Appetite 78 (2014) 172–178

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Research report

Effect of whey protein and glycomacropeptide on measures of satiety in normal-weight adult women☆ Sylvia M.S. Chungchunlam a,*, Sharon J. Henare a, Siva Ganesh b, Paul J. Moughan a a b

Riddet Institute, Massey University, Palmerston North 4442, New Zealand AgResearch Grasslands, Palmerston North 4442, New Zealand

A R T I C L E

I N F O

Article history: Received 9 November 2013 Received in revised form 24 March 2014 Accepted 27 March 2014 Available online 31 March 2014 Keywords: Satiety Food intake Appetite Whey protein Glycomacropeptide Preload

A B S T R A C T

Protein is the most satiating macronutrient and dairy whey protein is thought to be more satiating than other protein sources. The purported satiating effect of whey protein may be attributable to the presence of glycomacropeptide (GMP). The objective of this study was to investigate the role of GMP in the satiating effect of whey protein. Isoenergetic (~1600 kJ) preload drinks contained GMP isolate (86% GMP, “GMP”), whey protein isolate (WPI) with 21% naturally occurring GMP, WPI with 2% naturally present GMP, or maltodextrin carbohydrate (“carbohydrate”). Satiety was assessed in 22 normal-weight adult women by determining the consumption of a test meal provided ad libitum 120 min following ingestion of a preload drink, and also by using visual analogue scales (VAS) for rating feelings of hunger, desire to eat, prospective consumption and fullness (appetite). The ad libitum test meal intake was significantly different between the preload drinks (p = 0.0003), with food intake following ingestion of both WPI preload drinks (regardless of the amount of GMP) being ~18% lower compared with the beverages enriched with carbohydrate or GMP alone. There were no significant differences (p > 0.05) in the VAS-rated feelings of appetite among the four preload drinks. GMP alone did not reduce subsequent food intake compared with a drink enriched with carbohydrate, but whey protein had a greater satiating effect than carbohydrate. The presence of GMP in whey does not appear to be the cause of the observed effect of whey protein on satiety. © 2014 Elsevier Ltd. All rights reserved.

Introduction The role of dietary protein in enhancing satiety and reducing food intake to a greater extent compared with other macronutrients has been the subject of several scientific reviews (Anderson & Moore, 2004; Eisenstein, Roberts, Dallal, & Saltzman, 2002; Halton & Hu, 2004; Westerterp-Plantenga, Nieuwenhuizen, Tome, Soenen, & Westerterp, 2009). The source of protein may influence the satiating effect and dairy whey protein appears to be more satiating than other protein sources (Luhovyy, Akhavan, & Anderson, 2007; Veldhorst et al., 2008). During cheese making, whey is the soluble protein component of milk that is separated from the casein curd. This whey also con-

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Acknowledgements: The authors wish to gratefully thank the participants in this study. We thank New Zealand Starch Ltd. for providing the maltodextrin (Avondex 10); Fonterra Co-operative Group Ltd. for supplying the whey protein isolates; Davisco Foods International Inc. for providing the glycomacropeptide isolate (Bio-Pure GMP). We acknowledge Heleen van Dijke, Chanapha Sawatdeenaruenat and Natascha Strobinger for technical assistance. The research was funded by the Riddet Institute, a New Zealand government supported Centre of Research Excellence. * Corresponding author. E-mail address: [email protected] (S.M.S. Chungchunlam). http://dx.doi.org/10.1016/j.appet.2014.03.027 0195-6663/© 2014 Elsevier Ltd. All rights reserved.

