Natural antioxidants from collard greens (Brassica oleracae var. acephala): effects on the storage stability and quality of meatballs

Natural antioxidants from collard greens (Brassica oleracae var. acephala): effects on the storage stability and quality of meatballs | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Natural antioxidants from collard greens (Brassica oleracae var. acephala): effects on the storage stability and quality of meatballs Lubowa Muhammad, Nalweyiso Lailah, Shin-Yong Yeoh, Muwonge Abubakar, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4672436/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Synthetic antioxidants have been used to enhance the storage stability of meat by preventing lipid oxidation. However, concerns about their health risks and toxicity have led to increased interest in natural alternatives. Collard greens, known for their health benefits and appealing taste, are rich in bioactive compounds such as antioxidants, fiber, minerals, and vitamins. Their safety and nutritional profile make them ideal for use as preservatives or functional food ingredients in the food industry. This study evaluated the effects of collard greens extract (CGE) on the quality and storage stability of meatballs. Meatballs were formulated with CGE at 500 and 325 mg/kg and compared with those containing green tea extract (GTE) and a control group. The samples were stored under refrigeration, and oxidative stability was assessed over 30 days using TBARS. CGE demonstrated a significant lipid oxidation inhibitory effect comparable to GTE. Meatballs with 500 mg/kg of extract maintained significantly lower TBAR values ( P 0.05) and received positive sensory scores of the meatballs. Biological sciences/Biochemistry Biological sciences/Biophysics Natural antioxidants lipid oxidation collard greens extract meat quality plant extracts green tea extract Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 Introduction The quest for food quality and safety is a cornerstone of food science research and industry practices. In today's world, where global meat consumption is on the rise due to demographic shifts, economic growth, and changing dietary preferences, the meat industry faces the formidable task of meeting consumer demands for high-quality, safe, and stable products. One of the critical challenges in the meat industry is lipid oxidation. This process significantly degrades the sensory qualities, nutritional value, and shelf life of meat, leading to substantial economic losses and posing health risks through harmful oxidation byproducts (Domínguez et al., 2019 ; Dave & Ghaly, 2011 ). Historically, the meat processing industry has depended on synthetic antioxidants to combat the adverse effects of oxidation (Mani‑López et al., 2016; Rodil et al., 2012 ). However, growing consumer awareness about synthetic additives' safety and health implications has driven demand for natural alternatives (Feknous et al., 2023 ; Olvera-Aguirre et al., 2023 ). This shift reflects a broader societal movement towards more sustainable and health-conscious food production practices (Herrero et al., 2023 ; FAO, 2023 ). Synthetic antioxidants and natural alternatives differ markedly in terms of safety and efficacy (Fadhil et al., 2023 ). While synthetic antioxidants are effective, their effectiveness and convenience come at a price. There's a growing debate regarding their safety and potential health implications (Thakur et al., 2019 ; Yu et al., 2021 ). Long-term intake has been linked to skin allergies, gastrointestinal issues, increased cancer risk, DNA damage, and premature aging (Muthukumar et al., 2020 ; Xie et al., 2023 ; Błaszczyk et al., 2013 ; Shahidi & Zhong, 2010 ). Compounds, such as sulfites, BHA, and BHT, have raised concerns due to their allergenic or carcinogenic properties, while others, like nitrates and nitrites, can form potentially harmful nitrosamines when exposed to high heat (Muthukumar et al., 2020 ; Xie et al., 2023 ). As a result, consumer apprehensions regarding consuming chemically treated food have led to a shift away from these compounds. Conversely, natural antioxidants are generally considered safe and increasingly preferred by consumers seeking clean-label products. Natural antioxidants, such as tocopherols, ascorbic acid, and rosemary extract, offer nutritional value, safety, and stability in food products (Feknous et al., 2023 ; Gutiérrez-Del-Río et al., 2021 ; Brewer, 2011 ; Pokorný, 2007 ). These compounds, rich in phenolics, vitamins, and carotenoids, not only act as antioxidants but also possess antimicrobial properties and influence food flavors and textures. Thus, choosing between synthetic and natural antioxidants hinges on safety, efficacy, consumer preferences, and regulatory guidelines. Collard greens ( Brassica oleracea var. acephala ) stand out among the natural resources explored. This leafy vegetable, indigenous to East Africa, is rich in bioactive compounds like antioxidants, flavonoids, glucosinolates, and phenolics (Kuete, 2017 ). Phytonutrients in collard greens include phenols like caffeic and ferulic acid, flavonoids like quercetin and kaempferol, and glucosinolates like glucobrassicin and glucoraphanin as well as hydroxycinnamic acids (Velasco et al., 2011 ; Olsen, Aaby, & Borge, 2009 ). Kaempferol glycosides, acylgentiobiosides, and quercetin glycosides are the major phenolic antioxidants present, while derivatives of p-coumaric, ferulic, sinapic, and caffeic acid are some of the minor phytochemicals present (Lin & Harnly, 2009 ; Olsen, Aaby, & Borge, 2009 ; Picchi et al., 2020 ). Other phytonutrients in collard greens include indole-3-carbinol, diindolylmethane, sulforaphane, isothiocyanates, and carotenoids. Renowned for their culinary versatility and health benefits, collard greens present a promising solution to the meat industry's oxidation challenge (Kahlon et al., 2008 ; Kuete, 2017 ). Despite the known advantages of collard greens and other natural antioxidants, a significant gap exists in their application for meat preservation. Specifically, there is a lack of comprehensive studies exploring the efficacy of CGE in enhancing meat quality and storage stability without compromising sensory attributes. This gap highlights the need for research that not only substantiates the antioxidant capabilities of CGE but also evaluates their impact on the physicochemical, textural, and sensory characteristics of meat products. Building on existing research, this study aims to deepen our understanding of CGE's effects on meat quality and storage stability. By incorporating these extracts into meatballs—a widely consumed and commercially significant product—the research offers valuable insights into natural food preservatives and addresses practical food technology and product development challenges. By comparing the antioxidant effects of CGE with GTE, known for their antioxidant properties (Falla et al., 2021 ; Luo et al., 2020 ; Senanayake, 2013 ; Carrizo et al., 2016 ), this study assesses the impact of these natural antioxidants on the physicochemical, textural, and sensory attributes of meatballs over a specified storage period. Moreover, this research explores the broader implications of integrating bioactive compounds from plants like collard greens into the food industry. It highlights the shift towards functional foods that provide health benefits beyond essential nutrition, aligning with contemporary dietary trends and consumer expectations. In doing so, the study addresses immediate issues of meat product quality and safety and contributes to the evolving discussion on the role of natural antioxidants in the food industry. 2 Materials and methods 2.1 Materials All chemicals and reagents used were of analytical grade. Water used in all experiments was purified using a Milli-Q water purification system. Methanol (80% v/v) and butanol were utilized for the extraction processes and determination of total phenolic content and total antioxidant capacity, respectively. Sodium carbonate and Folin-Ciocalteu phenol reagent were specifically employed to determine total phenolic content, following a slightly modified method from Andressa Blainski et al. ( 2013 ). For the total antioxidant capacity (TAC) assay, DPPH (2,2-diphenyl-1-picrylhydrazyl) was used to evaluate the antioxidant activity of the extracts. Thiobarbituric acid, used to determine thiobarbituric acid reactive substances (TBARS), was sourced and prepared according to Pokorny ( 1989 ), with butanol as the solvent. These substances were pivotal in assessing the physicochemical properties and antioxidant activity of the meatball samples incorporated with CGE and GTE. 2.2 Preparation of plant extracts 2.2.1 CGE Collard greens were acquired from a local farmer in Uganda and transported (cooling boxes to avoid transpiration and respiration) to the Makerere University School of Food Technology for processing, which involved washing, trimming, and size reduction. Approximately 14.5 kg of the prepared collard greens leaves were dried using a solar dryer for seven days, with the leaves turned thrice daily. The dried sample was ground into powder and subjected to water extraction. During extraction, 14 g of powder was combined with 100 mL of water and extracted at 85°C for 30 min in a water bath, with occasional stirring, as described by Reihani et al. ( 2014 ). The mixtures were filtered, and the filtrates were dried and ground into powder. The extracts were stored in zip-lock plastic bags in a freezer until needed for the meatball formulation. 2.2.2 GTE Green tea, specifically the So Fine China brand, was purchased from Senana Supermarket, Kampala. The green tea leaves were ground to a fine powder using an electric power grinder (Model DE-100g, China). For the extraction, 14 g of the green tea powder was mixed with 100 mL of water and subjected to heat in a water bath at 85°C for 30 min. The mixture was then filtered through a cheesecloth to separate the residue from the filtrate. The filtrate was placed in silver plates and dried overnight in an oven (Gallenkamp, UK, Model: OHF09.XX1.5) at a temperature setting of 80°C. The dried filtrate was ground into powder using the same grinder and stored in zip-lock plastic bags in a freezer until further use. 2.3 Preparation of meatballs The meatballs were formulated using minced beef as the primary ingredient. The beef was obtained from a local butcher to ensure freshness and quality. The experimental treatments used previously prepared CGE and GTE (Section 2.2 ). To enhance the meatball mixture, onions and fat were finely chopped and melted, respectively. Eggs were whisked to aid in binding the mixture, while breadcrumbs, prepared from dried and ground bread, garlic powder, and salt, were added for flavor and textural purposes. The meatball mixture was evenly divided to incorporate the different extract concentrations, with 500 and 325 mg/kg for the CGE, alongside a comparable green tea extract concentration serving as a positive control. A batch without any extracts acted as the negative control. Each portion of the mixture was carefully shaped into balls weighing approximately 100 g, packaged in airtight zip-lock plastic bags, and labeled according to their specific formulations for further analyses, including lipid oxidation and pH measurement. These samples were stored under refrigeration temperatures. Samples for sensory evaluation were placed on baking sheets. The meatballs were then cooked in a preheated oven at 190°C for 25 min until they reached a safe internal temperature and acquired a golden-brown crust. Following baking, the meatballs were cooled to room temperature, packaged in airtight zip-lock plastic bags, and labeled according to their specific formulations, as shown in Table 1 : Table 1 Quantity of meat and extracts used in the meatball formulations. Samples Minced beef (g) Quantity of extract (mg) CC 1000 CGE-500 mg/kg 1000 500 CGE-325 mg/kg 1000 325 GTE-500 mg/kg 1000 500 GTE-325 mg/kg 1000 325 CC - Negative control doesn't have any extract; CGE - Contains collard greens extract; GTE - Positive control contains the green tea extract. The values attached stand for the quantities of extract. 