Danish University Colleges Health and immune responses in a rat model after intake of organically or conventionalle grown foods Jensen, Maja Melballe

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Danish University Colleges

Health and immune responses in a rat model after intake of organically or conventionalle grown foods

Jensen, Maja Melballe

Publication date:

2012

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Citation for pulished version (APA):

Jensen, M. M. (2012). Health and immune responses in a rat model after intake of organically or conventionalle grown foods.

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Health and immune responses in a rat model after intake of organically or conventionally grown foods.

Ph.D. thesis by Maja Melballe Jensen

Section for Immunology and Microbiology Department of Animal Science Faculty of Science and Technology

Aarhus University Denmark

Main supervisor: Senior Scientist Charlotte Lauridsen Co-supervisor: Senior Scientist Henry Jørgensen Project supervisor: Senior Scientist Helle R. Juul-Madsen

October 2012

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List of original manuscripts

This PhD thesis is based on the following original manuscripts

Maja Melballe Jensen, Henry Jørgensen, Ulrich Halekoh, Bernhard Watzl, Kristian Thorup-Kristensen and Charlotte Lauridsen. Health biomarkers in a rat model after intake of organically grown carrots.

Journal of The Science of Food and Agriculture, 2012; [Epub ahead of print] DOI 10.1002/jsfa.5705

Maja Melballe Jensen, Henry Jørgensen, Ulrich Halekoh, Jørgen E. Olesen and Charlotte Lauridsen. Can Agricultural Cultivation Methods Influence the Healthfulness of Crops for Foods?

Journal of Agricultural and Food Chemistry, 2012; 60, 6383-6390.

Maja Melballe Jensen, Ulrich Halekoh, Christopher R. Stokes and Charlotte Lauridsen.

Effect of maternal intake of organically or conventionally produced feed on immune development in offspring rats

Submitted to Journal of Agricultural and Food Chemistry

Maja Melballe Jensen (Jacobsen), Helle R. Juul-Madsen and Charlotte Lauridsen.

Development of an oral tolerance model in rats for investigation of bioactive food components.

Proceedings of the Nutrition Society, 2010; DOI 10.1017/S0029665110000789 and abstract for poster presentation at the 3^rd International Immunonutrition Workshop, 21-24- October 2009, Girona, Spain.

Other scientific contributions

Maja Melballe Jensen, Alicja Budek Mark, Charlotte Lauridsen and Susanne Bügel.

Effects of organic and conventional cultivation systems on plasma antibody status in humans.

Abstract for poster presentation at the 6^th International Immunonutrition Workshop, 15-17 October 2012, Palma de Mallorca, Spain.

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Preface

The present PhD project was conducted from November 2008 to October 2012 (including 11 months maternity leave during 2010/2011) at the Department of Animal Science, Faculty of Science and Technology, Aarhus University (AU). The PhD project was part of a large collaborative project entitled “Content, Bioavailability and Health Effects of Trace Elements and Bioactive Components of Food Products Cultivated in Organic Agricultural Systems (OrgTrace)” under the program

“Research in Organic Food and Farming” (DARCOF III 2006-2010). The three animal studies which are the core of this PhD dissertation were carried out under the OrgTrace project and the CoreOrg project. The "Coordination of European trans-national research in organic food and farming" (CoreOrg) was initiated as a part of the European Commission’s ERA-NET Scheme. The CoreOrg animal study in the current PhD project was carried out under the pilot project “QACCP:

Quality analysis of critical control points within the whole food chain and their impact on food qualiy, safety and health” (2007-2010).

Senior Scientist Charlotte Lauridsen, Department of Animal Science, Faculty of Science and Technology, AU was main supervisor, senior scientist Henry Jørgensen was co-supervisor and senior scientist Helle R. Juul-Madsen was project supervisor.

The OrgTrace project was funded by the Ministry of Food, Agriculture and Fisheries and coordinated by the International Centre for Research in Organic Food Systems (ICROFS)

(http://www.icrofs.dk/ ). The CoreOrg research was funded by the EU FP6 ERA-net CORE organic (contract no ERAC-CT-2004-011716) and coordinated by ICROFS. The PhD project was partly funded by the OrgTrace Project and partly by SAFE, the Graduate School of Agriculture, Food, and Environment, AU.

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Acknowledgements

A large number of people have contributed to the accomplishment of the three animal studies and the completion of this PhD project and thesis. First of all, I would like to thank my main supervisor Charlotte Lauridsen for giving me the opportunity to conduct this thesis, for guiding me through the entire PhD project while having trust in me and letting me stand on my own two feet at the same time and for always having time to discuss problems and ideas. Secondly, I would like to thank my co-supervisor Henry Jørgensen who has been invaluable in formulating the diets for the animal experiments and always taking time for discussions and advice. A special thanks to Ulrich Halekoh, my statistics guru, for the statistical work conducted in all three experiments and for patiently guiding me in the statistical challenges. Also thanks to my project supervisor Helle R. Juul-Madsen for the good ideas and discussions on the immunological issues in the thesis.

I am very grateful to lab technician Inger Marie Jepsen, who has been a great help and personal support during the whole project. Additionally, I would like to thank all the colleagues from D23 who were involved in care of the animals, especially Pernille Vendelboe. Thank you to Jens Laurids Sørensen, Mette Würtz Reeh, Elsebeth Lyng Pedersen and Carsten Berthelsen for their assistance regarding specific laboratory analysis. Also I wish to thank the rest of my colleagues at the Department of Animal Science. I would also like to thank Emese Kápolna and Jette Jakobsen regarding specific laboratory analysis and Jørgen E. Olesen, Kristian Thorup-Kristensen, Susanne Bügel and Alicja Budek Mark for their scientific contributions.

