Frank Welle
email: welle@ivv.fraunhofer.de

consumer poly(ethylene terephthalate) recycling concept

Keywords: Chromatography, Extraction, Risk assessment Additives/Contaminants: Food contact materials, Packaging, Packaging -migration modelling, Packaging -recycling PET recycling, analytical screening, challenge test, food packaging, migration

Investigation into the decontamination efficiency of a new post-consumer poly(ethylene terephthalate) recycling concept

Introduction

Since the early 1990's several recycling process technologies for post-consumer recycled poly(ethylene terephthalate) (PCR PET) have been developed. A few years later the first PCR PET for direct food contact applications entered the market. From a consumer safety point of view the concern is that post-consumer substances or compounds from the possible misuse of PET bottles, e.g. the storage of household chemicals, might migrate from the recyclate containing PET bottle into the packed food. Therefore several studies have investigated the principles of migration from post-consumer PET into food (simulants) [START_REF] Begley | Recycled polymers in food packaging: migration considerations[END_REF][START_REF] Komolprasert | Migration of residual contaminants from secondary recycled poly(ethylene terephthalate) into food-simulating solvents, aqueous ethanol and heptane[END_REF][START_REF] Bayer | The threshold of regulation and its application to indirect food additive contaminants in recycled plastics[END_REF][START_REF] Bayer | Polyethylene terephthalate recycling for food-contact applications: testing, safety and technologies: a global perspective[END_REF]. On the other hand, studies of the sorption of compounds in the PET materials has been published [START_REF] Pierce | Analysis of contaminants in recycled poly(ethylene terephthalate) by thermal extraction gas chromatography/mass spectroscopy. Plastics, Rubber[END_REF][START_REF] Huber | Studies on contamination of post consumer plastics from controlled resources for recycling into food packaging applications[END_REF], Franz and Welle 1999[START_REF] Bayer | Polyethylene terephthalate recycling for food-contact applications: testing, safety and technologies: a global perspective[END_REF][START_REF] Begley | Evaluating the potential for recycling all PET bottles into new food packaging[END_REF], Franz et al 2004b).

The key issue for any super-clean recycling process has to be the decontamination of postconsumer compounds or substances from misuse to levels down to the range of impurities of virgin PET materials. The first step is recollection of the post-consumer PET bottles. Subsequently the PET bottles were sorted and ground into PET flakes. A washing process removed surface contamination as well as dust and paper fibres from the labels. In addition using swim-sink separation techniques, the polyolefin closures were removed from the PET flakes. The final product of the sorting and washing steps are surface cleaned PET flakes, which are suitable for non-food packaging applications. If the post-consumer PET should be reused in food packaging further deep-cleansing steps should be applied. Most of the so-called super-clean recycling processes using melt phase filtration, high temperature and vacuum treatment of the flakes and pellets or surface treatment of the washed flakes. Using such treatment post-consumer contaminants from the first use of the PET packaging as well as compounds from possible misuse of PET bottles for storage of e.g. household chemicals were efficiently removed. However before using the super-cleaned recyclates for direct food contact applications, the cleaning efficiency of the super-clean recycling process has to be determined. Under consideration of the input concentrations of potential compounds for the first use of the packaging materials as well as for compounds coming from misuse of the PET bottles, the ability for the reuse of the final product for direct food contact can be evaluated [START_REF] Franz | Recycling of post-consumer poly(ethylene terephthalate) for direct food contact application -a feasibility study using a simplified challenge test[END_REF], Franz and Welle 1999[START_REF] Franz | Post-consumer Poly(ethylene terephthalate) for Direct Food Contact Application -Challenge-Test of an Inline Recycling Process[END_REF]. Provided that the cleaning efficiency is high enough in relation to the input concentrations of post-consumer compounds, the final product of such super-clean processes is from a migration point of view similar to virgin PET and can be reused in food packaging in direct food contact.

Typically, the state-of-the-art super-clean recycling processes are separated from the virgin PET production. Recycling lines are built up far away from the virgin PET production lines as well as from the preforms and bottle manufacturing plants, which were using a certain amount of supercleaned recycled PET for the bottles. A new development in the poly(ethylene terephthalate) PET recycling technologies is a so-called Flake To Resin (FTR  ) process, which combines the virgin PET production with the super-clean recycling in one process step. The technology is similar to the so-called Melt-To-Resin (MTR  ) processes, where PET pre-polymers are further reacted to the final polymer in only one step without a solid stating process step. The investigated FTR  process starts -similar to nearly every super-clean recycling process -with conventionally recycled postconsumer PET flakes. The flakes were re-molten and mixed with PET precursors (oligomer), which were typically used for the polymerisation process of virgin PET. In one process step the mixture was further condensed to the final PET product and transformed into PET pellets (or PET preforms). Due to the fact, that during the polymerisation process of the pre-polymer under high way into the PET pellet production process (in principle up to 100%). The PET producer is therefore able to react on the actual demands of the (recycling) market.

The cleaning efficiency of the investigated FTR recycling process was determined by carrying out a challenge test according to the principles recommended by United States FDA (FDA 1992, FDA 2006) and European Guidelines (ILSI 1998, Franz et al 2004a) in order to evaluate whether the output material is suitable for re-use in packaging materials with direct food contact.

Experimental

Input Material for the Recycling Process

The investigated FTR  process uses conventionally recycled post-consumer PET flakes as a recycling material source. Typically the input material for conventional recycling processes is postconsumer food PET containers from deposit systems as well from curbside collections. However some PET containers previously might have been used for non-food applications. The amount of the non-food PET container fraction depends on the re-collection system. A general statement about the percentage of the non-food fraction is therefore not possible. However, the amount of the non-food container fraction might be not higher than 20%. As far as we know all PET containers for food applications as well as for non-food applications are manufactured from food grade PET material thus being in compliance with 21 CFR 177.1630 as well as with 2002/72/EC.

Recycling Process

The investigated FTR  process starts with PET monomers which are chemically combined in a specific reactor. In this reactor the esterification and pre-polycondensation processes take place and generate the virgin pre-polymer which is used for the main process stream of the FTR  process. In a second line, conventional recycled post-consumer PET flakes are molten and partly depolymerised with ethylene glycol to a defined degree of polymerisation. The degree of polymerisation is brought into a range, which corresponds to the virgin pre-polymer in the main product stream. The depolymerisation process of the post-consumer PET allows a fine filtration of polymer to remove solid contaminants like fibres, metals or sand. Subsequently the depolymerised post-consumer PET is blended into the virgin pre-polymer from the main process stream without a pre-drying step. This is done at an appropriate point of a continuous polycondensation process right upstream of the last reaction stage. The applied melt-phase polymerisation technology allows the increase of the viscosity to levels required for direct use in PET bottle applications. In addition to the viscosity increase and carboxyl-group regulation, post-consumer contaminants and polymerisation by-products are removed by high vacuum (approx. 100 Pa), high temperature (above 270 °C) and residence time (more than 60 min) treatment in a highly surface active DISCAGE  reactor. Following polycondensation reaction the polymer melt is filtered and converted into PET pellets by granulation. The pellets are used directly for PET preform manufacturing after having passed an acetaldehyde degassing system. The process is also capable to produce preforms directly from the melt phase without the granulation step.

Challenge-Tests

The cleaning efficiency was determined by a challenge test by artificial contamination of PET flakes with model contaminants (surrogates). According to the generally accepted contamination protocols for challenge tests (FDA 1992, FDA 2006, ILSI 1998, Franz et al 2004a), post-consumer PET flakes should be soaked in a solution of the surrogates. Subsequently the contaminated material should be recycled with the investigated recycling process. In the special case of the investigated FTR  process, the surrogates can be injected directly into the polymer melt while introducing non-contaminated pre-polymers and conventionally recycled post-consumer PET flakes. For the contamination the solid model compounds were dissolved in the liquid chemicals without using an additional solvent to give a neat solution of the surrogates. This solution was pumped into the extruder directly to the polymer melt. This procedure allows the introduction of much higher concentrations of the surrogates in comparison to PET soaking contamination procedures as well as a flexible variation of the concentration during the challenge tests. The in Table 1 andTable 2. Place here Table 1 Place here Table 2

Surrogates

The cleaning efficiency of the recycling process was determined by introducing surrogates into the FTR  process. The surrogates were chosen in accordance with US FDA (FDA 1992, FDA 2006) and in agreement with EU-relevant criteria (ILSI 1998, Franz et al 2004a) such that they covered the whole spectrum of physical/chemical properties. The surrogates are in compliance with the four general categories of organic compounds: high volatile and polar, high volatile and non-polar, low volatile and polar as well as low volatile and non-polar. In addition, the surrogates used in the challenge test represent a broad variety of functional groups in order to reflect the different chemical nature and physical properties of possible real-life contaminants e.g. aliphatic and aromatic hydrocarbons, chlorinated hydrocarbons and carbonyl or ester functional groups. Also the molecular weight was important for the selection of the surrogates. Chemicals with a molecular weight up to approximately 300 g mol -1 are relevant for migration from and into PET. Substances with a molecular weight >300 g mol -1 have a very low migration potential in the case of low diffusivity plastics such as PET (Franz et al 2004a

Determination of the Cleaning Efficiencies

During the challenge tests samples were drawn from the final product as well as from intermediate process stages. In these samples the surrogate concentrations were determined using an extraction method after swelling of the polymer matrix with help of 1,1,1,3,3,3-hexafluoro-isopropanol (HFIP). HFIP is a well-known very aggressive solvent for PET. The extracts of the challenge test samples were analysed using gas chromatography with flame ionisation detector (FID) or electron capture detector (ECD). From each PET sample two sub-samples were drawn and extracted. The extracts were injected three times into the gas chromatograph. The cleaning efficiencies of the investigated FTR  process were determined for each surrogate from the concentration in the PET sample under consideration of the initial concentrations led into the recycling process according to Equation 1. The results were verified with another independent method (headspace gas chromatography, details see below). The headspace gas chromatograms were evaluated only on a relative basis, e.g. to verify the absence of the surrogates in some samples.

Place here Equation 1

HFIP-Extraction of the PET Material

Each PET material sample was analysed twice in the following way: 1.0 g of each PET sample was placed in a 5 ml glass vial. 1.0 ml 1,1,1,3,3,3-hexafluoro-iso-propanol (HFIP) was given to the PET material and stored for 1 d at 60 °C in order to swell the PET matrix. Then 2.0 ml iso-propanol was added for 1 d at 60 °C to extract the swollen matrix. The extract was decanted from the polymer and stored for 8 h at 4 °C. Subsequently the extracts were decanted again from the precipitate and analysed by GC/FID and GC/ECD. .