2.4 Measurement of lipid oxidation The extent of lipid oxidation in meatball samples was quantitatively determined using the TBARS assay, a widely recognized method for assessing secondary oxidation products, primarily malondialdehyde (MDA), which indicates lipid peroxidation. To prepare the samples for analysis, approximately 2 g of meatball from each formulation was homogenized with 15 mL of distilled water. The homogenate was then mixed with 2 mL of a 20% trichloroacetic acid solution to precipitate proteins, followed by centrifugation at 3000 rpm for 10 min to clarify the solution. The supernatant was subjected to the TBARS assay by adding 2 mL of the supernatant to 2 mL of a 0.02 M thiobarbituric acid solution in a test tube, which was then heated in a boiling water bath for 30 min to allow the formation of the TBA-MDA complex, characterized by its pink color. After cooling to room temperature, the absorbance of the solution was measured at 532 nm using a spectrophotometer. The TBAR values were calculated using a standard curve of 1,1,3,3-tetraethoxypropane, expressed in milligrams of MDA per kg sample. This measurement was conducted on meatballs stored under refrigeration for 0, 7, 14, 21, and 28 days to evaluate the oxidative stability of the meatballs over time. The measurements were done in triplicates. 2.5 Cooking yield The cooking yield of the meatball samples was determined as a measure of weight loss during cooking, which indicates moisture retention and fat content changes. These factors significantly affect the final product quality. For this purpose, each meatball formulation was weighed before and after baking in the oven at 190°C for 25 min. The initial weight (W1) was recorded immediately before cooking, and the final weight (W2) was noted after the meatballs had cooled to room temperature, ensuring consistency in measurement. The cooking yield was calculated using the formula: Cooking yield (%) = (W2/W1) × 100%. Cooking yield measurements were taken for all experimental groups to assess the impact of the natural extracts on the cooking performance of the meat products. Measurements were done in triplicates. 2.6 Physicochemical properties (pH) For pH determination, a representative sample of approximately 10 g from each meatball formulation was homogenized with 50 mL of distilled water using a laboratory blender to ensure thorough mixing. After homogenization, the slurry was allowed to settle for 5 min, and the pH was measured directly in the supernatant using a calibrated pH meter (Model INE-PHS-3E, MRC, Israel). The electrode was carefully rinsed with distilled water between measurements to prevent cross-contamination. The procedure was performed in triplicate for each sample, and the results were averaged to obtain the final pH value for each formulation. Measurements were conducted on samples immediately after cooking and after 7, 14, 21, and 28 days of refrigerated storage to evaluate changes in pH over time. 2.7 Texture profile analysis (TPA) TPA was performed using a TA.XTplus Texture Analyzer (Stable Micro Systems, UK) equipped with a 50 mm cylindrical probe and a 5 kg load cell. Before analysis, meatball samples were sectioned into uniform cylindrical pieces of approximately 20 mm height, ensuring consistency across all measurements. The textural attributes measured included hardness and cohesiveness, which are critical determinants of the sensory quality of meat products. The TPA procedure involved a double compression test, where each sample was compressed to 50% of its original height at specified speeds: a pre-test speed of 1.0 mm/s, a test speed of 1.7 mm/s, and a post-test speed of 10.0 mm/s. The interval between the first and second compression cycles was fixed at 5 s, mimicking the biting action to evaluate the sample's textural behavior comprehensively. Hardness was quantified as the peak force encountered during the first compression cycle, representing the sample's resistance to deformation. Cohesiveness, a measure of the sample's ability to withstand a second deformation relative to its initial structure, was calculated as the ratio of the area under the second compression curve to that under the first. Measurements were conducted in triplicate for each formulation, and the results were processed and analyzed using Texture Exponent software. 2.8 Analysis of microbiological stability In this study, an in vitro antimicrobial assay was performed using the plate count method to determine the total number of aerobic microorganisms in control samples and samples incorporated with CGE and GTE. Microbial counts were performed at 7-day intervals over the 28-day storage period (4 weeks), following the method described by Jałosińska and Wilczak ( 2009 ). At each interval, 5 g of each sample was weighed and placed into a Stomacher bag with 45 mL of sterile peptone water (Biocar Diagnostics) added for dilution. The samples were homogenized for 2 min at a standard speed using a Stomacher 400 apparatus. A series of tenfold dilutions were prepared from the homogenized samples, and 1 mL from each of the three consecutive dilutions was inoculated onto nutrient agar plates (Biocar Diagnostics) in triplicate. The inoculated plates were incubated at 37°C for 48 h, and colonies were counted to determine the total number of aerobic microorganisms. The antimicrobial effectiveness of the CGE and GTE was assessed by comparing the microbial counts of the treated samples with those of the control samples over the storage period. 2.9 Sensory evaluation Sensory evaluation was performed only on the samples incorporated with CGE and the control. A panel of 50 trained sensory evaluators from the Department of Food Science and Technology at Makerere University participated in the assessment. The panelists were selected based on their sensory analysis experience and ability to discern subtle differences in food samples. The meatballs were served at a controlled temperature of approximately 37°C to mimic typical consumption conditions. Each panelist was given samples coded with random three-digit numbers to prevent bias. Water and unsalted crackers were available to cleanse the palate between samples. The evaluation was structured around a 9-point hedonic scale, where 1 represented "dislike extremely" and 9 signified "like extremely." Panelists were asked to rate each sample based on the aforementioned sensory attributes. The scores for each attribute were then averaged to provide a composite score for each meatball formulation. To ensure the reliability of the sensory data, the evaluation session was conducted in a well-lit, odor-free room with individual booths for each panelist to prevent discussion and influence among participants. 2.10 Statistical analysis Analysis of variance (ANOVA) and Tukey's Honestly Significant Difference (HSD) were used to analyze the data. All statistical analyses were performed using SPSS Statistics software (Version 26, IBM Corp). Figures were generated using Python's matplotlib library (Version 3.3.2) to visually represent the data and highlight significant findings from the statistical analyses. Microbiological count data, presented as colony-forming units per milliliter (cfu/mL), were log-transformed before analysis to stabilize variances and improve the approximation to a normal distribution. The statistical significance level was set at P < 0.05 for all tests. 3 Results and discussion The current study provides insightful contributions to the burgeoning field of natural food preservatives, particularly focusing on the impact of CGE and GTE on the quality and stability of meatballs stored under refrigeration. The results encompass various critical aspects, including lipid oxidation, physicochemical properties, cooking yield, sensory attributes, texture, and microbiological stability, offering a comprehensive overview of the efficacy of natural extracts as meat preservatives. 3.1 Lipid oxidation stability The TBARS assay, employed to assess lipid oxidation stability in meatball samples stored over 28 days under refrigerated conditions, revealed distinct trends in oxidative stability (Fig. 1 ). Throughout the storage period, all samples demonstrated an increasing trend in TBARS values, indicative of progressive lipid peroxidation. However, the rate of increase varied significantly between different treatments. The ANOVA results highlight a marginally significant effect of treatment type on TBARS values (p = 0.053), suggesting that the type of extract used—whether CGE at 325 mg/kg and 500 mg/kg or GTE at the same concentrations—may influence lipid oxidation, though modestly. The most profound effect observed was time (p = 0.0006), confirming that lipid oxidation naturally escalated over the 28 days. Moreover, the interaction between sample types and time was highly significant (p = 0.0001), indicating that the efficacy of different treatments in inhibiting lipid oxidation varied at various storage times. Notably, samples treated with CGE-500 mg/kg and GTE-500 mg/kg generally displayed slower rates of increase in TBARS values, suggesting these higher concentrations are more effective at delaying lipid peroxidation. This is evident in the mid-storage data, where these treatments maintained lower TBARS values than their lower concentration counterparts and the control. By the end of the 28 days, however, all samples reached higher TBARS levels, with those treated with extracts consistently showing marginally better oxidative stability than the control. The application of CGE and GTE significantly reduced lipid oxidation in meatballs, clear from the TBARS values observed over the 28-day storage period. Collard greens contain high levels of natural antioxidants such as flavonoids, phenolic acids, and vitamins (e.g., vitamins C and E) (Picchi et al., 2020 ; Villalobos-Delgado et al., 2019 ; Mir, 2019 ; Olsen, Aaby, & Borge, 2009 ). These antioxidants can slow lipid oxidation in meatballs, preventing rancidity and off-flavors common in meat products. Specific compounds in CGE, such as kaempferol and quercetin, are likely behind this antioxidative effect. These flavonoids are renowned for scavenging free radicals and chelate metal ions, effectively halting the oxidation processes that lead to lipid degradation (Villalobos-Delgado et al., 2019 ). The presence of these compounds in CGE aligns with various research findings, which have identified kaempferol and quercetin in collard greens as powerful antioxidants (Picchi et al., 2020 ; Mir, 2019 ; Olsen, Aaby & Borge, 2009 ). These compounds contribute significantly to oxidative stability in various food matrices. For instance, Liu ( 2013 ) discussed the cellular antioxidant activity of common vegetables, highlighting the crucial roles of quercetin and kaempferol. Similarly, Ağagündüz et al. ( 2022 ) reviewed the antioxidant activity of cruciferous vegetables, including collard greens, noting their considerable impact on lipid oxidation stability. Furthermore, a comprehensive review of the nutritional composition and bio-active compounds of kale, a close relative of collard greens, detailed the antioxidative properties of kaempferol and quercetin. This review emphasized their role in enhancing the shelf-life and safety of food products by minimizing the formation of potentially harmful oxidation products (Satheesh, Fanta, & Yildiz, 2020 ). This antioxidant activity is crucial not just for extending the shelf-life of meat products but also for improving their safety by reducing the formation of harmful oxidation products. 