Evaluation of Nisin and Cultured Dextrose as Clean Label Preservatives in Braised Chicken Breast: Antibacterial Activity and Quality Preservation

Abstract

Meat products face microbial safety challenges, while growing consumer demand for “clean label” options discourages the use of synthetic preservatives. Although Nisin and cultured dextrose (CD) are known natural antimicrobials, their combined application in meat systems has not been fully assessed. Herein, we systematically evaluated the antibacterial activity of CD in combination with Nisin against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) using a uniform design. While regression modeling of the in vitro data indicated a potent synergistic interaction at lower concentrations, the optimal practical combination for meat preservation was identified as 0.02% Nisin and 0.5% CD. This combination was applied to braised chicken breast stored at 0–4 °C, with microbial counts, pH, color, and sensory quality monitored over 28 days. The results indicated that this Nisin-CD combination significantly suppressed the growth of total viable bacteria, S. aureus, and E. coli, stabilized pH, minimized color variation, and maintained sensory acceptability. Therefore, the 0.02% Nisin and 0.5% CD combination is recommended as an effective clean-label strategy to extend the shelf life of meat products by enhancing microbial safety and quality without synthetic preservatives.

1. Introduction

In recent years, the “clean label” trend has gained significant momentum in the food industry, driven by consumer demand for healthier, more natural, and minimally processed products. Although no official regulatory definition exists, the term generally refers to products formulated with fewer, simpler, and more recognizable ingredients that consumers perceive as natural and wholesome [1]. Consumers often associate “clean label” products with the absence of synthetic additives and the inclusion of familiar, transparent ingredients, reflecting a strong link between ingredient recognition, trust, and perceived health benefits [2]. This shift in consumer preferences has placed increasing pressure on meat processors to reformulate products in line with clean label expectations [3].

Despite its growing importance, implementing clean label strategies in meat products poses considerable challenges. Traditional meat preservation relies heavily on functional additives such as nitrites, phosphates, and synthetic antioxidants, which ensure microbial safety, color stability, and desirable sensory properties [1]. However, these additives are frequently perceived as artificial or unhealthy, leading to a negative consumer perception of processed meats. The lack of universally accepted natural replacements that can fully replicate these functions complicates reformulation efforts [4]. Although natural antimicrobials such as organic acids, essential oils, and bacteriocins have been explored as alternatives [5], their direct use may be constrained by strong flavor, color changes, or reduced stability [6]. For example, recent studies on poultry by-products have shown that while natural antimicrobials such as buffered vinegar or cultured dextrose fermentate can reduce microbial loads, their efficacy may be more limited compared with conventional chemical interventions, highlighting the practical difficulties of fully replacing synthetic additives. As a result, the meat industry faces the dual challenge of meeting consumer expectations for “naturalness” while safeguarding product safety, shelf-life, and quality. This challenge is particularly acute for ready-to-eat meat products like braised chicken breast, which is highly susceptible to microbial spoilage and pathogen growth due to its high moisture and protein content, as well as the heat treatment that eliminates competitive flora. Ensuring the safety and extending the shelf life of such products typically requires robust antimicrobial hurdles. Under refrigerated storage, the shelf life of braised chicken breast is often limited to approximately 7 to 14 days, primarily constrained by the growth of spoilage microorganisms (e.g., lactic acid bacteria, Brochothrix thermosphacta) and potential pathogens such as Listeria monocytogenes [7]. Therefore, identifying effective clean-label antimicrobials that can meet these stringent safety and shelf-life requirements is critical for product development.

Within this context, natural antimicrobials such as Nisin and cultured dextrose (CD) are gaining attention as promising clean label alternatives. Nisin, a bacteriocin widely recognized for its efficacy against Gram-positive bacteria, has been extensively applied in food preservation but demonstrates limited activity against Gram-negative bacteria [8]. CD generally refers to a fermentation-derived ingredient produced by fermenting dextrose (or a dextrose-containing substrate) with selected microorganisms (commonly lactic acid bacteria). The commercial material, often marketed as a “cultured dextrose fermentate” or similar term, is a complex mixture containing organic acids (e.g., lactic and acetic acids), low-molecular-weight peptides and other antimicrobial metabolites, inactivated microbial biomass, and residual medium components. This fermentate has shown effective inhibition of a broad range of spoilage and pathogenic microorganisms, acting similarly to other natural biopreservatives by acidifying the environment and disrupting microbial membranes or metabolism [9]. Moreover, as highlighted in clean-label research, fermentation-derived antimicrobials produced by lactic acid bacteria are well aligned with consumer expectations due to their natural origin and multifunctional preservative roles [10]. Both ingredients are considered label-friendly and are generally well accepted by consumers due to their natural origin. Importantly, when applied in combination, Nisin and CD may exert synergistic effects, broadening antimicrobial efficacy while reducing the concentrations required, thereby minimizing potential sensory impacts [11].