Quantification of Lindane

The surrogate lindane was determined by FID with the following equipment stating from the extracts. Gas chromatograph: Hewlett Packard 6890, column: DB 1 -30 m -0.25 µm i.d. -0.25 µm film thickness, temperature program: 50 °C (2 min), rate 10 °C min -1 , 340 °C (2 min), flame ionisation detection, pressure: 50 kPa hydrogen, split: 10 ml min -1 . Quantification of lindane was achieved by external calibration using the standard addition method.

Detection Limits

The detection limits for the above mentioned analytical methods are given in Table 3. The detection limits were determined using stock solutions of the surrogates. From the slope of the calibration curves the detection limits were calculated for each surrogate according to DIN 32645.

Place here

Results and Discussion

Determination of the Cleaning Efficiencies

For the challenge tests the extruder was fed with the PET pre-polymer (pellets) as well as with a certain amount of post-consumer PET flakes. Three challenge tests were performed with different amounts of PCR PET flakes as well as with different spiking levels of the surrogates. PCR PET in amounts of 15%, 25% and 50% as flakes referred to the total polymer were introduced. The surrogate mixture was directly fed into the extruder for re-melting of the PCR PET flakes and PET virgin pellets so that a homogeneous mixture of PET and surrogates was introduced into the FTR  process. The artificially introduced surrogate levels calculated for the amount of PCR PET flakes are summarized in Place here Table 4 Place here Table 5 Place here Table 6 Place here Table 7

Screening of the Challenge Test Samples for Migration Relevant Compounds

The screening for migration relevant compounds in the PET materials and bottles was carried out using headspace gas chromatography. This method detects substances up to a molecular weight of about 300 g mol -1

. The detection limit of the applied method is below 0.5 mg/kg (Franz et al 2004b). A representative headspace gas chromatogram of virgin PET pellets suitable for PET bottle manufacturing is shown in Figure 1 (reference material). Examples of the headspace gas chromatograms of the investigated samples are shown in Figure 2 and 3. In Figure 2 a sample of the melt after contamination is shown. Figure 3 shows the corresponding pellet sample after deep cleansing (final product of the investigated process).

In the virgin PET reference sample (figure 1) only PET typical compounds like acetaldehyde (retention time R t = 1.7 min), 2-methyl-1,3-dioxolane (R t = 2.4 min) and ethylene glycol (R t = 2.7 min) could be determined. Acetaldehyde is a thermal degradation product of poly(ethylene terephthalate) whereas ethylene glycol is one of the residual monomers. 2-Methyl-1,3-dioxolane is the condensation product of acetaldehyde and ethylene glycol. These compounds could be determined in every PET pellet sample, independent whether post-consumer PET is introduced or not. In Figure 2 several additional substance peaks could be determined. The artificially introduced surrogates were identified by mass spectrometry as well as by comparison of the retention time of neat standard substances: chloroform (R t = 2.2 min), toluene (R t = 3.6 min), chlorobenzene (R t = 4.4 min), methyl salicylate (R t = 9.5 min), phenyl cyclohexane (R t = 10.7 min), benzophenone (R t = 12.8 min) and lindane (R t = 13.6 min). The other substance peaks are by-products or impurities of the applied surrogates. The surrogates could only be determined in the samples drawn from the melt before the melt-phase reactor used for decontamination and IV increase (Figure 2). In this process stage, the contaminated melt is mixed with the melt from the pre-polymers. Therefore the concentration of the spiked surrogated is reduced by the introduction of the virgin pre-polymers (e.g. by 50% if 50% virgin pre-polymer is used). In the pellet samples after decontamination (Figure 3) surrogate concentrations below the analytical detection limits were determined. This result indicates the high decontamination effect of the applied process. Only the PET degradation product acetaldehyde has higher concentrations than found in the virgin PET samples used as reference.

Place here Figures 1 to 3

The experimentally determined concentrations in the final product are far below the maximum concentrations given by Franz et al (2004a). Based on migration models [START_REF] Begley | Recycled polymers in food packaging: migration considerations[END_REF][START_REF] Baner | The application of a predictive migration model for evaluation the compliance of plastic materials with European food regulations[END_REF][START_REF] Piringer | Plastic packaging materials for food -barrier function, mass transport, quality assurance and legislation[END_REF][START_REF] Piringer | Evaluation of migration models[END_REF][START_REF] Begley | Evaluation of migration models that might be used in support of regulations for food-contact plastics[END_REF] Franz et al calculated the maximum residual concentration c 0 of contaminants (surrogates) in the bottle wall for a 1 l bottle with 600 cm 2 surface area ("EU cube") which correspond to the migration limit of 10 µg/kg in PCR PET bottled foodstuff (FDA 1992, ILSI 1998, BgVV 2000). Toluene for example would allow a maximum concentration of 4.5 mg/kg of storage of 10 d at 40 °C for olive oil as food simulant. The higher molecular weight compound benzophenone allows a maximum concentration of 8.6 mg/kg in the bottle wall. Therefore the investigated process fulfils the requirements on the migration limits for the applied surrogates under worst case contamination conditions.

Conclusions

The experimental results obtained from the challenge-tests show that all applied surrogate compounds are efficiently removed by the investigated FTR  recycling process. It could be shown that the melt phase polycondensation reactor plays a crucial role in the decontamination of the post-consumer recyclates (PCR). The cleaning efficiencies for the applied surrogates are above or far above 99.9%.The high cleaning efficiencies are due to the high diffusion rates of compounds in For methyl salicylate, the low concentrations determined in the PET melt are most probably due to a trans-esterfication reaction which decreases the concentration of the surrogate by a chemical reaction and not by evaporation from the polymer melt. Methyl salicylate could therefore not be used as an indicator for the surrogates removal potential of the investigated recycling process.

This result indicates that methyl salicylate is not a useful surrogate for recycling processes which are based on a decontamination step in the melt phase. In solid stating processes, however, methyl salicylate seems to be a good surrogate for the simulation of non-food PET contaminants (Franz et al 2004a). In principle, the possible side reaction of this surrogate shows, that no general procedure exists for a challenge test, which could be applied to any recycling PET process.

Moreover, the applied surrogates and the input concentration should be chosen in relation to the investigated recycling process as well as in relation to the concentration of post-consumer contaminants in the input material.

In the final product of the investigated process, which are either PET pellets or preforms, no surrogates could be determined either at standardized contamination levels or freely selected several thousands mg/kg. Such high initial concentrations represent an extremely high contamination level of the PET which most probably could not be established in real life. Postconsumer compounds as well as compounds from misuse of PET containers are on average present in the lower mg/kg range (Franz andWelle 2003, Franz et al 2004b). This already follows from purely statistical considerations regarding the frequency of return of highly contaminated bottles and the correspondingly very high dilution factor.

The high cleaning efficiency was verified using a second, independent screening method. The results of both methods are in good agreement. Except the PET typical substances like acetaldehyde and ethylene glycol, the final products of the challenge test were -from a migrational point of view -comparable to virgin PET. Under the assumption that the measured residual surrogate concentrations in the final product would be the same as in the bottle produced thereof, the maximum concentration in the bottle wall corresponding with the generally accepted migration limit of 10 µg/kg are established for all kinds of foodstuffs. The investigated FTR  recycling process is therefore in a position to produce pellets and preforms with up to 50% recycled PET which fully meets European (ILSI 1998, BgVV 2000) as well as US regulations (FDA 1992) and is therefore capable to recycle post-consumer PET material for the sensitive applications of direct contact packaging for foodstuff. Therefore, the applied melt phase decontamination of post-consumer PET provides an interesting process for the effective decontamination of post-consumer PET. Ethylene glycol [a] 873 873 22460

[a]Ethylene glycol was used to reduce the IV during processing. Ethylene glycol was not used as a surrogate.

  temperature and vacuum conditions, the post-consumer PET should be also deep-cleansed similar to other super-clean PET bottle-to-bottle recycling processes. The advantage of such a process is, that the same production line can produce PET pellets in a flexible way either from 100% virgin pre-polymers or from a mixture of virgin PET and post-consumer PET flakes. The amount of conventional recycled post-consumer (PCR) PET flakes can be introduced in a flexible

  the different challenge tests and the spiking levels of the surrogates are given

  Analysis and GC/ECD AnalysisThe extracts of the challenge test samples were analysed according to the concentrations of all surrogates except lindane by gas chromatography (FID). Chloroform was monitored by an electron capture detector (ECD). For this purpose a gas chromatograph with a simultaneous detection by an FID and ECD detector was used. Quantification was achieved by external calibration using the standard addition method. Parts of a standard solution of the surrogates in iso-propanol were added to uncontaminated PET flakes and were analysed together with the PET samples of the contamination experiments. Gas chromatograph: HP 5890II, column: SE 10 -30 m -0.32 mm i.d. -0.32 µm film thickness, temperature program: 40 °C (5 min), rate 15 °C min -1 , 240 °C (15 min), pressure: 50 kPa hydrogen, split: 10 ml min -1

  . These cleaning efficiencies are similar or higher in comparison to other PET super-clean recycling technologies based on decontamination of PET flakes or pellets[START_REF] Franz | Recycling packaging materials[END_REF].

Figure 1 :Figure 2 :Figure 3 :

 123 Figure 1: Headspace gas chromatogram of virgin PET (reference sample)

Table 3

 3 Headspace Screening of the Challenge Test Samples for Verification Purposes 1.0 g of the PET samples was weighed into a headspace vial and analysed via headspace GC/FID. Gas chromatograph: Perkin Elmer AutoSystem XL, column: ZB 1 -30 m -0.25 mm i.d. -

	0.32 µm film thickness, temperature program: 50°C (4 min), rate 20°C min -1 , 320°C (15 min),
	pressure: 50 kPa helium, split: 10 ml min -1 . Headspace Autosampler: Perkin Elmer HS 40 XL,
	oven temperature: 200°C, needle temperature: 210 °C, transfer line: 210°C, equilibration time: 1 h,
	pressurization time: 3 min, injection time: 0.02 min, withdrawal time: 1 min. For identification, F another 1.0 g sample was drawn and analysed via headspace GC/MS. Gas chromatograph: o r Hewlett Packard 6890, column: Optima 1 MS -30 m -0.25 mm i.d. -0.25 µm film thickness,
	P temperature program: 50°C (4 min), rate 20°C min -1 , 320°C (15 min), full scan, m/z 40-500.
	e
	e r
	R
	e
	v i e
	w
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Headspace autosampler: Perkin Elmer HS 40 XL, oven temperature: 200 °C, needle temperature: 210°C, transfer line: 210°C, equilibration time: 1 h, pressurization time: 3 min, injection time: 0.04 min, withdrawal time: 1 min. The mass spectra that were obtained were identified by comparison with spectra in the NIST spectral library.