3.2 Microbiological stability of meatball samples The microbiological stability of meatballs incorporated with CGE and GTE was assessed through microbial count analysis over a four-week storage period, with values expressed in a logarithmic scale (log CFU/mL). Initial results showed relatively low microbial counts for all samples, indicating good baseline product hygiene, with the GTE-325 mg/kg treatment demonstrating the most potent initial antimicrobial effect, yielding the lowest counts, approximately at 5.37 log CFU/mL (Fig. 2 ). By the seventh day, microbial counts were increased across all groups, consistent with expected microbial growth under refrigeration. However, samples treated with higher concentrations of extracts, specifically CGE-500 mg/kg and GTE-500 mg/kg, exhibited notably lower counts, around 6.80 log CFU/mL, compared to control samples, which reached about 7.75 log CFU/mL. This trend persisted through day 14 and day 21, with treated samples continuing to show slower microbial growth, affirming the sustained antimicrobial properties of the extracts. By day 28, control samples had the highest microbial counts exceeding 10.00 log CFU/mL. In contrast, the extract-treated samples, particularly those with higher concentrations, maintained significantly lower levels, approximately 9.00 log CFU/mL. These observations highlight the effectiveness of CGE and GTE in inhibiting microbial growth, potentially enhancing the safety and extending the shelf-life of meat products. The antimicrobial efficacy of the extracts, evidenced by the slower microbial growth in treated meatballs, highlights their potential to enhance food safety and extend the microbial shelf-life of meat products. Polyphenol compounds and glucosinolates and their hydrolysis products (isothiocyanates and indoles) found in CGE are likely behind these antimicrobial properties (Bhat & Al-Daihan, 2014 ; Mourtzinos et al., 2019 ). Various studies have identified these compounds as potent antimicrobial agents that inhibit the growth of pathogenic bacteria (Mourtzinos et al., 2019 ). They achieve this probably through mechanisms like disrupting microbial membranes and interfering with microbial enzyme systems (Ahmad, Hassan & Azim, 2017 ). This capability not only curtails microbial proliferation but also contributes to the oxidative stability of the product, showcasing the dual functionality of these natural compounds. Horbańczuk et al. ( 2019 ) and Nur et al. (2021) highlighted the antimicrobial activities of plant extracts in meat products, emphasizing the efficacy of natural compounds in inhibiting microbial growth. Similarly, Kumar et al. ( 2023 ) underscored the role of glucosinolates and their hydrolysis products in providing antimicrobial activity. Additionally, Wali et al. ( 2024 ) confirmed the significant antimicrobial properties of these extracts, particularly against Gram-negative bacteria. The ability of these extracts to curb microbial proliferation while enhancing oxidative stability demonstrates their dual functionality. This aligns with the findings of Jałosińska and Wilczak ( 2009 ), who highlighted the role of natural antioxidants in inhibiting microbial growth and exerting oxidative stability in foods. Şimşek, Şimşek, and Kılıç ( 2017 ) also underscored these properties, showing the potential of natural extracts, like those from collard greens, to serve as comprehensive food preservatives. These extracts enhance the safety of meat products and prolong their longevity. 3.3 Physicochemical properties (pH stability over 28 days) Throughout the 28-day storage period, all meatball formulations exhibited a consistent downward trend in pH, indicative of ongoing acidification (Fig. 3 ). The initial measurements positioned the pH values near neutrality, reflecting a uniform starting quality among the samples. By the seventh day, we observed minor yet discernible decreases in pH across most samples, except for the control group, which remained relatively stable. The disparity in pH trajectories became more evident by day 14. Samples treated with CGE demonstrated significant reductions ( P < 0.05), with pH levels diminishing to approximately 5.80 ± 0.01 for the 500 mg/kg concentration and 6.00 ± 0.01 for the 325 mg/kg concentration. In comparison, while also showing declines, the GTE groups maintained slightly higher pH levels, underscoring a less aggressive acidification pathway. Continuing through day 21, all formulations showed further pH reductions, with the CGE-500 mg/kg treatment reaching a pH of 5.60 ± 0.01, illustrating the most pronounced decline among the treatments. This continued decline in pH suggests ongoing biochemical reactions within the meatballs, possibly linked to bioactive compounds in the natural extracts, microbial metabolism, or enzymatic activities associated with the extracts and meat. By the conclusion of the study on day 28, the downward trend persisted, culminating in the lowest pH values recorded for the CGE treatments at 5.48 ± 0.01 and 5.51 ± 0.01, respectively. These values represent a significant modulation of the acidification process by the natural extracts compared to the control, which concluded the period with a pH of 5.90 ± 0.03. This result highlights the effectiveness of the extracts not only in influencing pH reduction and potentially extending the microbial stability of the meat products. The observed decline in pH across all samples reflects the biochemical and microbial processes affecting the stability of the meatballs, a phenomenon similarly reported by various studies. The more pronounced pH reduction in samples treated with higher concentrations of CGE and GTE suggests that these extracts might influence the acidification process, possibly through their antimicrobial properties that affect microbial metabolism (Nor et al., 2010 ). Research has shown that plant extracts, including those from collard greens and green tea, can significantly impact the pH of meat products. For example, Horbańczuk (2019) discussed how natural antioxidants from plant extracts, like green tea, can alter the physicochemical properties of meat, including pH levels. The acidification process facilitated by CGE and GTE can be attributed to compounds such as glucosinolates and their hydrolysis products (isothiocyanates and indoles), which possess antimicrobial properties. These compounds disrupt microbial membranes and interfere with microbial enzyme systems, inhibiting microbial growth and reducing pH. This dual functionality curtails microbial proliferation and contributes to the overall stability and safety of meat products. Such findings are consistent with those reported by Zhou et al. ( 2023 ), Falowo et al. (2016), Shawky et al. ( 2018 ), and Jałosińska and Wilczak ( 2009 ), where the addition of plant extracts lowered the pH of meat products compared to control. The potential of natural extracts, like those from collard greens, to serve as comprehensive food preservatives is underscored by their ability to modulate pH and inhibit microbial growth, thus enhancing the safety and longevity of meat products. 3.4 Cooking yield The cooking yield of meatball samples treated with varying concentrations of CGE and GTE was analyzed, as depicted in the corresponding bar graph (Fig. 4 ). The results illustrate statistically significant differences in cooking yields among the formulations (ANOVA, F-Statistic: 1645.415, P-Value: 4.815 x 10 14 ). The control samples consistently demonstrated lower cooking yields than all treated samples. Specifically, the cooking yield was markedly higher in samples treated with 325 and 500 mg/kg of CGE, with increases of approximately 14.48 and 12.52%, respectively, compared to the control. Similarly, GTE treatments at 325 and 500 mg/kg increased yields by 6.80 and 8.85%, respectively. Tukey's HSD test further confirmed the significant disparities between the control and treated groups and among different concentrations of CGE and GTE. For instance, CGE-325 mg/kg showed statistically significant higher yields than CGE-500 mg/kg, and also when compared to both concentrations of GTE. Conversely, GTE-500 mg/kg, despite its lower yield compared to CGE-325 mg/kg, significantly outperformed GTE-325 mg/kg. These findings suggest that adding CGE and GTE contributes positively to the cooking yield of meatballs, enhancing moisture retention compared to the control samples. The increase in cooking yields is associated with the addition of natural extracts corroborates with the research by Stojanović‑Radić et al. (2018), Kilic et al. ( 2021 ), and Zheng et al. ( 2023 ), highlighting that natural plant extracts can enhance water retention, which is pivotal for increasing the cooking yield and decreases the purge loss, leading to better meat tenderness, juiciness and palatability of cooked meats. This functional benefit improves sensory acceptance and aligns with consumer preferences towards healthier and 'cleaner' label meat products. 3.5 Textural properties As shown in Fig. 5 , hardness values varied significantly among the groups (ANOVA F-statistic: 5.530, P-Value: 0.013), with the control exhibiting the lowest average hardness at 0.11 N ± 0.02. In contrast, meatballs treated with 500 mg/kg of CGE and GTE showed the highest hardness, averaging 0.17 N ± 0.02, suggesting that higher concentrations of these extracts effectively increased the firmness of the meatballs. Furthermore, the cohesiveness of the meatballs, indicating their ability to withstand deformation without rupturing, showed no significant differences across treatments (ANOVA F-statistic: 0.958, P-Value: 0.471), with values narrowly ranging from 0.025 ± 0.01 in the control group to 0.037 ± 0.01 in the highest CGE and GTE concentrations. This consistency implies that despite the hardened texture, the structural integrity and cohesiveness of the meatballs are maintained across all formulations. These findings highlight that while CGE and GTE can significantly enhance the hardness of meatballs, potentially influencing consumer perceptions of textural quality, they do not compromise cohesiveness, ensuring the meatballs remain integral during consumption. This balance is crucial for maintaining consumer satisfaction and suggests that these extracts can effectively modify texture without adversely affecting product integrity. The findings related to texture, where higher concentrations of extracts were associated with increased hardness without affecting cohesiveness, suggest that CGE and GTE can effectively tailor the textural properties of meat products. This aspect is crucial for consumer satisfaction, as texture directly influences the perceived quality and enjoyment of meat products (Mahfuz, Shang, & Piao, 2021 ). These results are consistent with those of Zheng et al. ( 2023 ), in which the textural properties of refrigerated chicken meat were improved after incorporating natural plant extracts from Oregano. The polysaccharides and fibers in collard greens could potentially contribute to this enhancement effect. 