protein and soy products, reported in the literature [21,22], for long time.
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In the present paper our results also show a strong cholesterol lowering effect in the liver of rats fed diets containing 20% of either SPI or WPC for 45 days (Fig. 2). These results also deviate, in various aspects from Sautier et al [6], and Nagaoka et al [7], who were not able to demonstrate statistical differences for total liver cholesterol concentration when casein, soy protein and whey protein were used in rat diets. On the other hand Nagaoka et al [8], using a hypercholesterolemic diet in a short (14 days) feeding experiment showed an equal liver cholesterol raising effect for casein and soy protein but a significant cholesterol lowering effect for whey protein. These investigators also demonstrated a relative increase in HDL-cholesterol in the rat serum for casein and whey protein but a lowering effect for soy protein. Therefore, the ratio of total cholesterol to HDL-cholesterol decreased for rats on casein and whey protein diets but increased in the rats on soy protein diet. Our results (Fig. 3) on rat serum triacylglycrols also seem to differ from the findings of Sautier et al [6], and Nagaoka et al [7], who showed no differences in serum concentrations of triacylglycerols when either casein, soy protein or whey protein was used in the rat diet. Although presenting considerable variation, the data of Fig. 3 show statistical differences in serum triacylglycerol concentrations at the various time intervals of sampling. The above investigators did not report on the reference (T0) values in their papers, therefore these comparisons can not be made. The literature on the effect of dietary vegetable and animal proteins, on the blood and liver lipidemia of experimental animal and humans, is still highly controversial. According to Kritchevsky [23], Beynen [24], Nagaoka et al [7], Carrol and Kurowska [5], the nature and concentration of dietary protein, of both animal and vegetable origin, may affect the concentration of blood serum cholesterol. Proteins of animal origin are generally thought as being hypercholesterolemic when compared with proteins of vegetable origin, which tend to be hypocholesterolemic, Carrol and Kurowska [5]. Yoshida et al [25] and Norton et al [26] reported on a lowering effect of serum cholesterol by milk whey protein. Stahelin and Ritzel [27] failed to demonstrate any effect of whey protein on blood serum cholesterol. On the other hand, Lovati et al [28] found an elevation of blood serum cholesterol by feeding whey protein to the rabbit. Zhang and Beynen [29] demonstrated that whey protein decreased liver cholesterol but not blood serum cholesterol, when given to rat at 15% concentration of the diet, however at 30% concentration both liver and serum cholesterol were reduced. Yuan and Kitts [30] studied the effect of milk products and milk protein fractions, compared with soy protein isolate, on the levels of liver and serum total cholesterol and triacylglyerols, using Wistar rats fed either casein, whey protein or defatted powdered milk as source of dietary protein. They found no effect of the dietary treatments on the concentrations of cholesterol and triacylglycerols, both in the liver and in the blood serum. According to Norum [31], the serum triacylglycerol levels is going to depend on the type and quality of dietary fat. There will be a post-prandial elevation of serum triacylglycerols which will last longer after ingestion of saturated than polyunsaturated fats. The type and quantity of dietary fat will also affect serum triacylglycerol levels in fasting periods. The present discussion reveals that the knowledge of how dietary proteins, both of animal and vegetable origins, affect experimental animal and human lipidemia, are still fragmentary and controversial.
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It is also not well established the mechanisms by which cholesterol and other lipids may, more effectively, be eliminated from the body circulation. In summary, the present study permitted to conclude that commercial casein and soy protein isolate promoted higher food intake and better rat growth than the whey protein concentrate. Both, soy protein isolate and whey protein concentrate, were effective in preventing blood serum cholesterol to rise, when hypercholesterolemic diets were fed to the rats. Soy protein isolate exhibited a stronger hypocholesterolemic effect than whey protein concentrate. On the other hand casein was hypercholesterolemic raising serum cholesterol to levels significantly higher than both soy protein isolate and whey protein concentrate. Casein also raised liver cholesterol to a level significantly higher than both soy protein isolate and whey protein concentrate, after 45 days on the diets. Rat serum triacylglycerols was significantly elevated for all three dietary proteins, as a function of time in the hypercholesterolemic diets.
