Hydrolyzed soy protein contains bioactive peptides that release - - PowerPoint PPT Presentation

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

• verweight with a 17% global
• besity 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

10 20 30 40 50 60 70 80 90 1 2 3 4 5 6 7 8 9 10

%Degree of Hydrolysis %Solubility +/- s.d.

DH vs. Viscosity and Solubility

1 10 100 1000 10000 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

DH (%)

Viscosity (cps, 10% P)

50 60 70 80 90 100 NSI (%)

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

Hydrolysis of soy protein can lead to improvements in viscosity

Viscosity Solubility

Intestinal Villus

• Peptides / hydrolysates*
• BSA & media controls
• PMA‡ positive control

CCK measured in cell media by ELISA (4 hrs)

STC-1 murine enteroendocrine cell line

Enteroendocrine Cell-based Screen for CCK Release

* Peptides/hydrolysates soluble in PBS

‡ PMA = phorbol 12-myristate 13-acetate

(activator of Protein Kinase C)

Hydrolyzed soy stimulates CCK release from Enteroendocrine cells

Optimal conditions for CCK inducing peptide generation are enzyme dependent

n = 4

50 100 150 200 250

1 2 3 4 5 6 7 8 9 10 11

Catalyst # CCK Release (% PMA control) +/- s.d. Catalyst Max CCK release (% control) 1 100.0 2 79.6 3 127.4 4 244.1 5 106.8 6 138.2 7 169.8 8 106.7 9 230.8 10 202.6 11 38.1

0.2 0.4 0.6 0.8 1 10 20 30

%DH CCK Release (ng/mL)

Enzyme 1 Enz 1+ Enz 2 Enzyme 2 Enzyme 3 Enzyme 4 Enzyme 5

Correlation between %Degree of Hydrolysis & CCK Release is Enzyme Dependent

Solae Confidential

Unique peptides are created with different enzymes & processing conditions

CCK release by hydrolysates is dose-dependent

EC50 = 0.86 R2 = 1.0 Select hydrolysates have been shown to stimulate release of CCK in a dose- dependent fashion, suggesting a true physiological response.

0.01 0.1 1 20 40 60 80 100 120

CCK Release (5 control) Dose (mg/ml)

In vitro digestion of soy hydrolysate does not destroy CCK inducing bioactivity

0.1 0.2 0.3 0.4 0.5 0.6 0.7 2 4 6 8 10

Protein (mg/mL) CCK Released (ng/mL)

H1 -pepsin H1 - pepsin + pancreatin H1 - undigested Pepsin Control Pancreatin control

Basal CCK PMA Ctrl

CCK release induced by <10K fraction of control hydrolysate

A B C D

200 K 45 K 6.5 K

Scanlon, Tulk

0.05 0.1 0.15 0.2 0.25

2 4 6 8 10 mg/mL Protein (w/v) CCK Released (ng/mL) PMA [+ve control]

Baseline

(10-100K) (< 10K) Whole control (>100K) hydrolysate #2, sample 1 Hydrolysate #2, sample #2) Hydrolysate #3, sample #1 Hydrolysate #3, sample #2

CCK-releasing hydrolysates contain substantial amts of <10 kDa peptides

200 400 600 800 1000 1200 1400 1600 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) A215

10 100 1000 10000 100000 1000000 10000000

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.

Thank you! Questions?