Agricultural and Food Science 4:2011 A G R I C U L T U R A L A N D F O O D S C I E N C E Lisiewska, Z. et al. Oxalates in root vegetables Vol. 20(2011): 305–314. 305PB © Agricultural and Food Science Manuscript received March 2011 Effect of processing and cooking on total and soluble oxalate content in frozen root vegetables prepared for consumption Zofia Lisiewska*, Piotr Gębczyński, Jacek Słupski, Katarzyna Kur University of Agriculture in Krakow, Department of Raw Materials and Processing of Fruit and Vegetables, Balicka 122, 30-149 Krakow, Poland *e-mail: rrlisiew@cyf-kr.edu.pl The oxalate content of beetroot, carrot, celeriac and parsnip after freezing by traditional and modified methods (the latter resulting in a convenience food product), and after the preparation of frozen products for consumption was evaluated. The highest content of total and soluble oxalates (105 and 82 mg 100 g-1 fresh matter) was found in beetroot. The lowest proportion (55%) of soluble oxalates was noted in celeriac; this proportion was higher in the remaining vegetables, being broadly similar for each of them. Blanching brought about a significant decrease in total and soluble oxalates in fresh vegetables. Cooking resulted in a higher loss of oxalates. The level of oxalates in products prepared for consumption directly after freezing approximated that before freezing. Compared with the content before freezing, vegetables prepared for consumption by cooking after frozen storage contained less oxalates, except for total oxalates in parsnip and soluble oxalates in beetroot and celeriac. The highest ratio of oxalates to calcium was found in raw beetroot; it was two times lower in raw carrot; five times lower in raw celeriac; and eight times lower in raw parsnip. These ratios were lower after technological and culinary processing. The percentage of oxalate bound calcium depended on the species; this parameter was not significantly affected by the procedures applied. The true retention of oxalates according to Judprasong et al. (2006) was lower than retention cal- culated taking its content in 100 g fresh matter into account. Key words: root vegetables, oxalates, pretreatment before freezing, freezing; preparation for consumption A G R I C U L T U R A L A N D F O O D S C I E N C E A G R I C U L T U R A L A N D F O O D S C I E N C E Lisiewska, Z. et al. Oxalates in root vegetables 307306 Introduction Numerous studies have shown that vegetables con- tribute to cancer prevention by supplying various biologically active compounds to the diet (Noonan and Savage 1999). Hence, the consumption of these products is promoted by dieticians. However, veg- etables also contain potentially harmful compounds. Included among these are oxalates, which in the digestive tract can limit the nutritional availability of calcium and, to a lesser extent, iron and magnesium (Noonan and Savage 1999, Weaver et al. 1997). The occurrence of oxalates in plants is com- mon; they appear in soluble form producing salts with potassium, sodium and NH4 ions, and also in insoluble form producing insoluble salts chiefly with calcium, but also with iron and magnesium (Savage et al. 2000). Numerous publications discuss the effect of genetic, agro-technical and morphological factors on the level of oxalates; however, only a few works deal with factors asso- ciated with the processing of vegetables or prepar- ing them for consumption (Catherwood et al. 2007, Kmiecik et al. 2004, Oscarsson and Savage 2007). Many vegetables, including those with a particu- larly high oxalate content, are generally consumed after culinary or technological processing. Since this is carried out with the use of water, the content of soluble oxalates in the vegetable is reduced as they are washed out into the medium; however, this decrease is negated if the water containing the leached oxalates forms part of the cooked product. The investigations usually concern vegetable spe- cies with high accumulations of these compounds. With the current tendency to reduce meat consump- tion, the increased proportion of vegetables in the diet may result in higher oxalate intake. The aim of the work was to evaluate changes in the oxalate content of root vegetables after freez- ing by traditional and modified methods, the latter resulting in a convenience food product. A further aim was to evaluate oxalate content in