Presentations - WUR...Protein for Life Conference Ede, 25 th October 2016 Lonneke JanssenDuijghuijsen ([email protected]) IPOP Customized Nutrition Hsuan Chen • Protein digestion

Presentations

Day 2 Session 5,6

DAY 2 Session 4;5;6

Session 4 Topic: Protein and human/animal health

Annemarie Rietman Associations between dietary factors and markers of NAFLD in a general Dutch adult population

Marco Mensink True ileal amino acid digestibility of a lamb meat hydrolysate and its postprandial metabolic utilization in elderly subjects

Soumya Kar Use of multi omics approaches in the search for alternative dietary protein sources”

Meike Bouwhuis The influence of low protein diets on growth performance and carcass characteristics of gilts or entire boars fed ad libitum

Session 5 1. Learnings from human and animal studies

Lonneke Janssen Duijghuijsen Mitochondrial ATP production and intestinal epithelial permeability – an in vitro model

Hsuan Chen Protein digestion kinetics of different protein sources in pigs

Janne Beelen Effects of a 12-week intervention with protein-enriched foods and drinks on protein intake and physical performance of older patients during the first 6 months after hospital release

Lotte van der Zanden Novel protein foods: what do elderly need, what do elderly want?

Session 6 2. Side-streams and insects

Marcel Hulst In vivo and in vitro study on the mode of action of spray dried plasma when used as feed additive

Natasja Gianotten Sustainable protein production using lesser mealworms

DAY 2, SESSION 5 Session 5 Topic: 1. Learnings from human and animal studies Presentations available: • Lonneke Janssen Duijghuijsen • Hsuan Chen • Janne Beelen • Lotte van der Zanden

3

Lonneke Janssen Duijghuijsen • Mitochondrial ATP production and intestinal epithelial permeability –

an in vitro model

Novel proteins and intestinal permeability: An in vitro model

Protein for Life Conference

25th October 2016, Ede

Lonneke Janssen Duijghuijsen

IPOP Customized Nutrition

Background

6

Novel proteins

Digestion and

absorption Intestinal

permeability Screening method

Background

7

Novel proteins

Digestion and


permeability Screening method

Transcellular Paracellular

Background

8

Novel proteins

Digestion and


permeability Underlying mechanism

Transcellular Paracellular

Background

9

Novel proteins

Digestion and


permeability Underlying mechanism

In vitro model Caco-2 cells

Digestion and


permeability Screening methods

Underlying mechanism intestinal permeability

10

In vitro model Caco-2 cells

Digestion and


permeability Screening methods

Role of mitochondrial energy production

11

High glucose in cell culture media



12


‘Crabtree effect’ (Crabtree 1929)



13



Energy production by glycolysis



14



Energy production by glycolysis

Substrate Glucose

Galactose



(Rossignol 2004; Marroquin 2007; Aguer

2011; Kase 2013)

15

Respirometry (OROBOROS and Seahorse)

More ‘aerobic’ = more O2 consumption

Glucose versus galactose

“Aerobic switch”

16

Respirometry (OROBOROS and Seahorse)

More ‘aerobic’ = more O2 consumption



17

Western blot

More ‘aerobic’ = more mitochondrial OXPHOS complexes



18

Western blot

More ‘aerobic’ = more mitochondrial OXPHOS complexes



19

Mitochondrial staining

More ‘aerobic’ = more (green) mitochondria



20

Complex I inhibitor (Piericidin A; PA)

Cellular energy status

Mitochondrial dysfunction

Mitochondrial energy production

21


Cellular energy status

Mitochondrial energy production


22


Decreased cellular energy status

Intestinal permeability

TEER and FS flux Markers for permeability


FS

23

Complex I inhibitor

Intestinal permeability

TEER and FS flux Permeability increased


Decreased cellular energy status

FS

Caco-2 intestinal model

Take home message (1)

Glucose Galactose

O2 O2

O2

O2

O2

O2 O2 O2

O2 O2

O2 O2

O2

O2

O2

O2 O2

O2 O2

“In vivo-like” Caco-2 intestinal model 24

25

“In vivo-like” Caco-2 intestinal model

Take home message (2)

Functional mitochondria

Dysfunctional mitochondria

FS

FS FS

FS FS

FS

Presenter

Presentation Notes

Novel in vitro model to: - study mechanisms of intestinal permeability - screen novel protein sources Effect on intestinal permeability Intestinal permeability towards (novel) protein and (novel) protein-derived peptides

Novel proteins and intestinal permeability An in vitro model

Wageningen University and Research

Jaap Keijer (HAP)

Sander Grefte (HAP)

Dorien van Dartel (HAP)

Harry Wichers (FBR)

Jurriaan Mes (FBR)

Klaske van Norren (HNE)

Renger Witkamp (HNE)

26

Protein for Life Conference Ede, 25th October 2016 Lonneke JanssenDuijghuijsen ([email protected])

IPOP Customized Nutrition

Hsuan Chen • Protein digestion kinetics of different protein sources in pigs

FAST OR SLOW Protein digestion kinetics of protein sources in pigs

Hsuan Chen, 2016-10-25

29

Current feed evaluation

The kinetics of protein digestion is not taken into account

• Current evaluation

• Composition of indispensable amino acids

• Digestibility at ileal level

Global shortage of protein sources Increased price of protein sources Increase utilization efficiency of protein sources

What is protein digestion kinetics?