Table 4 .

 4 The concentrations of the surrogates in the investigated samples determined by HFIP extraction are summarised in Table5 to Table 7.

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
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Table 1 :

 1 Parameters used for the challenge tests

	Equation 1:	cleaning	efficiency	=	1  -  	initial sample	conc. conc.	   	•	100%
		F o r
	Trial (recyclate content)	Amount P e e Prepolymer r PET PCR PET			Surrogate cocktail (dosage)
	Trial 1 (25%) 75 kg h -1 (1250 g min -1 ) Trial 2 (50%) 50 kg h -1 (833 g min -1 ) Trial 3 (15%) 85 kg h -1 (1417 g min -1 )		R 25 kg h -1 (417 g min -1 ) e v Cocktail A (5 ml min -1 ) 50 kg h -1 (833 g min -1 ) Cocktail A (10 ml min -1 ) i e w 15 kg h -1 (250 g min -1 ) Cocktail B (10 ml min -1 )
										O n l
										y

Table 2 :

 2 Amounts of surrogates used for the spiking cocktails

	Surrogate	Amount [g]
		Cocktail A	Cocktail B
	Toluene	226.5	151.0
	Chloroform	357.0	238.0
	Chlorobenzene	86.3	57.5
	Phenyl cyclohexane	22.5	15.0
	Methyl salicylate	69.0	46.0
	Benzophenone	60.8	40.5
	Lindane	53.3	35.5
	Ethylene glycol [a]	60.0	583.5
	[a] Ethylene glycol was used to reduce the IV during processing. Ethylene glycol was
	not used as a surrogate

Table 3 :

 3 Analytical detection limits of the surrogates (HFIP Method)

	surrogate	detection limit
		[ppm]
	Toluene	2.7
	Chloroform	0.8
	Chlorobenzene	0.9
	Methyl salicylate	1.0
	Phenyl cyclohexane	0.2
	Benzophenone	0.2
	Lindane	0.8

Table 4 :

 4 Initial concentrations of the surrogates in relation to the amount of PCR PET

	flakes
	Surrogate	Initial concentration [ppm]
		Trial 1	Trial 2	Trial 3
		Cocktail A, 5 ml min -1	Cocktail A, 10 ml min -1	Cocktail B, 10 ml min -1
		25% PCR PET	50% PCR PET	15% PCR PET
	Toluene	3295	3295	5812
	Chloroform	5194	5194	9161
	Chlorobenzene	1255	1255	2213
	Phenyl	327	327	577
	cyclohexane
	Methyl salicylate 1004	1004	1771
	Benzophenone	885	885	1559
	Lindane	775	775	1366

Table 5 :

 5 Concentrations (determined using the HFIP extraction method) of the surrogates in the investigated PET samples of

	Page 23 of 25			Food Additives and Contaminants
	Trial 1 (Cocktail A at 5 ml min -1 , 25% PCR flakes)
		Concentration [ppm]
		Toluene	Chloroform Chloro-	Phenyl	Methyl	Benzophenone Lindane
				benzene	cyclohexane	salicylate
	before	<2.7	<0.8	<0.9	<0.2	<1.0	<0.2	<0.8
	contamination
	calculated	3295	5194	1255	327	1004	885	775
	contamination conc.
	Before deep-	852 ±23	1453 ±32	296 ±21	81 ±3	<1.0	165 ±2	66 ±2
	cleansing
	After deep-cleansing	<2.7	<0.8	<0.9	<0.2	<1.0	<0.2	<0.8
	(final product)

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Table 6 :

 6 Concentrations (determined using the HFIP extraction method) of the surrogates in the investigated PET samples of Trial 2

				Food Additives and Contaminants		Page 24 of 25
	(Cocktail A at 10 ml min -1 , 50% PCR flakes)
		Concentration [ppm]
		Toluene	Chloroform Chloro-	Phenyl	Methyl	Benzophenone Lindane
				benzene	cyclohexane	salicylate
	calculated	3295	5194	1255	327	1004	885	775
	contamination conc.
	Before deep-	1999 ±28	3075 ±47	655 ±9	163 ±2	<1.0	345 ±1	133 ±1
	cleansing
	After deep-cleansing	<2.7	<0.8	<0.9	<0.2	<1.0	<0.2	<0.8
	(final product)

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Table 7 :

 7 Concentrations (determined using the HFIP extraction method) of the surrogates in the investigated PET samples of

	Page 25 of 25
	Trial 3 (Cocktail B at 10 ml min -1 , 15% PCR Flakes)
		Concentration [ppm]
		Toluene	Chloroform Chloro-	Phenyl	Methyl	Benzophenone Lindane
				benzene	cyclohexane	salicylate
	calculated	5812	9161	2213	577	1771	1559	1366
	contamination conc.
	Before deep-	826 ±1	1142 ±107 301 ±7	80 ±2	<1.0	175 ±1	64 ±1
	cleansing
	After deep-cleansing	<2.7	<0.8	<0.9	<0.2	<1.0	<0.2	<0.8
	(final product)

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Food Additives and Contaminants

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Rafael Rodríguez-Acuña

María Del Carmen Pérez-Camino

Wenceslao Moreda
email: wmoreda@ig.csic.es

Arturo Cert

CONTAMINATION SOURCES OF VIRGIN OLIVE OILS BY POLYCYCLIC AROMATIC HYDROCARBONS

Keywords: PAH, Process contaminants -PAH' polycyclic aromatic hydrocarbons, virgin olive oil, olive fruit, contamination sources, PAHs VOO, Virgin Olive Oil, PAHs, Polycyclic Aromatic Hydrocarbons, IARC, International High Performance Liquid Chromatography, GC, Gas Chromatography, FLD, Fluorescence Detector, MS, Mass Spectrometry, SIM, Single Ion Monitoring, BaP, benzo(a)pyrene, Chr, chrysene, BeP, benzo

The presence of polycyclic aromatic hydrocarbons (PAHs) in virgin olive oils is caused by contamination on the skin of olives, and also contamination of the oil during processing in the oil mill can occur. Contamination of olive fruits occurs on the olive skin, and it depends directly on the environmental pollution level and inversely on the fruit size. In the olive oil mill, the PAHs content can be increased by contamination of the oil during the extraction process if combustion fumes pollute the environment. Other factors during the extraction process, such as olive washing and talc addition, did not modify the PAHs levels of the oils. Very high concentrations of PAHs in oils were only found as a consequence of accidental exposure to contamination sources, such as the direct contact of olives with a diesel exhaust and oil extraction into a high polluted environment. Determination of 9 PAHs was carried out by isolation of the hydrocarbon fraction and subsequent clean-up by solid phase extraction, followed by RP-HPLC analysis using a programmable fluorescence detector.

INTRODUCTION

Polycyclic aromatic hydrocarbons (PAHs) are a group of contaminants that are widely present in the environment, known to be cancer causing agents: several of them are classified by the International Agency of Research on Cancer (IARC) in 2A and 2B groups [START_REF] Iarc | Polynuclear aromatic compounds, part 1: Chemical, environmental and experimental data in[END_REF]. They are generated by incomplete combustion of organic matter arising, in part, from natural combustion (forest fires, volcanic eruptions) and, for the most part, from human activities (engine exhaust, industrial production, coal derived products, petroleum distillates, waste incineration, tobacco smoke) [START_REF] Grova | Detection of polycyclic aromatic hydrocarbon levels in milk collected near potential contamination sources[END_REF].

Air pollution with dust and particle containing large quantities of PAHs may contaminate the plants via atmospheric fallout during its growing period and most of this superficial contamination can be transferred to the final product [START_REF] Lee | Analytical Chemistry of Polycyclic Aromatic Compounds[END_REF][START_REF] Bories | Tossicologia e Sicurezza degli Alimenti[END_REF][START_REF] Dennis | Factors affecting the polycyclic aromatic hydrocarbon content of cereals, fats and other food products[END_REF][START_REF] Bernd | Biomass burning: a review of organic tracers for smoke from incomplete combustion[END_REF]. This fact is much more important in industrial areas and highways than in rural areas, where contamination of vegetables can be ten time higher. [START_REF] Derache | Toxicidad de los hidrocarburos aromáticos policíclicos y de los productos de pirólisis en Toxicología y Seguridad de los Alimentos[END_REF][START_REF] Grova | Detection of polycyclic aromatic hydrocarbon levels in milk collected near potential contamination sources[END_REF]. On the other hand, PAHs may also be formed directly in food as a result of some heat processes (charcoal grilling, roasting, smoke drying, and smoking) [Lijinsky y Shubik, 1965a,b;[START_REF] Guillén | Food as a source of polycyclic aromatic carcinogens[END_REF][START_REF] Moret | Polycyclic aromatic hydrocarbons in edible fats and oils: occurrence and analytical methods[END_REF]Šimko, 2002].

PAHs have been found in many different foods, including edible vegetable oils that, because of their lipophilic nature, can be easily contaminated with these substances. Two main routes of PAH contamination in vegetable oils have been suggested: the contact with polluted environment and the drying process of the raw matter, using combustion fumes of organic matter [START_REF] Gertz | Investigation and legal evaluation of polycyclic aromatic hydrocarbon in vegetable fats and oils[END_REF][START_REF] Moret | Polycyclic aromatic hydrocarbons in edible fats and oils: occurrence and analytical methods[END_REF][START_REF] Bernd | Biomass burning: a review of organic tracers for smoke from incomplete combustion[END_REF]EC, 2002]. However, in virgin olive oils (VOOs) only 3 the contact with the polluted air must be taken in account since the raw matter is not subjected to any drying process.

Virgin olive oils (VOO) are usually obtained by a process involving several successive steps: olive harvest by manual or mechanical procedures, transport of fruits from olive grove to olive oil mill, piling of olives in the storage area, olive washing, crushing of olives in a hammer crusher, mixing of the olive paste in a thermobeater and separation of the oil by centrifugation or pressing. During the mixing step, oil drops combine, facilitating the release of the oil during centrifugation. However, some olive varieties and, in general, fruits at the unripe stage cause emulsions. In this case, 1-3% of micronized talc (hydrated magnesium silicate) is added to the olive paste in the thermobeater inlet improving the centrifugation effectiveness with no loss of oil quality [START_REF] Cert | Effects of talc addition and operation mode on the quality and oxidative stability of vigin olive oils obtained by centrifugation[END_REF]]. The talc is eliminated during the centrifugation step together with the olive pomace.