Acknowledgments The authors acknowledge the financial support for this research given by FAPESP (Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo, Brasil).
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[13] Abell LL, Levy BB, Kendall FE. A simplified method for the estimation of total cholesterol in serum, and demonstration of its specificity. J Biol Chem 1952;195:357– 66. [14] Fossati P, Prencipe L. Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide. Clin Chem 1982;28:2077– 80. [15] Bradford MM. A rapid, and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248 –54. [16] Gomes FP. Curso de Estatı´stica Experimental, 10th ed. Sa˜o Paulo: Nobel, 1982. 430p. [17] FAO/WHO. Food and Agriculture Organization/World Health Organization. Report of a Joint FAO/WHO Expert Consultation on Protein Quality Evaluation, 1990, Bethesda, Maryland. [18] Boirie Y, Dangin M, Gachon P, Vasson M-P, Maubois J-L, Beaufre`re B. Slow and fast dietary proteins differently modulate postprandial protein accretion. Proc Natl Acad Sci USA 1997;121:14930 –5. [19] Fru¨hbeck G. Slow and fast dietary proteins. Nature 1998;39:843–5. [20] Trigazis L, Pang MB, Cheng I, Anderson GH. Carbohydrate composition effects on food intake and energy compensation in rats. Nutr Res 1998;18:863–74. [21] Nagata Y, Tanaka K, Sugano M. Further studies on the hypocholesterolemic effect of soybean protein in rats. J Braz Nutr 1981;45:233– 41. [22] Potter SM. Overview of proposed mechanisms for the hypocholesterolemic effect of soy. J Nutr 1995;125: 606S–11S. [23] Kritchevsky D. Dietary protein, cholesterol and atherosclerosis: a review of the early history. J Nutr 1995;35:589S–93S. [24] Beynen AC. Comparison of the mechanisms proposed to explain the hypocholesterolemic effect of soybean protein versus casein in experimental animals. J Nutr Sci Vitaminol 1990;36:587–93. [25] Yoshida K, Yahiro M, Ahiko K. Effects of addition of arginine, cystine, and glycine to bovine milksimulated amino acid mixture to the level of plasma and liver cholesterol in rats. J Nutr Sci Vitaminol 1988;34:567–76. [26] Norton SA, Beames CG, Maxwell CV, Morgan GL. Effect of dietary whey upon the serum cholesterol of the pig. Nutr Rep Int 1987;36:273–9. [27] Stahelin HB, Ritzel G. Effect of whey on plasma lipids. Int J Vitaminol Nutr Res 1979;50:229 –30. [28] Lovati MR, West CE, Sirtori CR, Beynen AC. Dietary animals proteins and cholesterol metabolism in rabbits. Brit J Nutr 1990;64:473– 85. [29] Zhang X, Beynen AC. Influence of dietary fish proteins on plasma and liver cholesterol concentrations in rats. Br J Nutr 1993;69:767–77. [30] Yuan YV, Kitts DD. Milk product and constituent protein effects on plasma and hepatic lipids and systolic blood pressure of normotensive rats. Food Res Int 1993;26:173–9. [31] Norum KR. Dietary fat and blood lipids. Nutr Rev 1992;50:10 –37.