30

Digestion = Hydrolysis + Transition + Secretion + Absorption + Fermentation

Digestion Kinetics

Hydrolysis rate Passage rate

Absorption rate and location

Post-absorption metabolism

31

Lumen

Blood

• Oxidation to produce energy • Protein synthesis for secretion

• Protein synthesis for muscle • Oxidation to produce energy

Free AA Hydrolysis

Protein-bound AA

absorption absorption

Amino acids absorption and metabolism

Energy source

Hydrolysis

absorption

32

Aim of the study

To determine the protein digestion kinetics of protein sources

differing in ileal digestibility in pigs

Digestion Kinetics

Post-absorption metabolism

Increase utilization efficiency of protein sources

33

Experimental setup- Design

Soybean meal (SBM)

Only source of dietary protein TiO2 (2.5 g/kg feed) was included

Wheat gluten (WG)

Rapeseed meal (RSM)

Porcine plasma protein meal

(PPM)

Transition (5 days)

Adaptation (14 days)

Dissection (2 days)

2.5 times the maintenance requirement for energy Twice a day in equal amount Feed: Water = 1:3

34

Parameters- protein digestion kinetics

Postprandial amino acids and peptides in

plasma

Determination of protein digestion kinetics

Digestibility and mean retention time

Peptide molecular weight distribution

35

Results- Total plasma AAs and peptides

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

-60 0 60 120 180 240 300 360 420 480

Plas

ma

amin

o ac

ids a

nd p

eptid

es (m

mol

e/l)

Postprandial time (min)

RSM

SBM

WG

Plasma

Diet (P < 0.01) Time (P < 0.01) Interaction (P < 0.01)

36

Results- Total plasma AAs and peptides Parameter estimates characterising the kinetics of postprandial concentration of amino acids (AAs) and peptides in systemic blood

ka (min-1) k10 (min-1) t1/2ka(min) t1/2k10(min)

RSM 0.0330 - 21 -

SBM 0.0046 0.0039 151 177

WG 0.0319 0.0053 22 130

PPM 0.0062 0.0056 113 124

ka (min-1) is the increase rate constant k10 (min-1) is the decrease rate constant t1/2ka (min) is the time where half of maximal concentration of AAs and peptides in systemic blood is reached t1/2k10 (min) is the time where half of maximal concentration of AAs and peptides in systemic blood is removed

37

Results- Area under the curve

0

200

400

600

800

1000

1200

120 240 480

Plas

ma

amin

o ac

ids a

nd p

eptid

es (

mm

ole/

l)

Postprandial time (min)

RSMSBM

a

b b b

a a

b b

b b

ab

a Diet (P < 0.001)

38



plasma




0

50

100

150

200

250

300

350

400

450

Stomach 1st SI 2nd SI 3rd SI 4th SI Total SI

Mea

n re

tent

ion

time

(min

)

RSM

SBM

WG

PPM

39

Results- Mean retention time

ab ab b a

a ab

b b

Diet (P < 0.05)

a ab

b b

40

Results- CP Digestibility

0

10

20

30

40

50

60

70

80

90

100

1st SI 2nd SI 3rd SI 4th SI Total tract

Dige

stib

ility

coe

ffici

ent (

%)

RSM

SBM

WG

PPM

c

bc

a

ab

d

c

a b

a a

b

c

Diet (P < 0.001)

41

Results- Digestion curve

0.010.020.030.040.050.060.070.080.090.0

100.0

0 100 200 300 400 500 600

Dige

stib

ility

Coe

ffici

ent (

%)

Time (min)

SBM

0.010.020.030.040.050.060.070.080.090.0

100.0

0 100 200 300 400 500 600

Dige

stib

ility

Coe

ffici

ent (

%)

Time (min)

WG

0.010.020.030.040.050.060.070.080.090.0

100.0

0 100 200 300 400 500 600

Dige

stib

ility

Coe

ffici

ent (

%)

Time (min)

RSM

0.010.020.030.040.050.060.070.080.090.0

100.0

0 100 200 300 400 500 600Di

gest

ibili

ty C

oeffi

cien

t (%

) Time (min)

PPM

42

Results- Digestion curve

Parameter estimates characterising the kinetics of crude protein digestion in the small intestine of pigs Dmax (%) k (min-1) t1/2 (min) RSM 61 0.0136 63 SBM 78 0.0154 58 WG 94 0.0157 45 PPM 86 0.0338 20 Dmax (%) is potentially digestible CP (asymptote); k (min-1) is the rate constant; t1/2 (min) is the time where half of Dmax is reached.

43



plasma




44

Results- Molecular weight distribution

0

100

200

300

400

500

600

Diet Stomach 1st SI 2nd SI 3rd SI 4th SI

Abso

rban

ce a

t 214

nm

SBM

0

100

200

300

400

500

600


Abso

rban

ce a

t 214

nm

RSM

0

100

200

300

400

500

600


Abso

rban

ce a

t 214

nm

WG

0

100

200

300

400

500

600


Abso

rban

ce a

t 214

nm

PPM

45

Conclusions • Protein sources differ in protein digestion kinetics up to

the end of the small intestine in pigs. Plasma AAs and peptides: RSM>WG>PPM>SBM

CP digestion rate up to ileum: PPM>WG>SBM>RSM

Molecular distribution: ??