3.6 Sensory properties The sensory evaluation results showed significant differences among the samples for various attributes (Table 2 ). The appearance scores for CGE-500 mg/kg were significantly higher than the control and CGE-325 mg/kg ( P < 0.05). Similarly, color and taste scores for CGE-500 mg/kg were significantly higher than the other treatments ( P < 0.05). The aroma and texture scores also exhibited significant differences, with CGE-500 mg/kg outperforming CGE-325 mg/kg ( P < 0.05). The general acceptance scores for all samples were above the threshold of 5, indicating a positive reception across all tested formulations. Overall, the CGE-500 mg/kg treatment significantly improved the sensory attributes of the meatballs, enhancing their overall acceptability ( P < 0.05). The favorable sensory ratings across all treated samples, particularly at higher extract concentrations, reflect consumer acceptance and the potential of these extracts to enhance flavor profiles without compromising natural taste. As Reddy Maheswara D, Bhaskar Reddy, and Mandal (2018) noted, integrating natural extracts into meat products can subtly enhance inherent flavors while masking any undesirable notes associated with meat oxidation. Several studies have reported that natural plant extracts can improve the overall sensory acceptability of meat products (Noori et al., 2018 ; Kilic et al., 2021 ; Kim, 2020 ). The improvement in the overall sensory acceptability of meatballs with CGE can be attributed to their antioxidant properties, flavor enhancement, texture improvement, added nutritional value, and the positive perception of natural ingredients. These combined effects make the meatballs appeal to consumers in taste and health benefits. Table 2 Mean of hedonic scales for panelist's sensory acceptability of meatball samples incorporated with CGE. Sensory attributes Sample Appearance Aroma Taste Flavor Texture Overall Acceptability CC 7.05 ± 0.02 6.92 ± 0.02 7.25 ± 0.01 7.01 ± 0.02 7.12 ± 0.02 7.38 ± 0.01 CGE–325 mg/kg 7.14 ± 0.01 7.14 ± 0.01 7.11 ± 0.01 6.83 ± 0.01 6.84 ± 0.01 7.11 ± 0.01 CGE–325 mg/kg 7.24 ± 0.01 6.91 ± 0.01 7.72 ± 0.01 7.25 ± 0.01 7.14 ± 0.01 7.80 ± 0.01 F- Statistic 11.53 11.47 36.47 20.88 23.04 93.77 P- Value 0.007 0.007 0.0001 0.001 0.001 0.00001 Results display mean values ± standard deviations ( n = 3) values. CC—control, CGE—collard greens extract 4 Conclusion This study reveals the potential of CGE in meatball formulations. These natural antioxidants significantly boosted oxidative stability, improved physicochemical properties, enhanced cooking yield, and maintained texture. They also improved sensory attributes and microbiological safety. By replacing synthetic additives, CGE promises the potential to cater to the rising consumer demand for healthier and more sustainable food options. The findings encourage further exploration into their use across various meat products. Embracing these natural preservatives promises to improve food quality and aligns with broader health and sustainability objectives. Declarations Funding declaration This research was made possible due to the financial support the author received in the form of an IU-ISDB grant from the Islamic Development Bank. Acknowledgements The authors acknowledge the School of Food Science, Nutrition and Bioengineering of Makerere University (Uganda) and the Department of Food Science and Nutrition of the Islamic University in Uganda for testing facilities and support. Author contributions Lubowa Muhammad: Writing - Review & Editing, Validation, Formal analysis, Investigation, Methodology, Data Curation, Conceptualization, Visualization, Resources, Funding acquisition, Project administration. Nalweyiso Lailah: Writing - original draft, Formal analysis, Investigation, Methodology, Data Curation. Shin-Yong Yeoh: Writing - Review & Editing, Validation, Formal analysis, Methodology, Data Curation, Visualization. Muwonge Abubakar: Writing - Review & Editing. Mubajje Muhammad Shaban: Writing - Review & Editing. Competing Interest declaration. The authors declare that they have no competing interests. Data availability statement Data sharing is not applicable to the main text. The data supporting the findings of this study are available on request from the corresponding authors. Ethics approval and consent to participate The study was conducted in accordance with the University human ethics committee of the Islamic University in Uganda. 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Effect of natural plant extracts on the quality of meat products: A meta-analysis. Food Materials Research, 3, 15. https://doi.org/10.48130/FMR-2023-0015 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4672436","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":332480768,"identity":"70ad84e2-60b5-4a0f-8cc4-0286bae24835","order_by":0,"name":"Lubowa Muhammad","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYDACdjBpwWDAwHwAyJCQIayFGUQkSAC1sCWAtPCQooXHAMQkrMWcmfnZg48/JBK385/5/OpGjQUPA/vhoxvwabFsZjM3nJEgkbhzRu4265xjQIfxpKXdwKfF4DCDmTQPUMuGG7zbjHPYgFokeMwIaGH/Jv0HpOX8mWfGOf+I0sJjJs0A0nIgh/lxbhsRWiybecoke9IkjHfOSDNjzu2T4GEj5Bdz9vZtEj9sbGS38x9+/DnnW50cP/vhY/gdhsRmkwCT+JSja2H+QEj1KBgFo2AUjEwAABxeQvXSKUXQAAAAAElFTkSuQmCC","orcid":"","institution":"Islamic University in Uganda","correspondingAuthor":true,"prefix":"","firstName":"Lubowa","middleName":"","lastName":"Muhammad","suffix":""},{"id":332480769,"identity":"92ed1511-daef-4306-8486-e2350cd78596","order_by":1,"name":"Nalweyiso Lailah","email":"","orcid":"","institution":"Islamic University in Uganda","correspondingAuthor":false,"prefix":"","firstName":"Nalweyiso","middleName":"","lastName":"Lailah","suffix":""},{"id":332480772,"identity":"4f584e99-8f08-4a0f-a75b-f1853d2d5e4d","order_by":2,"name":"Shin-Yong Yeoh","email":"","orcid":"","institution":"Universiti Sains Malaysia, USM","correspondingAuthor":false,"prefix":"","firstName":"Shin-Yong","middleName":"","lastName":"Yeoh","suffix":""},{"id":332480773,"identity":"d1eb4151-73a0-461d-a322-7693617105f0","order_by":3,"name":"Muwonge Abubakar","email":"","orcid":"","institution":"Islamic University in Uganda","correspondingAuthor":false,"prefix":"","firstName":"Muwonge","middleName":"","lastName":"Abubakar","suffix":""},{"id":332480776,"identity":"12bdd8bc-56d9-4027-894e-e0deb381b87c","order_by":4,"name":"Mubajje Muhammad Shaban","email":"","orcid":"","institution":"Islamic University in Uganda","correspondingAuthor":false,"prefix":"","firstName":"Mubajje","middleName":"Muhammad","lastName":"Shaban","suffix":""}],"badges":[],"createdAt":"2024-07-02 07:57:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4672436/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4672436/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61411312,"identity":"f8c413be-91ad-4db1-b1cf-e444be1e0386","added_by":"auto","created_at":"2024-07-30 12:02:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":97765,"visible":true,"origin":"","legend":"\u003cp\u003eLipid oxidation (TBARS values) in meatball samples over 28 days of refrigerated storage. Error bars represent standard deviations (\u003cem\u003en \u003c/em\u003e= 3). Legends: CC—control, CGE—collard greens extract, GTE—green tea extract.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4672436/v1/c948f5371a8c5f465914015c.png"},{"id":61411302,"identity":"1a8936ac-bdb5-4ac8-ad5f-67b257827449","added_by":"auto","created_at":"2024-07-30 12:02:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":100801,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicrobial growth trends in meatball samples over 28 Days \u003c/strong\u003epresented in log CFU/ml. Error bars indicate standard deviation (\u003cem\u003en \u003c/em\u003e= 3). Legends: CC—control, CGE—collard greens extract, GTE—green tea extract.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4672436/v1/18b71b748646e547ed624513.png"},{"id":61411304,"identity":"ef240fb6-e8ba-4a18-9215-4d718220e356","added_by":"auto","created_at":"2024-07-30 12:02:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":102654,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003epH Changes in meatball samples over 28 days. \u003c/strong\u003eError bars indicate standard deviation (\u003cem\u003en\u003c/em\u003e = 3). Legends: CC—control, CGE—collard greens extract, GTE—green tea extract.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4672436/v1/3eac2c62b3e2ce4721fb4cec.png"},{"id":61411401,"identity":"0eb8756b-a097-4ac4-8c61-a84d31c2cc25","added_by":"auto","created_at":"2024-07-30 12:02:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":46953,"visible":true,"origin":"","legend":"\u003cp\u003eComparative cooking yields of meatball samples with natural extracts. Bar graph showing cooking yields for meatballs treated with plant extracts. Legends: CC—control, CGE—collard greens extract, GTE—green tea extract. Error bars indicate standard deviation (\u003cem\u003en \u003c/em\u003e= 3), and distinct letters denote significant differences identified by Tukey's HSD test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4672436/v1/5ed582255712811dea8b4d28.png"},{"id":61411303,"identity":"7cc0750f-63af-42d3-8887-de87c20a9851","added_by":"auto","created_at":"2024-07-30 12:02:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":127847,"visible":true,"origin":"","legend":"\u003cp\u003eTextural properties (hardness and cohesiveness) of the formulated meatball samples. Error bars denote standard deviation, illustrating variability within each measurement. Legends: CC—control, CGE—collard greens extract, GTE—green tea extract\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4672436/v1/8ab09f022c9f82a6e8a94bb6.png"},{"id":66736738,"identity":"d1580163-c6ba-47db-9447-a179d76b2e79","added_by":"auto","created_at":"2024-10-16 05:01:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1043308,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4672436/v1/1dd501d7-8849-44b3-983a-bfc3cdb59f6f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Natural antioxidants from collard greens (Brassica oleracae var. acephala): effects on the storage stability and quality of meatballs","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eThe quest for food quality and safety is a cornerstone of food science research and industry practices. In today's world, where global meat consumption is on the rise due to demographic shifts, economic growth, and changing dietary preferences, the meat industry faces the formidable task of meeting consumer demands for high-quality, safe, and stable products. One of the critical challenges in the meat industry is lipid oxidation. This process significantly degrades the sensory qualities, nutritional value, and shelf life of meat, leading to substantial economic losses and posing health risks through harmful oxidation byproducts (Dom\u0026iacute;nguez et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Dave \u0026amp; Ghaly, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHistorically, the meat processing industry has depended on synthetic antioxidants to combat the adverse effects of oxidation (Mani‑L\u0026oacute;pez et al., 2016; Rodil et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, growing consumer awareness about synthetic additives' safety and health implications has driven demand for natural alternatives (Feknous et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Olvera-Aguirre et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This shift reflects a broader societal movement towards