tains glycomacropeptide (GMP), a 64 amino acid soluble peptide cleaved from the action of rennet (chymosin) on κ-casein proteins. GMP refers to the glycosylated form of caseinomacropeptide (CMP) and contains varying amounts of oligosaccharides, mostly sialic acid (N-acetylneuraminic acid), galactosamine, and galactose (Walstra, Wouters, & Geurts, 2006). Human studies investigating the effect of GMP on food intake and satiety have resulted in mixed findings. Veldhorst et al. (2009a) showed a decrease in food energy intake at a subsequent meal 180 min after consumption of a test breakfast containing whey protein with GMP compared with whey protein without GMP. However, in studies where isolated GMP or CMP was tested, no difference in food energy intake at a subsequent test meal was found relative to a control preload (water, basal mixture, carbohydrate or whey protein) (Burton-Freeman, 2008; Clifton et al., 2009; Gustafson, McMahon, Morrey, & Nan, 2001; Keogh et al., 2010; Poppitt, Strik, McArdle, McGill, & Hall, 2013). With respect to subjective measures of satiety, Clifton et al. (2009), Gustafson et al. (2001), Keogh et al. (2010) and Poppitt et al. (2013) reported no effects, while Burton-Freeman (2008) found that ratings of satiety were greater after consumption of whey-containing drinks (whey protein with and without GMP) compared with a milk-based mixture and a GMP (0.8 g) drink in 10 women, although such a change in satiety ratings was not observed in men.

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Previous studies from our own group showed that the ingestion of preload drinks enriched with whey protein containing naturally present GMP resulted in subjects consuming a lower energy intake at a subsequent meal and reporting a greater feeling of fullness compared with a maltodextrin carbohydrate-enriched control beverage (Chungchunlam, Moughan, Henare, & Ganesh, 2012; Lam, Moughan, Awati, & Morton, 2009). The natural presence of GMP in whey confounds investigation of the satiating effect of whey protein, and the influence of GMP alone on satiety and food intake remains to be elucidated. We hypothesise that the greater satiety observed (Chungchunlam et al., 2012) when whey protein including naturally occurring GMP was consumed is related to GMP content. The aim of the presently reported study was to compare the effects of preload drinks enriched with WPI with a high (21%) and minimal (2%) level of naturally present GMP relative to GMP (86% GMP) alone on measures of satiety in normal-weight adult women. Maltodextrin carbohydrate was used as a comparator. Possible mechanisms underlying the effects on subsequent food intake and rated feelings of appetite were not studied. Subjects and methods Subjects Healthy women aged 18 to 40 years were recruited by public advertisement. Smokers, trained athletes, pregnant or lactating women and women not consuming breakfast everyday were excluded from the study. Subjects were also excluded if their body weight had changed over the past 6 months (weight gain or loss > 3 kg), if they had a history of menstrual irregularities, a gastrointestinal disorder or were taking any medication known to affect gastrointestinal motility or appetite. All subjects attended an information session about the study procedure, signed a consent form, had their height and weight measured to ensure that they met the body mass index (BMI) criteria (BMI range: 20–25 kg/m2), completed the Three Factor

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Eating Questionnaire (Stunkard & Messick, 1985) and tasted the preload drinks and test meal. The Massey University Human Ethics Committee (Application no. 11/47) approved the study. Twentytwo women completed the study, with 14 subjects being white Caucasian and eight subjects of Asian descent. The participants had a mean age of 23.8 ± 0.9 years, mean weight of 62.9 ± 1.3 kg and mean BMI of 23.1 ± 0.4 kg/m2. According to the Three Factor Eating Questionnaire, subjects scored low in restraint (8.9 ± 1.0), disinhibition (5.9 ± 0.4) and perceived hunger (4.5 ± 0.7). Preload drinks and test meal Preloads (Table 1) were approximately isovolumetric (~280 ml) and isoenergetic (~1600 kJ) drinks that comprised a basal mixture of skim milk powder, sucrose, natural sweetener, vanilla flavour, yellow colouring and water, and either GMP isolate containing 86% GMP (“GMP”), whey protein isolate (WPI) containing 21% naturally occurring GMP (“WPI-high GMP”), WPI containing minimal 2% GMP (“WPI-low GMP”), or maltodextrin carbohydrate (“carbohydrate”). The skim milk powder contained a minimal amount of whey protein (6.7%) and GMP (0.2%), contributing 1.005 g of whey protein and 0.03 g of GMP per 300 g serve of preload drink. The remaining 14% of the fully glycosylated GMP isolate (86% GMP) comprised 0.4% fat, 6.2% ash, 5.9% moisture and 1.5% undetermined material. Although the WPI-low GMP powder was anticipated to contain no GMP, protein analysis using reverse-phase high performance liquid chromatography showed that the GMP content in the WPI was 2%, likely arising from contamination during processing. The “protein” preload drinks were high in protein (66–76% of metabolisable energy) but also contained some carbohydrate (22–31% of metabolisable energy) while the “carbohydrate” preload drink mainly comprised of carbohydrate (89.5% of metabolisable energy) but also contained some protein (7.2% of metabolisable energy). The preload beverages of mixed macronutrient composition were deemed more acceptable to an independent group of sensory panellists than preload drinks

