THE EFFECT OF CHIPS PROCESSING ON THE CONTENT OF TOXIC COMPOUNDS

Headnote

ABSTRACT

The aim of the experiment was focused on studying the content of glycoalkaloids, nitrates and acrylamide in raw materials, semi and final products coming directly from the processing line of fabricated chips in snacks' factory. On the ground of the results it was stated that there was found less glycoalkaloids and nitrates by ca. 50% in obtained dough after mixing of dry components and water. The quantity of nitrates in fried chips was lower by 84% comparing to the content of those substances in used raw materials, and glycoalkaloids by 89%. The process of fabricated chips affected on farther quantities of acrylamide formation. In raw materials the content of this toxic component was determined in ranges from 32 µg kg^sup -1^ (flakes) up to 65 µg kg^sup -1^ (granules) but chips contained ten times more acrylamide content, i.e. about 397 µg kg^sup -1^.

- Keywords: fabricated chips, glycoalkaloids, nitrates, acrylamide -

INTRODUCTION

Chips made from potato dough owed their popularity to the large variety of flavors, crisp texture and attractive potato smell. They are especially popular among European and Asian consumers. In the USA the production of chips made from dehydrated potato products started in the 1970s (WEISS et al, 1977); in 1997 "Pringles" chips made from potato dough accounted for 12% of the total sales of crisps in the Country. In Europe, the production of such chips began after their commercial success in the United States and it was based on the technology developed in the USA. Dehydrated cooked potatoes and water are mixed in suitable proportions to achieve about 40% moisture, then rolled, cut into various shapes and fried. This method of production allows a wide range of different forms and flavors of the final product.

The main component of crisps are dehydrated potatoes, influencing on the organoleptic characteristics of the finished crisps and the content of toxic and anti-nutritional components, related to their initial amount in the tubers. Some of the naturally occurring potato toxins are glycoalkaloids: a-chaconine and a-solanine, potent poisons (FRIEDMAN et al, 1991; FRIEDMAN, 2006; DONALD, 2008; HAASE, 2010; LERI et al., 201 1) with toxicity similar to strychnine or arsenic (JECFA, 1992; HAASE, 2010); at 3-5 mg per kilogram of body weight they are lethal to humans. Potatoes used for direct consumption or processing in food industry should contain less than 10 mg glycoalkaloids per 100 g of potatoes (FRIEDMAN and MCDONALD, 1997; SPEIJERS, 1998; PERSA et al, 2002; KNUTHSEN et al, 2009). Importantly, processes used in the manufacture of dehydrated potato products, French fries or chips, do not remove all of the toxic compounds. According to the literature (RYTEL et al. , 2005; FRIEDMAN 2006; PÇKSA et al, 2006; TAJNER-CZOPEK et al, 2008), fried potato products still have about 10-20% of the initial content of glycoalkaloids.

Apart from glycoalkaloids, potatoes and potato products may contain nitrates, which are classified as anti-nutritional compounds. They are not toxic to humans, but intestinal microflora may reduce them to class 3 nitrites which can participate in the production of carcinogenic nitrosamines. The permissible amount of class 3 nitrates in potatoes should not exceed 200 mgN03/kg (HILL, 1999) and toxic nitrites should be below 5 mg NaNO3 kg1 (CIESLIK, 1995). Potato products usually contain about 20-30% of the initial amounts of these compounds in potatoes (CIESLIK, 1995; RYTEL et al., 2005; PERSA étal., 2006).

Dehydrated potato products also contain acrylamide, which may additionally be produced during frying Chipsletten. Acrylamide is mainly found in fried and baked high starch foods exposed to temperatures above 120°C (FRIEDMAN, 2003). In French fries and chips acrylamide content may vary from 30 to 2,300 µg kg"1 (KITA et al, 2009). The production of acrylamide in the final product depends on the amount of reducing sugars and amino acid (asparagine) in the raw material, the temperature and duration of drying or frying (FRIEDMAN, 2003). A potential mechanism for the formation of acrylamide in foods is associated with the reaction between asparagine and reducing sugars during exposure of high starch products to high temperature (FRIEDMAN, 2003). This mechanism is accompanied by a Maillard reaction occurring between free amino groups and carbonyl groups of reducing sugars (glucose and fructose) during baking, frying or drying (FRIEDMAN, 2003).

