Biogenic amines in food products on the Belgian market

139 Pages·2016·4.75 MB·English

by  Lisa Miclotte

Faculty of Bioscience engineering
Academic year 2015 – 2016

Biogenic amines in food products on the Belgian market

Lisa Miclotte
Promotoren: Prof. dr. ir. Liesbeth Jacxsens & Frank Devlieghere
Tutor: Markus Eriksson

Master thesis submitted in partial fulfillment of the requirements for the
degree of Master of Science in bio-engineering - Food Science and
Nutrition
ACKNOWLEDGEMENTS
At the start of this master thesis, I was still a full blown student, in the sense that I knew how to
decipher courses and how to make sure I controlled the material completely by the time the exams
began. During the past year, however, I've had to learn to do so much more than to study courses.
This master thesis taught me so many aspects of what it means to be a researcher and it gave me a
glimpse of what an adult’s working life looks like. I have grown in so many different ways during the
course of this master thesis and have come into contact with so many expertized people, to whom I
owe my thanks.
First of all, I wish to thank my promotor professor Liesbeth Jacxsens, who has been a competent
mentor with great expertise in terms of risk assessment and food safety. She proposed the structure
of this thesis, gave constructive pieces of advice concerning lab experiments, presentations and
reporting and was always present to answer questions and give feedback on my writings. Equally, I
wish to thank professor Frank Devlieghere and Bruno Demeulenaere for their support of this project
and their fruitful cooperation.
Special and explicit thanks also to my tutor Markus Eriksson, who has been a more than competent
mentor and partner in the lab and whom I could always turn to for advice. I would always receive
more detailed explanations than I could ever have hoped or wished for. On the other hand, working
together in the lab frequently turned into tease and laughter, making it an enjoyable experience for
me and even my fellow thesis students. I consider you as a friend, Markus, and I wish you the best
with your new job, your wife and your soon-to-be-born first child.
I would also like to thank Emmanuel Abatih for lending me his time and his expertise in the field of
statistical analysis using R. Our frequent meetings at the close of this thesis resulted in valuable
outcomes, without which my dissertation would not have been complete. Moreover, his calm and
friendly character and his multicultural approach of work and life have resulted into very pleasant
conversations. Thank you, Emmanuel.
Furthermore, I also thank all members of staff in the chemical and microbial lab for their patience
with the clumsiness that is typical of all thesis students, for their help in finding lab materials, for
their advice and for the many interesting conversations, which were always proof of their support.
And lastly, I thank my friends and family for their continuous support throughout this year. They
listened to my nagging and my joys, they advised me where they could and encouraged me in all my
endeavours, within and outside of this thesis. Moreover, I thank my parents for making this 5-year
journey of study possible by supporting me both financially and morally. I can never thank them
enough.
Table of contents
LIST OF ABREVIATIONS ............................................................................................................................. i
GLOSSARY .................................................................................................................................................ii
SUMMARY ................................................................................................................................................ v
SAMENVATTING ..................................................................................................................................... vii
1. INTRODUCTION ............................................................................................................................... 1
2. LITERATURE STUDY .......................................................................................................................... 4
2.1 BIOGENIC AMINES ................................................................................................................... 4
2.1.1 Occurrence in food .......................................................................................................... 4
2.1.2 Production by microorganisms........................................................................................ 5
2.1.3 Detection methods .......................................................................................................... 6
2.2 TOXICOLOGY ............................................................................................................................ 6
2.2.1 General ............................................................................................................................ 6
2.2.2 Detoxification .................................................................................................................. 6
2.2.3 Histamine ......................................................................................................................... 7
2.2.4 Tyramine and trace amines ............................................................................................. 8
2.2.5 Putrescine, cadaverine and polyamines .......................................................................... 9
2.3 QUALITY INDICATOR .............................................................................................................. 11
2.4 FORMATION OF EXOGENIC BIOGENIC AMINES .................................................................... 12
2.4.1 Substrate availability ..................................................................................................... 12
2.4.2 Presence of microorganisms ......................................................................................... 13
2.5 Conditions affecting MO-growth, decarboxylase production and decarboxylase activity ... 13
2.5.1 Temperature .................................................................................................................. 13
2.5.2 pH .................................................................................................................................. 14
2.5.3 Other factors ................................................................................................................. 14
2.6 CONTROL OF BA-CONTENTS .................................................................................................. 15
2.6.1 Raw material handling ................................................................................................... 16
2.6.2 Fermentation process.................................................................................................... 16
2.7 EXPOSURE AND RISK ASSESSMENT ....................................................................................... 17
