Food Dig. (2013) 4:16 DOI 10.

1007/s13228-012-0028-7

Fast and Slow Proteins: Modulation of the Gastric Behavior of Whey and Casein In Vitro
Tim T. Lambers & Willeke G. van den Bosch & Saskia de Jong

Received: 5 June 2012 / Accepted: 5 December 2012 / Published online: 23 March 2013 # Springer Science+Business Media New York 2012

Abstract Ingestion of whey protein rather than casein is associated with a number of enhanced physiological benefits including satiety, glycemic control, and muscle synthesis. Many of these differences can be attributed to apparent differences in digestion and absorption kinetics between these proteins. By simulation of gastric phase digestion, we show that casein coagulates and forms a protein network upon gastric acidification, resulting in an enhanced time viscosity profile, whereas whey proteins stay within solution indicating that altered gastric behavior of these proteins underlies the apparent differences in digestion and absorption kinetics. Moreover, by applying industrially relevant protein modification strategies, the behavior during the gastric phase of these protein sources could be modified. By hydrolysis and Maillard-based modification, protein network formation of casein and concomitantly increased viscosity under simulated gastric conditions could be prevented, likely resulting in faster digestion and absorption kinetics in vivo. On the other hand, cold gelling whey protein aggregates could be prepared that coagulated and formed a temporary protein network under simulated gastric conditions, possibly delaying the overall digestion and absorption kinetics in vivo. Furthermore, combinations of whey proteins or whey protein aggregates and polysaccharides displayed an altered behavior under simulated gastric conditions, most likely as a result of complex coacervation. Overall, the results from this study demonstrate the possibilities within food technology to control the gastric behavior of whey and casein determining the overall digestion and absorption kinetics of these protein sources.

Keywords Protein digestion . Microstructure formation . Muscle synthesis . Satiety . Glycemic control

Introduction As one of the macronutrients, protein is an important factor in our daily diet, affecting numerous physiological processes including tissue synthesis and repair, upregulation of defense systems, and acting as stimulus for key sites in metabolic pathways. Within nutrition, protein quality is mostly defined by the ability of a protein source to supply essential amino acids (composition) for human needs, taking into account its digestibility as measured by nitrogen balance. Protein quality is best determined by protein digestibility-corrected amino acid score procedures [5, 18]. However, nowadays, it is becoming clear that the digestion kinetics of protein, affecting the rate of amino acid appearance and coherent peak levels in plasma, is an independent regulating factor of postprandial protein retention and has a profound role in the overall metabolic response to protein intake [4]. Pioneering within this area of research was the discovery of the principle of fast and slow proteins [2]. Where ingestion of whey protein resulted in a postprandial plasma amino acid profile displaying an early peaked pattern, the profile of casein was more attenuated over time. The underlying mechanism of this difference is the behavior of these proteins under gastrointestinal conditions. Under gastric conditions during acidification of the gastric content, casein coagulates around its isoelectric point and forms a protein network resulting in a reduced accessibility to gastric digestive enzymes and delayed gastric emptying. On the other hand, (native) whey proteins that stay within solution are fully accessible to the gastric digestive enzymes and can empty into the duodenum without delay for further hydrolysis and absorption.

T. T. Lambers (*) : W. G. van den Bosch : S. de Jong NIZO Food Research B.V., P.O. Box 20, 6710 BA, Ede, The Netherlands e-mail: tim.lambers@gmail.com

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Later, these differences in absorption kinetics were associated with a number of physiological benefits. When given as pre-load whey reduced ad libitum food intake, coinciding with an increase in perceived satiety and plasma gut hormone levels [7]. Results from an additional dose-dependent study suggest that differences in appetite ratings between these types of proteins appear when plasma concentrations of certain amino acids are above and below particular threshold values [21], thus underlining the physiological relevance of the digestion and absorption kinetics of proteins. In type 2 diabetes patients, the ingestion of whey resulted in a higher postprandial beta-cell secretion as concluded from elevated plasma levels of insulin, pro-insulin, and Cpeptide [20]. Although these responses may partly rescue the reduced beta-cell responses in these individuals postprandial, it remains to be established whether these changes due to whey intake can be maintained in the long term. From a range of studies with amino acids and protein preparations, it has previously been demonstrated that the muscle synthesis stimulatory effect of protein is almost linear with the range of plasma amino acid concentrations typically observed after meal intake [3, 17]. This is especially relevant for the elderly as their muscle protein synthetic response to food intake may be impaired [11, 15]. Whey protein has been shown to stimulate postprandial muscle synthesis more effectively than casein [16]. Faster digestion and absorption kinetics in combination with higher leucine content in whey were suggested to underlie these observations. The majority of studies with fast and slow proteins use noncomplex beverages mostly consisting of water, standard whey, or casein preparations and in particular cases in combination with carbohydrates or alternative sweeteners. The effect of protein modification strategies and ingredient interaction within complex formulations on the overall gastrointestinal behavior of whey and casein is, however, still unknown. Therefore, the aim of the present study was to test the effects of a range of protein modification strategies and ingredient combinations on the gastric behavior of the dairy proteins whey and casein. By a comprehensive approach applying simulation of physiological gastric digestion and protein modification technology, we show that protein modification strategies applied in food technology can be utilized to control the gastric behavior known to determine the overall digestion and absorption kinetics of the dairy proteins whey and casein.

