Vitamin a and E Homologues Impacting the Fate of Acrylamide in Equimolar Asparagine-Glucose Model System

antioxidants Article Vitamin A and E Homologues Impacting the Fate of Acrylamide in Equimolar Asparagine-Glucose Model System Su Lee Kuek 1, Azmil Haizam Ahmad Tarmizi 2,* , Raznim Arni Abd Razak 2 , Selamat Jinap 1,3 and Maimunah Sanny 1,3,* 1 Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, UPM, Serdang 43400, Selangor, Malaysia; [email protected] (S.L.K.); [email protected] (S.J.) 2 Product Development and Advisory Services Division, Malaysian Palm Oil Board, 6, Persiaran Institusi, Bandar Baru Bangi, Kajang 43000, Selangor, Malaysia; [email protected] 3 Laboratory of Food Safety and Food Integrity, Institute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, UPM, Serdang 43400, Selangor, Malaysia * Correspondence: [email protected] (A.H.A.T.); [email protected] (M.S.); Tel.: +603-8769-4214 (A.H.A.T.); +603-9769-8363 (M.S.) Abstract: This study aims to evaluate the influence of Vitamin A and E homologues toward acrylamide in equimolar asparagine-glucose model system. Vitamin A homologue as β-carotene (BC) and five Vitamin E homologues, i.e., α-tocopherol (AT), δ-tocopherol (DT), α-tocotrienol (ATT), γ- tocotrienol (GTT), and δ-tocotrienol (DTT), were tested at different concentrations (1 and 10 µmol) and subjected to heating at 160 ◦C for 20 min before acrylamide quantification. At lower concentrations (1 µmol; 431, 403, 411 ppm, respectively), AT, DT, and GTT significantly increase acrylamide. Except for DT, enhancing concentration to 10 µmol (5370, 4310, 4250, 3970, and 4110 ppm, respectively) caused significant acrylamide formation. From linear regression model, acrylamide concentration Citation: Kuek, S.L.; Tarmizi, A.H.A.; demonstrated significant depreciation over concentration increase in AT (Beta = −83.0, R2 = 0.652, Abd Razak, R.A.; Jinap, S.; Sanny, M. p ≤ 0.05) and DT (Beta = −71.6, R2 = 0.930, p ≤ 0.05). This study indicates that different Vitamin Vitamin A and E Homologues A and E homologue concentrations could determine their functionality either as antioxidants or Impacting the Fate of Acrylamide in pro-oxidants. Equimolar Asparagine-Glucose Model System. Antioxidants 2021, 10, 993. https://doi.org/10.3390/ Keywords: acrylamide; Vitamin A; Vitamin E; asparagine-glucose model system; heating antiox10070993 Academic Editor: William L. Stone 1. Introduction Received: 4 May 2021 Acrylamide is considered a heat-induced contaminant generated from carbohydrate- Accepted: 16 June 2021 based foods when subjected to thermal treatments. This unwanted compound was first Published: 22 June 2021 discovered in foods in 2002 and is currently classified as a probable carcinogen (Group 2A) by the International Agency for Research on Cancer (IARC) [1]. Acrylamide can be Publisher’s Note: MDPI stays neutral developed through Maillard reaction in the presence of two precursors, namely free as- with regard to jurisdictional claims in paragine and reducing sugars when food is prepared at high temperature [2]. Carbonyl published maps and institutional affil- scavenging is proposed to effectively reduce or prevent acrylamide formation by inter- iations. rupting the critical steps or scavenging key intermediates on the pathways to acrylamide formation [3]. Antioxidants may scavenge these free radicals and decrease the formation of acrylamide. Vitamin E is one of the natural fat-soluble vitamins present in vegetable oils and fats. This phytonutrient can be classified into two isomers, namely tocopherols and Copyright: © 2021 by the authors. tocotrienols [4]. Tocopherols mainly consist of four homologues, i.e., α-tocopherol (AT), β- Licensee MDPI, Basel, Switzerland. tocopherol, γ-tocopherol, and δ-tocopherol (DT). Tocotrienols can be also categorized into This article is an open access article four homologues encompassing α-tocotrienol (ATT), β-tocotrienol, γ-tocotrienol (GTT), distributed under the terms and and δ-tocotrienol (DTT) [5]. Vitamin E plays an essential role in inhibiting oxidation by conditions of the Creative Commons slowing down the rate of hydroperoxides formation (primary oxidation) and subsequently Attribution (CC BY) license (https:// reduces the formation of secondary constituents such as aldehyde [6]. Vitamin A is another creativecommons.org/licenses/by/ form of the fat-soluble vitamin in vegetable oils and fats. Such vitamin is often found in 4.0/). Antioxidants 2021, 10, 993. https://doi.org/10.3390/antiox10070993 https://www.mdpi.com/journal/antioxidants Antioxidants 2021, 10, 993 2 of 9 crude palm oil at a considerable level between 500 and 1000 ppm [7]. In the form of α- and β-carotenes (BC), Vitamin A has been shown to have the ability to remove singlet oxygen and reduces peroxyl radical [8]. BC exhibits twice the Vitamin A activity (expressed in retinol equivalent) of alpha-carotene [7]. Kushairi et al. [9] coined that tocotrienols and BC have shown to possess potential health-promoting effects on humans. Most studies in the literature have focused on the eliminating effects of water-soluble antioxidants on acrylamide [3,10]. Although research on the effect of fat-soluble antioxidants on acrylamide formation is scarce, few studies reported using fat-soluble antioxidants to minimize acrylamide formation. Li et al. [11] showed that the addition of Vitamin E (0.1 mg kg−1) to a cookie formula reduced acrylamide formation by 49.6%. In another study, Kamarudin et al. [12] reported that the effect of the fat-soluble antioxidants in Carotino red palm oil was more apparent when a prolonged frying time was used for 10 consecutive frying sessions of French fries. Further, Kuek et al. [13] observed acrylamide concentration in French fries decreased up to the 48th frying cycle, using red palm super olein (industrial grade). It is believed that fat-soluble antioxidants (i.e., BC and Vitamin E) affect acrylamide formation, which can be attributed to the ability of fat-soluble antioxidants with different structures or functional groups to react with acrylamide precursors, with intermediates of the reaction or with acrylamide itself, leading to either reducing or promoting effects [14]. Acrylamide is generated through a complex process that forms free radicals and many intermediate products during the reaction; therefore, the presence of antioxidants helps to prevent the free radical formation or hinder the attack of acrylamide from free radicals through extinction resulting in inhibition of acrylamide formation [11]. However, the discordant effect of antioxidants on the occurrence of acrylamide has been reported in the literature. Although Vitamin E has shown inhibitory effects on acrylamide in a cookie model, it was found to promote the meat model and beef nuggets [11,15,16]. Another study shows that Vitamin E, in the form of AT, distorted the acrylamide formation in the fried snack model [17]. The authors concluded that Vitamin A in the form of retinol exhibited a slight inhibitory effect on acrylamide. Nevertheless, the effect of BC on acrylamide has yet to be elucidated in any context. There are a few reported studies utilizing model systems with known substrates or intermediate products to understand the effect of antioxidants on acrylamide behavior [18,19]. The advantage of model systems is that relevant information is obtained about the actual mechanisms of the reaction, before proceeding to work on more realistic systems (i.e., food system) [20]. Antioxidants could limit the accumulation of carbonyls and thus inhibit acrylamide formation [18]. Inequality in the role of Vitamin E towards acrylamide behavior can be due to the different concentrations and homologues applied in the model system. As such, the reactivity of individual homologues, in their simplest form, at various concentrations requires further investigation to attest to acrylamide formation in the model system. Therefore, the objective of this study was to examine the effect of different concentrations of Vitamin A and E homologues on the formation of acrylamide in asparagine-glucose model system. 2. Materials and Methods 2.1. Chemicals and Reagents Labeled [13C3]-acrylamide (99% isotopic purity, 1 mg/mL) was purchased from Cambridge Isotope Laboratories Inc. (Andover, MA, USA). Acrylamide (purity > 99%), D- (+)-glucose (purity ≥ 99.5%), L-asparagine (purity ≥ 98%), (+)-α-tocopherol (purity ≥ 96%), β-carotenes (purity ≥ 97%), and δ-tocopherol (purity ≥ 97%) were obtained from Sigma- Aldrich (St. Louis, MO, USA). The α-tocotrienol (purity 88%), δ-tocotrienol (purity 59%), and γ-tocotrienol (purity 95%) were obtained from ExcelVite Sdn. Bhd. (Chemor, Malaysia). Bromine (purity 99.99%), triethylamine, ethyl acetate, hexane, sodium sulphate anhydrous, silica gel 60 (0.063–0.200 mm, 70–230 mesh, ASTM), and sodium thiosulphate were acquired from Merck (Darmstadt, Germany). Silicone oil was purchased from Systerm (Shah Alam, Malaysia). All chemicals were of analytical grade. Ultra-pure water, i.e., 18.2 MW-cm, Antioxidants 2021, 10, 993 3 of 9 was utilized (Purelab Classic U.V., ELGA LabWater, High Wycombe, UK) throughout the research. Acrylamide stock solution (1000 µg/mL) and 13C3-labeled acrylamide (4 µg/mL) were prepared following the dilution procedure. Two working acrylamide standards at 1 and 10 µg/mL were prepared from the stock solution (1000 µg/mL). These working standards were then used to prepare series of calibration standards at 0, 10, 50, 100, 250, and 500 ng/mL. Internal standard comprising 200 ng/mL of 13C3-labeled acrylamide was prepared from a stock solution of 4 µg/mL. One liter of bromination reagent was obtained by mixing 200 g of potassium bromide in a solution containing 160 mL of saturated bromine water, 10 mL of hydrobromic acid, and 0.5 L of ultra-pure water. All reagents were kept in a capped dark amber bottle at 4 ◦C. 2.2. Asparagine-Glucose Model System Equimolar asparagine-glucose model system was established following the procedure from Ou et al. [19] with slight modification. The model system was first prepared from a mixture containing 10 µmol of each asparagine and glucose, and 1 µmol of BC was homogenized with 1 g of silica gel.

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