ACRYLAMIDE CONTENT AND ANTIOXIDANT CAPACITY IN THERMALLY PROCESSED FRUIT PRODUCTS

Acrylamide as a known processing contaminant was determined in various heat-treated plum products purchased from a local market using LC/ESI-MS-MS. The highest level of acrylamide in the range up to 60 μg/kg was detected in a plum stew known as a “povidla“, and in prunes, respectively. These products typically undergo intensive heat treatment that may take from several hours to days. Using a fruit dehydrator in home production of prunes, a low level of acrylamide under LOQ (15 μg/kg) was detected in comparison to most commercial products. Only in one of the prune samples from the market was the acrylamide content near to LOQ. The highest content of acrylamide (46 μg/kg) was detected in the Slovak sample of prune originated in Nitra region. High acrylamide content, in the range from 23 to 45 μg/kg, was observed in prunes from South America. In the rest of analysed heat-treated plum products such as plum juice, plum compote or baby food with plum puree, acrylamide was not detected due to moderate conditions during thermal processing: temperature below 120 °C and a shorter time of thermal exposure. The total phenolic content and antioxidant capacity of prunes were analysed using a UV-VIS-NIR spectrophotometer and an electron paramagnetic resonance (EPR) spectroscopy. Homeprepared prunes were characterized by the highest content of phenolics (4780 mg GAE/kg) and antioxidant capacity (14.6 mmol TEAC/kg). Commercial samples of prunes reached phenolics in the range from 1619 to 3461 mg GAE /kg, and antioxidant capacity was observed between 6.1 and 12.1 mmol TEAC/kg. Antioxidant capacity of prunes strongly correlated with total phenolic content and yellow and red colours measured in a CIELab system. However, no significant correlation between the acrylamide and antioxidative or organoleptic properties of prunes was observed. Moreover, it was noticed that bio production of plums did not demonstrate any positive impact on final acrylamide content or antioxidant capacity in comparison to conventional technology.


INTRODUCTION
Thermal processing is frequently used in food manufacturing to obtain safe products with prolonged shelf-life and has a strong impact on the final quality of products.One of the purposes of thermal input is to improve the sensory properties, palatability and to extend the intensity of colour, tastes, aromas and textures of food.However, undesired effects due to various chemical reactions being Maillard reaction, caramelization and lipid oxidation are the most prominent (Capuano and Fogliano, 2011).On the other hand, it is well known that some substances arising from the heating processes can play a positive role on human health.Many neo-formed compounds showing antioxidative, antimicrobial, antiallergenic effects or modulation activity in vitro have been observed ( van Boekel et. al., 2010).In addition to these positive effects, some detrimental consequences of thermal processes must be carefully evaluated, such as the loss of thermolabile compounds (vitamins, essential amino acids -lysine, tryptophan) or the formation of undesired tastes and off-flavours.Moreover, a major concern arising from heating processes comes from the formation of compounds that are not naturally present in foods, but which may develop during heating or preservation processes and which reveal harmful effects such as mutagenic, carcinogenic and cytotoxic effects known as neo-formed contaminants.Well-known examples of these compounds are heterocyclic amines, nitrosamines, polycyclic aromatic hydrocarbons (Knize et al., 1999) and recently discovered acrylamide with a high toxicological potential.
Acrylamide is formed during thermal processing of many types of foods.The highest acrylamide levels have been found in fried potato products, bakery wares and coffee (Ciesarová, 2013)

ABSTRACT
Acrylamide as a known processing contaminant was determined in various heat-treated plum products purchased from a local market using LC/ESI-MS-MS.The highest level of acrylamide in the range up to 60 μg/kg was detected in a plum stew known as a "povidla", and in prunes, respectively.These products typically undergo intensive heat treatment that may take from several hours to days.Using a fruit dehydrator in home production of prunes, a low level of acrylamide under LOQ (15 μg/kg) was detected in comparison to most commercial products.Only in one of the prune samples from the market was the acrylamide content near to LOQ.The highest content of acrylamide (46 μg/kg) was detected in the Slovak sample of prune originated in Nitra region.High acrylamide content, in the range from 23 to 45 μg/kg, was observed in prunes from South America.In the rest of analysed heat-treated plum products such as plum juice, plum compote or baby food with plum puree, acrylamide was not detected due to moderate conditions during thermal processing: temperature below 120 °C and a shorter time of thermal exposure.The total phenolic content and antioxidant capacity of prunes were analysed using a UV-VIS-NIR spectrophotometer and an electron paramagnetic resonance (EPR) spectroscopy.Homeprepared prunes were characterized by the highest content of phenolics (4780 mg GAE/kg) and antioxidant capacity (14.6 mmol TEAC/kg).Commercial samples of prunes reached phenolics in the range from 1619 to 3461 mg GAE /kg, and antioxidant capacity was observed between 6.1 and 12.1 mmol TEAC/kg.Antioxidant capacity of prunes strongly correlated with total phenolic content and yellow and red colours measured in a CIELab system.However, no significant correlation between the acrylamide and antioxidative or organoleptic properties of prunes was observed.Moreover, it was noticed that bio production of plums did not demonstrate any positive impact on final acrylamide content or antioxidant capacity in comparison to conventional technology.
food products other than those listed in the report of EFSA such as hazelnuts, almonds, olives or dried fruits (Amrein et al., 2007).Due to a lack of relevant information in literature and databases, the presented study focused on acrylamide analysis in thermally processed plum products available on the local market and consequently in correlation with beneficial properties of thermally processed fruit such as total phenolic content and antioxidant capacity.