Generally, the PAH content in crude vegetable oils can be reduced by refining using activated carbon together with activated clays during the bleaching step [Patterson, 1992;[START_REF] Moret | HPLC determination of polycyclic aromatic hydrocarbons in olive oils[END_REF]Texeira et al. 2007]. However, refining is not allowed for edible virgin olive oils [EC, 2001;IOOC, 2003] since organoleptic properties and chemical composition changes during this process. Due to the PAHs levels in edible virgin olive oils cannot be reduced, the aim of this study was to detect and evaluate the usual contamination sources by PAHs during the VOO obtaining process in order to prevent the contamination hazard and obtain VOOs with the lowest PAHs levels, following the European Commission recommendations [EC, 2005a,b].

MATERIALS AND METHODS

Samples

To determine PAHs contamination in olive fruits and oils, various sets of samples were collected:

1.-Olive fruits of Koroneiki, Arbequina, Picual, Lechín, Hojiblanca, and Ascolano varieties, all being exposed to the same environmental pollution, and harvested at the same time from an olive grove located in a medium polluted area of Seville (Spain). Moreover, olive fruits of Picual and Arbequina varieties were harvested from the of the Instituto de la Grasa's olive garden (Seville, Spain), located near the city centre.

2.-Olive fruits of Picual variety exposed to different environmental pollution were harvested at the same time from the same wide orchard in Jimena (Jaén, Spain). The orchard includes trickle irrigated and unirrigated groves growing in mountainous area and irrigated groves near a main road.

Materials

For oil extraction, tap water and micronized talc (Talcoliva ® , Luzenac, France) were used.

For sample clean-up procedure, Si and NH 2 Bondesil ® adsorbents (Varian, California, USA), nhexane and toluene Uvasol © grade (Merck, Darmstadt, Germany), and alkanes mixture of boiling point 65-70 ºC reagent grade (Scharlau, Barcelona, Spain), distilled using a Vigreux column, were used.

For the HPLC analysis, acetonitrile HPLC super purity solvent 190 (ROMIL, Cambridge, U.K.), and water purified with a Milli-Q system (Millipore, Bedford, MA, USA) were used.

For PAH identification, individual standard PAHs were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany) at concentrations of 10 ng/µL BaA, Chr, BeP, BbF, BkF, BaP, DahA, BghiPE and IP in acetonitrile. A stock solution containing: BaA 0.50 µg/mL, Chr 0.50 µg/mL, BeP 1.0 µg/mL, BbF 0.50 µg/mL, BkF 0.125 µg/mL, BaP 0.25 µg/mL, DahA 0.25 µg/mL, BghiP 0.50 µg/mL and IP 1.75 µg/mL of the PAHs was prepared in acetonitrile and stored at 4 ºC in darkness.

Apparatus

The HPLC equipment comprised a vacuum degasser for the mobile phase solvents Gastorr 154 (Flom, Japan), auto-sampler System Gold 508, binary pumping unit System Gold 126, Mistral peltier column thermostat unit (Beckman-Coulter, Fullerton, CA, USA) and a programmable fluorescence detector LAChrom L-7485 (Hitachi-Merck, Japan). A reverse phase C-18 HPLC column (250 x 4.6 mm i.d.) packed with Inertsil ODS-P (5 µm particle size) (GL Sciences Inc., Tokyo, Japan) was used together with a reverse phase C-18 high performance guard column (10 x 2.1 mm i.d.) packed with TP-201 (5 µm particle size) (Vydac, CA, USA). The data were processed using 32 Karat Gold v. 5.0 acquisition software (Beckman-Coulter, Fullerton, CA, USA). The GC equipment comprised a Trace GC2000 gas chromatograph coupled to a GCQ/Polaris ion trap mass spectrometer equipped with an AS2000 autosampler (ThermoFinnigan, Austin, TX, USA) operating in single ion monitoring (SIM) mode for identification purposes. The column used was a DB-5ms (J&W Scientific, CA, USA) fused silica capillary column (30 m long x 0.25 mm I.D. x 0.25 µm film thickness) coated with a non polar stationary phase (5% phenyl-methyl polysiloxane). The data were processed using Xcalibur v. 1.4 acquisition software (ThermoFinnigan, Austin, Texas, USA).

Analytical Procedures

One of the key points in the determination of PAHs is the cleaning of the material used for its determination, to avoid contamination, all glassware was cleaned several times with n-hexane Uvasol © grade before use and the purity of the solvents was checked by HPLC-FLD analysis of the concentrates.

PAH extraction from the olive skin

PAHs were extracted from the olive skins by rinsing the fruits with n-hexane in an ultrasonic bath as follows: The olives were weighed (500 g), placed into a 500 mL beaker (except Ascolano variety that was placed into an 800 mL beaker), and then hexane was added until the fruits were covered.

The beaker was subjected to ultra sound for 5 min at maximum power at room temperature. The hexane was then poured into a graduated cylinder and the olives washed again with hexane. The volume of combined extracts of hexane were measured and transferred to amber bottles, which were stored at -20 ºC in darkness. Half the hexane volume was concentrated in a rotary evaporator at room temperature under vacuum down to 2.5 mL volume, approximately. Then, the solution was analyzed following the procedure set for the olive oil samples.

PAHs extraction from the talc

Micronized talc (20 g) was placed into a 100 mL Erlenmeyer flask together with 50 mL of hexane and the mixture was shaken vigorously. The flask containing the suspension was placed in an ultrasonic bath for 5 min. at maximum power at room temperature. The suspension was allowed to settle and the upper layer was collected with the aid of a pipette. The hexane volume was measured, and stored in an amber bottle at -20 ºC in darkness until analysis. The hexane was then concentrated in a rotary evaporator at room temperature under vacuum down to 2.5 mL volume, approximately, and analyzed following the procedure set for the olive oil sample.

Oil extraction method

The Abencor (MC2 Ingeniería y Sistemas, Seville, Spain) method was used to obtain the olive oils at laboratory scale, this system, which simulates the industrial process of olive oil production [START_REF] Martinez | Report about the use of "Abencor" analyzer[END_REF]]. The olive fruits (700 g) were milled in a hammer crusher and the olive paste was mixed in a thermobeater at 25 ºC during 20 min, then 300 ml of hot water (40 ºC) was added and the mixture mixed again for 10 min. The olive paste was centrifuged for 1 min and the liquid phase was poured onto a 500 mL graduated cylinder. The solid phase remaining in the centrifuge was centrifuged again after the addition of 100 mL of hot water. The combined liquid phases were left to settle and the upper layer was separated, filtered through filter paper, and stored in glass bottles at 5 ºC.

Analysis of PAHs

Analysis of PAHs in oils and hexane extracts were carried out following the procedure previously described [START_REF] Moreda | Determination of high molecular mass polycyclic aromatic hydrocarbons in refined olive pomace and other vegetable oils[END_REF]]. The method involves isolation of the hydrocarbon fraction by solid phase extraction through silica gel phase (Si), subsequent clean up of PAHs using solid phase extraction through modified silica gel phase with NH 2 groups, and quantification of PAHs by RP-HPLC using a programmed fluorescence detector. The HPLC system is set up maintaining the column temperature at 20ºC and using a gradient of acetonitrile/water as mobile phase at a flow rate

Confirmation of PAHs identities

An extract obtained by rinsing olive fruits with hexane was purified by solid-phase extraction using silica gel and amino phases, as described in the analytical method [START_REF] Moreda | Determination of high molecular mass polycyclic aromatic hydrocarbons in refined olive pomace and other vegetable oils[END_REF]]. The residue was re-dissolved in 50 µL of heptane and aliquots were analyzed by GC-MS in SIM mode.

The GC operating conditions were: helium as a carrier gas at 1 mL/min in constant flow mode.

Injector temperature was 285 ºC and "splitless" mode injection was used, being 1 minute the "splitless time". The oven temperature was programmed as follow: the initial temperature was held for 3 min at 60 ºC and then from 60 to 295 ºC at 5 ºC/min and maintained for 10 min.

The MS operation conditions were the following: ion source and transfer line temperatures 200 and 300 ºC, respectively. The instrument was tuned in Electronic Impact (EI) positive mode using

RESULTS AND DISCUSSION

The identity of each PAH in the hexane solution, obtained by rinsing the olive fruit surface and extraction of talc, was confirmed using both HPLC-FLD and GC-MS analysis by comparison of the retention times of each peak between samples and standard solution to avoid wrong identification of the chromatographic peaks . Once ensured the PAH identities, only HPLC-FLD analyses with quantitative purpose were made.

PAH Contamination of olives

Contamination of olives fruits during growth on the olive tree is a factor to be considered in the PAHs content in the oil, since the olive skin contacts with the oil during the crushing and mixing processes in the oil mill allowing the transfer of PAHs from the skin to the oil. Therefore, olive size, environmental pollution, and accidental contamination were taken into account in the present study.

Effect of environmental pollution during the olive growing

To establish the influence of the olive fruit size on the PAHs oil levels, olive fruits of several varieties were harvested at the same time from an olive orchard nearby to an airport. Arbequina, Picual, Lechín, and Hojiblanca varieties were selected because they are the most abundant in Andalusia, and Koroneiki and Ascolano varieties because their fruits were the smallest and biggest, respectively (Table 1). Arbequina and Picual olive fruits from trees growing in an urban area were also harvested. The PAHs present on the olive fruit surface were extracted with hexane in an ultrasonic bath and analyzed, being the PAH level per fruit proportional to its surface. Consequently, the PAHs content per Kg of olives is in inversely relation to the fruit size. Assuming an average oil content in fruits around 20% [IOOC, 1996], since samples were harvested at the same time in different ripeness stage, and the total transfer of PAHs from the surface to the oil, the theoretical PAH contamination of oils would be inversely proportional to the olive size (Table 1). This hypothesis was confirmed by the PAH contents in olive oils obtained from the olives using the Abencor system (Table 1). The comparison between the real PAH concentrations in oils and those calculated from the hexane extracts suggests that the contamination of the oils is mainly in the skin of the fruits, which is transferred to the oil during the extraction process. These results are in 8 agreement with those obtained by [START_REF] Cejpek | Changes in PAH levels during production of rapeseed oil[END_REF] in rapeseed oil who found no significant differences in PAH concentrations determined by means of a procedure including seed rinsing with chloroform in an ultrasonic bath and that with chloroform extraction of the ground seed.

[Insert Table 1]

To establish the influence of environmental pollution on the olive grove, oils obtained by the Abencor system from Picual olives harvested from different places of the same olive orchard, exposed to different levels of pollution, were analyzed. The PAH concentrations were very low (the sum of PAHs was below of 1 µg/kg) in oils coming from trees growing in mountainous area, whereas those obtained from trees located near the roads were higher (Table 2). No differences in PAHs concentration were found in oils coming from trickle irrigated and unirrigated olive trees from the mountainous areas. These results indicated that the air pollution level on the tree is a significant contamination source in olive oils.

