Alexandre Esteban

Lourdes Abarca

Rosa Bragulat

Javier Cabañes

Effect of

Keywords: Aspergillus niger, coffee, grapes, raisins, ochratoxin A, pH

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Tel: 34 93 5811749 Fax: 34 93 5812006 e-mail: Javier.cabanes@uab.es Introduction Among the different mycotoxins which merit special concern for the hazard they represent in food commodities, ochratoxin A (OTA) deserves particular attention.

OTA is a nephrotoxic mycotoxin which also possesses carcinogenic, teratogenic, genotoxic and immunosuppressive potential [START_REF] O'brien | Ochratoxin A: the continuing enigma[END_REF]. The International Agency for Research on Cancer [START_REF] Iarc | Some naturally occurring substances: Food items and constituents, heterocyclic aromatic amines and mycotoxins[END_REF] has classed OTA as a possible human carcinogen (group 2B). The European Commission has established limits for OTA in cereals, dried vine fruits, wine, coffee and infant foods. These limits will be reviewed in the short term and maximum limits for more products such as cocoa, beer, meat products, spices or liquorice will be included (Commission Regulation 2002, 2004[START_REF] Montville | Approximate pH of foods and food products[END_REF].

OTA was until recently believed to be produced only by Aspergillus ochraceus and by Penicillium verrucosum. The description of OTA production by two species belonging to Aspergillus section Nigri, Aspergillus niger var. niger [START_REF] Abarca | Ochratoxin A production by strains of Aspergillus niger var. niger[END_REF] and Aspergillus carbonarius [START_REF] Horie | Productivity of ochratoxin A of Aspergillus carbonarius in Aspegillus section Nigri[END_REF], may contribute to explain the widespread OTA contamination reported in some food and feed products. This distribution was unclear only taking into account the incidence and natural occurrence of the previously described OTA-producing species.

Within Aspergillus section Nigri it is known that isolates included in the A. niger aggregate can produce a range of different toxic metabolites [START_REF] Frisvad | Mycotoxin production by common filamentous fungi[END_REF] but it is the ability of some of their members to produce OTA what deserves special concern. It is considered that in substrates such as grapes, raisins and wine the source of OTA contamination detected is due to A. carbonarius and some species included in the A. niger aggregate [START_REF] Abarca | Taxonomy and significance of black aspergilli[END_REF]). Furthermore, it is worth noting that A. niger products hold the GRAS (Generally Regarded as Safe) status from the United States, Food and Drug Administration and are used as a source of enzymes and organic acids for food processing, despite the reported ability to produce OTA [START_REF] Bigelis | Fungal enzymes and primary metabolites used in food processing[END_REF].

The taxa included in the A. niger aggregate are extremely difficult to distinguish one from each other by morphological means. In an attempt to solve the classification of these fungi, several molecular techniques have been carried out in order to clarify the taxonomy and divide these taxa into two or more species [START_REF] Abarca | Taxonomy and significance of black aspergilli[END_REF]). On the basis of their ITS-5.8S rDNA restriction fragment length polymorphism (RFLP), the strains were grouped in two patterns, designated N and T [START_REF] Accensi | New PCR method to differentiate species in the Aspergillus niger aggregate[END_REF]. To date the OTA-producing isolates with RFLP pattern known are classified as type N [START_REF] Accensi | Distribution of ochratoxin A producing strains in the A. niger aggregate[END_REF][START_REF] Cabañes | What is the source of ochratoxin A in wine?[END_REF][START_REF] Abarca | Aspergillus carbonarius as the main source of ochratoxin A contamination in dried vine fruits from the Spanish market[END_REF][START_REF] Leong | Occurrence of fruit rot fungi (Aspergillus section Nigri) on some drying varieties of irrigated grapes[END_REF][START_REF] Esteban | Effects of temperature and incubation time on production of ochratoxin A by black aspergilli[END_REF].

Little is known about the environmental factors which can affect the OTA production by species of the A. niger aggregate. In a previous study on the effect of temperature, the A. niger aggregate strains showed the highest levels of OTA in Yeast Extract Sucrose (YES) agar at 20-25ºC [START_REF] Esteban | Effects of temperature and incubation time on production of ochratoxin A by black aspergilli[END_REF]).

As little is known about the influence of pH, the aim of this study was to establish the effect of pH on growth and OTA production by different species belonging to the A. niger aggregate at 25ºC over a period of 30 days. The strains were selected to

Material and methods

Fungal strains and inocula preparation

Twelve isolates of the A. niger aggregate were used in this study (ten of N RFLP pattern and two of T pattern). The reported OTA production, source of isolation and RFLP pattern is shown in Table I. The strains were grown on malt extract agar [START_REF] Pitt | Fungi and Food Spoilage[END_REF] for 7 days at 25ºC. Conidia suspensions of each isolate were prepared in an aqueous solution of 0.05% Tween 80. After filtering through sterile cheesecloth they were adjusted to approximately 10 6 -10 7 conidia per ml as determined by a counting chamber.

Culture conditions

OTA production was determined on two culture media: yeast extract sucrose (YES) agar and Czapek yeast extract (CYA) agar [START_REF] Pitt | Fungi and Food Spoilage[END_REF]. The pH of the culture media was varied from 2 to 10. Adjustment of pH was performed before autoclaving by adding HCl (1 and 10 N) and NaOH (1 N). The pH value was measured using a pHmeter GLP21 (Crison Strumenti S.p.A., Carpi, Italy). The sterilization of the media adjusted at pH 2, 3 and 4 was carried out by autoclaving the agar separately from the rest of components in order to avoid solidification problems.

The pH of each media was checked after sterilization. Each plate was point inoculated with 1 µl of the adjusted suspension. Plates were incubated at 25ºC. Each assay was performed in duplicate. OTA production was determined after 5, 10, 15, 20 and 30 days of incubation at each pH value following a previously described high-pressure liquid chromatography (HPLC) screening method [START_REF] Bragulat | An easy screening method for fungi producing ochratoxin A in pure culture[END_REF], being efficient at the range of pH tested. On each sampling occasion, three agar plugs were removed from different points of the colony and extracted with 0.5 ml of methanol. The extracts were filtered and injected into the HPLC. OTA detection and quantification was made by a Waters LCM1 chromatograph with a fluorescence detector Waters 2475 (excitation wavelength: 330 nm/emission wavelength: 460 nm). Twenty µl of each extract were applied. The mobile phase, with a flow rate of 1 ml/min, consisted of the following isocratic program: acetonitrile, 57%; water, 41% and acetic acid, 2% [START_REF] Bauer | Ochratoxin A in der Nahrungsmittelkette[END_REF]. The extracts with the same retention time as OTA (around 6.8 min), were considered positive. Confirmation was made through derivatization of OTA in its methyl-ester [START_REF] Hunt | Confirmation of ochratoxin A by chemical derivatisation and high-performance liquid chromatography[END_REF]. The detection limit of the extraction procedure and the HPLC technique was 0.02 ng OTA and the quantification limit of HPLC technique with the extraction procedure was 0.01 µg g -1 for this mycotoxin.

Data analysis

Data obtained were analyzed statistically by means of the one-way analysis of variance test and Student's test. All statistical analyses were performed using SPSS software (Version 12.0). The twelve strains grew in CYA and YES media adjusted from pH 2 to10. According to our results, the pH range for growth in Aspergillus niger was previously reported to be from 1.4 to 9.8 [START_REF] Panasenko | Ecology of microfungi[END_REF].

Results and discussion

F

Six out of the 12 strains tested produced detectable levels of OTA in both media.

Tables II andIII show the concentration detected at each pH value and incubation time in CYA and YES medium respectively. In CYA medium most of the strains produced OTA at all the pH values tested. The strain CBS 618.78 produced OTA from pH 4 to 8 and A-943 was a weak OTA-producing strain with detected values very close to the detection limit of the technique at some pH values. In YES medium, the strains produced OTA from pH 2 (strains A-942, A-943 and CBS 139.52) or pH 3 (strains A-75 and A-136) to pH 10. However, this range was lower for the strain CBS 618.78 which produced OTA from pH 5 to 8.

Mean OTA concentration produced at each pH value by the six positive strains is shown in Figure 1. The concentration achieved by all the strains in YES was higher than in CYA medium except at pH 3 (Figure 1). At this pH value the strains showed a slight higher OTA production in CYA medium. The total mean OTA concentration produced in YES medium (4.42 µg g -1 ) was significantly higher (Student's t P<0.01) than in CYA medium (1.38 µg g -1 ) which supports that YES is better than CYA medium for OTA production. This result was in accordance with previous studies which reported the suitability of YES medium to test OTA production by A. niger aggregate strains [START_REF] Bragulat | An easy screening method for fungi producing ochratoxin A in pure culture[END_REF][START_REF] Esteban | Effects of temperature and incubation time on production of ochratoxin A by black aspergilli[END_REF]). On the contrary, CYA medium has been reported to be more suitable than YES medium in A. carbonarius [START_REF] Bragulat | An easy screening method for fungi producing ochratoxin A in pure culture[END_REF][START_REF] Esteban | Effects of temperature and incubation time on production of ochratoxin A by black aspergilli[END_REF][START_REF] Esteban | Influence of pH and incubation time on ochratoxin A production by Aspergillus carbonarius in culture media[END_REF]. In both media most of the strains produced OTA after only 5-10 days of incubation and showed different optimal values for OTA production. In CYA medium (Table 2) the strains showed the optimal OTA production within the range pH 3-8. In YES medium (Table 3) the optimal conditions for OTA production were reached within the range pH 5-10 and always after 5-10 days of incubation. In both media, the strain A-943, isolated from grapes, showed the lowest OTA production in comparison with the rest of strains studied.

In aggrement with previous studies [START_REF] Esteban | Effects of temperature and incubation time on production of ochratoxin A by black aspergilli[END_REF]) the OTA content decreased with increasing incubation time. It has been suggested that this may be due to the fact that the strains remove and assimilate the phenylalanine moiety from the OTA molecule as other nitrogen sources become exhausted [START_REF] Téren | Immunochemical detection of ochratoxin A in black Aspergillus strains[END_REF].

