Milk as a source of bioactives in health foods

The example of functional juices

STELA-FIL 2013 "Innovating for the future

of the dairy sector" May 13-16, 2013 Montreal, Canada

Élise Méric, Charles Lavigne, Simone Lemieux, Sylvie Turgeon and Laurent Bazinet Institut sur la nutrition et les aliments fonctionnels (INAF) Centre de recherche en sciences et technologie du lait (STELA) Laval University Quebec, Canada, G1V 0A6 E-mail: Laurent.Bazinet@fsaa.ulaval.ca

Introduction •Type 2 diabetes

•Socioeconomic context and solutions Review of the Literature

•Glycemia •Protein sources

Problem and Objectives

Methodology, Results and Conclusion

• In vitro study • Nutritional study

INTRODUCTION

Type 2 diabetes

Genetic disease

Physiological anomalies: tissue insulin resistance, insufficient production of insulin by the pancreas, and excessive production of glucose by the liver

Genetic predisposition, excess weight and lack of physical activity contribute to

its appearance

Serious long term complications: cardiovascular disease, cerebral embolism, blindness, amputations, kidney failure, death

Socioeconomic Context

It is currently a major problem

in most developed countries

A sharp increase in obesity can be observed in increasingly young populations

There are 365 million diabetics in the world, and this number is expected to double by 2025. Diabetes is a new epidemic.

It represents a huge economic and social burden:

2 billion dollars every year (Quebec)

Existing treatments (which can influence glycemic and/or insulin

response):

Nutritional-hygienic = diet and physical activity

Pharmacological = use of medication (oral anti-diabetic drugs)

Food ingredients: polysaccharides (Casiraghi et al., 2006), plant extracts (Williams et al., 2007) proteins, promising but still experimental! =>

Creation of functional foods

Solutions?

REVIEW OF THE LITERATURE

Glycemia

Definition

Level of sugar in the blood

Depends on the nutritional state (fasting blood glucose, postprandial glucose), age, and physiological condition (especially pregnancy)

Ranges from 0.7 g/l to 1.1 g/l (normal level 0.85g/l)

If too high = hyperglycemia (a sign of diabetes)

If too low = hypoglycemia

Glycemia

Regulation

Pancreas: hormonal regulation cells α (elevate blood glucose = glucagon)

cells β (lower blood glucose = insulin)

Insulin: enhances glucose uptake by muscles and peripheral tissues, and reduces liver glucose production (inhibited glycogenolysis)

Regulation of the pancreas: incretin hormones (GLP-1 and GIP) in enterocytes

Blood glucose +++

Enterocytes (L and K cells)

Small intestine

Insulin production

Pancreas

Regulation ---

Regulation +++

Incretin production GIP / GLP1

Storage

Glycemia: global regulation system

Liver and peripheral tissues (muscles)

Protein sources

Observed Effects

Effect on satiety (Luhovyy et al., 2007)

Insulinotropic effect (Frid et al., 2005 / Claessens et al., 2007)

Reduced glycemic response (Von Post-skagegard et al., 2006)

Increased plasmatic concentration of incretin => reduced glycemic response (Hall et al., 2003)

A global mechanism has yet to be identified.

Protein sources

Impact of form, composition, quantity and co-ingestion

The hydrolyzed form produces better results than the whole protein (Calbet et al., 2002 and 2004)

Amino acids with proven effects: leucine, phenylalanine, tyrosine…

(Van Loon et al., 2000) Co-ingestion: association of proteins + carbohydrates (Manders et al., 2006 and

2006) and/or amino acids (Nuttall et al., 2006) => Potentiation of effects

Effective at a theoretical minimum of 12g/drink = 4% of proteins (V/P)

PROBLEM AND OBJECTIVES

PROBLEM AND HYPOTHESIS

When choosing a protein ingredient, what are the most important

parameters to consider in order to most effectively influence the glycemic metabolism in vivo, and is it possible to predict effects in vitro?

The effects of protein ingredients in a digestate or a fruit-based drink on the glycemic mechanism (in vitro and/or in vivo) depend on dosage, form

and source.

1. To study in vitro the effects and impact of the amino acids content, dosage, and type, and of the source of protein ingredients on the

secretion of GLP-1

2. To clinically validate (in vivo with humans) the effects of various protein ingredients added to a fruit-based beverage on the glycemic and

insulin responses and on the secretion of GLP-1, and to analyze the impact of the dosage, the type and the source of these ingredients on

these responses.