[Insert Table 2]

Contamination during Olive Harvesting

In the crop 2004/2005, several VOOs obtained in an olive oil mill showed high PAHs content (up to 30 µg/kg of BaP). These concentrations were rather high and the possible contamination sources were investigated. After checking the lack of pollution problems, the olive harvesting process was then examined. The hexane extracts obtained by rinsing from various olive sets were analyzed: olives harvested by hand, olives mechanically harvested, and olives harvested by hand after that the combine had passed over the trees. Olives harvested by hand prior to passing the combine showed very low PAH levels, the olives harvested after passing the combine showed a slightly increase in the PAH level. The olives harvested mechanically by the combine reached very high PAH levels (Table 3). When the exhaust pipe of the combine was enlarged, the PAH concentration diminished.

These results are in agreement with the fact that the combine circulate over the olive trees and the harvested olives are carried to a hopper located close to the exhaust pipe outlet. When the exhaust pipe was enlarged the concentration diminishes significantly. Therefore, the exposure to the diesel exhaust fumes is the most important source of contamination. An appropriate design of the engine is required to minimize the olive contamination.

[Insert Finally, to confirm that the PAHs are deposited mainly in the olive skin, olives contaminated by the combine exhaust where rinsed with hexane in an ultrasonic bath and then, the oil extracted using an Abencor system. The oil showed a low BaP content (1.8 µg/kg) in comparison with the oil obtained from unwashed olives (27.0 µg/kg).

As expected, the PAHs composition in olive fruits depended on the contamination source (Table 4).

[Insert Table 4]

VOO CONTAMINATION DURING THE EXTRACTION PROCESS

The factors that could affect the PAHs concentration during the olive oil extraction process were also studied. These factors include, olive washing, talc addition, scale of the extraction process and environmental pollution in the olive oil mill.

Olive washing

Olive washing is a previous step in the process of olive oil extraction in order to remove earth particles from the olive surface. The elimination of solid particles from the fruit surface might reduce the contamination level. In order to check this hypothesis, two sets of highly contaminated olive fruits were rinsed with hexane and tap water respectively. The latter was dried at 100ºC and extracted with hexane. The extracts were analyzed showing similar PAHs concentration. These facts indicate that washings with water do not eliminate PAHs from the fruit skin.

Talc Addition

Nowadays, micronized talc is the only technical coadjutant allowed by EC legislation in the VOO extraction process. In the hexane extract of talc, the PAHs concentrations were very low (ΣPAHs = 0.05 µg/kg). Assuming that the olive paste yield around 20% of oil and the talc is usually added in proportion of 1-3%, the theoretical talc contribution to the final PAH level is negligible. To verify the low effect of talc addition in the final PAH content, VOOs were obtained by an Abencor system from Arbequina and Picual varieties both with and without addition of talc (2.8%). The total PAHs concentrations were 1.0 µg/kg for Arbequina and 0.4 µg/kg for Picual, both with and without talc. In conclusion, talc addition had no effects in VOO PAHs content.

Scale of the Oil Extraction Process

To study the influence of the industrial oil extraction process on PAHs concentration in VOOs, olive fruits were processed in the experimental oil mill of the Instituto de la Grasa, located in a low 10 polluted area, using a Pieralisi M-1 system (Pieralisi España S.L., Zaragoza, Spain), tap water, 1% of talc, and following the good manufacturing practices. The olive oil mill was composed by a hammer crusher, a thermomixer at 31 ºC, and horizontal and vertical centrifuges. In Table 5, the results

shows similar PAHs content in oils obtained at industry and laboratory scale whereas differences were found according to olive origin confirming the influence of olive contamination.

[Insert Table 5]

Environmental Pollution over the Olive Mill

To evaluate this factor, an olive oil mill placed in the same area of an olive pomace oil extraction plant was examined. In this industrial plant, exhausted olive pomace paste was burn as solid fuel for drying the olive pomace and, consequently, the olive oil mill was surrounded by smoke and the VOOs were obtained in a high polluted environment. In seasons before (2001/2002 and 2002/2003), some samples contains very high PAHs levels, up to a 3.6 µg/kg of BaP and 39.6 µg/kg of total PAHs. In this olive mill, the obtained olive oils were stored in outdoor tanks with ventilation shafts, where the smoke could come into. In the season 2003/2004, every single tank was exhaustively cleaned before starting the harvest season, and to avoid the incoming of the polluted air, a valve in their ventilation shafts was installed. After the adoption of these measures, the PAHs concentrations in VOOs were variable, but they did not reach those high levels which were observed in previous crops (Table 6). The PAHs levels in the VOOs stored in the oil tanks remained constant even three month after obtention. Then, the combustion fumes seem to be the main source of contamination of the VOOs in the olive mills.

[Insert

  harvested by hand and by combine from an orchard located at Zaragoza (Spain) were used to establish the influence of diesel exhaust on the olive contamination and the effect of olive washing.4.-The effect of talc addition and the scale of oil extraction process were studied in VOOs obtained from Picual and Manzanilla varieties growing in Córdoba (Spain), Seville (Spain) and Huelva (Spain).5.-To study the effect of the environmental pollution during the oil obtaining process, VOOs were obtained by centrifugation in an olive oil mill close to an olive pomace oil extraction plant with high environmental pollution.

  of 1 mL/min. The analyzed PAHs were chrysene (Chr), one the most abundant PAH in olive oils, and those required by the Spanish legislation: benzo(a)anthracene, BaA; benzo(e)pyrene, BeP; h)anthracene, DahA; benzo(g,h,i)perilene, BghiPE and indene(1,2,3-c,d)pyrene, IP[BOE, 2001].

  -43) according to manufacturer's recommendations in order to achieve the maximum sensitivity. Parameters such as automatic gain control (AGC) and multiplier (1150 V, 10E5 gain) were set by automatic tuning. The electron energy was 70 eV and the emission current 250 µA. The optimized parameter of buffer gas was set to 0.3 mL/min helium. For the single ion monitoring, the molecular ions of each PAHs were selected: m/z 228 for BaA and Chr; m/z 252 for BbF, BkF, BaP and BeP; m/z 276 for BghiP and IP; m/z 278 for DahA.

Table 3 ]

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Table 6 ]

 6 CONCLUSIONSThe PAHs content of olive fruits depends on the environmental pollution on the olive grove and olive size. The PAHs are deposited on olive skin and they are transferred to the oil during the oil extraction. The VOO extraction process does not increase the normal PAH background if it is carried out in a clean environment. The high PAHs levels in the VOOs are due to high environmental pollution by combustion fumes, both in the olive grove and the olive mill. Moreover, accidental and significant contaminations may occur by exposure of olives to engine exhausts. Therefore, a correct fruit harvest, and a suitable storage of the obtained VOOs are needed to avoid a virgin olive oil contamination by PAH. Basic components and procedures, in Bleaching and purifying fats and oils: Theory and practise / Henry Basil Wilberforce Patterson. AOCS Press, Champaign, IL, USA. Šimko P. 2002. Determination of polycyclic aromatic hydrocarbons in smoked meat products and smoke flavouring food additives. Journal of Chomatography B 770:2-18.

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Table 1 :

 1 Morphological characteristics and PAHs concentrations (µg/kg) in olive fruits of different varieties exposed to the same pollution level.

				Morphological characteristics		[Σ Σ Σ ΣHAPs] 1
	Zone		Variety	Olives/100 g (approx.)	Medium weight (g/fruit)	Medium size (mm)	µ µ µ µg/Olive	in Olives (µg/kg)	in Oils* (µg/kg)	in Oils (µg/kg)
			Koroneiki	126	0.795	15 x 11	0.00019	0.239	1.2	1.3
			Arbequina	40	2.486	18 x 17	0.00046	0.185	0.9	0.9
	Airport		Picual	25	4.018	28 x 19	0.00051	0.142	0.7	0.8
	surrounding	Lechín Hojiblanca	24 21	4.152 4.878	25 x 20 30 x 21	0.00057 0.00079	0.137 0.162	0.7 0.8	0.8 0.8
			Ascolana	7	13.648	37 x 29	0.00140	0.103	0.5	0.5
	Urban		Arbequina	107	0.93	13 x 12	0.00019	0.203	1.0	1.0
			Picual	26	3.78	22 x 17	0.00048	0.124	0.6	0.5
	•	Calculated assuming an oil yield of 20%; 1 concentration as an average of two determinations

Table 2 :

 2 PAHs content (µg/kg) in VOOs obtained from olive fruits of different areas in the same olive orchard.

	Zone	Irrigation	Area	Variety [Σ [Σ [Σ [ΣPAHs]
			Near Road	Picual	0.9
		Trickle irrigation	Near Road	Picual	0.8
	Rural		Mountain	Picual	0.5
			Mountain	Picual	0.5
		Unirrigated
			Mountain	Picual	0.5

Table 3 :

 3 PAHs levels (µg/kg) in oils coming from olives harvested by different

	methods,

Table 4 :

 4 Ranges of PAHs compositions (%) in olives exposed to different environments

	Page 17 of 19	Food Additives and Contaminants
	PAHs Airport Surrounding Urban Area Harvested by Combine
	BaA	11.5 -13.2	7.5 -8.3	4.8 -6.1
	Chr	57.3 -46.5	20.9 -24.0	6.3 -9.2
	BeP	7.6 -10.3	14.8 -15.5	18.5 -27.0
	BbF	13.5 -16.9	17.5 -18.6	16.1 -20.0
	BkF	5.3 -6.2	7.0 -7.3	4.5 -7.3
	BaP	2.5 -3.0	5.5 -6.4	9.6 -13.7
	DahA	ND	ND	1.6 -2.4
	BghiPE	2.1 -3.3	14.8 -16.5	13.7 -18.8
	IP	2.6 -3.9	7.1 -8.3	9.9 -13.5
		ND: non detected

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Table 5 :

 5 Comparison between the most abundant PAHs (µg/kg) in VOOs samples obtained at laboratory and industrial scales.

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Table 6 :

 6 PAH content (µg/kg) in VOOs samples from olive mill under high polluted environment.

	Crop	2001/2002	2002/2003	2003/2004
	BaA	0.5 -1.9	0.3 -4.8	0.2 -0.7
	Chr	1.5 -2.6	0.9 -7.7	0.5 -1.5
	BeP	0.9 -3.2	< LOQ -7.7	ND -1.3
	BbF	0.6 -3.1	< LOQ -6.7	< LOQ -1.0
	BkF	0.3 -1.0	< LOQ -2.1	< LOQ -0.4
	BaP	0.5 -1.4	0.1 -3.6	< LOQ -0.6
	DahA	ND -0.4	< LOQ -0.9	ND -< LOQ
	BghiP	0.6 -1.7	0.5 -3.8	< LOQ -0.7
	IP	ND -1.0	ND -2.3	ND -< LOQ
	Σ Σ Σ ΣPAHs	4.9 -16.1	1.8 -39.6	0.7 -6.2

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Sample


Andrea Catalina Galvis-Sánchez

Antonio Barros

Ivonne Delgadillo

Andrea ; Galvis-Sánchez

Andrea C Galvis-Sánchez

Detection of OTA contaminated

Keywords: screening, sultanas, origin, ochratoxin A (OTA), FTIR-ATR spectroscopy

come

Introduction

Mycotoxin-producing mould species are extremely common; they can grow on a large range of substrates and under a wide range of environmental conditions. Ochratoxin A (OTA) was discovered as a metabolite of Aspergillus ochraceus in 1965 and subsequently the toxic effects of (OTA) have been reported [START_REF] Bennett | Mycotoxins[END_REF].