The production process of Chipselleten may induce changes in the content of anti-nutritional and toxic compounds in the raw material or dehydrated potatoes, and also may increase the content of acrylamide in the finished product. For this reason it is important to determine technological factors that have an effect on the content of toxic compounds in the semi and final potato products.

The aim of the experiment was focused on determined the content of glycoalkaloids, nitrates and acrylamide in raw materials, semi and final products coming directly from the processing line of Chipselleten in the snacks' factory.

MATERIAL AND METHODS

Material

Potato flakes, granules and starch were used in the experiment as the raw materials. Samples were taken directly from the processing line of formed potato chips. Samples were obtained from snacks factory three times yearly in the period of 2009-2010, from the following stages of the process: raw material (granules, flakes and modified potato starch) before dough preparation, formed dough pieces and readyto-eat fried chips (before flavouring).

Potato samples preparation for the analysis

The content of dry matter, total sugars, reducing sugars, nitrates, glycoalkaloids (a-solanine i a-chaconine) and acrylamide were determined in raw material. The content of nitrates, glycoalkaloids (a-solanine i a-chaconine) and acrylamide were determined in freeze dried dough and ready fried non-defatted chips.

The scheme of analytical studies is presented below (Scheme 1).

Glycoalkaloids analysis

Apparatus

A high-pressure liquid Chromatograph HPLC (pro Star) was used, made by Varian company (Walnut Creek, CA, USA), equipped with UV detector - 310 type, analytical column Mikrosorb NH2 (25x46 cm LD) (firm- Rainin Instrument, Woburn, Ma, USA) and a computer system monitoring the Chromatograph (Varian Chromatography System).

Conditions of glycoalkaloids separation

As an eluent, a mixture of the tetrahydrofuran (firm Merck, Germany), acetonitrile, water 50:20:30 + KH2P04 (1.02 g) per liter was used. The process was carried out at the temperature of 35°C, with the speed of flow of 2 cm3 min1 and pressure of 1 1,3 MPa, applying the light wavelength of 208.

Sample preparation for chromatographic analysis

1 g of potato flakes, granules, starch, freezedried dough and non-defatted chips were homogenized with 4 cm3 of water and 30 cm3 of methanol (by Labscan, Ireland) for 2 minutes, followed by filtration. The filtrate was brought to a final volume of 50 cm3 with methanol. A 5 cm3 aliquot of extract was cleaned up on the SPE column (Bond Elut C18; 500 mg; 6.0 mL firm Varian, USA). Glycoalkaloids were rinsed with methanol and evaporated to dryness in vacuo at the temperature of 50°C. The residue formed was dissolved in 1 cm3 of THF:ACN:H20 - 50:20:30. Before application into the column, the sample was cleand up by using filters of 0.45 µp?. The volume of the injection was 10 µp?. Standard solutions (1 mg/cm3) were prepared by dissolving 10 mg of a-solanine and a-chaconine (Sigma) in 10 cm3 of methanol; 10 µ?^, containing from 1 to 50 µg/cm3for both a-solanine and a-chaconine, were injected.

Acrylamide analysis

Reagents

All reagents were of analytical grade unless otherwise stated. Acrylamide (2-propene amide) and d3-acrylamide (standard solution) were purchased from Fluka, Switzerland. Methanol HPLC -grade (methanol) was purchased from Merck (Darmstadt, Germany), water Milli-Q (deionized water) from a Milllpore purification system (Millipore, Bedford, MA, USA) and HPLCgrade acetonitrile from Lab-Scan (Ireland). Two Supelco SPE columns (Bellfonte PA, USA) were used for purification of the samples. The upper one (MCAX) of 300 mg and 3 mL in volume was placed directly on the lower one (DSC C- 18 of 1 g and 6 mL in volume). Two columns were connected with each other by an adapter.

Standard solutions and calibration standard solutions

Preparation and calibration of a standard solution were performed by modifying the method described by (ROSEN and HELLENÄS, 2002; AURAND and TRINH, 2005). The stock solutions of acrylamide and o^-acrylamide-standard solution as well as calibration standard were prepared in acetonitrile concentration of 500 mgxL1 (500 ppm) (Fluka Co.). The standard solution was protected from light and stored in a refrigerator. The stock solutions of standards 500 mgxL1 (500 ppm), were prepared in acetonitrile and refrigerated. A working standard solution, for spiking samples as well as for the standard curve, was obtained by dilution in acetonitrile. Concentrations for the standard curve were 0, 1, 5, 10, 30, 50, 100, 500, 1,000, 1,500 and 2,000 ngxmL1, all with acrylamide-d3 at 50 ngxmL1 for potato chips.