WG and PPM: once major release of AAs and peptides SBM and RSM: continuous release of AAs and peptides • Nutrient synchronization increase utilization efficiency

of protein sources

Contact: [email protected]

TAKE HOME MESSAGE Protein digestion kinetics differs among protein sources in pigs different timing of postprandial appearance of amino acids and peptides in blood

Janne Beelen • Effects of a 12-week intervention with protein-enriched foods and

drinks on protein intake and physical performance of older patients during the first 6 months after hospital release

Effects of a 12-week intervention with protein-enriched foods and drinks on protein intake and physical performance of older patients during the first 6 months after hospitalization

Janne Beelen

Protein for Life

25 Oct 2016

Background

Protein requirements higher in older adults who are

acutely or chronically ill 1:

1.2 – 1.5 g protein/ kg body weight/ day

On average protein intake of older patients:

<0.9 g/kg/d

New strategies needed!

1 ESPEN Expert Group (Deutz, 2014) &

PROT-AGE Study Group (Bauer, 2013)

Study objective

To study the effectiveness of supplementing a hospital and

home menu with a protein-enriched assortment of

familiar foods and drinks

in reaching a protein intake of 1,2-1,5 g/kg/d

& in improving physical recovery during 12 weeks after

hospital stay in older patients

* Beelen et al. 2016, JNHA

Intervention products

Step 1: Interviews with elderly patients and dietitians

Step 2: Product

development

Step 3: Sensory evaluation

and Pilot study*

Study design

1 October 2014 – 1 October 2015

Pulmonary & Geriatric Medicine New patients: n = 860

Included: n= 147 Continued at home: n = 75

Exclusion criteria: - < 65 year - Expected hospital stay < 4 days - Delirious - Dietary protein restrictions - eGFR < 30 - Terminally ill

Exclusion criteria: - Living in nursing home - Cognitive impaired

Measurements at day before discharge and 2, 6, 12 and 24 weeks after hospitalization

Presenter

Presentation Notes

Ongeveer 1700 tasjes bezorgd

Subjects: characteristics at admission

Controle (n=39) Intervention (n=36)

Age (mean ± SD) 77.2 ± 7.2 76.5 ± 6.7

Female/Male (n) 22/17 20/16

BMI (mean ± SD) 28.2 26.9

Medical diagnosis (n) - COPD, Asthma - Lung infection/inflammation - Other pulmonary diseases - Other infection (not lung) - Malaise or other

16 7 7 2 7

13 4 12 1 6

MUST score (n) - 0 - 1 - ≥2

33 2 4

26 3 7

Presenter

Presentation Notes

112 van de opnames waren vanwege longziektes: COPD, longinfectie/ontsteking, embolisme, klaplong MUST score moeten we nog goed naar kijken of dit ook echt verschilt, of dit invloed heeft.

Dietary assessment – Protein intake

• 24hr recall during home visit • Food record as a memory aid • Dutch Food Composition Table 2013

Results Protein intake

Control Cater with Care

Average intake during intervention period (wk. 2, 6, 12)

Control Intervention

Protein intake (g/kg/d) 1.0 ± 0.4 1.5 ± 0.6*

Reached intake of 1.2 g/kg/d 31% 72% * P < 0.01

Physical function: Short Physical Performance Battery

Balance (3 positions)

Gait speed (4 meter)

Chair rise time (5x)

Results Physical function

Control Cater with Care

Max score = 12 Linear Mixed Models:

No group or interaction effects

Same for other outcomes: Gait speed, Chair rise, Leg strength, Hand grip, Body weight, MNA, ADL, Physical Activity

Post-hoc analysis based on protein intake: below or above 1.2 g/kg/d

- Time (P<0.01) & Group effects (P=0.003)

- No interaction effect (P>0.05)

- Same letter on 2 time points: significant difference between these 2 time points within a group

Presenter

Presentation Notes

(e.g. in the group with an intake above 1.2 g/kg/d, week 0 and 6 are significantly different).

Take Home message

Protein-rich and -enriched familiar foods and drinks successfully increased protein intake of older

patients, but did not improve physical recovery.

However, we do not rule out a beneficial role of these products in combination with exercise in older adults

that normally have low protein intakes.

More information: www.alliantievoeding.nl

[email protected]

Thank you! Any questions?

Acknowledgements

All participants

Cater with Care project partners

Astrid Doorduijn & Renske Geers

Dr. Emmelyne Vasse

Nancy Janssen

André Janse, MD.

Medical and Nursing staff, Hospital Gelderse Vallei

Cater with Care students

Dr. Nicole de Roos

Prof. Lisette de Groot

http://www.alliantievoeding.nl/

http://www.zgv.nl/

Lotte van der Zanden • Novel protein foods: what do elderly need, what do elderly want?

Imagine...

