There is increasing evidence to support that a high-protein diet may promote weight loss and prevent weight (re)gain better than a low-protein diet, and that the effect is due to higher diet-induced thermogenesis (DIT) and increased satiety. However, data on the effect of different types of protein are limited. In the present study we compare the effect of whey, casein and milk on DIT and satiety.
Seventeen slightly overweight (29±4 kg/m2) male subjects completed the study. The study had a randomized, crossover design, where the effect on 4 h postprandial energy expenditure (EE), substrate oxidation and subjective appetite sensation of three isocaloric test meals containing either a whey drink, a casein drink or skim milk was examined. Energy intake (EI) at a subsequent ad libitum lunch was also measured.
There was no significant effect on subjective appetite sensation, but EI at lunch was lower after the milk test meal than after the casein (9%; P=0.0260) and the whey (9%; P=0.0258) test meals. Postprandial lipid oxidation was significantly higher after the casein test meal compared with the whey test meal (P=0.0147) when adjusted for baseline values. There was no significant difference in effect on EE, protein oxidation or carbohydrate oxidation.
Milk reduced subsequent EI more than isocaloric drinks containing only whey or casein. A small but significant increase in lipid oxidation was seen after casein compared with whey.
There is accumulating evidence that a high protein intake increases weight loss and prevents weight (re)gain.1, 2, 3, 4 The large multicenter European study Diet, Obesity and Genes (Diogenes) have recently shown that even a modest increase in protein intake combined with a modest reduction in glycemic index can prevent weight regain after a weight loss5 and reduce the prevalence of overweight among children.6 The beneficial effect of a high protein intake seems to be due to a higher diet-induced thermogenesis (DIT), increased satiety and decreased hunger.7, 8, 9 It has been speculated that different protein sources may affect satiety and DIT differently, but only very limited data from human studies on this topic is available.10
Dairy products are rich in protein. Milk contains approximately 32 g protein per litre, the protein content consisting of 80% casein and 20% whey. The digestion and absorption of whey and casein differs as casein, unlike whey, coagulates in the stomach, owing to its precipitation by gastric acid. Therefore, the gastric emptying time for casein is delayed compared with whey, resulting in a slower release and absorption of amino acids from casein.11, 12 In 1997, Boirie et al.11 introduced the concept ‘slow’ and ‘fast’ protein to describe these differences in digestion and absorption of whey and casein. It has been suggested that a ‘fast’ protein like whey may be more satiating than a ‘slow’ protein like casein. However, data from studies examining this is inconclusive. Hall et al.13 found that a preload drink of whey was more satiating than an isoenergetic casein drink, and that it increased postprandial cholecystokinin (CCK) and glucagon-like peptide (GLP)-1 response more than the casein drink, indicating that CCK and GLP-1 may be mediators of the increased satiety response to whey. Interestingly, Diepvens et al.14 found that the unique combination of whey and casein found in milk stimulates CCK and GLP-1 more than whey alone. Others have reported no difference in the effect of whey and casein on satiety,15 whilst some have reported that casein is more satiating than whey.16, 17
The aim of the present study was to compare the effect of whey, casein and milk, consumed as part of a mixed meal, on satiety, DIT and substrate oxidation.
Subjects and methods
Twenty two male subjects were recruited through advertising at universities in Copenhagen and on the internet. The subjects were healthy, between 18- and 50-year-old and moderately overweight (body mass index: 25–31 kg/m2). Exclusion criteria were lactose intolerance, smoking, diseases or taking medicines known to affect appetite regulation or energy expenditure (EE), elite athletes and participation in other intervention studies. Subjects were instructed not to change their diets or activity habits during the study. The study was carried out at the Department of Human Nutrition, LIFE, Frederiksberg, Denmark. The study was conducted according to the guidelines laid down in the Declaration of Helsinki, and all procedures involving human subjects were approved by the Municipal Ethical Committee of Copenhagen and Frederiksberg (H-A-2008-091). Written informed consent was obtained from all subjects. The study was registered at http://www.clinicaltrails.gov (NCT01133964). Subjects received an honorarium of ∼340 $ on completion of all tests.