Very warm thanks to Prof. Chris Stokes from Infection and Immunity, School of Clinical Veterinary Science, University of Bristol and Prof. Dr. Bernhard Watzl from the Department of Physiology and Biochemistry of Nutrition, Federal Research Institute of Nutrition and Food, for giving me the opportunity to work in their laboratories and for the review of the manuscripts.

A warm thanks to my “køregruppe” and my office mate Sine Nygaard Langerhuus for fun times, good discussions and patience during ups and downs.

My deepest thanks to my closest family and friends for your support. A heartfelt thanks to my boyfriend Christian Melballe Jensen and our son Hans for your love and for constantly reminding me of more important things in life.

Maja Melballe Jensen

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Summary

The consumption of organic foods has been increasing during the last decade and organic products are becoming more visible on the market. Consumer perceptions of organic foods being of better quality, more nutritious and healthier are some of the main reasons driving the organic market. Scientific research on organic foodstuffs is very contradictory and conclusive research on possible impact on human and animal health is lacking. The definition of health and potential biomarkers of health is also debated heavily. One aspect is the fact that the immune system can be modulated by nutrients and that the immunological defense system is imprinted during gestation and the early lifespan. So optimization of plant-derived food by cultivation system could be a very cost effective method for disease prevention, since diet-induced health improvements would carry no costs for the health sector. The objective of this PhD thesis is to study if the intake of diets produced of crops from well-controlled field experiments leads to differences in biomarkers characterizing health and well-being using a rat model. Furthermore, the hypothesis that the intake of organically produced diets during pregnancy and lactation/suckling can affect the immunological status and function of both the mothers and the offspring is also investigated.

To investigate the potential health effects of eating organic foods, it was decided to follow the whole food chain from field to fork, using a rat model for measuring the potential health benefits. Food products were obtained from continuing field studies and two long-term feeding study with rats were performed to study the influence of cultivation system, year, and location on the nutritional composition of the crops and health- related biomarkers. The results from these two studies are presented in Paper I and Paper II. A single food item, carrots was used in Paper I and it was found that nitrate and protein in carrots was affected by

cultivation system, while the measured health biomarkers in rats were not affected. In Paper II complete diets were used and it was found that cultivation system, harvest year and locations had a little impact on the nutritional quality. Besides from plasma IgG levels, none of the measured health biomarkers were affected by cultivation system. Paper III investigated if there was an effect of maternal consumption of organically or conventionally produced feed on the modulation of immunological tolerance in offspring. The first step was to develop an oral tolerance model (pilot study), which was employed on second generation rats who’s mothers originated from the study described in Paper II. Except from PGE2, cultivation system had little effect on the immunological parameters studied in either generation and no difference between the offspring of rats fed diets of organically versus conventionally grown crops in their response to a novel dietary antigen was observed.

In conclusion, it was found that cultivation system, harvest year and location had a small effect on the nutritional quality of the foods and that cultivation system had no clear effect on the measured health-related or immunological biomarkers.

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Dansk resumé

Efterspørgslen af økologiske fødevarer har været stigende i det sidste årti og økologiske fødevarer bliver mere og mere synlige i butikkerne. Mange forbrugere har en forventning om, at økologisk producerede fødevarer har en bedre kvalitet, er mere rige i næringsstoffer og er sundere. Forskningsresultater omkring økologiske fødevarer er dog meget divergerende og der mangler forskning omkring indflydelsen på dyr og menneskers sundhed. Definitionen af sundhed og potentielle biomarkører for sundhed debatteres også kraftigt. Et af aspekterne er, at immunsystemet kan moduleres af næringsstoffer og at immunsystemet præges under foster tilstanden og i den tidlige levetid. Derfor kunne optimeringen af plante-baserede fødevarer ved brug af forskellige dyrkningssystemer være en meget omkostnings-effektiv metode til sygdomsforebyggelse, da kost-inducerede sundhedsmæssige forbedringer ikke ville betyde nogen omkostninger for

sundhedssektoren. Formålet med denne afhandling er, at undersøge om indtaget af afgrøder fra forskellige dyrkningssystemer kan føre til forskelle i biomarkører for sundhed ved brug af en rotte model. Desuden testes hypotesen om, at indtagelse af økologisk producerede fødevarer under graviditet og amning/diegivning kan påvirke den immunologiske status og funktion i både mødre og afkom.

For at undersøge de potentielle sundhedsrelaterede effekter af at spise økologiske fødevarer, blev det besluttet at følge hele fødekæden fra jord til bord, med brug af en rotte model til måling af de potentielle sundhedsrelaterede effekter. Afgrøderne blev høstet fra længerevarende mark-forsøg. To fodringsforsøg med rotter blev udført for at undersøge indflydelsen af dyrkningssystem, år, og lokalitet på den næringsmæssige sammensætning af afgrøderne og på sundhedsrelaterede biomarkører efter indtagelse af diæterne.