CHALLENGES IN MARKETING TO ELDERLY CONSUMERS Illustrated with the case of protein-enriched foods

Lotte van der Zanden, Ellen van Kleef, Rene de Wijk & Hans van Trijp October 26th, 2016 Wageningen University

Background

Source: IeC Analysis; Un population division

Source: UN World Population Prospects (2002) Eurostat Demographic Projections (2004)

Background

Background

Background

24%

76%

SuccessFailure

Background

Someone aged….

45+ (e.g. Weijters & Geuens, 2003)

50+ (e.g. Sudbury & Simcock, 2009)

55+ (e.g. Moschis & Friend, 2008)

60+ (e.g. Delaney & McCarthy, 2009)

64 (The Beatles, 1967)

65+ (e.g. Mattila, Karjaluoto & Pento, 2000)

Background

Source: www.projectcartoon.com

Background

This presentation

Methods

Methods

Focus groups

Methods

Narrative review

Methods

Segmentation

Methods

Field study

This presentation

Results (Focus groups)

“Not for us!”

Aged 58-93

Results (Focus groups)

“For pregnant

women, children, ill people, and . . .

for old people”

“Not for us!”

Aged 58-93

ID SUB ACC STA RES aCOM aDSG

cognitive age +- +- +- ++ ++ +- --

life course -- ++ +- ++ ++ -- --

time perspective +- -- -- +- ++ ++ ++

demographics ++ ++ ++ ++ -- -- --

general food beliefs -- ++ -- +- ++ +- +-

food choice motives +- ++ -- ++ +- ++ ++

attributes & benefits -- ++ -- +- ++ ++ ++

past purchase +- ++ +- ++ -- -- +-

Results

abbreviations: ID, identifiability; SUB, substantiality; ACC, accessibility; STA, stability; RES, responsiveness; aCOM, actionability for communication; aDSG, actionability for design

(Narrative review)

Total Group 1 Group 2 Group 3 Group 4 Group 5 Group 6

Mean age 67.0 66.5 66.6 67.3 66.4 69.7 67.1


Mean age 67.0 66.5 66.6 67.3 66.4 69.7 66.3


Percentage 100% 31% 27% 21% 12% 6% 10%

R-squared 0.361 0.560 0.511 0.798 0.435 0.755 n.a.


Percentage 100% 31% 27% 21% 12% 6% 3%

R-squared 0.361 0.560 0.511 0.798 0.435 0.755 0.385

Results (Segmentation)


Mean age 67.0 66.5 66.6 67.3 66.4 69.7 66.3


Percentage 100% 31% 27% 21% 12% 6% 3%

R-squared 0.361 0.560 0.511 0.798 0.435 0.755 0.385



Mean age 67.0 66.5 66.6 67.3 66.4 69.7 66.3


Percentage 100% 31% 27% 21% 12% 6% 3%

R-squared 0.361 0.560 0.511 0.798 0.435 0.755 0.385



Healthiness 1.03 1.09** 0.85** 1.59** 1.09** 0.35* -.68*

Novelty -0.22 0.46** -.80** n.s. n.s. -1.56** -.61*

Meal type 0.40 0.72** 0.59** 0.43** -.42* n.s. -.67*



Healthiness 1.03 1.09** 0.85** 1.59** 1.09** 0.35* -.68*

Novelty -0.22 0.46** -.80** n.s. n.s. -1.56** -.61*

Meal type 0.40 0.72** 0.59** 0.43** -.42* n.s. -.67*

Results

“Healthy products that we consume

frequently” (Focus groups)

(Segmentation)


Healthiness 1.03 1.09** 0.85** 1.59** 1.09** 0.35* -.68*

Novelty -0.22 0.46** -.80** n.s. n.s. -1.56** -.61*

Meal type 0.40 0.72** 0.59** 0.43** -.42* n.s. -.67*


“Healthy products that we consume

frequently” (Focus groups)


Healthiness 1.03 1.09** 0.85** 1.59** 1.09** 0.35* -.68*

Novelty -0.22 0.46** -.80** n.s. n.s. -1.56** -.61*

Meal type 0.40 0.72** 0.59** 0.43** -.42* n.s. -.67*



Healthiness 1.03 1.09** 0.85** 1.59** 1.09** 0.35* -.68*

Novelty -0.22 0.46** -.80** n.s. n.s. -1.56** -.61*

Meal type 0.40 0.72** 0.59** 0.43** -.42* n.s. -.67*



Healthiness 1.03 1.09** 0.85** 1.59** 1.09** 0.35* -.68*

Novelty -0.22 0.46** -.80** n.s. n.s. -1.56** -.61*

Meal type 0.40 0.72** 0.59** 0.43** -.42* n.s. -.67*



Healthiness 1.03 1.09** 0.85** 1.59** 1.09** 0.35* -.68*

Novelty -0.22 0.46** -.80** n.s. n.s. -1.56** -.61*

Meal type 0.40 0.72** 0.59** 0.43** -.42* n.s. -.67*



Tailored formats

appropriateness 5.20 5.22 5.31 5.53 4.68 5.06 3.63

trial purchase 4.92 4.50 5.26 5.20 4.59 5.40 4.06

repeat purchase 4.18 3.31 5.15 4.57 3.38 4.95 2.44

Results


All formats appropriateness 3.45 3.06 3.65 3.38 4.86 4.00 3.74

trial purchase 3.17 2.83 3.23 3.37 3.33 3.51 3.41

repeat purchase 2.58 2.35 2.65 2.80 2.60 2.71 2.52

Measured on a scale from 1 to 7

(Segmentation)