It is not known yet why the percentage of OTA-producing strains in the A. niger aggregate is low [START_REF] Abarca | Taxonomy and significance of black aspergilli[END_REF]), nor if this is due to a genetic based feature. To clarify this fact it is necessary to study the effect of different environmental conditions on OTA production. In this study, the six strains initially considered as OTA negative did not produce the mycotoxin at any of the pH values tested for the period of 30 days. In a previous study, these six strains did not produce OTA at any of the temperatures tested [START_REF] Esteban | Effects of temperature and incubation time on production of ochratoxin A by black aspergilli[END_REF]). The strains with the RFLP pattern of type T did not produce OTA in the range of pH values assayed. To date OTA production has never been demonstrated in type T strains [START_REF] Accensi | Distribution of ochratoxin A producing strains in the A. niger aggregate[END_REF][START_REF] Cabañes | What is the source of ochratoxin A in wine?[END_REF][START_REF] Abarca | Aspergillus carbonarius as the main source of ochratoxin A contamination in dried vine fruits from the Spanish market[END_REF][START_REF] Leong | Occurrence of fruit rot fungi (Aspergillus section Nigri) on some drying varieties of irrigated grapes[END_REF][START_REF] Esteban | Effects of temperature and incubation time on production of ochratoxin A by black aspergilli[END_REF]. There is no previous published work regarding the effect of pH on OTA production by A. niger aggregate strains. Nevertheless, in the case of A. carbonarius it has been recently reported that the strains are able to produce OTA in a broad pH range (pH 2 to 10) [START_REF] Esteban | Influence of pH and incubation time on ochratoxin A production by Aspergillus carbonarius in culture media[END_REF]. The results obtained here with the A. niger aggregate strains state that they are also able to grow at a wide range of pH values (pH 2-10). This would explain why A. niger is so commonly isolated from a great variety of food commodities and is considered the most common Aspergillus species responsible for the post-harvest decay of fresh fruit [START_REF] Pitt | Fungi and Food Spoilage[END_REF]. The black aspergilli belonging to the A. niger aggregate are the predominant Aspergillus species isolated from grapes. They are isolated along the different developmental stages of berries although it is at harvesting when the highest levels are observed [START_REF] Battilani | Occurrence of ochratoxin A-producing fungi in grapes grown in Italy[END_REF]Serra et al. 2003;[START_REF] Bau | Ochratoxigenic species from Spanish wine grapes[END_REF]. The black spores provide protection from sunlight and UV light, providing a competitive advantage in such habitats [START_REF] Pitt | Fungi and Food Spoilage[END_REF]. It is worth mentioning that the pH of grapes increases during ripening, achieving a final pH range of 3-4 [START_REF] Splittstoesser | Fruits and fruit products[END_REF]FDA 2003). Aspergillus niger has been also reported to be the predominant fungi in dried vine fruits [START_REF] Heenan | Ochratoxin A production by Aspergillus carbonarius and A. niger isolates and detection using coconut cream agar[END_REF][START_REF] Abarca | Aspergillus carbonarius as the main source of ochratoxin A contamination in dried vine fruits from the Spanish market[END_REF] which show pH ranges of 3.8 to 4.1 (FDA 2003).

Aspergillus niger aggregate strains have been also isolated from different stages of coffee beans, before and after harvesting [START_REF] Taniwaki | The source of ochratoxin A in Brazilian coffee and its formation in relation to processing methods[END_REF]. The pH of unfermented coffee beans can range from 5.4 to 6.4 [START_REF] Thompson | Cocoa and coffee[END_REF] and therefore, the time before the arrival of the cherries at the factory may represent a high risk for OTA production [START_REF] Bucheli | Research on the origin, and on the impact of postharvest handling and manufacturing on the presence of ochratoxin A in coffee[END_REF]. The remarked conditions could support growth and also the production of OTA on these substrates. It is worth noting that our results show A. niger aggregate strains can grow and produce OTA in a wide pH range. Moreover significant amounts of OTA can be produced in only 5 days. This would lead to confirm their contribution in the OTA contamination of substrates, either in the field or after harvesting, due to the presence of the proper environmental conditions for the production of the mycotoxin.

Further studies on the effect of environmental factors on growth and OTA production are necessary. A better knowledge of the ecology of these species will provide us with management strategies to reduce the OTA contamination of the substrates where they are usually isolated. a,b,c,d,e Values with the same superscript within each strain and incubation time are not significantly different (p < 0.05); nd: not detected (limit of detection 0.01 µg g -1 ).

OTA a,b,c,d,e Values with the same superscript within each strain and incubation time are not significantly different (p < 0.05); nd: not detected (limit of detection 0.01 µg g -1 ).

  on ochratoxin A production by A. niger aggregate species A Esteban, ML Abarca, MR Bragulat, FJ Cabañes* Grup de Micologia Veterinària, Departament de Sanitat i d'Anatomia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona. i d'Anatomia Animals Facultat de Veterinària, Universitat Autònoma de Barcelona 08193 Bellaterra (Barcelona), Spain.

  , different reported ochratoxigenic ability and different ITS-5.8S rDNA RFLP patterns.

Fig. 1 .

 1 Fig.1. Mean OTA concentration produced by the OTA-producing strains at each pH value and culture media tested.

Table I .

 I Aspergillus niger aggregate strains used in this study.

			Food Additives and Contaminants
	Species	Strain (source) a	OTA production reported (Ref.)	RFLP pattern (Ref.)
	A. niger var. niger	A-75 (feedstuffs, CCFVB)	+ (Abarca et al. 1994; Esteban et al. 2004)	N (Accensi et al. 2001)
	A. niger var. niger	A-136 (soy, CCFVB)	+ (Abarca et al. 1994; Esteban et al. 2004)	N (Accensi et al. 2001)
	A. niger var. niger	A-942 (raisins, CCFVB)	+ (Abarca et al. 2003; Esteban et al. 2004)	N (Abarca et al. 2003)
	A. niger var. niger A. awamori A. foetidus A. niger A. niger A. niger var. niger A. niger A. tubingensis A. niger var. niger F o r P e e r R e v i e w A-943 (grapes, CCFVB) + (Esteban et al. 2004) CBS 139.52 (kuro-koji) + (Ono et al. 1995; Esteban et al. 2004) CBS 618.78 (unknown) + (Téren et al. 1996; Esteban et al. 2004) CECT 2088 (unknown ) -(Varga et al. 2000; Esteban et al. 2004) CBS 554.65 (tannin-gallic acid fermentation) -(Accensi et al. 2001; Esteban et al. 2004) A-946 (coffee, CCFVB) -(Esteban et al. 2004) CBS 121.55 (otomycosis) -(Esteban et al. 2004) CBS 134.48 (unknown) -(Accensi et al. 2001; Esteban et al. 2004) A-947 (grapes, CCFVB) -(Cabañes et al. 2002; Esteban et al. 2004) O n l y	N (Esteban et al. 2004) N (Accensi et al. 2001) N (Accensi et al. 2001) N (Esteban et al. 2004) N (Accensi et al. 2001) N (Esteban et al. 2004) N (Esteban et al. 2004) T (Accensi et al. 2001) T (Cabañes et al. 2002)
				14

a Abbreviations: CFFVB, Culture Collection of Veterinary Faculty of Barcelona, Bellaterra (Barcelona), Spain; CBS, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; CECT, Colección Española de Cultivos Tipo, Valencia, Spain.

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table II .

 II OTA concentration (mean and mean standard error) produced by the six OTA-producing strains of A. niger aggregate in CYA medium at 25ºC at each pH value tested.

Table III .

 III OTA concentration (mean and mean standard error) produced by the six OTA-producing strains of A. carbonarius in YES medium at 25ºC at each pH value tested.