Impact of different dietary protein on rat growth, blood serum lipids and protein and liver cholesterol Helaine B. Jacobucci, Valdemiro C. Sgarbieri*, Na´dia F.G.P. Dias, Patrı´cia Borges, Cristina Tanikawa Institute of Food Technology (ITAL), Av. Brasil, 2880, CEP 13073-001, Campinas, SP, Brazil Received 21 July 2000; received in revised form 15 December 2000; accepted 31 January 2001
Abstract The purpose of this research was to comparatively investigate the effect of whey protein concentrate (WPC), soy protein isolate (SPI) and casein on the growth and lipid concentration on rat liver and blood serum in a 45 days feeding experiment. Hypercholesterolemic diets containing 6% coconut fat plus 1% cholesterol and 20% protein from whey protein concentrate (WPC), soy protein isolate (SPI) and commercial casein (CC) were prepared. The remaining of the diets composition was identical with AIN (AIN-93G) recommendation. After 45 days of feeding CC and SPI diets promoted higher intake and better growth than the WPC diet. Blood serum and liver cholesterol was significantly higher (p ⬍ 0.05) for rats on the casein diet than for the rats on SPI and WPC diets. SPI diet was relatively more hypocholesterolemic than WPC diet. None of the dietary protein was able to maintain blood serum triacylglycerols at or near reference levels, with an increase of nearly two fold for all three dietary treatments. Blood serum total protein increased nearly two and a half fold, and no statistical differences were found among treatments. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Whey protein concentrate; Casein; Soy protein isolate; Hypercholesterolemic diet; Hypocholesterolemia
1. Introduction Milk as food has thousands of years of history. It is considered to be the ideal food for the new born of their species. Isolated milk protein products, as food ingredients, have become
* Corresponding author. Tel.: ⫹55-19-3241-5222 R 213; fax: ⫹55-19-3242-4585. E-mail address: [email protected] (V.C. Sgarbieri). 0271-5317/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 1 ) 0 0 2 9 7 - 4
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commercially available only in this century. In bovine milk 80% of the proteins are caseins and 20% whey proteins. Caseins are traditionally defined as the milk proteins which precipitate from raw skin milk at pH 4.6 and 20°C whereas whey proteins are those proteins which remain in the serum or whey under the same conditions [1]. Caseins are well known for their good nutritive value and excellent functional properties for food formulation [2]. The industrial interest for the milk whey proteins is much newer than for the caseins. Whey proteins are increasingly being used for nutritional purposes because they consistently score high in traditional tests of protein quality. The contents of total essential amino acids and branched-chain amino acids is superior in whey protein than in most dietary proteins. Potter et al [3], considered dietary vegetable protein as an important factor influencing blood serum lipid. According to Carrol [4], Carrol and Kurowska [5], protein of animal origin, such as casein, are generally hypercholesterolemic and atherogenic, when compared with vegetable proteins, as for example soybean protein, in both experimental animals and in humans. On the other hand, Sautier et al [6], and Nagaoka et al [7–8] reported on the hypolipidemic effect of milk whey protein comparatively to casein and soybean protein. Therefore, controversy still exists in the literature regarding the role of vegetable versus animal proteins on blood and liver cholesterol concentrations, particularly with respect to casein and whey protein compared with soybean protein. This was the motivation to investigate a WPC prepared in our pilot plant, with minimum heat-treatment, aiming at preserving the structural and physiological functional properties of the proteins. In this paper, the impact of feeding to Wistar rats, diets containing 20% of casein, whey protein concentrate or soybean protein isolate on growth, lipids and total protein concentration in blood serum, and liver cholesterol, is evaluated.
2. Materials and methods 2.1. Material of study Commercial casein was acquired from the local market (M. Cassab Company), soy protein isolate–Samprosoy 90 NB, was furnished by the company “Ceval Alimentos”, and the whey protein concentrate (WPC) was produced in our pilot plant. 2.2. Preparation of WPC WPC (80 – 85% protein) was produced in our pilot plant applying the following operations: defatted and pasteurized milk (72°C, 15 sec) was obtained in a milk farmer’s cooperative near our laboratory. On arriving at the pilot plant, the milk was immediately coagulated (Chymosin, 34°C, 45 to 60 min), following the separation of whey by filtration, after breaking the casein coagulum. The whey was concentrated by ultrafiltration (Kock Membrane Systems Inc., MW cut-off 10 kDa), to a concentration factor of 10. The operation was completed by diafiltration of the retentate, by using 15 times the retentate volume of deionized water (15 cycles). The retentate was then frozen and freeze-dried. All operations were performed at around or below 40°C to prevent protein denaturation.