vegetables prepared for consumption from frozen products directly after freezing and after 12-month storage at −20 oC. Materials and methods Materials The investigated material consisted of four root species: beetroot – Beta vulgaris L. (Czerwona Kula cv.); carrot – Daucus carota L. (Koral cv.); celeriac – Apium graveolens L. (Dukat cv.) and parsnip – Pastinaca sativa L. (Fagot cv.). The content of components was determined in fresh vegetables (A), after blanching (B), after cooking in 2% brine to consumption consistency (C), and in frozen products after storage at −20 oC and then prepared for consumption. Frozen products from sample B were cooked in brine, this yielding sample D (after 0 months of storage) and sample F (after 12 months of storage). Frozen products from sample C were defrosted and heated in a microwave oven, yielding sample E (after 0 months of storage) and sample G (after 12 months of storage). Production of the raw material The vegetables were grown in the experimental field of the Department carrying out the investigation. The field was in good horticultural condition; it lies on the western outskirts of Krakow in southern Poland. The cultivation was conducted on brown soil with the mechanical composition of silt loam in the third year after manure fertilization. The pH of the soil in H2O was 7.08, with a humus content of 1.66%, nitrogen NO3 24 mg dm -3, phosphorus 53 mg dm-3, potassium 101 mg dm-3, and calcium 1020 mg dm-3. The fertility of the soil and the nutritional re- quirements of the crops having been taken into account, and doses of mineral fertilizers for all vegetables were as follows: nitrogen 60 kg (80 kg for celeriac) as ammonium nitrate 34%N, phospho- rus P2O5 60 kg as superphosphate 46% P2O5, and potassium K2O 150 kg ha -1 as potassium chloride 60% K2O. Cultivation measures included sprinkler watering, mechanical weed control and, where nec- A G R I C U L T U R A L A N D F O O D S C I E N C E Lisiewska, Z. et al. Oxalates in root vegetables Vol. 20(2011): 305–314. 307306 Table 1. Pre-treatment times of vegetables before freezing (blanched – B, cooked – C) and preparing frozen vegetables for consumption (from B by cooking – D and F; from C by defrosting and heating in a microwave oven – E and G). Species Vegetables before freezing Frozen vegetables B C D and F E and G Beetroot 15 min 35 min –a 8 min 15 sec Carrot 2 min 45 sec 12 min 6 min 8 min 15 sec Celeriac 2 min 30 sec 8 min 5 min 8 min 15 sec Parsnip 2 min 30 sec 10 min 4 min 30 sec 8 min 15 sec a The blanched frozen red beetroot was not cooked since it was regarded as a semi-finished product to be used in pre- paring beetroot soup (borsch); in this case it would be cooked in a different proportion to water while, and the vegeta- ble and the fluid fractions would be utilized. essary, protective treatments against diseases and pests. Vegetables were harvested in September and October 2009. Directly after harvest, mean samples repre- senting the whole batch of the material, 25–30 kg out of about 100 kg, were taken for analysis and preparation of frozen products. All the vegetables were cleaned in water. The investigation covered the following vegetables: beetroot about 6 cm in diameter; carrot 3–4 cm; celeriac 13–25 cm and parsnip 4–5 cm in diameter. The carrot, celeriac and parsnip were peeled; the carrot and celeriac were cut into cubes 10×10×10 mm and the parsnip into matchsticks 30×10×10 mm. After the process- ing described above (washing and appropriate cut- ting), the vegetables were ready for blanching or cooking. Before freezing, the raw vegetables (sam- ples A) were blanched (samples B) or boiled (sam- ples C). Beetroot was blanched and also cooked whole in the skin to protect it from loosing juice and natural color; then the beetroots were peeled and coarse grated. Preparation of frozen products Two methods of processing the raw materials before freezing were used. In method I, the traditional technology of blanching the raw material was ap- plied; after freezing and refrigerated storage, the frozen product was cooked to consumption consist- ency. In method II, the raw material was cooked to consumption consistency to obtain a ready-to-eat product which, after freezing and refrigerated stor- age, only required to be defrosted and heated in a microwave oven. In method I, the fresh material was blanched in a stainless steel