1. Introduction
The global production of white meat products, especially poultry meat, has increased because of its sensory (e.g., color, odor, flavor, and texture) attributes, and the consumer belief that white meat is healthier than red meat. One of the main goals of meat manufacturers is to provide consumers with fresh food in terms of color, flavor, and odor. However, white meat products are highly sensitive to spoilage and damage; for example, different methods of meat preparation, such as grinding, blending, and heating, cause fat oxidation [
1,
2,
3]. Fat oxidation is the most important factor affecting the quality of meat. It can help develop pleasant aromas in some circumstances such as processing, handling, and storage [
4]. Indeed, it is well recognized that chemicals formed from lipid oxidation play a key role in the production of the characteristic aromas associated with meat products, which are highly valued by consumers [
5]. Lipid oxidation is a complex process which occurs in three different ways, each of which involves a series of complex reactions: autoxidation, enzymatic-catalyzed oxidation, and photo-oxidation. Autoxidation, which is a continuous free radical chain reaction, is the predominant process-causing lipid oxidation in meat [
5]. Unsaturated fatty acids and oxygen interact mostly through autoxidation [
6,
7]. Oxygen must be activated, resulting in the formation of a singlet oxygen (
1O
2) or a reactive oxygen species, such as hydrogen peroxide (H
2O
2), a superoxide anion (O
2•−), or a hydroxyl radical (OH•) [
7]. Initiation occurs as hydrogen is abstracted from an unsaturated fatty acid. The resulting alkyl radical tends to be stabilized by double-bond rearrangement to form a conjugated diene or triene [
8]. These alkyl radicals are the first free radicals that initiate lipid oxidation [
9]. The alkyl radical produced during the initiation phase reacts with the molecular oxygen to form peroxy radicals (a radical coupling with an oxygen molecule). They are highly reactive and abstract hydrogen from adjacent lipids. A hydroperoxide and an alkyl radical are produced due to this reaction. The process is repeated when the new alkyl radical interacts with molecular oxygen to create new peroxy radicals [
6]. Hydroperoxides and aldehydes, which are formed due to this reaction, are the most important breakdown products, and the main contributors to the volatile flavors in meat [
10].
Oxidation leads to considerable changes in food properties, such as color, reduction in nutritional value [
11], damage to the product, vitamin and unsaturated fatty acid depletion, generation of free radicals, development of unpleasant flavors, and shelf-life reduction [
12]. Thus, oxidation results in the production of different compounds, which have adverse effects on the quality of meat and meat products [
1,
13]. This has led to a demand for techniques to increase the shelf-life, safety, and quality of poultry meat [
14,
15]. One of the methods used to address these issues involves the addition of antioxidants to food during manufacturing [
16]. Butylhydroxytoluene (BHT) and butyl hydroxyanisole represent the most important chemical agents used to reduce fat oxidation; however, their use is only permitted within acceptable limits because they have toxic effects in addition to the odor imparted by phenols [
17,
18]. One important advantage of natural antioxidants is that they do not require safety tests before use. In addition, they may be more efficient than synthetic agents [
19]. Therefore, the demand for natural antioxidants has increased in recent years. Consequently, customers are increasingly interested in using natural products instead of synthetic products [
20]. Plants are the most important source of natural antioxidants because they contain bioactive compounds, such as flavonoids, carotenoids, tocopherols, and polyphenolic substances [
21,
22]. These contribute to the preservation and improvement of the quality of meat and meat products [
1].
Moringa oleifera (Moringa) leaves and seeds are valuable sources of bioactive compounds [
23]. Moringa is widely cultivated in Southeast Asia, mainly in Thailand, India, the Philippines, and Pakistan [
24]. The compounds in Moringa, in addition to their important medicinal properties [
23], function as effective antioxidants and inhibitors of bacterial and fungal growth. Moringa is rich in phenolic compounds that have been found to considerably inhibit oxidation in food [
25,
26]. The leaves of
Moringa oleifera contain 11 phenolic acids (gallic acid, caffeic acid, chlorogenic acid, o-coumaric acid, p-coumaric acid, ellagic acid, gentisic acid, sinapic acid, and syringic acid) [
27,
28], flavonoids (primarily flavonol and glycoside: quercetin, rhamnetin, campferol, apigenin, and myricetin), and their derivatives (coumaroylquinic acids and their isomers, feruloylquinic, and caffeoylquinic) [
29]. In addition, amino acids, minerals, vitamins, and beta-carotene are present in Moringa leaves and seeds [
30,
31].
