DEGRADATION OF OCHRATOXIN A BY LACTOBACILLUS ACIDOPHILUS K1

Jarosław Mazurkiewicz
Department of Biotechnology, Human Nutrition and Science of Food Commodities, University of Life Sciences in Lublin, Poland

ABSTRACT

Degradation of ochratoxin A (OTA) was performed in 16 and 24-hour long incubation in PBS of various pH (4 to 7). Standard OTA was dissolved in acetonitrile and added to PBS. The concentration of mycotoxin was determined with the use of direct competence ELISA tests. Dead cells of bacteria were obtained by the thermal inactivation in PBS. The dead cells of Lactobacillus acidophilus K1 strain have the highest ability to degrade OTA after 24 hours of incubation in  PBS at pH 5. At these conditions OTA was degraded by 79%. Lowering of pH caused an increase in the OTA degradation. After 16 hours of incubation at pH 4 the residue of mycotoxin amounted to 25%. The elongation of incubation time caused lowering of mycotoxin concentration. At the shorter time of incubation, live cells showed higher ability to eliminate OTA.

Key words: Ochratoxin A, degradation, ELISA, Lactobacillus acidophilus.

INTRODUCTION

Ochratoxins occur in a wide range of food commodities such as cereals, coffee, cocoa beans, dried fruits, spices, as well as beer, grape juice and wine [10]. Ochratoxin A (OTA) shows the strongest biological effect in this group of mycotoxins. The effect of other ochratoxins is much weaker (e.g. ochratoxin B) or has not yet been proven. OTA has neurotoxic effects, teratogenic, carcinogenic (causing kidney cancer) and may cause inflammation of the intestines [1].

There are many well known methods which are applied for mycotoxins detoxification. They can be classified as: physical, chemical and biological [7]. Many accessible physical and chemical methods are limited due to problems with assuring safety of the products exposed to these interventions, possible losses of the nutritional quality of food products, limited efficiency, as well as the costs of use. Therefore, there is a search for safer methods to protect food products from mycotoxins. Biological methods, for instance by using microorganisms to degrade mycotoxins, can be the solution [2,5]. The bacteria of lactic fermentation can be used effectively to degrade mycotoxins [6].

Lactic acid bacteria (LAB) are widely used for the production of fermented foods. These bacteria are also the part of the gastrointestinal microflora. Many investigations confirm that they have the ability to degrade various mycotoxins and thus the use of LAB may become an alternative to the other methods of mycotoxins degradation [4].

Also the processes of fermentation cause the reduction of the mycotoxins' toxicity. For example, ochratoxin A in barley can be destroyed by ethanol fermentation, whereas strains of Aureobasidium pullulans can lower ochratoxin A contamination in wine grapes [3,5].

The aim of the investigations was to evaluate the biodegradation degree of ochratoxin A (OTA)  caused by live and dead cells of lactic fermentation bacterium – Lactobacillus acidophilus K1 strain.

MATERIALS AND METHODS

The Lactobacillus acidophilus K1 strain was taken from the culture  collection of the Department of Biotechnology, Human Nutrition and Science of Food Commodities, University of Life Sciences in Lublin. OTA came from SIGMA ALDRICH.

The cultures were performed  MRS liquid medium (4.75 mL) inoculated with cell suspension (0.25 mL) and incubated for 24 hours at 37°C (TERMOMETA incubator). After incubation the bacterium concentration was estimated by OD measurement at 600 nm (SMARTSPEC PLUS, BIORAD) spectrophotometer. The liquid cultures with OD = 2 were used for investigations. The cultures were  centrifuged (SIGMA 4K15) at 3000 rpm, at 5°C for 10 min. The supernatant was removed and cells were washed with PBS  (5mL) and were centrifuged (CENTRIFUGE MPW-365, MPW, Poland) for 10 min. at 5000 rpm, 5°C. Subsequently, the liquid above pellet was removed, and 2.5 mL of PBS buffer of pH from 4 to 7 was added. The tubes were centrifuged again in the same conditions. These operation was repeated twice. After that  1 mL of PBS of proper pH was added to the tubes. Dead cells were obtained by  keeping   suspended bacteria  (prepared as above) at 75°C for 1 hour.