(Sigma) were used. For the coacervation of whey, HM pectin (CPKelko) and gum arabic (Brenntag, The Netherlands) were used. Methods Casein Modification For the hydrolyzed casein, a commercially available casein hydrolysate (FrieslandCampina) was used. For glycation, casein was coupled to lactose or dextran by the Maillard reaction. Dried mixtures of casein (40 g/l) and lactose (12 g/l) or dextran (12 g/l) were heated for 3 days at 60 C and 60 % RH. After glycation, samples were dialyzed against Milli-Q water to remove non-bound carbohydrates. Whey Modification Whey protein aggregates were prepared by different methods. Cold gelling aggregates were prepared as previously described [1]. In short, a 3 % (w/w) WPI solution was heated for 22 h at 68.5 C (0.5) before concentration and drying. Microparticulated whey protein aggregates were prepared as previously described [8]. In short, 12 % (based on protein content) WPI solutions at pH 3.4 were batch-wise heated for 3 h at 80 C. Simulation of Gastric Digestion with In-Line Rheology Measurements Measurements were performed using a controlled stress rheometer (AR2000, TA Instruments, New Castle) equipped with a four blade stainless steel vane geometry (stator inner diameter 53.7 mm, height 78.0 mm; vane diameter 50.0 mm, height 60.0 mm). Protein samples (3 % protein w/v) were tested under steady shear conditions (3 h at 75 s1, 37 C) at which the viscosity (plotted logarithmic) was recorded as a function of time. After an initial 5 min of measuring, 5 N HCl was titrated such that within approximately 15 min, the solution was acidified from pH 7 towards gastric pH (pH 2), after which pepsin from porcine gastric mucosa (Sigma-Aldrich) in simulated gastric buffer (NaCl (3.1 g/l, Merck), KCl (1.1 g/l, Merck), Na2CO3 (0.6 g/l, BDH), and CaCl22H2O (0.11 g/l, Merck), pH 2) was added to a final concentration of pepsin of 81.6 mg/l (per gram protein 12 mg pepsin).

Materials and Methods Results and Discussion Materials Whey protein isolate (WPI Bipro, Davisco) and casein (sodium caseinate, FrieslandCampina) were used as protein sources. For the glycation of casein, dextran (Fluka, 6 kDa) and lactose Differences in gastrointestinal behavior of casein and whey underlie the distinct absorption kinetics of these proteins [2]. We sought to further characterize these differences under simulated gastric digestion in vitro to separate changes in the

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composition of the bolus by acidification and by protease activity (Fig. 1). For 5 min, baseline viscosity was recorded before gastric acidification towards gastric pH (within 15 min) was started and pepsin (after 20 min) was added. Throughout this simulated gastric digestion, the whey solution remained clear and viscosity, relative to that of the casein solution, remained low although a small increase after 10 min was visible. Upon acidification of the casein solution, large lumps were formed as a result of coagulation and network formation by the individual casein proteins. Overall, this resulted in an elevated viscosity profile, probably the result of the rheometer vane pushing the protein lumps. Upon addition of pepsin, viscosity was reduced, likely by the (partial) hydrolyses of casein although, relative to the whey solution, viscosity remained high, and after 100 min, a small increase in viscosity could be observed. In vivo, the concerted action of protease activity and gastric grinding will result in complete breakdown of the casein lumps before emptying in the duodenum, further breakdown and absorption. However, as in this particular in vitro setting, antral grinding is not simulated and the mixing as delivered by the rheometer vane is insufficient; breakdown of the casein lumps under these conditions was not complete, likely explaining the prolonged elevated viscosity over time. We next tested a range of industrially applicable casein protein modification strategies to prevent coagulation and network formation under gastric conditions to ultimately enhance absorption kinetics. Protein hydrolysis is a straightforward and well-accepted protein modification strategy applied within the food industry. Compared to its intact counterpart, the casein hydrolysate displayed a viscosity profile comparable to that of whey protein (Fig. 2). Moreover, this particular casein hydrolysate no longer formed a protein network as lumps were not observed. This is in line with recent observations where digestion kinetics of an