In addition to being a human nephrotoxin, studies involving animals indicated that OTA is a liver toxin, an immune suppressant, a potent teratogen, and a carcinogen. The occurrence of OTA in baby food, barley, corn, beer, coffee and wine as well as the analytical methods used for its detection have been well documented by [START_REF] Gilbert | Validation of analytical methods for determining mycotoxins in foodstuffs[END_REF]. Mycotoxins can enter the food chain in the field, during storage or at later points [START_REF] Delage | Ocurrence of mycotoxins in fruit juices and wines[END_REF]. The incidence of Ochratoxin A (OTA) in grapes was correlated with the presence of OTA in wine [START_REF] Sage | Fungal flora and ochratoxin A production in grapes and must from France[END_REF].

For the detection of food contamination, rapid, simple and sensitive methods with high degree of automation which can meet the specific necessities of food industries are required. Routinely methods for detection and determination of the levels of mycotoxins make use of various analytical techniques as thin-layer chromatography (TLC), high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (CG-MS) which provide reliable results but are expensive and time consuming methodologies [START_REF] Mossoba | Analysis of trichthecene mycotoxins in contaminated grains by gas chromatography/matrix isolation/Fourier transform infrared spectroscopy and gas chromatography/mass spectrometry[END_REF]. [START_REF] Krska | Advances in the analysis of mycotoxins and its quality assurance[END_REF] reported the most recent advances in the area of rapid methodologies for mycotoxins detection with their principal applications and their limitations as the 'false positive' or 'false negative' results often occurred with the ELISA method or the overestimation of the contamination when applied in some food matrixes. Until now the main applications reported for the near infrared (NIR) and the Fourier transform infrared spectroscopy (FTIR) in the food industry were for authentication of food products [START_REF] Defernez | Use of infrared spectroscopy and chemometrics for the authentication of fruit purees[END_REF][START_REF] Briandet | Discrimination of Arabica and Robusta in instant coffee by Fourier transform infrared spectroscopy and chemometrics[END_REF]) and for compositional information [START_REF] Bruno-Soares | Use of near infrared reflectance spectroscopy (NIRS) for the prediction of the chemical composition and nutritional attributes of green crop cereals[END_REF]. However the possibility of the application of spectroscopic techniques as the near infrared (NIR) and the Fourier transform infrared spectroscopy (FTIR) as a tools for screening fungal infection in agricultural commodities showed promising results (Gordon et al. 1997, Petterson and[START_REF] Pettersson | Near infrared spectroscopy for determination of mycotoxins in cereals[END_REF]. Results of the application of mid-infrared diffuse reflection (DR) and attenuated total reflection (ATR) spectroscopy for the detection of fungal infection in corn were reported by [START_REF] Kos | A comparative study of mid-infrared diffuse reflection (DR) and attenuated total reflection (ATR) spectroscopy for the detection of fungal infection on RWA2-corn[END_REF]; the outcomes of the ATR were very optimistic For any of the methodologies mentioned previously for the determination of the contamination sample preparation represents a potential problem as the heterogeneous distribution of the mycotoxin in the sample can introduce high variability on the results (Kos et al. 2003, Möller and[START_REF] Möller | Ochratoxin A in raisins and currants: basic extraction procedure used in two small marketing surveys of the occurrence and control of the heterogeneity of the toxins in samples[END_REF].

In the present work the main objectives pursued were (i) The identification of the main features of sultana's FTIR spectra by screening samples from different origins (ii) A study of the factors that could be involved in the reproducibility of the spectra and (iii)

The separation of samples contaminated with OTA at different levels of concentration from the uncontaminated ones.

Materials and methods

Samples preparation

Sultanas of different origins (viz. Chile, South Africa and Turkey) and brands were purchased at the local market in Portugal; most of the samples were bought as packages of 250 g. A total of 5 samples were analysed which included samples from Turkey (N=2), from Chile (N=2) and from South Africa (N=1).

Initially spectra of sultanas from different origins were acquired in order to recover their principal spectra characteristics. A puree of each origin was prepared separately using 5 g of fruit; this amount was selected as the heterogeneous distribution of any contamination in the sample could be masked in higher amounts. Spectra reproducibility was studied using only the samples from Chile. Since spectra reproducibility was a concern and most of the samples were under protected packed conditions, the sultanas from Chile were introduced inside four chambers preconditioned with salts of different water activities (a w ) and allowed to get equilibrium for posterior analysis. The salts used for precondition treatments were ammonium nitrate (NH 4 NO 3 ) a w = 0.62, sodium chloride (NaCl) a w = 0.75, potassium chloride (KCl) a w = 0.84 and barium chloride di-hydrate (BaCl 2 .2H 2 O) a w = 0.90 all from Sigma Chemicals, St. Louis, MO. Control 1 and Control 2 were samples without any treatment. A standard of 10 µg OTA ml -1 (Sigma Chemicals, St. Louis, MO) was used for the preparation of a calibration curve in which the standard was directly added on no contaminated sample, the volumes added in the samples cover a range of C1 = 10 C2 = 20 and C3 = 40 µg OTA kg -1 .

Instrumental Setup

All spectra were acquired using a spectrometer (Brüker IFS 55, Germany) set up for mid-infrared measurements equipped with a horizontal one single reflection ATR Golden Gate (Specac, Geramany). The software OPUS v. 5.0 (Brüker, Germany) was programmed to record each spectrum between 4000 and 600 cm -1 at a resolution of 8 cm -1 . Each spectrum was made up of 845 data points. Samples and background measurements were made by adding together128 scans for each spectrum before Fourier transformation. The interferometer was operated at a laser frequency of 10 kHz and in the single-sided directional mode. Fourier transformation was done with a Mertz phase correction, a Triangular apodization function, with a zero-filling factor of 2. At least fifteen spectrum replicates were recorded for each sample according to their origin, storage precondition and concentration of OTA.

Data analysis

Data analysis was performed using the program CATS 97 [START_REF] Barros | Contribution à la sélection et la comparaison de variables caractéristiques[END_REF]. Principal component analysis (PCA) was used for reducing the dimensionality of the data and to extract the main sources of variability. For calculations all data were arranged in a way that absorbance values were displayed in columns and each raw of data represented one sample. Outliers were detected and removed using the Mahalanobis criteria before PCA final analysis, and the datasets were SNV (standard normal deviate) corrected.

The PLS1 algorithm was used for modelling the regression. Standard errors of calibration and cross-validation were calculated to asses the quality of the regression.

Results and discussion

Figure 1 shows typical spectra of sultanas from three origins and different brands. The analysis of the whole spectra show small differences specially in the regions between 3600 to 2850 cm -1 which correspond to the -O-H and -C-H groups (I), in the region of 1725 to 1570 cm -1 which show bands arising largely from pectins and in the fingerprint region from 1250 to 1000 cm -1 (II). The PCA analysis of the sultanas shows a separation between the samples according to their origin (figure 2). The loading profile of PC1 (figure 3a) shows that the separation of the sultanas by their origin is due mainly to water, fat and sugar contents with bands located at 3568, 1720 and 1022 cm -1 , respectively. The loading profile of PC2 (figure 3b) confirms that samples from South Africa had high water content with a positive PC1 and PC2 (figure 1). Samples from Chile were characterized by a band at 1720 cm -1 , negative PC1, corresponding to the fat content. Finally sultanas from Turkey had higher sugar content than those from South Africa; the former ones having a negative PC2, with a band characteristic of fructose at 1049 cm-1 (figure 3b).

[Insert figure 2 and 3 about here] A lack of reproducibility was observed in the spectra of sultanas acquired on different days of analysis. Because some changes could occur once fruit were removed from the package, contributing to the problems of spectral reproducibility, the pre-condition of sultanas with different water activity (a w ) was assessed so that the methodology could avoid or reduce this artefact, allowing the separation of the contaminated samples from the uncontaminated ones. Furthermore, this precondition step may allow the addition of new samples to build the model.

In order to see the effect of the precondition treatment, only samples from Chile were used. The spectral reproducibility was much better in samples pre-conditioned with water activities set at 0.62 and 0.75 than in those with the a w set at 0.84 and 0.90 and in the control ones, which showed higher degree of dispersion in the cluster formation (figure 4). The PCA analysis also showed a separation of the samples according to the water activity treatment, sultanas from Chile with a w of 0.75 or higher were separated from those with lower a w and from the ones used as control (figure 4). The samples with an a w of 0.75 or higher were characterised by two bands, one located at 3537 cm -1 and the other found at 1639 cm -1 , corresponding to the -O-H group shown in figure 5.

On the other hand, the samples treated with ammonium nitrate (a w = 0.62) and the control samples were characterised by the band placed at 1010 cm -1 corresponding to glucose (Figure 5). For the PCA analysis of samples pre-conditioned at different a w various spectral regions were analysed, the 1250-1000 cm -1 region was chosen as giving the best results in terms of the introduction of new samples to an existing set of data. As it can be seen in figure 6 the scores for the scatter plot of sultanas from Chile analysed on two different days showed that the precondition of the samples to an a w of 0.62 allowed their introduction into a set of samples previously analysed (control 1 and 2). It is important to note that in this region the main features arise mainly from coupled C-O modes corresponding to fructose and glucose carbohydrates, the former one is a very hygroscopic compound and by the pre-condition treatment at 0.62 this carbohydrate might be stable (Figure 7a,b).

[Insert figure 6 and 7 about here]

The PCA analysis of samples from Chile contaminated with different concentrations of OTA showed a separation of the samples with concentrations of OTA of C2 = 20 and C3 = 40 µg kg -1 from those with the lowest concentration C1 = 10 µg kg -1 and from the control samples (Figure 8). The intensities of bands located at 3160 cm -1 , and 1550 cm - 1 and in the fingerprint region were very high as is shown in the figure 9.

