Explore
Communities in English
Advertise on Engormix

Patulin And Citrinin In Portuguese Apples

Patulin And Citrinin In Portuguese Apples With Rotten Spots

Published: September 5, 2004
By: Alberto Gimeno
Technical Consultant of SPECIAL NUTRIENTS, INC., 1394 Coral Way, Miami, Florida, 33145 USA.
The author refers to a part of the article "Co-occurrence of patulin and citrinin in Portuguese apples with rotten spots" ublished by M.L Martins, A.Gimeno, H.M.Martins and F.Bernardo in Food Additives and Contaminats, 2002, Vol.19, No.6, 568-574

The partial reproduction of the original article was done with the permission of the authors previously mentioned.


Abstract
Patulin and citrinin are mycotoxins produced by certain fungi mainly belonging to Penicillium and Aspergillus and may be detectable in mouldy fruits and fruit products. The data presented in this study refer the simultaneous occurrence of patulin and citrinin in 351 samples of seven different varieties of apples with small rotten areas (Casanova, Golden Delicious, Red Delicious, Reineta, Richared, Rome Beauty and Starking). It used a rapid multi-detection thin layer chromatography (TLC) method. The minimum detectable concentrations of the patulin and the citrinin were 120-130 micrograms/kg and 15-20 micrograms/kg respectively. The percentage of contamination with patulin only was higher ( 68.6 %) than the percentage of contamination with citrinin only (3.9 %). Patulin and citrinin (19.6%) were also detected simultaneously. The highest mean patulin content was 80.50 mg/kg for the Richared variety, but the mean level of citrinin was lower. The lowest mean contaminations of patulin were found in Rome Beauty, Red Delicious and Reineta, ranging from 3.06 to 5.37 mg/kg. All analyzed apples varieties had low citrinin contamination, ranging from 0.32 to 0.92 mg/kg. These findings indicate that there may be a risk of human exposure to patulin through the consumption of juices and jams manufactured with apples with small rotten areas.
Key words : Patulin, citrinin, TLC, co-occurrence, apples.



Introduction
Patulin and citrinin are toxins produced by moulds of the genera Aspergillus and Penicillium (Steiman et al. 1989), including A. clavatus, P. patulum and P. expansum (Scott 1994). They can be detected in mouldy fruit, such as apples, pears (Harwig et al. 1973, Scott 1974, Ciegler et al. 1977, Frank 1977, Burdaspal and Pinilla 1979, Mortimer et al. 1985, Martins 1987), and in cereals (Harwig et al. 1977, Lopez-Diaz and Flannigan 1997). Patulin is particularly associated with brown rot in apples (Krogh 1987). The minimum aw value for patulin production are 0.99 and 0.95 and the temperature range were 0 to 24ºC and 4 to 31ºC for P. expansum and P. patulum, respectively (Northolt et al. 1978).

Patulin (4-hydroxy-4H-furo[3,2-c]pyran-2(6H)-one) is a toxic substance with suspected carcinogenic properties (McKinley et al. 1982). Pohland and Allen (1970) reported that patulin rapidly reacts with sulfur dioxide (SO2) so that it could be destroyed when the latter is used as an antioxidant or antimicrobial agent. Its biological activity is decreased in an alkaline medium and in presence of molecules comprising sulfhydryl group such as cysteine and glutathione (Lindroth 1980). From experiments it has been demonstrated that while the patulin is eliminated during fermentation in wines (Ough and Corison 1980), its content is decreased only by about 20% during the usual technological process of fruit juice production (Harrison 1989). Bissessur et al. 2001 showed that patulin was significantly reduced in apple juice when clarification procedures were employed. Therefore, pressing followed by centrifugation resulted in an average toxin reduction of 89%. The total toxin reduction using filtration, enzyme treatment and fining were 70, 73 and 77%, respectively. Clarification was successful in the reduction of patulin in apple juice. Nevertheless, clarification resulted in high levels of patulin in the pressed pulp after filtration and centrifugation, and this could be harmful if they were used as animal feeds (Bissessur et al. 2001). Patulin was originally described as an antibiotic and exhibits strong antibiotic activity against different Gram-positive and Gram-negative bacteria including Mycobacterium tuberculosis. It also possesses antifungal activity and it is highly toxic to plant and animal cells in tissues (Sorenson et al. 1985). Several studies indicate the immunosuppressive action of patulin, which is known for producing ulceration, congestion, and haemorrhagic lesions, particularly in the gastrointestinal tract (McKinley et al. 1982).

