Hydrolyzed soy protein contains bioactive peptides that release cholecystokinin from enteroendocrine cells Sarah L. Martin, Barry M. Tulk, Nancy McGraw, Jia Li, Nida Napawan, Dustie Butteiger, Jason Lombardi, Zebin Wang, Kelly Moore, Elaine Krul; Solae LLC, St. Louis, MO, USA
Presentation Outline 1. Introduction • Obesity and Health • Weight Management • Soy protein and nutrition 2. Increasing protein functionality 3. Satiety peptides 4. Summary
Growing Obesity Creating Need for Better Weight Management Solutions • Approximately half the world’s population is overweight with a 17% global obesity rate, led by the U.S. (34%), Mexico (30%) • Prevalence of obesity in the U.S. has doubled over the last 15 years • Cost of obesity healthcare in the U.S. $350B by 2018 or 21% of total healthcare spending Sources: American Journal of Preventative Medicine, Packaged Facts, Organization for Economic Cooperation and Development (OECD)
Weight Management • Many products exist in the marketplace to help people manage their weight. • There are three main strategies used in weight management products: – Increase energy expenditure – Maintain lean body mass – Induce satiety • Protein consumption leads to all three phenomena.
Satiety is a key component of weight management strategies • Regulation of food intake is complex. Gut hormones play a major role. • Protein consumption is associated with satiety. Soy protein is as good as milk or animal proteins at inducing satiety.
Soy Protein Consumption results in lower weight gain in ob/ob mice • Body weight gain and food intake: Peptides from black soybeans (BSP) shown to reduce both total body weight gain and food intake in an acute study using leptin-deficient (ob/ob) mice. Taken from Jang et al (2008) Intl J. Obesity, 32:1161
Soy Proteins • The only nutritionally complete vegetable protein • Used in a variety of food applications, but can be difficult to work with due to solubility and viscosity issues. • Hydrolysis can oftentimes improve functionality, with the added benefit that it releases bioactive peptides.
Hydrolysis of soy proteins improves solubility at acid pH %DH vs. Solubility at pH 4 90 80 70 %Solubility +/- s.d. 60 50 40 30 20 10 0 0 1 2 3 4 5 6 7 8 9 10 %Degree of Hydrolysis
Hydrolysis of soy protein can lead to improvements in viscosity DH vs. Viscosity and Solubility 75-Visc 77 NS-Visc 77 SO3-Visc 16-Visc(OldTL1) 82 SO3-Visc 89 SO3-Visc 90-Viscc 75-NSI 77 NS-NSI 82 SO3-NSI 77 SO3-NSI 89 SO3-NSI 90-NSI 100 10000 Viscosity (cps, 10% P) 90 Solubility 1000 80 NSI (%) 100 70 Viscosity 10 60 1 50 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 DH (%)
Enteroendocrine Cell-based Screen for CCK Release • Peptides / hydrolysates* STC-1 murine • BSA & media controls enteroendocrine • PMA ‡ positive control cell line CCK measured in cell media by ELISA (4 hrs) * Peptides/hydrolysates soluble in PBS ‡ PMA = phorbol 12-myristate 13-acetate (activator of Protein Kinase C) Intestinal Villus
Hydrolyzed soy stimulates CCK release from Enteroendocrine cells CCK Release (% PMA control) 250 Max CCK release 200 Catalyst (% control) 1 100.0 150 +/- s.d. 2 79.6 3 127.4 4 244.1 100 5 106.8 n = 4 6 138.2 50 7 169.8 8 106.7 0 9 230.8 1 2 3 4 5 6 7 8 9 10 11 10 202.6 Catalyst # 11 38.1 Optimal conditions for CCK inducing peptide generation are enzyme dependent
Correlation between %Degree of Hydrolysis & CCK Release is Enzyme Dependent 1 CCK Release (ng/mL) Enzyme 1 0.8 Enz 1+ Enz 2 0.6 Enzyme 2 Enzyme 3 0.4 Enzyme 4 Enzyme 5 0.2 0 0 10 20 30 %DH Unique peptides are created with different enzymes & processing conditions Solae Confidential
CCK release by hydrolysates is dose-dependent 120 Select hydrolysates EC 50 = 0.86 have been shown to CCK Release (5 control) R 2 = 1.0 100 stimulate release of 80 CCK in a dose- dependent fashion, 60 suggesting a true 40 physiological response. 20 0.01 0.1 1 Dose (mg/ml)
In vitro digestion of soy hydrolysate does not destroy CCK inducing bioactivity 0.7 H1 -pepsin CCK Released (ng/mL) 0.6 H1 - pepsin + 0.5 pancreatin PMA Ctrl 0.4 H1 - undigested 0.3 Pepsin Control 0.2 Basal CCK 0.1 Pancreatin control 0 0 2 4 6 8 10 Protein (mg/mL)
CCK release induced by <10K fraction of control hydrolysate 0.25 (10-100K) 0.2 control] CCK Released (ng/mL) PMA [+ve (< 10K) Whole control (>100K) 0.15 200 K hydrolysate #2, sample 1 45 K 0.1 Hydrolysate #2, sample #2) Hydrolysate #3, sample #1 Hydrolysate #3, sample #2 Baseline 0.05 6.5 K A B C D 0 0 2 4 6 8 10 Scanlon, Tulk mg/mL Protein (w/v)
CCK-releasing hydrolysates contain substantial amts of <10 kDa peptides 1600 10000000 1400 1000000 1200 100000 1000 A215 800 10000 600 1000 400 100 200 0 10 0 1 2 3 4 5 6 7 8 9 10 11 1213 1415 16 1718 19 2021 22 2324 2526 27 2829 30 Time (min) MW range Retent time AUC Area % >20 kDa 7.7-14.44 371867 37.53% 10-20 kDa 14.44-15.3 144766.9 14.61% 5-10 kDa 15.3-16.17 168930.5 17.05% 2-5 kDa 16.17-17.3 191685.6 19.34% 1-2 kDa 17.3-18.17 65223.69 6.58% <1kDa 18.17-20 48470.9 4.89%
Summary • Obesity is a significant global issue that needs to be addressed. • Weight management strategies that include protein are most likely to be successful • Soy is an excellent source of nutritionally-complete protein. • Enzymatic processing of soy protein: – can improve functionality, thus making it easier to incorporate soy into different food forms – Releases bioactive peptides that may be useful in, e.g., maintenance of lean body mass, or inducing satiety • Bioactive peptides derived via hydrolysis of soy can release CCK from enteroendocrine cells in a dose-dependent fashion, and a significant portion of the bioactive portion survives digestion. • The majority of this bioactivity is found in the <10 kDa fraction. • Future work will focus on determining whether the bioactive peptides exert an enhanced satiating effect in vivo
Path Forward • Fractionate bioactive hydrolysate(s) to identify peptide(s) responsible for CCK release. • Look at the effect of soy peptides on the release of other satiety hormones.
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