vessel in water, the proportion of water to the raw material being 1:5 and the blanch- ing temperature 95–98 oC. The blanching time for the different species is given in Table 1. The blanching parameters applied enabled a decrease in the activity of catalase and peroxidase to a level not exceeding 5% of the initial activity. After blanch- ing, the material was immediately cooled in cold water and left to drip on sieves for 30 min. A G R I C U L T U R A L A N D F O O D S C I E N C E A G R I C U L T U R A L A N D F O O D S C I E N C E Lisiewska, Z. et al. Oxalates in root vegetables 309308 In method II, the vegetables were cooked in a stainless steel vessel in brine containing 2% added salt (NaCl), the proportion by weight of the raw material to brine being 1:1. The vegetables were placed in boiling water, and the cooking time, measured from the moment when the water came to the boil again, is given in Table 1. After cooking to consumption consistency, the material was left on sieves and cooled in a stream of cold air. The materials from blanched and cooked sam- ples were placed on trays and frozen at −40 oC in a Feutron 3626-51 blast freezer (Ilka Feutron, Germany). The time required for the inside of the product to reach −20 oC was 90 min. The frozen vegetables were then packed in 500 g polyethylene bags and stored for 12 months. Preparation of frozen products for evalu- ation Samples of vegetables blanched before freezing (sample B) were cooked in 2% brine, the proportion by weight of the brine to the raw material being 1:1. As was the case when cooking fresh vegetables, the frozen product was placed in boiling water. The cooking time, measured from the moment when the water came to the boil again, is given in Table 2. After cooking, the water was immediately drained; the product was cooled to 20 oC (samples D and F), and analyzed. Samples of vegetables cooked before freezing (sample C) were defrosted and heated in a Panasonic NN-F621 microwave oven (samples E and G). For microwave heating, a 500 g portion was placed in a covered heatproof vessel. The time required to defrost and heat the material to consumption temperature is given in Table 2. The samples were then cooled to 20 oC and analyzed. All cooking experiments were carried out in duplicate. Chemical analyses All chemical analyses as well as cooking experi- ment were carried out in duplicate. Samples of fresh and processed vegetables were homogenized and frozen at −30 oC and freeze-dried. The freeze-dried vegetables were stored in hermetically sealed vials at −80 oC until analysis. The level of dry matter was determined by the method given in AOAC (1990) to allow the calculation of oxalate content per 100 g dry matter. Soluble and total oxalate contents of each sam- ple of vegetable were extracted using the procedure described by Savage et al. (2000). For soluble ox- alic acid extraction, 1–2 g samples of finely ground freeze-dried plant material were weighed into 250 ml beakers and 50 ml distilled water was added. The beakers were placed in a shaking water bath at 80 oC for 15 min. The extract was allowed to cool and then transferred quantitatively to a 100 ml volumetric flask and made up to volume with distilled water. For total oxalic acid, 1−2 g samples of the freeze-dried material were weighed into a 250 ml beaker and 50 ml 2M HCl was added. The beakers were placed in a shaking water bath at 80 oC for 15 min. The extract was allowed to cool and then transferred quantitatively to a 100 ml volu- metric flask and made up to volume with 2M HCl. An enzymatic method (Trinity Biotech 2008) proposed for the determination of oxalate content in urine was used to determine the soluble oxalate in raw and processed root vegetables. The same method was used in plant studies for example by Ilarslane et al. (1997) and Quinteros et al. (2003). This method is based on the oxidation of oxalate by oxalate oxidase action producing hydrogen per- oxide. The H2O2 then reacted with 3-methyl-2-ben- zothiazolinone hydrazone (MBTH) and 3-(dimeth- ylamino) benzoic acid (DMAB) in the presence of peroxidase to yield an indamine dye which has an absorbance maximum at 590 nm. An oxalate kit for the quantitative, enzymatic determination of oxalate in vegetables was used according to pro- cedure No. 591 (Trinity Biotech 2008). Deionized water from a Millipore-MilliQ apparatus was used in the study. The content of calcium was determined in mineralized, diluted and filtered samples by in- ductively coupled argon plasma emission spec- trometry using JY 238 Ultrace apparatus (Jobin Yvon, France). The content of calcium was used A G R I C U L T U R A L A N D F O O D S C I E N C E Lisiewska, Z. et al. Oxalates in root vegetables Vol. 20(2011): 305–314. 