more sustainable and health-conscious food production practices (Herrero et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; FAO, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSynthetic antioxidants and natural alternatives differ markedly in terms of safety and efficacy (Fadhil et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). While synthetic antioxidants are effective, their effectiveness and convenience come at a price. There's a growing debate regarding their safety and potential health implications (Thakur et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yu et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Long-term intake has been linked to skin allergies, gastrointestinal issues, increased cancer risk, DNA damage, and premature aging (Muthukumar et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Xie et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Błaszczyk et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Shahidi \u0026amp; Zhong, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Compounds, such as sulfites, BHA, and BHT, have raised concerns due to their allergenic or carcinogenic properties, while others, like nitrates and nitrites, can form potentially harmful nitrosamines when exposed to high heat (Muthukumar et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Xie et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). As a result, consumer apprehensions regarding consuming chemically treated food have led to a shift away from these compounds. Conversely, natural antioxidants are generally considered safe and increasingly preferred by consumers seeking clean-label products. Natural antioxidants, such as tocopherols, ascorbic acid, and rosemary extract, offer nutritional value, safety, and stability in food products (Feknous et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Guti\u0026eacute;rrez-Del-R\u0026iacute;o et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Brewer, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Pokorn\u0026yacute;, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). These compounds, rich in phenolics, vitamins, and carotenoids, not only act as antioxidants but also possess antimicrobial properties and influence food flavors and textures. Thus, choosing between synthetic and natural antioxidants hinges on safety, efficacy, consumer preferences, and regulatory guidelines.\u003c/p\u003e \u003cp\u003eCollard greens (\u003cem\u003eBrassica oleracea var. acephala\u003c/em\u003e) stand out among the natural resources explored. This leafy vegetable, indigenous to East Africa, is rich in bioactive compounds like antioxidants, flavonoids, glucosinolates, and phenolics (Kuete, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Phytonutrients in collard greens include phenols like caffeic and ferulic acid, flavonoids like quercetin and kaempferol, and glucosinolates like glucobrassicin and glucoraphanin as well as hydroxycinnamic acids (Velasco et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Olsen, Aaby, \u0026amp; Borge, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Kaempferol glycosides, acylgentiobiosides, and quercetin glycosides are the major phenolic antioxidants present, while derivatives of p-coumaric, ferulic, sinapic, and caffeic acid are some of the minor phytochemicals present (Lin \u0026amp; Harnly, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Olsen, Aaby, \u0026amp; Borge, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Picchi et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Other phytonutrients in collard greens include indole-3-carbinol, diindolylmethane, sulforaphane, isothiocyanates, and carotenoids. Renowned for their culinary versatility and health benefits, collard greens present a promising solution to the meat industry's oxidation challenge (Kahlon et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Kuete, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite the known advantages of collard greens and other natural antioxidants, a significant gap exists in their application for meat preservation. Specifically, there is a lack of comprehensive studies exploring the efficacy of CGE in enhancing meat quality and storage stability without compromising sensory attributes. This gap highlights the need for research that not only substantiates the antioxidant capabilities of CGE but also evaluates their impact on the physicochemical, textural, and sensory characteristics of meat products.\u003c/p\u003e \u003cp\u003eBuilding on existing research, this study aims to deepen our understanding of CGE's effects on meat quality and storage stability. By incorporating these extracts into meatballs\u0026mdash;a widely consumed and commercially significant product\u0026mdash;the research offers valuable insights into natural food preservatives and addresses practical food technology and product development challenges. By comparing the antioxidant effects of CGE with GTE, known for their antioxidant properties (Falla et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Luo et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Senanayake, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Carrizo et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), this study assesses the impact of these natural antioxidants on the physicochemical, textural, and sensory attributes of meatballs over a specified storage period. Moreover, this research explores the broader implications of integrating bioactive compounds from plants like collard greens into the food industry. It highlights the shift towards functional foods that provide health benefits beyond essential nutrition, aligning with contemporary dietary trends and consumer expectations. In doing so, the study addresses immediate issues of meat product quality and safety and contributes to the evolving discussion on the role of natural antioxidants in the food industry.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eAll chemicals and reagents used were of analytical grade. Water used in all experiments was purified using a Milli-Q water purification system. Methanol (80% v/v) and butanol were utilized for the extraction processes and determination of total phenolic content and total antioxidant capacity, respectively. Sodium carbonate and Folin-Ciocalteu phenol reagent were specifically employed to determine total phenolic content, following a slightly modified method from Andressa Blainski et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). For the total antioxidant capacity (TAC) assay, DPPH (2,2-diphenyl-1-picrylhydrazyl) was used to evaluate the antioxidant activity of the extracts. Thiobarbituric acid, used to determine thiobarbituric acid reactive substances (TBARS), was sourced and prepared according to Pokorny (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1989\u003c/span\u003e), with butanol as the solvent. These substances were pivotal in assessing the physicochemical properties and antioxidant activity of the meatball samples incorporated with CGE and GTE.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation of plant extracts\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 CGE\u003c/h2\u003e \u003cp\u003eCollard greens were acquired from a local farmer in Uganda and transported (cooling boxes to avoid transpiration and respiration) to the Makerere University School of Food Technology for processing, which involved washing, trimming, and size reduction. Approximately 14.5 kg of the prepared collard greens leaves were dried using a solar dryer for seven days, with the leaves turned thrice daily. The dried sample was ground into powder and subjected to water extraction. During extraction, 14 g of powder was combined with 100 mL of water and extracted at 85\u0026deg;C for 30 min in a water bath, with occasional stirring, as described by Reihani et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The mixtures were filtered, and the filtrates were dried and ground into powder. The extracts were stored in zip-lock plastic bags in a freezer until needed for the meatball formulation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 GTE\u003c/h2\u003e \u003cp\u003eGreen tea, specifically the So Fine China brand, was purchased from Senana Supermarket, Kampala. The green tea leaves were ground to a fine powder using an electric power grinder (Model DE-100g, China). For the extraction, 14 g of the green tea powder was mixed with 100 mL of water and subjected to heat in a water bath at 85\u0026deg;C for 30 min. The mixture was then filtered through a cheesecloth to separate the residue from the filtrate. The filtrate was placed in silver plates and dried overnight in an oven (Gallenkamp, UK, Model: OHF09.XX1.5) at a temperature setting of 80\u0026deg;C. The dried filtrate was ground into powder using the same grinder and stored in zip-lock plastic bags in a freezer until further use.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Preparation of meatballs\u003c/h2\u003e \u003cp\u003eThe meatballs were formulated using minced beef as the primary ingredient. The beef was obtained from a local butcher to ensure freshness and quality. The experimental treatments used previously prepared CGE and GTE (Section \u003cspan refid=\"Sec4\" class=\"InternalRef\"\u003e2.2\u003c/span\u003e). To enhance the meatball mixture, onions and fat were finely chopped and melted, respectively. Eggs were whisked to aid in binding the mixture, while breadcrumbs, prepared from dried and ground bread, garlic powder, and salt, were added for flavor and textural purposes. The meatball mixture was evenly divided to incorporate the different extract concentrations, with 500 and 325 mg/kg for the CGE, alongside a comparable green tea extract concentration serving as a positive control. A batch without any extracts acted as the negative control. Each portion of the mixture was carefully shaped into balls weighing approximately 100 g, packaged in airtight zip-lock plastic bags, and labeled according to their specific formulations for further analyses, including lipid oxidation and pH measurement. These samples were stored under refrigeration temperatures. Samples for sensory evaluation were placed on baking sheets. The meatballs were then cooked in a preheated oven at 190\u0026deg;C for 25 min until they reached a safe internal temperature and acquired a golden-brown crust. Following baking, the meatballs were cooled to room temperature, packaged in airtight zip-lock plastic bags, and labeled according to their specific formulations, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e:\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eQuantity of meat and extracts used in the meatball formulations.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSamples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMinced beef (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eQuantity of extract (mg)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCGE-500 mg/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCGE-325 mg/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e325\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGTE-500 mg/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGTE-325 mg/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e325\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eCC - Negative control doesn't have any extract; CGE - Contains collard greens extract; GTE - Positive control contains the green tea extract. The values attached stand for the quantities of extract.