3. MATERIALS AND METHODS .......................................................................................................... 20
3.1 EXPERIMENTAL OUTLINE ....................................................................................................... 20
3.1.1 Storage tests .................................................................................................................. 20
3.1.2 Screening tests .............................................................................................................. 21
3.2 EXPERIMENTAL ANALYSES ..................................................................................................... 23
3.2.1 Free amino acids ............................................................................................................ 23
3.2.2 Biogenic amines ............................................................................................................. 25
3.2.3 Microbiology .................................................................................................................. 28
3.3 STATISTICAL ANALYSES .......................................................................................................... 30
3.3.1 Objectives ...................................................................................................................... 30
3.3.2 Preparation of the database.......................................................................................... 30
3.3.3 Part 1: T-tests fermented vs. non-fermented and animal vs. plants ............................ 31
3.3.4 Part 2: ANOVA-tests for difference between food groups ............................................ 32
3.3.5 Part 3: Correlation analysis ............................................................................................ 32
4. RESULTS AND DISCUSSION ............................................................................................................ 34
4.1 INTRODUCTION ..................................................................................................................... 34
4.2 POTENTIAL FOR BA-FORMATION IN TUNA AND MARINATED PORK .................................... 35
4.2.1 Storage test tuna ........................................................................................................... 36
4.2.2 Storage test marinated pork ......................................................................................... 39
4.3 STORAGE TEST OF DIFFERENT TYPES OF MARINATED MEAT................................................ 43
4.4 SCREENING OF MEAT PRODUCTS AND PREPARATIONS FROM THE BELGIAN MARKET........ 47
4.4.1 Salami and dried sausages ............................................................................................. 47
4.4.2 Cooked hams ................................................................................................................. 50
4.4.3 Raw hams (dried and cured) ......................................................................................... 52
4.4.4 Meat preparations ......................................................................................................... 54
4.4.5 Concluding remarks on fresh meat, meat preparations and meat products ................ 56
4.5 DESCRIPTION OF BA-DATA ASSEMBLED FOR OTHER FOOD GROUPS ................................... 56
4.5.1 Fruit and vegetables ...................................................................................................... 57
4.5.2 Chocolate ....................................................................................................................... 57
4.5.3 Beer ............................................................................................................................... 57
4.5.4 Meat products and preparations .................................................................................. 58
4.5.5 Dairy .............................................................................................................................. 58
4.6 STATISTICAL ANALYSIS ........................................................................................................... 59
4.6.1 PART 1: T-tests fermented vs. non-fermented and animal vs. plants ........................... 59
4.6.2 PART 2: ANOVA-tests for difference between food groups .......................................... 61
4.6.3 PART 3: Correlation analysis .......................................................................................... 64
5. CONCLUSIONS ............................................................................................................................... 67
6. FUTURE INVESTIGATIONS .............................................................................................................. 69
7. REFERENCES .................................................................................................................................. 70
8. APPENDICES ................................................................................................................................... 74
Appendix 1: Design of the 9-day storage test for marinated pork................................................ 74
Appendix 2: Design of the 9-day storage test for 3 types of marinated meat, stored at 7°C and
MAP-packaged (70/30 O /CO ). .................................................................................................... 74
2 2
Appendix 3: Reagents used during the chemical analyses. ........................................................... 75
Appendix 4: Materials and Machines used for the chemical analyses. ........................................ 76
Appendix 5: Tables required during the calculation of the BA-concentrations. ........................... 77
Appendix 6: Materials used during the microbial analyses. ......................................................... 78
Appendix 7: Pictures of tuna samples during 6-day storage test. ................................................. 80
Appendix 8: BA-concentrations on the last day of analysis of the storage test for marinated
pork:. Mean values of the two samples on the last day of analysis for each condition and each
BA are plotted. .............................................................................................................................. 82
Appendix 9: Pictures of marinated pork during the 9-day storage test. ....................................... 83
Appendix 10: Results of the screening tests on salami’s and dried sausages. .............................. 85
Appendix 11: Results of the screening tests on cooked hams. ..................................................... 87
Appendix 12: Results of the screening tests on raw dried and cured hams and beef products. .. 90
Appendix 13: Results of the screening tests on meat preparations. ............................................ 93
Appendix 14: Preparation methods of cooked and raw dried and cured ham samples as
designated on the package. .......................................................................................................... 97
Appendix 15: Overview of the BA-concentrations in different food groups and their subgroups.