Table 1 Composition of the preload drinks containing either glycomacropeptide isolate (GMP), whey protein isolate containing 21% glycomacropeptide (WPI-high GMP), whey protein isolate with 2% glycomacropeptide (WPI-low GMP) or maltodextrin carbohydrate (carbohydrate). GMPa Ingredient (g per 300 g serve) Skim milk powder Sucrose Natural sweetener (Stevia) Vanilla flavour Yellow colour Glycomacropeptide isolatea Whey protein isolate-high glycomacropeptideb Whey protein isolate-low glycomacropeptidec Maltodextrin carbohydrated Water Nutrient (per 300 g serve) Metabolisable energy (ME)e (kJ) Fat (g) (% of ME) Carbohydrate (g) (% of ME) Protein (g) (% of ME) GMP (g) a

15 9 0.06 1.5 0.09 60 – – – 214.35 1599

WPI-high GMPb 15 9 0.06 1.5 0.09 – 60 – – 214.35 1623

WPI-low GMPc 15 9 0.06 1.5 0.09 – – 60 – 214.35 1558

Carbohydrated 15 9 0.06 1.5 0.09 – – – 60 214.35 1650

1.3 3

1.2 3

1.0 2

1.4 3

29.3 31

22.8 23

20.4 22

88.4 90

63.5 66 51.4

71.6 74 12.8

70.6 76 1.5

7.2 7 0

Glycomacropeptide isolate containing 86% glycomacropeptide (BioPURE-GMP™, Davisco Foods International Inc., Le Sueur, MN, USA). Whey protein isolate containing 21% glycomacropeptide (WPI 894, Fonterra Co-operative Group Ltd, Palmerston North, New Zealand). c Whey protein isolate containing 2% glycomacropeptide (WPI 895, Fonterra Co-operative Group Ltd). d Maltodextrin carbohydrate (Avondex 10, New Zealand Starch Ltd, Auckland, New Zealand). e Metabolisable energy (ME) calculated from the energy-containing food components using the energy conversion factors of 16.7 kJ/g for protein and available carbohydrate and 37.7 kJ/g for fat. b