Resultaterne fra de to studier findes i artikel I og II. I artikel I fandt vi, at nitrat og protein i gulerødderne blev påvirket af dyrkning system, mens de målte sundhedsmæssige biomarkører i rotterne var upåvirkede. I studie II blev der brugt komplette diæter og resultaterne viste, at dyrkningssystem, høstår og lokalitet havde en lille effekt på den ernæringsmæssige kvalitet. Til gengæld blev der ikke fundet nogen forskel i de målte sundheds biomarkører på grund af dyrkningssystem, udover et højere plasma IgG i rotter der havde fået den konventionelle diæt. I artikel III blev det undersøgt, om der var en effekt af moderens indtag af økologisk eller konventionelt producerede afgrøder på modulationen af immunologisk tolerance hos afkommet. Det første skridt var, at udvikle en oral tolerance model (pilotforsøg), som blev anvendt på anden-generations rotter af mødre fra studiet beskrevet i artikel II. Bortset fra PGE2, havde dyrkningssystem lille virkning på de immunologiske parametre undersøgt i begge generationer og der var ingen forskel på afkom af rotter fodret med økologisk eller konventionelt dyrkede afgrøder i deres respons mod et nyt diæt introduceret antigen.

Det kan konkluderes, at dyrkningssystem, høstår og lokalitet kun havde en lille indflydelse på næringsindholdet af afgrøderne og at dyrkningssystem kun i ringe grad havde en virkning på de sundhedsrelaterede eller immunologiske biomarkører målt i rottemodellen.

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Abbreviations

AU Aarhus University

APC Antigen presenting cells

C Conventional

CD Cluster of differentiation

CLA Conjugated linoleic acid

CO Control

DC Dendritic cells

DHEA Dehydroepiandrosterone

DON Deoxynivalenol

ELISA Enzyme-linked immunosorbent assay

Fla Flakkebjerg

Fou Foulum

GALT Gut-associated lymphoid tissue

GI Gastro-intestinal

GM Genetically modified

ICROFS International Centre for Research in Organic Food Systems IFOAM International Federation of Organic Agriculture Movements

Jyn Jyndevad

KOALA study Dutch acronym for Child, Parent and health: Lifestyle and Genetic constitution

LPS Lipopolysaccaride

MLN Mesenteric lymph nodes

NK cells Natural killer cells

NSP Non-starch polysaccharides

OA Organic A

OB Organic B

OVA Ovalbumin

PARCIFAL study Prevention of Allergy—Risk Factors for Sensitization Related to Farming and

Anthroposophic Lifestyle

PP Peyers Patches

ROS Reactive oxygen species

SIgA and SIgM Secretory IgA and secretory IgM

TDP True digestibility of protein

TFA Trans fatty acids

TVA Tran- vaccenic acid

WHO World Health Organization

ZEA Zearalenone

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Table of content

List of original manuscripts ... 1

Preface ... 2

Acknowledgements ... 3

Summary ... 4

Dansk resumé ... 5

Abbreviations ... 6

Table of content ... 7

PART I: Introduction ... 9

Chapter 1: Cultivation system practices and nutritional quality of plant foods ... 10

Comparison of organic and conventional farming ... 10

Effect of cultivation system on nutritional quality and safety of foodstuffs ... 11

Other factors affecting the nutritional quality ... 16

Chapter 2: Cultivation systems impact on health of consumers ... 16

In search for biomarkers of health ... 17

Effect of cultivation systems on biomarkers of health ... 18

Epidemiological and human intervention studies ... 19

Chapter 3: Nutritional influence on the immune system ... 22

The immune system in health and disease ... 22

Cultivation system influence and nutrient modulation of the immune system ... 23

Effect of cultivation system on nutrient modulation of the immune system in infants ... 26

Chapter 4: Oral tolerance ... 27

The mucosal immune system ... 28

Oral tolerance ... 29

The mucosal immune system and oral tolerance in neonates ... 30

Concluding remarks ... 31

PART II: Hypothesis and objectives ... 32

PART III: Study design considerations ... 33

Diet preparation and animal study design approaches ... 33

The rat as a model for humans ... 37

Development of an oral tolerance model in rats ... 38

Oral tolerance study design ... 40

PART III: Manuscripts... 42

Paper I... 42

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Paper II ... 43

Paper III ... 44

Peer reviewed-abstract ... 45

PART IV: General discussion ... 46

PART V: Conclusion and perspectives ... 54

References ... 56

Appendix ... 65

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PART I: Introduction

In recent years, a rapidly expanding organic market has been observed throughout Europe and USA. ^1,2 There appears to be several reasons for why consumers buy organic products and some of the main ones are the belief that organic products have a better nutritional quality than conventional products and hence make organic products healthier. ^3,4 In general, limited research has been

performed to study the potential health effects of organic foodstuffs, and reliable physiological markers or health biomarkers are needed to measure any possible influence on the health status. ^5-7 Problems for investigating if organically produced foodstuffs are healthier than conventionally produced foodstuffs are having solid experimental designs, i.e. well described food products, randomized controlled trials in humans or animals, with sufficient sample size, long and realistic dietary exposures and finally accurate and objective measurements of dietary intake and relevant health outcomes. ⁵

The main objective of the current thesis is to follow selected bioactive compounds all the way from the plant and soil system, to absorption and to possible health effects measured in a rat model.

The present thesis is based on a literature review of peer-reviewed, English language studies from the last two decades investigating the effect of cultivation system on the nutritional quality and biomarkers of health. Other factors that can have an influence, such as year and location are also discussed to some extent. Figure 1 outlines the thesis delimitation on impact of cultivation system on nutritional quality and health effects. The thesis is limited to discuss the product quality of crops and vegetables used in the current thesis studies, and do not cover other products such as livestock products, except for a section on milk quality in relation to the immune system and health related biomarkers. In evaluating the product quality, the focus in the current studies and thesis is on the nutrient content and to some degree toxin contamination of the products. The thesis is focusing on the rat as a model for humans in nutritional research and farming practice, however, studies on other animal models and human studies are also discussed. Finally, several possible biomarkers of health can be evaluated and it varies greatly in prior studies what has been measured. In the current thesis, the biomarkers listed in Figure 1 are discussed; however a specific focus is given to immune system parameters and the early imprinting of the immune system since they convey a large interest area.