Three different isocaloric breakfast meals including either milk or a drink containing only whey or casein was tested in a randomized single-blind, crossover design. The subjects were randomly assigned by lottery to the sequence of the test meals. There was a washout period of ⩾1 week between test days, and each test day lasted 6.5 h. The subjects were instructed not to drink alcohol or perform hard physical activities 48 h prior to each test day. The evening before the test day the subjects consumed a standardized meal supplied by the study, which consisted of spaghetti bolognese, orange juice and crackers (4.5 MJ: protein 18 energy percent (E%), fat 22 E% and carbohydrate 60 E%). Subjects were not allowed to eat or drink anything other than a half litre of water after the meal. On the morning of the test days, the subjects travelled to the Department of Human Nutrition by car, bus, train or slowly walking, to arrive at 8 a.m. On arrival they were weighed wearing only underwear and after emptying their bladder. Weight was measured in kilograms with one decimal (Tanita WB-110 MA, Frederiksberg, Denmark). After 15 min rest in a bed, resting EE were measured by indirect calorimetric in a ventilated hood system (HOOD; Jaeger Oxycon Pro, Cardinal Healthcare GmbH, D-97204 Hoechberg, Germany). Before the test meal was served, the subject's appetite sensation was assessed by visual analog scales (VAS).18 The subjects were instructed to consume the test meal within 10 min from commencement, and drink and eat bread by turns to ensure the meal was properly mixed. They subsequently completed VAS on palatability of the meal and on appetite sensation. In the following 4 h, resting EE was measured in periods of 25 min with 5 min break between each measurement. The first measurement started 15 min after the subject started consuming the meal. Appetite sensation was assessed every 30 min by VAS (between the resting EE measurements). Subjects were not allowed to eat or drink anything else during the test period. They were allowed to hear quiet music during the measurements but they were not allowed to sleep. At the end of the test day, an ad libitum lunch consisting of pizza (17E% protein, 34E% fat and 49E% carbohydrate) and water (300 ml) was served. The subjects were instructed to eat until comfortably satisfied. Food intake was registered and energy intake (EI) calculated afterwards. Subjects completed a VAS on the palatability of the meal and on appetite sensation after the ad libitum lunch.
Subjects were asked to collect all urine during the test day, and the nitrogen content was measured as a marker for protein oxidation. Any variation from the exact time schedule was recorded in a protocol and repeated next test day.
The three isocaloric test breakfast meals were provided to the subjects in a randomized order on three different days. To mimic a normal breakfast meal, the test meal contained bread, butter, jam and a test drink. The test drinks were a drink based on whey, a drink based on casein or ordinary skim milk. The nutrient content of the test meals is presented in Table 1. Both the whey and casein drink was based on intact proteins (WPI, Lacprodan DI-9213 and Miprodan 40, Arla Foods Ingredients Group P/S, Viby, Denmark) The content of macronutrients and micronutrients in each meal was estimated by using Dankost 3000 dietary assessment software (Danish Catering Center, Herlev, Denmark) and information from Arla Foods Ingredients Group P/S. The test meals were prepared at the Department of Human Nutrition. Portions were matched to each subject's individual energy requirement adjusted to the nearest 1 MJ. Each test meal contained 20% of the subject's daily energy requirement. The energy requirement of each subject was calculated as basal EE * physical activity level.19 Subject's physical activity level was estimated according to their full time job and level of leisure-time physical activity.19, 20 The subjects were instructed to maintain their habitual activity level throughout the study.
Subjects were instructed to empty their bladder on arrival at the Department of Human Nutrition in the morning of the test day. All urine was thereafter collected throughout the rest of each test day. Urine volume was measured and samples were stored at −20 °C until further analysis. Nitrogen was analyzed according to the Dumas analysis principle using an Elementar Vario Max CN (Hanau, Germany). The CVintra% and CVinter% was 2.03 and 4.68, respectively.
All values are expressed as mean±s.e.m., unless otherwise stated. Composite appetite score was calculated using the following equation: composite appetite score=(satiety+fullness+(100−prospective food consumption)+(100−hunger))/4
Repeated measurements analysis of variance was used to assess the effect of time, meal, and interaction of meal and time. In order to adjust for differences in baseline values, these were included as covariate in all analyses. The analysis was performed in proc mixed. The Gaussian model of spatial correlation was chosen for covariance structure. As the study had a crossover design, subjects were included as a random effect. Sequence of the test days was not significant in any of the analyses and was therefore not included as covariate.