					Food Additives and Contaminants
							concentration(µg/g)
		days	2	3	4	5	6	7	8	9	10
	A-75	5	nd a	1.78±1.02 a	1.92±1.12 a	7.82±0.55 b	1.47±0.27 a	0.74±0.07 a	1.33±0.77 a	0.93±0.42 a	0.64±0.16 a
	A-136 A-942 A-943 CBS 139.52 CBS 618.78	10 15 20 30 5 10 15 20 30 5 10 15 20 30 5 10 15 20 30 5 10 15 20 30 5	1.70±0.49 a 0.30±0.06 a 0.57±0 a 0.18±0.05 a 0.03±0.04 a 3.68±0.71 a 1.14±0.28 a 1.50±1.08 ab 1.24±0.71 ab 0.42±0.18 a 3.51±1.84 ab 0.22±0.01 a 0.16±0.01 a 0.36±0.01 ab nd a 0.10±0.01 a 0.19±0.04 a nd a nd a 4.87±1.99 ab 14.58±2.02 a 2.07±0.54 ab 1.80±1.37 a 2.14±0.59 a nd a F o r P e e r R e v i e w 1.57±0.66 a 1.59±0.49 a 1.19±0.03 a 0.82±0.13 a 3.09±0.89 b 1.38±0.08 a 1.10±0.31 a 0.89±0.01 a 1.09±1.01 a 1.44±0.07 a 0.41±0.06 a 0.92±0.02 a 1.22±1.20 a 1.55±0.04 a 0.95±0.60 a 1.21±0.91 a 4.00±1.13 b 2.79±0.35 ab 9.31±2.12 c 2.44±0.47 ab 2.93±0.20 a 1.98±0.03 b 2.01±0.57 b 0.77±0.23 c 4.16±0.54 b 2.00±0.05 c 1.83±0.28 c 1.06±0.12 a 2.86±0.19 c 1.98±0.09 bc 0.71±0.06 ab 1.09±0.01 ab 2.80±0.05 c 1.80±0.20 b 1.67±0.31 ab 1.41±0.78 ab 7.24±1.66 b 4.23±0.43 c 3.86±2.28 ac 1.21±0.04 ac 4.90±0.66 b 2.91±0.64 ac 1.04±0.02 c 0.86±0.05 c 8.53±0.29 b 1.92±0.21 c 0.89±0.06 de 1.06±0.13 e 7.00±0.38 b 2.62±0.61 c 1.28±0.21 d 1.07±0.01 de 4.59±0.31 c 1.37±0.10 d 0.49±0.06 ab 0.76±0.21 b 0.14±0.01 b nd a nd a 0.05±0 c 0.26±0.06 b nd c nd c 0.03±0.04 c 0.22±0 a nd b nd b nd b 0.07±0.09 a nd a nd a 0.03±0.04 a 0.08±0.11 a nd a nd a 0.05±0.06 a 10.50±0.21 c 1.21±1.03 a 3.85±0.62 ab 2.90±1.02 ab 7.77±2.90 b 0.65±0.91 c 2.35±0.90 c 1.87±0.48 c 8.63±1.22 c 0.42±0.28 a 1.55±0.22 ab 2.30±0.86 ab 15.35±4.59 b 0.63±0.80 a 1.41±0.43 a 2.04±0.37 a 7.69±2.23 b 0.39±0.55 a 3.01±0.37 a 1.76±0.02 a nd a nd a 0.12±0.01 ab 0.17±0.01 b	0.69±0.08 a 0.81±0.01 a 0.62±0.27 a 0.61±0.13 a 1.92±0.82 ab 1.17±0.03 bc 0.67±0 a 1.02±0.01 ab 0.81±0.18 ab 0.88±0.09 ac 1.00±0.17 c 0.92±0.36 de 0.87±0.07 ade 0.81±0.11 b nd a nd c nd b nd a nd a 2.06±0.41 ab 1.87±0.33 c 1.95±0.25 ab 2.02±0.42 a 1.56±0.18 a 0.28±0.12 c O n l y 0.79±0.23 a 0.89±0.06 a 1.00±0.30 a 0.87±0.13 a 1.54±0.20 ab 0.90±0.21 c 0.69±0.27 a 0.82±0.25 ab 0.59±0.13 ab 1.32±0.34 ac 1.10±0.37 c 1.07±0.07 e 1.22±0.20 d 1.28±0.05 d nd a nd c nd b 0.06±0.01 a nd a 2.68±1.47 ab 2.02±0.52 c 3.14±0.53 b 2.57±1.22 a 2.56±0.50 a 0.08±0.01 ab	0.39±0.03 a 0.35±0.05 a 0.32±0.04 a 0.45±0.08 a 1.50±0.13 ab 0.43±0.19 c 0.54±0.26 a 0.50±0.27 a 0.33±0.09 a 0.99±0.31 ac 0.84±0.11 c 0.91±0.15 de 0.65±0.04 ade 0.52±0.16 ab nd a nd c 0.09±0.05 c 0.08±0.04 a nd a 5.69±1.4 b 3.24±1.34 c 2.79±0.78 b 3.64±1.71 a 4.16±2.79 a nd a	0.41±0.06 a 0.21±0 a 0.31±0.01 a 0.21±0.06 a 1.67±0.12 ab 0.58±0.06 c 0.51±0.11 a 0.46±0.17 a 0.30±0.06 a 0.44±0.14 a 0.52±0.10 c 0.37±0.04 ad 0.31±0.06 ae 0.25±0.04 a nd a nd c nd b nd a nd a 3.81±0.42 ab 3.06±0.57 c 1.95±0.12 ab 3.78±1.58 a 2.82±0.15 a nd a
		10	nd a	nd a	nd a	1.04±0.50 a	0.25±0.09 a	0.52±0.39 a	1.84±2.47 a	nd a	nd a
		15	nd a	nd a	nd a	0.68±0.15 b	0.73±0.14 b	0.78±0.34 b	0.46±0.35 ab	nd a	nd a
		20	nd a	nd a	nd a	0.07±0.02 a	0.49±0.11 b	0.55±0.03 b	0.25±0.20 a	nd a	nd a
		30	nd a	nd a	0.05±0.06 a	0.96±0.56 b	0.27±0.06 a	0.31±0.09 a	0.04±0.06 a	nd a	nd a
											15

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

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

Acknowledgements

This research was supported by the Ministerio de Educación y Ciencia of the Spanish Government (AGL2004-07549-C05-03) and by the grant 2002 SGR 00079 from the DURSI, Generalitat de Catalunya.


Claudia Ruiz-Capillas
email: claudia@if.csic.es

Jimenez Colmenero

Francisco Jimenez-Colmenero

Determining preservatives in meat products by flow injection analysis (FIA): A review

Keywords: Additives general, Nitrate, Nitrite, Sulfite Meat products, additives, preservatives, nitrates and nitrites, sulfites, sorbates, benzoates and p-hydroxybenzoate esters, flow injection analysis

Various preservatives are added to meat products to extend their shelf-life stability and enhance their safety. Determining preservatives is essential for purposes of legislation and consumer safety. The analytical methodologies based on flow injection analysis (FIA) can be highlighted among those available. FIA methodologies offer attractive advantages compared with other procedures, including versatility, precision, low cost, speed and ease of automation. This review considers the present status of published FIA methodology available for the determination of preservatives in meat products. The techniques are described with their application to different preservatives (nitrates and nitrites, sulfites, sorbates, benzoates and p-hydroxybenzoate esters), emphasizing aspects

Introduction

Meat and meat products are highly perishable because they contain significant amounts of the nutrients needed for micro-organism growth. The development of bacteria, yeast and molds is one of the main agents responsible for the deterioration of these food products, limiting their stability with important effects on the product quality and safety levels, and relevant economic implications. Various preservation procedures are used to protect meat from microbial spoilage, including those based on additives known as preservatives. These are chemical compounds added to foodstuffs which prolong their shelf-life by protecting them (by prevention or inhibition) from deterioration caused by microorganisms and thereby enhance food safety.

The control and regulation of the use of preservatives in meat products are very important because they are effective and ensure consumer safety. The acceptable daily intake (ADI) for preservatives has been calculated, just as for other food additives, since if they are consumed in inappropriate quantities they may have adverse effects on human health. This is why legislation on the use of additives is needed to establish specific conditions for their use. Food additive legislation adopted by the European Union is included in several European Parliament and Council Directives (Directives 95/2/EC and 2006/52/EC). The authorized preservatives for meat products are shown in Table I. To be able to evaluate whether they are being used in the specified quantities and/or foods , it is essential to have appropriate analytical methods available.

Numerous methodologies for determining preservatives in foodstuffs are cited in the relevant literature, including specific references to meat products. They are based on separation, identification and quantification processes using a wide variety of spectrophotometric, chromatographic and electrophoretic techniques etc. ( [START_REF] Karovicová | Determination of synthetic phenolic antioxidants in food by high-performance liquid chromatography[END_REF][START_REF] Wood | Analytical Methods for Food Additives[END_REF][START_REF] Ruiter | Biogenic amine formation and nitrite reactions in meat batter as affected by high-pressure processing and chilled storage[END_REF][START_REF] Colmenero | Additives: Preservatives. In: Handbook of Processed Meats and Poultry Analysis[END_REF]. Among the methodologies, those based on flow injection analysis (FIA) are of particular interest and have developed significantly in recent years because of the many advantages they offer. FIA is a very versatile, flexible, economical technique, suitable for the fast, routine analysis of a large number of samples with high precision. Affordably priced and easy to use, it requires minimum intervention of the operator and a lower consumption of samples and reactants. This is especially important when working with toxic or expensive reactants.

It can be installed on-line and is compatible with a large number of detectors (potentiometric, amperometric, spectrophotometric, fluorimetric, etc.). This methodology responds to growing demands for the mechanization and automation of analysis made by many laboratories faced with the increasing number of routine analyses. This, along with its advantageous cost/efficiency relationship, favors the use of this method over the alternative procedure for determining additives [START_REF] Valcarcel | Flow injection Análisis. Principles and Applications[END_REF][START_REF] Sullivan | Determination of total sulfite in shrimp, potatoes, dried pineapple, and white wine by flow-injection analysis -collaborative study[END_REF][START_REF] Ferreira | Simultaneous assay of nitrite, nitrate and chloride in meat products by flow injection[END_REF][START_REF] Pinho | FIA evaluation of nitrite and nitrate contents of liver pates[END_REF][START_REF] Kazemzadeh | Sequential flow injection spectrophotometric determination of nitrite and nitrate in various samples[END_REF][START_REF] Andrade | A flow-injection spectrophotometric method for nitrate and nitrite determination through nitric oxide generation[END_REF][START_REF] Frenzel | Characterisation of a gas-diffusion membrane-based optical flow-through sensor exemplified by the determination of nitrite[END_REF][START_REF] Penteado | FIA-spectrophotometric method for determination of nitrite in meat products: An experiment exploring color reduction of an azo-compound[END_REF].

Early studies of FIA focused on the determination of contaminants in water (a matrix generally easy to work with and with little interference except in waste water), but by the mid-80s the applications of FIA had been extended to other fields (food, biomedicine, control of industrial processes etc.), in more complex matrices [START_REF] Valcarcel | Flow injection Análisis. Principles and Applications[END_REF]. Although FIA has been trialed to determine some preservatives in meat products no in-depth critical analysis has been made of the use of this methodology and the possibilities it offers, as far as the authors are aware. This review considers the current status of the available published flow injection analysis methodology for the determination of authorized preservatives in meat products. To do this, different aspects are analyzed, related to extraction, separation, detection and quantification procedures in complex muscle matrices.

Brief description of FIA

The original concept of FIA was established by Ruzicka and Hansen in 1975, and has been continuously modified since then. Flow injection analysis is based on the injection of a liquid sample into a moving, non-segmented continuous carrier stream of a suitable The simplest FIA system (see Figure 1) consists of a tube through which the carrier stream moves towards the flow-through detector. Briefly, it consists of the following basic elements:

-Propulsion system. This is generally a peristaltic pump with a variable number of channels, depending on the moving or reactive (carrier) phases which intervene in the reaction. It provides a constant flow of a carrier solution.

-Injection system. This is normally a low pressure Rheodyne valve which allows a small volume of the sample, usually measured in micro liters, to be inserted into the flow of the carrier solution. The use of valves with various ports allows the simultaneous determination of various analytes.

-Reaction coils and complementary components. The most frequently used reactors are tubes made from various materials such as polytetrafluorethylene or plastic tubing of different sizes and diameters (e.g. ID 0.8 mm), which can be coiled, knitted or knotted. The purpose of this geometric deformation is to decrease the zone dispersion. These coils can be thermostatized. Sometimes the system can also have other integrated elements in series or in parallel (Figures 2 and3) such as columns (e.g. to cause chemical reactions -as with cadmium for the transformation of nitrates to nitrites or to immobilize reactants), or gas diffusion cells (e.g. for separating SO 2 or NO 2 ).

