Impulse Program

Food-Related Health Hazards

(1990 - 1995)

Final Report

The following text presents research results of the Belgian Incentive Program "Health Hazards - Food", initiated by the Belgian state - Prime Minister's Services - Federal office for scientific, technical and cultural affairs (OSTC). The scientific responsability is assumed by its authors.

Apart from the question of a balanced and/or appropriate diet (protecting health), technological innovations in the field of agro-food production (such as processing or conservation processes) and new products (e.g. oligofructoses) must also be examined from the point of view of public health. For instance, effective microbiological and epidemiological controls call for not only better methods for identifying and fingerprinting pathogenic microorganisms but also responses to recent challenges in the field of the production and distribution of fresh products and prepared meals.

The links between diet, pollution and health constitute another field of action. In this respect, we must not only determine the volume of contaminants - such as heavy metals - ingested through foodstuffs but also pay more attention to the growing problem of food allergies.

These aspects, among others, form the subject of the impulse programme "Food-Related Health Hazards" managed by the Federal office for scientific, technical and cultural affairs (1990-1995), in which four research fields have been identified:
- Microbiological contamination
- Chemical contamination
- Functional foods
- Food consumption

The results of this programme constitutes the rational basis for recommendations, in the fields of public health, agro-food production and distribution and R&D policy.

For further information about these research projects, apply to the authors or to the OSTC:

Prime Ministers's Office - Scientific, Technical and Cultural Affairs
8 rue de la Science
B-1000 Brussels
Tel.: (02) 238 34 11
Fax: (02) 230 59 12
E-mail: vdpu@belspo.be
http://www.belspo.be

The following text presents research results of the Belgian Impulse Program "Health Hazards - Food", initiated by the Belgian state - Prime Minister's Service - Federal office for scientific, technical and cultural affairs (OSTC). The scientific responsability is assumed by its authors.

Contents

A. Microbiological contamination

A1. P. Tobback:
Mathematical models for risk analysis (HACCP) and total system analysis in food preservation (Refrigerated Processed Foods of Extended Durability)

A2. G. Waes:
Development of quick, specific and sensitive identification methods for Listeria monocytogenes

A3. M. Devleeschouwer & P. André:
Food contamination by L. monocytogenes: Assessment of the situation and study of technical improvements in the manufacture

A4. A. Hughebaert & R. De Wachter:
Development of rapid methods for identification and quantification of microbiological contaminants in food

B. Chemical contamination

B1. C. De Meester & B. Rollmann:
Development of analytical methods forthe quantitative determination of Mutagenic and carcinogenic contaminants in food

B2. S. Srebrnik-Friszmann:
Residues of polychlorinated biphenyls in market basket products and total diets

B3. W. Dejonckheere:
Residues of polychlorinated biphenyls in market basket products and total diets

B4. J. Staessen:
A study on how to prevent the toxic effects of cadmium in the population at large

C. Functional foods

C1. M. Roberfroid:
Development of a new method to assess safety of non digestible dietary oligosaccharides

C2. G. Dandrifosse:
Alimentary allergies and intolerances: Preventive effect of ingested polyamines

D. Food consumption

D1. H. Deelstra, D.L. Massart & C. Van Peteghem:
An action oriented food monitoring programme

D2. R. Van Havere:
Belgian Extension on Nutrition Education and Data Input, Control, Training, Exchange (BENEDICTE)

A. Microbiological contamination

A1. Mathematical models for risk analysis (HACCP) and total system analysis in food preservation (Refrigerated Processed Foods of Extended Durability)

Prof. P. Tobback, KUL - Fac. Landbouw, Afdeling Levensmiddelen en Microbiële Technologie
Kardinaal Mercierlaan 92, 3001 Heverlee

In this study, the potential of mathematical models to describe the evolution of microbial and visual quality during the product life cycle of minimally processed vegetables was evaluated in a case study on cut endive (Cichorium endivia). The study includes (i) measurement of residence time (distribution) and temperature conditions in the chill chain from producer to consumer, (ii) microbial growth kinetics, (iii) kinetics of visual quality evolution, and (iv) analysis of existing distribution practices through simulation.

Time and temperature conditions of minimally processed vegetables in the Belgian chill chain were analysed. Temperature abuses were found during transport and unloading at the supermarket, storage and display in cabinets, and finally, in domestic refrigerators. Residence time and residence time distribution of three categories of minimally processed vegetables in retail display cabinets of two local supermarkets were analysed using a stimulus‑response technique. This technique was proposed to calculate mean residence time and standard deviation. It was found that more than 50% of the packages were sold during the first day of display.

The kinetics of microbial growth was first approached by measuring and modelling growth of indicator organisms. Emphasis was on model selection, parameter, estimation and model validation. Growth of Pseudomonas marginalis and Lactobacillus plantarum was modelled as a function of time, temperature and gas composition (CO2). It was possible to predict microbial evolution under dynamic temperature conditions using the model parameters obtained under constant temperature. In contrast to the sigmoidal growth curve of indicator organisms in a liquid synthetic medium, the evolution of the intrinsic microflora of cut endive (pseudomonads; psychrotrofic, Gram‑negative population; and Mesophilic aerobic count) was described using a modified model for restricted growth. This explicit model was differentiated with respect to time to Mathematical models for risk analysis and total system analysis in food preservation predict growth under dynamic temperature conditions. Predicting microbial growth on cut endive under dynamic temperature conditions was more difficult. After a second temperature shift, the number of pseudomonads and the Mesophilic aerobic count were underestimated.

It was suggested that this phenomena could be related to the complete breakdown of the vegetable tissue (e.g. Ieading to additional nutrient release and matrix change) so that next to temperature, other factors become growth controlling.

Visual quality deterioration of cut endive was measured in a quantitative descriptive test. An extensive statistical analysis revealed that a linear weighted least squares regression on the logarithmic transformed normalised data was the appropriate way to fit the panel responses. Temperature was found to be the most important rate controlling factor. In contrast to microbial growth, the raw materials used (bleaching) significantly influenced visual quality deterioration rate. Visual shelf‑life of cut endive was evaluated by an independent consumer panel. Where a maximum allowable microbial limit unambiguous defines the end of microbial shelf‑life, the end of visual shelf‑life depends on a proportion of the consumer panel rejecting the sample for consumption. The rejection curve was described using a logistic function. Therefore, the visual shelflife could be related to a certain drop in mean response of the QDA panel. Predicting quality loss under fluctuating temperature conditions using the model parameters obtained under constant temperature was only limited successful. Differences in predicted and observed mean responses of the QDA panel were attributed to poor model parameter estimates. Predictions of visual shelf‑life determined by the consumer panel under dynamic temperature conditions were somewhat better.

Using the models and model parameters described above, the effect of existing time and temperature conditions in the chill chain on the microbial and visual quality evolution was simulated. The modified atmosphere was approached by a controlled atmosphere and no synergistic effect of the gas composition at low storage temperature was considered. It was found Mathematical models for risk analysis and total system analysis in food preservation that the microbial and visual shelf‑life of cut endive stored under air was not attained under average time and temperature conditions in the Belgian chill chain. For an hypothetical combination of maximum storage temperatures with maximum residence time in each part of the chill chain; both microbial and visual shelf‑life will be exceeded before the products are consumed. Reduction of the initial microbial load and the addition of a controlled gas composition of 19% CO2 (with 2% CO2), resulted in a further extension of the microbial shelf‑life.

These observations reinforce the general agreement that good temperature and residence time control are necessary to maintain quality of these minimally processed vegetables.

Open dating of refrigerated products is meaningless and gives nothing but a false sense of safety and quality if temperature abuses have occurred. Time‑Temperature Integrators (TTls) are proposed to monitor on‑line the time‑temperature history of the food product and to display the actual status of the quality or safety attribute of interest. However, for minimally processed vegetables other microbial growth controlling intrinsic and extrinsic factors must be incorporated to exactly mimic the growth of the spoilage microflora. It was concluded that additional research on the kinetics of microbial growth was necessary to develop and select the appropriate Product History Integrator. As an alternative, the visual quality evaluation was proposed as a valuable intrinsic Product History Integrator. Furthermore, when it really comes to consumption, other quality characteristics of the food product, such as aroma, have to be evaluated, leaving the visual evaluation not as the sole decision criterion.