Olive trees (
Olea europaea L.), which are native to the Mediterranean Basin, have spread throughout the world and adapted to a variety of climatic conditions, despite the fact that the Mediterranean region is the most important in terms of olive production [
32].
O. europaea is now found throughout Asia, America, and Oceania, owing mostly to the olive oil industry. Olive oil is a major component of the Mediterranean diet, is used in medicine, and is a source of lamp fuel. It now has wide applications in the modern cosmetic sector and in nutrition. However, regardless of the field in which it is used, the olive oil industry produces large amounts of waste, especially during the agricultural phase, which includes harvesting and oil production. Olive pulp and leaves are common by-products linked to pollution of soil and water [
33]. The demand for olive leaves, as both whole and extract forms, have increased dramatically in the use of foodstuff to increase the value of the food and as a preservative of unsaturated fat-rich foods [
34]. Phenolic compounds such as oleuropein, hydroxytyrosol, oleuropein, verbascoside, ligostroside, tyrosol, and tocopherol are present in large quantities in mature olive leaves [
35,
36,
37]. These compounds contain groups of phenolic compounds that serve as antioxidants by chelating metals like copper and iron, which catalyze free radical production reactions, such as lipid oxidation [
38]. In addition, olive leaves show antimicrobial activity against bacteria, fungi, and mycoplasma [
39,
40].
In recent years, the number of local and international consumers of chicken burgers has increased rapidly during the past ten years, and this food item has become highly preferred by consumers [
2]. The substitute of red meat with chicken in the burger industry is gaining popularity because of their high-fat content and because there are no cultural or religious restrictions on eating poultry [
41]. In accordance with The World Cancer Research, eating a lot of red meat (more than 500 g per week) can be harmful to health [
42]. Poultry meat remains the simplest, quickest, and most cost-effective way to obtain high-quality animal protein. Chicken is high in protein and a good source of all necessary amino acids, with a lower saturated fat content than beef fat and no carbohydrates, making it an excellent choice for people trying to reduce weight or who suffer from conditions like cardiovascular disease. In addition, According to the Collaborative Research Support Program in Nutrition, the largely plant-based diets of children in rural areas of Egypt, Kenya, and Mexico were found to be significantly lower in micronutrients: vitamin A, vitamin B12, riboflavin, calcium, iron, and zinc. Chicken meat is a very rich source of all these elements, and when added to the diet, it can greatly enhance the nutritional content [
43]. However, chicken and other meats have limited shelf stability [
1,
2]. The purpose of this study was to evaluate the effect of Moringa and olive leaves, and their extracts, on fat oxidation in chicken burgers under refrigerated storage, and to study the physical, chemical, and sensory attributes of chicken burgers. Four different treatments of Moringa and olive leaves and their extracts (powder and methanolic extract) were compared. This is the first study to evaluate the effects of Moringa and olive leaf preparations on chicken burger quality. The findings of this study will improve our understanding of using Moringa and olive leaves in the preservation of chicken burgers, and other processed meat products.
2. Materials and Methods
2.1. Materials
Methanol, sodium nitrite (NaNO2), BHT, aluminium chloride (AlCl3), sodium hydroxide (NaOH), Folin-Ciocalteu reagent, 2,2-diphenyl-1-picryhydrazyl radical (DPPH), gallic acid, sodium carbonate (Na2CO3), distilled water, magnesium oxide, boric acid, Tashiro indicator, HCl, filter paper, antifoaming agent, chloroform, acetic acid, potassium iodide, starch, and sodium thiosulfate (Na2S2O3.5H2O) were purchased from Sigma-Aldrich (Riyadh, Saudi Arabia). Moringa (oleifera) leaves were obtained from a local market in Al-Ahsa, Saudi Arabia. Olive leaves (Olea europaea L. var. koroneiki) were collected after harvesting fruits in October 2020, from olive tree farms in Al-Jouf, northwest Saudi Arabia.