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Measurement of lipid oxidation\u003c/h2\u003e \u003cp\u003eThe extent of lipid oxidation in meatball samples was quantitatively determined using the TBARS assay, a widely recognized method for assessing secondary oxidation products, primarily malondialdehyde (MDA), which indicates lipid peroxidation. To prepare the samples for analysis, approximately 2 g of meatball from each formulation was homogenized with 15 mL of distilled water. The homogenate was then mixed with 2 mL of a 20% trichloroacetic acid solution to precipitate proteins, followed by centrifugation at 3000 rpm for 10 min to clarify the solution. The supernatant was subjected to the TBARS assay by adding 2 mL of the supernatant to 2 mL of a 0.02 M thiobarbituric acid solution in a test tube, which was then heated in a boiling water bath for 30 min to allow the formation of the TBA-MDA complex, characterized by its pink color. After cooling to room temperature, the absorbance of the solution was measured at 532 nm using a spectrophotometer. The TBAR values were calculated using a standard curve of 1,1,3,3-tetraethoxypropane, expressed in milligrams of MDA per kg sample. This measurement was conducted on meatballs stored under refrigeration for 0, 7, 14, 21, and 28 days to evaluate the oxidative stability of the meatballs over time. The measurements were done in triplicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Cooking yield\u003c/h2\u003e \u003cp\u003eThe cooking yield of the meatball samples was determined as a measure of weight loss during cooking, which indicates moisture retention and fat content changes. These factors significantly affect the final product quality. For this purpose, each meatball formulation was weighed before and after baking in the oven at 190\u0026deg;C for 25 min. The initial weight (W1) was recorded immediately before cooking, and the final weight (W2) was noted after the meatballs had cooled to room temperature, ensuring consistency in measurement. The cooking yield was calculated using the formula: Cooking yield (%) = (W2/W1) \u0026times; 100%. Cooking yield measurements were taken for all experimental groups to assess the impact of the natural extracts on the cooking performance of the meat products. Measurements were done in triplicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Physicochemical properties (pH)\u003c/h2\u003e \u003cp\u003eFor pH determination, a representative sample of approximately 10 g from each meatball formulation was homogenized with 50 mL of distilled water using a laboratory blender to ensure thorough mixing. After homogenization, the slurry was allowed to settle for 5 min, and the pH was measured directly in the supernatant using a calibrated pH meter (Model INE-PHS-3E, MRC, Israel). The electrode was carefully rinsed with distilled water between measurements to prevent cross-contamination. The procedure was performed in triplicate for each sample, and the results were averaged to obtain the final pH value for each formulation. Measurements were conducted on samples immediately after cooking and after 7, 14, 21, and 28 days of refrigerated storage to evaluate changes in pH over time.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Texture profile analysis (TPA)\u003c/h2\u003e \u003cp\u003eTPA was performed using a TA.XTplus Texture Analyzer (Stable Micro Systems, UK) equipped with a 50 mm cylindrical probe and a 5 kg load cell. Before analysis, meatball samples were sectioned into uniform cylindrical pieces of approximately 20 mm height, ensuring consistency across all measurements. The textural attributes measured included hardness and cohesiveness, which are critical determinants of the sensory quality of meat products. The TPA procedure involved a double compression test, where each sample was compressed to 50% of its original height at specified speeds: a pre-test speed of 1.0 mm/s, a test speed of 1.7 mm/s, and a post-test speed of 10.0 mm/s. The interval between the first and second compression cycles was fixed at 5 s, mimicking the biting action to evaluate the sample's textural behavior comprehensively. Hardness was quantified as the peak force encountered during the first compression cycle, representing the sample's resistance to deformation. Cohesiveness, a measure of the sample's ability to withstand a second deformation relative to its initial structure, was calculated as the ratio of the area under the second compression curve to that under the first. Measurements were conducted in triplicate for each formulation, and the results were processed and analyzed using Texture Exponent software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Analysis of microbiological stability\u003c/h2\u003e \u003cp\u003eIn this study, an in vitro antimicrobial assay was performed using the plate count method to determine the total number of aerobic microorganisms in control samples and samples incorporated with CGE and GTE. Microbial counts were performed at 7-day intervals over the 28-day storage period (4 weeks), following the method described by Jałosińska and Wilczak (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). At each interval, 5 g of each sample was weighed and placed into a Stomacher bag with 45 mL of sterile peptone water (Biocar Diagnostics) added for dilution. The samples were homogenized for 2 min at a standard speed using a Stomacher 400 apparatus. A series of tenfold dilutions were prepared from the homogenized samples, and 1 mL from each of the three consecutive dilutions was inoculated onto nutrient agar plates (Biocar Diagnostics) in triplicate. The inoculated plates were incubated at 37\u0026deg;C for 48 h, and colonies were counted to determine the total number of aerobic microorganisms. The antimicrobial effectiveness of the CGE and GTE was assessed by comparing the microbial counts of the treated samples with those of the control samples over the storage period.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Sensory evaluation\u003c/h2\u003e \u003cp\u003eSensory evaluation was performed only on the samples incorporated with CGE and the control. A panel of 50 trained sensory evaluators from the Department of Food Science and Technology at Makerere University participated in the assessment. The panelists were selected based on their sensory analysis experience and ability to discern subtle differences in food samples. The meatballs were served at a controlled temperature of approximately 37\u0026deg;C to mimic typical consumption conditions. Each panelist was given samples coded with random three-digit numbers to prevent bias. Water and unsalted crackers were available to cleanse the palate between samples. The evaluation was structured around a 9-point hedonic scale, where 1 represented \"dislike extremely\" and 9 signified \"like extremely.\" Panelists were asked to rate each sample based on the aforementioned sensory attributes. The scores for each attribute were then averaged to provide a composite score for each meatball formulation. To ensure the reliability of the sensory data, the evaluation session was conducted in a well-lit, odor-free room with individual booths for each panelist to prevent discussion and influence among participants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Statistical analysis\u003c/h2\u003e \u003cp\u003eAnalysis of variance (ANOVA) and Tukey's Honestly Significant Difference (HSD) were used to analyze the data. All statistical analyses were performed using SPSS Statistics software (Version 26, IBM Corp). Figures were generated using Python's matplotlib library (Version 3.3.2) to visually represent the data and highlight significant findings from the statistical analyses.\u003c/p\u003e \u003cp\u003eMicrobiological count data, presented as colony-forming units per milliliter (cfu/mL), were log-transformed before analysis to stabilize variances and improve the approximation to a normal distribution. The statistical significance level was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for all tests.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results and discussion","content":"\u003cp\u003eThe current study provides insightful contributions to the burgeoning field of natural food preservatives, particularly focusing on the impact of CGE and GTE on the quality and stability of meatballs stored under refrigeration. The results encompass various critical aspects, including lipid oxidation, physicochemical properties, cooking yield, sensory attributes, texture, and microbiological stability, offering a comprehensive overview of the efficacy of natural extracts as meat preservatives.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Lipid oxidation stability\u003c/h2\u003e \u003cp\u003eThe TBARS assay, employed to assess lipid oxidation stability in meatball samples stored over 28 days under refrigerated conditions, revealed distinct trends in oxidative stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Throughout the storage period, all samples demonstrated an increasing trend in TBARS values, indicative of progressive lipid peroxidation. However, the rate of increase varied significantly between different treatments. The ANOVA results highlight a marginally significant effect of treatment type on TBARS values (p\u0026thinsp;=\u0026thinsp;0.053), suggesting that the type of extract used\u0026mdash;whether CGE at 325 mg/kg and 500 mg/kg or GTE at the same concentrations\u0026mdash;may influence lipid oxidation, though modestly. The most profound effect observed was time (p\u0026thinsp;=\u0026thinsp;0.0006), confirming that lipid oxidation naturally escalated over the 28 days. Moreover, the interaction between sample types and time was highly significant (p\u0026thinsp;=\u0026thinsp;0.0001), indicating that the efficacy of different treatments in inhibiting lipid oxidation varied at various storage times. Notably, samples treated with CGE-500 mg/kg and GTE-500 mg/kg generally displayed slower rates of increase in TBARS values, suggesting these higher concentrations are more effective at delaying lipid peroxidation. This is evident in the mid-storage data, where these treatments maintained lower TBARS values than their lower concentration counterparts and the control. By the end of the 28 days, however, all samples reached higher TBARS levels, with those treated with extracts consistently showing marginally better oxidative stability than the control.