Fig. 2 Viscosity profile over time of modified casein sources during simulated gastric digestion. a The casein hydrolysate (circle) was subjected to simulated gastric digestion and its viscosity profile over time was compared to that of non-modified casein (square). b Casein carbohydrate conjugates prepared with either lactose (diamond) or dextran (circle) were subjected to simulated gastric digestion and their viscosity profiles over time were compared to that of non-modified casein (square). After an initial 5 min of measuring gastric acidification was initiated (open arrow), pepsin in simulated gastric buffer was added (closed arrow)

Fig. 1 Viscosity profile over time of whey and casein during simulated gastric digestion. WPI (triangle) and casein (square) were subjected to simulated gastric digestion and viscosity was recorded over time. After an initial 5 min of measuring gastric acidification was initiated (open arrow), pepsin in simulated gastric buffer was added (closed arrow)

extensive casein hydrolysate were enhanced and comparable to that of whey proteins [10, 16]. Although these studies used a casein hydrolysate rich in dipeptides and tripeptides which will contribute to the enhanced absorption kinetics, the results from the current study illustrate that hydrolysis is an effective strategy to, at least partially, enhance the digestion and absorption kinetics of casein by prevention of network formation under gastric conditions. Casein hydrolysis is, however, often associated with the formation of bitter peptides due to the high proline content in casein which upon hydrolysis will result in the formation of proline-rich peptides known to decrease overall organoleptic properties [9]. Maillard-based modification (glycation) of intact proteins results in the formation of proteincarbohydrate conjugates that display altered physiochemical properties [12]. Caseincarbohydrate conjugates with a low molecular weight carbohydrate source (lactose) and high molecular

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weight source (dextran) were prepared and subjected to simulated gastric digestion (Fig. 2). Network formation was no longer observed with these conjugates, and compared to nonmodified casein viscosity, it was decreased; however, relative to whey viscosity, it remained higher. Although still to be confirmed in vivo, these results may suggest that the absorption kinetics of casein might be enhanced upon glycation as under gastric conditions protein network formation is prevented by these protein modifications. On the other hand, glycation as a result of processing might decrease overall digestibility of protein [6, 14]. The effects of industrially applicable whey protein modification strategies were tested to change the gastrointestinal behavior of whey and ultimately delay absorption kinetics. Whey protein aggregation strategies are well-established procedures to modify the physiochemical properties of whey proteins within food formulations [1, 8]. Cold gelling whey protein aggregates increased baseline viscosity which further increased during simulated gastric acidification (Fig. 3). Upon gastric acidification, small (relative to those observed with casein) lumps were formed, possibly explaining the increase of viscosity during simulated gastric digestion. Upon addition of the gastric enzymes, viscosity decreased, coinciding with the disruption and solubilization of the observed protein network. Although remaining to be confirmed in vivo, these results suggest that these temporal changes in physical appearance may reflect changes contributing to a delay in overall digestion and absorption kinetics of the whey proteins as, similar to the situation with casein, these changes may result in a reduced accessibility to pepsin and gastric emptying. A completely different profile was observed with microparticulated whey, which did change in viscosity during simulated gastric digestion, however, without the formation of a protein

network (Fig. 3). Currently, it is unknown if and how microparticulation will effect whey protein digestion and absorption. Complex coacervation of protein and polysaccharides is an example of ingredient interactions within more complex formulations induced by, amongst others, changes in pH [19, 22]. As complex coacervation may also occur during gastrointestinal digestion, we subjected mixtures of whey proteins and polysaccharides to simulated gastric digestion and studied their overall viscosity profiles (Fig. 4). Compared to the profile of whey protein and gum arabic alone, and relative to the increases observed with casein or whey protein aggregates, a small and temporary increase in viscosity was observed with the mixture of whey and gum arabic. These findings suggest that, at least under these simulated conditions, complex coacervation between whey and gum arabic may occur under gastric conditions, possibly

Fig. 3 Viscosity profile over time of reactive whey protein aggregates during simulated gastric digestion. Cold gelling whey protein aggregates (circle) and microparticulated whey aggregates (diamond) were subjected to simulated gastric digestion and their viscosity profiles over time were compared to that of non-modified whey (triangle). After an initial 5 min of measuring gastric acidification was initiated (open arrow), pepsin in simulated gastric buffer was added (closed arrow)