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Figure 1: Overview of the primary steps in studying farming practices (organic versus conventional) effect on product quality and health. The boxes outline the areas investigated in the current studies and discussed in the

thesis.

Chapter 1: Cultivation system practices and nutritional quality of plant foods Comparison of organic and conventional farming

The potential benefits from organic farming has often been discussed and studied however, investigating this can be very complex. First of all, organic and conventional farming practices are not well defined and can be performed in very different ways, and clear definitions are therefore needed. ⁸ The basic principles of organic farming have been described by the International

Federation of Organic Agriculture Movements (IFOAM)(2003-2005) by the four ethical principles, to make it more visible what the goal for organic farming is;

1) The principle of health: Organic agriculture should sustain and enhance the health of soil, plant, animal, human and planet as one and indivisible.

2) The principle of ecology: Organic agriculture should be based on living ecological systems and cycles, work with them, emulate them and help sustain them.

3) The principle of fairness: Organic agriculture should build on relationships that ensure fairness with regard to the common environment and life opportunities.

4) The principle of care: Organic agriculture should be managed in a precautionary and responsible manner to protect the health and well-being of current and future generations and the environment.

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In general, it is said that organic farming uses “natural” methods and differs from conventional farming in a number of ways although the difference in the actual practices of both cultivation systems can vary substantially between different countries/ regions. In conventional farming, inorganic fertilizers (NPK) are used to increase the yield, while organic farming makes use of growing intercrops or cover crops, uses organic wastes and compost, crop rotation and bio- fertilizers to keep and build good soil structure and fertility and supply nutrients to the plants. To get rid of pests and weeds, organic farming makes use of biological pest control, crop rotation, use of resistant seed, natural pesticides, hand picking of weeds or tilling and bio-pesticides. The conventional farming method uses pesticides, insecticides, herbicides and fungicides. ^9,10 Before a pesticide can be used commercially its safety must be tested and the law requires that only

pesticides approved by the individual government regulators can used. Different countries have different rules on this area, and in Denmark, EU legislation (Council regulation (EC) No 834/2007) together with specific Danish amendments are the rules in force and organic products from other countries sold in Denmark have to live up to these standards as well. Pesticide usage has been subject to intense legislative and regulatory scrutiny to reduce pesticide residues in foods and the debate continues on how low the concentrations have to be, to not impose any health risks. ⁹ Hence, during the last three decades the quantities of pesticides used in conventional agriculture have declined as the industry is developing more efficient methods of delivery and more pest specific pesticides. ¹¹ In addition, conventional farming practices also makes use of genetically modified (GM) crops, where organic farming exercises a zero-tolerance approach towards GM crops. It is still debated in scientific communities what the potential effects of changing the make-up of genes are, the safety and nutritional implications and if any, the long-term implications of GM food. ¹² Effect of cultivation system on nutritional quality and safety of foodstuffs

A vast amount of studies have been made on establishing the nutrient content of crops and foodstuffs grown under organic or conventional farming practices, and several reviews have been completed to try to get an overview on the topic. ^11,13-16 Some of the consistent findings of these non-systematic and systematic reviews are that conventionally produced crops contain more nitrogen/nitrate, while organically produced crops contain more phenolic compounds, magnesium, phosphorus, zinc, titratable acidity, flavonoids, sugars, vitamin C and have a higher dry matter content. ^13,16-18 Most studies on nutritional quality have focused primarily on the nutrient content of single crops or vegetables and not complete diets, making comparisons between individual studies difficult. Table 1 comprises a literature based review on studies from the last two decades,

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comparing nutrient content of the organically or conventionally grown crops and vegetables utilized in the current thesis studies: carrots, potatoes, winter wheat, spring barley and rapeseed oil. The focus of the table is only on nutrient content and nutritional compounds that potentially could have an effect on health, no studies on preference, sensory quality or undesirable compounds or

contaminants have been included. Some of the studies did not observe an overruling effect of cultivations system, but in general there appeared to be a higher level of nitrogen/nitrate and protein level in the conventionally grown produce than the organic produce, while the organic produce was high in phosphorous and vitamin C. Apart from the general chemical composition (i.e. protein, lipids, starch, sugars, and dietary fibers), micronutrients, trace elements, bioactive secondary metabolites and vitamins are also known to have potential health effect. They have become

increasingly more investigated in relation to cultivation system and the literature overview in Table 1 did show some differences in some secondary metabolites due to cultivation system. A review by Hunter et al. ¹⁹ evaluated the micronutrient composition of plant foods grown either organically or conventionally and found that the micronutrients was reported more frequently to be higher in organically produced vegetables and legumes. A study by Laursen et al. ²⁰ making multielement fingerprinting of potatoes, fava bean, wheat and barley (originating from the same field experiment as crops for the diets in Paper II and III in the current thesis) reported no systematic difference between organic and conventional crops or factors such as year and location in the content of essential plant nutrients.

Table 1: Literature overview of studies on cultivation system practices (organic versus conventional) effect on the nutritional quality of carrots, potatoes, wheat, barley and rapeseed (oil).

Reference Type of study design

Study design Main effects of cultivation system on nutrient compounds.

Carrots Paoletti et al.²¹ Field study

One conventional and three organic cultivation systems.

Grown in one location (three plots) in two consecutive years. (VegQure ²²)

No clear differentiation in nutritional or sensory outputs between carrots from different cultivation systems.