\u003c/p\u003e \u003cp\u003eThe application of CGE and GTE significantly reduced lipid oxidation in meatballs, clear from the TBARS values observed over the 28-day storage period. Collard greens contain high levels of natural antioxidants such as flavonoids, phenolic acids, and vitamins (e.g., vitamins C and E) (Picchi et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Villalobos-Delgado et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mir, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Olsen, Aaby, \u0026amp; Borge, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). These antioxidants can slow lipid oxidation in meatballs, preventing rancidity and off-flavors common in meat products. Specific compounds in CGE, such as kaempferol and quercetin, are likely behind this antioxidative effect. These flavonoids are renowned for scavenging free radicals and chelate metal ions, effectively halting the oxidation processes that lead to lipid degradation (Villalobos-Delgado et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The presence of these compounds in CGE aligns with various research findings, which have identified kaempferol and quercetin in collard greens as powerful antioxidants (Picchi et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mir, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Olsen, Aaby \u0026amp; Borge, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). These compounds contribute significantly to oxidative stability in various food matrices. For instance, Liu (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) discussed the cellular antioxidant activity of common vegetables, highlighting the crucial roles of quercetin and kaempferol. Similarly, Ağag\u0026uuml;nd\u0026uuml;z et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reviewed the antioxidant activity of cruciferous vegetables, including collard greens, noting their considerable impact on lipid oxidation stability. Furthermore, a comprehensive review of the nutritional composition and bio-active compounds of kale, a close relative of collard greens, detailed the antioxidative properties of kaempferol and quercetin. This review emphasized their role in enhancing the shelf-life and safety of food products by minimizing the formation of potentially harmful oxidation products (Satheesh, Fanta, \u0026amp; Yildiz, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This antioxidant activity is crucial not just for extending the shelf-life of meat products but also for improving their safety by reducing the formation of harmful oxidation products.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Microbiological stability of meatball samples\u003c/h2\u003e \u003cp\u003eThe microbiological stability of meatballs incorporated with CGE and GTE was assessed through microbial count analysis over a four-week storage period, with values expressed in a logarithmic scale (log CFU/mL). Initial results showed relatively low microbial counts for all samples, indicating good baseline product hygiene, with the GTE-325 mg/kg treatment demonstrating the most potent initial antimicrobial effect, yielding the lowest counts, approximately at 5.37 log CFU/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). By the seventh day, microbial counts were increased across all groups, consistent with expected microbial growth under refrigeration. However, samples treated with higher concentrations of extracts, specifically CGE-500 mg/kg and GTE-500 mg/kg, exhibited notably lower counts, around 6.80 log CFU/mL, compared to control samples, which reached about 7.75 log CFU/mL. This trend persisted through day 14 and day 21, with treated samples continuing to show slower microbial growth, affirming the sustained antimicrobial properties of the extracts. By day 28, control samples had the highest microbial counts exceeding 10.00 log CFU/mL. In contrast, the extract-treated samples, particularly those with higher concentrations, maintained significantly lower levels, approximately 9.00 log CFU/mL. These observations highlight the effectiveness of CGE and GTE in inhibiting microbial growth, potentially enhancing the safety and extending the shelf-life of meat products.\u003c/p\u003e \u003cp\u003eThe antimicrobial efficacy of the extracts, evidenced by the slower microbial growth in treated meatballs, highlights their potential to enhance food safety and extend the microbial shelf-life of meat products. Polyphenol compounds and glucosinolates and their hydrolysis products (isothiocyanates and indoles) found in CGE are likely behind these antimicrobial properties (Bhat \u0026amp; Al-Daihan, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Mourtzinos et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Various studies have identified these compounds as potent antimicrobial agents that inhibit the growth of pathogenic bacteria (Mourtzinos et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). They achieve this probably through mechanisms like disrupting microbial membranes and interfering with microbial enzyme systems (Ahmad, Hassan \u0026amp; Azim, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This capability not only curtails microbial proliferation but also contributes to the oxidative stability of the product, showcasing the dual functionality of these natural compounds. Horbańczuk et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and Nur et al. (2021) highlighted the antimicrobial activities of plant extracts in meat products, emphasizing the efficacy of natural compounds in inhibiting microbial growth. Similarly, Kumar et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) underscored the role of glucosinolates and their hydrolysis products in providing antimicrobial activity. Additionally, Wali et al. (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) confirmed the significant antimicrobial properties of these extracts, particularly against Gram-negative bacteria. The ability of these extracts to curb microbial proliferation while enhancing oxidative stability demonstrates their dual functionality. This aligns with the findings of Jałosińska and Wilczak (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), who highlighted the role of natural antioxidants in inhibiting microbial growth and exerting oxidative stability in foods. Şimşek, Şimşek, and Kılı\u0026ccedil; (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) also underscored these properties, showing the potential of natural extracts, like those from collard greens, to serve as comprehensive food preservatives. These extracts enhance the safety of meat products and prolong their longevity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Physicochemical properties (pH stability over 28 days)\u003c/h2\u003e \u003cp\u003eThroughout the 28-day storage period, all meatball formulations exhibited a consistent downward trend in pH, indicative of ongoing acidification (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The initial measurements positioned the pH values near neutrality, reflecting a uniform starting quality among the samples. By the seventh day, we observed minor yet discernible decreases in pH across most samples, except for the control group, which remained relatively stable. The disparity in pH trajectories became more evident by day 14. Samples treated with CGE demonstrated significant reductions (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with pH levels diminishing to approximately 5.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 for the 500 mg/kg concentration and 6.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 for the 325 mg/kg concentration. In comparison, while also showing declines, the GTE groups maintained slightly higher pH levels, underscoring a less aggressive acidification pathway. Continuing through day 21, all formulations showed further pH reductions, with the CGE-500 mg/kg treatment reaching a pH of 5.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01, illustrating the most pronounced decline among the treatments. This continued decline in pH suggests ongoing biochemical reactions within the meatballs, possibly linked to bioactive compounds in the natural extracts, microbial metabolism, or enzymatic activities associated with the extracts and meat. By the conclusion of the study on day 28, the downward trend persisted, culminating in the lowest pH values recorded for the CGE treatments at 5.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 and 5.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01, respectively. These values represent a significant modulation of the acidification process by the natural extracts compared to the control, which concluded the period with a pH of 5.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03. This result highlights the effectiveness of the extracts not only in influencing pH reduction and potentially extending the microbial stability of the meat products.\u003c/p\u003e \u003cp\u003eThe observed decline in pH across all samples reflects the biochemical and microbial processes affecting the stability of the meatballs, a phenomenon similarly reported by various studies. The more pronounced pH reduction in samples treated with higher concentrations of CGE and GTE suggests that these extracts might influence the acidification process, possibly through their antimicrobial properties that affect microbial metabolism (Nor et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Research has shown that plant extracts, including those from collard greens and green tea, can significantly impact the pH of meat products. For example, Horbańczuk (2019) discussed how natural antioxidants from plant extracts, like green tea, can alter the physicochemical properties of meat, including pH levels. The acidification process facilitated by CGE and GTE can be attributed to compounds such as glucosinolates and their hydrolysis products (isothiocyanates and indoles), which possess antimicrobial properties. These compounds disrupt microbial membranes and interfere with microbial enzyme systems, inhibiting microbial growth and reducing pH. This dual functionality curtails microbial proliferation and contributes to the overall stability and safety of meat products. Such findings are consistent with those reported by Zhou et al. (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), Falowo et al. (2016), Shawky et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and Jałosińska and Wilczak (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), where the addition of plant extracts lowered the pH of meat products compared to control. The potential of natural extracts, like those from collard greens, to serve as comprehensive food preservatives is underscored by their ability to modulate pH and inhibit microbial growth, thus enhancing the safety and longevity of meat products.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Cooking yield\u003c/h2\u003e \u003cp\u003eThe cooking yield of meatball samples treated with varying concentrations of CGE and GTE was analyzed, as depicted in the corresponding bar graph (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The results illustrate statistically significant differences in cooking yields among the formulations (ANOVA, F-Statistic: 1645.415, P-Value: 4.815 x 10\u003csup\u003e14\u003c/sup\u003e). The control samples consistently demonstrated lower cooking yields than all treated samples. Specifically, the cooking yield was markedly higher in samples treated with 325 and 500 mg/kg of CGE, with increases of approximately 14.48 and 12.52%, respectively, compared to the control. Similarly, GTE treatments at 325 and 500 mg/kg increased yields by 6.80 and 8.85%, respectively. Tukey's HSD test further confirmed the significant disparities between the control and treated groups and among different concentrations of CGE and GTE. For instance, CGE-325 mg/kg showed statistically significant higher yields than CGE-500 mg/kg, and also when compared to both concentrations of GTE. Conversely, GTE-500 mg/kg, despite its lower yield compared to CGE-325 mg/kg, significantly outperformed GTE-325 mg/kg. These findings suggest that adding CGE and GTE contributes positively to the cooking yield of meatballs, enhancing moisture retention compared to the control samples.\u003c/p\u003e \u003cp\u003eThe increase in cooking yields is associated with the addition of natural extracts corroborates with the research by Stojanović‑Radić et al. (2018), Kilic et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and Zheng et al. (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), highlighting that natural plant extracts can enhance water retention, which is pivotal for increasing the cooking yield and decreases the purge loss, leading to better meat tenderness, juiciness and palatability of cooked meats. This functional benefit improves sensory acceptance and aligns with consumer preferences towards healthier and 'cleaner' label meat products.