-Detection systems. A wide variety can be used, including potentiometric, amperometric, spectrophotometric, fluorometric, etc.

-Data collector system. This is normally carried out from a PC linked to the system or to the detector, with signal integration software.

Nitrites and nitrates

Nitrites and nitrates are used in processed meat because of their effect on its organoleptic characteristics (color stabilization, flavor development), oxidative stability of lipids and inhibition of pathogenous micro-organisms such as Clostridium botulinum or Listeria monocytogenes [START_REF] Cassens | Reaction of nitrite in meat[END_REF][START_REF] Cassens | Residual nitrite in cured meats[END_REF][START_REF] Efsa | The effects of Nitrites/Nitrates on the microbiological safety of meat products[END_REF]. Although the positive effect of nitrite on human health has been indicated (protective role of nitric oxide), the interest in nitrite and its reaction is mainly centered on the potential formation of nitrosamines (on reaction with secondary amines), compounds with teratogenic, mutagenic and carcinogenic effects, highly dangerous for human health [START_REF] Cassens | Residual nitrite in cured meats[END_REF]. For this reason and for some time now, the possibility has been put forward of reducing or even avoiding the use of these additives in the food industry.

In the EU, potassium and sodium nitrite and nitrate are authorized for use in different meat products, and maximum amounts (150 mg kg-1 for nitrite and 300 mg kg-1 for nitrate) are established for all products as well as maximum residual levels for some of them (Directives 95/2/EC and 2006/52/EC). Current regulations on the use of nitrite and nitrate in the US vary depending on the curing method used and the product to be cured.

For comminuted products, the maximum ingoing nitrite and nitrate limits are 156 mg kg-1 and 1718 mg kg-1, respectively. For immersion-cured and massaged or pumped products those limits are 200 mg kg-1 and 700 mg kg-1, respectively. In dry cured products nitrite is limited to 625 mg kg-1 and nitrate to 2187 mg kg-1 (USDA 2007).

The ADI for nitrites, as nitrite ion, is 0.07 mg kg-1 body weight, and 3.7 mg kg-1 for nitrates, as nitrate ion (FAO/WHO 2007a, b).

Different methodologies have been used to determine nitrates and nitrites, including ion chromatography, capillary electrophoresis, differential pulse voltammetry, enzymatic reduction and electrochemistry (biosensors with amperometric monitoring among others). For these, different detection systems based on UV, mass spectroscopy, fluorescence, chemiluminiscence or electrochemical detection have been used (Usher and Telling 1975;[START_REF] Wood | Analytical Methods for Food Additives[END_REF][START_REF] Ruiter | Biogenic amine formation and nitrite reactions in meat batter as affected by high-pressure processing and chilled storage[END_REF][START_REF] Colmenero | Additives: Preservatives. In: Handbook of Processed Meats and Poultry Analysis[END_REF]. Among these methodologies a prominent position is occupied by those based on flow analysis injection with their unquestionable advantages, which have been applied in numerous meat products (see Table II). Generally speaking, the determination of nitrates and nitrites by FIA can be divided into two phases, as is also the case with other preservatives. The first phase is the extraction process of these compounds from the meat matrix and the other is its evaluation per se using FIA methodology. Both these phases are analyzed below.

Nitrate and nitrite extraction

The extraction of nitrate and nitrite from meat products is the most critical in terms of obtaining adequate recoveries for the nitrates and nitrites and to reduce possible interferences. When nitrite is added to a biological material such as meat products, it reacts with various components present naturally in the matrix, which means that the levels of nitrite detectable analytically (known as residual) necessarily vary. This means that the levels of residual nitrite fall during the different stages of processing, storage, preparation and consumption depending on various factors related to the type of product and the conditions of each of the phases mentioned above. [START_REF] Cassens | Reaction of nitrite in meat[END_REF][START_REF] Cassens | Residual nitrite in cured meats[END_REF][START_REF] Efsa | The effects of Nitrites/Nitrates on the microbiological safety of meat products[END_REF]. Different procedures have been used in the extraction of nitrate and nitrite from foods including processed meats.

Just as with other analytical methods, most of the nitrite and nitrate extraction processes by FIA determination use hot water and different systems for blending or homogenizing the sample and filtration (see Table II). An extraction system for cured meat based on microwave cooking of the samples following cold extraction (water and NH 4 Cl at different pH) in a nitrogen atmosphere has been reported by [START_REF] Van Staden | Spectrophotometric determination of nitrite in foodstuffs by flow injection analysis[END_REF]. Some compounds present in meat products (ascorbic acid, fat, protein, etc.) which can interfere in the determination of nitrates and nitrites, may be present in the extract. This is why it has been emphasized that purification or clarification steps need to be followed. Several clearing techniques have been used for the analysis of nitrite and nitrate in food especially in meat products. These may be based on de-proteinisation and clarifying agents using reagents such as potassium aluminum sulfate, borax (sodium tetraborate anhidra), mercuric chloride, zinc sulfate, or potassium hexacyanoferrate (Carrez I) plus zinc acetate (Carrez II), the most commonly used clarifying agents for aqueous extracts of meat samples (see Table II). Other clearing techniques may also be used such as active carbon or gel filtration on Sephadex G25 -this last only used for [START_REF] Torro | Flow-injection biamperometric determination of nitrate (by photoreduction) and nitrite with the NO 2 -/Ireaction[END_REF]). Some of these reagents have been used to precipitate proteins from meat extract and also to fix the pH between 6.0 and 6.5, to avoid nitrate and nitrite loss. One of the major criticisms leveled at the use of Carrez is that the pH of the final solution (approximately pH 5) tends to promote nitrite loss. However, this deproteinization procedure of samples was also complicated because it caused cloudy filtrates and as a result serious experimental errors (Usher and Telling 1975).

Measurements of nitrite

In the FIA analytical methods the extracted samples are generally injected through the injection system valve into the corresponding carrier streams (see Figure 2; see Table II); this is generally a reagent composed of ammonium chloride and EDTA [START_REF] Van Staden | Spectrophotometric determination of nitrite in foodstuffs by flow injection analysis[END_REF][START_REF] Oliveira | Spectrophotometric determination of nitrite and nitrate in cured meat by sequential injection analysis[END_REF]Ruiz-Capillas et al. 2006) or ammonium chloride, sodium tetraborate and EDTA (Pinho et al. 1996;[START_REF] Ferreira | Simultaneous assay of nitrite, nitrate and chloride in meat products by flow injection[END_REF] or a buffer 7.5 [START_REF] Ruiz-Capillas | Application of flow injection analysis to determine protein-bound nitrite in meat products[END_REF]). These masking and buffering reagents draw the injected extracted solution into the coil of different lengths; then it is mixed directly with the color forming reagent (in most cases, since most of the methods used for nitrite determination by FIA in meat products are colorimetric) in a second reaction coil (see Figure 2; see Table II). The color forming reagents are mainly those used in the traditional diazo-coupling reaction (the Griess reaction), generally composed of sulphanilamide, (N-(1-naphthyl)ethylenediamine dihydrochloride -NED) and phosphoric acid [START_REF] Ferreira | Simultaneous assay of nitrite, nitrate and chloride in meat products by flow injection[END_REF]Pinho et al. 1996;[START_REF] Van Staden | Spectrophotometric determination of nitrite in foodstuffs by flow injection analysis[END_REF]Ruiz-Capillas et al. 2006, 2007) or other acids such as HCl [START_REF] Oliveira | Spectrophotometric determination of nitrite and nitrate in cured meat by sequential injection analysis[END_REF]Frensel et al. 2004). These procedures are based on the well-known ability of nitrite to convert aromatic amines (sulphanilamide) into diazonium ions, which are coupled to another aromatic compound (NED) in order to produce an azo-dyamine which is detected spectrophotometrically at 520-540 nm. This FIA system based on the Griess reaction has been also used for the determination of protein-bound nitrite in frankfurter and dry sausages [START_REF] Ruiz-Capillas | Application of flow injection analysis to determine protein-bound nitrite in meat products[END_REF]).

Although the Griess reaction presents important advantages, including excellent sensitivity, it also has certain limitations in relation to the control requirements of the reaction conditions, stability of the reactants, interferences or the use of some reactants which present carcinogenic effects. Many of these problems were also reported when the FIA version of the Griess reaction was applied to meat matrices. E.g. van Staden and Makhafola (1996) reported that for certain samples, mainly from an acidic origin (cured meat products), the experimental nitrite value obtained was lower than the expected value. To solve these problems it has been recommended that the nitrite samples should always be kept under nitrogen atmosphere during preparation and they must always be analyzed immediately after preparation or stored under nitrogen atmosphere. The nitrite solution was also standardized against a permanganate solution treated with a few drops of chloroform and kept in the refrigerator [START_REF] Van Staden | Spectrophotometric determination of nitrite in foodstuffs by flow injection analysis[END_REF]. SO 2 can also interfere with nitrite determination [START_REF] Ahmed | Simultaneous spectrophotometric determination of nitrite nitrate by flowinjection analysis[END_REF]. [START_REF] Haghighi | Flow injection analysis of nitrite by gas phase molecular absorption UV spectrophotometry[END_REF] showed the importance of the dye instability, so that the color reaction must be time controlled as it is affected by numerous oxidation reactions.

Over recent years, and as the result of these drawbacks, a number of other amines have been proposed as potential reactants for nitrite and potential coupling agents applied to the analysis of food extracts by FIA spectrophotometric determination (see Table II).