309308 to calculate the oxalate:calcium ratio and calcium bound as calcium oxalate. On the assumption that all the insoluble oxalate is in the calcium oxalate, it was calculated as follows: the content of insoluble oxalates was converted into the quantity of oxalate acid moles, this corresponding to the quantity of calcium moles bound in the form of calcium oxa- late. The quantity of grams of calcium bound in the form of oxalates was then calculated and converted into the percentage of bound calcium in relation to the calcium content in a sample. The percent true retention (%TR) was also calculated. This value was determined as the amount of oxalates remaining in the vegetables after technological and culinary treatments were applied. The calculation was carried out according to the formula given by Judprasong et al. (2006). For each sample, the raw material weight of the fresh vegetable and the weight after technological and culinary processing were determined. Statistical analysis All experiments were carried out in two independent experimental replications, and all chemical analyses were carried out in two parallel replications. The statistical analysis, which enabled a comparison of the oxalate content in the fresh raw material − in blanched and cooked material and in frozen material after preparation for consumption − was carried out using single-factor analysis of variance (ANOVA) on the basis of the Duncan test calculated at the probability level p < 0.05. The Statistica 6.1 (Stat- Soft Inc., Tulsa, OK, USA) programme was used. Results and discussion Content of total and soluble oxalates According to Franceschi and Nakata (2005), the formation of oxalates is genetically controlled and hence different vegetable species contain different amounts of these compounds. In the investigated root crops, the highest content of total and soluble oxalates was found in fresh and dry matter of beetroot (Table 2). Noonan and Savage (1999) and Santamaria et al. (1999) classified this species as an oxalate-accumulating vegetable. However, the values quoted in the literature vary considerably. Santamaria et al. (1999) report that the total content of oxalates in beetroot varied from 540–1088 mg 100 g-1 fresh matter, while according to Savage et al. (2000), the content in beetroot was only 46 mg oxalates in 100 g fresh matter. Lower amounts of oxalates in carrot and celeriac than those found in the present investigation were reported by Hönow and Hesse (2002) and higher amounts by Santamaria et al. (1999). The accumulation of oxalates in plants is as- sociated with the activity of photosynthesis and, as the observations show, different tendencies in this process depend on the species and age of the plant as well as on the phase of growth (Franceschi and Nakata 2005, Poeydomenge and Savage 2007). Moreover, the oxalate content within a particular species depends on the conditions and year of growth (Bakr and Gawish 1997, Jaworska 2005a), as well as on the usable part of the plant (Kmiecik et al. 2004). The accumulation of oxalates is also influenced by the form of nitrogen fertilization: if the nitrogen is supplied in nitrate form, it has to undergo reduction through nitrate reductase before it is utilized by the plant. This process results in the production and accumulation of organic acids, including oxalic acid (Libert and Franceschi 1987). Investigations into different forms of fertilization revealed that a higher content of calcium in the sub- strate results in a reduced level of soluble oxalates in plants (Bakr and Gawish 1997). The lowest proportion of soluble oxalates in total oxalates was found in celeriac (55%); this proportion was higher in the remaining vegetables, being broadly similar for each of them (68–78%). Savage et al. (2000) and Chai and Liebman (2005) reported that in many species the level of solu- ble oxalates exceeded that of insoluble oxalates. Soluble oxalates are potentially more harmful than insoluble since they reduce the nutritional