Combined reproductive toxicity, long-term toxicity/carcinogenicity studies in rats have established a provisional maximum tolerable daily intake (PMTDI) of 0.4 micrograms/kg bw , based on a no-effect level (NOEL) of 43 micrograms/kg bw/day ( JECFA,1995).

Citrinin (3R-trans)-4,6-dihydro-8-hydroxy-3,4,5-trimethyl-6-oxo-3H-2-benzo-pyran-7-carboxylic acid (Cole and Cox 1981) was first identified as a secondary metabolite of P. citrinum, from which it derived its name. The site of effects of citrinin is primarily the kidneys and liver; within a few hours after citrinin administration, DNA, protein, and glutathione (GSH) content in these tissues are decreased (Cheeke and Shull 1985). Campbell et al. (1981) demonstrated that citrinin appears to be immunostimulatory rather than immunosuppressive; however, it remains to be determined whether ingestion of feed contaminated with low levels of citrinin produces responses similar to intraperitoneal exposure. Citrinin has been suggested as a causative factor in renal disease among livestock, poultry, pigs, rats and dogs (Reiss 1977, Cheeke and Shull 1995,). Citrinin was strongly implicated as the cause of pruritis, pyrexia and haemorragic syndrome in cattle (Griffiths and Done 1991). The renal system of humans is affected the most and the mitochondrial respiratory chain was identified as a possible sensitive target for this mycotoxin (Ammar et al 2000). It is commonly found in grain samples along with ochratoxin A and these two mycotoxins have been suggested as the cause of porcine nephropathy in Denmark (Betina 1989). Citrinin is a primarily known as nephrotoxin, and few studies have addressed its potential for immunotoxicity (Sharma 1993).
Several countries have set limits for the content of patulin in foods of 50 micrograms/kg or 50 micrograms/L (FAO 1997). There is no specific legislation for citrinin; nevertheless, the FAO (1997) refers to a level of zero for ´all mycotoxins` in preserved food in Hungary, legume products in The Netherlands and ice cream in Trinidad.

The aim here was to provide information on the occurrence and levels of patulin and citrinin in different varieties of apples with small brown rotten areas in a significant sampling. The ratio of weight of the rotten area to the whole apple was about 1/3. We determined these mycotoxins using a rapid multidetection TLC method (Gimeno and Martins 1983, Gimeno 1984)



Results and Discussion
A total of 351 apples with different-sized brown rotten areas and of different varieties (44 Casanova, 60 Golden Delicious, 28 Red Delicious, 62 Reineta, 40 Richared, 42 Rome Beauty and 75 Starking) were collected randomly from markets and supermarkets in Portugal for determination of patulin and citrinin by a rapid multi-detection TLC method. The whole apple and the respective rotten area were weighed and it was found that from 351 samples the rotten area represented an average of 32 % of the whole apple; they ranged from 25 to 42 % (SD = 22). There was no correlation between the concentration of patulin and/or citrinin and the percentage of the rotten area in the whole apple, e.g. apples with a lower percentage of rotten area than the others showed a significantly higher mycotoxin concentration than the others, and apples with a higher percentage of rotten area than the others showed a significantly lower mycotoxin concentration than the others.

A total of 241 samples of the 351 analyzed (68.6 %) contained patulin only (table1). Fourteen samples (3.9%) were contaminated with citrinin only and 69 samples (19.6%) were simultaneously contaminated with patulin and citrinin. Twenty-seven (7.6%) were negatives. (table 1). All apples analyzed had a higher incidence of patulin contamination compared with that of citrinin; and of the seven analyzed varieties, the Red Delicious and Reineta showed greater frequency of contamination (table 1).

The highest mean patulin content was 80.5 mg/kg for the Richared variety; nevertheless the citrinin contamination was the lowest (0.32 mg/kg) (table 2). Casanova and Starking varieties had mean patulin concentrations of 33.10 and 13.67 mg/kg; Reineta, Red Delicious, Rome Beauty and Golden Delicious showed low contamination, ranging from 5.37 to 3.05 mg/kg. (table 2).