A. Microbiological contamination

A2. Development of quick, specific and sensitive identification methods for Listeria monocytogenes

Dr. G. Waes, Rijkszuivelstation, Brusselsesteenweg 370, 9230 Melle

The classical bacteriological method for the detection of Listeria monocytogenes in dairy products, includes an enrichment in fluid medium (2 days), a cultivation on selective agar medium (2 days) in order to obtain colonies, followed by a set of confirmation assays which takes typically 8 to 10 days to complete.

The time for confirmation of the species and genus identity can be decreased by the application of a DNA identification. A commercial DNA probe assay (GenProbe, San Diego, CA) was evaluated for the identification of L. monocytogenes colonies, grown on the agarmedium. All results, obtained with the DNA probe assay were confirmed with the classical biochemical and morphological identification method. By this very elegant and non-radioactive method, L. monocytogenes is identified on half a day. The total assay takes 4,5 days. Four days are still necessary for bacterial culturing.

In order to simultaneously identify the colonies, grown on the agar plate, to the genus level (Listeria spp.) and to the species level (L. monocytogenes), a multiplex PCR method was developed. For the identification of Listeria spp., two specific primers were deduced from the 16S rRNA sequence. For the identification of L. monocytogenes, the specific primers were deduced from the invasion associated protein gene sequence. The two amplified DNA fragments, respectively 1003 bp and 593 bp, were separated by agarose gelelectrophoresis. The results, obtained by the multiplex PCR method were in agreement with these obtained by the classical bacteriological method for the 64 dairy samples with presumptive positives.

By means of PCR it is possible to shorten not only the identification procedure but also the bacterial culturing.

Starting from a pure bacterial culture, it is possible to detect 10 L. monocytogenes cells per ml. In order to test the sensitivity of PCR for detecting L. monocytogenes in cheese samples, an artifical contamination procedure was applied after the enrichment incubation of cheese samples. The PCR is strongly inhibited by cheese components and not by the ingredients of the enrichment broth. A variation of the detection limit from 10 till 106 cfu/ml was noticed, depending on the soft and semi-soft cheeses analyzed.

Therefore, PCR can only be applied for shortening of the bacterial culturing, when a decent sample preparation method is available. It is even possible to avoid bacterial cutluring and to directly detect L. monocytogenes in the food product by PCR.

A method for direct detection of L. monocytogenes in 25 ml of raw milk was developed. The detection limit can be situated between 5 and 10 CFU. The detection method is based on chemical extraction of the milk components and PCR amplification with two nested pairs of primers, specific for L. monocytogenes. the results of this method are obtained within one day.

By application of a direct detection method, the question arises if dead L. monocytogenes cells are detectable by PCR. Non culturable L. monocytogenes cells are detected by PCR after treatment with a variety of desinfectants and after different heat conditions. The sensitivity of this PCR detection is strongly dependent on the treatment applied. Dissolving L. monocytogenes cells in pure ethanol for 30 days only reduced the PCR sensitivity 100 times compared to untreated cells. Treatment with 1% HCl and sterilization at 124°C for 15 min prevented PCR detection after 1 hour. Because of the possible detection of dead L. monocytogenes cells by PCR, it is often advisable to opt for a short, overnight enrichment, followed by a simple sample preparation method and a PCR identification. So, it is possible to obtain a reliable results in 24-48 hour detection time.

By molecular typing methods, it is possible to identify a bacterial strain to the subspecies level. This is helpfull to trace a contamination source which is important for epidemiological studies and for the industry.

The molecular biological typing method RAPD (Randomly Amplified Polymorphic DNA Profile) was applied on 100 L. monocytogenes strains. RAPD is based on PCR with short arbitrary choosen primers. To evaluate the reproducibility of this method, we participate with the international WHO trial. This study showed that especially different thermocyclers and small variations in PCR buffer concentration are the cause of the poor reproducibility obtained.

Molecular typing of Listeria spp. and L. monocytogenes was also possible by repetitive element sequence based PCR. Families of repetitive elements are dispersed throughout the genome of different bacterial species. Two families were used, namely the 35 to 40 bp repetitive extragenic palindromic sequence (REP) and the 124 to 127 bp enterobacterial repetitive intergenic consensus sequence (ERIC)..

REP-PCR showed a higher discriminative potential than ERIC-PCR, comparable with RAPD, combining 3-4 primers. The advantage of this method is its simplicity and its high reproducibility.

In this project, a specific PCR identification method was developed for Brucella spp. the specific primers were deduced from the 16S rRNA sequence on the basis of a sequence alignment between Brucella abortus and Agrobacterium tumefaciens. 

A. Microbiological contamination

A3. Food contamination by L. monocytogenes: 
Assessment of the situation and 
study of technical improvements in the manufacture

Dr. P. Andre, IHE, Dép. de Microbiologie, Section Bactériologie, Rue J. Wytsman 14, 1050 Bruxelles

Dr. Devleeschouwer, ULB - Biocontaminants et Microbiologie et Hygiène, Inst. de Pharmacie - CP 205/2
Boulevard du Triomphe, 1050 Bruxelles

Prof J. Dony, ULB - Unité des Biocontaminants et risques physico-chimiques du milieu
Boulevard du Triomphe, 1050 Bruxelles

Aims

- To evaluate an eventual correlation between the contamination by Listeria monocytogenes and a lack of manufacturing hygiene.
- To establish a relation between strains isolated from food and those isolated from human listeriosis.
- To isolate ans identify inhibitory strains of Listeria monocytogenes from contamination-free cheese and to perform in vitro inhibitory tests.
- To formulate Public Health recommendations.

To reach this goals several approaches were considered:

(a) A study of cheese contamination by Listeria spp. This study performed on 1902 samples allowed us to compare three detection methods for Listeria. The aims of this part were:
- to evaluate the frequency of the cheese contamination
- to evaluate the relation between contamination rate and cheese type
- to evaluate the localisation of the contamination (crust etc...)
- to study the relation between Listeria and fecal contaminations.

Sample analysis and strain identification were performed by ULB. IHE confirmed the identifications and performed serotyping.

(b) A study of the characteristics of Belgian Listeria monocytogenes strains isolated from various foods and from sporadic episodes of human listeriosis. This part required from the IHE to develop a novel typing method based on esterase polymorphism. This study concerned 832 strains. A comparison of serotyping and esterase typing was performed on two strain populations (clinical and dairy strains) and discrimination indexes were calculated. The study showed that the population of Listeria monocytogenes isolated from cheese was different of that isolated from clinical cases. This could not be explained by differences of secretion of virulence marker PI-PLC neither by differences of pathogenicity in a murine model of these strains.

The esterase typing was for the first time used by the IHE to incriminate a contaminated food in a sporadic case of listeriosis.

Furthermore, ULB characterised 54 strains of Listeria monocytogenes, 27 human strains given by IHE and 27 cheese strains, by a study of hybridation polymorphism using ARNr 16s and 23s probes on restriction profiles generated by two endonucleases. This study was performed in collaboration of the University of Liege.

(c) A study aiming to reduce or eliminate the contamination by means of technical improvements. Inhibitory strains of Listeria monocytogenes were isolated from Listeria-free cheese. The strains were identified and the factors influencing the production of the inhibitor were studied more extensively for two inhibitory strains. Some properties of the inhibitory molecules were compared to those of nisin.

Summary of the research performed by ULB

The study has been divided in three parts. We first evaluated the contamination of Belgian and foreign cheeses by Listeria monocytogenes. The second part aimed to adapt to L. monocytogenes a ribotyping method used for Clostridium perfringens. The third part of the work was dedicated to the isolation of bacterial strains able to inhibit the growth of L. monocytogenes. We then characterized the antagonistic fractions secreted by these bacteria.