[Insert figure 8 and 9 about here] A cross validation was carried out using leave-3-out spectra and PLS1 model was performed using the spectra obtained from the contaminated samples from Chile as an attempt to obtain a calibration curve for OTA. The spectral region chosen which gave the best results for the calibration was located between 1550 and 600 cm -1 . The number of latent variables chosen for PLS1 was 4, because the root-mean-square error of cross validation, which is the measure of the quality of predicted values, reached a minimum there. Results of PLS1 model and B coefficients are displayed in the figure 10a andb, which shows a good correlation between the actual concentrations of the contaminated samples and by the FTIR-ATR predicted concentrations (Table 1). The RMSEC and RMSEP (Table 1) were high which could be by the inherent variability of the samples and by the lack of more levels of OTA concentration.

[Insert figure 10

Conclusions

The main outcomes of the methodology presented here show the possibility of use the FTIR-ATR as a simple procedure for screening OTA in dried sultanas samples: As for any methodology applied for the detection of contamination in food matrices there are parameters that need to be optimised in order to get meaningful results; as in this case the inherent variability of the samples due their origin and the way they were preserved were shown to be critical points that influenced the reproducibility of the spectra.

(3) (4) (a) 18.8 RMSEP (%) (b) 43.4

  on the classification of contaminated samples was very high, preceded by a simple methodology for sample preparation, and with the perspective of automatization introducing the likelihood of the on-line control during the process.

[

  Insert figure 4 and 5 about here]

Figure 1 .

 1 Figure 1. Typical spectra of sultanas from different origins and brands: (1) S. Africa, (2) Turkey A, (3) Turkey B, (4) Chile A and (5) Chile B.

Figure 2 .Figure 4 .Figure 8 .Figure 6 .

 2486 Figure 2. Scores scatter plot (PC1 vs. PC2) of sultanas according their origin.

Figure 3 .Figure 5 .

 35 Figure 3. PC1 (a) and PC2 (b) loading profile plots of sultanas according their origin.

Figure 7 .

 7 Figure 9. PC1 Loading plot of sultanas from Chile contaminated with OTA.

  about here]

	[Insert table 1 about here
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	7

Table 1 .

 1 PLS1 regression statistics of sultanas from Chile for an 4 Latent Variable OTA (µg kg -1 ) model.

	Latent Variables	4
	R 2	0.95
	RMSEC (%)

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants


Pavel Dostálek
email: pavel.dostalek@vscht.cz

Igor Hochel

Enrique Méndez

Alberto Hernando

D Gabrovská

Immunochemical determination of gluten in malts and beers

Keywords: Gluten, gliadin, peptide, protein, beer, malt, barley, wheat, prolamin content, coeliac disease

published or not. The documents may come

Introduction

Coeliac disease (CD) is a digestive disease that damages the small intestine and interferes with absorption of nutrients from food [START_REF] Lewis | Celiac Disease, Beer, and Brewing[END_REF]. People who suffer from CD cannot tolerate a protein called gluten, which is found in wheat, rye, barley, and possibly oats. Gluten consists generally from prolamin and glutelin proteins.

Wheat prolamin is known as gliadin and barley prolamin as hordein. When people 3 with CD eat foods containing gluten, their immune system responds by damaging the small intestine. Specifically, tiny finger-like protrusions, called villi, on the lining of the small intestine are lost. Nutrients from food are absorbed into the bloodstream through these villi. Without villi, a person becomes malnourished. Because the body's own immune system causes the damage, coeliac disease is considered as an autoimmune disorder [START_REF] Lewis | Celiac Disease, Beer, and Brewing[END_REF]. However, it is also classified as a disease of malabsorption because nutrients are not absorbed. If a person responds to the glutenfree diet (GFD), the physician will know for certain that the diagnosis of coeliac disease is correct. A GFD means avoiding all foods that contain wheat (including spelt and triticale), rye, barley, and possibly oats [START_REF] Poms | Methods for allergen analysis in food: a review[END_REF]). Whether there is some maximum tolerable daily intake of gluten which can be estimated for treated coeliacs is an important issue. There is a study which suggests that 10 mg should be considered as the maximum tolerable daily intake of gluten in treated coeliacs [START_REF] Catassi | The Italian study on gluten microchallenge: preliminary results[END_REF]. On the other hand, there is another study [START_REF] Biagi | A milligram of gluten a day keeps the mucosal recovery away: A case report[END_REF] which reports that even a milligram of gluten a day keeps the mucosal recovery away.

As each person suffering from CD has different tolerance to gluten ingestion, we cannot fix general safe daily intake limit for gluten.

With an annual consumption of almost 160 litres of beer per capita, the Czech Republic is the world's number one beer nation. Beer constitutes an important part of the diet for a significant section of the Czech population. Beer contains only about 0.3% proteins or polypeptides, the principal source of which is malt. Malt is produced mostly only from wheat or barley. These cereals, however, also comprise prolamins that are associated with CD. Knowledge about distribution and final levels of these proteins or their "toxic" fragments in beer is essential to ensure the safe consumption of this drink by coeliacs [START_REF] Ellis | Detection and estimation of the barley prolamin content of beer and malt to assess their suitability for patients with coeliac disease[END_REF][START_REF] Ellis | Demonstration of the presence of coeliac-activating gliadin-like epitopes in malted barley[END_REF][START_REF] Kanerva | Determination of Prolamins in Beers by ELISA and SDS-PAGE[END_REF]; This study has been undertaken to find out whether beer contains any gluten that could be toxic to coeliacs.

Materials and methods

Determination of gluten

Two sandwich enzyme-linked immunosorbent assay (ELISA) commercial kits were used for gluten determination, the RIDASCREEN ® Gliadin kit base on the monoclonal antibody R5 [START_REF] Valdés | Innovative approach to low-level gluten determination in foods using a novel sandwich enzyme-linked immunosorbent assay protocol[END_REF][START_REF] Hernando | Measurements of hydrolysed gliadins in malts, breakfast cereals, heated/hydrolysed foods, whiskies and beers by means of a new competitive R5 ELISA[END_REF]) (R-Biopharm AG, Darmstadt, Germany, prod. no. R 7001) and Gliadin ELISA kit based on two monoclonal antibodies [START_REF] Gabrovská | Gluten-free diet and the situation in the Czech Republic[END_REF][START_REF] Gabrovská | Collaborative study of a new developed ELISA kit for gluten determination[END_REF][START_REF] Gabrovská | Gluten determination by gliadine enzyme-linked immunosorbent assay kit: Interlaboratory Study[END_REF]) (Immunotech a Beckman Coulter Company, Prague, Czech Republic, prod.

No. IM3717) with a detection limit for gluten of 3 and 20 mg kg -1 respectively. The instructions manufacturers for their proper use were followed. As RIDASCREEN ® Gliadin kit is effective for intact prolamins but not for hydrolysed ones, hydrolysed peptides in beers were also analysed by a home-made competitive ELISA kit based on R5 antibody [START_REF] Ferre | Measurement of hydrolysed gliadins by a competitive ELISA based on monoclonal antibody R5: analysis of syrups and beers[END_REF][START_REF] Hernando | Measurements of hydrolysed gliadins in malts, breakfast cereals, heated/hydrolysed foods, whiskies and beers by means of a new competitive R5 ELISA[END_REF]. This system (Méndez, García and Ferre 2005) has a detection limit 6 ppm of gluten, reproducibility 7.7 %, repeatability 4.7 % and nearly quantitative recovery (90-96 %). Gliadin standard (PWG gliadin standard -candidate reference material -IRMM-480) was used for calibration ( [START_REF] Eckert | The PWG gliadin, a new reference material[END_REF].

Sample preparation

Five grams of grains or malts were grinded to powder. A 1.0 g amount of representative flour was extracted with 10 ml of 60 % (v/v) ethanol solution by 5 shaking for 30 minutes, followed by centrifugation at 2500 g for 30 minutes [START_REF] Valdés | Innovative approach to low-level gluten determination in foods using a novel sandwich enzyme-linked immunosorbent assay protocol[END_REF]. One ml of beer or brewing semiproducts (after removal of carbon dioxide) was mixed with 9 ml of ethanol solution (60% v/v). Beers with low prolamin concentrations were concentrated by freeze-drying.

Analyses of wheat, barleys, barley malts and malts from other cereals

Eleven commercial malts made from barley, wheat, spelt and rye were analysed for gluten content. These commercial malts were purchased from Weyermann Specialty Malting Company (Bamberg, Germany). Five barley commercial malts were prepared from five different barley varieties by the same malting technological process in Nymburk malt house (Malteries Soufflet ČR, Plc., Czech Republic). All samples were analysed for gluten content by RIDASCREEN ® Gliadin kit except of wheat and wheat malt samples, which were analysed by Gliadin ELISA kit.

Technological trials -brewing the beer

Analyses of beers

Nineteen commercial beers which represent all principle brands of beers available on the Czech market were analysed for gluten content. All beers were analysed for gluten content by RIDASCREEN ® Gliadin kit and only some beers with very low gluten content detected by RIDASCREEN ® Gliadin kit were also analysed by competitive ELISA kit based on R5 antibody. One commercial beer was additionally treated for removal of proteins by silica gel -Stabiquick strong and Stabifix super (2 g⋅l -1 ) from Stabifix Braeurei-Technik (München, Germany). This trial was done for confirmation, if there is a possibility to reduce the level of gluten in final beer

Results and discussion

Barley (wheat), hop, yeast and water are basic raw materials for brewing the beer.

Gluten or products of their hydrolysis detected in beer can come only from barley (wheat). Malt for production of beer is produced from barley and wheat by malting process, which consists of steeping, germination and kilning. The activities of amylase enzymes and proteolytic modification in grains increase during the malting process. Wheat and malt prepared from the wheat were analysed for gluten content.