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Søltoft et al.

(2010) ²³ and ²⁴

Field study

One conventional and three organic cultivation systems.

Grown in one location (three plots) in two consecutive years. VegQure ²²

Significantly higher content of nitrogen in the conventional carrots. No systematic difference in phenolic acids or

polyacetylene content due to cultivation system, across different growth years and geographical locations.

Lima et al.

(2008) ²⁵

Farm study

Conventional or organic farms.

Higher polyamines and total flavonoids in the organic produce than the

conventional. No difference in polyphenols.

Warman and Havard (1997) ²⁶

Field study

Conventional and organic cultivation system. Grown in three years using five replicates.

Macro- and micronutrient and vitamin content was not systematically affected over the three years.

Potatoes Søltoft et al.

(2010) ²⁷

Field study

One conventional and two organic cultivation systems.

Grown in three locations (two plots) in two consecutive years. CropSys ²⁸

A higher content of 5-CQA phenolic acid was measured in organically grown potatoes compared to the conventional, overruling geographical location and growth year.

Lima et al.

(2008) ²⁵

Farm study

Conventional or organic farms.

Higher polyamines and lower total flavonoids in the organic produce than the conventional. No difference in

polyphenols.

Hajšlová et al.(2005) ²⁹

Field study

Conventional and organic cultivation systems. Two different locations. Grown in four years.

Higher vitamin C and lower nitrate in the organic potatoes than in the conventional potatoes.

Warman and Havard (1998) ³⁰

Field study

Conventional and organic cultivation systems. Grown in three years using five

replicates.

P was lower in conventional potato tubes than organic and N was higher in conventional than organic potato leaf tissue. All other micro- and macro- nutrients did not give a systematic difference over the three years.

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Winter wheat

Ceseviciene et al. (2012) ³¹

Field study

Conventional and organic production systems. Grown in two years, using four

varieties.

Conventional winter wheat had higher protein and gluten content than organic winter wheat.

Arncken et al.

(2011) ³²

Field study

Two organic and one conventional farming system.

Three years.

(DOK experiment)

Wheat from both organic systems had a lower crude protein content than the conventional.

Zuchowski et al.

(2010) ³³

Field study

Conventional and organic management. Four varieties.

A significantly higher concentration of the phenolic secondary metabolites, ferulic and p-coumaric acid in organic kennels.

Mäder et al.

(2007) ³⁴

Field study

Two organic and two

conventional farming systems.

21 years. (DOK experiment)

The nutritional value (protein content, amino acid composition and mineral and trace element contents) was not affected by farming system.

Zörb et al.

(2006) ³⁵

Field study

Two organic and two

(DOK experiment)

52 metabolites was detected, of those only alanine, β-alanine, valine, myo- Inositol, glycerate, hydroglutarate, urea and vitamin B₅ was affected by cultivation system.

Langenkämper et al. (2006) ³⁶

Field study

Two organic and two

(DOK experiment)

Protein content was higher in the fertilized wheat, while no significant differences were found in the majority of the other measured conpounds.

Ryan et al.

(2004) ³⁷

Farm study

Conventionally and organically grown wheat, during three years.

Conventional wheat had lower Zn and Cu but higher P than organic grain.

Spring barley

Tsochatzis et al.

(2010) ³⁸

Field study

Twelve barley varieties grown organically or conventionally over two years.

An increase in tocotrienols and a decrease in tocopherols were observed for the organic wheat.

Rapeseed (oil)

Samman et al.

(2008) ³⁹

Basket study

Purchased at supermarkets, organic retail outlets and health food stores.

No consistent overall difference in fatty acid composition between the organic or conventional rapeseed oil.

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Besides the nutrient content of foodstuffs, the nutritive value is also determined by the bioavailability of the given nutrient. ^11,40 Only few studies have been made regarding the bioavailability of nutrients and energy according to cultivation system, even though this is an important step for nutrients to have any potential health effects. The pig is generally considered a good model for humans, but the laboratory rat has also been widely used and accepted as a model to investigate the basic principles of nutrient bioavailability. ⁴¹ In a study by Kristensen et al, ⁴² they concluded that organically grown foodstuffs did not contain more major or trace elements than conventionally grown foodstuffs, and there was no effect on the bioavailability measured in rats.

Another important point was made in the study; “if the content of the nutrients of interest was higher than the nutritional needs and lower than levels considered toxic, moderate differences in the content would not lead to any health consequences”. ⁴²

In addition, from the detection of possible desirable compounds, many studies have focused on the more undesired compounds in food, such as pesticide and insecticide residues. Significant

differences have been reported and as expected conventionally grown crops have been found to contain higher levels of pesticides than organically grown crops. ^9,10 Other possible undesired compounds that have been in focus are mycotoxins from moulds, toxins from plants or gram negative bacteria. The limited literature on mycotoxin contamination of foodstuffs does not give a clear picture of the influence of cultivation system. ^43,44 It is argued that without the use of

pesticides, organic crops develop a natural defense mechanism by producing protective substances, such as phenols that are antioxidants and actually have been known to protect the body from cancers and heart disease. ¹⁴ On the other hand, others argue that organic crops are more vulnerable to moulds than conventional crops if they are damaged by insects or the weather. A study by Schneweis et al. ⁴⁵ have show that conventionally produced wheat are more frequently

contaminated with Fusarium and have higher concentrations of the Fusarium toxins zearalenone (ZEA) and deoxynivalenol (DON) than organically produced wheat. However, they have also found that the performance and health status of pigs eating the wheat was not affected and they concluded that climatic conditions, soil quality or proceeding crops should be comparable to make these conclusions, since they are known to influence mycotoxin production. ⁴⁶ Another controversial topic in food safety when discussing organic versus conventional farming is pathogenic microbes.