For example, 3-nitroaniline, with NED, has been reported as a spectrophotometric reagent (color-forming reagent) for nitrite and nitrate determination in beef and pork sausages [START_REF] Ahmed | Simultaneous spectrophotometric determination of nitrite nitrate by flowinjection analysis[END_REF]. Safranine O (3,7-diamino-2,8-dimethyl-5phenylphenazinium chloride) has also been reported to form diazonium salts in an acidic solution which causes the reddish-orange dye color of the solution to change to blue in acidic media and which absorbed at 520 nm has been used for flow injection spectrophotometric determination of nitrite in beef and commercial sausages (see Table II). The reaction of safranine with the nitrite decreases the color of the dye instead of its appearance as in the case of diazodye [START_REF] Kazemzadeh | Sequential flow injection spectrophotometric determination of nitrite and nitrate in various samples[END_REF][START_REF] Penteado | FIA-spectrophotometric method for determination of nitrite in meat products: An experiment exploring color reduction of an azo-compound[END_REF]. [START_REF] Andrade | A flow-injection spectrophotometric method for nitrate and nitrite determination through nitric oxide generation[END_REF] also reported the development and application of FIA based on spectrophotometric methods for the determination of nitrite in foodstuffs (vegetables and meat products). This is based on the formation of FeSCNNO + in the flow injection system, with transient absorbance signal at 460 nm (see Table II). The method is based on the reduction of the nitrite to NO in a sulfuric acid medium and the reaction of nitric oxide with ferrous and thiocyanate ions forming FeSCNNO + .

Oxidation/reduction reaction has been also used in the determination of nitrites. [START_REF] Ensafi | Simultaneous determination of nitrite and nitrate in various samples using flow injection with spectrophotometric detection[END_REF] proposed a flow injection method with spectrophotometric II). [START_REF] Sen | Rapid and sensitive determination of nitrite in foods and biological-materials by flow-injection or high-performance liquidchromatography with chemiluminescence detection[END_REF] also developed a FIA method based on nitrite reduction with potassium iodine followed by chemiluminescence detection of the liberated NO using a thermal energy analyzer for the determination of nitrites in food (cured meat) and other biological materials.

Torró et al. ( 1998) developed a FIA method (tested on vienna sausages) that involved the photochemical reduction of nitrate in a photo reactor consisting of a low pressure mercury lamp. The nitrite produced is determined indirectly with the I 3 -/I -system using a flow-cell with two platinum (Pt) electrodes polarized at 100 mV (see Table II). [START_REF] Haghighi | Flow injection analysis of nitrite by gas phase molecular absorption UV spectrophotometry[END_REF] reported a flow injection method on the basis of gas phase molecular absorption for the determination of nitrite in meat sausages and mortadella and aqueous solutions (see Table II). The sample solution is injected into a stream of water which is then reacted with a stream of hydrochloric acid. The gaseous products (NO, NO 2 , HNO 2 and NOCl) are separated from the liquid stream by the home-made gas liquid separator and they are swept by the carrier O 2 gas into a homemade flow-through cell which has been positioned in the space of the cell compartment of a UV/VIS spectrophotometer. The transient absorbance of the gaseous phase is measured at 205 nm. The interference of anions in the methods proposed by [START_REF] Haghighi | Flow injection analysis of nitrite by gas phase molecular absorption UV spectrophotometry[END_REF] are overcome adding silver and sodium hydroxide solutions as the precipitating agent. This method is less sensitive than the Griess methods but there are also fewer limitations than in the Griess method. II), using the modified Griess reaction where sulfuric acid was used as a releasing agent to set HNO 2 and NO 2 free from the nitrite solutions. The color reagent was prepared by a dissolution of sulfanilamide, N-(1-naphthyl)-ethylenediamine dihydrochloride and concentrated hydrochloric acid in water. The main reason for developing the proposed sensor was the inherent selectivity enhancement due to analyte separation by the gas-diffusion process. Only a few analytes are sufficiently volatile to pass through the membrane and they are effectively separated from the sample matrix. In particular, colored and turbid samples can be analyzed without problems and interferences as extreme pH values or the presence of high salt content in the samples are eliminated so that the proposed sensor is almost nitrite specific. However, the relatively low sensitivity of the developed sensor (compared to the common spectrophotometric method (ISO 1996) limits its applicability [START_REF] Frenzel | Characterisation of a gas-diffusion membrane-based optical flow-through sensor exemplified by the determination of nitrite[END_REF].

Measurements of nitrates and nitrites

Although initially FIA was used only for nitrite determination, nitrate determination was also developed later. Since for nitrate analysis the previous reduction of nitrate to nitrite is required, FIA methodology included a reduction system. This means that in its application in meat products the nitrate is reduced on-line to nitrite in a cadmium column which is then treated with azo dye reagent and the absorbance due to the sum of nitrite and nitrate is measured: nitrate is determined from the difference in absorbance values (see Figure 2) (Ruiz-Capillas et al. 2006, 2007).

The simultaneous determination of nitrates and nitrites has been trialed since this substantially reduces analysis times. [START_REF] Ahmed | Simultaneous spectrophotometric determination of nitrite nitrate by flowinjection analysis[END_REF] determined nitrites and nitrates (beef and pork sausages) simultaneously using a selector valve to split the sample into two streams after it was injected into the carrier stream. The quantification was based on the 3-nitroaniline+NED color reaction (see Table II). For different meat products [START_REF] Ferreira | Simultaneous assay of nitrite, nitrate and chloride in meat products by flow injection[END_REF] and [START_REF] Pinho | FIA evaluation of nitrite and nitrate contents of liver pates[END_REF] also determined nitrate and nitrite simultaneously. The test was based on the splitting of the flow after injection and subsequent confluence of the flow before it reached the detector, allowing the reduction of nitrate to nitrite in part of the sample plug in an on-line copper cadmium redactor column. Since each channel has a different residence time two peaks were obtained for nitrite and for nitrite plus nitrate. The spectrophotometric determination was made after a diazotization coupling reaction. A similar system for simultaneous determination of nitrite and nitrate in various beef samples used flow injection with spectrophotometric detection based on a oxidation system where the nitrate is reduced to nitrite by using two sequential columns: copper and copper-coated cadmium [START_REF] Ensafi | Simultaneous determination of nitrite and nitrate in various samples using flow injection with spectrophotometric detection[END_REF]. [START_REF] Kazemzadeh | Sequential flow injection spectrophotometric determination of nitrite and nitrate in various samples[END_REF] reported similar procedure for the simultaneous determination of nitrate and nitrite but based on the reaction of nitrite with safranine O (see Table II). On the other hand, [START_REF] Oliveira | Spectrophotometric determination of nitrite and nitrate in cured meat by sequential injection analysis[END_REF]

Sulfites

Sulfites are used as preservatives in food and beverages to prevent oxidation and bacterial growth. In meat, sulfites also help to give a bright color. Appearance (colour) is one of the prime factors motivating consumer choice. In the EU the maximum permitted amount of added sulfite agents in different meat products is 450 mg kg-1, expressed as SO 2 (Directives 95/2/EC and 2006/52/EC). These compounds are not permitted for use in meat in the USA. Sulfites have been associated with allergic reactions and food intolerance symptoms. They are also known to degrade thiamine (vitamin B1), of which meat is a good source. The ADI for sulfite (expressed as SO 2 ) is 0.7 mg kg-1 body weight (FAO/WHO 2007c).

There are many methods for analyzing sulfites in beverages and foods. These procedures involve titrimetry, electrochemistry, fluorimetry, chemiluminesence spectrometry, colorimetry, gas-liquid chromatography, high preformed liquid chromatography, etc. [START_REF] Fazio | A Review of sulphites in foods: analytical methology and reported finding[END_REF][START_REF] Karovicová | Determination of synthetic phenolic antioxidants in food by high-performance liquid chromatography[END_REF][START_REF] Wood | Analytical Methods for Food Additives[END_REF][START_REF] Ruiter | Biogenic amine formation and nitrite reactions in meat batter as affected by high-pressure processing and chilled storage[END_REF][START_REF] Colmenero | Additives: Preservatives. In: Handbook of Processed Meats and Poultry Analysis[END_REF]. The most commonly used techniques are based on the classic Monier-Williams method.

Although this method demonstrates sufficient sensitivity, it also presents certain limitations because of the long analysis time (with important repercussions on the analytical capacity of the laboratory ) and the extreme control needed in some of the analytical phases (e.g. N 2 stream adjustment).

FIA procedures have been extensively researched for determining sulfites in beverages and foods, in an attempt to overcome these limitations and reduce the time and effort needed for those procedures. Most FIA procedures have been developed for liquid foods, with far fewer for solid foods and especially for muscle foods (see Table III). It has been reported that the FIA procedure is a more rapid, accurate and precise method for the determination of total sulfite in food than the Monier-Williams procedure [START_REF] Sullivan | Determination of sulfite in food by flow-injection analysis[END_REF][START_REF] Sullivan | Determination of total sulfite in shrimp, potatoes, dried pineapple, and white wine by flow-injection analysis -collaborative study[END_REF].

Sulfite extraction

When sulfiting agents are added to foods such as fish or meat products they can combine with some of their components (aldehydes, proteins etc.), so that they may be present as free, reversibly bound (probably as hydrosulphonate adducts) and irreversibly bound [START_REF] Wedzicha | Reactivity of sulfur-dioxide in comminuted meat[END_REF][START_REF] Pizzoferrato | Determination of free, bound and total sulphites in foods by indirect photometry-HPLC[END_REF]. The relative presence of each of these depends on factors linked to composition, processing and conservation. This is why the determination of sulfites in complex food matrices such as meat products requires the application of some procedure for removing and recovering the sulfites (free and reversibly bound both together called total sulfites). Since substantial loss of sulfites can occur during extraction, different alternative extraction conditions (distillation, acidic, alkaline extractant, etc.) have been tried, to obtain free and bound sulfite fractions. For analysis using FIA various extraction media have been trialed (including solutions containing acetone, formaldehyde and ethanol), but the most efficient extraction procedure in muscle foods for FIA analysis includes pH extraction with a sodium tetrachloromercurate (TCM) solution [START_REF] Sullivan | Determination of sulfite in food by flow-injection analysis[END_REF][START_REF] Sullivan | Determination of total sulfite in shrimp, potatoes, dried pineapple, and white wine by flow-injection analysis -collaborative study[END_REF]AOAC 2005) (see Table III). TCM helps to release the sulfite from solid foods and stabilize it forming a stable sulfite-mercuric complex, providing excellent sulfite recovery [START_REF] Sullivan | Determination of sulfite in food by flow-injection analysis[END_REF][START_REF] Ruiter | Biogenic amine formation and nitrite reactions in meat batter as affected by high-pressure processing and chilled storage[END_REF].

Measurements of sulfites: separation/detection systems.

Afterwards, the extracting solution obtained from muscle food matrices (meat and fish) containing the sulfiting agents is injected into the FIA system to proceed to the separation of the SO 2 followed by the corresponding detection process (see Figure 3).

