1. Introduction
Animal food products account for a high proportion of the Western diet, and high meat consumption has been widely debated recently due to its adverse impacts on the environment, animal welfare, and human health [
1]. Meat production is associated with several concerns, including excessive use of water resources and land, greenhouse gas emissions, as well as detrimental effects on aquatic and terrestrial biodiversity and animal welfare [
2]. Livestock farming significantly impacts the environment, contributing approximately 14.5% of human-induced greenhouse gas emissions (GHGs), it also consumes nearly 40% of arable land and 56% of agricultural freshwater resources [
3]. Eshel et al. suggested that replacing meat with plant-based alternatives could potentially reduce national dietary land use by 34%, nitrogen fertilizer by 47%, and GHG emissions by 38% in the USA [
4]. Moreover, prolonged consumption of red or processed meat has been associated with the development of cardiovascular diseases and type 2 diabetes [
2]. Concerns also extend to additional health risks such as colon cancer and breast cancer in women, which may be exacerbated by the use of hormones in meat production to enhance growth rates and yields [
5]. For these reasons, there has been a shift toward a more plant-based diet for increased sustainability, leading both the food industry and researchers to actively seek plant proteins as alternatives for animal-sourced proteins. The market for plant protein-based foods is expanding rapidly, driven by an increase in consumers following vegan, vegetarian, or flexitarian lifestyles [
6]. As part of this trend, flexitarian consumers expect that consuming plant-based products will help them lower their intake of cholesterol and saturated fatty acids associated with meat consumption [
7]. Texturized vegetable protein is widely recognized for its significant role in addressing two prominent food trends: the growing demand for high-quality, low-fat foods, and the expanding market of functional and nutraceutical foods [
7]. Soy-based texturized vegetable protein, as a meat substitute, offers various economic and functional advantages. It is a plant-based protein product characterized by low saturated fat and calories, a rich concentration of essential amino acids, and is cholesterol-free [
8], with meat analogs having 96% less saturated fatty acids, 58% less energy, 33% less sodium, no cholesterol, and double the amount of fiber than a meat burger [
9].
The term “meat analogs” refers to a group of plant protein-based products closely resembling animal whole-muscle meat in terms of appearance, texture, and flavor, as well as some restructured products that resemble processed meats, such as sausage and patties [
10]. It is also known as meat substitutes, meat alternatives, mock meat, or imitation meat [
11]. The major plant protein ingredients in the production of plant-based meat analogs are soy protein, pea protein, and wheat gluten [
11]. Although the processing technology of plant-based meat analogs has been well developed, differences still exist between meat analogs and traditional meat products in terms of appearance, taste, flavor, and texture [
12]. Among these challenges, the appearance of plant-based meat analogs, particularly the color, is a significant issue for researchers. Color plays a vital role in the sensory experience for meat analogs because the initial sensory experience of food starts with the sense of sight, which is crucial in shaping consumer expectations and influencing acceptance [
13].
Caramel color is one of the world’s most widely used food pigments, with strong coloring capacity and excellent water solubility [
14]. Typically, caramel color with heat stability can provide a brown color appearance to the final meat analog [
15]. It has been used in meat analog products variously, such as vegetarian meat substitutes, e.g., Bacon Bits from Augason Farms, Frieda’s plant-based Soyrizo™, and BOCA’s All American Veggie Burgers. Tomato powders are highly nutritious, providing essential nutrients to humans such as folate, vitamin C, and potassium. They are also rich in carotenoids, with lycopene being the most abundant; carotenoids contribute to the antioxidant activity of tomatoes, making them valuable sources of antioxidants [
16]. Additionally, tomatoes contain vitamin E, trace elements, flavonoids, phytosterols, and various water-soluble vitamins, further enhancing their health benefits [
16]. Carrot powder rich in carotenoids, flavonoids, polyacetylenes, minerals, and vitamins, offers a wide array of health and nutritional benefits. The carotenoids, polyphenols, and vitamins found in carrots serve as antioxidants, anticarcinogens, and immune enhancers, contributing to their significant health-promoting properties [
17]. Recently, natural pigments were experimented with in some meat analogs to mimic the color of real meat. For example, Sakai et al. [
18] developed a browning system for plant-based meat analog patties using sugar beet pectin and laccase to enhance the product appearance, making it more similar to real meat patties. Milani and Conti [
19] used caramel color in textured soy protein to improve the sensory quality of meat analog and soy burgers. Carrot juice extract was used in the veggie burgers from “Morning Star Farms™”, and Aksu and Turan [
20] also used carrot extract to improve the storage quality of vacuum-packaged fresh meat products. Lyu et al. [
21] investigated the effect of tomato powder in soy protein-based high-moisture meat analogs, and Savadkoohi et al. [
22] incorporated tomato pomace with soy protein isolate to produce a meat-free sausage. However, research on the effect of natural pigments on both the color and nutritional quality of meat analogs is still limited. Therefore, this research aimed to investigate the impacts of three colorants (caramel, carrot, and tomato powder) on the color and pH of soy protein meat analogs during refrigerated storage. The color, total phenolic content (TPC), antioxidant activity, lipid oxidation, and texture profile of the raw and cooked soy protein meat analogs were also compared. This research may provide a reference for the processing of plant protein-based meat products using natural colorants to mimic the real meat color, with potential health benefits.