2.2. Sample Preparation
Samples of fresh Moringa and olive leaves were cleaned by hand, washed with distilled water to remove dust and foreign contaminants, and dried at (35–40 °C). The dried leaves were crushed using an electric blender, sieved through a fine mesh, and stored in polyethylene bags at 4 °C, according to the method described by Singh and Immanuel [
44].
2.3. Methanol Extraction
An orbital shaker (from Light Duty Orbital Shakers, Parsippany, NJ, USA) was used for extraction preparation (10 min, 100 rpm). Dry powdered Moringa and olive leaves (100 g) were individually mixed with 1500 mL of 80% methanol at 30 °C for 5 h at 500 rpm. Extracts were filtered using a Whatman #41 filter paper. The methanolic extracts were evaporated under reduced pressure for 3 h at 40 °C, using a rotary evaporator. The methanolic extract obtained was placed in an oven for drying for 24 h at 50 °C to obtain the dried, residue extracts [
44].
2.4. Extraction Yield
After evaporation, the extracts were concentrated by oven drying and weighed to determine the extraction yield, which was calculated following Mahmoud et al. [
45].
2.5. Total Phenolic Content (TPC)
The TPC was determined using the Folin–Ciocalteu technique [
46]. Briefly, 2 g of each the dried Moringa, olive leaves, and their crude extracts were added to beakers, and 20 mL of methanol acidified with HCl (10%) was added, sonicated for 10 min in an ultrasonic bath, centrifuged for 10 min at 4000×
g, and filtered. Subsequently, the sample (0.5 mL) was placed into test tubes to which 1.5 mL of Folin–Ciocalteu reagent was added, and the mixture was allowed to rest for 5 min at 25 °C. After another 5 min, 2 mL of NaCO
3 (7%) was added, and the sample was incubated in the dark for 45 min at 25 °C. After incubation, the mixture turned blue, and 10 mL of distilled water was added to dilute the solution. The absorbance of the blue solution in various samples was determined at 715 nm, using a UV-18000 spectrophotometer (Shimadzu, China). The total phenolic content was calculated as mean ± SD (
n = 3) and expressed as mg/100 g of gallic acid equivalent (GAE) of the dried samples.
2.6. Total Flavonoid Content (TFC)
A colorimetric assay was used to determine the presence of flavonoids [
47]. Briefly, 4 mL of water was added to 1 mL of the methanolic of dried Moringa, olive leaves, and their crude extracts, separately. Subsequently, 0.3 mL of NaNO
2 (50 g/L,
w/
v) and 0.3 mL of AlCl
3 solutions (100 g/L) were added. After 5 min of incubation at room temperature, 2 mL of 1 M NaOH was added. The reaction mixture was immediately added to distilled water, to a final volume of 10 mL. The solution was vortexed, and the absorbance of the pink solution was measured at 510 nm using a UV-18000 spectrophotometer (Shimadzu, China). The TFC was calculated using the rutin calibration, and the results were presented as mg/100 of rutin equivalent (RE) of the dried sample (mg/100 g).
2.7. DPPH Radical-Scavenging Assay
Antioxidant activity was determined using the DPPH assay [
48]. Briefly, 1 mL of each methanolic solution of the Moringa, olive leaves, and their crude extracts at various concentrations (40, 80, 120, 160, and 200 mg/mL) was added to 3 mL of DPPH in methanol (0.33%). The absorbance at 517 nm was measured using a UV spectrophotometer after 30 min at 37 °C. In this assay, methanol served as a blank control, and all extracts and controls were examined in triplicate. The scavenging effect (%) was calculated using the following formula:
The scavenging activity of DPPH was plotted against the concentration, and the IC50 value (the extract concentration that scavenges 50% of the radicals) was calculated via linear regression.
2.8. Chicken Burger Preparation
The chicken burgers were prepared using chicken meat purchased from local markets. They comprised 10% breast meat, 55% thigh meat, and 15% fat from the chicken skin, according to Fishler [
49]. The chicken meat was minced and mixed with spices and cool water (13.8%). The completely homogenised chicken meat was divided into eight samples:
- I.
Control sample (C): chicken burgers without any treatment
- II.
Chicken burger + 0.01% BHT (BHT)
- III.