OTA was dissolved in acetonitrile at a concentration of 40µg/mL and   dosed (25µL) to the bottom of empty centrifuge tubes. Tubes were transferred to the vacuum chamber's (Varian model 400–1902) to evaporate acetonitrile.

L. acidophilus cells suspended in 1 mL PBS was added to the tubes with OTA. Then the tubes were vortexed and incubated at 37°C for 16 hours. The half of the obtained samples was centrifuged at 5000 rpm, at 5°C, for 10 min. and the supernatant was analysed for mycotoxin content. These operations were performed in the same way with the second part of samples after 24-hour incubation.

Control samples with OTA were also prepared in PBS of different pH without bacterial cells. The procedure for preparation and analysis of control samples was the same as for the appropriate ones with bacteria.

The concentration of mycotoxin was estimated using direct competence ELISA tests – AgraQuant Test Kit (Romer Labs). The analysis was made on the transparent plate with antibodies. The results of ELISA tests were read at a wavelength of 450 nm by using Sunrise (Tecan) reader. The measurement of absorbance was analysed using software Magellan (Tecan). The mycotoxin concentration was calculated from standard  absorbance curve  delivered by the manufacturer.

Data were analyzed statistically using the Tukey test to determine significant differences between the mean values of examined samples at significance level of P≤0.05.

RESULTS AND DISCUSSION

Significant differences in the degradation of ochratoxin A were found after 16 and 24 hours of incubation. Elimination of ochratoxin A by live and dead cells after 24-hour incubation was greater than after 16 hours (Fig. 1). The best results in the reduction of mycotoxins were achieved at pH 5 by dead cells after 24 hours of incubation, where ochratoxin A was degraded by 79%. Lowering pH caused an increase in the degradation of OTA, as after 16-hour incubation with dead cells, the residue of mycotoxin increased from 5.5% in PBS at pH7 to 35.2% at pH4 degraded by live cells. However, after 24 hours the lowest concentration of mycotoxins in PBS at pH 5 was received, which amounted to 208 ng/mL (Table 1). Dead cells were characterized by better degradation after 24 hours of incubation, but after 16 hours OTA was degraded more significantly by living cells. Incubation at 75°C for an hour destroys enzymes effectively. Therefore, the lower content of mycotoxin may be due to the fact that OTA was adsorbed on the remains of cells. OTA could be bound by the cell walls of the damaged cells.

a)

b)

The highest content of OTA (946 ng/mL) was detected in the sample after 16-hour incubation with thermal inactivated cells at pH7. After degradation by live cells, the highest mycotoxin residue amounting to 924 ng/mL was detected in the buffer at pH 6 after 24 h of incubation. Incubation time and the pH have become significant factors influencing the elimination rate of the analyzed toxin.

The lowest concentration of mycotoxins (25% of the initial concentration) obtained by live cells of bacteria in the sample resulted from the application of the substrate with the lowest pH (pH 4). However, dead cells more significantly decreased the content of the mycotoxin in PBS with a higher pH equal to 5 (20%).

In studies conducted by Fush et al. [4] the most effective strain of lactic acid bacteria of the species Lactobacillus, which caused the greatest decrease in the quantity of ochratoxin A (OTA), was Lactobacillus acidophilus VM 20. That strain caused 97% reduction of OTA from liquid medium after 4 hours of incubation at 37°C. The most effective Bifidobacterum strains were Bifidobacterium longum LA 02 and Bifidobacterium longum VM 14. Those strains caused about 50% degradation of ochratoxin A. The pH optimum at which the largest OTA reduction was observed was 5.0. The amount of bacteria added to a mixture of mycotoxins strongly influenced the elimination of ochratoxin from liquid medium.

