Mycotoxin Prevention and Control in Agriculture - American Chemical

Chapter 4

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The fate of ochratoxin A in soy milk and bean curd (tofu) productions Maria H. Iha1,*, Mary W. Trucksess2, Jeanne I. Rader2 1

Seção de Bromatologia e Química, Instituto Adolfo Lutz – Laboratório I de Ribeirão Preto, Ribeirão Preto, São Paulo, Brazil 2 Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, Maryland, United States

Soybeans are a good source of protein. Soy products are widely consumed, particularly in vegetarian diets. The aim of the study was to determine the distibution of OTA in bean curd (tofu), soy milk and by- products during tofu and soy milk production. The effects of washing, soaking and cooking on OTA in soybean were also determined. By-products collected from each step before and after cooking were analyzed for OTA. Soy milk and tofu were prepared and all by-products were collected and analyzed for OTA. OTA was quantified using a method consisting of methanol-sodium bicarbonate extraction, dilution with buffer, immunoaffinity column cleanup, liquid chromatographic separation and fluorescence detection. The results show that washing, soaking and cooking can reduce the OTA in final products if the water used is discarded. Approximately 50 - 80% of incurred OTA in soybeans could be reduced during the production of soy milk and tofu.

Ochratoxins are a group of chemically similar (isocoumarin derivatives) mycotoxins, produced by Aspergillus ochraceus and related species as well as by Penicillium species. The major mycotoxin in this group is ochratoxin A (OTA) (1). OTA is nephrotoxic in animals, and exerts immunotoxic, neurotoxic and teratogenic effects at higher dose levels. (2) OTA has been found in a wide © 2009 American Chemical Society In Mycotoxin Prevention and Control in Agriculture; Appell, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

59


60 variety of food products such as coffee, grapes, paprika (3), sultanas (4), rice (5), dried fruits (6), wine (6, 7), beer (8), and cereal grains (9, 10). Soybeans are an excellent source of protein. They contain little saturated fat and significant amounts of dietary fibre (11). Dietary soy protein has been shown to have beneficial effects on obesity (12). However, soybeans can become moldy in the field or during storage, and thereby become contaminated with mycotoxins. Schollenberger et al., (13) studied the natural occurrence of mycotoxins in soy food marketed in Germany. The results demonstrated the possibility of a multi-mycotoxin contamination in these products. Numerous reports have shown that OTA is commonly found in corn and cereal grains worldwide. OTA is not the predominant mycotoxin in soybean products. Climate changes can influence fungi and mycotoxin contamination, therefore, it is important to study this mycotoxin. In the Republic of Croatia, Domijan et al. (14) investigated the presence of seed-borne fungi and OTA in dried beans and found OTA at low concentrations. In our laboratory, we found in two kinds of dried beans samples: one pink bean and one green bean (Phaseolus vulgaris L.) were contaminated with OTA at levels of 60 and 110 µg/kg (personal communication). The pink beans showed no apparent damage but the green beans showed seed coat damaged and had high level of split. We concluded that high initial levels in beans from field, the lack of suitable extended storage facilities and optimum storage conditions could cause molds to grow and increase the OTA production in the dried beans. In our previous work (15), we investigated the effects of washing, soaking and cooking on OTA content in dried beans. The study provided evidence that discarding the washing, soaking and cooking water led to a significant reduction of OTA contamination in residual dried beans. Soybeans can be processed in various manners and these result in productions of soy milk, okara, and tofu. Soy milk, a beverage made from soybeans, is a stable emulsion of oil, water and protein. The dried soybeans are soaked in water, ground, and cooked. The beans are then separated from the soy milk. The residual beans that are separated from soy milk are called “okara” or soy pulp. This white by-product resembles wet sawdust. Okara, which is high in protein and fiber (16), is used in Japanese cooking for soups, vegetable dishes and even salads. Tofu is prepared from soy milk. Calcium chloride or magnesium chloride is added to the soy milk and is heated to produce tofu. Tofu is high in protein and is one of the most popular oriental food ingredients (16). The objective of this study was to determine the distribution of OTA in soy milk, tofu and by products during their production from soybeans.