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2.3. Analytical determinations Total solids, ashes and protein (N x F) were determined by the procedures described in the A.O.A.C. [9]. The appropriate factor (F) was applied to each protein source. Total lipids were determined by the method of Bligh and Dyer [10], and carbohydrate calculated by difference to 100%. Amino acids were determined after acid hydrolysis (6N HCl, 110°C, 22h), under vacuum, by cation exchange chromatography (Dionex-300 Amino Acid Analyser), and the amino acids quantified by post-column reaction with ninhydrin. Tryptophan was determined in a pronase hydrolysate by the colorimetric method of Spies [11]. 2.4. Rat assays, animals and diets A 45-days rat assay was performed using 64 weanling male rats of the Wistar strain, (30 to 35 g) initial body weight. The animals were furnished by the Central Animals Breeding Facilities of the University of Campinas, Sa˜o Paulo, Brazil, and were specific pathogen-free (SPF). Ten rats were sacrificed at the start of the experiment (T0) for determination of initial reference values. The remaining rats were randomly distributed in three groups to receive diets containing 20% protein diets from commercial casein (CC), soy protein isolate (SPI) or whey protein concentrate (WPC). Other dietary components were according with AIN-93G recommendation, reported by Reeves et al [12], except for the 7% vegetable oil in the AIN-93G which was replaced by 6% coconut fat plus 1% cholesterol, to make the diets more cholesterolemic. The animals were kept individually in stainless steel screen cages and received diets and water ad libitum. The temperature of the assay room was kept at 22 ⫾ 2°C with periods of light-dark alternating every 12 hours. Samples of blood and liver were collected at 15, 30 and 45 days of feeding, after anesthesia with ethyl ether. 2.5. Blood collection and analysis Blood collection was performed after 16 h fasting by total bleeding. The animals were anesthetized with ethyl ether, the axillary veined plexus was opened and the axillary vein cut. Blood was collected by using a Pasteur pipette. After collection the animals were immediately killed by cervical dislocation. The coagulated blood was maintained in water-bath (37°C, 15 min) followed by ice-bath (4°C, 15 min). Blood serum was obtained by centrifugation (1500 g, 15 min). Total cholesterol was determined by the Liebermann Burchard reaction using a Laborlab kit [13]. Triacylglycerols were determined by the enzymatic method of Fossati and Prencipe [14], by specific Laborlab kit. Total protein in the blood serum was determined by the Coomassie Brilliant Blue complexation method of Bradford [15]. 2.6. Liver collection and analysis After blood collection and the rats killed, the livers were collected, weighed and kept into saline solution prior to analysis. Liver extracts for analysis were prepared on isopropyl alcohol from 10% w/v suspensions of liver tissue. The cells suspensions were transferred to
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Table 1 Proximate percent composition (dry basis) of three dietary protein sources Component (%)
WPC1
SPI2
CC3
Protein (N ⫻ F) Total lipids Ash Carbohydrate (difference)
83.8 4.5 2.8 8.9
92.0 0.5 4.0 3.5
84.3 1.4 3.5 10.8
1
Milk whey protein concentrate (F ⫽ 6.38); 2 Soy protein isolate (F ⫽ 6.25); 3 Commercial casein (F ⫽ 6.38).
centrifuge tubes and let them stand for 48 h and then centrifuged (3000 g, 15 min). The supernatant was used for cholesterol [13] and triacylglycerol [14] determinations. 2.7. Statistical analysis The experimental results were submitted to analysis of variance and to the Tukey test of means at a confidence level of 95% (p ⬍ 0.05), according to Gomes [16]. The Statistica: Basic Statistics and Tables Program was used.
3. Results The proximate composition of the three protein sources under study is shown in Table 1. WPC and commercial casein (CC) showed greater similarity in terms of gross composition, except for total lipids which was considerably lower in CC. Soy protein isolate (SPI) showed very low total lipids, a lower carbohydrate and a higher protein concentration, compared with WPC and CC. The essential amino acid profiles of the three proteins are shown in Table 2, in comparison with the FAO/WHO [17] recommendation for children 2 to 5 years old. The amino acid Table 2 Essential amino acid profiles of three dietary protein sources and amino acid scores based on the FAO/WHO theoretical profile Amino acid (g/100gP)
WPC1
SPI2
CC3
FAO/WHO
Threonine Valine Methionine⫹Cystine Isoleucine Leucine Phenylalanine⫹tyrosine Lysine Histidine Tryptophan AAS4
6.40 5.04 3.43 5.29 9.81 6.15* 9.27 5.15 1.12 0.98
3.60 4.50 2.10* 4.30 7.80 9.50 6.50 2.70 1.00 0.84
4.15 6.16 2.17* 4.62 8.87 9.92 7.67 2.84 1.96 0.87
3.4 3.5 2.5 2.8 6.6 6.3 5.8 1.9 1.1
1
2
3
4
—
Milk whey protein concentrate; Soy protein isolate; Commercial casein; Amino acid score, FAO/WHO [17]; * limiting essential amino acids.