Area under the curve (AUC) was calculated as the total increase above zero. Only postprandial measurements were included in the calculation. The recording of appetite sensation measured after intake of the ad libitum lunch was not included in AUC calculation or any of the analyses. Analyses of covariance were used to examine the effect of meal on AUC. Analyses of covariance were performed in proc mixed. Subject was included as a random effect and baseline values as a covariate in all analyses. Sequence of the test days was not significant in any of the analyses, and therefore not included as covariate. Analyses of variance were used to examine the effect of protein source on the palatability of the meals and ad libitum EI. Analyses of variance was performed in proc mixed, with subject included as a random effect. Model control for all analyses was performed in proc mixed and data was transformed before analysis if necessary. Least squares means were used to estimate the adjusted means. Pairwise comparisons were performed by paired t-test. All statistical analyses were performed using Statistic Analysis Package, SAS version 9.1 (SAS Institute, Cary, NC, USA). The level of significance was set at P<0.05.
Seventeen subjects completed the four test meals (mean±s.e.m.: age 31±9 years, body mass index: 29±4 kg/m2). Only data from the subjects completing the study were included in the data analyses.
No significant difference in overall palatability of the test meal was found (mean±s.e.m.: casein 53.5±4.6; milk 46.4±6.3; whey 52.8±6.5 P=0.642). In agreement taste, aftertaste, smell and visual appeal was scored similar for the test meals (data not shown).
Postprandial responses and baseline-adjusted AUC for appetite sensation are shown in Figure 1. There was no significant difference in baseline score of satiety, fullness, prospective food consumption or hunger. With regards to postprandial response, repeated measurement analyses showed no significant effect of test meal or test meal*time on any of the measurements, either unadjusted or adjusted for baseline score. In agreement there was no significant effect of test meal on AUC for any of the measurements, either unadjusted or adjusted for baseline score.
At the end of the test, the subjects were served an ad libitum lunch and EI was registered (Figure 2). There was a significant difference in ad libitum EI (P=0.0374). EI was lower after the milk test meal than after the casein (9%; P=0.0258) or the whey test meal (9%; P=0.0260). Ad libitum EI was similar after the casein and the whey test meals. Palatability of the ad libitum lunch with regards to taste, aftertaste, smell, visual appeal or overall palatability was not affected by the test meal (data not shown).
Postprandial responses and baseline-adjusted AUC for EE, carbohydrate and lipid oxidations and AUC for protein oxidation are shown in Figure 3. There was no significant difference in baseline EE, lipid oxidation or carbohydrate oxidation.
Repeated measurement analysis showed no significant effect of test meal or test meal*time on postprandial EE or carbohydrate oxidation, either unadjusted or adjusted for baseline values. In agreement, there was no significant effect of test meal on AUC for postprandial EE or carbohydrate oxidation, either unadjusted or adjusted for baseline values. In addition, there was no significant effect on AUC for protein oxidation. But according to the repeated measurement analyses, there was a significant effect on lipid oxidation (P=0.0475) when adjusted for baseline oxidation. Lipid oxidation was significantly higher after the casein test meal compared with the whey test meal (P=0.0147). There was no significant difference between the other meals. In agreement, there was a significant effect of protein source on AUC for lipid oxidation (P=0.0388) when adjusted for baseline values. AUC after the casein test meal was significantly higher than after the whey test meal (P=0.0138).
There is accumulating evidence that a high-protein diet may promote weight loss and prevent weight (re)gain better than a low protein diet,1, 2, 3, 4, 5, 6 and that this may be because of a high-protein diet inducing higher DIT, increased satiety and decreased hunger.7, 8, 9 However, data on the effect of different types of protein sources are limited. Although whey has been found to be more satiating than casein in some studies,21, 22 others have found no difference between the satiating effect of whey and casein,15 and others again have found that casein is more satiating than whey.16, 17 In the present study, whey, casein and milk had similar effects on acute appetite sensation, but ad libitum EI at the subsequent lunch was significantly decreased by milk compared with the casein and the whey beverages. There was no significant difference in EI after the whey and the casein beverages. Thus, our results do not support that whey is more satiating than casein.
There are several possible explanations for the inconsistencies in the outcome of studies comparing the effects of casein and whey. It has been suggested that the amount of the protein given may have a role. Veldhorst et al.22 found that whey decreased hunger (measured by appetite ratings) more than casein when given at 10E%, but not when given at 25E%. They suggest that a difference in appetite ratings between the proteins only appears when plasma concentration of certain amino acids are above or below particular threshold values. In their study, plasma concentration of threonine, isoleucine, tryptophan, leucine and lysine were increased more after the meal with 10E% whey than after the meal with 10E% casein. All of these amino acids have been suggested to be involved in appetite regulation or body weight regulation.22 However, after the meal with 25E% plasma concentration of these amino acids seems to be above the threshold value for both types of protein. In our study the test meal contained 23E% protein, which is similar to the high protein meal used by Velhorst et al.22 However, Hall et al.13 reported a significantly lower EI at an ad libitum meal, 90 min after intake of a drink containing 48E% whey compared with an similar casein drink.