All analyzed apples varieties had lower citrinin contamination compared with patulin contamination. Rome Beauty variety had the highest mean level of citrinin (0.92 mg/kg) but a low mean level of patulin. The others varieties had mean citrinin levels ranging from 0.32 to 0.76 mg/kg. (table 2).

In this study we also verified that the patulin had spread to the areas not affected by rot. We did not found citrinin in these areas, and this may be explained by the lower content of this mycotoxin in apples analyzed.

Surveillance studies of patulin in apples and apple-based food were reported by Beretta et al. (2000). Their data showed lower contamination with levels ranging from 0.71 to 1170 micrograms/kg. Patulin was also found by Frank (1977) in 50% of different varieties of apples and pears with brown-rotten areas (about 120 samples). Fritz et al. (1979) found patulin concentrations ranging from 0.002 to 0.3 mg/L in commercial apple juice and from 0.3 to 42 mg/kg in the brown-rotten portions of apples. In Brazil, in 30 test samples of apple juice, only one was found to contain patulin at 17 micrograms/L, (deSylos and Rodriguez-Amaya 1999). Viñas et al. (1993) studied the citrinin producing capacity of 122 Penicillium expansum strains isolated from apples from Lleida (Spain) and verified that among the strains examined 46% produced citrinin in a culture medium (glucose yeast agar[GYA]) and 73 % of these strains were isolated from decayed apples.

The mean levels of patulin and citrinin found in the present survey were higher than those presented by the others researchers mentioned above. This may be due to the superior toxigenic potential of the indigenous Penicillium strains, to the substrate composition or to the physical ecological conditions (temperature, pH ). The values show that if apple juice and apple-based foods are prepared with low-quality fruit the presence of patulin and citrinin can be higher than the safe limits established by international committees and this could be true even when the brown rotten area is removed from the apples. According to FAO (1997), limits of 50 micrograms/L of patulin have been set for fruit derivatives and the data presented in this study indicate mean concentration levels above this. According to the same reference (FAO 1997) there are no specific levels established for citrinin in foods or feeds.




References
AMMAR, H., MICHAELIS, G., and LISOWSKY, T, 2000, A screen of yeast repiratory mutants for sensitivity against the mycotoxin citrinin identifies the vacuolar ATPase as an essential factor for the toxicity mechanism. Current Genetics, 37, 277-284.
BERETTA, B., GAIASCHI, A., GALLI, C.L., and RESTANI, P, 2000, Patulin in apple-based foods: ocurrence and safety evaluation. Food Additives and Contaminants, 17, 399-406.
BETINA, V., 1989. Mycotoxins. Chemical, Biological and Environmental Aspects. (New York: Elsevier).
BISSESSUR, J., PERMAUL, K., and ODHAV, B, 2001, Reduction of patulin during apple juice clarification. Journal of Food Protection, 64, 1216-1219.