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Table 3 Average diet consumption (DC), protein consumption (PC), body weight gain (BWG) and BWG/PC ratio, for rats fed three different sources of dietary protein at 20% concentration, for a 45 days period Determined Index1
Diet consumption (g) Protein consumption (g) Body weight gain (g) Body weight gain/protein consumption 1
Dietary protein (20% w/w) WPC2
SPI3
CC4
822.2 ⫾ 26.2b 164.4 ⫾ 5.2b 159.2 ⫾ 17.2b 0.96
940.0 ⫾ 40.3a 188.0 ⫾ 8.0a 188.0 ⫾ 8.0a 1.00
972.0 ⫾ 61.8a 194.4 ⫾ 12.4a 199.9 ⫾ 11.5a 1.02
Means ⫾ SD of four rats; 2 Whey protein Concentrate; 3 Soy protein isolate; 4 Commercial casein. Different superscript letters (lines) indicate statistical different results (p ⬍ 0.05).
a,b
composition of SPI and CC are quite similar, except for higher concentrations of valine and tryptophan in CC, compared to SPI. WPC presents higher concentrations of threonine, sulfur-containing amino acids (methionine ⫹ cystine), branched-chain amino acids (valine, leucine, isoleucine), lysine and histidine, and lower concentrations of the aromatic chain amino acids (phenylalanine ⫹ tyrosine), in comparison with SPI and CC. The essential amino acid scores, compared with FAO/WHO [17] is higher (essentially 1.0) for WPC and lower (0.84 and 0.87) respectively, for SPI and CC. Table 3 illustrates the values for some indices of growth promotion and dietary protein utilization for the three protein sources under study. SPI and CC diets had a better performance than WPC diet for all the indices determined. Although no statistical treatment was given to the ratio of body weight gain/protein consumption it is believed that no difference should exist between these ratios for the three dietary proteins. Therefore it is suggested that the lower body weight gain for the rats on WPC should be attributed to lower food intake, rather than difference in protein quality. Results of total serum cholesterol are shown in Fig. 1. Statistical differences for means (p ⬍ 0.05) were found among the dietary treatments for each time interval. Total serum cholesterol was consistently higher for the rats on CC diet and consistently lower (p ⬍ 0.05) for the rats on WPC and SPI diets. After 30 and 45 days on the diets (T30 and T45) serum cholesterol was lower for the rats on SPI than for the rats on WPC diet. Changes in the liver total cholesterol are illustrated in the Fig. 2. A net increase in liver cholesterol, compared with the initial value (T0) can be noticed for all three dietary treatments. After the first 15 days, liver cholesterol concentration was identical for the three dietary treatments. After 30 days (T30), cholesterol was higher and identical for the groups on CC and WPC and significantly lower (p ⬍ 0.05) for SPI diet. At the end of the experiment (T45), cholesterol was higher for the rats on CC diet but lower for the rats on SPI and WPC, which did not differ statistically among themselves. Changes in rat serum triacylglycerols, as a function of time and dietary protein type are shown in Fig. 3. Contrary to serum cholesterol, triacylglycerols did not follow a definite pattern for any one of the dietary treatment, alternating statistical differences at the three time intervals studied. Compared with the starting value (T0) all three dietary treatments promoted a pronounced rise in the blood serum triacylglycerols. At the end of the experiment (T45) the
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Fig. 1. Blood serum cholesterol of rats fed on hypercholesterolemic diets containing 20% (w/w) protein of the following sources: WPC, whey protein concentrate; SPI, soy protein isolate; CC, commercial casein. a,b,cDifferent letters indicate statistical differences among treatments at each time interval (p ⬍ 0.05).