To reach to goals of the first part of the study, we adapted and compared three detection methods of Listeria: the direct method, the method by twofold enrichment and the cold enrichment method. Serology and confirmation of the identification of the strains of Listeria monocytogenes have been made at the National Reference Centre for Listeria, Institute of Hygiene and Epidemiology (Doctor P. ANDRE). In the second part of the work, we studied the restriction profiles of some Listeria strains, profiles obtained with the EcoR1 and HindIII endonucleases. Using those profiles, we have done hybridization using either a rRNA 16S probe from Bacillus subtilis or a rRNA 23S probe from Clostridium perfringens. These experiments were done at the microbiology laboratory of the faculty of veterinary medicine of the "Université de Liège" (Professor KAECKENBEECK and Doctor G. DAUBE). In the third part of the work, we have isolated, from cheeses not contaminated by Listeria sp., strains that inhibit Listeria sp. We further studied the antagonistic properties of strains whose the inhibition was not due to the production of acid metabolites or hydrogen peroxide.

The first step of the study involved, during three successive periods, the analysis of 1902 cheese samples (1520 of Belgian origin and 382 imported ones). Listeria sp. have been isolated from 446 samples (23.4%) and Listeria monocytogenes from 159 samples (8.4%). We have recorded a diminution of the contamination rate of L. monocytogenes during the study. Indeed, this contamination reached 11% (period June 1988 to September 1990), then 6.9% (period March 1991 to February 1992) and finally 6.4% (period April 1992 to February 1993). In 1995, 52 new samples have been analysed. This last sampling suggests that the contamination persist for one producer. The two most frequently isolated species were L. innocua and L. monocytogenes. Serotype 1/2a represented 77.9% of the strains of L. monocytogenes and serotype 4b 12.6%.

The soft cheeses are the most often contaminated. The contamination is located mainly in the surface layer or in the whole cheese. A significant relation could be found between the contamination by Listeria and the presence of faecal coliforms. The numbers of L. monocytogenes isolated per gram of cheese range from less than 100 to one million.

The restriction profiles obtained with the two endonucleases show too many strips to be easily used. Nevertheless, the ribotypings, that associate an EcoR1 restriction and a hybridization with a rRNA 23S probe from C. perfringens, or a HindIII restriction with a hybridization with a rRNA 16S probe from B. subtilis, gave the best discrimination of the strains, particularly in association with serotyping. The combination of serotyping and ribotyping has allowed us to show that two different cheeses from the same producer were relatively homogeneously contaminated, while there was a heterogeneous contamination of a third cheese. Ribotyping is thus a useful technique when studying contamination of dairy products.

In the third part of our work, we hold, on a total of 1320 tested strains, 25 inhibitory strains, namely, 22 Enterococcus faecalis strains, 2 Lactococcus lactis and one Pediococcus pentosaceus strain. An Enterococcus and the Pediococcus strains have been selected to further study the properties of their antagonistic fraction. Several parameters have been studied: production conditions of the antagonist, its sensitivity to proteolytic enzymes and to heat, and the influence of the pH and of casein on the inhibitory action. These experiences showed that these strains secreted molecules with the properties of bacteriocins. Like nisin, they are bactericidal and thermostable. Unlike nisin, they have a narrow bactericidal spectrum. The production of the antagonist fraction (enterocin B57) is plasmid dependent. Attempts to transfer this plasmid to lactic bacteria did however not succeed.

We recommend a regular control of the dairy products for L. monocytogenes. Soft cheeses in particular should be regularly controlled and cheeses should no longer be made with raw milk but made only with pasteurized milk. Improvement of manufacturing hygiene could be reached by implementation of the HACCP (hazard analysis critical control point) system and by using normalised fast detection methods, adapted to each specific pathogen and each specific product.

The cheese protection could be ensured by using pure bacteriocins added before ripening instead of using the antagonist strains themselves. On the other hand, the natural lactic bacteria, into which the plasmids producing the bacteriocins are transferred, could be used for the production of safer cheeses.

Summary of the research performed by IHE

Listeria monocytogenes strains recovered in Belgium, fom different foodstuffs and from human sporadic cases of listeriosis, were analysed. The distribution of serovars differed in each of these populations. The bacteria isolated from cheeses and from human cases of listeriosis were further studied by a newly developed typing method. We have analysed L. monocytogenes esterases by starch gel electrophoresis. Five esterases, numbered EST 1 to EST5 in order of decreasing anodal migration, were identified. The EST1, EST 3, EST 4, and EST 5 set was most active toward a-naphtyl propionate, while EST2 was most active toward a-naphtyl acetate. Results from inhibitor studies suggest that all of these esterases were EC class 3.1.1.1 carboxylesterases, except that EST 1 and EST 3 also showed some sensitivity to parahydroxymercuribenzoate. Polymorphism of these five esterases was observed and used to developed a new typing method (esterase typing). Twenty esterase patterns were defined in the population of strains originating from cheeses and from human sporadic cases of listeriosis. These 20 esterase patterns were not equally distributed in these two populations. The secretion of the virulence determinant phosphatidylinositol-specific phospholipase C, and the pathogenicity level of strains in immunocompromised mice couid not explain the different distribution of esterase types. The discrimination of esterase typing (Dl = 0.868) was compared with that of serotyping (Dl = 0.666) and with that of the two combined methods (Dl = 0.899).

A. Microbiological contamination

A4. Development of rapid methods for identification and quantification of microbiological contaminants in food

Prof. A. Huyghebaert, RUG, Fak. Landbouwwetenschappen, Technologie levensmiddelen, 
Coupure links 653, 9000 Gent

Prof. R. De Wachter, UIA, Dep. Biochemie, Universiteitsplein 1, 2610 Wilrijk

Over the last 15 years there has been an increased interest in Campylobacter spp. within the Food Industry. The number of reported infections caused by the intake of Campylobacter spp. by food increases every year. In several developed countries the incidence of campylobacteriosis exceeds even the combined infection rates of Salmonella and Shigella. Campylobacters are commensal microflora of the intestinal tracts of warm-blooded animals for example cattle, swine, sheep and poultry. During the slaughtering process and further processing steps, campylobacters can easily be spread from the intestinal contents to the carcasses. Reported prevalence studies have shown that up to 100% of all poultry products sold at retail are contaminated with campylobacters. The consumer can become infected by inadequate cooking practices or by cross-contamination; moreover, the minimum dose of infection for Campylobacter is very low (approximately 500 cells).

In a screening test for the presence of Campylobacter spp. on a large number of chicken and poultry products, sampled in slaughterhouses, wholesale and retail stores, more than 30% of the samples were found positive for Campylobacter spp. Most of the strains were identified as C. jejuni. The conventional detection methods for Campylobacter spp. are time-consuming and labour-intensive and because of this two commercially available DNA testkits for the detection of Campylobacter in foods were evaluated on speed, specificity and sensitivity. Both kits showed a specificity of almost 100%. To obtain a positive signal on the other hand, the presence of more than 107 Campylobacter cells per ml seemed to be necessary, which means the need of a prolonged enrichment stage. In this way, the high speed of the detection kit (45 min. to 3 h) is disposed by an enrichment period of 2 days. Above all, an adequate technique to shorten this enrichment stage is necessary. In this research project we chose the application of the immunomagnetic separation (IMS) technique. In this technique microorganisms are concentrated and isolated out of a food sample by binding to specific antibodies coated on magnetic particles. By the application of a magnetic field, these particles together with the bounded organisms are separated and detected by plating or by using them in a rapid detection method. In this research project we used the commercially available Dynabeads© M-280 tosylactivated and coated them with the commercially available goat anti-Campylobacter antibodies. The tests established of a good binding between Dynabeads and the antibody on the one hand and between the antibody and the antigen (Campylobacter) on the other hand.