The separation phase in these matrices is based on the use of a gas diffusion process [START_REF] Sullivan | Determination of sulfite in food by flow-injection analysis[END_REF]AOAC 2005). For determination of total sulfites previous to this separation in the gas diffusion system a hydrolysis treatment is applied with NaOH aimed at the release of most of the bound sulfites from the extract solution which is complete above pH 8 [START_REF] Kim | Analysis of free and total sulfites in food by ion chromatography with electrochemical detection[END_REF][START_REF] Sullivan | Determination of total sulfite in shrimp, potatoes, dried pineapple, and white wine by flow-injection analysis -collaborative study[END_REF]AOAC 2005) The separation of SO 2 itself is carried out in the gas diffusion cell (see Figure 3). Two liquid streams converge in this cell: the first is a donor stream which transports a strong acidic donor solution intended to provoke the release of the SO 2 present in the test solution. Although other donor reagents may be used in gas diffusion systems, sulfuric acid is what is used in muscle based food [START_REF] Sullivan | Determination of sulfite in food by flow-injection analysis[END_REF][START_REF] Sullivan | Determination of total sulfite in shrimp, potatoes, dried pineapple, and white wine by flow-injection analysis -collaborative study[END_REF]AOAC 2005).

This choice is closely linked to the detection system. The second is an acceptor stream (indicator which collects the released SO 2 ), a solution containing an acid-base indicator separated by a permeable membrane (generally made of teflon). The SO 2 released from the donor diffuses through the membrane and is dissolved into the acceptor solution.

The membrane is not only necessary to separate the strong acidic donor solution from the acceptor solution but also forms a barrier for potential interferences in the sample.

Different colorimetric reagents (iodine, bromocresol green etc.) can be found among the acceptor solutions used, although in the determination of SO 2 by FIA in solid matrices (muscle based food) a malachite green solution is used [START_REF] Sullivan | Determination of sulfite in food by flow-injection analysis[END_REF][START_REF] Sullivan | Determination of total sulfite in shrimp, potatoes, dried pineapple, and white wine by flow-injection analysis -collaborative study[END_REF]AOAC 2005) (see Table III). The degree of discoloration of malachite green is proportional to the amount of sulfite in the test solution and it is measured spectrophotometrically at 615 nm. Spectrophotometric detection seems more appropriate as it is more robust and it is usually available in routine control laboratories.

The possible interferences in the determination of SO 2 by FIA with a gas diffusion unit are related to the presence of compounds able to pass through the membrane, including nitrite, sulfide and cyanide (Frenzel and Hillman 1995). Although these interferences have been established in beverages, their incidence is plausible under any circumstances in solid matrices such as meat products.

Sorbates, benzoates and p-hydroxybenzoate esters.

Sorbic acid, benzoic acid and the methyl and ethyl esters of 4-hydroxybenzoic acid (parabens) or their (Na, K, or Ca) salts are organic acids used as meat product preservatives (see Table I). In the EU they are allowed in the surface treatment and jelly coating of many processed meats, used singly or in combination (sorbates, benzoates and parabens in concentrations ranging from 1000 mg kg-1 to quantum satis) and in pâtés (sorbates and parabens used singly or in combination), up to a maximum level of 1000 mg kg-1. (Directives 95/2/EC and 2006/52/EC). The acceptable daily intake is 25 Given that these compounds present strong UV absorption, the method most commonly used to determine organic preservatives in foods is spectrophotometry. Other methods have also been reported including thin-layer chromatography, gas chromatography, high performance liquid chromatography and capillary electrophoresis among others [START_REF] Karovicová | Determination of synthetic phenolic antioxidants in food by high-performance liquid chromatography[END_REF][START_REF] Wood | Analytical Methods for Food Additives[END_REF][START_REF] Ruiter | Biogenic amine formation and nitrite reactions in meat batter as affected by high-pressure processing and chilled storage[END_REF][START_REF] Colmenero | Additives: Preservatives. In: Handbook of Processed Meats and Poultry Analysis[END_REF].

The application of FIA method for the determination of sorbic acid, benzoic acid and parabens in meat products has not been found in the literature. A sorbic acid determination method by FIA in wine has been proposed [START_REF] Molina | Spectrophotometric flow-injection method for determination of sorbic acid in wines[END_REF]). The advantages offered by the traditional spectrophotometric methods in terms of simplicity and specificity may help to understand the lack of interest in the application of other methodologies, including FIA, to the determination of these compounds in foods. There is no methodological reason which limits the application of FIA in the determination of these preservatives in meat products. The procedure requires, just as for other methods, an extraction process (for the extraction of the compound and, where relevant, the elimination of interferences related to the nature of the matrix) before it is introduced into the equipment, where the color reaction is developed and later detection, both visible and UV, takes place. In this case a basic advantage of the FIA system is the possibility of automation, which would mean an increase in the volume of samples analyzed and reduced handling.

CONCLUSIONS

The application of FIA to determine preservatives in meat is an important alternative to more conventional methods and presents several advantagesn in routine laboratory analysis for official bodies and industry. FIA is a method which allows a simple, precise, fast determination of small amounts of sulfite with low reagent consumption, small volumes of samples required and the use of different detection systems. However, more studies are necessary in order to develop new FIA procedures for the benzoates etc.). It is also necessary to improve the present procedure in order to reduce the quantity of toxic reagent that is used in some of them. More studies of the extraction and cleaning procedures will be necessary for the application of FIA in complex matrices such as muscle based foods. Therefore preservative recovery should be improved and the reduction of possible interference associated with both natural matrix components and other food additives should be studied. Studies regarding the automatization of all the procedures must also be carried out, as well as reviewing the stabilising reagent for the extraction solution and its conservation to avoid substantial losses of preservatives. In depth studies will also be necessary to apply FIA procedures to new products (including meat based functional foods), which have been developed using new non-meat ingredients (e.g. from plant and marine sources).

  injected sample forms a zone which is then transported towards a detector that continuously records phisical parameter such as the absorbance, electrode potential, or other physical parameter, changing as the sample material passes through the flow cell. The FIA response curve (FIAgram) is the result of two kinetic processes: the physical process of dispersion of the sample zone within the carrier stream and the chemical process of formation of a chemical species. These two processes occur simultaneously and together with the dynamic characteristics of the detector, they yield the FIA response curve.

  of nitrite in beef samples based on the catalytic effect of nitrite on the oxidation of gallocyanine by bromate in acidic media and the decrease in absorbance of the system at 530 nm (see Table

Frenzel

  et al. (2004) use a system of gas diffusion membrane-based optical flow through optical sensor for the determination of nitrite in waste waters and meat extracts (see Table

  interfere in the Griess reaction are therefore almost entirely excluded.

  the sulfiting agent from the food and the other related to evaluating it.

  Figure3).

  sorbic acid (FAO/WHO 2007d) and 5 mg kg-1 body weight for benzoic acid (FAO/WHO 2007e). The EFSA has established a full-group ADI of 10 mg kg-1 body weight for the sum of methyl and ethyl p-hydroxybenzoic acid and esters and their sodium salts (Directive 2006/52/EC).

  preservatives present in meat and meat products (e.g. sorbates,

  Figure 1.-Diagram of a basic Flow Injection Analyser

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

Acknowledgements

This research was supported under projects AGL2007-61038/ALI of the Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I+D+I) and the Consolider CSD2007-00016, Ministerio de Ciencia y Tecnología.

- -----------------------


Agustin Ariño

Marta Herrera

Eloisa Langa

Javier Raso

Antonio Herrera

Ochratoxin A in licorice as affected by processing methods

A study of the effect of several processing methods on the ochratoxin A (OTA) 2 concentration in licorice and derived products was carried out. The effect of sorting, 3 washing and peeling of fresh licorice roots was investigated, as well as the production 4

oat a laboratory scale of licorice extract and block licorice from dry roots. Finally, the 5 thermal stability of OTA was assessed. The OTA content was analyzed by HPLC-6 fluorescence and confirmed by methyl ester formation. The OTA level in licorice 7 extract was stable to heat treatment at 150ºC for 60 min. The OTA concentration was 8 unaffected by sorting or washing, but it was much reduced by peeling (53.1% 9 reduction). A great reduction in the OTA level was found during the production of 10 licorice extract (78.6%) and block licorice (91.8%). 11 Penicillium verrucosum and Aspergillus ochraceus, but also A. carbonarius and A. 4 niger aggregate species [START_REF] Esteban | Effect of water activity on 23 ochratoxin A production by Aspergillus niger aggregate species[END_REF]. OTA contaminates foods such as cereals, 5 coffee, dried fruit, grapes, cocoa and wine, and it can also occur in animal products. 6

Licorice and derived products may also contribute to human exposure to OTA, 7 particularly for high consumers of these foods, as it was noted in a recent international 8 conference (Fink-Gremmels 2005). Ochratoxin A is a mycotoxin with carcinogenic 9 (class 2B of IARC), nephrotoxic, teratogenic, and immunotoxic properties, and it has 10 been linked to nephropathy in humans [START_REF] Creppy | Update of survey, regulation and toxic effects of mycotoxins in 12 Europe[END_REF]. 11

12

The European Food Safety Authority considered that it would be prudent to reduce 13 exposure to ochratoxin A as much as possible, establishing a Tolerable Weekly Intake 14 Generally Recognized as Safe (GRAS) status in the USA (Anonymous 1985). Licorice 1 is also a popular ingredient in herbal tea and in some botanicals, consumed to improve 2 overall health and to alleviate a wide range of diseases [START_REF] Fiore | A history of the 4 therapeutic use of liquorice in Europe[END_REF]). The natural 3 presence of ochratoxin A in licorice was first reported in Germany by Bresch et al. 4 (2000) and [START_REF] Majerus | Ochratoxin A in Sübholz, Lakritze 12 und daraus hergestellten Erzeugnissen[END_REF], who detected a high percentage of positive samples. A 5 recent survey conducted in our laboratory showed that all samples of licorice and 6 derived products contained ochratoxin A, and some of them had extremely high 7 concentrations up to 253 ng/g of OTA [START_REF] Ariño | High levels of ochratoxin A in 1 licorice and derived products[END_REF]). Therefore, every effort 8 should be made with regard to research and prevention measures to reduce the presence 9 of OTA in licorice as much as possible. 10 11 A typical traditional process for licorice production, as outlined by Gabriele et al. 12 (2001), is essentially based on four different unit operations: grinding of dry licorice 13 roots, extraction with steam or hot water, filtering/centrifugation of the extract, and 14 concentration. The initial extraction and filtering yields the licorice extract, which is 15 further dehydrated to obtain a dark brown material called block licorice. In our previous 16 study of licorice derivatives and foods containing licorice, OTA was abundant in block 17 licorice (39.5 ng/g), as well as in licorice-confectionery (3.8 ng/g) [START_REF] Ariño | High levels of ochratoxin A in 1 licorice and derived products[END_REF]. 18 However, very little is known about the effects of licorice processing on the OTA 19 concentrations and the feasibility of decontamination methods for OTA. 20