Tailored formats


trial purchase 4.92 4.50 5.26 5.20 4.59 5.40 4.06

repeat purchase 4.18 3.31 5.15 4.57 3.38 4.95 2.44

Results



trial purchase 3.17 2.83 3.23 3.37 3.33 3.51 3.41

repeat purchase 2.58 2.35 2.65 2.80 2.60 2.71 2.52

Measured on a scale from 1 to 7 Or, roughly speaking, 1-2-3 4 5-6-7

(Segmentation)


Tailored formats

appropriateness 5.20 5.22 5.31 5.53 4.68 5.06 n.a.

trial purchase 4.92 4.50 5.26 5.20 4.59 5.40 n.a.

repeat purchase 4.18 3.31 5.15 4.57 3.38 4.95 n.a.



trial purchase 3.17 2.83 3.23 3.37 3.33 3.51 1.00

repeat purchase 2.58 2.35 2.65 2.80 2.60 2.71 1.01 Total Group 1 Group 2 Group 3 Group 4 Group 5 Group 6

Tailored formats


trial purchase 4.92 4.50 5.26 5.20 4.59 5.40 4.06

repeat purchase 4.18 3.31 5.15 4.57 3.38 4.95 2.44

Results



trial purchase 3.17 2.83 3.23 3.37 3.33 3.51 3.41

repeat purchase 2.58 2.35 2.65 2.80 2.60 2.71 2.52

Measured on a scale from 1 to 7 Or, roughly speaking, 1-2-3 4 5-6-7

(Segmentation)

Results (Field study)

“There is nothing extra in there, it is all just marketing.”

(Focus groups)

Control (N = 93) Prompt (N = 115) % fruit quark 6.5% 45.2%

Grams of protein 24.0 30.5

Would you like some

fruit quark?


iNspire

Discussion

• age is irrelevant

• protein is also irrelevant?

• the “average” elderly does exist, but...

• attractive ≠ purchaseable

• tailoring increases acceptance

• reaching rejecters likely the hardest

Presenter

Presentation Notes

Thank you for listening.

Discussion

• focus on preferences

• protein is also irrelevant?





Presenter

Presentation Notes


Discussion


• like calcium is for bones





Presenter

Presentation Notes


Discussion



• focus groups VS market segmentation




Presenter

Presentation Notes


Discussion




• distinguish between measures of acceptance



Presenter

Presentation Notes


Discussion





• sometimes hiding it is better


Presenter

Presentation Notes


Discussion





• sometimes hiding it is better

• inspire initial use and/or purchase

Presenter

Presentation Notes


Segmentation van der Zanden, van Kleef, de Wijk & van Trijp (2015). Examining heterogeneity in elderly consumers’ preferences towards carriers for enriched food. Food Quality and Preference, 42, 130-138.

Field study van der Zanden, van Essen, van Kleef, de Wijk & van Trijp (2015). Using a verbal prompt to increase protein consumption in a hospital setting: A field study. International Journal of Behavioral Nutrition and Physical Activity 2015, 12, 110.

Focus groups van der Zanden, van Kleef, de Wijk & van Trijp (2014). Knowledge, perceptions and preferences of elderly regarding protein-enriched functional food. Appetite, 80, 16-22.

Narrative review van der Zanden, van Kleef, de Wijk & van Trijp (2014). Understanding heterogeneity among elderly consumers: An evaluation of segmentation approaches in the functional food market. Nutrition Research Reviews, 27, 159-171.

Thank you!

DAY 2, SESSION 6 Session 6

Topic: 2. Side-streams and insects Presentations available: • Marcel Hulst • Natasja Gianotten

105

Marcel Hulst • In vivo and in vitro study on the mode of action of spray dried plasma

when used as feed additive

IN VIVO AND IN VITRO STUDY ON THE MODE OF ACTION OF SPRAY DRIED PLASMA WHEN USED AS FEED ADDITIVE

HULST, MARCEL1,KAR, SOUMYA1,2 VAN VUURE, CARINE3; VAN DOREMALEN, ANKE3; HERES, LOURENS3; DE WIT, AGNES2; JANSMAN, ALFONS2; SMITS, MARI1,2

1 Wageningen UR Livestock Research, Wageningen, The Netherlands. 2 Host-Microbe Interactomics, Wageningen University, Wageningen, The Netherlands. 3 Darling Ingredients, Son, The Netherlands.

Presenter

Presentation Notes

In the IPOP Customised Nutrition research program a pig and mice feeding experiment was performed with protein-rich diets of plant origin and animal origin, experiments of PHD researcher Sam Kar. Spray dried plasma powder prepared from the blood of pig was the protein-rich additive from an animal source. This protein source was also tested in an pig enterocyte cell line in cooperation of the producer Darling ingredients.