In the present study, EI at the ad libitum lunch was 9% lower after intake of milk than after intake of casein or whey. Although there was no significant difference, the subjects did score prospective food consumption slightly lower after intake of milk compared with intake of casein and whey, and fullness and satiety slightly higher. Diepvens et al.14 have previously shown that a shake (25E% protein) with milk proteins (80% casein and 20% whey) induces a significantly higher postprandial response in CCK and GLP-1 than a similar shake with only whey protein. They have no plausible explanation for this difference but suggest that it may be because of the unique combination of the ‘fast’ whey and the ‘slow’ casein in milk proteins.14 However, it is also possible that milk contains other, as yet unidentified, bioactive components that affect appetite regulation. It has been shown by others that whey induces a significant higher response in CCK and GLP-1 than casein.13 In contrast to our study Diepvens et al.14 did not see any significant difference in EI at an ad libitum meal 180 min after intake of the shakes.
It is well documented that proteins have a greater thermogenic effect than carbohydrate or fat.7 However, less is known about the possible differences in effect of proteins from various sources. Mikkelsen et al.23 found that animal proteins (pork) stimulated 24 h EE more than vegetable proteins (soy). Karst et al.24 has previously reported, based on data from a very small study, that a casein drink produce a higher DIT than isocaloric drinks with either gelatine, egg white or starch. Acheson et al.16 found a greater thermogenic effect after a meal containing whey (50% protein) than after casein and soy meals, and a greater effect after the whey, casein and soy meals than after a high carbohydrate meal. Cumulative lipid oxidation was greater after the whey, casein and soy meals than after the high carbohydrate meal, and tended to be greater after the whey meal than after the soy meal (P=0.097). By contrast, Alfenas et al.17 observed a significantly higher DIT after a breakfast meal containing soy compared with a breakfast meal containing carbohydrate, but no significant difference in DIT after breakfast meals with soy, whey and casein. The respiratory quotient was significantly low after whey compared with soy, whereas casein was not significantly different from either whey or soy. We observed no significant differences in postprandial EE, but a small increase in lipid oxidation was observed after the casein drink compared with the two other drinks (significantly different only in relation to the whey drink). As far as we know, we are the first to report such differences in humans. Hochstenbach-Waelen et al.25 reported that 25E% casein induced significantly higher lipid oxidation than 10E% casein, but as their study did not include another protein source it is not possible to conclude whether the effect is simply because of an increased protein intake, as observed by others, or is specific to casein. The difference observed in lipid oxidation after casein and whey may be because of differences in postprandial insulin response (not measured). Although Acheson et al.16 observed similar lipid oxidation after casein and whey they did observe a lower postprandial insulin response after casein compared with whey. As insulin is known to suppress lipid oxidation a lower postprandial increase in insulin could be expected to lead to a higher postprandial lipid oxidation, as observed in the present study. It has to be emphasized that we only examined the acute effect in the present study, and our results therefore cannot predict whether there will be a long-term effect. More studies are needed to examine whether casein simulates lipid oxidation, and to establish possible plausible mechanisms.
In conclusion, our data do not add support to that suggestion that whey is more satiating than casein, as reported by others. In fact, we found that milk is more satiating than whey or casein alone. This may be because of the unique combination of a ‘slow’ and a ‘fast’ protein found in milk. No significant effect on postprandial EE was observed, but a small but significant increased lipid oxidation was observed after casein compared with whey.
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We are grateful to the laboratory and kitchen staff at the department for their assistance, especially Søren Andresen, Jane Jørgensen, Charlotte Kostecki and Yvonne Rasmussen, and to Tina Cuthbertson for proof reading the manuscript. JK Lorenzen, C Hoppe and A Astrup designed the study. R Frederiksen, R Hvid and JK Lorenzen were responsible for collection of data and data analysis. All authors participated in the discussion of the results and preparation of the manuscript. The study was financed by Arla Foods Amba Viby, Denmark and the Danish Ministry of Food, Agriculture and Fisheries, Copenhagen, Denmark.
A Astrup is a member of Global Dairy Platform, Chicago and received an honorarium for each board meeting.
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Lorenzen, J., Frederiksen, R., Hoppe, C. et al. The effect of milk proteins on appetite regulation and diet-induced thermogenesis. Eur J Clin Nutr 66, 622–627 (2012). https://doi.org/10.1038/ejcn.2011.221
- milk protein
- diet-induced thermogenesis
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