....................................................................................................................................................... 99
Appendix 16: Box-plots of BA-concentrations belonging to Part 1 of the statistical analysis. ... 107
Appendix 17: Tables representing the p-values resulting from the mean comparisons (ANOVA) of
the BA-concentrations in the different food groups. .................................................................. 112
Appendix 18: Correlation triplots resulting from redundancy analyses (RDA) executed to analyze
the correlations between BA, FAA and microbial concentrations. ............................................. 115
Appendix 19: Results of the multiple linear regressions (MLR) executed during the analysis of the
correlations between FAA, BA and microbial counts. ................................................................. 123
LIST OF ABREVIATIONS
AA Amino acids
ACN Acetonitrile
AO Amino oxidase
BA Biogenic amines
BAI Biogenic amine indicator
CCP Critical control point
CE Capillary Elektrophoresis
DAO Diamino oxidase
FAA Free aminoacids
FSMS Food Safety Management System
GC Gas Chromatography
GHP Good Hygiene Practices
Gr +/- Gram positive /negative
GMP Good Manufacturing Practices
HACCP Hazard Analysis Critical Control Points
HPLC High Pressure Liquid Chromatography
LOD Limit Of Detection
LOQ Limit Of Quantification
MAO Monoamine oxidase
MAOI Monoamine oxidase inhibitors
MAP Modified atmosphere packaging
MLR Multiple linear regression
MO Microorganisms
MRS De Mann, Rogosa and Sharpe agar
MSA Mannitol Salt Agar
NOAEL No Observable Adverse Effect Level
OPA Ortho-Phtalic aldehyde
PA Pseudomonas Agar
PCA Plate Count Agar
PRP Prerequisite Program
RDA Redundancy analysis
RE Rapid Enterobacteriaceae agar
SB Slanetz and Bartley agar
SMS Safety management system
TLC Thin Layer Chromatography
VRBG Violet Red Bile Glucose agar

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GLOSSARY
24h-recall Quantitative research method used in nutritional assessment in the form of an interview,
in which individuals are asked to recall all foods and beverages they consumed in the
twenty-four hours prior to the interview. It may be self-administered or administered by
a trained professional.
Aminogenic Generating amino acids
Arrhythmia Cardiac arrhythmia is group of conditions in which the heartbeat is irregular, too fast, or
too slow. It originates from a change from the normal sequence of electrical impulses.
Brachycardia A form of cardiac arrhythmia, in which the heartbeat is too slow (less than 60 beats per
minute).
Brain hemorrhage A brain hemorrhage or cerebral hemorrhage is a type of stroke. It is caused by
an artery in the brain bursting and causing localized bleeding in the surrounding tissues.
This bleeding kills brain cells (= hemorrhagic stroke).
Bronchospasm Abnormal contraction of the smooth muscle of the bronchi, resulting in
an acute narrowing and obstruction of the respiratory airways, which causes difficulty to
breathe.
Catecholamines Organic compound containing a catechol (benzene with two hydroxyl side groups) and
a side-chain amine. Examples are epinephrine (adrenaline), norepinephrine
(noradrenaline), and dopamine, all of which are produced from phenylalanine and
tyrosine
Cell proliferation The process that results in an increase of the number of cells, and is defined by the
balance between cell divisions and cell loss through cell death or differentiation. Cell
proliferation is increased in tumours.
Crohn's disease Crohn's disease is an inflammatory bowel disease (IBD). It causes inflammation of the
lining of your digestive tract, which can lead to abdominal pain, severe diarrhoea,
fatigue, weight loss and malnutrition. Inflammation caused by Crohn's disease can
involve different areas of the digestive tract in different people.
Endogenous Originating or produced within an organism, tissue,or cell: endogenous hormones.
Exogenous Originating or produced from outside a cell, tissue,or organism: exogenous antioxidants.