Some argue that organic crops might carry a relative higher risk of microbial contamination compared with conventional crops, due to increased presence of pathogens via manure and fewer decontamination alternatives, such as irradiation and pasteurization in the food processing steps.

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However, the bulk of available studies have not shown any significant differences in the bacterial count or endotoxin status. ⁴⁴

Other factors affecting the nutritional quality

As stated in a recent systematic review by Dangour et al, ¹³ both the individual studies on organic versus conventional agricultural methods and the reviews on the literature have some problems or factors that one should be aware of when interpreting the results. First of all, the individual studies are most often carried out on different single food items which probably contribute little on a daily basis/whole menu and very few studies have investigated complete diets, which might actually be more comparable to the real world than single food items. Often specific compounds are also singled out as the compounds of interest, but one could argue that a more holistic perspective is needed on the subject, meaning that it is important to think of the foodstuff as a whole food and a dynamic, hierarchically organized unit and not only consider it as chemical compounds. ⁷ Secondly, when setting up a comparative study between organic and conventional cultivation systems on the nutritional quality and eventual health effects hereof, one also has to consider what kind of design one would like to use. The so-called “basket”-design is based on buying the source products from retailers. This reflect realistic conditions, however, it is not possible to know much about the

growth, harvest or processing conditions of the products. The “farm “-design can also be considered fairly realistic and a level of more knowledge on the product is added. It is possible to choose farms close to each other to try to even out soil and climatic variations and information can be collected from the individual farmers. However, the practices between individual farms can still be very different and influence the outcome. The final experimental design that can be used is the “field”- design, where the produce of interest is grown in controlled field trials and several factors can be taken into account; ⁴⁷ The use of different varieties between different studies or between organic and conventional cropping systems within the same study can give rise to different results, so this has to be considered. ^48,49 Harvest time, year, location, and soil type, are also known factors to influence the nutritional content of crops and vegetables and these should also be included in the

“field”-design experimental setup. Most studies also suffer from the fact that only one replication per food produce was used, making it impossible to take field variation into account. 20,40,47-50

Chapter 2: Cultivation systems impact on health of consumers

The organic market is increasing in Denmark and throughout the Western world, and an interest has been put on the character of the demand for organic food ^1,2. As reviewed by Yiridoe et al. ⁴,

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several consumer perceptions of attributes of organically produced food products lead to the

increased purchase of organic food products; health benefits, animal welfare, food safety, impact on the environment and the production process. For consumers, the health argument is a very important reason for buying organic products, and thus the scientific question whether consumption of organic food indeed results in better health is highly relevant.

In search for biomarkers of health

A primary goal of nutrition research is to optimize health and prevent or alleviate diseases.

However, identifying biomarkers which can be used to determine health has proven difficult.

Biomarkers are considered indicators of biological processes and the most cited biomarker definition is: “A biomarker is a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmaceutical responses to therapeutic intervention”. ⁵¹ The current WHO definition of health describes health as “a state of complete physical, mental and social well-being and not merely an absence of disease and infirmity”, which is a tricky entity to measure, and more operational definition are needed for measurement purposes, research, and evaluating intervention. ^52,53 It is not enough to measure health as a reduction or absence in disease, and health should be considered as a more dynamic state of well-being, with a potential to meet different challenges and to be able to handle health threats and restore homeostasis. Hence, the ability of the individual to adapt to specific stressors could be an important operational aspect of measuring health and a stressor or challenge could be applied to connect with the principles of organic agriculture of striving to maximize resilience, robustness and adaptability in order to increase health. ⁵² Table 2, which is adapted from an article by Huber et al.

52 on the challenge of evaluating effects of organic food, shows a list of suggestions of physiological parameters that could be used for investigating health effects.

Table 2: Physiological responses that can be used for measuring stress (adapted from Huber et al. ⁵²).

Parameter Physiological markers for measuring stress

Muscoskeletal Muscle tension, breathing patterns, activity, heat production

Neuroendocrine Corticosteroids, catecholamines, testosterone, prolactin, growth hormone, insulin, DHEA

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Cardiovascular Heart rate, heart rate reserve, heart rate variability, cardiac arrhythmias, cardiac output, stroke volume, myocardial contractility, pulse transit time, blood pressure, total peripheral resistance, regional blood flow, coronary blood flow, transient myocardial ischemia, renal blood flow, glomerular filtration rate, sodium excretion, platelet aggregation and adhesion

Electrodermal Sympathetic nerve activity, skin conductance, skin potential, sweat gland control Gastrointestinal tract Salivation, gastrointestinal transit time, fat metabolism, total cholesterol,

cholesterol fractions, triglycerides, free fatty acids, blood glucose levels, amino acids, bioavailability

Immune related Specific and unspecific immunoglobulins, lymphocyte subsets, natural killer cell activity, mitogen-induced lymphocyte proliferation, interleukins, cytokines, lipid mediators