Materials and Methods Reagents and Materials The following chemicals and supplies were obtained from various suppliers: LC grade methanol, acetonitrile (EM Science, Gibbstown, NJ, USA); NaHCO3 (J. T. Baker, Phillipsburg, NJ); ACS grade acetic acid (Merck, Darmstadt,

In Mycotoxin Prevention and Control in Agriculture; Appell, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


61 Germany); phosphate buffered saline, pH 7.4 (PBS, Sigma P-3813, SigmaAldrich, St. Louis, MO); Tween 20 (Sigma, P-5927); Clorox (6.25% sodium hypochlorite, Clorox, Oakland, CA); magnesium chloride (Merck, Darmstadt, Germany), glass microfiber filter paper (Whatman 934AH, Whatman, Inc., Clifton, NJ); OchraTest column (G1017, Vicam, Watertown, MA). A. ochraceus ATCC 22947 was purchased from the American Type Culture Collection (Manasas, VA, USA).The ochratoxin immunoaffinity columns (IAC) contain monoclonal antibodies cross reactive towards OTA and should have a minimum binding capacity of not less than 100 ng OTA, and should give a recovery of not less than 80%. OTA standard – O1877 (Sigma-Aldrich Chemie Gmbh, Steinheim, Germany). An OTA solution at approximately 100 µg/mL in methanol was prepared and then 1 mL of the solution was pipetted into a 3 mL volumetric flask and diluted to volume with methanol. Absorbance was determined at 333 nm. A molar absorptivity of 6330 was used to calculate concentration of the second stock standard solution which should be approximately 30 µg/mL. The stock solutions were stored at -18o C. Working OTA Calibrant Solution were prepared daily in methanol at concentrations of 4, 2, 1, 0.5 and 0.25 ng/mL. The extraction solution was methanol - 1% sodium bicarbonate (7 + 3 v/v). The diluting solution was prepared by dissolving 1 package PBS in 1 L of water and adding 1 mL Tween 20. Apparatus The apparatus used in our experiments included the following: liquid chromatography (LC) system, Waters Model 2690 Alliance separate system (Waters, Milford, MA), Waters Model 2475 fluorescence detector; or , LC system from Shimadzu Instruments (Kyoto, Japan) with a fluorescence detector, a Rheodyne L.P. injector with a 50 μL loop (Rheodyne, Cotati, CA, USA); LC column (Beckman, catalogue #235332, Ultrasphere, 4.6 x 250 mm, 5 µm (Beckman Instruments, Inc., Fullerton, CA), or equilvalent; grinder (Blixer 3, Robot Coupe USA Inc., Ridgeland, MS); centrifuge (Allegra X – 22R Centrifuge, VWR International, Bridgeport, NJ); blender (Waring Laboratory, Torrrington, CT); vortex (Vortex 2 Genie, Scientific Industries, Bohemia, NJ); and orbital shaker (DS-500E, VWR, West Chester, PA). Materials OTA incurred soybeans (Glicine max L.) and soybeans were purchased from local markets and were analyzed for OTA. The soybeans were found to be free of OTA contamination (Limit of detectiton 0.1 µg/kg). Twenty- five g soybeans and 100 mL of 10% Clorox solution were placed in a sterile wide mouth flask. After 1 minute, the flask was emptied and the soybeans were rinsed 3 times with 200 mL sterile water. An appropriate amount of sterile water was added to result a total water content of 25 g (the sum of water absorbed by the beans from washing and water added). After storing at 5o C for 14 hours, the beans were inoculated with A. ochraceus suspension and kept at ambient


62 temperature (about 25o C) for 3 days. Beans used to process soybean test sample were disinfected by soaking in 50 mL methanol overnight and rinsing 3 times with water. Beans were examined for mold growth and no A. ochraceus was found. The beans were analyzed for OTA production and were dried at 70o C for 5-6 h until the weight of the beans was reduced to 25 g. Methods


Processing of soybean test samples A) Washing, soaking and cooking soybeans Liquid was collected from soybeans after 3 different processing procedures: washing, soaking and cooking (Figure 1). Six replicate test samples were carried through each processing procedure. (a) Washing: 5 g soybeans were washed with 25 mL water for 60 min. The washing water and the beans were separated and OTA analyses were performed for both test samples; (b) Soaking: 5g soybeans were soaked with 25 mL water for 60 min. The soaking water and the beans were separated and OTA analyses were performed for both test samples; (c) Cooking: 5 g soybeans were cooked with 25 mL water for 60 min. After cooking, the liquid was separated from the beans and OTA analyses were performed for both test samples.