Chicken burger + 1% Moringa leaf powder (MLP1%)
- IV.
Chicken burger + 2% Moringa leaf powder (MLP2%)
- V.
Chicken burger + 0.02% Moringa leaf extract (MLE)
- VI.
Chicken burger + 1% olive leaf powder (OLP1%)
- VII.
Chicken burger + 2% olive leaf powder (OLP2%)
- VIII.
Chicken burger + 0.02% olive leaf extract (OLE)
Each chicken burger weighed 100 g and was shaped by hand using a mold plate (112 mm diameter × 2 cm height, HP 112; Picelli, Rio Claro, Brazil). Subsequently, the burgers were individually wrapped in polyethylene plastic and stored in a refrigerator at 4 ± 1 °C. All samples were stored for 20 days, and oxidation measurements were performed on Day 0 (at production), Day 10, and Day 20 (end of storage).
2.9. Total Volatile Nitrogen (TVN)
To measure the TVN, samples (10 g) were added to 100 mL distilled water and rinsed into a distillation flask containing 100 mL distilled water, and then 2 g of magnesium oxide and an antifoaming agent were added. A micro-Kjeldahl distillation device (Kjeltec system 2020 digestor) was used to dissolve the mixture. In total, 25 min of distillation was performed in 25 mL boric acid (4%), containing 5 drops of Tashiro indicator. To calculate the TVN in the sample, in terms of mg TVN/100 g, the solution was titrated with 0.1 M HCl (Pearson, 1976). The TVN concentration was calculated using the following formula [
50]:
Vi = Volume of 0.01 M hydrochloric acid solution in mL for sample; V0 = Volume of 0.01 M hydrochloric acid solution in mL for blank; M = Weight of sample in g.
2.10. Peroxide Value (PV)
The PV was calculated using the following technique (A.O.A.C.2000, Number 965.33), with certain modifications. First, 15 mL of hexane was added following centrifugation at 10,000 rpm for 10 min, which was performed twice to obtain de-fatted chicken burgers. Hexane was removed by heating, and the recovered oil was used to calculate the PV. A 2 g sample of oil was dissolved in 20 mL chloroform: acetic acid (1:2,
v/
v), and then 1 g of potassium iodide was added following heating in a water bath for 1 min to extract the oil. Subsequently, 20 mL potassium iodide solution (5%) and 50 mL of distilled water were added. In the presence of 0.5 mL of starch solution as an indicator, the iodine released was titrated with sodium thiosulfate (0.1 N). PV was measured in milliequivalents per kilogram, and was calculated using the following equation:
S = mL Na2S2O3 of the sample; B = mL Na2S2O3 of blank; N = normality of Na2S2O3; G = sample weight.
2.11. pH Value
The pH was determined by homogenising 10 g of material in 50 mL distilled water, after which the pH was measured using a digital pH meter (Benchtop pH Meters Hanna Instruments, Italy) [
51].
2.12. Drip Loss
The difference between the weight of a completely frozen and an identical burger after thawing was used to calculate drip loss. Drip loss was calculated as a percentage of weight change [
52].
2.13. Cooking Loss
The cooking loss of the prepared chicken burgers was calculated using the following method: [
53]
2.14. Cooking Yield
The cooking yield (%) was determined as specified by Zargar et al. (2014) [
54]. The weight of each chicken burger was recorded before and after cooking. The following formula was used to calculate the cooking yield, which was then represented as a percentage:
2.15. Sensory Evaluation
Chicken burgers were prepared based on standard specifications for burgers prepared in the Kingdom of Saudi Arabia [
49]. Color, odor, flavor, texture, and general acceptability were evaluated by ten panellists on a ten-point scale as follows: excellent = 9–10, very good = 7–8, good = 5–6, acceptable = 3–4, and barely acceptable = 1–2, following Mahdi et al. (2016) [
55].
2.16. Statistical Analysis
All measurements were performed thrice, and the results were presented as the mean standard deviation of triplicated experiments. The statistical analyses were carried out using the Statistical Package for the Social Sciences (SPSS) software, version 26 (IBM SPSS statistics, USA). Data were analyzed using ANOVA for all samples, and the results were compared using Duncan’s Test at a 5% significance level (p < 0.05).