Glucono-δ-lacton A food additive with the E-number E575, used as a sequestrant, an acidifier, or a
curing, pickling, or leavening agent. GDL is neutral, but hydrolyses in water to gluconic
acid which is acidic, adding a tangy taste to foods, though it has roughly a third of the
sourness of citric acid.
Hypertensive crisis Hypertensive crisis is an umbrella term for hypertensive urgency and hypertensive
emergency. These two conditions occur when blood pressure becomes very high (over
180 systolic or 110 diastolic), possibly causing organ damage.
Hypoxia Diminished availability of oxygen to the body tissues.
Immunomodulation Alteration of the body's immune response
Isoflavones Isoflavones are a type of often naturally occurring isoflavonoids, many of which act as
phytoestrogens in mammals. Some are termed antioxidants because of their ability to
trap singlet oxygen. Isoflavones are produced almost exclusively by the members of the
Fabaceae (i.e., Leguminosae, or bean) family.
Lockjaw Trismus, or Lockjaw, refers to reduced opening of the jaws caused by spasm of the
muscles of mastication, or may generally refer to all causes of limited mouth opening.
Lymphocytes A lymphocyte is one of the subtypes of white blood cell in a vertebrate's immune system.
Lymphocytes include natural killer cells (NK-cells) which function in cell-
mediated, cytotoxic innate immunity, T-cells (for cell-mediated, cytotoxic adaptive
immunity), and B-cells (for humoral, antibody-driven adaptive immunity). They are the
main type of cell found in lymph, which prompted the name lymphocyte.
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Mass spectrometry Mass spectrometry (MS) is an analytical technique that ionizes chemical species and
sorts the ions based on their mass to charge ratio. In simpler terms, a mass
spectrum measures the masses within a sample. Mass spectrometry is used in many
different fields and is applied to pure samples as well as complex mixtures
Mast cell A type of white blood cell. Although best known for their role in allergy and anaphylaxis,
mast cells play an important protective role as well, being intimately involved in wound
healing, angiogenesis, immune tolerance, defence against pathogens, and blood–brain
barrier function
Mesophilic A mesophile is an organism that grows best in moderate temperature, neither too hot
nor too cold, typically between 20 and 45 °C (68 and 113 °F).
Oedema Oedema is the medical term for swelling. It is a general response of the body to injury or
inflammation. Oedema results whenever small blood vessels become "leaky" and release
fluid into nearby tissues. The extra fluid accumulates, causing the tissue to
swell. Oedema can be isolated to a small area or affect the entire body. Medications,
infections, pregnancy, and many medical problems can cause oedema.
Organoleptic Organoleptic properties are the aspects of food, water or other substances that an
individual experiences via the senses—including taste, sight, smell, and touch.
Paresis Weak form of paralysis. It is condition typified by a weak or partial loss of voluntary
movement or by impaired movement. When used without qualifiers, it usually refers to
the limbs, but it can also be used to describe the muscles of the eyes (ophthalmoparesis),
the stomach (gastroparesis), and also the vocal cords (Vocal cord paresis). Neurologists
use the term paresis to describe weakness, and plegia to describe paralysis in which all
voluntary movement is lost.
Pathogenesis The biological mechanism (or mechanisms) that lead to the diseased state.
Peripheral blood The peripheral vascular system consists of the veins and arteries not in the chest or
vessels abdomen (i.e. in the arms, hands, legs and feet). The peripheral arteries supply
oxygenated blood to the body, and the peripheral veins lead deoxygenated blood from
the capillaries in the extremities back to the heart.
Piperidine This heterocyclic amine consists of a six-membered ring containing five methylene
bridges (-CH2-) and one amine bridge (-NH-).
Pyrrolidine Cyclic secondary amine, which is miscible with water and behaves alkalic.
Polycation Polymer whose repeating units contain at least one positive charge. DNA, as a polymer of
nucleotides, is an example.
Potentiators Enhancers of a certain effect. In the context of biogenic amines: compounds which
enhance the toxic effects of BA by enhancing their absorption and/or diminishing AO-
activity.
Pruritus Itching
Psychrotropic Psychrotrophic bacteria are bacteria that are capable of surviving or even thriving in a
cold environment.