Spray dried (porcine) plasma (SDPP) is widely applied in weaning diets for piglets. Beneficial effects; shortens the period and reduce the severity of Post Weaning Diarrhea Objective > understanding in the mode of action of SDPP > can we use in vitro data to complement in-vivo data ? Approach; 1 -gene expression in ileum piglets after feeding a diet supplemented with SDPP 2- gene expression in porcine enterocyte cell line (IPEC-J2) exposed to SDPP 3 - overlapping in vivo-in vitro pathways/processes -“putative” effector genes/proteins expressed by enterocytes (IPEC-J2) 4- integration of in-vitro data (IPEC-J2) with in vivo data (piglets)

IN VIVO AND IN VITRO EXPERIMENT WITH SPRAY DRIED PORCINE PLASMA (SDPP)

Presenter

Presentation Notes

Several in vivo studies in pigs with SDPP iin weaned piglets indicate that this additive shortens the period and severity of weaning and therefore it could be an could alternative for growth promoting antibiotics during weaning. Nothing is know about the mechanisms how SDPP works when added to a weaner diet. To tackle this we combined in vivo and in vitro data In vivo, pigs were exposed for a long period to plasma powder, in vitro, exposure was short (maximal 6 hours)SDPP data. So we were also interested whether these short-term data may complement in-vivo data to study the mode of action of SDPP

enterocytes produce effector molecules (proteins/compounds) in response to changes in the composition of the luminal content of the ileum

Digestion of proteins in gastro-intestinal tract

Partially or Undigested Proteins (e.g. IgG)

Presenter

Presentation Notes

Enterocytes are among the first cells that sense changes in the intestinal environment and respond by producing signals ino the or the lamina propia and periphery, by effector proteins and chemical substances (for example cytokines and leukotrienes).

Gene-expression analysis / microarray

• Measures activity of all genes (messenger-RNAs) in cells/tissues • 28.000 pig genes • Comparison treated versus control cells or tissues (ratio treated/non-treated; Fold Change)

mRNA +

1 2

3 4

5

60k expression profiles

“Which proteins are activated and which are

silenced “

Presenter

Presentation Notes

What delivers whole genome gene expression analysis using micro arrays... Which genes/proteins are active and which are silenced. For the specialists; Affimetrics porcine arrays were used for in vivo experiments and Agilent arrays for the in-vitro experiments

List of response genes /Datamining / Functional analysis

Gene symbol FC S/M 2hr up/down

Gene symbol

FC ([M-S] vs [M]) up/down

CXCL2 53.23 up GPR158 -2.00 DOWN CXCL2 39.64 up RNF115 -2.00 DOWN IL1A 21.25 up TMEM151B -2.00 DOWN

CDK17 20.24 up SEC22C -2.00 DOWN CSF2 17.19 up HEATR4 -2.02 DOWN IL1A 13.88 up HYLS1 -2.03 DOWN

MAP3K8 13.41 up KLF13 -2.03 DOWN SLC2A5 13.28 up KLF13 -2.03 DOWN DDIT4 13.11 up FAM189A2 -2.03 DOWN CHAC1 12.75 up BEST2 -2.03 DOWN

TNFAIP3 12.11 up PIGV -2.03 DOWN SNAI1 8.48 up SYNGR3 -2.04 DOWN

IL8 7.42 up SIT1 -2.05 DOWN INHBA 6.41 up Pfn2 -2.05 DOWN CYP1A1 5.53 up CCNL1 -2.05 DOWN

IER3 5.51 up NOSTRIN -2.05 DOWN

Analysis list of IPEC-J2 response genes using bioinformatics programs

(FC: Fold change = ratio treated over control(s)

Pig Feeding experiment; Gene set enrichment analysis (GSEA), a statistical method to identify significantly enriched or depleted groups of genes associated with Gene Onthology terms (function).

IPEC-J2 experiment; differentially expressed genes with a Fold change (FC) of > 2.0 up- or down regulated (p< 0.05) and selection of up-regulated effector genes/proteins using phenotyping program.

Pathway >>

Associations with compounds

Enriched -pathways

-compounds

Presenter

Presentation Notes

What can you reveal from list of response genes; Bioinformatics programs retrieve pathways and associations enriched in response genes using statistical algorisms. This provides insight in biological processes possibly activated or silenced in the intestine; for example >>> all genes/proteins encircles this scheme steer the process of mineral absorption by enterocytes.

Pig feeding experiment with protein diets (Kar, Soumya et. al.)

Animal source -Spray dried porcine plasma protein (SDPP; Proglobulin Darling -ingredients) Plant source -Wheat gluten meal (WGM) -Rapeseed meal (RSM). -Soybean meal (SBM). Feeding experiment piglets -Six-week old pigs -3-weeks fed with experimental diets -Gene expression analysis of mucosal scrapings ileum. -Comparison results of SDPP diet to “plant protein” diets >> SDPP specific genes

Dietary protein level of 160 g/kg for protein sources. “No large differences in weight gain between groups”

Presenter

Presentation Notes

This was the design of the pig feeding experiment from which we used the gene expression data. Most work was performed by Sam Kar of the IPOP project.. Groups of pigs were fed for 3 weeks with meals enriched with proteins from 3 plant sources and gene expression was measured in mucosal scrapings of the ileum and compared to that of scrapings dissected from pigs with SDPP. Genes responding specific, thus only to SDPP were extracted fro gene-list and analysed using bioinformatics programs to identify biological processes.