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containing pure forms of carbohydrate or protein. The preload beverages were freshly prepared 1–3 days prior to the study day and stored in a refrigerator. Preload drinks were thoroughly mixed prior to serving. Subjects received a hot lunchtime test meal provided in excess of the amount to be consumed (ad libitum) 120 min after ingestion of the preload drinks. The test meal consisted of minced chicken meat, eggs, peas, corn, carrots, chicken stock, salt, sugar and vegetable oil, mixed and fried. The chicken fried rice test meal contained 910 kJ metabolisable energy (ME), 29.5 g available carbohydrate, 9.7 g crude protein, 6.4 g crude fat and 2.1 g total dietary fibre per 100 g. Water was available with the test meal. Experimental procedure The study was a single-blind, completely randomised block design. Women participated during the menstrual period and follicular phase of their menstrual cycles (Buffenstein, Poppitt, McDevitt, & Prentice, 1995; Dye & Blundell, 1997) and were tested in four sessions with a minimum of 2 days in between each session. On the day prior to each study session, subjects were instructed to abstain from alcohol and to consume only water from 2200 h onwards. On the study day, participants consumed a subjectspecific breakfast that consisted of toasted wholemeal bread with margarine and/or strawberry jam and either water or a hot drink (coffee or tea, with or without sugar and/or milk). The breakfast meal (recorded using food diaries on each study day) provided on average 1326 kJ of ME with 58.4% of the ME derived from available carbohydrate, 11.3% from protein and 30.3% from fat. Each participant was instructed to consume the same subject-specific breakfast at least 3 h before the test session appointment, followed only by water as desired. On each study day, participants reported to the laboratory between 1200 and 1300 h and filled out a questionnaire to check for compliance with the restrictions on the evening before and the morning. Two out of the 22 subjects did not comply once with the experimental protocol and were scheduled for another test day. Each subject completed a questionnaire for her baseline assessment of appetite (hunger, desire to eat, prospective consumption, and fullness) and nausea using 10-cm visual analogue scales (VAS). Subjects were provided with one of the four preload drinks (~280 ml) immediately followed by 50 ml of water to wash away any aftertaste, all to be consumed within 5 min. After complete ingestion of the preload and water (0 min), subjects filled in a questionnaire assessing palatability of the preload drink, appetite and nausea on a 10-cm VAS. Subsequent appetite and nausea ratings were completed at 15, 30, 45, 60, 75, 90 and 120 min after preload ingestion. After the 120 min VAS rating, subjects were seated in individual cubicles and offered the lunchtime test meal and water to be consumed within 15 min. Subjects were instructed to consume as much or as little as they desired and additional food and water were provided if requested. The test meal was provided in excess (600 g) and none of the subjects finished the offered test meal completely. After the test meal and water were taken away, subjects rated the likeability of the test meal with the use of a 10-cm VAS. Appetite and nausea ratings were taken at 15 and 30 min after consumption of the lunch test meal and subjects were then allowed to leave the laboratory. Measures The amount of the chicken fried rice test meal and water consumed at lunch were measured before and after consumption using an electronic scale, to the nearest 0.01 g. Subjects rated the palatability of the preload drink (overall likeability, pleasantness of taste, likeability of texture and overall sweetness) and overall likeability

of the fried rice test meal using a 10-cm visual analogue scale (VAS) immediately after consumption. The VAS was end-anchored with “dislike extremely” and “like extremely” for ratings of overall likeability and likeability of texture, and with “not at all” and “extremely” for ratings of pleasantness of taste and sweetness. Subjective feelings of hunger, desire to eat, prospective consumption, fullness and nausea were also rated on a 10-cm VAS (Flint, Raben, Blundell, & Astrup, 2000; Parker et al., 2004) labeled at each end with extremes: “not at all” and “extremely” for hunger, fullness, and nausea, “very weak” and “very strong” for desire to eat, and “nothing at all” and “a large amount” for prospective consumption. VASrated appetite and nausea scores were collected immediately before consuming the test preload drink (baseline), during a 120 min period following consumption of the preload drink (0, 15, 30, 45, 60, 75, 90 and 120 min), and 15 and 30 min following consumption of the lunchtime ad libitum test meal and water.