avail- ability of calcium in the organism and contribute A G R I C U L T U R A L A N D F O O D S C I E N C E A G R I C U L T U R A L A N D F O O D S C I E N C E Lisiewska, Z. et al. Oxalates in root vegetables 311310 Table 2. Dry matter content (DM, g 100g-1), total oxalate and soluble oxalate content (mg 100 g-1 of fresh matter – FM or dry matter – DM) and retention (%) in root vegetables (raw material – A; material before freezing: blanched – B, cooked – C; product prepared for consumption after freezing: from B by cooking – D, from C by defrosting and heating in microwave oven – E; product prepared for consumption after frozen storage: from B by cooking – F, from C by de- frosting and heating in microwave oven – G). Item DM Total oxalate Soluble oxalate FMa % DMa % FMa % DMa % Beetroot A 14.2 105 ± 10d 100 739 ± 69c 100 82 ± 10b 100 577 ± 69b 100 B 14.1 90 ± 6c 86 638 ± 43b 86 60 ± 7a 73 426 ± 49a 74 C 13.5 77 ± 6ab 73 570 ± 47ab 77 52 ± 7a 63 385 ± 55a 67 D 14.0 86 ± 9bc 82 614 ± 61b 83 55 ± 6a 67 393 ± 44a 68 E 14.2 74 ± 6a 70 521 ± 40a 71 49 ± 8a 60 345 ± 54a 60 F 14.0 73 ± 3a 70 521 ± 24a 71 48 ± 6a 59 343 ± 44a 59 G 14.1 76 ± 5ab 72 539 ± 37a 73 50 ± 7a 61 355 ± 51a 61 Carrot A 13.1 74 ± 6c 100 565 ± 43e 100 50 ± 5d 100 382 ± 41c 100 B 10.8 50 ± 5b 68 463 ± 42d 82 25 ± 4c 50 231 ± 32b 61 C 12.3 45 ± 6ab 61 366 ± 49b 65 19 ± 3ab 38 154 ± 27a 40 D 10.5 48 ± 5ab 65 457 ± 43cd 81 22 ± 3bc 44 210 ± 30b 55 E 12.9 46 ± 5ab 62 357 ± 35b 63 17 ± 3ab 34 132 ± 21a 34 F 10.4 42 ± 3a 57 404 ± 32bc 71 16 ± 3a 32 154 ± 25a 40 G 13.6 41 ± 3a 55 301 ± 24a 53 18 ± 2ab 36 132 ± 16a 35 Celeriac A 16.9 40 ± 4b 100 237 ± 23c 100 22 ± 4c 100 130 ± 22c 100 B 15.4 31 ± 4a 78 201 ± 29b 85 16 ± 3b 73 104 ± 17b 80 C 18.1 28 ± 3a 70 155 ± 18a 65 11 ± 2a 50 61 ± 10a 47 D 16.7 29 ± 3a 73 174 ± 15ab 73 16 ± 3b 73 96 ± 16b 74 E 18.8 30 ± 5a 75 160 ± 26a 67 13 ± 3ab 59 69 ± 14a 53 F 16.5 25 ± 3a 63 152 ± 18a 64 11 ± 2a 50 67 ± 10a 51 G 19.1 27 ± 3a 68 141 ± 16a 60 13 ± 2ab 59 68 ± 10a 52 Parsnip A 20.7 25 ± 2c 100 121 ± 8c 100 18 ± 2c 100 87 ± 8c 100 B 17.7 20 ± 2b 80 113 ± 10bc 93 12 ± 2b 67 68 ± 10ab 78 C 19.1 19 ± 2b 76 99 ± 12ab 82 10 ± 2ab 56 52 ± 11a 60 D 15.5 18 ± 2b 72 116 ± 14bc 96 12 ± 3b 67 77 ± 17bc 89 E 20.5 20 ± 2b 80 98 ± 9a 81 12 ± 3b 67 59 ± 13a 67 F 15.5 14 ± 2a 56 90 ± 15a 75 8 ± 2a 44 52 ± 10a 59 G 20.5 21 ± 3b 84 102 ± 13abc 85 11 ± 3b 61 54 ± 13a 62 a Means with different letters in the same column, within one species of vegetable, indicate significant differences (p < 0.05). A G R I C U L T U R A L A N D F O O D S C I E N C E Lisiewska, Z. et al. Oxalates in root vegetables Vol. 20(2011): 305–314. 311310 to the formation of renal calculi (Noonan and Sav- age 1999). Soluble salts are produced when oxa- lates bind with potassium, sodium or magnesium, magnesium salts being less soluble than potassium or sodium salts. Insoluble salts are formed when oxalates bind with calcium or iron (Noonan and Savage 1999). Thermal processing with the use of water caus- es many changes due to the leaching of soluble con- stituents to the medium; the absorption or release of water; and the shrinking of the raw material, result- ing in absolute or relative changes in the content of the constituents. In general, the above reactions are characterized by changes in the weight and content of dry matter. In the case of the investigated spe- cies, blanching in water brought about a significant decrease in the content of total and soluble oxalates both in fresh and dry matter, with the exception of total oxalate content in the dry matter of parsnip. The loss of total oxalates varied from 20–32% and that of soluble oxalates from 27–50% converted into fresh matter, and 7–18% and 20–39% re- spectively converted into dry matter. The results obtained by Bakr and Gawish (1997) showed that blanching in solutions of calcium citrate and so- dium ascorbate instead of tap water brought about a higher loss of oxalates. The above authors claim that this was due to differences in the permeability of plant tissue in these solutions. Cooking, a process which takes longer than blanching, increased the losses of total oxalates; however, these were only significant in the fresh matter of beetroot and the dry matter of carrot and celeriac. This may have been due to fact that in the latter two species cooking caused a much greater increase in the level of dry matter than