Journal of Food Composition and Analysis 32 (2013) 6–11
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Original Research Article
Determination of total oxalate contents of a great variety of foods commonly available in Southern China using an oxalate oxidase prepared from wheat bran Quan-Yuan Ruan a, Xue-Qin Zheng a, Bai-Ling Chen a, Yang Xiao a, Xin-Xiang Peng a, David W.M. Leung b, E-E Liu a,* a
Laboratory of Molecular Plant Physiology, South China Agricultural University Key Laboratory of Ministry of Education for Rice Fertility Development and Resistance, College of Life Sciences, South China Agricultural University, Guangzhou 510642, PR China b School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 April 2012 Received in revised form 22 August 2013 Accepted 22 August 2013
The total oxalate contents of various vegetables, legume seeds, cereals, fruits and nuts commonly available in Southern China were determined using an improved enzymatic method. Spinach, xeric water spinach (grows on moist soil), amaranth, bamboo shoot, ginger, Chinese wolfberry, rice bean and black glutinous rice contained more than 100 mg oxalate/100 g FW, but leaf mustard, white radish, broccoli, cauliflower, cabbage, onion, gourd except bitter melon, tomato, pepper, chufa, kelp, mushroom, soybean sprout and cowpea contained less than 10 mg oxalate/100 g FW. The oxalate contents of star fruit and dragon fruit were 111.4 and 97.1 mg/100 g FW, respectively, and those of other fruits except strawberry and black plum were less than 20 mg/100 g FW. Almond, cashew, hazel, pine nut and abalone fruit contained more than 150 mg total oxalate/100 g FW, and the oxalate levels in Chinese torreya fruit, peanut, pistachio and walnut ranged from 54.1 to 83.1 mg/100 g FW. Based on the results obtained, water spinach, Chinese wolfberry, black glutinous rice, dragon fruit, rice bean, abalone fruit and Chinese torreya fruit should also be considered as high oxalate-foods. The enzymatic method used in the present study enabled us to perform a large-scale quantitative investigation of oxalate contents of different foods. ß 2013 Elsevier Inc. All rights reserved.
Keywords: Cereals Enzymatic analysis Fruits Legume seeds Nuts Oxalate content Oxalate oxidase Vegetables Food safety Food composition Food analysis
1. Introduction Oxalic acid is a dicarboxylic acid commonly found in microorganisms, plants and animals. Besides dietary intake oxalate in the human body may also be derived from metabolism of ascorbic acid and glyoxylate (Williams and Wandzilak, 1989). Consumption of a large amount of oxalate could be fatal to humans because of oxalosis or the formation of calcium oxalate deposits in vital tissues or organs of the body (Sanz and Reig, 1992). The patients with kidney stone problems should control dietary oxalate intake to less than 40– 50 mg per day (recommended by the American Dietetic Association, 2005). Therefore, determination of oxalate content in foods is very important to patients with kidney stone problems. The methods for determination of oxalate are titration (Baker, 1952), capillary electrophoresis (CE) (Trevaskis and Trenerry,
* Corresponding author. Tel.: +86 020 85280194; fax: +86 020 85282023. E-mail address:
[email protected] (E-E Liu). 0889-1575/$ – see front matter ß 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jfca.2013.08.002
1996), gas chromatography (Ohkawa, 1985), high performance liquid chromatography (HPLC) (Savage et al., 2000; Yu et al., 2002) and enzymatic analysis (Kasidas and Rose, 1980; Trevaskis and Trenerry, 1996; Chai and Liebman, 2005; Okombo and Liebman, 2010). Oxalate oxidase-based colorimetric determination of oxalate is a simple and fast method without the need for any special apparatus yielding results that are consistent with those obtained using the CE method (Trevaskis and Trenerry, 1996; Chai and Liebman, 2005). The oxalate oxidase-based method has, however, been mainly used to measure oxalate in urine, and might have limited use for oxalate determination in foods because substances such as ascorbate in foods might interfere with the colorimetric estimation of H2O2 in the final step of the procedure (Holmes and Kennedy, 2000). The oxalate decarboxylase-based procedure could also be affected by an incomplete removal of dissolved carbon dioxide from the sample solution leading to artifactually high oxalate values (Ho¨now and Hesse, 2002). Moreover, enzymatic determination of oxalate in plants may be subject to interference by carbohydrates extracted from
Q.-Y. Ruan et al. / Journal of Food Composition and Analysis 32 (2013) 6–11