Apparatus

The acrylamide content of potato flakes, granules, starch, freeze-dried dough and non-defatted chips was obtained using LC-MS-MS 1200 L (Varian, Walnut Creek, CA, USA) performed using a triple quadruple with interfacial electrospray (ESI) a HPLC system Model a ProStar of a Varian, Walnut Creek, USA. HPLC-MS-MS 1200L system was equipped with one ProStar 210 Pump and ProStar 430 Autosampler. (Nitrogen generator, domnick hunter, model G 4510 E, UK). The samples were separated by the Pursuit XRs 3 u C-18 column (150 ? 2.0 mm) - with a Metaguard Pursuit XRs 3 u C18 column (2.0 mm, Varian, Walnut Creek, CA, USA), using the mobile phase of 0.5% methanol in aqueous 0. 1% acetic acid, at a flow rate of 0.2 mL-min1. The volume of each injected sample was 20 µL.

MS-MS condition for the acrylamide determination

The LC-MS-MS was operated in the positive electrospray mode, needle voltage 5000V, nebulizing gas (compressed air or N2) 54 psi, capillary voltage 25V, drying gas (N2, 99.5%, 400°C, 22 psi). The collision cell gas (Ar 99.999%) pressure was 1.86 mTorr) and the detector voltage was set to 1,300 V. Acrylamide was determined by multiple reaction monitoring (MRM). The MRM mode was performed by monitoring the 72-54.9 m/z transition for acrylamide (collision energy- 10V) and 75-58 m/z transition for acrylamide-d3 (collision energy -10 V). In all the MRM transitions, the dwell and inter scan delay times were 1 sec, respectively, SIM width 0.7 atm. The methods detection of limit values were calculated for S/N = 3 for the summed signals of both fragment ions.

Sample preparation for the LC-MS-MS analysis

The samples were prepared by modifying the methods described by (ROSEN and HELLENÄS, 2002; AURAND and TRINH, 2005; PEDRESCHI et ed., 2007). Samples of potato flakes, granules, starch, freeze-dried dough and non-defatted chips weighed 2 g (0.0001 g). The samples (dough and chips) were ground using a GM 200 cutting mill (Retsch, GmbH, Germany) at a speed of 2,000 rev· min1 for 16 seconds (2 ? 16 seconds). The samples were next placed in 50 mL Falcon centrifuge tubes and added 40 mL of deionised water. Parallel, the samples containing 200 µg/L of internal standard (akrylamidd3), at a concentration of 10 µg·mL~1 were prepared. The samples were shaken using a Multi Reax shaker (Heidolph Instruments GmbH & Co. KG. Germany) for 10 minutes. Next, the samples were centrifuged in 3 KI 5 centrifuge (Sigma) at a speed of 9,500 revmin1 and 4°C for 15 minutes.

Solid phase extraction (SPE). Solid phase extraction (SPE) was performed by modifying the method described by AURAND and TRINH (2005) and KIM et cd. (2007). 1 mL of transparent (clarified) solution (IA) was placed in SPA columns, which had been previously conditioned with 1 mL of methanol and 1 mL of deionized water, and vacuum-dried (Vac Elut 20 Manifold, Varian, Walut Creek, CA, USA). The samples in the columns were washed with 1 mL of deionized water. The upper SPE columns (MCAX) and the filtrate were removed. Acrylamide from the lower SPE columns (C- 1 8) were eluted with 2 mL of methanol and the filtrate obtained was nitrogendried (at 30°C). The dry residue was dissolved in 500 µ? (0.5 mL) of deionized water and placed in an autosampler using liquid chromatographytandem mass spectrometry (LC-MS-MS). The final acrylamide content of the finished product was expressed in ^gkg1] = [ppb].