Piotrowska and Żakowska [8] found that the Lactobacillus acidophilus (K1, VM 20) strains have the largest ability to degrade ochratoxin A as well as Bifidobacterium longum (LA 02, VM 14), which degraded OTA in 50%. The lowest residue of OTA was observed at pH 5. In conducted investigations, pH 5 was also the most proper for ochratoxin A degradation. Taking into account the influence of the thermal inactivation of cells on the degradation, live cells were characterized by the greater ability to deactivate OTA than the thermal inactivated cells. The authors obtained similar results after 16 hours of incubation, but after 24 hours dead cells showed greater ability to biodegrade ochratoxin A.

Other microorganisms were also taken into account in the studies on the biodegradation of OTA [2]. The Aspergillus strains have that ability. Strains of Aspergillus fumigatus and Aspergillus niger were found to detoxify the culture medium from ochratoxin A. After two days of incubation the degradation amounted to 20%, however, after 7 days this value reached 100% [9].

CONCLUSIONS

1. The dead cells of Lactobacillus acidophilus K1 strain have the highest ability to degrade ochratoxin A after 24 hours incubation on PBS at pH5. In these conditions ochratoxin A was degraded in 79%.

2. Low pH improved  the degree of degradation. After 16 hours of incubation at pH 4 the residue of mycotoxin amounted to 25%. The longer time of incubation influenced the lower concentration of mycotoxin.

3. The elongation  of incubation time caused further decrease of mycotoxin concentration. In the initial stage of incubation, live cells showed greater ability to eliminate ochratoxin A.

4. The highest residue of mycotoxin was detected after 16 hours of incubation in pH6 PBS with live cells, and after 16 hours of incubation in pH7 with thermally inactivated cells.

REFERENCES

1. Bräse S., Encinas A., Keck J., Nising C.F., 2009. Chemistry and Biology of Mycotoxins and Related Fungal Metabolites., Chemical Reviews 109(9), 3903–3990.

2. Ciconova P., Laciakova A., Mate D., 2010. Prevention of Ochratoxin A contamination of food and ochratoxin A detoxification by microorganisms-a review. Czech J. Food Sci. 28(6), 465–474.

3. Felice D.V., Solfrizzo M., De Curtis F., Lima G., Visconti A., Castoria R., 2008. Strains of Aureobasidium pullulans Can Lower Ochratoxin A Contamination in Wine Grapes. Postharvest Pathology and Mycotoxins 12, 1261–1270.

4. Fuchs S., Sontang G., Stidl R., Ehrlich V., Kundi M., Knasmuller S., 2008. Detoxification of patulin and ochratoxin A, two abundant mycotoxin, by lactic acid bacteria., Food and chemical Toxicology 46, 1398–1407.

5. Kabak B., Dobson A.D.W., Var I., 2006. Strategies to prevent mycotoxin contamination of food and animal feed. Critical Reviews in Food Science and Nutrition 46, 593–619.

6. Niderkorn V., Boudra H., Morgavi D.P., 2006. Binding of fusarium mycotoxins by fermentative bacteria in vitro. Journal of Applied Microbiology 101, 849–856.

7. Park B.J., Kosuke T., Yoshiko S.K., 2007. Degradation of mycotoxins using microwave-induced argon plasma at atmospheric pressure. Surface & Coatings Technology 210, 5733–5737.

8. Piotrowska M., Żakowska Z., 1997. Metody oznaczania mikotoksyn [Methods for the determination of mycotoxins]. Przemysł Spożywczy 12, 16–18 [in Polish].

9. Varga J., Rigo K., Teren J., 2000. Degradation of ochratoxin A by Aspergillus species. International Journal of Food Microbiology 59(1), 1–7.

10. Zimmerli B., Dick R., 1996. Ochratoxin A in table wine and grape juice: Occurrence and risk assessment. Food Addit. Contam. 13, 655–668.

Accepted for print: 3.06.2011

Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed 'Discussions' and hyperlinked to the article.