Design of plant foods for optimal iron and zinc bioavailability

Ann-Sofie Sandberg

Dept of Biology and Biological Engineering

Chalmers University of Technology

Chicago Nutrition-2015

Retards brain

development in

infants

Increase morbidity

of mother and child

Increase

premature

deliveries

Iron

deficiency

Iron deficiency (WHO)

Hb <120 mg/l

S-transferrin receptors

>8.5mg/l

S-ferritin <15 µg/l

2 billion with iron deficiency

(ID) and 1 billion with iron

deficiency anemia (IDA)

Plant based diets in low-income countries major risk factor

Ann-Sofie Sandberg

Increased

morbidity Poor

pregnancy

outcome

Zinc

deficiency

Prevent

normal

child growth

Immune

function

Cognitive

function

Plant based diets in low income countries major risk factor

2 billion estimated to be zinc deficient

Ann-Sofie Sandberg

Two generalised dietary patterns are likely to be major factors in the aetiology of dietary zinc deficiency

• Cereal and legume-based diets which are not fermented are potentially high in phytate, a potent inhibitor of zinc absorption.

• Diets based on starchy roots and tubers have a low total zinc content. When these diet types are combined with a low intake of meat, poultry or fish, the intake of absorbable zinc is likely to be inadequate.

(Zinc and Human Health,

Conference in Stockholm June 2000) Ann-Sofie Sandberg

Design of foods targeting optimal bioavailability of iron and zinc

Oral-gastrointest. processing Optimal duodenal bioavailability

Biological processing techniques - malting - fermentation - add enzymes

Breeding genetic engineering - high iron - low phytate

Home processing

Agricultural production

Process Retailing Consumption Biological response

Ann-Sofie Sandberg

Ann-Sofie Sandberg

Oral-gastrointestinal processing of raw and extruded bran product

Raw bran

Extruded bran

Ann-Sofie Sandberg

Ann-Sofie Sandberg Sandberg et al

Dietary factors inhibiting/enhancing iron and zinc absorption

Inhibitors

IRON Phytate

Polyphenols

Calcium

Legume protein

ZINC Phytate

Enhancers

Ascorbic acid

Muscle protein

Organic acids

Animal protein,

amino acids

Ann-Sofie Sandberg

Bioavailability: the absorption and utilization of iron/zinc for normal metabolic processes.

Phytate – major inhibitor of iron and zinc absorption Dose dependent effect

0

20

40

60

80

100

0 50 100 150 200 250 300

InsP3-InsP6 P (mg)

fibre rich breads

phytate + white rolls

Brune, Rossander, Hallberg, Gleerup, Sandberg J Nutr 1992

Hallberg et al 1989

Re

lati

ve i

ron

ab

s %

Iron Zinc

Sandström & Sandberg 1992, Sandberg 1991, Fredlund et al JTEMB 20:49, 2006

Ann-Sofie Sandberg

Ann-Sofie Sandberg

Hunt JR. Int J Vitam Nutr Res 2005;75:375-84

Food factors Iron solubility/complex (phytic acid, polyphenols ascorbic acid, organic acids)

Food factors (Fe , AA, Ca)

Host factors Iron status (hepcidin) Infection, inflammation

Food factors affecting iron bioavailability

Lumen Blood Enterocyte

Intracellular effects: Influence of Fe and Ca • Fe decrease DMT-1(Arrendondo et al 1997,Tallkvist et al 2000, Sharp et al. 2002) and ferroportin

expression thereby downregulating the absorption of iron (Thompson et al.

2010)

• Ca decrease DMT-1 expression at the apical cell membrane downregulating iron uptake into the Caco-2 cell (Thompson et al. 2010).

Control Fe Ca Fe+Ca

Treatment

Ad

just

ed c

ou

nt

inte

nsi

ty

Ann-Sofie Sandberg

Intracellular effects:influence of ascorbic acid (AA) status

• AA influence the expression of proteins involved in iron uptake and transport in the Caco2-cell model in the absence of iron.

• Short-term effect upregulation of DMT-1 (Nramp2), Dcytb, ferritin and ferroportin, long-term downregulation

• AA induced iron transport through ferroportin involves IRP2 and HIF2α

Picture borrowed from: homepages.strath.ac.uk

Scheers & Sandberg, Eur J Nutr 47:401, 2008 Scheers & Sandberg, Nutrients 6:249, 2014