Statistical analysis All statistical analyses were performed using the SAS software program for WINDOWS, version 9.2 (SAS Institute Inc., Cary, NC, USA). The design of the study was a single-blind randomised factorial design with subject as the blocking variable (Ott & Longnecker, 2010). According to a power analysis for a randomised block design (Kastenbaum, Hoel, & Brown, 1970) and based on our previous study using a similar experimental procedure with women subjects (Chungchunlam et al., 2012), a sample size of 13 subjects was deemed sufficient to detect a difference of 792 kJ in ad libitum test meal intake with 80% power at a significance level of 0.05. Primary analyses were undertaken to assess the normality of the distributions of data and to test for the presence of statistical outliers. VAS ratings for palatability of the preload drink and test meal (cm), test meal and water intake (g) and energy intake of test meal (kJ ME) were analysed using an analysis of variance (ANOVA) with preload drink as experimental factor and subjects as blocks. PROC GLM in SAS was utilised for this analysis. PROC GLM was also used to compare the effect of study session days on test meal intake (kJ ME). For the ratings of hunger, desire to eat, prospective consumption, fullness and nausea, a repeated-measures ANOVA (using PROC GLM) was performed to examine the effect of preload drink and time of rating and their interaction (preload × time). If a significant interaction was found, this effect was further examined using the least squares means of the preload drink and time of rating combinations. When the interaction was not significant (p > 0.05), the main effects of preload drink and time of rating were examined using the least squares means of the preload drink and time of rating separately. Both procedures were conducted via Tukey’s multiple comparison tests. The VAS ratings were further analysed by adjusting post-preload scores (0 to 120 min) for baseline measurements, by fitting them as covariates in the model. The nausea data were found to have a skewed distribution and were log transformed. The net incremental (area above and below the baseline rating) area under the curve (AUC) (Gannon, Nuttall, Westphal, Neil, & Seaquist, 1989) following consumption of the preload drinks (0 to 120 min) was calculated using the trapezoid rule and analysed for an effect of preload drink using a one-way ANOVA. To examine the relationship between energy intake at the test meal (kJ ME) and palatability of the preload drink as well as VAS ratings of appetite and nausea from 0 to 120 min, Pearson’s correlation analysis was performed. Results were considered statistically significant at a p-value of less than 0.05. Tukey’s multiple range test procedure was applied for multiple pairwise treatment comparisons. Results are reported as means and their standard errors (mean ± sem).

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Table 2 Palatability scores measured on a 10-cm visual analogue scale for the four preload drinks containing either glycomacropeptide isolate (GMP), whey protein isolate containing 21% glycomacropeptide (WPI-high GMP), whey protein isolate with 2% glycomacropeptide (WPI-low GMP) or maltodextrin carbohydrate (carbohydrate). Palatability (cm)

Likeability Pleasantness of taste Likeability of texture Sweetness

Preload drink

p-value

GMP

WPI-high GMP

WPI-low GMP

Carbohydrate

3.0 ± 0.5c 3.0 ± 0.6c 3.2 ± 0.5b 3.1 ± 0.5b

5.3 ± 0.5b 5.3 ± 0.6b 6.5 ± 0.4a 3.6 ± 0.5b

5.5 ± 0.5b 5.5 ± 0.6b 6.4 ± 0.6a 3.6 ± 0.5b

6.7 ± 0.6a 6.7 ± 0.6a 7.2 ± 0.5a 5.2 ± 0.6a

WPI-low GMP, consistent with WPI affecting satiety. Tukey’s multiple comparison tests revealed that GMP alone increased subsequent food intake more than both whey protein-containing drinks, thereby negating the notion that the consumption of GMP alone exerts a stronger suppressive effect on food intake. In addition, Tukey’s tests suggest that the satiating effect of consumption of the preload drink enriched with WPI containing a high level of GMP did

not differ from the satiating response from ingestion of the preload drink enriched with WPI containing minimal GMP. There were significant differences between preload drinks in total energy intake (the addition of energy intake from the preload drink and test meal) (F(3,63) = 7.85, p = 0.0002) (Table 3). Following consumption of the carbohydrate and GMP preloads, total energy intake was significantly greater than following ingestion of the two whey protein preloads.