IPEC-J2 in-vitro assay

LTB; Lymphotoxin Beta (cytokine) BTG2; B-Cell Translocation Gene 2 (cytokine) IL6; Interleukin 6 (cytokine) IL8; Interleukin 8 CXCL2; Chemokine (C-X-C Motif) Ligand 2 CSF2; Colony stimulating factor 2 (cytokine) enzymes metabolising chemical immune modulators! HPGD; Hydroxy-prostaglandin Dehydrogenase 15 (prostaglandines > regulation of inflammation/cytoprotection/platelet activation)

-Bioinformatics analysis of list with response genes >> pathways -Selection (secreted) effectors from lists of SDPP response genes.

Example immune-modulating effector genes

IL8 ↑ CSF2 ↑↑ IL6 ↓ CXCL2 ↑ LTB ↑ IL1A ↑ BTG2↓ HPGD ↑

IPEC-J2 cells

Exposure to 2 different concentration of SDPP

2 and 6h mRNA Microarray

Cultured IPEC-J2 cells grown for 6 -7 days form an enterocyte-like layer

Presenter

Presentation Notes

In this slide the design of the IPEC in-vitro test is shown; strait-forward; Examples are given for a set of immune modulating effector genes regulated by SDPP in enterocytes

Approach summarized; integration of in vitro – in vivo gene expression data

114

In-vivo pig experiment

enrichment analysis (GSEA); sets of

response genes specific for SDPP in the ileum (comparison to SBM,

WGM, RSM data).

Association of SDPP-specific

response- genes with

pathways and compounds

In-vitro IPEC-J2 experiment; SDPP

IPEC-J2 response genes with a FC>2 up-and

down (2 and 6h).

Participation of 28 IPEC-J2 EFFECTOR GENES in in-vivo

pathways

in-vitro IPEC-J2 experiment SDPP Genes with FC>2.

up-regulated; EFFECTOR GENES >> secreted genes

coding for cytokines-chemokines-hormones-growth factors- immune regulators-proteases and

inhibitors.

Association of IPEC-J2

response genes with

pathways and compounds

Overlapping compounds, with IPEC-J2 effector

Overlapping pathways, with IPEC-J2 effector

Presenter

Presentation Notes

Summerized our approach in one scheme; -In vivo response genes specific for SDPP and significant pathways and compounds extracted from these set of genelists -IPEC-J2 effector genes that respond in IPEC-J2 cells to SDPP; mainly genes secreted proteins, growth factors/cytokine/chemokines/hormones, but also enzymes involved in, for example leukotriene synthesis. -Analysis of full list of IPEC-J2 response genes and pathways compounds extracted from these set of genes. -in vitro _ in vivo overlapping compounds and pathways , in which an IPEC-J2 effector acts. -

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Results (1) IPEC-J2 effector genes participating in in vivo pathways/processes

28 effector genes; up-regulated expression in response to SDPP (FC>2) CASP7 apoptosis inducer (non-secreted) TNFAIP3 apoptosis inhibitor CXCL10 chemokine CXCL2 chemokine IL8 chemokine IL1A cytokine CSF2 cytokine IL1RN cytokine VEGFA endothelial growth factor EDN3 endothelial growth factor NRG1 growth factor INHBA hormone/growth factor NPPB hormone/growth factor HBEGF smooth muscle cell proliferation/growth factor ITGA2 focal adhesion MMP13 Metalloendopeptidase NGF Metalloendopeptidase Inhibitor Activity/growth factor TIMP3 Metalloendopeptidase Inhibitor Activity PLAT Serine-type Endopeptidase activity F3 Serine-type Endopeptidase activity COL7A1 Serine-type Endopeptidase Inhibitor Activity CRISPLD2 Serine-type Endopeptidase Inhibitor Activity SERPINE1 Serine-type Endopeptidase Inhibitor Activity SLPI Serine-type Endopeptidase Inhibitor Activity PRDM1 Transcription repressor / Drives the maturation of B-lymphocytes (non-secreted) HMOX1 anti-inflammatory proteins whenever oxidation injury takes places PPARG steroid/thyroid hormone coactivator receptor (non-secreted) PTGS1 Prostaglandin synthesis (Prostaglandins; secreted)

Presenter

Presentation Notes

Lets look at the set of 28 up-regulated effector genes selected from the IPEC dataset using the phenotype program VarElect We identified several functional clusters; immunological, structural, and growth promoting effectors

Results (2) IPEC-J2 effector genes participating in in vivo pathways

term Overlapping in-vivo/in-vitro pathways (SDPP specific genes) IPECJ2-effectors part of in-vivo pathway

angiogenesis Angiogenesis (CST) HMOX1, TIMP3 apoptosis Apoptotic Pathways PPARG, ITGA2, NRG1, HBEGF, NGF, TIMP3, CASP7