Objectives

METHODOLOGY,

RESULTS, AND DISCUSSION

Objective 1

To study in vitro the effects and impact of the amino acids content, the

dosage, the type and the source of protein ingredients on the secretion

of GLP-1

3 ingredients and 4 amino acids + controls

Enzymatic digestions with PEPSINE AND PANCREATINE (Ma & Xiong 2009)

Enterocytes (Nci-H716) : incretin production (GLP-1)

1 concentration of protein ingredient or amino acids (2%)

1 contact time (2 h) 2 digestions (repetitions)

5 tests per digestion on different cells

Methodology

*

* *

* *

*

pM /

μg

* *

*

* * *

Standardized secretion of GLP-1 on Nci-H716 cells

Significant increase of GLP-1 secretion in relation to "normal" secretion, regardless of protein ingredient (double the control)

Generated secretions were similar regardless of the source or type of protein ingredient

used

There is a difference in GLP-1 secretion at the basal level and after contract with a protein ingredient

*

* *

* * * *

IPS IPL HPL

Soya: glycine x2

Whey: leucine x2

Unlike alanine and aspartate, leucine and glycine can increase the basal secretion of GLP-1.

Amino acids composition is an important factor to take into account because it

influences the intensity of GLP-1 secretion.

Effect of amino acids composition

Objective 2

To clinically validate (in vivo with humans) the effects of various

protein ingredients added to a fruit-based beverage on the glycemic and

insulin responses and on the secretion of GLP-1, and to analyze the

impact of the dosage, the type and the source of these ingredients on

these responses.

4 fortified beverages + 1 unfortified control beverage ⇒ 5 beverages to test

Selection of 25 volunteers: Men ranging in age from 18 to 55 AND in good health

Determination of the basal constants: sampling blood at -15 minutes and time 0 Ingestion of 300 ml of beverage at time 0 after blood sampling

AND Determination of the effects caused by the beverages: blood sampling at times 30, 60, 90, 120, 150 and

180 minutes

Methodology

Analysis of quantities of insulin, C-peptide, glucose and GLP-1 in each of the 8 blood samples

Beverages tested in vivo:

Two sources = whey and soya

Two types for whey = IPL and HPL

Choice of two concentrations to verify the quantitative effect of the ingredient which can most easily be used industrially = IPL

At the outset, 4 protein ingredients:

6% IPS / 6% HPL / 6% IPL / 2.6% IPL

Methodology

0 1 2 3 IPL

0 1 2 3 IPS

a

bc

ab

c

bc

HPL 6% IPL 6% IPS 6% IPL 2.6% Matrix

time

pM

Nutritional study: insulin secretion in vivo

HPL 6% IPL 6% IPS 6% IPL 2.6% Matrix

time

mM

b

b

a

b

ab

Nutritional study: glycemia in vivo

The effect of the beverage containing 2.6% IPL was not significantly

different than the effect of the matrix alone

=> there is therefore a quantitative effect related to the glycemic and insulin responses

A significant effect occurs only above a minimum

threshold

Ingredients containing 6% IPL perform significantly better than ingredients containing 6% HPL or IPS.

⇒ There is therefore a qualitative effect:

the source and type of ingredient used are also parameters which influence physiological

responses in vivo

a

ab

b

ab

a

IPL 6% HPL 6% IPS 6% IPL 2.6% Matrix

time

pM a

a

b

b

b

Nutritional study: GLP-1 secretion in vivo

At 6%, ingredients produce a significantly different secretion in vivo

than that produced by the matrix alone.

At 2.6%, the ingredient is not significantly different than the matrix.

⇒ There is therefore a quantitative effect:

The GLP-1 response in vivo is affected by the dosage ingested

In vivo and in vitro responses for the protein ingredients which were tested were similar.

Nutritional study: GLP-1 secretion in vivo

CONCLUSION

The amount of protein ingredient ingested has an important impact on in vivo physiological effects. There is a threshold of response to protein ingredients.

THE IN VIVO GLYCEMIC METABOLISM IS INFLUENCED BY THE DOSE INGESTED,

AS WELL AS THE SOURCE AND TYPE OF PROTEIN INGREDIENT USED.

There is an vivo/in vitro correlation between the GLP-1 responses of all the protein ingredients.

IN VITRO TESTS CAN IDENTIFY

WHEY OR SOYA BASED INGREDIENTS WHICH WILL HAVE AN IMPACT ON THE GLP-1 RESPONSE IN VIVO.

LASSONDE and NSERC

Joseph Makhlouf

Diane Gagnon, Pascal Dubé and Monica Araya-Farias

Bruno Marcotte, Yannève Rolland, Simon Peletier

Acknowledgements

Web Site: http://www.laurentbazinet.fsaa.ulaval.ca

THANK YOU

Experimental design of the study of GLP-1 secretion of Nci-H716 cells 1. Experiment (Chen&Reimer 2009 ; Anini&Brubaker 2003 ; Reimer 2001)

Basal control: KRB + 0.2% BSA Positive control: KRB + 0.2% BSA + DMSO Negative controls: KRB + 0.2% BSA + 2% leucine+ Wortmannin KRB + 0.2% BSA + Wortmannin 3 ingredients and 4 amino acids to test at 2% (Wt/Vol): KRB + 0.2% BSA + 2% leucine KRB + 0.2% BSA + 2% glycine KRB + 0.2% BSA + 2% aspartate KRB + 0.2% BSA + 2% alanine KRB + 0.2% BSA + 2% ING (IPL / HPL / IPS)

C+ C- C-

IPL

AA1

IPS

AA2 AA4 AA3

HPL

C