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Textural properties\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, hardness values varied significantly among the groups (ANOVA F-statistic: 5.530, P-Value: 0.013), with the control exhibiting the lowest average hardness at 0.11 N\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02. In contrast, meatballs treated with 500 mg/kg of CGE and GTE showed the highest hardness, averaging 0.17 N\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, suggesting that higher concentrations of these extracts effectively increased the firmness of the meatballs. Furthermore, the cohesiveness of the meatballs, indicating their ability to withstand deformation without rupturing, showed no significant differences across treatments (ANOVA F-statistic: 0.958, P-Value: 0.471), with values narrowly ranging from 0.025\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 in the control group to 0.037\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 in the highest CGE and GTE concentrations. This consistency implies that despite the hardened texture, the structural integrity and cohesiveness of the meatballs are maintained across all formulations. These findings highlight that while CGE and GTE can significantly enhance the hardness of meatballs, potentially influencing consumer perceptions of textural quality, they do not compromise cohesiveness, ensuring the meatballs remain integral during consumption. This balance is crucial for maintaining consumer satisfaction and suggests that these extracts can effectively modify texture without adversely affecting product integrity.\u003c/p\u003e \u003cp\u003eThe findings related to texture, where higher concentrations of extracts were associated with increased hardness without affecting cohesiveness, suggest that CGE and GTE can effectively tailor the textural properties of meat products. This aspect is crucial for consumer satisfaction, as texture directly influences the perceived quality and enjoyment of meat products (Mahfuz, Shang, \u0026amp; Piao, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These results are consistent with those of Zheng et al. (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), in which the textural properties of refrigerated chicken meat were improved after incorporating natural plant extracts from Oregano. The polysaccharides and fibers in collard greens could potentially contribute to this enhancement effect.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Sensory properties\u003c/h2\u003e \u003cp\u003eThe sensory evaluation results showed significant differences among the samples for various attributes (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The appearance scores for CGE-500 mg/kg were significantly higher than the control and CGE-325 mg/kg (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similarly, color and taste scores for CGE-500 mg/kg were significantly higher than the other treatments (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The aroma and texture scores also exhibited significant differences, with CGE-500 mg/kg outperforming CGE-325 mg/kg (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The general acceptance scores for all samples were above the threshold of 5, indicating a positive reception across all tested formulations. Overall, the CGE-500 mg/kg treatment significantly improved the sensory attributes of the meatballs, enhancing their overall acceptability (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThe favorable sensory ratings across all treated samples, particularly at higher extract concentrations, reflect consumer acceptance and the potential of these extracts to enhance flavor profiles without compromising natural taste. As Reddy Maheswara D, Bhaskar Reddy, and Mandal (2018) noted, integrating natural extracts into meat products can subtly enhance inherent flavors while masking any undesirable notes associated with meat oxidation. Several studies have reported that natural plant extracts can improve the overall sensory acceptability of meat products (Noori et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kilic et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kim, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The improvement in the overall sensory acceptability of meatballs with CGE can be attributed to their antioxidant properties, flavor enhancement, texture improvement, added nutritional value, and the positive perception of natural ingredients. These combined effects make the meatballs appeal to consumers in taste and health benefits.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean of hedonic scales for panelist's sensory acceptability of meatball samples incorporated with CGE.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e \u003cp\u003eSensory attributes\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAppearance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAroma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTaste\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFlavor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTexture\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOverall Acceptability\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCGE\u0026ndash;325 mg/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCGE\u0026ndash;325 mg/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF- Statistic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e36.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e23.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e93.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP- Value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.00001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eResults display mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3) values. CC\u0026mdash;control, CGE\u0026mdash;collard greens extract\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Conclusion","content":"\u003cp\u003eThis study reveals the potential of CGE in meatball formulations. These natural antioxidants significantly boosted oxidative stability, improved physicochemical properties, enhanced cooking yield, and maintained texture. They also improved sensory attributes and microbiological safety. By replacing synthetic additives, CGE promises the potential to cater to the rising consumer demand for healthier and more sustainable food options. The findings encourage further exploration into their use across various meat products. Embracing these natural preservatives promises to improve food quality and aligns with broader health and sustainability objectives.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was made possible due to the financial support the author received in the form of an IU-ISDB grant from the Islamic Development Bank.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the School of Food Science, Nutrition and Bioengineering of Makerere University (Uganda) and the Department of Food Science and Nutrition of the Islamic University in Uganda for testing facilities and support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLubowa Muhammad: Writing - Review \u0026amp; Editing, Validation, Formal analysis, Investigation, Methodology, Data Curation, Conceptualization, Visualization, Resources, Funding acquisition, Project administration. Nalweyiso Lailah: Writing - original draft, Formal analysis, Investigation, Methodology, Data Curation. Shin-Yong Yeoh: Writing - Review \u0026amp; Editing, Validation, Formal analysis, Methodology, Data Curation, Visualization. Muwonge Abubakar: Writing - Review \u0026amp; Editing. Mubajje Muhammad Shaban: Writing - Review \u0026amp; Editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest declaration.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData sharing is not applicable to the main text. The data supporting the findings of this study are available on request from the corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted in accordance with the University human ethics committee of the Islamic University in Uganda. Informed consent was obtained from all human participants.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAğag\u0026uuml;nd\u0026uuml;z, D., Şahin, T. \u0026Ouml;., Yılmaz, B., Ekenci, K. D., Duyar \u0026Ouml;zer, Ş., \u0026amp; Capasso, R. (2022). Cruciferous vegetables and their bioactive metabolites: From prevention to novel therapies of colorectal cancer. Evidence-based Complementary and Alternative Medicine, 2022, 1534083. https://doi.org/10.1155/2022/1534083\u003c/li\u003e\n \u003cli\u003eAhmad, Z., Hassan, S. S., \u0026amp; Azim, S. (2017). 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Food Materials Research, 3, 15. https://doi.org/10.48130/FMR-2023-0015\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Natural antioxidants, lipid oxidation, collard greens extract, meat quality, plant extracts, green tea extract","lastPublishedDoi":"10.21203/rs.3.rs-4672436/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4672436/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSynthetic antioxidants have been used to enhance the storage stability of meat by preventing lipid oxidation. However, concerns about their health risks and toxicity have led to increased interest in natural alternatives. Collard greens, known for their health benefits and appealing taste, are rich in bioactive compounds such as antioxidants, fiber, minerals, and vitamins. Their safety and nutritional profile make them ideal for use as preservatives or functional food ingredients in the food industry. This study evaluated the effects of collard greens extract (CGE) on the quality and storage stability of meatballs. Meatballs were formulated with CGE at 500 and 325 mg/kg and compared with those containing green tea extract (GTE) and a control group. The samples were stored under refrigeration, and oxidative stability was assessed over 30 days using TBARS. CGE demonstrated a significant lipid oxidation inhibitory effect comparable to GTE. Meatballs with 500 mg/kg of extract maintained significantly lower TBAR values (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) than control samples over one month, showing up to a 30% reduction in lipid oxidation. The addition of CGE significantly influenced the texture and cooking yields (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) and received positive sensory scores of the meatballs.\u003c/p\u003e","manuscriptTitle":"Natural antioxidants from collard greens (Brassica oleracae var. acephala): effects on the storage stability and quality of meatballs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-30 12:02:27","doi":"10.21203/rs.3.rs-4672436/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a6c08c79-f555-4396-93ea-88811aeed9ca","owner":[],"postedDate":"July 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":35205886,"name":"Biological sciences/Biochemistry"},{"id":35205887,"name":"Biological sciences/Biophysics"}],"tags":[],"updatedAt":"2024-10-16T04:53:38+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-30 12:02:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4672436","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4672436","identity":"rs-4672436","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}