In tests with pure cultures of Campylobacter spp. in liquid media we tried in the first place to optimize the IMS technique by examining the most important process parameters. By the determination of the process sensitivity and specificity with pure cultures, recovery values up to 35% were achieved, while under optimal conditions a detection limit of approximately 200 Campylobacter cells was obtained. By using the IMS technique in mixed cultures, the occurence of cross-reactivity and the formation of non specific binding with other organisms were established. Finally, the IMS technique has been applied to 90 naturally contaminated poultry samples. After a preenrichment and enrichment (7 h) period, the IMS technique was performed in parallel with the conventional detection methode for Campylobacter. After separation the beads were streaked onto a number of selective isolation media for Campylobacter spp. on the one hand and applied in a specific PCR assay on the other hand. Screening of the 90 poultry samples with the conventional method resulted in 44 positive samples (49%). With the combination of IMS and plating only 12% of the samples were found positive, while the combination of IMS with PCR gained 57 positive samples (63%). A specific concentration technique (IMS) in combination with a sensitive and very specific identification method (PCR) not only detected more positive samples, moreover the results were obtained in a shorter time. While the conventional detection methode gives a presumptive result at the earliest after 72 h, the combination of IMS - PCR gives a specific result within 30 h and under optimal conditions even within 12 h.

The conventional methods used for the detection and identification of campylobacters are time demanding, tedious, and sometimes even unreliable. An alternative fast and sensitive detection method is the polymerase chain reaction or PCR. Our first approach was a method based on 16S rDNA. On the basis of available and new 16S rDNA sequences determined at our laboratory, universal PCR primers and group specific oligonucleotide probes were developed. PCR amplification and subsequent hybridisation, results in a group specific detection of entheropathogenic campylobacters. Because the 16S rDNA did not allow us to develop species specific detection methods, we switched to the use of 23S rDNA.

Based on the 23S rDNA, a method was developed for the specific detection of Campylobacter and Arcobacter. On the basis of approximately 40 sequences of a variable area of the 23S rDNA, determined at our laboratory, specific PCR primers were developed for the (a) group specific detection of thermophilic campylobacters, (b) species specific detection of thermophilic campylobacters, (c) species specific detection of nonthermophilic campylobacters, (d) group specific detection of arcobacters, and (e) species specific detection of arcobacters.

Using the thermophilic and nonthermophilic Campylobacter specific PCR primers, several clinical isolates that could not be identified with other methods, could be identified.

The PCR assay for the group specific detection of thermophilic campylobacters was used for the detection of Campylobacter spp. in enriched food samples. 144 poultry samples were investigated, of which approximately 46% gave a positive result with the PCR assay and of which approximately 43% gave a positive result with the conventional methods. Contradictory results were obtained for approximately 34% of the food samples. Further investigations showed that these discrepancies were not the result of the detection of dead Campylobacter cells by the PCR assay or of a difference of the detection limit between both methods.

An alternative method for the detection of PCR products, PCR ELISA, was tested. The advantage of PCR ELISA, as opposed to the conventional analyses of PCR products, is not its sensitivity which seems to be approximately the same, but the possibility to perform an automatised detection, which allows a simultaneous handling of a large number of samples.

B. Chemical contamination

B1. Development of analytical methods for the quantitative determination of mutagenic and carcinogenic contaminants in food

Dr. C. De meester & Dr. Rollmann, UCL - Fac. de Médecine, Unité de Mutagénèse et de Teratogénèse,
Av. E. Mounier 72, 1200 Bruxelles

A family of food contaminants belonging to the chemical class of heterocyclic aromatic amines (HAAs) is produced during the heat treatment of protein-rich foods. These compounds have marked genotoxic properties; some of them behave as the most potent bacterial mutagens and were shown to be carcinogenic in rodents and non human primates. Different epidemiological informations seems to indicate that they could be implied as the etiological agents of colorectal cancer and also may be in other forms of tumors.

Therefore, accurate quantification methods are required for the human risk assessment of cooked foods contaminated by these substances. This problem remains a challenge because HAAs are generally found at very low levels (ng/g) in the heated food matrices like cooked meat or fish products

For these reasons, extensive fractionations must be applied, before the analysis. In the past, different solvent extraction procedures were proposed, including complex schemes with subsequent poor recoveries. It is actually recognised that the method developped by G.A. Gross (1990) is the most reliable, with the main advantage of a "on line" working scheme avoiding losses between the usual transfers, whereas the recovery of some compounds like PhlP remains unsatisfactory. The method which is based on a tandem solid phase extraction method with HPLC analysis is simple enough to be widely recommended as a routine procedure. It comprises an efficient sample clean-up through the combination of several modular purification steps using commercially available diatomaceous earth, cation exchange and reversed phase extraction cartridges. Multiple standard addition is necessary because of variable and sample dependent extraction efficiencies of the different HAAs, with TriMelQx as internal standard.

Quantification is done via HPLC with a reverse phase column and UV or electrochemical detection (EC). The most currently used methods are based on UV detection.

The aim of our research which started in 1991, was to develop EC detection in order to improve not only the sensitivity but also the specificity of the analysis.

For these reasons it was necessary to look carefully to a number of analytical parameters which could influence the response of the EC detection after the HPLC separation. The following factors have been investigated:
- pKa of the HAAs - pH of the solvent
- composition of the buffer
- effect of ion pairing agents - ionic strength
- use of a ternary instead as binary mobile phase
- choice of the internal standard
- choice of the column
- choice of the potential of the electrode

A particular interest was given to the comparative sensitivity of the EC compared to the UV detection; in the actual state of the project, we obtaineed an increase of sensitivity with a factor of about 5 for EC detection. In a more fundamental point of vue, the mechanism of the electrochemical oxidation was also investigated, in order to explain the mechanism of the oxydation reactions which occurs on the electrode.

On the other hand, parameters which could influence the chromatographic separation were also investigated: the efficiency of the reverse phase column was compared with a cation exchange stationary phases. These results were compared with capillary electrophoresis, which has been proposed as an alternative separation technique.

Finally, the method proposed by G.A. Gross was aplied with different food matrices, in order to validate a procedure which could be recommended as a routine method. The detection limits of four HAs: IQ, MelQ, MelQx and 4,8DiMelQx were quite similar, with figures around 5 p moles, with our UV detection system. The separation of the compounds is satisfactory, with recoveries between 70 and 80%, after a complete extraction procedure on a mixture of the pure compounds. Different heated meat products have been already tested: beef extract, fried sausage and fried ground beef; it appeared from these assays that the recoveries of the HAs under investigation were quite different.

The UV detection system has been compared with the EC after HPLC analysis with an isocratic solvent on three heat processed meat products:
- In commercial food grade beef extract, the following amounts of MelQx, IQ and MelQ have been calculated to be respectively: 13,9 - 8,5 and 1,25 ng/g, with UV detection. With EC detection, these figures are respectively: 27,7 - 17,6 and 2,9 ng/g.
- In fried ground beef, cooked under different degree of doneness, amounts of MelQx, IQ and MelQ ranged from respectively: 2,16 - 2,49 /13,1 - 27,3 /1,72 2,5 ng/g with UV detection. The EC detection has given very similar figures.
- In a commercial fried mixed meat sample ("pitta") MelQx, IQ and MelQ have been found at levels of respectively: 1,97 - 16,4 and 2,2 ng/g (UV) or 2,5 - 18,5 and 2,7 ng/g (EC). These last results reveal that the simultaneous UV and EC detection after the HPLC analysis provides very similar figures.

The dispersion of the data obtained reveals that the reproducibility of the method can be improved; more particularly for IQ which is poorly separated. Under the actual state of these investigation, we can conclude that the EC detection is reliable, compared to the classical UV system, whereas some discrepancies are observed, depending of the sample analysed.

The original HPLC method which has been developed after ion exchange separation (IEC) present different important advantages over the classical reverse phase separation:
- good separation of the most important HAAs: IQ, MelQ, MelQx, 4,8DiMelQx and PhlP
- solvent easy to prepare (isocratic elution)
- important reduction of the time of analysis
- reduction of the tailing of the peaks separated
- reduction of the cost of the column needed
- simplification of the HPLC pump necessary

Moreover the IEC/EC and IEC/UV methods have been validated by an intercomparison study with a methanolic solution of unknown composition.