Fig. 4 Viscosity profile over time of mixtures of whey protein and polysaccharides during simulate gastric digestion. a A mixture of whey protein and gum arabic (diamond) was subjected to simulated gastric digestion and its viscosity profile over time was compared to those of whey (triangle) and gum arabic alone (circle). b A mixture of whey protein and pectin (diamond) was subjected to simulated gastric digestion and its viscosity profile over time was compared to those of whey (triangle) and pectin alone (circle). After an initial 5 min of measuring gastric acidification was initiated (open arrow), pepsin in simulated gastric buffer was added (closed arrow)

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delaying the overall digestion kinetics of whey protein. With the combination of whey protein and pectin, a different profile was observed. During simulated gastric acidification, pectin displayed a pH-sensitive viscosity profile in line with the known rheological behavior of pectin [13]. In combination with whey, the viscosity seamed lower, although temporarily aggregation (turbidity) could be observed. Furthermore, combinations of whey protein aggregates and polysaccharides were studied under the simulated gastric conditions (Fig. 5). Baseline viscosity for the mixture of cold gelling whey protein aggregates and gum arabic was lower than for the aggregates alone. However, viscosity increased upon simulated gastric acidification and relative to the starting viscosity, a larger increase was observed than with the cold gelling aggregates alone. Whether these effects

are explained by network formation of the aggregates themselves or by electrostatic interaction between the aggregates and gum arabic remains to be established. The effect of gum arabic on the viscosity profile of microparticulated whey is more pronounced. Viscosity of the more complex formulation was elevated and, as compared to microparticulated whey alone, remained high for a longer time. Formulation of cold gelling aggregates with pectin enhanced the viscosity as observed with the aggregates or pectin alone. Besides baseline viscosity, the viscosity upon gastric acidification was also elevated as compared to the profile of the individual ingredients. The latter thus suggests that interactions between the cold gelling aggregates induced by acidification may enhance microstructure formation which potentially delays the digestion and absorption

Fig. 5 Viscosity profile over time of mixtures of whey protein aggregates and polysaccharides during simulated gastric digestion. a A mixture of cold gelling whey protein aggregates with gum arabic (diamond) was subjected to simulated gastric digestion and its viscosity profile over time was compared to that of non-modified whey (triangle), cold gelling whey protein aggregates (multiplication sign), and gum arabic (circle) alone. b A mixture of microparticulated whey protein aggregates with gum arabic (diamond) was subjected to simulated gastric digestion and its viscosity profile over time was compared to that of non-modified whey (triangle), microparticulated whey protein aggregates (multiplication sign), and gum arabic (circle) alone. c A

mixture of cold gelling whey protein aggregates with pectin (diamond) was subjected to simulated gastric digestion and its viscosity profile over time was compared to that of non-modified whey (triangle), cold gelling whey protein aggregates (multiplication sign), and pectin (circle) alone. d A mixture of microparticulated whey protein aggregates with pectin (diamond) was subjected to simulated gastric digestion and its viscosity profile over time was compared to that of non-modified whey (triangle), microparticulated whey protein aggregates (multiplication sign), and pectin (circle) alone. After an initial 5 min of measuring gastric acidification was initiated (open arrow), pepsin in simulated gastric buffer was added (closed arrow)

