Intestinal health benefits of bovine whey proteins after simulated gastrointestinal digestion
Graphical abstract
Introduction
Bovine whey proteins are rich in essential amino acids, have a high nutritional value and contain bioactive peptides encrypted in their sequences (Corrochano, Buckin, Kelly, & Giblin, 2018). It has been suggested that these bioactive peptides have several health benefits including antidiabetic (Nongonierma & FitzGerald, 2013), weight management (Chaudhari et al., 2017) and reduction of cellular oxidative stress (Corrochano et al., 2018). Whey peptides can also give rise to food intolerances particularly notable in the infant gut (Brill, 2008). The protein component of bovine whey is composed of β-lactoglobulin (β-LG, 50–60%), α-lactalbumin (α-LA, 15–25%), bovine serum albumin (BSA, 6%), immunoglobulins (10%) and lactoferrin (LF, <3%) (Corrochano et al., 2018). Commercial whey products differ in their protein content from 95% (whey protein isolate, WPI) to 34% (whey protein concentrate) (Corrochano et al., 2018) and are commonly used as food ingredients especially in the sports nutrition sector. Once ingested, intestinal cells are the first point of contact and where whey proteins are most likely to exert their greatest effect.
The mechanism by which whey proteins may have a positive effect on ameliorating type 2 diabetes or aiding weight management is in their ability to increase the enteroendocrine incretin hormone glucagon-like peptide-1 (GLP-1) (Geraedts, Troost, Fischer, Edens, & Saris, 2011) and inhibit the ubiquitous dipeptidyl peptidase IV (DPP-IV) (Lacroix & Li-Chan, 2014). GLP-1 is produced by L-cells in the gut and functions to stimulate insulin production, increase satiety, influence appetite and regulate gastric emptying and ileal brake. There is conflicting data on whether consumption of whey can promote postprandial GLP-1 levels. Whey protein supplementation (25% energy intake) significantly increased postprandial plasma GLP-1 (425 ± 135 pmol/L h) compared to casein supplementation (161 ± 90 pmol/L h) in 25 healthy individuals (Veldhorst et al., 2009). In contrast, 14 type 2 diabetic subjects who consumed 36.4 g whey protein per day did not show increases in postprandial serum GLP-1 levels compared to those who consumed a reference diet without whey (Frid, Nilsson, Holst, & Björck, 2005). Certainly, 10 mg/mL of intact whey protein concentrate can stimulate GLP-1 secretion 1.3 fold from the enteroendocrine cells line STC-1 (Power-Grant et al., 2015). Whether whey proteins are pre hydrolysed or intact also appears to influence GLP-1 levels in vitro and in vivo (Gillespie, Calderwood, Hobson, & Green, 2015).
The enzyme DPP-IV is a ubiquitous protease, which is produced and secreted by intestinal cells (Gu et al., 2008) and inactivates GLP-1 by cleavage of N-terminal proline and alanine (Gu et al., 2008). To prolong the antidiabetogenic effect of GLP-1 and reduce diabetes progression, the treatment with DPP-IV inhibitors is being used as antidiabetic therapy (Ahren et al., 2004). Power-Grant et al. (2015) showed that 50% inhibition of the DPP-IV enzyme can be achieved with commercial whey hydrolysates at concentrations 1.5 and 1.1 mg/mL.
Intestinal cells are routinely exposed to exogenous molecules, which can trigger the formation of free radicals and damage the intestinal epithelium and mucus (Bhattacharyya, Chattopadhyay, Mitra, & Crowe, 2014). Bovine whey products and their hydrolysates have demonstrated antioxidant activity in vitro by chelating metals (Gad et al., 2011, Peng et al., 2010), decreasing lipid peroxidation (de Castro & Sato, 2014), reducing ferric ion (Lin, Tian, Li, Cao, & Jiang, 2012), scavenging radicals (peroxyls (Adjonu, Doran, Torley, & Agboola, 2013), hydroxyls (Kerasioti et al., 2014) and superoxides (Zhang et al., 2012)) and also neutralizing synthetic radicals (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (Torkova et al., 2016) and 1,1-diphenyl-2-picrylhydrazyl (Mohammadian & Madadlou, 2016)). Whey products (0.02–2.00 mg/mL) can protect against cellular oxidation and boost intracellular antioxidant markers such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) in lung fibroblasts, hepatocytes and endothelial cells (Kong et al., 2012, O'Keeffe and FitzGerald, 2014, Pyo et al., 2016). In the human epithelial colorectal adenocarcinoma cell line, Caco-2, Piccolomini, Iskandar, Lands, & Kubow (2012) reported that a 23 h treatment with digested WPI (0–2 mg/mL) protected H2O2-stressed cells against free radical formation and increased the ferric reducing antioxidant power of cellular supernatants.
However, when considering the physiological benefits of whey, it is important to be cognizant that whey proteins do not reach the intestine in their intact form. As they transit the conditions of the upper gut, they will be extensively hydrolysed with a digestibility score of 1.09 (Rutherfurd, Fanning, Miller, & Moughan, 2015).
We therefore pose the question of what happens to the bioactivities of commercial WPI and individual whey proteins after simulated upper gastrointestinal digestion (GID).
Section snippets
Materials
Bovine WPI from pasteurized milk (≥72 °C, 26 s) used in cheese manufacturing (Isolac, 91.4% protein content) was purchased from Carbery Food Ingredients (Cork, Ireland). The proteins β-LG (92.1% β-LG content) and α-LA (93% α-LA content) were obtained from Davisco Foods International, Inc. (Minnesota, USA). The BSA (98% protein content) was purchased from Sigma-Aldrich (Dublin, Ireland) and LF (Bioferrin 2000, which contains 95% of LF and 0.02% of iron) was donated by Glanbia Nutritionals, Inc.
Identification of peptides released after simulated gastrointestinal digestion of whey proteins
Protein powders (WPI, β-LG, α-LA, BSA and LF) were subjected to a simulated in vitro GID by the protocol described by Minekus et al. (2014). Samples were separated by UPLC, and the peptides were identified by HR-MS(/MS). The sequences of the peptides identified in each of the samples are listed in Table 1 and Supplementary Table S1. GID WPI revealed 47 peptides with 14 from β-LG (18.4 kDa), 3 from α-LA (14.4 kDa), 2 from BSA (66.5 kDa) and 2 from LF (80 kDa) (Table 1). Surprisingly, 26 peptides
Discussion
The whey fractions β-LG and α-LA exposed to the conditions of the upper gut were able to protect HT-29 cells from free radical formation. GID LF also increased the amount of the antioxidant proteins SOD1, SOD2 and TRX1 present in Caco-2 cells. All GID whey samples inhibited the protease DPP-IV activity. However, the hydrolytic conditions of the upper gut destroyed the ability of whey proteins to increase secretion of GLP-1. The peptide profiles revealed several sequences common to those
Conclusions
Conditions of the gut modulated the bioactivity of WPI and individual whey proteins. In particular in vitro evidences demonstrated that WPI lost its ability to stimulate GLP-1 but gained DPP-IV inhibition suggesting digestion facilitates or impedes bioactivity. Some antioxidant benefits were noted but these observations were intestinal cell model dependant. Whether some or all of the peptides identified were capable of crossing the intestinal barrier to subsequently have a health benefit to
Acknowledgement
This work was supported by the Department of Agriculture, Food and the Marine (Dublin, Ireland; FIRM project 13 F 354-WheyGSH). A. R. Corrochano is in receipt of a Teagasc Walsh Fellowship.
Conflict of interest
None.
Ethics statement
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