2. Materials and Methods
2.1. Materials
Uncolored texturized soy protein (TSP) was obtained from Bob’s Red Mill Co. Ltd. (Milwaukie, OR, USA). Wheat gluten flour (Vegan Grocery Store Pty. Ltd., Melbourne, Australia), food-grade methylcellulose (Delices d‘Oz Pty. Ltd., Port Augusta, SA, Australia), coconut oil (Woolworths Group Ltd., Mel, VIC, Australia), and table salt (Woolworths Group Ltd., Mel, VIC, Australia) were purchased. Caramel powder, whole tomato powder, and carrot powder were obtained from Maple Lifesciences Co. Ltd. (Haryana, India), Spice Masters Australia (Kingsgrove, NSW, Australia), and Opera Foods Pty. Ltd. (Warners Bay, NSW, Australia), respectively. All chemicals were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia).
2.2. Sample Preparation
2.2.1. Preparation of Soy Protein Meat Patties (SPMPs)
The meat patties were made from TSP (25.8%), boiled water (54.2%), wheat gluten flour (12%), coconut oil (6%), methylcellulose (1%), and salt (1%) [
23]. Briefly, the uncolored dry TSP was immersed in boiling water (1:6,
w/
v) for 15 min for rehydration. The excess water was squeezed out from the swollen TSP to achieve a TSP-to-water ratio of 1:2.1. Then the rehydrated TSP was mixed thoroughly with the coconut oil first and other ingredients. The uncolored SPMPs were used as the control group, and another nine colored SPMPs were prepared with the addition of caramel, carrot, and tomato powder by different weight percentages (
Table 1), based on our preliminary experiments on the color similarity of SPMPs to real meat. Finally, the formulated mixtures were molded to a diameter of 6.5 cm, a thickness of 1.2 cm, and a 50 g patty. Three independent batches were prepared.
The SPMPs were wrapped with plastic wrap, packaged in sterile airtight bisphenol A-free bags, and stored at 4 °C for 0, 1, 4, 7, and 10 days for cooking and analysis.
2.2.2. Cooking Condition
The SPMPs were cooked individually in a frypan at approximately 170 °C and turned over every 1 min until the internal temperature reached 75 °C by inserting a digital thermometer (NORONIX 600 N 50 °C to + 200 °C, Mitchell Park, Australia) into the geometric center of the patties. Then, the patties were taken out of the frypan, cooled to room temperature, and covered with plastic wrap for further analysis.
2.3. Color Measurement
The color of the SPMPs within 10 days of storage was measured before and after cooking by using a Minolta Colorimeter with a C illuminant and 8 mm aperture size (Model CR-400, Minolta Co., Ltd., Osaka, Japan) [
24]. The instrument was calibrated with a standard white tile (L* = 93.80, a* = 4.84, b* = −0.80) before use and the CIELAB color system for L* (lightness), a* (redness), and b* (yellowness) values were recorded. Each patty was measured at six different locations. The color difference (ΔE) was calculated to describe the changes in the color of the raw SPMPs and cooked SPMPS, and the color change during the 1, 4, 7, and 10 days of storage (4 °C) compared to day 0, which was calculated using the following formula [
22]:
2.4. pH Measurement
Three grams of SPMP sample were homogenized with 27 mL of milli-Q water using an IKA-T25 Ultra-Turrax homogenizer (IKA-Werke GmbH & Co, Staufen, Germany) at 8400 rpm for 2 min in an ice water bath [
25]. A pH meter (HI1230, Hanna Instruments, Smithfield, RI, USA) was used to measure the pH of the resulting homogenate at room temperature. All tests were performed in triplicate.
