Effects of Roselle Extract, Potato Peel Flour, and Beef Fat on the Formation of HCA of Beef Patties Studied by Response Surface Methodology

: Heterocyclic amines (HCA) are compounds formed by heating meat at a high tempera-ture. Formation of HCA in meat products should be minimized by applying different approaches. The objective of this study was to investigate the effects of roselle extract (RE, 0%–1%), potato peel flour (PP, 0%–2%), and beef fat (BF, 0%–15%) on the formation of HCA in beef patties using response surface methodology. IQx, IQ, MeIQx, MeIQ, 4,8-DiMeIQx, and PhIP were identified and quantified by HPLC. Values from 0 to 18.09 ng IQx, 0.20 to 11.66 ng IQ, 0.29 to 3.93 ng MeIQx, 0.19 to 11.51 ng MeIQ, 0.65 to 1.72 ng 4,8-DiMeIQx, and 1.87 to 14.7 ng PhIP per gram of sample were obtained. All models were significant ( p < 0.05), with no lack of fit ( p > 0.05) and showed a determination coefficient >0.70. Roselle extract had a negative linear effect on the formation of the total HCA, while PP and BF had a positive quadratic effect. The optimal formula that minimizes the formation of HCA included 0.80% RE, 0.85% PP, and 7.54% BF. Roselle extract and potato peel are foods that can be used as ingredients to minimize the formation of HCA in beef patties.


Introduction
A higher incidence of cancer has been reported when the intake of fruits and vegetables is low [1], when the consumption of foods with an elevated glycemic index increases [2], or when the meat intake is high [3]. With respect to the latter one, genotoxic compounds, such as heterocyclic amines (HCA), may form during cooking at high temperatures from Millard reactions between creatine, free amino acids, and carbohydrates present in meat [4]. The HCA ingested through foods and, subsequently absorbed into the body, are converted in genotoxic metabolites. These metabolites react with the DNA of various tissues to form adducts that lead to cancer problems.
The increase of the risk of cancer due to ingestion of HCA from food is an important issue in which it is necessary to seek solutions, by reducing the formation of these compounds during meat preparation. The use of vegetable tissue or extracts, as an antioxidant source, is one of the best strategies for the reduction of HCA. This is because the polyphenolic compounds, present in some vegetables, inhibit the formation of free radicals, preventing HCA formation. The addition of calyx or roselle extract, to meat products, has  been researched as a source of natural antioxidants. However, few articles have thoroughly studied their impact on HCA formation [5]. On the other hand, potato peel contains polyphenolic compounds, which, in aqueous extracts, have shown antioxidant activity [6]. Finally, beef fat is susceptible to oxidation, which is accelerated during cooking, producing free radicals that may be involved in the formation of HCA [7]. The objective of this research was to evaluate the effects of roselle extract, potato peel, and fat addition on the formation of HCA using response surface methodology.

Beef Patties Preparation
All ingredients were mixed according to Table 1, and homogenized for 3 min. In addition, 70 g of the resulting batter were manually shaped into beef patties. Each beef patty was cooked for 4 min per side at 190 ± 10 °C.

Extraction of Heterocyclic Amines
The extraction of heterocyclic amines was according to Messner et al. [8] with slight modifications. One gram of lyophilized sample was homogenized in 12 mL 1 M NaOH. The suspension was shaken for 1 h at 300 rpm and at room temperature. Subsequently, 13 g of diatomaceous earth (Extrelut NT, Merck, Darmstadt, Germany) was added, homogenized, and poured into a column of Extrelut NT20. Extraction was performed by washing the column with 75 mL of ethyl acetate, which was passed through an ion exchange (Waters, Milford, MA, USA) Oasis MCX cartridge (3 cm 3 /60 mg). The cartridge was washed with 2 mL 0.1 M HCl and 2 mL MeOH. The analytes were recovered with 2 mL ammonium hydroxide (25%) and MeOH (1/19, v/v) and frozen at −30 °C until further use. The samples were evaporated in a water bath at 40 °C with a constant nitrogen flow. The extracts were finally dissolved in 100 µL of MeOH for further quantification.