concentration was lower (p ⬍ 0.05) for the CC diet and significantly higher, although not different among themselves, for the SPI and WPC diets. Net total blood serum protein raised around three fold during the 45 days experiment, as
Fig. 2. Liver cholesterol of rats fed on hypercholesterolemic diets containing 20% (w/w) protein of the following sources: WPC, whey protein concentrate; SPI, soy protein isolate; CC, commercial casein. a,bDifferent letters indicate statistical differences among treatments at each time interval (p ⬍ 0.05).
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Fig. 3. Blood serum triacylglycerols of rats fed on hypercholesterolemic diets containing 20% (w/w) protein of the following sources: WPC, whey protein concentrate; SPI, soy protein isolate; CC, commercial casein. a, b, c Different letters indicate statistical differences among treatments at each time interval (p ⬍ 0.05).
illustrated in Fig. 4. There was no statistical differences for blood serum protein, among the dietary treatments.
4. Discussion The essential amino acid composition and the amino acid scores [17], for the three protein sources, permitted to anticipate a higher growth for the WPC diet, in comparison with the CC and SPI diets. However, food intake and growth (Table 3) was lower for WPC diet compared with the SPI and CC diets. It is not apparent what could have caused lower food intake which resulted in lower growth for the rats on WPC diet. Work reported by Boirie et al [18] and Fru¨hbeck [19] demonstrated that the speed of amino acid absorption after protein ingestion has a major impact on the postprandial metabolic response to a single protein meal. They showed that casein was absorbed slowly and promoted postprandial protein deposition by inhibition of protein breakdown without excessive increase in blood amino acid concentration. On the other hand, whey protein was absorbed very fast and rapidly stimulated protein synthesis but also amino acids oxidation. These investigators classified casein and whey protein, respectively as slow and fast metabolizing proteins. There is a possibility that the rapid and high influx of amino acids into the blood from ingestion of whey protein may have triggered two simultaneous physiological processes, i.e.:
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Fig. 4. Blood serum total protein of rats fed on hypercholesterolemic diets containing 20% (w/w) protein of the following sources: WPC, whey protein concentrate; SPI, soy protein isolate; CC, commercial casein. a, b Different letters indicate statistical differences among treatments at each time interval (p ⬍ 0.05).
inhibition of appetite and food intake by a momentary accumulation of free amino acids in the blood; secondly the fast stimulation of protein synthesis and breakdown, all above the dietary casein, might be responsible for an energy deficit relative to casein diet that might be responsible for the lower body weight gain of rats on whey protein diets. The inhibition of food intake by a fast and high influx of free amino acids in the rat blood could be compared to what happened when carbohydrates were given to the rat as a single meal [20]. The voluntary food intake suppression was proportional to the dose and dependent on the type of carbohydrate. The results described in this paper differed from published data [6 – 8], which reported no differences in food intake and growth of rats fed on diets containing similar but not identical concentrations of the proteins under study, in the present paper. Differences not only in protein concentrations but also in the nature and proportions of other dietary components might have influenced diet intake by the rats. With regard to blood serum total cholesterol, our results also show some differences with reported data. Sautier et al [6] using diets with 23% protein from casein, whey protein and soy protein, in experiment of 49 days of duration, demonstrated a cholesterol lowering effect of whey protein, compared with casein and soy protein. Compared with casein they could not demonstrate a cholesterol lowering effect of soy protein in the rat serum. Nagaoka et al [7,8] arrived essentially to the same conclusions as Sautier et al [6]. These authors also demonstrated a cholesterol lowering effect for whey protein and a cholesterol raising effect of both casein and soy protein, but no statistical significant difference for casein and soy protein. These authors, particularly Nagaoka et al [8] questioned the suggested hypocholesterolemic property of soy protein and soy products, reported in the literature [21,22], for long time.