Food Dig. (2013) 4:16 6. Dupont D, Mandalari G, Moll D, Jardin J, Rolet-Rpcaud O, Duboz G, Lonil J, Mills CE, Mackie AR (2010) Food processing increases casein resistance to simulated infant digestion. Mol Nutr Food Res 54(11):167789 7. Hall WL, Millward DJ, Long SJ, Morgan LM (2003) Casein and whey exert different effects on plasma amino acid profiles, gastrointestinal hormone secretion and appetite. Br J Nutr 89(2):239248 8. Hudson HM, Daubert CR, Foegeding EA (2000) Rheological and physical properties of derivitized whey protein isolate powders. J Agric Food Chem 48(8):31123119 9. Kilara A, Panyam D (2003) Peptides from milk proteins and their properties. Crit Rev Food Sci Nutr 43(6):607633 10. Koopman R, Crombach N, Gijsen AP, Walrand S, Fauquant J, Kies AK, Lemosquet S, Saris WH, Boirie Y, van Loon LJ (2009) Ingestion of a protein hydrolysate is accompanied by an accelerated in vivo digestion and absorption rate when compared with its intact protein. Am J Clin Nutr 90(1):106115 11. Koopman R, van Loon LJ (2009) Aging, exercise, and muscle protein metabolism. J Appl Physiol 106(6):20402048 12. Li CP, Enomoto H, Ohki S, Ohtomo H, Aoki T (2005) Improvement of functional properties of whey protein isolate through glycation and phosphorylation by dry heating. J Dairy Sci 88(12):41374145 13. Lofgren C, Guillotin S, Hermansson AM (2006) Microstructure and kinetic rheological behavior of amidated and nonamidated LM pectin gels. Biomacromolecules 7(1):114121 14. Luz Sanz M, Corzo-Martnez M, Rastall RA, Olano A, Moreno FJ (2007) Characterization and in vitro digestibility of bovine betalactoglobulin glycated with galactooligosaccharides. J Agric Food Chem 55(19):791625 15. Narici MV, Maffulli N (2010) Sarcopenia: characteristics, mechanisms and functional significance. Br Med Bull 95:139159 16. Pennings B, Boirie Y, Senden JM, Gijsen AP, Kuipers H, van Loon LJ (2011) Whey protein stimulates postprandial muscle protein accretion more effectively than do casein and casein hydrolysate in older men. Am J Clin Nutr 93(5):9971005 17. Rennie MJ, Bohe J, Smith K, Wackerhage H, Greenhaff P (2006) Branched-chain amino acids as fuels and anabolic signals in human muscle. J Nutr 136(1 Suppl):264S268S 18. Schaafsma G (2000) The protein digestibility-corrected amino acid score. J Nutr 130(7):1865S1867S 19. Schmitt C, Sanchez C, Desobry-Banon S, Hardy J (1998) Structure and technofunctional properties of protein-polysaccharide complexes: a review. Crit Rev Food Sci Nutr 38(8):689753 20. Tessari P, Kiwanuka E, Cristini M, Zaramella M, Enslen M, Zurlo C, Garcia-Rodenas C (2007) Slow versus fast proteins in the stimulation of beta-cell response and the activation of the entero-insular axis in type 2 diabetes. Diabetes Metab Res Rev 23(5):378385 21. Veldhorst MA, Nieuwenhuizen AG, Hochstenbach-Waelen A, van Vught AJ, Westerterp KR, Engelen MP, Brummer RJ, Deutz NE, Westerterp-Plantenga MS (2009) Dose-dependent satiating effect of whey relative to casein or soy. Physiol Behav 96(45):675682 22. Weinbreck F, de Vries R, Schrooyen P, de Kruif CG (2003) Complex coacervation of whey proteins and gum arabic. Biomacromolecules 4(2):293303

kinetics of whey proteins. Similar accounts for the combination of microparticulated whey and pectin. As compared to the profile of the individual ingredients, the viscosity profile of microparticulated whey was similarly enhanced by pectin as with cold gelling aggregates. Overall, the results from this study illustrate the potential of protein modification strategies to control gastrointestinal behavior and concomitant digestion and absorption kinetics. Although only confirmed by in vitro digestion, by studying microstructure formation and viscosity profiles under simulated gastric digestion, coagulation of casein under gastric conditions underlying the slower gastrointestinal absorption could be prevented by protein technology. These findings suggest that the absorption kinetics of casein can be modulated by food technology; this has been partly confirmed in vivo by others for casein hydrolysis. Conversely, whey proteins could be modified such that microstructures were formed and viscosity profiles were elevated, changes that potentially will delay digestion and absorption kinetics in vivo. Whether absorption kinetics and overall effects on physiological processes such as glycemic control, muscle synthesis, and satiety can be modulated by these protein modification strategies remains to be established in vivo.

References
1. Alting AC, Weijers M, de Hoog EH, van de Pijpekamp AM, Cohen Stuart MA, Hamer RJ, de Kruif CG, Visschers RW (2004) Acid-induced cold gelation of globular proteins: effects of protein aggregate characteristics and disulfide bonding on rheological properties. J Agric Food Chem 52(3):623631 2. Boirie Y, Dangin M, Gachon P, Vasson MP, Maubois JL, Beaufrere B (1997) Slow and fast dietary proteins differently modulate postprandial protein accretion. Proc Natl Acad Sci USA 94(26):1493014935 3. Brooks GA (1987) Amino acid and protein metabolism during exercise and recovery. Med Sci Sports Exerc 19(5 Suppl):S150 156 4. Dangin M, Boirie Y, Garcia-Rodenas C, Gachon P, Fauquant J, Callier P, Ballevre O, Beaufrere B (2001) The digestion rate of protein is an independent regulating factor of postprandial protein retention. Am J Physiol Endocrinol Metab 280(2):E340348 5. Darragh AJ, Hodgkinson SM (2000) Quantifying the digestibility of dietary protein. J Nutr 130(7):1850S1856S