21

The prevention of mycotoxin contamination should include all phases of food 22 production, because mycotoxins may be synthesized in the field, during storage, as well 23 as in transport. All methods of decontamination increase the cost of production and 24 should be used only in cases when preventive measures in the field have failed. With 25 washing, peeling and heat processing. Chemical detoxification may be very effective, 3 but it does not meet the EC requirements, because some compounds leave behind their 4 toxic metabolites and others reduce the nutritional value of treated foodstuffs. 5

Therefore, foodstuffs containing mycotoxins shall not be deliberately detoxified by 6 chemical treatments (EC 2006a). 7 8The objective of the present study was to obtain data on the effect of several processing 9 methods in the OTA concentration in licorice and derived products. First, the effect of 10 sorting, washing and peeling of fresh licorice roots was investigated. Then, licorice 11 extract and block licorice were produced at laboratory scale to assess the transfer rate of 12 OTA from the starting material (dry licorice roots). Finally, the thermal stability of 13 OTA in licorice extract was assayed. 14 15

Materials and methods 16 17

Licorice samples 18

The samples of fresh licorice root (for the study of sorting, washing and peeling) were 19 provided from local growers of Zaragoza, Spain. These fresh root samples were 20 of Zaragoza, Spain. The licorice roots were ground in an IKA laboratory mill to a coarse 1 root powder, and kept refrigerated at 4ºC until processing. 2 3

Effect of washing and peeling of fresh licorice roots 4

To assess the effect of first-stage processing of licorice roots on OTA contamination, 5 eight batches of freshly-collected roots were subjected to washing and peeling. Washing 6 was done in a suitable container using tap water with no detergent aid. Peeling was 7 carried out manually with a knife, removing the outer part of the licorice root. The peel: 8 root ratio (as a percentage of peel removed over total root weight) was around 16% in 9 all batches. 10 11

Transfer of ochratoxin A into licorice extract and block licorice 12

In order to evaluate the transfer rate of OTA from dry licorice roots to derived products, 13 experiments were carried out at laboratory scale to simulate the industrial processing of 14 licorice. First, licorice extract was obtained by hot water extraction in an Italian coffee 15 maker. For this, the container was filled with distilled water and the sample holder with 16 12 g of ground licorice root. The instrument was placed over a low flame and the 17 licorice extract leaching through the roots was collected and filtered through a Whatman 18 1 filter (volume ~ 120 ml). The extraction time was 1 minute and the temperature was 19 between 93-100ºC. Then, the licorice extract was subjected to drying in a heater at 20 105ºC during 12 h to obtain block licorice (~ 2.5 g). A total of 4 batches of dry licorice 21 roots were processed in this way into the corresponding derived products. The thermal stability of ochratoxin A was carried out in a thermo-resistometer TR-SC 1 as previously described [START_REF] Raso | Influence of temperature and pressure on the 18 lethality of ultrasound[END_REF]). The corresponding treatment medium was 2 licorice extract (350 ml) with an OTA concentration of 7.7 ng/ml. The heat treatment 3 was conducted isothermally at 150ºC, once the temperature had attained stability. At 4 preset intervals (15, 30 and 60 min), samples for each treatment time were collected and 5 analyzed for ochratoxin A by LC-FLD. Experiments were performed in duplicate and 6 results were expressed as the average value. 7 8 Apparatus and reagents for ochratoxin analysis 9 HPLC grade acetonitrile and methanol were purchased from Lab-Scan (Dublin, Ireland) 10 and HPLC grade acetic acid and hydrochloric acid (37%) from Merck (Darmstadt, 11 Germany). Ultrapure water was obtained from a Milli-Q Plus apparatus from Millipore 12 (Milford, MA). The immunoaffinity columns RIDA Ochratoxin A were supplied by R-13

Biopharm AG (Darmstadt, Germany). Ochratoxin A standard was provided by Sigma 14 (St. Louis, MO) and stock solutions (0.01 mg/ml) were prepared in methanol and stored 15 at -21ºC. Reagents for sodium bicarbonate buffer pH 8.1 (0.13M) and phosphate buffer 16 saline pH 7.4 (20 mM) were provided by Panreac (Barcelona, Spain). 17

Analysis of ochratoxin in licorice and derived products 1

The analytical method for ochratoxin has been previously described elsewhere (Ariño et 2 al. 2006), and it is based on immunoaffinity column/HPLC method EN 14132:2003 for 3 ochratoxin A in cereal grains and roasted coffee of the European Committee for 4

Standardization (CEN, 2003). Briefly, 3 g of licorice roots (12 ml for licorice extract, 1 5 g for block licorice) was extracted with 30 ml sodium bicarbonate 0.13 M and methanol 6 (9:1 v/v). The extract was centrifuged at 3500 rpm during 15 min, and 10 ml of it was 7 purified through the RIDA ochratoxin A immunoaffinity columns. The ochratoxin-8 containing eluate was evaporated to dryness in a heating block under a gentle stream of 9 nitrogen, and re-dissolved in the injection solvent, a mixture of methanol, distilled water 10 and acetic acid (30:69:1 v/v). Aliquots of 20 µl were injected into the LC-FLD system. 11

The ochratoxin residues were confirmed by production of their methyl-ester derivatives, 12 as described later. 13 14 The analytical method was validated in-house with respect to precision and recovery. 15

The average recovery and relative standard deviation (RSDr, repeatability) obtained by [START_REF] Ec | [END_REF] the described method for ochratoxin A were 90.6% (RSD 3.8%). The performance 17 characteristics were within the acceptable margins indicated in the Commission 18 Regulation No 401/2006 (EC 2006b). The study of sensitivity indicated that the limit of 19 quantification (LOQ) for ochratoxin A was 0.5 ng/g. 20

21

Confirmation procedure 22

The identity of OTA was confirmed by methyl ester formation according to Li et al. 23 (1998). To an amber vial containing 25 µl of OTA residue, 75 µl of hydrochloric acid 24 (6N) and 400 µl of methanol were added. The vial was shaken, closed and left for 24 h 25 The results from OTA analyses obtained by the LC-FLD method were subjected to 6 descriptive and comparative statistics according to Sachs (1978). Calculations were 7 performed on StatView™ SE+Graphics (Abacus Concepts, Inc. 1988, Berkeley, CA) 8

for Macintosh personal computers. 9 10

Results and discussion 11

Occurrence of OTA in healthy and damaged fresh licorice roots 12 Healthy fresh licorice roots were uniformly shaped and bright-yellow coloured while 13 damaged roots were unevenly shaped with some brownish parts. However, no samples 14 were moldy in appearance. All samples tested positive for OTA, with mean levels 15 ranging from 10.4 ng/g in healthy roots to 10.0 ng/g in damaged roots. The subsequent 16 statistical analysis revealed no significant differences between samples (p>0.05), 17 indicating that first-stage sorting of licorice based on appearance may not be effective in 18 separating out contaminated samples. However, [START_REF] Bresch | Ochratoxin A in food containing liquorice[END_REF] reported that visual 19 analysis of licorice indicated that homogeneous bright yellow samples were less 20 contaminated by OTA than those containing brownish material and being less 21 homogeneous in shape. The natural occurrence of OTA in botanical roots has been also 22 reported by [START_REF] Trucksess | Determination of 21 aflatoxins and ochratoxin A in ginseng and other botanical roots by immunoaffinity 22 column cleanup and liquid chromatography with fluorescence detection[END_REF], who found OTA concentrations in kava-kava (6 23 ng/g) and licorice (20 ng/g). [START_REF] Ariño | High levels of ochratoxin A in 1 licorice and derived products[END_REF] 25

Effect of washing and peeling of licorice roots on OTA contamination 1

Eight batches of fresh licorice roots were subjected to water washing and manual 2 peeling to assess for OTA removal (Table I). The mean OTA level in the starting 3 material was 10.2 ng/g, and remained essentially unchanged after washing (11.0 ng/g, 4 p>0.05). However, in all of the samples, the peeling process caused a reduction of the 5 OTA level. Thus, peeling significantly removed the 53.1% of the OTA present in fresh 6 roots (p<0.05), as initial levels dropped from 10.2 to 4.9 ng/g after peeling. This shows 7 that on average, nearly half of the OTA contamination in licorice roots is found in the 8 outer root layer. However, the distribution of OTA in the roots is not homogeneous, as 9 peeling caused varying reductions from 12 to 85% even though the peel: root ratio was 10 around 16% in all batches. [START_REF] Majerus | Ochratoxin A in Sübholz, Lakritze 12 und daraus hergestellten Erzeugnissen[END_REF] analyzed commercial samples and 11 reported that peeled licorice roots were much less contaminated by OTA than unpeeled 12 roots. Similarly, in the processing of cocoa beans, shelling has been reported to cause 13 more than 50% reduction in OTA levels [START_REF] Amezqueta | Occurrence of 19 ochratoxin A in cocoa beans: Effect of shelling[END_REF]). 14 II. The 5 level of OTA contamination in dry roots varied from 53 to 253 ng/g, in licorice extract 6 ranged from 1.1 to 5.8 ng/ml, and concentrations in block licorice were between 20.4 to 7 98.4 ng/g. On average, 21.4% of the OTA present in dry roots was transferred to the 8 corresponding licorice extract, what is to say that 78.6% of OTA was retained in the 9 brewed dry licorice root powder during extraction. The subsequent concentration of the 10 licorice extract further reduced OTA level by 91.8%, as only 8.2% of the initial amount 11 was carried over into block licorice. In comparison, a 32.1% reduction of the OTA level 12 was attained by brewing roasted coffee in an Italian coffee maker (Perez de Obanos et 13 al. 2005). The different percentage of OTA transfer in licorice extract (21.4%) and 14 coffee brew (67.9%) could be explained in terms of the higher fibre content of licorice 15 roots (contain root bark) and the fact that coffee is roasted, which may have facilitated 16 the transfer of OTA into the corresponding brew. 17