2.5. Total Phenolic Content (TPC)
The TPC of raw and cooked SPMPs was analyzed using a modified Folin–Ciocalteu assay [
26]. Briefly, 25 mL of methanol and a 5 g SPMP sample were mixed and shaken for one hour on a platform mixer (Ratek Instruments Pty Ltd., Boronia, Australia). After centrifugation of the mixture at 4000×
g for 10 min at 4 °C, the supernatant was collected and filtered with a Whatman No. 1 filter paper; 0.1 mL of filtrate and 0.5 mL of Folin–Ciocalteu solution were mixed for 10 min. The new mixture was added with 0.4 mL of 7.5% sodium carbonate solution, followed by a vortex and incubation for 30 min at room temperature in the dark. The absorbance of the resulting solution was measured at 760 nm with a UV-Vis spectrophotometer (Multiskan GO, Thermo Scientific, Vantaa, Finland). Gallic acid (0.05–0.25 mg/mL) was used as a standard. The TPC was expressed as mg gallic acid equivalent (GAE)/100 g sample. All tests were performed in triplicate.
2.6. DPPH Radical Scavenging Activity
The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay was used to estimate the antioxidant activity of the SPMPs [
27]. To prepare the DPPH stock solution, 24 mg of DPPH was dissolved in 100 mL methanol and stored in the dark at −20 °C. The DPPH working solution (absorbance of 1.1 ± 0.02 units at 517 nm) was prepared freshly by diluting about 8 mL of stock solution in 40 mL of methanol [
28]. An aliquot of 1 mL of the above-filtrated sample and 4 mL of fresh DPPH working solution were mixed and kept in the dark for 30 min. The absorbance of the resulting solution was measured at 517 nm with the Multiskan GO UV-Vis spectrophotometer. Trolox (12.5–100 mg/L), methanol, and the DPPH working solution were used as the standard, blank, and control, respectively. All tests were performed in triplicate. Radical scavenging activity (RSA (%)) was calculated using the following formula:
2.7. Lipid Oxidation (TBARS Assay)
Lipid oxidation of the raw and cooked SPMP samples was analyzed based on the 2-thiobarbituric acid reactive substances (TBARS) assay, according to the method of Xiong et al. (2022), with some modifications. About 5 g of SPMP sample in 15 mL of 10% trichloroacetic acid (TCA) solution was homogenized using a homogenizer (Polytron PT 10–35 GT, Kinematica AG, Luzern, Switzerland) at 8400 rpm for 60 s. The mixture was centrifuged at 3500×
g and 4 °C for 8 min. Two milliliters of supernatant were collected and mixed with 2 mL of 20 mM 2-thiobarbituric acid (TBA) solution and incubated at 95 °C for 30 min in a water bath. Then the sample was cooled to room temperature in cold water and centrifuged at 3000×
g 4 °C for 10 min [
29]. The absorbance of the supernatant was measured at 532 nm with the Multiskan GO UV-Vis spectrophotometer. The chemical 1,1,3,3-tetraethoxypropane (0–20 μM) was used as the standard. The TBARS value was expressed as a milligram malondialdehyde equivalent per kilogram (mg MDA/kg) sample. All tests were performed in triplicate.
2.8. Cooking Loss
After cooking the samples as described in
Section 2.2.2, the proportion of fluid lost, which includes water, lipids, proteins, and minerals, was defined as the cooking loss and calculated using the formula below [
30]:
2.9. Texture Profile Analysis
The texture profile analysis (TPA) of the SPMPs was conducted by a double-bite compression test using a single-column universal testing machine (Lloyd LS5 universal testing machine, Ametek Inc., Berwyn, PA, USA), fitted with a cylindrical probe (20 mm diameter), as described by Xiong, Zhang, Warner, Hossain, Leonard, and Fang [
25], with modifications. The raw and cooked samples were cut into square shapes in dimensions of 15 × 15 × 10 mm. The raw and cooked sample was double-compressed to 50% of the original height at a constant speed of 1 mm/s and 1 s time interval between the two cycles. Nine replicates (
n = 9) were performed for each sample. The TPA parameters of hardness, cohesiveness, gumminess, springiness, and chewiness were calculated using the machine software.
2.10. Statistical Analysis
All data were expressed as mean ± standard deviation (SD), and one-way analysis of variance (ANOVA) was conducted using Minitab software (Minitab Windows, version 17, Sydney, Australia). Tukey’s post hoc test at a 95% confidence level was used to compare the differences between different SPMP formulations, and the T-test (p < 0.05) was used to compare raw and cooked samples in the same formulation.