Ca2+ Ca-dependent Events NRG1, SERPINE1, VEGFA, F3 ECM Cell Adhesion / ECM Remodeling MMP13, PLAT, SERPINE1, HBEGF, TIMP3, IL8, ECM Endochondral Ossification MMP13, PLAT, VEGFA, TIMP3 ECM Integrin Cell Surface Interactions ITGA2 IL2 IL-2 Pathway IL1A, NRG1, HBEGF

NFKB NF-kappa B Signaling Pathway HMOX1, PRDM1, IL1RN, PTGS1, CXCL10, TNFAIP3 Antigen recognition Nucleotide-binding Domain, LRR Containing Receptor (NOD) PRDM1,TNFAIP3, CASP7, CXCL2, IL8 Antigen recognition Toll-like Receptor Signaling Pathway SERPINE1, CXCL10, IL8

TGF TGF-beta Signaling Pathway INHBA B-cell B Cell Receptor Signaling Pathway (KEGG) IL1A, CSF2

Presenter

Presentation Notes

In this slide a list of most significant overlapping pathways called significant by the Gene-analytics bioinformatics program for both in-vivo and in-vitro. And the IPEC-effector genes acting in these pathways. Clusters of important pathways; pathways involved in Extracellular matrix remodelling (intregity of the epithelial layer; many up-regulated growth factors) and antigen recognition; and also important; activation of B-cells.

Results (3) Common compounds, association with IPEC-J2 effectors (most important)

TERM – Matching COMPOUND – function (Enterocyte effector genes)

Angiogenesis -VEGF - endothelial growth factors (VEGFA, EDN3, HMOX1, TIMP3)

Coagulation - Heparin - Serine-type Endopeptidase (inhibitor) activity (PLAT, F3, SERPINE1)

Inflammation – Dexamethasone, anti-inflammatory drug – (cytokines, chemokines, HMOX1)

ECM - Cysteine - Metalloendopeptidase (Inhibitor) activity (e.g. MMP13, TIMP3, NGF)

ECM-Serine -Serine-type Endopeptidase activity/Inhibitor (SLPI, CRISPLD2, COL7A1, SERPINE1)

Immune modulation- Pge2 - Prostaglandin synthesis (PTGS1, HPGD )

Overlapping compounds, with IPEC-J2

effector Pge2 -Inflammation -cytoprotection -platelet activation

Presenter

Presentation Notes

In this slide a list of in-vitro and in-vivo shared associations of SDPP response genes with compounds, and participation of IPEC effectors in these compound-clusters Prostaglandin-E2 feedbac mechanism to inactivate this group of chemical immune-modulators.

-Overlap in SDPP-induced pathways/processes between the ileum and in-vitro cultured enterocytes. -Short-term response in IPEC-J2 enterocytes identified a set of effector genes/proteins providing insight in how biological processes imposed by a SDPP-rich diet in the ileum are regulated. > Remodeling and repair of the extracellular matrix. > Stimulation of the innate (via Toll- and NOD-like receptors) and adaptive (B-cell) immune system. Conclusion Insight in the mode of action how the protein-rich additive SDPP may support homeostasis in the intestine of piglets.

Summary and Conclusion

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Thank you for your attention

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Natasja Gianotten • Sustainable protein production using lesser mealworms

SUSTAINABLE PROTEIN PRODUCTION USING LESSER MEALWORMS

PROTEIN FOR LIFE CONFERENCE

Natasja Gianotten Proti-Farm R&D BV

COPYRIGHTS Copyright 2016-2017 PROTI-FARM. All Rights reserved. This material contains confidential and proprietary information. Any disclosure, reproduction, dissemination or distribution of the material contained herein is strictly prohibited without written permission from the author.

60% increase 1+ Billion 840M people

18% of all GHG 70% of world’s 25% of world’s

Children suffer from malnutrition

Emissions come from livestock farming

Arable land is used for livestock farming

Don’t have enough to eat today

People without fresh water in 2050

Of meat consumption till 2050

GLOBAL CHALLENGES

More iron than spinach

More calcium

than milk

Natural vitamins & minerals Healthy

fats

High protein content

All nine essential amino

acids

WHY INSECTS - NUTRITIONAL

WILD CAUGHT INSECTS

FARMED INSECTS

Mealworm Tenebrio Molitor 1-2

cm

Giant mealworm Zophobas Morio 2,5-

3cm

Lesser Mealworm Alphitobius diaperinus 0,7 cm

PROTI-FARM’S MEALWORMS

Proti-Farm production facilities The Netherlands Fully mechanised and automated Controlled environment Vertical, sustainable farming

LARGE SCALE UP PRODUCTION OF LMW

Low Feed Conversion Ratio

Limited GHG Impact

Low Land Use

Low Water Use

Feed / raw material choice

INSECTS - SUSTAINABILITY

Feed /raw materials / FCR

Make use of by-products from food industry or food grade ‘rest streams’

Improve FCR by feeding method,

feed quality

High productivity => good quality feed

INSECTS - SUSTAINABILITY

PROTI-FARM HOLDING NV

The Netherlands [email protected]

Thank you for your attention

mailto:[email protected]

Documents

Presentations - WUR...Protein for Life Conference Ede, 25 th October 2016 Lonneke JanssenDuijghuijsen ([email protected]) IPOP Customized Nutrition Hsuan Chen • Protein digestion