blanch- ing. Compared with blanching, cooking resulted in significant decreases in soluble oxalate content in the fresh and dry matter of carrot and celeriac. When Mosha et al. (1995) prolonged thermal pro- cessing, they observed increased losses in oxalates, as was also found in the present study; however, these were significant only in a few cases. It can be assumed that this was due to the low solubil- ity of these compounds and their resistance to the effect of temperature (Baldwin et al. 1986). How- ever, by prolonging the soaking time in water by several dozen times, Savage and Dubois (2006) were able to show a progressive decrease in soluble oxalates from marginal quantities to 26% of their initial content. The blanching parameters applied in the present investigation were selected in order to minimize the activity of peroxidase and catalase. The cooking parameters were selected in order to obtain optimal consistency and to ensure that the frozen product after storage had all the traits of a convenience food; but also to retain the greatest possible quantities of antioxidative constituents (Gębczyński 2005, 2006a, 2006b). Compared with the raw material before freez- ing, the level of the investigated compounds did not change significantly either in the traditional frozen product prepared for consumption by cooking in brine, or in the “convenience” frozen product after defrosting and heating to 70 oC in a microwave oven. Frozen storage drastically curtails biochemical and chemical processes, and after 12 months of storage at −20 oC no regeneration of catalase and peroxidase was recorded. However, chemical reac- tions and changes in the permeability of cell mem- branes taking place during processing and storage may have increased the leaching of oxalates, the traditionally frozen product when prepared for con- sumption following frozen storage: after cooking it contained 12–22% less total oxalates and 13–43% less soluble oxalates compared with vegetables cooked directly after freezing. In the case of sam- ples prepared for consumption in a microwave oven the changes varied from +5 to −11% and from +6 to −9% respectively. This variation could be evoked by increase in dry matter content and from the other side by the leaching with cell sap after heating. This change was not always significant. Compared with vegetables before freezing, oxalate content in the fresh matter of frozen products obtained using the traditional method was significantly reduced, ex- cept for total oxalate content in celeriac and soluble oxalate content in beetroot and parsnip. In products obtained using the modified method, a significant decrease in the content of oxalates was noted only in the dry matter of carrot. At the same time there were observed only small changes in calcium con- tent (Table 3). The solubility of calcium could be A G R I C U L T U R A L A N D F O O D S C I E N C E A G R I C U L T U R A L A N D F O O D S C I E N C E Lisiewska, Z. et al. Oxalates in root vegetables 313312 limited by such components of plants like phytic acid, some proteins or polyphenols (Hurrell 2003, Vitali et al. 2008). Products obtained using the traditional method and prepared for consumption retained 57% and 32% (carrot) and 70% and 59% (beetroot) of total and soluble oxalates respectively compared with the content in the raw material. Products of the convenience food type usually contained slightly more oxalates, significant differences being found only in the case of total and soluble oxalates in the fresh matter of parsnip. Gębczyński (2005, 2006a, 2006b) reported that the retention of antioxidative compounds and the sensory evaluation were simi- lar for the two types of product or slightly lower for frozen products of the convenience food type. A much higher reduction in oxalates can be ob- tained if a high-calcium product, such as milk, is added during vegetable processing (Brogren and Savage 2003, Savage et al. 2009). Water with a high calcium content can play a similar role. The tap water used in the present experiment for pro- cessing and preparing vegetables for consumption contained 130–150 mg Ca dm-3, which, accord- ing to the data given by Belitz et al. (2004), can be classed as hard water containing 325–375 mg CaCO3 dm -3. Oxalate:calcium ratio, calcium bound as calcium oxalate and oxalate true reten- tion Fresh beetroots contained the