1 2 3

Ferropor n

β-ac n

Ann-Sofie Sandberg

Strategies to improve iron and zinc absorption from plant foods

1. Increase the amount of iron and zinc

2. Decrease the content of inhibitors (phytate, polyphenols)

3. Increase the content of enhancers (ascorbic acid, combine with animal protein sources)

4. A combination of 1-3

Ann-Sofie Sandberg

Design of foods targeting optimal bioavailability of iron and zinc

Oral-gastrointest. processing Optimal duodenal bioavailability

Biological processing techniques - malting - fermentation - add enzymes

Breeding genetic engineering - high iron, ferritin - low phytate

Home processing

Agricultural production

Process Retailing Consumption Biological response

Ann-Sofie Sandberg

Ann-Sofie Sandberg

Iron biofortified staple food crops (cross-breeding, agronomic biofortification, genetic engineering)

Iron Beans

• Republic Kongo

• Rwanda

Iron Pearl Millet

• India

Rice

• Phillipines

HarvestPlus

Strategy: Grow and consume the same foods - improve iron intake. Results: In general - higher total iron absorption rates

Frano et al Nutrition Reviews 2014 Ann-Sofie Sandberg

Design of foods targeting optimal bioavailability of iron and zinc

Oral-gastrointest. processing Optimal duodenal bioavailability

Biological processing techniques - malting - fermentation - add enzymes

Breeding genetic engineering - high iron, ferritin - low phytate

Home processing

Agricultural production

Process Retailing Consumption Biological response

Ann-Sofie Sandberg

Ann-Sofie Sandberg

The use of natures own processing methods to improve bioavailability of Fe

Biotechnological processing techniques

• Malting

• Fermentation

• Addition of phytase

• Overexpression of phytase in yeast

Controlled degradation of inositol

phosphates improves Fe absorption

0

20

40

60

80

100

0 50 100 150 200 250 300

InsP3-InsP6 P (mg)

fibre rich breads

phytate + white rolls

Brune et al J Nutr 122:442, 1992

Ann-Sofie Sandberg

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20 25 30 35 40

8

7

65

4

3

2

1

9

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1112

13

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15 16

1718

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20

2 1

22 23 24

25

min

AU

Ann-Sofie Sandberg

Reduction of phytate content in tempe fermented whole grain barley

Meal 1:

Tempe fermented barley

(InsP3-6 41 µmol/meal or 6.5 mg phytate-P/meal d.m.)

Meal 2:

Boiled barley

(InsP3-6 452 µmol/meal or 72.3 phytate-P/meal)

Charlotte Eklund-Jonsson et al Open J Nutr 2:42,2007

Ann-Sofie Sandberg

Phytase producing microorganisms for nutritious vegetarian food

Tempeh fermentation process

• Pearling 5 min

• Soaking 6+4 h, 0.3% LAc

• Boiling 10 min

• Pearling residue brought back

• Inoculation of Rhizopus oligosporus 27 h at 35°C

• Inactivation of fungus by owen baking

Charlotte Eklund-Jonsson et al J Cereal Sci 44:154 2006 Ann-Sofie Sandberg

Absorption of iron

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

BBM TM

% o

f to

tal

Human iron abs.

Extrinsic tag

55Fe 59Fe (n=8)

3.0±0.7%

Boiled barley

5.5±1.5%

Barley tempe

p = 0.05

Eklund-Jonson, Sandberg, Hulthén, Alminger , Open J Nutr 2:42, 2007

Iron absorption improved by tempe fermentation with Rhizopus oligosporus

Ann-Sofie Sandberg

Dephytinization of whole grains by hydrothermal process

• Optimization of phytate degradation in barley

• 95-96 %, T1=48, T2=48-50

• Lactic acid concentration 0.8%

Ann-Sofie Sandberg

Fredlund et al. J Cereal Sci 25:83, 1997 Bergman et al. J Cereal Sci 29:261, 1999

Zn absorption from single meals improved by hydrothermal process. (65Zn, 47Ca)

Test meal A (n=12 ): 200 g milk porridge barley - high phytate, 111 mg P - Zn 32 μmol; 5.8 mmol Ca - PA:Zn 17:1 Test meal B (n= 12): 200 mg milk porridge hydrothermal barley - low phytate, 28 mg P - Zn 31.5 μmol; 5.8 mmol Ca - PA:Zn 2:1 *Optimised process T140⁰ C,

T250⁰ C, 0.8 % v/w lactic acid

Ann-Sofie Sandberg

0

5

10

15

20

25

30

35

Zn Ca

% a

bso

rpti

on

A

B

Fredlund et al. EJCN 2002

Isolation of yeasts from Tanzanian togwa

Preparation of togwa (based on red finger millet, sorghum, maize, cassava)

Togwa fermentation and sampling

Isolation of yeasts on selective media

Purification of strains

Ann-Sofie Sandberg

Identification of many yeast strains in Togwa

Strain Togwa

origin

Identification method

API PCR + sequensing TY22 Sorghum ? Candida glabrata

TY14 Sorghum Kloeckera spp. Hanseniaspora guilliermondii

TY15 Sorghum Kloeckera spp.