MATERIAL AND METHODOLOGY
Samples of various thermally processed fruit products produced from plum (Prunus domestica) were purchased from a local market in the Slovak Republic with focus on dried plums (prunes) and plum stew, moreover in juice, compote and baby food.A total of 8 samples of prunes with various origin were analysed listed in detail in Tab. 1.Three samples of traditional plum stew known as 'povidla' produced by different companies from the Czech Republic and the Slovak Republic (represents one sample with declared 5% content of apples), plum compote (halved, pitted, sterilized) produced in Hungary, plum juice with 25% of fruit juice content (from plum puree and plum juice concentrates) originated in Austria, two samples of baby food with various content of plums (80% plums, 20% apples) from the Czech Republic, and baby food originated from Hungary with 50% plum content and with declared bio-production and starch addition.
Samples were collected for acrylamide and colour analysis and further evaluations of total phenolic content and antioxidant capacity.

Acrylamide determination
Acrylamide (ACR) was extracted from samples into water and pre-extracted into ethylacetate with an internal standard D3-acrylamide addition according to Bednáriková and Ciesarová (2012) and Ciesarová et al. (2009).Acrylamide was analysed by LC/ESI-MS-MS using an HPLC system 1200 Series (Agilent Technologies, USA) with positive electrospray ionization (ESI+) and mass spectrometer 6460 Triple Quad detection with LOQ of 15 μg/kg.The analytical separation was performed on Atlantis dC18 column (100 mm x 2.1 mm, 1.8 μm particle size; Waters, Milfor, MA, USA) using isocratic mixture of 1 % of methanol and 0.2 % of glacial acetic acid in water at flow rate 0.4 ml/min at ambient temperature.
Antioxidant capacity was measured by an EPR spectroscopy and the results were expressed as Trolox equivalent using a standard solution of ABTS cation radical (TEAC ABTS

Colorimetric Analysis
Measurement of product colour was carried out using a UV-3600 spectrophotometer (Shimadzu, Japan) in reflectance mode with 10° Observer, D65 Illuminant and wavelength range 380 -780 nm.CIELAB parameters L * , a * , b * were collected and a hue angle as an attribute of a visual sensation was calculated according to the formula  =  − (  *  * )

Statistical Analysis
All results presented are means of three replicates along with standard deviations.Correlation coefficients were determined between colour parameters, antioxidant capacity, phenolic compounds and acrylamide content.

RESULTS AND DISCUSSION
Acrylamide content in thermally processed fruit products are not restricted till now by the law, however the European Commission have proposed an indicative value of acrylamide for baby foods of 50 μg/kg, and for products containing prunes of 80 μg/kg, respectively (EC Recommendation, 2013).In our study, acrylamide content below LOQ (<15 μg/kg) was detected in the products of plum juices, canned sterilised plum products and baby food in general.On the other hand, a significant content of acrylamide was detected in plum stew and prunes which were consequently subjected to further deeper study focused on an evaluation of beneficial properties and correlations with visual parameters measured in a CIELab system.

Acrylamide content in plum stew
The highest content of acrylamide was detected in traditional stew prepared from plums known in Czech, Slovak or Polish market as a 'povidla'.This type of product produced by traditional technology is characterized with dark colour and a very thick consistency that is most suitable as a filling of dumplings.Traditional 'povidla' is prepared by long term boiling without any addition of sugar or other additives from ripe fruits, harvested as late as possible, ideally after the first frosts, in order to ensure they contain enough sugar.For this moderate thermal treatment the authentic samples of plum stew were characterized by presence of acrylamide in concentration of 60 μg/kg ±5 μg/kg (data not shown).On the other hand, acrylamide content in the commercial sample of plum stew with declared 5% addition of apples was not detected.