plant tissues (Zarembski and Hodgkinson, 1962). To overcome these problems, Ho¨now et al. (1997) developed a very sensitive and selective HPLC-enzyme reactor method (HPLC-ER). Recently we described an enzymatic method which is sensitive, less timeconsuming and relatively inexpensive (Liu et al., 2009). In this method, a lower concentration of hydrochloric acid (HCl) was used to extract oxalate to avoid inadvertently extracting interfering substances and in preliminary trials it was found that the recovery of the added oxalate to soybean leaf samples during oxalate extraction ranged from 96.1% to 101.4% (mean was 99.8%). More importantly it is simple to perform without the need to access expensive specialist equipment, and a large quantity of oxalate oxidase can be prepared from wheat bran, which is a more practical source of the enzyme than the sources used in previous studies, for example moss and banana fruit peel. It was also found that the wheat bran oxalate oxidase method was not as susceptible as other published enzyme methods to interfering substances such as ascorbic acid, which is likely to have been degraded during sample processing. Moreover, there is a good agreement between the results obtained using this method and the HPLC method. This is an important practical requirement for routine monitoring or any large-scale investigations of oxalate contents of foods. In this study, we showed that our oxalate determination method based on oxalate oxidase prepared from wheat bran could be applied to investigate the total oxalate contents of a great variety of foods including vegetables, legume seeds, cereals, fruits and nuts commonly available in Southern China. This study might be adapted in different developing countries as a new, easy to perform, relatively low cost, large-scale monitoring of oxalate contents in produce grown locally. 2. Materials and methods 2.1. Samples and preparation Vegetables, legume seeds, cereal, fruits and nuts were purchased from local markets in Guangzhou, China. Once purchased, vegetables and fruits were washed with tap water to eliminate any unwanted debris or other substances. Legume seeds
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were soaked overnight. Rice, black glutinous rice, glutinous rice and edible part of nuts were chopped into small pieces. Triplicate samples (1 g each) were taken from edible parts of nuts and leafy vegetables except watercress, red cabbage, cilantro, garlic leaf, napp cabbage, leaf mustard and cabbage for determination of oxalate content. For other foods, the oxalate contents in triplicate samples (2 g each) were also determined (see Tables for identification of all samples). 2.2. Total oxalate extraction and determination Total oxalate content was measured using the enzyme method as described by Liu et al. (2009). Each sample (1.0 or 2.0 g) was homogenized with 1.6 mL 0.5 mol/L HCl first, and then diluted with 1 mL distilled water. The homogenate was transferred into 10 mL graduated tubes and heated in a boiling water bath for 20 min. After cooling, distilled water was added to each tube to bring the volume of the homogenate up to 10 mL. The next day, about 1 mL of the homogenate was clarified by centrifugation (12,000 g, 10 min) at 4 8C. After this, 0.016 mL NaOH (2 mol/L) was added accurately to 0.5 mL supernatant. This mixture was called an oxalate extract here. For determination of oxalate content in the oxalate extracts, about 20 mg oxalate oxidase in the form of dry powder prepared from wheat bran were first placed in a 2-mL test tube. Then 0.06–0.11 mL distilled water, 0.8 mL color reagent (10 mg 4-aminoantipyrine (Sigma–Aldrich, Shanghai, China), 25 mL N,N-dimethylaniline (Sigma–Aldrich, Shanghai, China) per 100 mL of 125 mmol/L succinate–NaOH buffer with 75% alcohol (V/V), pH 4.0) and 0.04 mL (150 U/mL) horseradish peroxidase (Bio Life Science & Technology Co., Shanghai, China) were added before 0.05–0.1 mL oxalate extract was added to initiate the reaction. After incubation at room temperature for 90 min, the absorbance at 555 nm of the reaction mixture was read in a spectrophotometer (Unico UV/VIS 2802PCS, Shanghai, China) and oxalate content was determined with reference to the standard curve which was prepared by adding 0, 2, 4, 6, 8, 10 mg oxalic acid (Sigma–Aldrich, Shanghai, China. Purity: 99+%) into 1 mL reaction system, respectively. The data are presented as mean mg oxalate/ 100 g FW food material.
Table 1 Total oxalate contents of leafy vegetables.a Samples (scientific name)
Spinach (Spinacia oleracea L.) Chinese wolfberry (Lycium chinense Mill.) Amaranth (Amaranthus tricolor L.) Xeric water spinach (Ipomoea aquatica Forsk.) Aquatic water spinach (Ipomoea aquatica Forsk.) Sweet potato leaves (Ipomoea batatas (L.) Lam.) Chinese chive (Allium tuberosum Rottler ex Spreng.) Flowering Chinese cabbage (Brassica parachinensis Bailey) Watercress (Nasturtium officinale Aiton) Red cabbage (Brassica oleracea L.var. Capitata f. rubra DC) Cilantro (Coriandrum sativum L.) Potherb mustard (Brassica juncea (L.) Czern. et Coss. var. multiceps Tsen et Lee) Lettuce (Lactuca sativa L. var. ramosa Hort.) Romaine lettuce (Lactuca sativa L. var. longifolia Lam.) Green pak choi (Brassica rapa L. var. chinensis (L.) Kitamura) Malabar Spinach (Basella alba L.) Milk pak choi (Brassica campestris L. ssp. chinensis (L.) Makino) Endive (Cichorium endivia L.) Garlic leaf (Allium sativum L.) Napp cabbage (Brassica rapa L. var. glabra Regel) Leaf mustard (Brassica juncea (L.) Czern. et Coss. var. foliosa Bailey) Cabbage (Brassica oleracea L. var. capitata L.) a
Oxalate content (mg/100 g FW) Leaf (present study)
Petiole (present study)
Ho¨now and Hesse (2002)
Massey (2007)
490.5 69.0 337.4 1.6 274.7 22.6 104.8 4.0 79.2 3.2 58.3 2.7 25.3 0.5 20.2 2.5 19.5 4.3 18.9 0.1 18.0 0.4 16.7 2.3
217.9 8.2 – 36.8 3.3 23.1 1.4 25.4 3.3 35.9 0.6 – 74.0 5.1 – – – 5.4 0.3
364 (boil)
400–900
16.1 1.1 16.1 0.8 15.3 1.6 13.9 0.3 13.8 1.5 13.7 0.4 12.1 0.1 4.0 0.1