These two methods were shown to be suitable for the routine analysis of heat treated foods like commercial beef extract or fried beef.

B. Chemical contamination

B2. Residues of polychlorinated biphenyls 
in market basket products and total diets

Dr. S. Srebrnik-Friszman, IHE, Serv. Denrées Alimentaires, Rue J. Wytsman 14, 1050 Bruxelles

During these decades, Pesticides have been used in large quantities in agriculture and horticulture. The industry as well, has utilized great quantity of chlorinated compounds such as polychlorbiphenyl. This intensive usage has had a significant impact on environment and also on the elements of the life chain.

Due to the toxicity of all these products, the governments have limited, controlled and also prohibited their usage. Despite these actions, the stability of these products is such that we still detect them in the environment. Nevertheless the concentrations have gradually decrease to their todays level.

The actual study has been conducted to measure the concentrations of the organochlorine pesticides and polychlorbiphenyls in the different current food products, and to see if these concentrations may have a negative impact on the public health.

We have analysed 2399 samples from different nature, commonly used in the normal diet of the household (samples of meat, fish, milk and oil products). These analysis have pointed out that all measured concentration are weak. We have not been able to see a significant difference of concentrations between fresh products or prepared products.

Each result has been compared to the maximal authorized value from the Belgian law to ensure that residue concentration were not in excess with the official tolerance level. In these legislation there is no maximal value for organochlorine pesticides in fish, and nothing exists for polychlorobiphenyls. All other results stay below the tolerance level.

To examine possible effect on health, average, median and maximal values have been compared to Acceptable Daily Intake value (A.D.I.).

The following table gives an overview of the percent of A.D.I. for an average residue concentration, for an average ingestion of all food and for a 60 kg person weight.

Average consommation of all foods			
Pesticides	A.D.I. (mg/kg/day)	Percent of A.D.I.	
a-HCH
b-HCH
Lindane
Hexachlorobenzene
Heptachlor epoxide
Dieldrin
DDT
Polychlorobiphényl	8
8
8
6
0.1
0.1
20
5	0.070
0.110
0.158
0.167
6.354
11.591
0.134
3.000	

This table shows that the A.D.I. values recommended by FAO/WHO are respected. The highest values of the table are due to more strictly recommended values for this pesticide.

In conclusion, Belgian legal tolerances for organochlorine pesticides are respected. The percent of A.D.I. for an average residue concentration, an average ingestion of food and for a 60 kg person weight are lower than 11.6% of the recommended value by the international bodies.

To confirm the results obtained and continue to observe an eventuel variation of pesticide or polychlorobiphenyl concentration it will be suitable to maintain a study of food stuffs.

B. Chemical contamination

B3. Residues of polychlorinated biphenyls 
in market basket products and total diets

Prof. W. Dejonckheere, RUG - Fac. Landbouw, Lab. Fytopharmacie, Coupure Links 653, 9000 Gent

The use of pesticides in agriculture and horticulture is officially permitted by the Ministries of Agriculture and Public Health. The prescribed rules with respect to the use of pesticides are such that they can be applied efficiently and that, upon harvesting the edible product, the residues are toxicologically acceptable.

In accordance with the rules of Good Agricultural Practice (GAP) the use of pesticides is therefore only permitted if it yields an effective control of pests and diseases and if the maximum residue limits, which are based on toxicological data, are not exceeded.

In this study it was primarily investigated whether plant-based food commodities and the possible products derived from them are in conformity with the legally fixed maximum residue limits, i.e. whether the amount of residues in these commodities was not too high at the moment of purchase for consumption. In order to do this, a total of 3698 samples covering 21 kinds of vegetables, 15 kinds of fruit and 7 kinds of other commodities were taken over a period of 2 years, 1 April 1991 - 31 March.

On these samples a variety of analyses were performed, using both multi-residue methods, in which several pesticides are detected in one analysis, as well as specific methods, in which only one kind of pesticide is detected. For the whole of the research project twelve analytical methods were available and about 130 different pesticides or metabolites could be detected. On the basis of information concerning both the use of pesticides and the detected residues, those analytical methods were applied that enable the detection of pesticides which are applied to a given crop in normal circumstances, and which could be expected to result in residues.

The percentages of exceeded maximum residue limits detected with the help of these analyses are as follows:

	Percentage of samples not conforming with the MRLs	Percentage of samples with residues without MRLs	Total percentage of non-conforming samples	
Vegetables
Fruit
Other	6.9
0.7
1.2	2.5
1.4
0.0	9.4
2.1
1.2	
Total	4.2	1.8	6.0	

The samples that did not conform with the legal maximum residue limits mainly concern leafy vegetables of which the whole plant is harvested and in which residues of all applied pesticides may therefore be present. For that reason, the risk of exceeding the tolerance limit is obviously greater than in the case of e.g. tomatoes or apples, where most of the pesticides are applied with the purpose of keeping the plant healthy and a proportionally smaller amount of pesticide ends up in the final harvested product, resulting in lower amounts of residues.

The samples with residues for which no maximum residue limits exists, are the result of using non-permitted products (therefore no residue tolerances are available, but the products may well be allowed in e.g. neighbouring countries) or the presence of residues in crops that are not grown in Belgium (grapes, oranges, bananas) but of which the use of pesticides is legally permitted in countries of production.

The secondary aim of the present study was to determine the amount of pesticides that is taken up through consumption. In order to do this each commodity in which residues could be detected in the first analysis was processed in the way that it is normally processed before consumption (washing, peeling, boiling, etc ...). Afterwards a new analysis was performed and in some cases a second or third culinary treatment was performed, each time with a subsequent new analysis.

In this way it was possible to gain an insight in the decrease in residue after normal culinary processing, which is clearly of importance to the consumer as a way of limiting the intake of pesticides through consumption. In Table 10 examples are given of the percentages with which the residues decreased after the above mentioned culinary processes.

In order to calculate the intake of pesticides, the amount of detected residues in the con-sumption-ready commodity was recalculated to values per 100 g of commodity and expressed as percentages of the ADI value. This is the toxicologically acceptable daily intake by a person of 60 kgs as determined by the World Health Organisation (WHO) and the World Food and Agriculture Organisation (FAO).

In order to determine the average intake of pesticides by means of the investigated food commodities it is necessary to know how much of each of these commodities is on average consumed daily per person. Since no official figures are available for this in Belgium an estimated survey was made on the basis of information on buying habits supplied by the National Institute of Statistics (NIS) and the Agricultural Economic Institute (LEI).

Next, a choice was made from the most obvious culinary processes that each commodity is subjected to, e.g. washing for lettuce, peeling for carrots. In some cases no processing was taken into consideration e.g. in the case of apples (no peeling after purchase). For each detected pesticide the percentage of the ADI was calculated that is taken up through the average amount of each food commodity per day.

Finally, the total percentage of the ADI was calculated for each pesticide in the whole food package under investigation.

Overall, in the most average situation, with a daily intake of the investigated food package, the following results were obtained:
- a total of 54 different pesticides are taken up
- the percentages of the ADI of only two pesticides are higher than 1% i.e. dithiocarbamates (1.9%) and bromide (1.6%).

As a general conclusion of this study it can be said that:
- the maximum residue limits are exceeded fairly in vegetables, more particularly in leafy vegetables available to the Belgian consumer and produced in Belgium but also in other countries as no distinction was made between domestic and imported food
- the processing of vegetables and fruit before consumption (e.g. peeling, washing, boiling) results in a significant reduction of the amounts of residue taken up
- the real uptake of pesticides through consumption is much lower than what may be considered toxicologically acceptable

Two remarks are in order here:

1. If a comparable study was made in other countries, the percentage of exceeded maximum residue limits might be lower than in Belgium. It should be clearly stated in this respect that the results of a residue investigation are largely dependent on the number of different analytical methods used. Usually only multi-residue methods are applied while specific methods are used to a much lower degree, which is in contrast to what was done in the present study (e.g. the anti-spouting agent chlorpropham used on potatoes, and bromides on leafy vegetables).