Analytical methods

The dry matter of potato flakes, granules, starch, dough, chips and freeze-dried dough was determined by the reduced weight after drying at 102°C until constant weight was achieved (AOAC, 1995). The content of total and reducing sugar were determined chemically. The nitrate content in potato granules, flakes, starch, dough and chips was determined by the used the colorimetrie method (RYTEL et al, 2005). The quantities of oc-solanine and a-chaconine were determined by the method of PERSA et cd. (2002) and SAITO et cd. (1990). All analyses were carried out in duplicate. Chosen Chromatograph of acrylamide standard, potato flakes, granules, dough and Chipsletten is presented on Fig. 1.

Statistical analysis

The data obtained in the study were analyzed statistically using the Statistica 9.0 software package. For comparison, the results obtained were analyzed using multi-way analysis of variance with the application of Duncan's test (P< 0.05). The results presented in the tables and figures are the mean values of two-years experiments.

RESULTS AND DISCUSSION

Changes in the content of glycoalkaloids and nitrates

The process of manufacturing chips made from potato dough had an impact on the content of glycoalkaloids and nitrates in semi and final products (Table 1). Semi and final potato products contain the same as potatoes proand anti-nutritional ingredients but in different amounts, depending on the product and enrichment with other components, such as fat (French fries, chips). The high content of glycoalkaloids in potatoes results in a high amount of these compounds in potato products, as the production process often does not remove them completely. Therefore dehydrated potato products should be made from varieties with low susceptibility to glycoalkaloid synthesis.

In this study, the amount of glycoalkaloids in dehydrated potato products averaged 8.61 mg 100 g-1 (Table 1). A high content of these compounds was found in potato granules- 5.37 mg 100 g1, flakes contained 3.57 mg 100 g1 while the potato starch did not contain any glycoalkaloids. According to the study of SMITH et cd. (1996), the content of potato glycoalkaloids varies depending on the type of products, with more TGA found in potato flour derived from cooked potatoes (from 6.5 to 7.5 mg 100 g1) than in potato flakes from 1.5-2.3 mg 100 g1. Also MÄDER et cd. (2009) observed a low amount of glycoalkaloids in potato flakes at 0.6 mg 100 g1, while FRIEDMAN and DAO (1992) noted a considerably higher level of these compounds in potato pancake powder at 4.5 mg 100 g1 level. All the aforementioned differences in concentration of glycoalkaloids in dehydrated potato products depend not only on the type of product but also on treatments during manufacture. Many Authors (FRIEDMAN, 2006; RYTEL et ed., 2005; PERSA et ed., 2006) argue that most thermal treatments used in the industry and households have little effect on decreasing the amount of glycoalkaloids due to the compounds' high thermal stability and low solubility at the temperatures below 170°C (FRIEDMAN, 2006).

In this study, in the analyzed raw materials used in Chipsletten production, the highest nitrate concentration was observed in granules ( 1 75 mg NaNOgkg- J) , then flakes ( 1 63 NaNOgkg1) , whereas in the starch nitrates it was not detected (Table 1). Technological procedures used during the production of dehydrated potato products do not remove the entire amount of nitrate contained in the potatoes, although many authors (CIESLIK, 1995; RYTEL et al, 2005; PÇKSA et al., 2006) report nitrites' good water solubility and low resistance to high temperatures used during thermal processes. The content in potatoes products (flakes, granules) during drying can only be decreased by very high temperatures, while the lack of leaching agents such as water may be the reason of a large amount of these compounds remain in the product (Table 1).

The analyzed potato dough taken from the Chipsletten technological line, a mixture of 40% flakes, 40% potato granules and 20% potato starch, contained about half as much glycoalkaloids and nitrates in comparison with raw materials (Figs. 2 and 3). The content of glycoalkaloids decreased from 5.37 (granules) and 3.24 (flakes) to 2.38 (dough) (Fig. 2), and nitrate content decreased from 175 (granules) and 163 (flakes) to 104 (dough) (Fig. 3). The decrease in the content of these compounds in the finished dough was due to the mixing of granules and flakes with potato starch which did not contain glycoalkaloids or nitrates. Furthermore, in order to obtain dough with an appropriate 40% moisture, the dry mixture was supplemented with large quantities of water which further reduced the concentration of these compounds. In the process of preparing dough from dried potato products, there is no factor that would result in leaching or degradation of these compounds. The content of toxic compounds in the dough depends mainly on the recipe used by the plant.