BURDASPAL, P.A., and PINILLA, I, 1979, Estudio de la contaminación natural por micotoxinas en manzanas y otros productos. Aplicación de un nuevo método de multidetección. VII Symposium Nacional de Microbiologia, Sociedad Española de Microbiologia, 24-27 de Septiembre, Cadiz, España, 35-37.
CAMPBELL, M.L., DOER, J.A., and WYATT, R.D., 1981, Immune status in broiler chickens during citrinin toxicosis. Poultry Science, 60, 1634-1636.
CHEEKE, P.R., and SHULL, L.R., 1995, Natural Toxicants in Feeds and Poisonous Plants. (Wesport, CT: AVI Publishing Company, Inc.), p.442-445.
CIEGLER, A., VESONDER, R. F., and JACKSON, L. K., 1977, Production and biological activity of patulin and citrinin from Penicillium expansum. Applied and Environmental Microbiology, 33, 1004-1006.
COLE, R. J., and COX, R. H., 1981, Handbook of Toxic Fungal Metabolites. (New York: Academic Press).
De SYLOS, C. M., and RODRIGUEZ- AMAYA, D. B., 1999, Incidence of patulin in fruits and fruit juices marketed in Campinas, Brazil. Food Additives and Contaminants, 16, 71-74.
FAO (FOOD AND AGRICULTURE ORGANIZATION). 1997, Worldwide Regulations for Mycotoxins, 1995. A compendium. FAO Food and Nutrition: Paper 64. (Rome: Food and Agriculture Organization of the United Nations).
FRANK, H. K., 1977, Occurrence of patulin in fruit and vegetables. Annales de la Nutrition et de L´Alimentation, 31, 459-465.
FRITZ, W., BUTHIG, C., and ENGST, R., 1979, Determination and the hygienic- toxicologic significance of patulin in fruit and fruit products. Nahrung, 23, 159-167.
GIMENO, A., and MARTINS, M. L., 1983, Rapid thin layer chromatographic determination of patulin, citrinin and aflatoxin in apples and pears, and their juices and jams. Journal of the Association of Official Analytical Chemists, 66, 85-91.
GIMENO, A., 1984, Determination of citrinin in corn and barley on thin layer chromatographic plates impregnated with glycolic acid. Journal of the Association of Official Analytical Chemists, 67, 194-196.
GRIFFITHS, I.B., and DONE, S.H, 1991, Citrinin as a possible cause of the pruritis, pyrexia, haemorragic syndrome in cattle. Veterinary Record, 129, 113-117
HARRISON, M. A., 1989, Presence and stability of patulin in apple products: a review. Journal of Food Protection, 9,147-153.
HARWIG, J., CHEN, Y.-K., KENNEDY, B.P.C., and SCOTT, P.M., 1973, Occurrence of patulin and patulin-producing strains of Penicillium expansum in natural rots of apples in Canada. Canadian Institute of Food Science and Technology Journal, 6, 22-25.
HARWIG, J., BLANCHFIELD, B.J., and JARVIS, G., 1977, Effect of water activity on disappearance of patulin and citrinin from grains. Journal of Food Science, 42, 1225-1228.
JOINT FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS/ WORLD HEALTH ORGANIZATION EXPERT COMMITTEE ON FOOD ADDITIVES (JECFA) (WHO), 1995. Evaluations of Certain Food Additives and Contaminants. WHO Technical Reports Series, 859 (Geneva: WHO).
KROGH, P., 1987, Mycotoxins in Food. (New York: Academic Press), pp. 54-62.
LINDROTH, S., 1980, Occurrence, formation and detoxification of patulin mycotoxin. Technical Research Centre of Finland. Materials and Processing Technology. Publication 24. ISBN 951-38-1096-8.
LOPEZ-DIAZ, T.M., and FLANNIGAN, B., 1997, Production of patulin and cytochalasin E by Aspergillus clavatus during malting of barley and wheat. International Journal of. Food Microbiology, 35, 129-136.
MARTINS, M.L., 1987, Patulina e citrinina em maçãs e pêras. Thesis. Laboratorio Nacional de Investigação Veterinária , Lisboa- Portugal.
MCKINLEY, E.R., CARLTON, W. W., and BOON, G. D., 1982, Patulin mycotoxicosis in the rat. Toxicology, pathology and clinical pathology. Food and Chemical Toxicology, 20, 289-300.
MORTIMER, D.N., PARKER, I., SHEPHARD, M.J., and GILBERT, J., 1985, A limited survey of retail apple and grape juices for the mycotoxin patulin. Food Additives and Contaminants, 2, 165-170.
NORTHOLT, M.D., van EGMOND, H.P, and PAULSCH,W.E., 1978, Patulin production by some fungal species in relation to water activity aqnd temperature. Journal of Food Protection,41, 885-890.
OUGH, C. S., and CORISON, C. A.,1980, Measurement of patulin in grapes and wines. Journal of Food Science, 45,476-478.
POHLAND, A. E., and ALLEN, R., 1970, Stability studies with patulin. Journal of the Association of Official Analytical Chemists, 53, 688-691.
REISS, J., 1977, Mycotoxins in foodstuffs. X. Production of citrinin by Penicillium chrysogenum in bread. Food and Cosmetics Toxicology,15, 303-307.
SCOTT, P.M., 1974, Patulin. Mycotoxins, edited by I.F.H Purchase (Amsterdam: Elsevier Scientific Publishing Company), pp. 383-403
SCOTT, P.M., 1994, Penicillium and Aspergillus toxins. Mycotoxins in Grain: Compounds Other Than Aflatoxin, edited by J.D.Miller and H.L.Trenholm (St Paul, MN: Eagan Press), pp.261-285.
SCOTT, P.M., and KENNEDY, B.P.C., 1973, Improved method for the thin layer chromatographic determination of patulin in apple juice. Journal of the Association of Official Analytical Chemists, 56, 813-816.
SHARMA, R.P., 1993, Immunotoxicity of mycotoxins. Journal of Dairy Science,76, 892-897.
SORENSON, W.G., SIMPSON, J., and CASTRANOVA, V., 1985, Toxicity of the mycotoxin patulin for rat alveolar macrophages. Environmental Research, 38, 407-416.
STEIMAN, R., SEIGLE-MURANDI, S., SAGE, L., and KRIVOBOK, S., 1989, Production of patulin by micromycetes. Mycopathologia, 115, 129-133.
VIÑAS, I., DADON, J., and SANCHIS, V., 1993, Citrinin-producing capacity of Penicillium expansum strains from apple packinghouses of Lerida (Spain). International Journal of Food Microbiology, 19, 153-156.
WOUTERS, M.F.A., and SPEIJERS, G.J.A., 1996, Patulin. Toxicological Evaluation of Certain Food Additives and Contaminants (Geneva: World Health Organization), pp. 337-402.