2. Because of the ultimate limitations on the available financial means and the analytical equipment and methods at our disposal, not all food commodities were investigated in this study and therefore it cannot be claimed that all conceivably present pesticides were detected.

All the results of this study have been collected as shown in great detail in 4 appendices which are available on diskette.

The titles of these appendices are:
1. Results calculated in terms of an amount of 100 g of the food commodity under investigation.
2. Results calculated in terms of the average daily intake of each food commodity.
3. Results calculated in terms of an amount of 100 g of the food commodity and for each pesticide.
4. Results calculated in terms of the average daily consumption and for each pesticide.

B. Chemical contamination

B4. A study on how to prevent the toxic effects 
of cadmium in the population at large

Prof. J. Staessen, KUL, UZ Gasthuisberg, Klinisch Lab. Hypertensie, Inwendige Geneeskunde-Cardio,
Herestraat 49, 3000 Leuven

Setting: The PheeCad study is a longitudinal population study, which investigated the health effects of environmental exposure to cadmium and lead. The participants constituted a random sample of the population of two areas, chosen in order to provide a wide range of environmental exposure to cadmium.

Transfer of cadmium: The transfer of cadmium was investigated in 230 subjects (age range: 20-83 years), who consumed vegetables grown in kitchen gardens on a sandy acidic soil (mean pH +/- 6.3). The study investigated the association between the cadmium levels in blood and urine and the cadmium concentration in the soil (range: 0.2 - 44 ppm). Seventy-six subjects were current smokers and 122 participants lived in a district with known cadmium pollution. Urinary cadmium in the 230 subjects averaged 8.7 nmol/24‑h (range: 1.3 to 47 nmol/24‑h) and was after adjustment for age positively correlated with the cadmium level in the soil; a two-fold increase of the soil concentration was accompanied by a 7% rise in urinary cadmium in men (r2=0.05; p=.04) and by a 4% rise in women (r2=0.02; p=.05).

In conclusion, in a rural population, consuming vegetables grown on a sandy acidic soil, 2 to 4% of the variance of urinary cadmium was directly related to the cadmium level in the soil.

Glomerular and tubular dysfunction related to cadmium exposure: The PheeCad study also explored whether across 10 land areas with differing cadmium pollution an association could be demonstrated between glomerular and tubular renal function and the internal (in urine) and external (in the environment) cadmium doses. Cadmium was measured in the soil and in vegetables in 10 districts, six of which bordered on non-ferrous smelters. Renal function and the concentrations of metals in blood and urine were measured in 703 randomly selected residents (response rate: 78%). The six polluted areas, compared with the four other districts, showed higher cadmium levels in the soil (4.86 vs. 0.81 ppm) and in locally grown vegetables, such as celery (2.43 vs. 0.68 ppm) and beans (0.42 vs. 0.15 ppm). Residents from the polluted areas had a raised urinary cadmium (10.5 vs. 7.9 nmol/24-h) and copper (0.16 vs. 0.14 mmol/24-h), higher serum creatinine (100 vs. 97 mmol/l) and elevated urinary excretions of ß2-microglobulin (109 vs. 95 mg/24-h), retinol-binding-protein (136 vs. 118 mg/24 h) and N-acetyl-ß-glucosaminidase (1.78 vs. 1.38 U/24-h).

The creatinine clearance (87 vs. 92 ml/min) was reduced in the six polluted areas. Across the 10 districts, cadmium in the soil was positively correlated with cadmium in celery (r=0.77), in beans (r=0.67) and in the residents' urine (r=0.76). The creatinine clearance was inversely correlated with cadmium in the soil (r=-0.78), in celery (r=‑0.90) and in beans (r=‑0.70). The correlations across the 10 areas between the cadmium doses and the excretion of microproteins and N-acetyl-ß-glucosaminidase were positive, but tended to be weaker than those observed with the creatinine clearance.

In conclusion, the past emissions from non-ferrous smelters gave rise to persistent contaminants in the environment, such as cadmium, which permeate into the food chain. These contaminants create the potential for exposure of the general population, leading to mild renal dysfunction and alterations in the zinc and copper homeostasis.

Exposure to inorganic arsenic: The exposure of the population to inorganic arsenic, a known carcinogen, was assessed in Belgium by measuring the 24-h urinary arsenic excretion in the CadmiBel (1985-1989) and PheeCad (1991-1995) surveys. In the former study measurements were obtained in industrialized urban areas (Liège: n=664, Charleroi: n=291), in a rural control area (Hechtel-Eksel: n=397) and in rural districts, in which the population had possibly been exposed via the emissions of nonferrous smelters (Western Noorderkempen: n=93, Eastern Noorderkempen: n=244). The CadmiBel results showed that after adjustment for gender, age and body mass index, the 24-h arsenic excretion in the Liège area (91 nmol) was lower (p<.001) than in Charleroi (155 nmol), Hechtel-Eksel (144 nmol) and the Western Noorderkempen (158 nmol), whereas the highest 24-h excretion was observed in the Eastern Noorderkempen (455 nmol). From 1991 to 1995, the rural areas (n=609) were re-examined, together with an urban control area (Leuven, n=152). In this period, the adjusted 24‑h urinary arsenic excretion in the rural areas (Hechtel-Eksel 99 nmol; Western and Eastern Noorderkempen 97 nmol) was lower than six years earlier and similar to the excretion Leuven (108 nmol). Longitudinal observations in 529 people residing in the rural areas showed that their 24‑h urinary arsenic excretion decreased (p<.001) from 222 to 100 nmol.

In conclusion, industry was identified as the most likely source of the increased exposure in the Eastern Noorderkempen from 1985 to 1989, (1) because the urinary arsenic excretion did not follow the regional differences in the arsenic content of the drinking water, (2) because an increased exposure was only observed down wind from the industrial sites, (3) because in individual subjects the urinary arsenic excretion was inversely correlated with the distance to the nearest smelter (4) and because the fall in the urinary arsenic excretion over time was accompanied by a decrease in the industrial activity.

An important observation was that the official network monitoring the arsenic concentration in airborne and precipitating dust, did not detect the increased exposure in the Eastern Noorderkempen between 1985 to 1989. These findings highlight the necessity of validating environmental monitoring programs by directly estimating the internal exposure of the population.

Lead exposure and blood pressure in a longitudinal survey: In order to re-evaluate in a prospective fashion the association between low-level lead exposure and blood pressure, a random population sample was studied in Belgium in 1985-1989 (n=728; 49% men; age range, 20-82 years) and re-examined in 1991-1995. At baseline and follow-up, blood pressure was measured by conventional sphygmomanometry (15 readings in total) and at follow-up also by 24‑h ambulatory monitoring. Exposure was estimated from lead and zinc protoporphyrin in blood. Multivariate analyses allowed for gender, age, body mass index, smoking and drinking habits, physical activity, exposure at work, social class, menopausal status, intake of medications, hematocrit and serum calcium and -glutamyltransferase. At baseline the conventional blood pressure averaged (systolic/diastolic ± SD) 130±17/77±9 mm Hg, blood lead 0.42 mmol/l (range: 0.08-3.50) and zinc protoporphyrin 1.0 mg/g hemoglobin (0.3-19.5). Over the 5.2 year (median) follow-up, lead dropped (p<.001) by 0.14 mmol/l (32%). Small but significant (p<.01) changes occurred in the conventionally measured systolic (-1.5 mm Hg) and diastolic (+1.7 mm Hg) blood pressures and in zinc protoporphyrin (+0.5 mg/g hemoglobin). Over follow-up, no consistent associations emerged between the changes in the conventional blood pressure and in blood lead or zinc protoporphyrin. In addition, blood lead or zinc protoporphyrin at baseline did not predict the development of hypertension in 47 patients (risk ratio associated with a doubling of the initial blood lead concentration: 1.2; CI: 0.7-2.0). In a time-integrated analysis, in which each person was characterized by all available measurements, the conventional blood pressure did not correlate with blood lead and zinc protoporphyrin concentrations.