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In the present paper our results also show a strong cholesterol lowering effect in the liver of rats fed diets containing 20% of either SPI or WPC for 45 days (Fig. 2). These results also deviate, in various aspects from Sautier et al [6], and Nagaoka et al [7], who were not able to demonstrate statistical differences for total liver cholesterol concentration when casein, soy protein and whey protein were used in rat diets. On the other hand Nagaoka et al [8], using a hypercholesterolemic diet in a short (14 days) feeding experiment showed an equal liver cholesterol raising effect for casein and soy protein but a significant cholesterol lowering effect for whey protein. These investigators also demonstrated a relative increase in HDL-cholesterol in the rat serum for casein and whey protein but a lowering effect for soy protein. Therefore, the ratio of total cholesterol to HDL-cholesterol decreased for rats on casein and whey protein diets but increased in the rats on soy protein diet. Our results (Fig. 3) on rat serum triacylglycrols also seem to differ from the findings of Sautier et al [6], and Nagaoka et al [7], who showed no differences in serum concentrations of triacylglycerols when either casein, soy protein or whey protein was used in the rat diet. Although presenting considerable variation, the data of Fig. 3 show statistical differences in serum triacylglycerol concentrations at the various time intervals of sampling. The above investigators did not report on the reference (T0) values in their papers, therefore these comparisons can not be made. The literature on the effect of dietary vegetable and animal proteins, on the blood and liver lipidemia of experimental animal and humans, is still highly controversial. According to Kritchevsky [23], Beynen [24], Nagaoka et al [7], Carrol and Kurowska [5], the nature and concentration of dietary protein, of both animal and vegetable origin, may affect the concentration of blood serum cholesterol. Proteins of animal origin are generally thought as being hypercholesterolemic when compared with proteins of vegetable origin, which tend to be hypocholesterolemic, Carrol and Kurowska [5]. Yoshida et al [25] and Norton et al [26] reported on a lowering effect of serum cholesterol by milk whey protein. Stahelin and Ritzel [27] failed to demonstrate any effect of whey protein on blood serum cholesterol. On the other hand, Lovati et al [28] found an elevation of blood serum cholesterol by feeding whey protein to the rabbit. Zhang and Beynen [29] demonstrated that whey protein decreased liver cholesterol but not blood serum cholesterol, when given to rat at 15% concentration of the diet, however at 30% concentration both liver and serum cholesterol were reduced. Yuan and Kitts [30] studied the effect of milk products and milk protein fractions, compared with soy protein isolate, on the levels of liver and serum total cholesterol and triacylglyerols, using Wistar rats fed either casein, whey protein or defatted powdered milk as source of dietary protein. They found no effect of the dietary treatments on the concentrations of cholesterol and triacylglycerols, both in the liver and in the blood serum. According to Norum [31], the serum triacylglycerol levels is going to depend on the type and quality of dietary fat. There will be a post-prandial elevation of serum triacylglycerols which will last longer after ingestion of saturated than polyunsaturated fats. The type and quantity of dietary fat will also affect serum triacylglycerol levels in fasting periods. The present discussion reveals that the knowledge of how dietary proteins, both of animal and vegetable origins, affect experimental animal and human lipidemia, are still fragmentary and controversial.
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It is also not well established the mechanisms by which cholesterol and other lipids may, more effectively, be eliminated from the body circulation. In summary, the present study permitted to conclude that commercial casein and soy protein isolate promoted higher food intake and better rat growth than the whey protein concentrate. Both, soy protein isolate and whey protein concentrate, were effective in preventing blood serum cholesterol to rise, when hypercholesterolemic diets were fed to the rats. Soy protein isolate exhibited a stronger hypocholesterolemic effect than whey protein concentrate. On the other hand casein was hypercholesterolemic raising serum cholesterol to levels significantly higher than both soy protein isolate and whey protein concentrate. Casein also raised liver cholesterol to a level significantly higher than both soy protein isolate and whey protein concentrate, after 45 days on the diets. Rat serum triacylglycerols was significantly elevated for all three dietary proteins, as a function of time in the hypercholesterolemic diets.
Acknowledgments The authors acknowledge the financial support for this research given by FAPESP (Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo, Brasil).
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