Table 1-Frequency of patulin and citrinin in apple

Variety of apple
Positiveª Patulin /n
%
Positive b Citrinin /n
%
Positivec
Patulin+Citrinin /n
%
Negatives
%
Casanova
25/44
56.8
0/44
0
12/44
27.3
7
15.9
   
nter">
     
Golden Delicious
50/60
8330
3/60
5.0
7/60
11.7
0
0
         
Red Delicious
25/28
89.3
0/28
0
3/28
10.7
0
0
         
         
Reineta
50/62
80.6
5/62
8.1
4/62
6.4
3
4.9
         
Richared
21/40
52.5
1/40
2.5
18/40
45.0
0
0
         
         
Rome Beauty
30/42
71.4
0/42
0
10/42
23.8
2
4.8
         
Starking
40/75
53.3
5/75
6.7
15/75
20.0
15
20.0
         
         
         
Total = 351
241/351
68.6
14/351
3.9
69/351
19.6
27/351
7.6
         

n - total number of apple variety;
ª patulin positive samples only;
b citrinin positive samples only;
c samples positive for both patulin + citrinin.




Table 2 - Levels of patulin and citrinin (mg/kg) in apples - statistical data

Variety of Apple
n
Patulin
Citrinin
  
Mean
mg/kg
Standard
deviation
Mean
mg/kg
Standard
deviation
Casanova
44
33.1
70.3
0.76
0.75
      
Golden Delicious
60
3.05
4.76
0.40
0.36
      
Red Delicious
28
4.37
10.1
0.37
0.62
      
Reineta
62
5.37
30.1
0.60
0.9
      
Richared
40
80.5
161
0.32
0.68
      
Rome Beauty
42
3.06
20.1
0.92
0.63
      
Starking
75
13.7
39.2
0.50
0.75

n-total number of apples




Patulin And Citrinin In Portuguese Apples With Rotten Spots - Image 1

Figure 1.- Qualitative analysis. Samples contaminated with patulin. TLC plate sprayed with methyl-benzothiazolinone hydrazone hydrochloride monohydrate (MBTH), heated at 130ºC for 15 min (Scott and Kennedy 1973) and observed under 366 nm UV light. (Left to right) Five samples: sample 1, sample 1 + internal standard; sample 2, sample 2 + internal standard; …………external standard.




Patulin And Citrinin In Portuguese Apples With Rotten Spots - Image 2

Figure 2 - Qualitative analysis. Some samples were contaminated with citrinin. The TLC plate was sprayed with 20% AlCl3 in methanol, heated 5 min at 105ºC and observed under 366 nm UV light. (left to right) Four samples: sample1, sample1 + internal standard; sample 2, sample 2 + internal standard; …………external standard.
Related topics
Authors:
ALBERTO GIMENO
Alberto Gimeno
Join to be able to comment.
Once you join Engormix, you will be able to participate in all content and forums.
* Required information
Would you like to discuss another topic? Create a new post to engage with experts in the community.
Create a post
Join Engormix and be part of the largest agribusiness social network in the world.
LoginRegister