Similarly, the 24-h blood pressure at follow-up, which averaged 119±11/71±8 mm Hg (n=684), did not show a consistent relation with blood lead or zinc protoporphyrin at baseline or at follow-up. Lead exposure, at the intensity studied, was not associated with increased conventional or 24‑h blood pressures in the general population or with an increased risk of hypertension.

In conclusion, these findings argue against the hypothesis that the current exposure levels would be associated with an excess cardiovascular morbidity and mortality.

C. Functional foods

C1. Development of a new method to assess safety of 
non digestible dietary oligosaccharides

Prof. M. Roberfroid, UCL - Fac. de Médecine, Unité de Bioch. Toxicol. et Cancérologique (BCTC 7369),
Av. E. Mounier 72, 1200 Bruxelles

The present work takes part in the development of new methodologies suitable for the evaluation of health risks associated with consumption of new food ingredients, in particular non digestible fructooligosaccharides. The objective of the study was, more precisely, the effect of fructooligosaccharides on lipid metabolism, as well as on the sensitivity of the liver to the steatogenic effects of some chemical agents which are likely to be eaten concomittantly.

Administration to rats of a diet supplemented with a particular fructooligosaccharide, namely oligofructose (10%), induces a significant reduction (30-50%) in serum triglycerides namely affecting VLDL secretion. Using hepatocytes isolated from oligofructose fed rats, it was further shown that, as compared to control rats, triglycerides synthesis and secretion were reduced.

Effect of oligofructose on the in vitro hepatotoxicity of chemicals has also been studied. Since its hypolipidemic effect made interesting to study the interaction with steatogenic substances, four substances have been tested: tetracycline (0,1 mM), sodium valproate (1 mM), BHT (0,1 mM) and aflatoxin B1 (0,01 mM). Aflatoxin, valproate and BHT induce cellular accumulation of triglycerides in hepatocytes from control but not oligofructose fed rats. These data demonstrate a positive effect (reduction of potential steatogenic effect) of the new food ingredient (oligofructose) and toxic potential of chemicals. This work opens the way to a better evaluation of nutritional toxicology because:
- it confirms the value of assessing toxic potential by measuring early metabolic changes predicitive of later toxic effects;
- it uses an in vitro methodology to identify these early modifications as well as an ex vivo methodology to demonstrate interaction (positive) between nutrition and toxic potential;
- it demonstrates, that a food ingredient might protect against a frequent toxicity: steatogenesis.

The last part of the project consisted in the development and validation of the liver slices model to analyse the steatogenic potential effect of drugs.

In conclusion, we propose that in vitro model, and particularly the model of liver slices, will be very useful to evaluate nutrition-toxicity interaction.

C. Functional foods

C2. Alimentary allergies and intolerances: 
Preventive effect of ingested polyamines

Prof. G. Dandrifosse, ULg - Sart Tilman, Faculté de Médecine, 
Bioch. et Physiol. gén., Institut de Chimie, 4000 Liège

The research project included the following tasks:

1. Estimation of polyamine content in human milk, powder milks for babies and animals
2. Study of the effect of polyamine ingestion on the pancreas and liver postnatal maturation.
3. Analysis of the effect of putrescine ingestion on the postnatal maturation of the intestine.
4. Estimation of the intestinal permeability towards macromolecules in adult- and suckling rats treated with or without spermine.
5. Analysis of the effect of milk polyamine content on the intestinal permeability towards proteins in premature infants.
6. Ontogenesis of the mucous immune system in suckling rats treated with or without spermine.
7. Effects of food pollutants ingestion by nursing rats on parameters characterising the intestine and the liver of their pups.
8. Analysis of the spermine action mechanism.
9. Epidemiological study.
10. Therapeutic action of oral polyamines towards intensities of reactions presented by food allergic patients.

Tasks 9 and 10 were added during the project realisation.

The main results and conclusions are as follows.

Putrescine ingestion did not modify the maturation degree of the intestine in suckling rats. On the contrary, the oral administration of spermine or spermidine induced this phenomenon in the same animals. This observation was confirmed by two independent laboratories. For examples, we showed that oral administered spermine controlled the intestinal permeability towards macromolecules in suckling rats. This observation was also made by an independent laboratory The ingestion of spermine induced liver and pancreas maturation in unweaned rats. It modulated immunological properties in the same animals. These observations support the hypothesis that exogenous spermine could play a preventive role towards food allergy.

This last possibility is currently under investigation.

The polyamine concentration of human milk was measured from 60 women during a period extending from the 1st week to the 6th month of lactation. The polyamine content of 18 infant formulas was estimated. The spermine and spermidine content of these powdered milks was lower that that of the human milk. This observation was confirmed by four foreign laboratories. It has led to the proposal that the possible protective effect of human milk against allergies, which has been reported in the literature, could be partly explained by its high level of polyamines, especially spermine.

Preliminary results indicate that the intestinal permeability to macromolecules could be different in premature babies depending on whether they receive maternal milk, powdered milk without addition of spermine or powdered milk supplemented with spermine.

An epidemiological study was undertaken on a small scale. Consequently, the obtained results do not allow to formulate definitive conclusions. They showed a tendency (not significant) indicating that breast fed children (having drunk a milk rich in polyamines) are less often allergic than the others.

Spermine modified the proliferation and differentiation of T lymphocytes isolated from child tonsils and cultured in media containing phytohemagglutinin L.

Otherwise, in the suckling rat, the analysis of the action mechanism of oral administered spermine was deeply analysed at the level of the liver, the intestine and the pancreas. This polyamine acts either directly on the enterocytes or indirectly on the latter or pancreas- and liver cells (activation of ACTH, corticosterone, bombesin and cytokine secretion). In our experimental conditions, spermine did not induce carcinogenous lesions.

In order to study the therapeutic action of polyamine ingestion towards the allergic reaction intensity of patients suffering of food allergy, we measured the intestinal permeability to macromolecules in children with different intestinal diseases. Gastro-resistant pellets containing human a-lactalbumin were prepared. Preliminary results prove that our technique allows to confirm the presence of food allergy in children and to follow up the treatment chosen.

All these observations and other not cited above indicate that it is highly probable that exogenous spermine and spermidine are molecules able to prevent food allergy. Nevertheless, additional research needs to be undertaken in one coherent approach with sufficient critical mass to arrive at some definite answers (as, for example, to the rationale of adding polyamines to infant formulas, both as trophic factors and as prophylaxis against food allergy). Without such an approach, these research questions will most likely also be addressed but in a dispersed way.

D. Food consumption

D1. An action oriented food monitoring programme

Prof. H. Deelstra, UIA, Fac. Geneeskunde & Farmacie, Lab. voor Bromatologie, 
Universiteitsplein 1, 2610 Wilrijk

Prof. D.L. Massart, VUB, Fac. Geneesk., Dienst Farmaceutische en Biomedische analyse,
Laarbeeklaan 103, 1090 Brussel

Prof. C. Van peteghem, RUG - Fac. Farmacie, Lab. voor Bromatologie, Harelbekestraat 72, 9000 Gent

The aims of the project were twofold. In a first part the concentration of different constituents of the food and foodstuffs consumed in Belgium were determined: on the one hand inorganic constituents i.e. 14 minerals and trace elements, on the other hand organic constituents namely total fat and the fatty acid profile. In order to ensure the reliability of the results obtained a thorough quality assurance plan was developed. In a second part mathematical and statistical methods were applied in order to draw relevant conclusions from the large amount of data produced. Moreover these mathematical techniques allow to relate existing data concerning cancer incidences in Belgium and Europe to the foods consumed, the constituents determined in these foods as well as their estimated daily intake.

The different parameters of the quality assurance plan were thoroughly investigated. A validation strategy was developed which was based on well defined method performance characteristics. For the validation of the different atomic absorption spectrometric methods a user‑friendly‑knowledge-based system was developed. The different analytical methods used were thoroughly validated before the analyses were carried out.