Wheat contained 50.4 ± 1.8 grams of gluten per kg and corresponding malt 68.0 ± 4.0 grams of gluten per kg. These gluten levels were determined by Gliadin ELISA kit in which the used antibody (against wheat gliadin) has cross-reactivity with barley prolamins only about 20% [START_REF] Gabrovská | Collaborative study of a new developed ELISA kit for gluten determination[END_REF][START_REF] Gabrovská | Gluten determination by gliadine enzyme-linked immunosorbent assay kit: Interlaboratory Study[END_REF]). The higher level of gluten in malt compared to original grain is fully confirmed by analysis of gluten in three barley varieties from two different growing areas and the corresponding malt samples produced under the same malting conditions (Figure 1). Gluten analysis of these samples was performed by RIDASCREEN ® Gliadin kit. In this case, the used monoclonal antibody detects the gliadin fractions from wheat and corresponding prolamins from barley and rye [START_REF] Valdés | Innovative approach to low-level gluten determination in foods using a novel sandwich enzyme-linked immunosorbent assay protocol[END_REF] equally. Content of gluten in cereals and malts are lower then the theoretical ones [START_REF] Belitz | Food Chemistry[END_REF]) due to used analytical technique. These results were found also in previous work [START_REF] Ellis | Detection and estimation of the barley prolamin content of beer and malt to assess their suitability for patients with coeliac disease[END_REF]). Table I shows gluten content in commercial cereal malts. The highest content of gluten was ascertained in wheat pale malt. The content of gluten in several kinds of barley malt varied from 18.8 to 45.0 g kg -1 . The gluten content in spelt and rye malt ranged approximately over the same interval.

[ Insert Figure 1 about here ] [ Insert Table I about here ] The gluten balance was examined from raw materials to final beer during the brewing process (Table II). Total gluten content in malt, used for brewing beer, was 100 % gluten source. Malt extract was transferred into solution during the mashing process in the brewing house. During the mashing process in the brewing house most of proteins were precipitated and only some proteins were spit into simple polypeptides [START_REF] Lewis | Celiac Disease, Beer, and Brewing[END_REF]. The majority of precipitated proteins remained in the spent grain after lautering process. Only a small part of gluten (1.75 %) passed from malt to sweet wort. Negligible consequent gluten depletion was noted in hopped wort after hop boiling (1.70 %). The pH decreased during the primary and secondary adsorption onto the yeast cell surface (only 0.21 % of the original gluten content remains in beer). After the filtration process beer is colloidally stabilised by removal of proteins and polyphenolic substances by adsorption on polyvinylpolypyrrolidone (PVPP) and kiesegel (silica gel). This process (less then 0.11 % of original content of gluten) resulted in decreasing of gluten content (Table II).

[ Insert Table II about here ]

As shown in Table III, gluten level of commercial beers from the Czech market are very variable. The gluten concentration increases from alcohol free beer (<3.0), lager beers (<3.0-8.7 mg l -1 ), stouts (9.0-15.2 mg l -1 ), to wheat beers (10.6-41.2 mg l - 1 ). The gluten content of some Czech lager beers analysed by sandwich RIDASCREEN ® Gliadin kit and by competitive ELISA kit based on R5 antibody are shown in Table IV. The both kits are based on R5 antibody. It is evident that not all Czech lager beers contain less than 20 mg of gluten per litre. Since 1996, the legislation threshold for foods gluten-free by nature is 20 mg kg -1 of gluten (10 mg kg - 1 of gliadin) [START_REF] Deutsch | Topic relating to Codex, Labelling and Symbol[END_REF]. The R5 antibody is capable of recognising several and small repetitive celiac-toxic epitopes QQPFP, LQPFP, QLPYP, QLPTP, QQSFP, QQTFP, PQPFP, QQPYP and PQPFP in prolamin molecules [START_REF] Osman | A monoclonal antibody that recognizes a potential coeliac-toxic repetitive pentapeptide epitope in gliadins[END_REF][START_REF] Kahlenberg | Monoclonal antibody R5 for detection of putatively coeliac-toxic gliadin peptides[END_REF]). The sandwich R5-ELISA needs at least two specific celiactoxic epitopes. Prolamins presented in beer are partially hydrolysed in fragments with one or a few celiac-toxic epitopes. Consequently these small fragments containing a single celiac-toxic epitope cannot be measured by the conventional sandwich R5- IV. The competitive R5-ELISA yields prolamin contents higher than the sandwich ELISA. The competitive R5-ELISA is a reliable assay for determination of gluten-free beers based on wheat or barley malt extract as had been demonstrated in some beers based on barley malt and approved by the Finnish Coeliac Society for consumption by coeliacs [START_REF] Hernando | Measurements of hydrolysed gliadins in malts, breakfast cereals, heated/hydrolysed foods, whiskies and beers by means of a new competitive R5 ELISA[END_REF]. Prolamins present in beer are partially hydrolysed and these small fragments always contain proline (Dale et al. 1989;[START_REF] Siebert | Effects of Protein-Polyphenol Interactions on Beverage Haze, Stabilization and Analysis[END_REF][START_REF] Siebert | Comparison of Methods for Measuring Protein in Beer[END_REF]. The haze-forming activity of a polypeptide in beer depends greatly on its proline content [START_REF] Siebert | Effects of Protein-Polyphenol Interactions on Beverage Haze, Stabilization and Analysis[END_REF]. Haze-forming polyphenols have at least two binding groups, each of which has at least two hydroxyl groups on an aromatic ring for binding of proline part of protein [START_REF] Siebert | Effects of Protein-Polyphenol Interactions on Beverage Haze, Stabilization and Analysis[END_REF]). As proline is structurally similar to pyrrolidone the PVPP can bind polyphenols [START_REF] Siebert | Comparison of Methods for Measuring Protein in Beer[END_REF][START_REF] Leiper | Optimising Beer Stabilisation by the Selective Removal of Tannoids and Sensitive Proteins[END_REF]. Measurement of prolamin concentration thus can represent excellent tool for prediction of colloidal beer stability [START_REF] Siebert | Effects of Protein-Polyphenol Interactions on Beverage Haze, Stabilization and Analysis[END_REF][START_REF] Siebert | Comparison of Methods for Measuring Protein in Beer[END_REF].

[ Insert Table III about here ] [ Insert Table IV about here ]

Conclusions

Two commercial sandwich enzyme-linked immunosorbent assays (Gliadin ELISA kit and RIDASCREEN ® Gliadin kit) and competitive ELISA kit were used for gluten determination in barley, wheat, malts and beers. Gliadin ELISA kit is applicable only for intact prolamins from wheat and wheat malts. RIDASCREEN ® Gliadin kit can be The gluten level ranges in barley wheat, rye and spelt malts were found to be 18.8-45.0, 44.0-68.0, 41.6, and 21.2 g kg -1 , respectively. The amount of these proteins, decreased from 100 % in malt to less then 0.11% in stabilized beer during the mashing process, primary and secondary fermentation and stabilization process.

Gluten concentrations were found to increase from alcohol free beer (<3.0), lager beers (<3.0-8.7 mg⋅l -1 ), stouts (9.0-15.2 mg⋅l -1 ), to wheat beers (10.6-41.2 mg⋅l -

  . 2005). Comprehensive quantitative data on the gluten content of beers is still lacking, the same situation is for gluten balance during the brewing process.

  Beer for gluten balance trial was prepared in brewery pilot plant (50 litres of wort) in the Department of Fermentation Chemistry and Bioengineering in the Institute of Chemical Technology Prague. Mashing process was double mash process with mashing-in temperature of 50 °C. Only barley malt of Pilsen type was used for mashing. Wort was separated by a classical lautering process. Hop boiling time was 90 minutes. Dried natural Czech Saaz hop was used only. Final wort bitterness was 35 BU (EBC bitterness units -mg of bitter substances per litre of wort). After hot trub separation fermentation was carried out with bottom-fermenting yeast strain 95, purchased from Research Institute of Brewing and Malting, Plc. (Prague, Czech Republic), using 14 .10 6 viable cells per ml. The primary fermentation process took place at 12 °C. A cold maturation was carried out for 21 days at 1 °C. After kieselguhr stabilised by PVPP (30 g hl -1 ) and by silica gel (Stabiquick strong -30 g hl -1 ).

  It caused precipitation of some polypeptides and their

  prolamins are always underestimated. The efficiency of the competitive R5-ELISA over the sandwich is superior in quantifying hydrolysed prolamins as demonstrated in the analysis of beers in Table

  prolamins from wheat, barley and rye, while the competitive R5-ELISA is ideal for testing hydrolysed wheat, barley and rye prolamins in beer.

Figure 1 .

 1 Figure 1. Content of gluten in some barley varieties and corresponding barley malts.

F

  deviation (n=3); b All samples were analysed by RIDASCREEN ® Gliadin kit.

  Content of gluten in some Czech lager beers (analysed by two methods)

Table I .

 I Mean content of gluten in some malt types

	_____________________________________________________________
	Malt type		Gluten (g kg -1 ) ac
	_____________________________________________________________
	Caraaroma (barley)		42.0 ± 0.2
	o r Melanoid (barley) Cararred (barley) F o r	20.4 ± 0.2 20.6 ± 0.2
	Carafa (barley)	P P	45.0 ± 0.2
		e e
	Pilsner (barley)	e r 18.8 ± 0.6 e r
	Smoked (barley)		R 20.8 ± 1.0 R
	Munich (barley)		e 20.2 ± 0.2 e
	Wheat chocolate (Wheat) b	v i e 44.0± 1.6 v i e
	Wheat pale (Wheat) b	68.0 ± 4.0	w w
	O n l y O n l _____________________________________________________________ Spelt chocolate (Spelt) 21.2 ± 0.4 Rye chocolate (Rye) 41.6 ± 1.4 a ±standard deviation (n=3); b Sample analysed by Gliadin ELISA kit y
	c All samples except of wheat samples were analysed by RIDASCREEN ® Gliadin kit
			16 17

Table II .

 II Balance of gluten in course of the brewing process

	_____________________________________________________________
	Raw material,	Gluten b (mg kg -1 )	(%) a
	semiproducts,
	product _____________________________________________________________ Malt 18780 100 F o r
	Sweet wort	49.4 P e	1.75
	Wort	48.0	e r	1.70
	Beer	6.0	R	0.21
	Stabilised beer _____________________________________________________________ <3.0 e <0.11 v i e
			w
			O n l
				y
			18

a Content of gluten in malt represents 100% b All samples were analysed by RIDASCREEN ® Gliadin kit.

Table III .

 III Content of gluten in beers from the Czech market

	_____________________________________________________________
	Beer	Gluten (mg l -1 ) ab

). Only 84 % of nineteen beer samples (analysed by RIDASCREEN ® Gliadin kit) was safe for consumption of coeliacs from point of view legislation limit (20 mg⋅l -1 ).The number of safe beer samples dramatically decreased to 30 %, if the samples were analysed by competitive ELISA. Nevertheless, there is no safe beer produced from barley or wheat regarding the maximum tolerable daily intake of gluten (10 mg) and different tolerance of the patients suffering coeliac disease to gluten level.

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Acknowledgement

This study was supported by the Ministry of Education, Youth and Sports of the Czech Republic (Research Centre 1M6215648902) and the Ministerio de Ciencia y Tecnología AGL2005-02721.