18 "[Insert Table II about here]" 19 20 In summary, the net effect of licorice processing showed a 91.8% reduction of the OTA 21 concentrations found in the starting material. However, due to the high OTA 22 concentrations detected in the roots, the mycotoxin was still present in its derivatives. 23 This is a cause for concern, as for example licorice sweets may contain between 5 to 24 10% of licorice derivatives. [START_REF] Majerus | Ochratoxin A in Sübholz, Lakritze 12 und daraus hergestellten Erzeugnissen[END_REF] detected OTA in 24 out of 26 samples 25 Where they are legally permitted, detoxification or decontamination processes will 5 involve degradation, destruction and/or inactivation of the mycotoxins. These processes, 6 while reducing the concentration of the mycotoxins to safe levels, should not produce 7 toxic degradation products or reduce the nutritional or palatability properties of the 8 commodities. In our study, licorice extract was subjected to heating to assess the effect 9 on OTA concentrations. A thermoresistometer instrument was loaded with licorice 10 extract containing OTA at 7.7 ng/ml, and tested at 150ºC at preset time intervals. The 11 results showed the extreme stability of the ochratoxin to heat, as the levels remained 12 constant during the process (Figure 1). Ochratoxin is a thermostable compound; in a 13 study of the stability in cereals during heating, the calculated half-lives (time to 50% 14 destruction of ochratoxin A) were 707 and 201 min, respectively, at 100 and 150ºC for 15 dry wheat, and 145 and 60 min, respectively, at 100 and 150ºC for wheat heated under 16 wet conditions [START_REF] Boudra | Thermostability of ochratoxin A in wheat under 4 two moisture conditions[END_REF]. The presence of water (50%) increased the 17 decomposition of OTA at 100 and 150ºC. 18 roots. Washing the roots was also ineffective in removing OTA, whereas peeling the 1 roots significantly reduced OTA contents by more than 50%. A great reduction in the 2 OTA level was attained during the production of licorice extract (21.4% remaining) and 3 block licorice (only 8.2% of the original OTA remains). Thus, the combined unit 4 operations of peeling, processing into extract and dehydration considerably reduced the 5 OTA concentrations in finished products. However, due to the high OTA concentrations 6 detected in the roots, the mycotoxin was still present in its derivatives. Further research 7 is needed for the prevention of the contamination by ochratoxin-producing molds in the 8 fields where the licorice is grown, during sun-drying and storage of the roots, and for 9 methods of selection of the raw material to effectively separate out contaminated roots.

7 Spain, 8 2 16 17 19 F

 781619 1 , M. HERRERA 1 , E. LANGA 2 , J. RASO 1 & A. HERRERA 1 , University of Zaragoza, c/ Miguel Servet 177, E-50013 Zaragoza, Agri-Food Research and Technology Center, Avda. Montañana 930, Ariño. E-mail: aarino@unizar.es 14 Phone: +34-976-761543; Fax: +34-976-761612 15 Running head: Effect of processing on ochratoxin A in licorice 18

12Keywords:

  Mycotoxins, ochratoxin A, HPLC, immunoaffinity, licorice root, licorice 13 extract, block licorice, licorice processing methods OTA) is a secondary metabolite of several species of fungi, notably 3

(

  TWI) of 120 ng/kg b.w. (EFSA 2006). Recent analyses of the dietary exposure of adult 15 European consumers to OTA revealed that at present the weekly exposure ranges from 16 15 to 60 ng OTA per kg b.w., but more data would be needed to assess exposure rates 17 of infants and children, taking into account their dietary preferences (EFSA 2006). 18 Under Regulation No. 1881/2006 (EC 2006a), maximum limits for ochratoxin A were 19 set in cereal grains and derived products, dried vine fruits, coffee products, wine, grape 20 juice, musts and certain infant and dietary foods. 21 22 Licorice (Glycyrrhiza glabra L., Leguminosae) is a common dietary supplement used as 23 flavouring and sweetening agent in confectionery and other food products, such as 24 beverages and chewing gum (Gabriele et al. 2001), and its derivatives have been given 25

  decontamination of foodstuffs, the European Community favours 1 the use of sorting procedures and physical decontamination processes, such as cleaning, 2

  classified into two types (healthy and damaged), distinguished by each other by the 21 presence of less uniformly shaped roots showing some brown coloration instead of the 22 typical bright yellow colour of the healthy roots. The samples of dry licorice root (for 23 the preparation of licorice extract and block licorice) were purchased from herbal stores 24

Food

  /mc.manuscriptcentral.com/tfac Email: tfac@csl.gov.uk

18 25 F

 25 The LC system consisted of an Agilent Technologies 1100 high-performance liquid 19 chromatogram coupled to an Agilent fluorescence detector at 333 nm (excitation) and 20 460 nm (emission). The LC column was Ace 5 C 18 , 250 x 4.6 mm, 5 µm particle size 21 (Advanced Chromatography Technologies, Aberdeen, Scotland). The isocraticLC 22 mobile phase was distilled water, acetonitrile and acetic acid (51:48:1 v/v); the flow 23 rate, 1 ml/min. 24

  at 25ºC. A 20 µl aliquot was analyzed using liquid chromatography 1 coupled with fluorescence detection, and compared with blanks and standards prepared

15"[ 16 17 3 Four

 163 Insert Table I about here]"Transfer of OTA from dry licorice roots to derived products 18 Licorice derivatives are largely used in different areas of food industry, such as 19 confectionery and beverages. Licorice trade is usually made with licorice extract 20 (liquid) or crude dried block licorice (solid). The few reports available on the OTA 21 contamination of licorice derivatives indicate a high degree of contamination, as the 22 mycotoxin was detected is almost all analyzed samples by LC-FLD and LC-MS 23[START_REF] Majerus | Ochratoxin A in Sübholz, Lakritze 12 und daraus hergestellten Erzeugnissen[END_REF] and by LC-FLD plus methyl-ester confirmation (Ariño et al.[START_REF] Ariño | High levels of ochratoxin A in 1 licorice and derived products[END_REF] However, very little is known about the effects of licorice processing into its 1 derivatives. 2 batches of dry licorice roots were processed into licorice extract and block licorice 4 at laboratory scale, and the effects on OTA concentrations are shown in Table

3

  licorice confectionery (candies and other sweets) at a mean level of 1.1 ng/g, and 1 Ariño et al. (2006) found OTA in 4 samples of licorice sweets averaging 3.8 ng/g. 2 Effect of thermal treatment on OTA levels in licorice extract 4

19 "[

 19 Insert Figure 1 about here]" 20 21 Licorice roots and derived products are frequently contaminated with OTA, and 22 licorice derivatives are largely used in different areas of food industry. OTA level in 23 licorice extract was stable to heat treatment at 150ºC during 60 m. Sorting of damaged 24 roots based on visual appearance has not proved useful to separate out contaminated 25

  supported by the Government of Aragón (Spain) through Projects 13 DGA/Grupos de Investigación Consolidados and PIP065/2005. We thank L. Martin and 14 F. Pons for their assistance in the laboratory work.

Table I .

 I Effect of washing and peeling of fresh licorice roots on ochratoxin A 3

	4	concentrations (ng/g).
	5
		Fresh root	After washing	After peeling	a Percentage
		Sample	(ng/g)	(ng/g)	(ng/g)	of reduction
		1	10.7	17.1	5.7	47.0 %
		2	8.1	10.4	7.1	12.0 %
		3	12.2	13.2	7.0	42.8 %
		4	14.7	11.5	8.6	41.3 %
		5	10.3	10.7	3.3	67.9 %
		6	9.9	8.5	3.1	68.2 %
		7	8.6	8.1	3.4	60.4 %
		8	7.3	8.7	1.1	84.7 %
		b Mean	10.2 A	11.0 A	4.9 B	53.1 %
		Standard error	± 0.9	± 3.0	± 0.9	± 7.9 %
	6
	7	a Percentage reduction of OTA from the starting material (fresh root) to the finished
	8	product (peeled root); b Means with different letter in a row are significantly different by
	9	ANOVA test (p < 0.05).
	10
	11

Page 16 of 18 http://mc.manuscriptcentral.com/tfac Email: tfac@csl.gov.uk Food Additives and Contaminants

	1
	2
	F o r
	P
	e
	e r
	R e v
	i e
	w
	O n l
	y

Table II .

 II Transfer of ochratoxin A into licorice extract and block licorice at laboratory 3 Transfer rate of ochratoxin A from the dry root to the licorice extract; b Transfer rate of 7 ochratoxin A from the dry root to the block licorice. 8

	4	scale.
	5
		Dry root (~ 12 g)	Licorice extract (~ 120 ml)	Block licorice (~ 2.5 g)
		OTA ng/g	OTA ng/ml	a Transfer (%)	OTA ng/g	b Transfer (%)
		253	5.8	22.9	98.4	8.1
		112	2.1	19.1	39.2	7.3
		88	2.0	22.6	39.7	9.4
		53	1.1	21.0	20.4	8.0
		Mean ± SE		21.4 ± 0.9		8.2 ± 0.4
	6
		a

9 10 Page 17 of 18 http://mc.manuscriptcentral.com/tfac Email: tfac@csl.gov.uk Food Additives and Contaminants

  Effect of thermal treatment on ochratoxin A heated at 150ºC in licorice extract during 4 60 min. Initial OTA concentration was 7.7 ng/ml.

	1
	2
	3	Figure 1.
	5
	6
	7
		F o r
		P
		e
		e r
	8 9 10 11	R e v i e
		w
		O n l
		y
		1

Page 18 of 18 http://mc.manuscriptcentral.com/tfac Email: tfac@csl.gov.uk Food Additives and Contaminants

http://mc.manuscriptcentral.com/tfac Email: tfac@csl.gov.ukFood Additives and Contaminants