To estimate the daily dietary intake of the specific constituents of food, duplicates of total (24 h) diets were analyzed. This approach was preferred over an estimation based on food consumption surveys because for the latter recent data are not available in Belgium. The 24 h diets were collected during seven consecutive days at five different locations in Belgium. For the study of the total fat and the fatty acid profile a separate sampling plan was elaborated which was based on the BIRNH‑study. A total of 150 individual foodstuffs, which are responsible for at least 90% of the daily fat intake, were analyzed.

Since minerals and trace elements are present in almost all foodstuffs the latter were grouped into 16 categories. From these categories about 300 foodstuffs were selected for the analysis of minerals and trace elements.

The analysis results for the minerals and trace elements are discussed, for the duplicate diets and for the individual foodstuffs. When the daily intake is compared with international and Belgian norms, no toxicologic problems are detected for the five non‑essential elements (cadmium, lead, chromium, arsenic and aluminium). For the essential minerals and trace elements there are no problems for the intake of copper, manganese and not even for potassium and sodium. The low calcium intake and the low iron intake by women are, on the other hand, important problems. The intake of magnesium, zinc and selenium are marginal. A comparison between the intake of inorganic components on the four different locations where the duplicate menus were collected shows important differences, due to different dietary habits and food preparation methods. For the duplicate menus the measured mineral concentrations are compared with those estimated by the food composition tables. This shows that those tables must be handled with care.

The fat amount and the fatty acid profile were determined in the double menus but also in 150 types of foodstuffs, which are responsible for 90% of the fat intake (BIRNH‑study). The results indicate that the total fat consumption in Belgium is very high, namely 42% of the total energy intake. The main categories of foodstuffs that are responsible for this are "fats, oils and sauces" (50%), "meat and poultry" (22%) and "cheese and eggs" (9%). Our study shows that the saturated, the mono‑ and the polyunsaturated fatty acids contribute for 17%, 14% and 8% to the total energy intake, respectively. When the intake of the fatty acids is compared between different provinces, one does not only observe differences between the French speaking and the Dutch speaking part, but also between the individual provinces.

The mean intake of fatty acids at the different locations where the duplicate menus are collected are of the same order as that computed by the BIRNH study. A comparison with international norms shows that the relative fat intake in Belgium is much higher than the recommended 15‑30%.

This is also true for saturated fat, namely 17% in stead of 10%. The mean intake of polyunsaturated fatty acids is also situated above the recommended 3‑7%. A comparison with recent EU‑recommen-dations shows that no deficiencies exist for the n‑6 fatty acids: 90% of the population has an intake higher than the PRI‑value (Population Reference Intake). The n‑3 fatty acid intake, on the other hand, is quite low. About 37% of the population has an intake lower than the PRI.

Two literature studies were performed to investigate the relation between the intake of the different components and the cancer incidence. It can be concluded that the high fat intake is clearly correlated with breast, colon and prostate cancer. High amounts of n‑6 fatty acids are positively correlated with different cancer types, while n‑3 fatty acids have a more protective effect. The literature study on minerals and trace elements indicated that increased intake of calcium, selenium and zinc would decrease the risk for different types of cancer, while an increased sodium intake could indirectly increase the risk of stomach cancer. When data from the literature are compared with the results that are obtained during this study, it must be concluded that in Belgium the intake of fat and n‑6 fatty acids is clearly high while the intake of n‑3 fatty acids is low. For the minerals and trace elements, one can state that the intake of calcium, zinc and selenium is low.

Multivariate statistical techniques were used to correlate specific cancer types with nutritional patterns. These techniques were first applied to European cancer and nutritional data. For each type of cancer the correlation with the intake of specific food groups is given. It is not possible to go here into all results, only examples can be given. It is found for instance that Belgium has one of the highest cancer rates in Europe for certain cancer types (colon, breast,...). Moreover, the Belgian diet is characterised by a high intake of animal fats and a lower intake of vegetables and cereals. The analysis of the Belgian data shows that different cancer patterns occur in different parts of the country and that these differences are not restricted to the north/south axis. For instance the highest cancer mortalities for colon and rectum cancer are found in East‑ and West‑Flanders, where meat intake is high and vegetable intake is low, while oesophagus/larynx cancer are most frequent in Hainaut, where the consumption of alcoholic beverages is high.

The correlation of our analysis results with cancer incidences in Belgium are made difficult because the consumption data date from the years '80‑'85 (BIRNH‑study), while the most recent mortality data date from 1989. The lag-time between the influence of nutrition and the development of cancer is statistically estimated at about 10 years.

The recommendations derived from this research project are directed towards all persons having responsibility for the formulation of policies concerning food and health. In addition to the food components studied here, there are many others that should be studied in relation with cancer. Continuously updated data for morbidity and mortality are needed. At a technical level only validated techniques should be used for completing food composition tables. The use of multivariate mathematical techniques should be stimulated. Food consumption data for the Belgian population should be obtained on a regular basis. Sensibilisation campaigns are required to improve nutritional habits to prevent disease. Food producing instances should take into account increasing knowledge about the relationship food‑diseases.

An active information policy is needed towards the consumer and particularly the young consumer, among others to allow him/her to read and understand nutritional labelling information. 

D. Foodconsumption

D2. Belgian Extension on Nutrition Education and 
Data Input, Control, Training, Exchange (BENEDICTE)

Dr. Van Havere, Ministerie Volksgezondheid, Eetwareninspectie,Rijksadministratief Centrum,
Pachecolaan 19 / bus 5, 1010 Brussel

The aims of the project "Belgian Extension on Nutrition Education and Data Input, Control, Training, Exchange" of the Impulse programme "Health risks and food" are the following:
- Extension of the food database using high quality analytical results obtained by means of analyses of foods for which nutritional data where missing up to now;
- Extension of the soft and hardware products (also applicable for other European countries) which was needed for the enlargement of the food composition table;
- Development of made to measure application software programmes and/or publications for potential users;
- The organisation of training workshops to familiarise users with the developped application programmes.

This project allows to conclude that a further cooperation is desirable between government, research and business (food industry and distribution), in order to further develop the database on nutrients in food in a scientific manner.

This project signified a true boost to collect and process the obtained data in a structured and scientific way. The legal regulation on nutrient labeling of prepacked foods (royal decree of 8 January 1992) is an important reference.

Within the legal context business can furnish an important contribution by supplying their own validated data for input in the NUBEL database.

Analytical results produced by accredited laboratories and validated by the Scientific Council have to be available and recognizable for business.

Indeed, let's not forget that the data are destined for the consumer. By means of the label, he will be informed about the composition of his food

The project offered the opportunity for NUBEL to, by means of the obtained analytical results, provide objective and scientific relevant information to:
- potential users, in casu food industry and distribution, who asked for certain data
- education, with the aim to teach the way to eat healthy.

So it is clear that a well elaborated database on nutrients is an important tool for a coherent food policy.

The National Food Council in Belgium just finished its recommendations on nutrient intake.

The Benedict-project of the Impulse-programme "Health risks and food" is connected perfectly to bring insight in food composition to allow an optimal choice to obtain an equilibrated diet.

A well structured cooperation between the different policy makers is in all this a must. When a true nutrient policy is to be reached, the responsable services on regional and federal level have to cooperate. Thanks to the significant efforts of NUBEL, the food composition table and its application programme "Diet Planner" is respected by regional authorities. The cooperation certainly can be called very well. Although NUBEL is a federal service, it's also active in regional initiatives.

Thanks to the FLAIR-project EUROFOODS-ENFANT, Belgium has caught up a big arrears on a European level. Moreover, the BENEDICTE-project was also part of the reason for this success.

The management system NIMS, that was followed on a European level via EURONIMS, was made possible by this project.

The fruitful exchange between both projects has learned Belgium, and especially NUBEL, a lot and made it pay its dues less.

However, we should not become too self-satisfied, there's still a long way to go.

So we strongly recommend that Belgium wouldn't miss this European link to the future.

NUBEL will also in the future try to keep the international connection.

To summarize, thanks to this project, NUBEL has got the unique opportunity to obtain a real breakthrough.

The management system NIMS, but also the analytical results in the database, were largely supported by this project. Moreover, the publication of the food composition table means an important breakthrough and recognition in the nutrient world.