B) Soy milk and tofu production Soy milk and tofu were prepared using a mixture of OTA-free and OTA incurred soybeans. Figure 2 shows the procedure used for production of soy milk and tofu. In our first trial (n=4), approximately 34 g of OTA-free and 34 g OTA-incurred soybeans were used, while in the 2nd and third trials (n=2), approximately 4 g OTA-incurred soybeans and 50 g of OTA-free soybeans were used. The beans were soaked in 150 mL water overnight (16 hours). The soaking water was separated from the beans and OTA was analyzed. The soaked beans were ground with 230 mL water, an aliquot was collected and OTA was analyzed. The ground beans were cooked for 10 min and filtered. The residual ground beans (okara) and an aliquot soy milk were analyzed for OTA. After warming to 70oC, 2 g magnesium chloride were added to the approximately 200 mL warm soy milk. The mixture was stirred gently. After tofu was formed, the whey was separated from the tofu. Both tofu and whey were analyzed for OTA.


63 Washing


Cooking

25 mL

25 mL

25 mL

5g soybean

5g

5g

Wash for 60 min

OTA

Soaking

Soak for 60 min

OTA analysis

Cook (boil) for 60 min

OTA analysis

Figure 1. Processing procedures (washing, soaking, and cooking) for soybean.


64 Soaking water OTA analysis

150mL H2O

Soaking water

OTA-incurred soybean + healthy soybean

16 hours Beans

Ground


230 mL H2O

Ground beans

Boiling 10 min Filter

Residual ground beans

Okara

Soy milk Soy milk 70oC

2g MgCl2

Tofu Tofu

Tofu whey

Tofu whey

Figure 2. Procedure to produce soy milk and tofu. Method of analysis OTA was determined using a published method (17). The method consists of methanol-sodium bicarbonate extraction, dilution in buffer, immunoaffinity column cleanup, and reversed phase liquid chromatographic/fluorescence detection, separation and determination. The mobile phase was acetonitrile:water:acetic acid (47:53:1, v/v) at a flow rate of 1 mL/min. The fluorescence detector was set at an excition wavelength of 333 nm and an emission wavelength of 460 nm. The limit of detection was 0.1 μg/Kg and recovery 86%.



65 Liquid separated from beans after washing, soaking and cooking, from tofu processing after each step, soy milk and residual beans and tofu were all analyzed for OTA. Details of analysis are described below: (a) Analysis of liquid separated from beans, soy milk, and tofu whey: Five mL liquid, 1 mL 5% NaHCO3 and 14 mL methanol were placed in 50 mL tubes, mixed on a vortex and centrifuged for 10 min (7000 rpm, g = 5323 mm s-2). The supernatant (5 mL) and 40 mL diluting solution were mixed and then filtered through a glass microfiber filter. Five milliliters of filtratrate were passed through an IAC. The column was washed with 5 mL diluting solution and 5 mL water. OTA was eluted 2 times with 1 mL MeOH. The eluate was collected into a 5-mL volumetric flask and diluted to volume with water. After mixing, the test extract was subjected to LC separation and quantitation. (b) Analysis of beans, ground bean, okara and tofu: Appropriate amounts of methanol and 3% NaHCO3 were added to 5 g test samples to result in a total volume of 25 mL extraction solvent containing a mixture of methanol:1%NaHCO3 (7+3). Test samples were shaken for 30 min on a shaker. Beans were homogenized with a polytron for 2 min. The extract was centrifuged for 10 min (7000 rpm, g = 5323 mm s-2. The remaining procedure was the same as above (a).