Blood serum Creatine, albumin, uric acid, heat shock proteins

Effect of cultivation systems on biomarkers of health

As for the studies on nutritional quality of conventionally and organically grown foodstuffs, studies on health and agricultural practice have a large variability in the design, the exposure and the measured outcomes, which makes it difficult to make clear cut conclusions. Several study types, such as human studies, in vitro and ex vivo studies in cell lines and serum and animal studies have been implemented in investigating the health effect of organic versus conventional farming and several health outcomes have been of interest. Especially in early studies, feeding experiments were performed with a clear focus on effect of fertilizer application on fertility, reproduction and egg production. ⁷ As reviewed in Velimirov et al., ⁷ some have reported a better survival rate of

offspring, better semen and higher egg production of animals (primarily rats, rabbits and chickens) fed low fertilizer diets than those fed mineral fertilizer diet, while some reported no difference. As discussed earlier, the usage of fertilizer and pesticides has been lowered and become more targeted over the years and toxicology testing is performed on any chemical used, so these lines of

experiments are not so frequent nowadays. In recent experiments on health benefits from organic products, the focus has shifted more towards investigating growth, antioxidant capacity and immunological parameters. In vitro experiments have come more into use in investigating among other things the antioxidant effects of organic food. In a study by Tarozzi et al. ⁵⁴ on the antioxidant properties of apples in Caco-2 cells, it was concluded that cultivation system had no effect. On the other hand, another study by Tarozzi et al. ⁵⁵ investigating the antioxidant effectiveness of

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organically or non-organically grown red oranges in cell culture systems, it was concluded that there was higher phytochemical content and intracellular antioxidant activity in organic than integrated red oranges. Olsson et al. ⁵⁶ evaluated the effects of organically or conventionally cultivated strawberries on inhibition of cancer cell proliferation. They showed higher anti- proliferative activity of extracts form organically produced strawberries, possibly due to higher concentration of secondary metabolites with anti-carcinogenic properties. Although in vitro experiments has some advantages, such as the possibility of controlling confounding variables animal feeding experiments are warranted for identifying health-related biomarkers suitable for further human intervention studies. In a long-term feeding trial with rats performed by Lauridsen et al. ⁵⁷, several possible health outcomes were evaluated i.e., biochemical and physiological

measurements (bioavailability of nutrients, metabolism and nutritional status in blood and tissue), growth, physical activity, post-mortem evaluation, physiological functions of organs and intestine and immunological analysis. Some notable differences appeared in liver metabolic function and liver lipid peroxidation and rats fed the “low fertilizer” diets had a lower daytime activity, a higher plasma IgG and 14% less adipose tissue that those fed the “high fertilizer” diet. Several other feeding studies have looked immune related parameters (Table 2) as possible interesting health biomarkers ^57-60 and these are discussed further in Chapter 3.

Epidemiological and human intervention studies

As described above, some studies have looked at the health effect of cultivation system, and many of these studies are in vitro studies or animal studies and they are not clearly transferrable to

humans. Both human and animal studies have some advantages and disadvantages in design and execution. In animal studies it is possible to use large population groups, which is also the case with epidemiological studies. However, epidemiological studies, such as the PARSIFAL project ⁶¹ and the KOALA study ⁶² are based on collected data/observational data and rely on people’s honesty and memory and it is not possible to control other health confounding factors. Intervention studies have the advantage to introduce the food object(s) of interest and other health factors than the diet can be controlled. The limitations are that it is possible only to have short intervention periods and a small population. Table 3 lists the epidemiological and human intervention studies performed on cultivation systems effect on health in the last ten years, excluding contaminant and occupational health studies. The human intervention studies have in general focused on antioxidant activity and no difference was observed in the health outcomes from the human intervention studies. ^27,63-66 This could be due to short intervention periods or a diluting effect of the subject’s habitual diets. The

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Grinder-Pedersen et al. ⁶⁷ intervention study did however result in a higher urinary excretion of quercetin and kaempherol and an increase in protein oxidation and a decreased total plasma antioxidant capacity.

The epidemiological studies have been interesting with regard to influences of cultivation system on the immune system and they are discussed in Chapter 3.

Table 3: Epidemiological and human intervention studies with organic and conventionally produced foodstuffs.

Reference Study type Treatment Time of treatment

Main effects of cultivation system

Health implications Stracke et

al. (2010)

65

Two human intervention studies

Six and 43 male subjects.

Organically or conventionally produced apples.

Immediate (2 x 1 day with one week washout period) and long-term (1 week

depletion and 4 weeks intervention).

No difference in the bioavailability of apple polyphenols between the cultivation system practices.

None, since no cultivation system effect was observed.

Søltoft et al. (2010)

27

Human cross-over intervention study

18 subjects.

Complete diets. Three cultivation systems in two consecutive years.

3 x 12 days with a washout period of minimum 2 weeks.

Growth system had no effect on the carotenoid content of the carrots or the plasma carotenoid concentrations in the humans.

Mueller et al. (2010)

68

KOALA birth cohort study

Lactating women (310) with a conventional or alternative lifestyle*.

- Lower

9(trans)18:1/11(trans)1 8:1-ratio in breast milk from women consuming organic meat and dairy products.

TFA are associated with effects on fatty acid metabolism, oxidative stress, cholesterol levels, and infant

development.

Kummelin g et al.

(2008) ⁶⁹

Children at 2 years of age with a conventional or alternative lifestyle*.

- Consumption of organic dairy products was associated with lower eczema risk. No effect of organic meat, fruit, vegetables or eggs.

A possible effect of organic dairy products on the development of atopic diseases in infancy.

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treatment

Health implications Stracke et

al. (2008)

64

Human intervention study

36 subjects.

Diet

supplementation with blanched carrots from cultivation systems.

4 weeks low carotenoid period and 2 weeks intervention.

Cultivation system did not affect carotenoid content of the carrots, nor the bioavailability of the carotenoids and on antioxidant,

antigenotoxic and immunological effects.

Rist et al.(2007) ⁷⁰

Lactating women (312) with a conventional or alternative lifestyle*.