Acrylamide content in prunes
Similarly to plum stew also prunes are typically processed by heat treatment, therefore in this type of plum products a significant acrylamide content was determined in concentrations comparable to plum stew.The drying process of prunes is intensive and slow, taking a long time to complete, up to 35 hours, depending on the drying conditions (Sabarez, 2012).
Results of acrylamide analysis of a sample of prunes obtained from the local market and a sample of homeprepared prunes using a kitchen dehydrator are summarized in Table 1.The lowest acrylamide content was determined in domestically prepared prunes as well as in the product of European origin (France) in concentration near to LOQ (<15 μg/kg).The rest of prune products obtained from market contained acrylamide in the range from 19 μg/kg (Turkey) to 46 μg/kg (Slovakia, obtained from marketplace in Bratislava, produced in Nitra region).Samples from South America were characterized by an acrylamide content in the range from 23 to 45 μg/kg.Higher acrylamide content led to an assumption of more intensive heat treatment of raw material or other specific technological procedure.Moreover, in prunes declared with bio origin, the acrylamide content was 33 and 36 μg/kg, respectively.It can be concluded that bioproduction declared on the packaging did not result in lower acrylamide content.

Total phenolic content and antioxidant capacity of prunes and their correlations
Prunes contain naturally high levels of fibre and have been shown to have one of the highest antioxidant levels of the common fruits and vegetables (Stacewicz- Sapuntzakis et al., 2001;Cantu-Jungles et al., 2014;Jarvis et al., 2015).Aforementioned beneficial properties of prunes expressed as antioxidant capacity and total phenolic content are summarized in Table 1.Homeprepared prunes were distinguished from commercial samples by the highest content of phenolics (4780 mg GAE/kg) as well as antioxidant capacity (14.6 mmol TEAC/kg).Commercial samples of prunes were low in both phenolics (from 1619 to 3461 mg GAE/kg) and antioxidant capacity (from 6.1 to 12.1 mmol TEAC/kg).Phenolics, commonly found in fruits, have been reported to exhibit antioxidant activity due to the reactivity of the phenol moiety, and have the ability to scavenge free radicals (Donovan et al., 1998).A correlation analysis presented in Table 2 pointed out that antioxidant capacity of prunes strongly correlated with total phenolic content (a correlation coefficient 0.8388).However, acrylamide content as a potentially harmful compound was not correlated with health beneficial properties such as the total phenolic content or antioxidant capacity of products (correlation coefficient 0.4816 and 0.5163, respectively).

Colour of prunes and correlations with beneficial and health hazardous compounds
Colour of prune samples obtained from market varied in visual colour sensation expressed as a hue angle in the range from 23.4 to 48.2.The hue value parameter was in a good correlation with the antioxidant capacity of samples (a correlation coefficient -0.7686).On the basis of individual colour parameters measured in the CIElab system it was observed that antioxidant capacity correlated with redness (a * ) and yellowness (b * ) (correlation coefficients of -0.7267 and -0.7458, respectively), although not with colour saturation (L * ) (a correlation coefficient -0.2392).On the other hand, statistical analysis presented in Table 2 did not show any significant correlations with neither acrylamide content nor colour of the product (a correlation of 0.0148 for hue angle), that excludes the possibility of a visual estimation of the risk of acrylamide formation in this type of product, which would be useful in practice.

CONCLUSION
The significant content of acrylamide was analysed in dried fruits from plums and plum stews in concentrations up to 60 μg/kg.Acrylamide content was not detected in thermally processed fruit products from plums, in sterilised canned plums, plum juice or baby food.Acrylamide, as a potentially harmful compound, was not correlated with health beneficial properties such as total phenolic content or antioxidant capacity of products.

.
Acrylamide levels in food monitored between 2007 and 2010 have been compiled by the European Food Safety Authority (EFSA) in the Scientific Report (

EFSA, 2010 and 2012).
On the basis of investigation results obtained during 2011 and 2012, and on the basis of the monitoring results obtained pursuant to Recommendations 2007/331/EC and 2010/307/EU, it was appropriate to modify certain indicative values provided for in the Annex to the Commission Recommendation 2013/647/EU.However, acrylamide has been found in

Table 1
Acrylamide, total phenolic compounds, antioxidant capacity and colour evaluation of prune samples.