highest amount of oxalates and the lowest amount of calcium, giving a high oxalate:calcium ratio (Table 3). In fresh carrot this ratio was almost halved, while in celeriac and parsnip it was five and eight times lower respectively. Nowadays, parsnip is not a popular species in spite of its attractive sensory traits and significant levels of antioxidative compounds (Gębczyński personal report). Parsnip’s favorable oxalate:calcium ratio should recommend it for wider use. The technological and culinary procedures ap- plied had only an insignificant effect on the content of calcium (Table 3); however, as Table 2 shows, they contributed to a decrease in oxalate content. Therefore, the oxalate:calcium ratio in the products prepared for consumption was always lower than in the raw material, the greatest decrease being noted in carrot. There is no clear evidence that oxalates in food preferentially bind with calcium since some solu- ble fractions can be also bound with dietary fiber (Savage et al. 2000). On the assumption, however, that insoluble oxalates in vegetables are calcium oxalates, it is possible to calculate the content of calcium bound in that molecule (Oscarsson and Savage 2007). The results given in Table 3 show that the percentage of bound calcium depended on the species, but technological and culinary process- ing did not significantly affect this parameter. Ac- cording to Brogren and Savage (2003), as much as 77% of calcium in spinach was bound in insoluble oxalates, while Oscarsson and Savage (2007) stat- ed that differences in the amounts of bound calcium in taro leaves depended on their age. As mentioned previously, the weight of veg- etables was affected by technological and culinary treatments. Judprasong et al. (2006) suggest that changes in weight can be utilized to calculate the true retention (TR) of oxalates. The values in Table 3 calculated according to the formula given by Jud- prasong et al. (2006) show that in samples having undergone technological and culinary processing, the true retention of both total and soluble oxalates was lower than when calculated taking the content in 100 g fresh matter into account (Table 2), with the exception of blanched parsnip. Similar results were reported by Murphy et al. (1975) since in their opinion the apparent retention overestimated the TR. The results presented are among the few which reveal changes in the level of oxalates in the whole process of freezing vegetables and preparing them for consumption (Kmiecik et al. 2004, Jaworska 2005b). Finally, hydrothermal processing, in this num- ber blanching and cooking, decreased significantly oxalate level in studied vegetables. It is especially important for the beetroot which is one of the most important root vegetable for European consumers. Products of the convenience food type usually con- tained slightly more oxalates, compared to tradi- tional products. A G R I C U L T U R A L A N D F O O D S C I E N C E Lisiewska, Z. et al. Oxalates in root vegetables Vol. 20(2011): 305–314. 313312 Table 3. Total calcium content, oxalate:calcium ratio, calcium bound as calcium oxalate and true retention of total and soluble oxalate in root vegetables (raw material – A; material before freezing: blanched – B, cooked – C; product prepared for consumption after frozen storage: from B by cooking – F, from C by defrosting and heating in microwave oven – G). Item Total calcium content (mg 100 g-1 fresh matter) Oxalate:Calcium ratio Calcium bound True retention of mg 100g-1 fresh matter % of total calcium total oxalates soluble oxalates Beetroot A 24.4 1.92 10.2 42 100 100 B 23.4 1.71 13.4 57 84 72 C 22.3 1.54 11.1 50 68 59 F 22.9 1.42 11.1 49 66 56 G 22.2 1.52 11.6 52 62 52 Carrot A 38.8 0.85 10.7 28 100 100 B 38.2 0.58 11.1 29 65 48 C 36.8 0.54 11.6 31 55 35 F 36.5 0.51 11.6 32 51 29 G 37.6 0.49 10.2 27 45 30 Celeriac A 46.1 0.39 8.0 17 100 100 B 45.9 0.30 6.7 15 77 72 C 41.3 0.30 7.6 18 66 47 F 38.2 0.29 6.2 16 59 47 G 42.3 0.28 6.2 15 58 51 Parsnip A 46.8 0.24 3.1 7 100 100 B 46.5 0.19 3.6 8 82 68 C 45.8 0.18 4.0 9 73 53 F 43.9 0.14 2.7 6 55 44 G 46.8 0.20 4.4 9 74 54 References AOAC. 1990. Official methods of analysis of the associa- tion of official analytical chemists. 15th ed. Washington: Association of Official Analytical Chemists. Bakr, A. A. & Gawish, R. A. 1997. 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