- Higher level of CLA

and TVA in the breast milk of women eating diets containing organic dairy and meat products, than in those eating conventional.

Positive effects of CLA on immune- modulation.

Could be relevant to asthma and allergy incidence in children.

Briviba et al. (2007)

63

6 subjects.

Organically or conventionally grown apples.

2 x 1 day with one week washout period.

No effect on antioxidant capacity.

Alfvén et al. (2006)

61

The

PARSIFAL study

Children (14000, 5 countries) at 5- 13 years from farm or anthroposophic families or families eating conventional food products.

- Growing up on a farm

or leading an

anthroposophic lifestyle was associated with a lower risk of atopic diseases.

Relevant for asthma and allergy incidences in children.

Caris- Veyrat et al. (2004)

66

Human intervention study

20 subjects.

96g/day of organic or convention tomato puree.

3 weeks with a following tomato product depletion period of 3 weeks.

Organic tomato puree had a higher content of vitamin C and phenols.

No difference was observed in plasma vitamin C or carotenoids.

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22 Reference Study type Treatment Time of

treatment

Health implications Akçay et

al. (2004)

71

8 subjects.

Organically or conventionally produced wine.

2 x 1 day with 6-week wash-out period.

Cultivation systems lead to inconsistent changes in antioxidant capacity.

Could be relevant due to the

antioxidant activity and possible anti- inflammatory effects.

Grinder- Pedersen et al.

(2003) ⁶⁷

16 subjects.

Organically or conventionally produced diets.

2 x 22 days with 3 weeks wash-out.

Cultivation system affected the quercetin content in the diets and resulted in differences in markers of oxidation and urinary excretion of major dietary

flavonoids.

Antioxidant properties of polyphenolic compounds.

Potential

beneficial health actions.

*Alternative lifestyle: Rudolf Steiner, use of organic food, anthroposophic lifestyle etc CLA: Conjugated linoleic acid. TFA: Trans fatty acids. TVA: Trans-vaccenic acid

Chapter 3: Nutritional influence on the immune system The immune system in health and disease

The immune system protects animals and humans against microbial invasion and is therefore essential for life. The four key roles for the immune system are; 1) to act as an exclusion barrier, 2) to discriminate between self and non self, 3) to ensure tolerance and to eliminate non-self and 4) to retain memory of immunological encounters. To be effective and reliable, multiple defense systems must be available. The first line of defense is the physical and physiological barriers, such as the skin and the mucosal membranes with the sticky mucus layer. Other factors like the pH, enzymes and commensal bacteria also help to keep out pathogens. The second line of defense in the battle against invading pathogens is the innate immune system, which reacts rapidly and consists of a non- specific cell defense and a non-cellular defense. The non-specific cell defense is unspecific and comprises neutrophilic granulocytes, macrophages and natural killer (NK) cells that are capable of recognizing and killing invading pathogens and presenting them to other cells. The non-cellular factors include interferons, acute-phase proteins and the complement system, which is a protein cascade that leads to killing of bacteria and also functions as chemotaxins and opsonins. The third line of defense is the acquired immune system, which is slower in onset, but is specific and has significant memory. It consists of a cell-mediated response and a humoral response and the cells in

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charge of this are T-lymphocytes and B-lymphocytes, respectively. CD8+ T cells generally functions as T cytotoxic cells. CD4+ T cells generally functions as T helper (Th) cells. Th cells either initiate a Th1 response that support inflammation and activates macrophages, certain T cells and macrophages and induce B-cell antibody class switching, or initiate a Th2 response that activates mainly B cells (plasma cells) that produce especially IgE and IgG. Antibodies (IgG, IgA, IgM, IgD, andIgE) are selectively distributed in the body and serve different functions; IgG and IgM predominate in plasma, while IgA predominates in secretions across epithelia and IgE is found mainly associated with mast cells just beneath epithelial surfaces (the respiratory tract,

gastrointestinal tract and skin). ^72,73

Cultivation system influence and nutrient modulation of the immune system

The role of nutrition in disease management has traditionally focused on the health benefits of supplementation for groups suffering of apparent nutrient deficiencies. ⁷⁴ However, recently an interest in nutritional therapy has evolved due to further knowledge about certain nutrients’

pharmacological properties that can influence disease processes and have a positive impact on both morbidity and mortality of certain diseases. Optimization of foodstuffs would be a very cost

effective method for disease prevention, since diet-induced health improvements would not carry any costs for the health sector. ⁷⁵ Therefore there has been an interest in finding means to modify immune cell responses; either to improve the host defense mechanisms thereby reducing the incidence and severity of infectious diseases or to reduce adverse immunological reactions to reduce autoimmune and allergic diseases. As listed in Table 2, there are several immune related physiological markers that could be used for evaluating the effect of cultivation system and some of these have been investigated in animal studies; A study by Finamore et al. ⁶⁰ observed a difference in lymphocyte function in rats due to cultivation system and a pilot experiment by Baranska et al. ⁷⁶ showed an increased lymphocyte proliferation in rats eating low input organic diets. In a recent study by Roselli et al. ⁵⁹, changes in lymphocyte populations were observed in mice eating Danish or Italian carrots grown organically or conventionally in two consecutive years suggesting a positive effect of organic products. Moreover, a study by Huber et al. ⁵⁸ evaluating health related outcomes in a chicken model where a stressor, an immunological challenge was introduced, it was concluded that both the specific and unspecific immune system was affected by cultivation system.

Chickens originating from the same feeding study were also reported to display differences in regulation of jejunal gene expression of among others genes involved in immunological processes.