TY26 Maize Candida glabrata Candida glabrata

TY24 Maize Candida glabrata

TY23 Sorghum Candida glabrata

TY20 Cassava Kloeckera spp. Hanseniaspora guilliermondii

TY08 Sorghum Saccharomyces cerevisiae Saccharomyces cerevisiae

TY27 Cassava Geotricum capitatum Pichia norvegensis

TY02 c Pichia kudriavzevii Pichia kudriavzevii

TY21 Cassava Pichia kudriavzevii

TY25 Maize Pichia kudriavzevii

TY05 Maize Pichia kudriavzevii

TY03 Sorghum Pichia kudriavzevii Pichia kudriavzevii

TY13 Sorghum Pichia kudriavzevii

TY18 Maize Pichia kudriavzevii

TY07 Cassava Candida pelliculosa

TY06 Maize Candida pelliculosa Pichia anomala

TY16 Maize Candida pelliculosa Pichia anomala

TY17 Maize ? Kluyveromyces marxianus

TY04 Sorghum ?

TY01 c Candida lucitaniae Pichia burtonii

TY11 Cassava Candida lucitaniae

TY09 Maize Candida guilliermondii Candida fermentati

TY19 Cassava ? Saccharomyces cerevisiae

TY12 Sorghum ? Saccharomyces cerevisiae

Screening for : 1) High phytase activity 2) Secreted activity 3) Good performance in

cereal-based media

Red strains shown particularly strong – selected

Int J Food Microbiol, 136:352-358 (2010) Hellström A., Vazquez-Juarez R., Svanberg U., Andlid T.

Ann-Sofie Sandberg Andlid et al.

Some excellent phytase producing yeasts found; TY13 expressing a truly extracellular phytase of

high activity

P. kudriavzevii TY13

S. cerevisiae

Halo surrounding

colony

Phytase assay performed with cell-free

supernatant of selected high phytase yeast

Halo shows secreted phosphatase activity,

subsequently confirmed as phytase activity

TY13 – strong secreted activity

Int J Food Microbiol (2012) 153:73-77 Hellström et al.

Ann-Sofie Sandberg Andlid et al.

High phytase yeast. Specific modifications in Baker’s yeast (Saccharomyces cerevisiae)

IP6 degradation during yeast cultivation in medium, in the presence or absence of inorganic phosphate (Pi)

Three categories of improved strains have been created: 1) Deletion of negative regulatory

genes

2) Overexpression of structural genes (the actual phytases)

3) Overexpression of transcriptional activators

All resulting in strains showing high

constitutive phytase activity

Andlid et al

0

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Me

diu

m IP

6 (m

M)

Time (h)

PHO-mutant; -Pi

PHO-mutant; +Pi

wild type (control); -Pi

wild type (control); +Pi

Traditional yeast (WT)

Improved high phytase

yeast

1. Andlid et al (2004) Int J Food Microbiol, 97(2):157-169 2. Veide and Andlid (2006) Int J Food Microbiol, 108 (1):60-67 3. Haraldsson, Veide , Andlid, Sandberg , 2005, JAFC, 53:5438 ,

Ann-Sofie Sandberg

0

5

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15

20

25

30

rice wheat Maize high tanninsorghum

rice wheat maize HT sorghum

Rel

ativ

e ir

on

ab

sorp

tio

n

Extrinsic tag radioiron, 8-11 adults/study, porridges from 50g cereal/300 ml water, 2.5 mg Fe, phytic acid degraded enzymatically

Complete dephytinization does not improve iron absorption from high tannin sorghum porridge

(Adapted from Hurrell et al. 2002 Br J Nutr 88:117)

Ann-Sofie Sandberg

PA mg/100g 160 120 260 870 0 0 0 0

% Fe abs. (± SE) corrected to low iron status (SF 12µg/L)

No effect of dephytinization when milk is added to wheat porridge

Ann-Sofie Sandberg

0

2

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6

8

10

12

A B C D

% F

e a

bso

rpti

on

Test meals

A. wheat, ascorbic acid, native phytate

B. wheat-milk, ascorbic acid, native phytate

C. wheat, ascorbic acid, dephytinized

D. wheat-milk, ascorbic acid, dephytinized

Adapted from Hurrell et al. AJCN 77:1213, 2003

Phenolic compounds in plant foods

• Phenolic compounds inhibitors of iron absorption

• Polyphenols occurs in cereals, legumes vegetables , spices

• Examples of foods with high content of iron binding polyphenols:

high tannin sorghum, millets, beans, herbs • Beverages: tea, coffee, red wine

Gallic acid Cathechin

Ann-Sofie Sandberg

Polyphenols in plant foods – major inhibitors of iron absorption (from Hurrell et al 1999)