Subjective ratings of appetite and nausea There was no significant interaction between preload drink and time for the VAS ratings of hunger (F(30,829) = 1.38, p = 0.08), desire to eat (F(30,830) = 1.32, p = 0.11), prospective consumption (F(30,820) = 1.08, p = 0.34) and fullness (F(30,829) = 0.66, p = 0.91) during the whole test period (Fig. 1). There was a significant main effect of time (p < 0.0001) for each rating of appetite, but no main effect of preload drink was observed (p > 0.05). Analysis of the VAS ratings of appetite following consumption of the preload drinks (from 0 to 120 min), adjusted for the baseline rating, revealed no significant interaction between preload drink and time of rating for the feelings of hunger (F(21,587) = 0.98, p = 0.48), prospective consumption (F(21,586) = 1.06, p = 0.38) and fullness (F(21,587) = 0.44, p = 0.98). There was no main effect of preload (p > 0.05) but there was a main effect of time (p < 0.0001) for these ratings. However, a significant interaction between preload drink and time of rating was found for desire to eat scores (F(21,587) = 1.77, p = 0.0184). Desire to eat ratings were significantly lower 90 min following consumption of the carbohydrate preload (3.3 ± 0.6 cm) compared with the GMP (4.9 ± 0.6 cm, p = 0.0362) and WPI-high GMP (5.3 ± 0.6 cm, p = 0.0006) preloads. When expressed as net incremental area under the curve (AUC) over the 120 min period, there was no significant main effect of preload drink on VAS ratings of hunger (F(3,63) = 1.34, p = 0.27), desire to eat (F(3,63) = 1.25, p = 0.29), prospective consumption (F(3,63) = 0.87, p = 0.46) and fullness (F(3,63) = 0.40, p = 0.75) (Fig. 1). Following ingestion of the preload drinks (from 0 to 120 min), there was no significant effect of preload drink on the VAS scores (F(3,419) = 1.79, p = 0.16) and net incremental AUC response (F(3,63) = 1.90, p = 0.13) for feelings of nausea (data not shown).

Table 3 Water intake, ad libitum test meal food (g) and energy (kJ ME) intakes and total (preload + test meal) energy (kJ ME) intake following consumption of preload drinks enriched with either glycomacropeptide isolate (GMP), whey protein isolate containing 21% glycomacropeptide (WPI-high GMP), whey protein isolate with 2% glycomacropeptide (WPI-low GMP) or maltodextrin carbohydrate (carbohydrate). Preload drink

Water intake (g) Test meal food intake (g) Test meal energy intake (kJ) Total energy intake (kJ)

p-value

GMP

WPI-high GMP

WPI-low GMP

Carbohydrate

407.0 ± 52.0 348.3 ± 27.3a 3169.6 ± 248.3a 4689.2 ± 242.9a

454.7 ± 49.2 290.2 ± 29.2b 2640.4 ± 266.0b 4121.9 ± 262.1b

441.9 ± 36.8 273.6 ± 32.3b 2490.0 ± 294.0b 3996.9 ± 292.3b

381.3 ± 39.7 338.9 ± 30.1a 3084.2 ± 273.5a 4606.7 ± 265.1a

Means in a row with different superscript letters were significantly different (Tukey’s test, p < 0.05).

0.06 0.0003 0.0003 0.0002

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Fig. 1. Subjective VAS ratings of hunger, desire to eat, prospective consumption, and fullness in response to ingestion of preload drinks enriched with glycomacropeptide isolate (GMP), whey protein isolate with high glycomacropeptide (WPI-high GMP), whey protein isolate with low glycomacropeptide (WPI-low GMP) or maltodextrin carbohydrate (carbohydrate). The preload drink ingestion (0 to 120 min) was preceded by a baseline (B) measurement. Ratings were also completed 15 and 30 min following consumption of the ad libitum lunchtime test meal. There was no statistically significant preload by time interaction (p > 0.05) and no significant effect of preload drink (p > 0.05), but an effect of time was found (p < 0.0001) for each appetite measure. The net incremental area under the curve (AUC) is shown for each variable. There was no significant main effect of preload drink on net incremental AUC for each subjective feeling (p > 0.05). Values are mean ± sem, n = 22.