The last stage was frying chips made of potato dough. Obtained chipsletten contained 35-37% fat. Ready-to-eat chips contained on average ca. 30% less chakonine (1,23 mg 100g1 a-chaconine) and ca. 9% less solanine (0,57 mg 100 g"1 a-solanine) than potato dough (1,75 mg 100 g1 a-chaconine and 0,63 mg 100 g1 a-solanine) (Fig. 2). Process of frying chips didn't influence significanltly on the loss of solanine, despite the high temperature. From the studies presented by PERSA et al. (2006) it results that loss of a-solanine after frying potato chips are smaller than a-chakonine. Frying had a significant impact on lowering the amount of toxic compounds, including glycoalkaloids. The obtained chipsletten contained 1.8 mg 100 g1 TGA, which represents 19% of the original content in raw material (Figs. 2 and 3). According to RYTEL et al (2005) after the 2nd stage of frying, French fries contained below 10% of glycoalkaloids and chips contained less than 18% compared to raw unpeeled potatoes (PERSA et al., 2006). In the process of frying potato chips or French fries, the high loss of toxic substances was due to the facilitated penetration of oil and high temperature (above 170°C). According to FRIEDMANN and MCDONALD (1997) during this process glycoalkaloids are removed from the material partly by leaching and partly through their degradation. Also the amount of nitrates reduced significantly after frying Chipsletten, to 16% of the initial content in dehydrated potatoes (Fig. 3). This is mainly due to their high solubility, especially at higher temperatures. According to PERSA et al. (2006) potato chips contain 18% of the original nitrate content in raw material; CIESLIK (1995) presents a higher level, at 20-30%.

Acrylamide

The amount of acrylamide increased during the production process of Chipsletten. The most significant factor that influenced on the amount of acrylamide in raw material, intermediates and crisps, was the content of reducing sugars. In this study the amount of reducing sugars in dehydrated potato samples was low, less than 0.2 g 100 g1 (Table 1). The content of reducing sugars in dried potato depends on the initial amount in the potato tubers. Due to the well demonstrated high toxicity of acrylamide (FREDMAN, 2003; PEDRESCHI et al., 2004), food manufacturers are trying to lower the reducing sugar content in potatoes by selection of varieties and appropriate storage conditions. The level of reducing sugars required by the producers of dehydrated potato and French fries is 0.3%, and during the production of chips even 0.2% (TAJNER-CZOPEK et al., 2008; KITA et al, 2009), which is beneficial not only for the colour of finished products but also due to the lower amount of acrylamide produced during frying.

Potato chips made of dough are prepared from intermediate products (granules, flakes), the quality of which should be subjected to thorough examination. In our experiment, potato granules contained 65 µg kg1 acrylamide and flakes 32 µg kg"1 (Table 1). In the dough the amount of acrylamide decreased by about 40% as a result of adding water during this stage. After frying, the level of acrylamide in chips increased significantly and reached 397 µg kg1 (Fig. 4). It was the result of frying in oil at the temperature of 185°C and the reaction of reducing sugars with asparagine.

The content of acrylamide in fried and dehydrated potato products may vary greatly; e.g. chips may contain from 50 to 1,900 µg kg1 acrylamlde (PEDRESCHI et ed., 2004; TAJNER-CZOPEK et ed., 2008) and crisps may contain 170 to 3,700 ng kg"1 acrylamide (FRIEDMAN, 2003). Hence the necessity of efforts of scientists and food manufacturers to induce substantial reductions in the quantities of acrylamide formed in these products.

It should be noted that dehydrated potato products are often a semi product that is added to food which undergoes further thermal treatment (baking, frying), making it very important to analyze potato products with regard to the content of toxic and anti-nutritional compounds.

CONCLUSIONS

On the ground of the results obtained it was stated that there was found less glycoalkaloids and nitrates by ca. 50% in obtained dough after mixing of dry components and water. The quantity of nitrates in fried chips was lower by 84% comparing to the content of those substances in used raw materials, and glycoalkaloids by 89%. The process of fabricated chips affected on farther quantities of acrylamide formation. In raw materials the content of this toxic component was determined in ranges from 32 µg kg1 (flakes) up to 65 µg kg"1 (granules) but chipsletten contained ten times more acrylamide content, i.e. about 397 µg kg1.

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Paper received January 27, Accepted June 5, 2012