• Iron absorption

(extrinsic tag radioiron

method)

• Dose dependent effect

• 20-50 mg PP/serving

reduced Fe absorption

by 50-70%

Ann-Sofie Sandberg

Bread meal with water (1.0) or polyphenol beverage

0

10

20

30

40

Untreated +Phytase +Phytase+PPO

Ph

en

oli

c c

on

ten

t (m

g/g

)

0.0

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In v

itro

avail

ab

le i

ron

(m

g/1

00g

)

Incubation of sorghum with phytase and PPO

0.8 u/g 900 u/g 0.8 u/g

Effect on the total phenolic content and on in vitro iron availability

Source: Matuschek et al. 2001 Ann-Sofie Sandberg

0

5

10

15

20

25Fe

ab

s (%

)

white wheat roll + 200g mixed vegetables (carrot, turnip, onion)

whole meal wheat roll +200g mixed vegetables (carrot, turnip, onion)

fresh

fermented

Lactic fermentation of mixed vegetables doubled iron absorption from bread meals (extrinsic tag, n=8 and 9 subjects resp.)

Scheers et al EJN 2015

Ann-Sofie Sandberg Ann-Sofie Sandberg

Ann-Sofie Sandberg

Why is iron bioavailability increased in the lactic fermented vegetables ?

• Degradation of inhibitors?

• Increase in ascorbic acid?

• Lactic acid produced- increased iron absorption?

• Was hepcidin release by liver cells influenced?

• Speciation of iron after digestion changed?

• Does Fe3+bind to mucus?

Scheers et al EJN 2015 Scheers , Almgren, Sandberg J Nutr Biochem 2014

Yes

No

Yes

Yes

Ann-Sofie Sandberg

Mukösa bägarceller (HT29-MTX-E12)

Tarmceller (HTB-37)

Ann-Sofie Sandberg

No

No

Ann-Sofie Sandberg

Design of foods targeting optimal bioavailability of iron and zinc

Oral-gastrointest. Processing Microbiol phytase Cereal phytase Optimal duodenal bioavailability

Biological processing techniques - malting - fermentation - add enzymes

Breeding genetic engineering - high iron - low phytate

Home processing

Agricultural production

Process Retailing Consumption Biological response

Ann-Sofie Sandberg

Ann-Sofie Sandberg

Dietary A.niger phytase increases Fe absorption in humans

Control White wheat rolls

butter

150 ml water

Test meals: Addition of

a) 1g raw bran

b) 1g phytase deactivated bran

c) 1g phytase deactivated bran +

A.niger phytase Extrinsic labelling 55Fe, 59Fe

0

20

40

60

80

100

120

WhiteWheat roll

+1g deactivbran

+1g rawbran

+1g deactivbran

+phytase

Re

lati

ve ir

on

ab

sorp

tio

n

Sandberg et al. J.Nutr 126:476,1996

Ann-Sofie Sandberg

Ann-Sofie Sandberg

Phytate degradation by dietary phytase active in the gastrointestine improved Zn absorption

Test meal C (n=10): 200 g milk breakfast cereals (barley) - high phytate - deactivated phytase –Zn 34 μmol; Ca 6.0 mmol - PA:Zn 15:1 Test meal D (n=10): 200 mg milk breakfast cereals (malted

barley) - high phytate - active phytase - Zn 27 μmol ; Ca 6.1 mmol - PA:Zn 13:1 *Labelled with 65Zn and 47Ca

0

5

10

15

20

25

30

35

Zn Ca

% a

bso

rpti

on

C

D

Fredlund et al. EJCN 57:1507, 2002 Ann-Sofie Sandberg

General Conclusions

• Phytic acid (and polyphenols) constitutes a nutritional problem in terms of iron and zinc supply in low-income countries where the diet is based on cereals and legumes.

• Use of biological processing techniques (malting, fermentation, addition of enzymes) to degrade inhibitors to improve iron and zinc nutrition is feasible. Processing has to be optimised and for iron the degradation almost complete.

• Promising enhancing effect of lactic fermentation associated with release of iron and change in speciation (ferric iron).

• Processing in the gut by active phytase to degrade phytate is also an option.

• Biological processing methods can be used in combination with breeding and genetic engineering for iron biofortification. Engineered yeast and lactobacillus strains for improved fermentation.

Ann-Sofie Sandberg

Food and Nutrition Science Chalmers Nathalie Scheers Nils-Gunnar Carlsson Marie Alminger Thomas Andlid Ulf Svanberg Andreas Hellström Charlotte Eklund-Jonsson Kerstin Fredlund Annette Almgren

Collaborators Novozymes Dan Pettersson Kathrine Pontoppidan Sahlgrenska Academy Lena Rossander Hultén

Acknowledgements

Ann-Sofie Sandberg