Chromatographic Separation
The identification and quantification of HCA followed the methodology described by Oz et al. [9] using HPLC equipment (Agilent Technologies, Santa Clara, CA, USA) with a diode array detector (Agilent, 1220 infinity DAD, LC, USA). The separation was carried out on a reversed phase analytical column, TSK-GEL ODS-80TM 5 µm

Statistical Analysis
The response surface methodology (RSM) was used to develop the prediction models. The analysis of variance (ANOVA), the determination coefficient (R 2 ), and the lack of fit of the models were determined using Design Expert (V.7.6.1, Stat-Ease, Inc., Minneapolis, MN, USA). The mathematical model corresponding to CCD is: where y are the response variables (formation of heterocyclic amines), β0, βi, βii, and βij are the coefficient of the intercept, liner, quadratic, and interaction, respectively, and Xi−j are independent variables (roselle extract, potato peel flour, and fat). The numerical optimization technique of the software Design-Expert was used for the simultaneous optimization of the multiple response. The formation of heterocyclic amines (IQ, IQx, MeIQ, MeIQx, 4,8-DiMeIQx, and PhIP) was kept as a minimum target.

Results and Discussion
Values for totals HCA are shown in Table 1 Table 2 shows the means and regression equations obtained for the prediction of the heterocyclic amines formed during cooking of beef patties prepared with roselle extract, potato peel flour, and fat. A greater effect by the incorporation of roselle extract was observed in the decrease of the studied HCA. Linear equations (p < 0.05) were adjusted in the formation of IQ, 4,8-DiMeIQx, and PhIP obtaining only a significant decrease by the roselle extract. While the polynominal equations of IQx, MeIQx, and MeIQ as well as quadratic equations were obtained, observing a negative linear and positive quadratic effect by the roselle extract. A positive quadratic effect was found from the effect of the incorporation of potato peel flour, allowing the formation of MeIQx and total HCA. On the other hand, a quadratic positive effect of the added fat was observed in the formation of MeIQx.
The observed values of HCA in our beef patties are similar to some reported studies, where the effect of other vegetable extracts were evaluated [10,11]. However, it is important to highlight that the quantity of amines formed depends on the type of meat, time, and type of cooking [12,13]. A significant effect of the decrease of the HCAs was found by the effect of the roselle extract. This is because the roselle is a flower rich in phenolic compounds that confers an antioxidant capacity [14]. It has been shown that antioxidants decrease the HCA formation due to antioxidants stabilizing the pirizine and/or pyrazyl radicals avoiding the union with creatinine. This way the aminoimidazole part of the amine is not formed [15]. On the other hand, the potato peel flour incorporation had a contrary effect to what was expected, increasing the MeIQx content. Potato peel flour contains a carbohydrate quantity of 275 to 1014 equivalents of glucose per gram of peel [16]. This causes an increase in glucose, which is a precursor of the HCA formation. This is causing a greater amount of MeIQx. The fat content in treatments does not have a significative impact on the HCA formation except in the MeIQx, where a positive quadratic effect was found. Fat was previously reported to promote the formation of heterocyclic amines [17]. However, it has been reported that a greater amount of HCA is formed in lean meat [18]. This is because, in meat with higher fat, there are fewer precursors, such as amino acids and glucose for the HCA formation. Some authors report that fattier meats have greater heat transmission, so foods cook faster, thus, allowing less amines formation [19,20]. On the other hand, optimization of the ingredients used for formulation of beef patties in this study was performed as described in the Materials and Methods section. The desirability function approach is one of the most utilized methods for the multiple response process optimization. In this study, the optimal formulation of beef patties that minimize HCA formation was estimated using the numerical optimization tool of the Design Expert software. According to the maximum desirability of multiple responses (0.92), the optimal formulation included 0.80% of roselle extract, 0.85% of potato peel, and 7.54% of beef fat. Predicted values for the HCA formation of the optimal formulation were: 0 ng IQx, 1.89 ng IQ, 0.29 ng MeIQx, 0.04 ng MeIQ, 0.85 ng 4,8-DiMeIQx, and 4.01 ng PhIP per gram of the sample.

Conclusions
The incorporation of roselle extract decreases HCA formation in beef patties, while the potato peel flour has a contrary effect. The incorporation of fat has no significate effect in the majority of the HCA, only in the MeIQx. Roselle extract and potato peel are foods that can be used as ingredients to minimize the formation of HCA in beef patties.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.