Results and Discussion LC chromatograms of OTA standard and soybean by products: soaking water, ground beans, soy milk, okara, tofu, and whey from tofu all showed a similar pattern. Figure 3 shows typical chromatograms of OTA standard solution, test extracts of soy milk and tofu. There were no interfering peaks at the area near or at the OTA peak. The mean recovery of OTA added in pink beans at 1 µg/kg was 86% and the standard deviation (SD) was +/-3%. Our data indicate that the method was applicable to dried beans. The soybeans used in this experiment were mixture of OTA-free and OTAincurred soybean. The incurred beans contained OTA at levels ranging from 2000 and 4000 µg/kg (on the basis of 5 separate analyses). It was not possible to prepare a homogeneous mixture of whole beans. Bean test samples at the beginning of each procedure could contain OTA at different levels. Therefore, the unit of expression for all data is presented as percentage of OTA distribution (i.e., % OTA in liquid = [total OTA in liquid/(total OTA in liquid + total OTA in beans)] x 100). Table I gives the distribution of OTA in soybeans and in liquid from washing, soaking and cooking. Approximately 10% of OTA partitioned from the bean into the liquid after washing for 60 min. We also obtained similar partition data for beans washed for only 2 min. Soaking for 60 min or washing for 60 min also gave similar yields of % OTA in water. Cooking was a more effective way to reduce % OTA in beans. These results are in agreement with our previous study with OTA distribution in dried beans during food processing (15). The partition of OTA from the beans into washing, soaking, and cooking water could reduce the amount of OTA contamination in soybean before consumption if the liquid is discarded.


66 There is some uncertainty to the absolute OTA distribution in liquid and beans. The nutrient compostion of OTA-naturally contaminated soybeans might be different from that of OTA incurred beans. The disinfection step in the treatment of the A. ochraceus infected beans probably denatured some of the protein on the surface of the beans. However, from our previous study on OTA distribution using OTA naturally contaminated bean flour, we obtained similar results to those obtained in this experiment during preparation of soy milk.

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A

B

C

0

2 2

0

4

4

6

6

8

8

10

10

12

12

14

14

16

min

Minutes

Figure 3. LC Chromatograms of (A) ochratoxin A standard solution (1 ng/mL), (B) soy milk test extract and (C) tofu test extract. Table I. Ochratoxin A distribution in liquid and residual soybeans after processing (5 g soybeans, 25 mL water). Procedure

time (min)

Wash Soak Cook

60 60 60

residual beans liquid separated from beans liquid (mL) OTA±SD* (%) OTA±SD* (%) 17 ± 1 13±2 87±2 17 ± 1 9±1 91±1 15 ± 3 32±4 68±4

* SD, standard deviation, n=6

Results of soy milk and tofu of trial 1 are somewhat different from trial 2 and trial 3 in Table II for two reasons. 1. The same procedure was used for the three trials except larger amounts of OTA-incurred soybeans were used for the first trial. And 2. Soy milk and tofu were prepared simulating home production. With use of filtration with cheese cloth rather than filter paper, it was not possible to measure the separated liquid accurately.


67 Table II. Ochratoxin A distribution during tofu processing By product Soybean Soaking water Ground beans Okara Soy milk Tofu whey Tofu

OTA total amount (μg) ± SD1 Trial 1 Trial 2 Trial 3 197.5 26.4±0.4 20.0±0.5 96.0 12.9±0.3 9.7±0.4 101.5±6.8 13.5±0.4 10.3±0.6 22.7±0.4 3.0±0.2 1.5±0.1 63.9±0.9 9.1±0.9 6.9±0.3 15.7±0.1 1.3±0.1 1.2±0.1 42.8±1.9 4.5±0.3 5.0±0.1

1


SD, standard deviation, trial 1 , n=4, trials 2 and 3, n=2.

In summary, if, during the preparation of soy milk and tofu, the soaking water and tofu whey are discarded, then the OTA level in the final products such as soy milk, okara, or tofu, could be reduced. According to this study only 20% of OTA in contaminated soybeans remained in tofu. Approximately 50% OTA that remained inside the beans after soaking and cooking were distributed in soy milk and okara, 34 and 10%, respectively. Further work could be required to support the distribution of OTA in these products. There are many ways of cooking soybeans and soybean products. Subsequently, the amount of OTA that remains in the final consumable portion could be reduced depending on the methods of food preparation.

Acknowledgements This project was supported by an appointment to the Research Participation Program at the Center for Food Safety and Applied Nutrition administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and U.S. Food and Drug Administration. The equipment used in part of this project was provided by Fundação de Amparo à Pesquisa do Estado de São Paulo. This work was partly supported by the Office of Dietary Supplements, National Institutes of Health, Bethesda, MD, USA.

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Mycotoxin Prevention and Control in Agriculture - American Chemical

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