Scombroid fish The Scombridae family of the mackerels, tunas, and bonitos includes many of the most
important and familiar food fishes. Scombrids are generally predators of the open ocean.
They contain a large amount of red muscle, which helps them maintain a high activity-
level.
Sympathomimetic Sympathomimetic drugs are stimulant compounds which mimic the effects of agonists of
the sympathetic nervous system such as the catecholamines (epinephrine (adrenaline),
norepinephrine (noradrenaline), dopamine, etc.) Sympathomimetic drugs are used to
treat cardiac arrest and low blood pressure, or even delay premature labor, among other
things.
Tachycardia A form of cardiac arrhythmia, in which the heartbeat is too fast (more than 100 beats per
minute).
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Ulcerative colitis A form of inflammatory bowel disease (IBD) that causes inflammation and ulcers in
the colon. What sets it apart from Crohn's disease is that ulcerative colitis only affects
the colon and rectum, rather than the whole GI tract.
Urticaria Commonly referred to as hives. It is a kind of skin rash notable for pale red, raised, itchy
bumps. Hives may cause a burning or stinging sensation. It is frequently caused
by allergic reactions, although there are also many non allergic causes.
Vasoconstriction Narrowing of the blood vessels resulting from contraction of the muscular wall of the
vessels
Vasodilation Widening of blood vessels

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SUMMARY
Currently, several biogenic amines (BA) are known cause food intoxication after ingestion of food
products containing excessive amounts. As such, histamine-poisoning (aka "scombroid poisoning")
and tyramine-poisoning ("cheese reaction") are well documented and their concentrations in the
most hazardous products (fish, cheese and fermented meat products) have been investigated before.
Nevertheless, little is known on the concentration of these biogenic amines in other, uncharacteristic
food groups. Likewise, insufficient information is available on the concentration of other biogenic
amines in different types of food, nor is there enough information on their harmful effects (1; 2).
When EFSA decided in 2010 to perform a risk assessment on biogenic amines present in all types of
food, the Belgian government was asked to provide data on the concentration of several biogenic
amines in food products on the Belgian market. As such data was insufficiently available, the
BIOGAMI-project was started in October 2013, a project aiming to assess the health risks associated
with biogenic amines in products part of the Belgian food market, via an elaborated risk assessment.
During this master thesis, a contribution to this envisioned risk assessment was made. As such, in a
first part of the thesis, the currently available toxicological data were investigated, as a part of the
hazard characterization. Then, in order to perform an exposure assessment (the third step in a risk
assessment), data on the concentration of 6 BA in food products on the Belgian market was gathered
via screening tests, according to a predefined sampling plan. These screening tests had started in
October 2014 and have been finished in April (2016) and during the course of this mater thesis
screenings were performed on yoghurt, beer, cheeses and meat (fresh meat, meat products and
meat preparations). Next to the screening tests, also some in depth storage tests have been
performed on fish and marinated meat to provide a preliminary view on the biogenic amine profile in
these animal products and on the influence of several storage conditions on the BA-formation during
storage. Subsequent to all these experimental analyses, statistical analysis of the results of the entire
body of screening tests were executed. First, some exploratory comparisons of the BA-profile were
made between different food groups, between fermented and non-fermented foods and between
animal and plant-based products. Furthermore, redundancy analysis combined with multiple linear
regression was performed in R, looking for the possible correlations between BA-concentrations,
microbial counts and concentrations of free amino acids. These last two types of data were gathered
during the screening tests as well.
The exploratory results of the storage tests showed that in tuna, histamine is the most dominant BA,
occurring in toxic concentrations when the product is misused (stored at too high temperatures). In
marinated meats (pork as well as chicken, beef and lamb), cadaverine and tyramine were the most
prominent BA. It was also concluded that MAP-packaging and refrigeration are effective techniques
to limit BA-formation.
The results of the screening tests for different meat products and preparations showed that tyramine
is always the most predominant BA in these kinds of products. The content of other BA depended
strongly on the type of product and its production method.
Screenings of food products in other food categories resulted in a database containing information
on their biogenic amine and free amino acid concentrations and their microbial contamination.
Exploratory investigation of these BA- data showed that the BA-profile differed strongly between
different food groups. Also some indications were found to state that in animal-based and fermented
products BA are formed differently than in plant-based and non-fermented products respectively.
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