Correlation analyses There was no significant overall correlation found between test meal energy intake and the VAS-rated scores for overall likeability (r = −0.07, p = 0.47), pleasantness of taste (r = −0.11, p = 0.27), likeability of texture (r = −0.08, p = 0.44) or overall sweetness (r = −0.14, p = 0.18) of the preload drinks. The palatability scores of the preload drinks and post-drink (from 0 to 120 min) ratings for hunger, desire to eat, prospective consumption, fullness and nausea were not overall correlated (p > 0.05). Although the palatability VAS ratings differed among preload drinks, none of these differences were related to subsequent ad libitum food intake or ratings of appetite or nausea in the subjects. Pearson’s correlation analysis of the relationship between energy intake at the ad libitum test meal and subjective ratings of appetite following consumption of the preload drinks (from 0 to 120 min) showed a significant but weak overall positive correlation with overall hunger (r = 0.25, p < 0.0001), desire to eat (r = 0.28, p < 0.0001) and prospective consumption (r = 0.38, p < 0.0001). This significant positive correlation was also observed 120 min following ingestion of the four preload drinks and immediately preceding the

consumption of the lunchtime test meal for hunger (r = 0.30, p = 0.0047), desire to eat (r = 0.36, p = 0.0004) and prospective consumption (r = 0.46, p < 0.0001). A significant but weak overall negative correlation was found between test meal energy intake and overall ratings of fullness (r = −0.34, p < 0.0001) as well as fullness ratings at 120 min (r = −0.36, p = 0.0005). There was no significant overall relationship between VAS ratings of nausea with test meal energy intake (r = −0.02, p = 0.78) or VAS-rated subjective feelings of appetite (p > 0.05). Discussion In the present study involving 22 normal-weight young adult women, consumption of a preload drink enriched with whey protein, regardless of the amount of GMP present (WPI-high GMP and WPIlow GMP), reduced energy intake at a subsequent ad libitum test meal to a greater extent than with a preload drink containing GMP alone or a maltodextrin carbohydrate-enriched preload drink control. There was no difference in food energy intake at the subsequent test meal between the GMP- and carbohydrate-enriched preload drinks. Ingestion of the two whey protein-enriched preload drinks also re-

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sulted in lower total energy intakes (preload + test meal energy intake) in comparison with the carbohydrate- and GMP-enriched preload drinks. The VAS ratings of appetite, however, did not differ significantly between preload drinks. The finding that ingestion of a preload enriched with whey protein led to a reduced food intake at an ad libitum test meal compared with a carbohydrate-enriched preload is in agreement with several other studies (Anderson, Tecimer, Shah, & Zafar, 2004; Bellissimo et al., 2007; Bertenshaw, Lluch, & Yeomans, 2008; Chungchunlam et al., 2012; Zafar, Waslien, AlRaefaei, Alrashidi, & AlMahmoud, 2013), though not all studies have shown an effect of whey protein versus carbohydrate (Bowen, Noakes, & Clifton, 2007; Burton-Freeman, 2008; Keogh et al., 2010). The main finding that whey protein resulted in a reduced subsequent food energy intake compared with GMP alone, which is in contrast to the results of other studies that showed similar food intakes (Burton-Freeman, 2008; Clifton et al., 2009; Keogh et al., 2010; Lam et al., 2009; Poppitt et al., 2013), is novel. The result was consistent for the two whey diets and may have been related to the dose of whey protein (51.0 g) and GMP (51.4 g) used in the presently reported study, which was higher than the amount of GMP (0.8–42.3 g) and whey protein (25.0–44.4 g) used in the previous preload studies (Burton-Freeman, 2008; Keogh et al., 2010; Lam et al., 2009; Poppitt et al., 2013). GMP alone does not appear to play an important role in satiety, and the satiating effect of whey protein may not be influenced by the presence or absence of GMP at a high protein level (>65% of energy). The presently reported work adopted a time delay interval between preload and test meal of 120 min based on a previous experiment by our own group, where we reported a stronger suppression in food intake when a time interval of 120 or 60 min was used compared with 30 min (Chungchunlam et al., 2012). In addition, the suppressive effect of protein on subsequent food intake and rated feelings of appetite is thought to occur 120 min after ingestion (Fischer, Colombani, & Wenk, 2004). Other investigators used shorter (75 min in the Burton-Freeman, 2008 study; 90 min in the Poppitt et al., 2013 study) and longer (180 min in the Keogh et al., 2010 study) time intervals, but no differential effect of whey protein and GMP on food intake was observed. There is some evidence of greater satiety and fullness ratings (VAS) following consumption of whey protein, but not GMP alone (Burton-Freeman, 2008; Lam et al., 2009) though the present observation that subjective VAS-rated measures of satiety did not differ between the preload drinks containing either whey protein or GMP, has also been reported by others (Clifton et al., 2009; Gustafson et al., 2001; Keogh et al., 2010; Poppitt et al., 2013; Veldhorst et al., 2009a). Nonetheless, and independent of the preload drink consumed, hunger, desire to eat and prospective consumption scores were positively correlated with energy intake at the ad libitum test meal, while fullness was negatively related to test meal energy intake. The effect of perceived sensory differences between preload drinks may mask the potential effect of the carbohydrate or type of protein under investigation (Bellisle et al., 2012; Rolls, 1991; Sorensen et al., 2012). Subjects perceived the preload drinks as being different in overall likeability, pleasantness of taste, likeability of texture and overall sweetness. In the current study, the carbohydrate beverages was rated as being more liked, pleasant in taste and sweeter compared with the other three protein preload drinks. However, there was no statistically significant correlation between the rated palatability of the preload drinks and subsequent food intake, suggesting that the differences in the perceived sensory properties of the preload drinks were not related to subsequent food intake. Although Veldhorst et al. (2009a) found that GMP as a component of whey protein (amount of GMP present not reported) in a custard breakfast led to a lower ad libitum energy intake 180 min later compared with a breakfast containing whey protein without

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GMP fraction, results from the present study showed that ad libitum food energy intake 120 min after consumption was not different for the two whey protein containing preload drinks (with high or low GMP contents). It is unlikely that the small amount of GMP present in the WPI-low GMP preload drink (1.5 versus 12.8 g GMP per 300 g serve in the WPI-low GMP versus WPI-high GMP preloads) would have contributed significantly to the suppressive effect on subsequent food intake of the whey protein-enriched preload drinks. Interestingly and in line with the present finding, Keogh and Clifton (2008) reported that daily consumption of 66 g of whey protein containing 90% GMP for 1 year did not induce a greater reduction in body weight loss compared with the consumption of skim milk powder in overweight subjects. These results suggest that the effect of whey protein on food energy intake and satiety is attributable to factors other than GMP content. The whey protein powders used in the present study contained beta-lactoglobulin (WPI-high GMP: 43% and WPI-low GMP: 64%) and alpha-lactalbumin (WPI-high GMP: 13% and WPI-low GMP: 16%) and as studies investigating the effects of these major components of whey protein on satiety are sparse (Poppitt et al., 2013; Veldhorst et al., 2009b), the relative contributions of these two proteins to the whey protein-induced satiety response, is unknown. In conclusion, ingestion of a preload meal containing whey protein with high or low amounts of GMP suppressed food energy intake at an ad libitum test meal compared with GMP alone. Total energy intake (preload + test meal) was also lower following consumption of the whey protein-enriched preloads than following ingestion of maltodextrin carbohydrate- or GMP-enriched preloads. Subjective measures of satiety (VAS) did not differ between preloads. GMP alone did not reduce subsequent food intake in comparison with carbohydrate or whey protein, and the presence of GMP does not appear to play an important role in the observed satiating effect of whey protein.

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