However, a significant scientific gap exists regarding the systematic optimization and practical application of this specific combination in ready-to-eat poultry products. While the individual effects of Nisin and CD are documented, their synergistic potential in a complex meat matrix like braised chicken breast has not been fully elucidated, particularly in terms of identifying an optimal ratio that balances efficacy with clean-label principles. Admittedly, Staphylococcus aureus (S. aureus) is recognized as a representative Gram-positive foodborne pathogen that can contaminate ready-to-eat poultry products through post-processing handling and is a common cause of food poisoning outbreaks. In addition, Escherichia coli (E. coli) is considered as a critical indicator of fecal contamination and general hygiene during meat processing. Its control is also vital for ensuring the overall sanitary quality of poultry products. Therefore, systematically investigating the combined antibacterial effect of Nisin and CD against both S. aureus and E. coli is essential to address the aforementioned gaps and to develop an effective preservation strategy for ready-to-eat poultry products.

In the present study, preliminary laboratory-scale assays employing a uniform experimental design were first conducted to systematically evaluate the antibacterial activity of Nisin and CD, individually and in combination, against S. aureus and E. coli, and to identify effective concentration ranges. Regression analysis was used to model the relationship between additive concentrations and bacterial inhibition rates, thereby identifying optimal combinations. Subsequently, the most effective combination was applied to braised chicken breast stored under refrigerated conditions (0–4 °C) to validate its practical efficacy. Throughout this storage study, microbial growth (total viable count, E. coli and S. aureus), pH, color, and sensory properties were monitored to assess preservative efficacy and product quality. This study aims to provide scientific evidence supporting the use of natural, clean label antimicrobial strategies in meat preservation, contributing to the development of safer and more consumer-acceptable products.

2. Materials and Methods

2.1. Materials

S. aureus (ATCC 6538) and E. coli (ATCC 2592) were obtained from Ningbo Testobio Co., Ltd. (Ningbo, China). Cultured dextrose (CD) and Nisin were supplied by Henan Miracle Food Technology Co. Ltd. (Zhengzhou, China). Sodium dehydroacetate was purchased from AoKai Food Industry Co., Ltd. (Zhengzhou, China).

2.2. Experimental Methods

2.2.1. Single-Factor Experiments

Single-factor experiments were performed to evaluate the individual antimicrobial effects of CD and Nisin against S. aureus and E. coli. Bacterial cultures were recovered from −80 °C glycerol stocks and grown in TSB to mid-log phase, washed and resuspended in sterile 0.85% saline, and adjusted to ~1.5 × 10⁸ CFU/mL using the 0.5 McFarland standard. The suspension was then diluted to a working concentration of (1.0 ± 0.2) × 10⁶ CFU/mL, which was verified by plate counting and used immediately. The inhibitory effects of CD at concentrations of 0.025%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4% and 0.5% and Nisin at concentrations of 0.0025%, 0.005%, 0.01%, 0.02%, 0.03%, 0.04% and 0.05% were systematically evaluated. These single-factor experiments were conducted to determine the concentration-dependent antibacterial efficacy and to identify the optimal concentration range for subsequent studies.

2.2.2. Uniform Design

Based on the above findings, the concentration ranges of CD and Nisin were selected for further evaluation using a uniform design approach. Within these ranges, seven gradient concentrations were selected as experimental factors. The antibacterial activity was assessed by measuring the inhibitory rates of S. aureus and E. coli at bacterial concentrations of 10⁵, 10⁶, and 10⁷ CFU/mL. A blank group without CD and Nisin served as the negative control, while a group supplemented with 0.05% sodium dehydroacetate (SDA) was included as the positive control group. This design enabled a comprehensive assessment of the inhibitory effects of CD and Nisin at different concentration levels. The factor levels of the uniform experiment design are shown in

Table 1. U7(7²) Uniform design.

Combination	Factor
	The Amount of Nisin (%)	The Amount of CD (%)
1	0.0500	0.400
2	0.0400	0.200
3	0.0300	0.050
4	0.0200	0.500
5	0.0100	0.300
6	0.0050	0.100
7	0.0025	0.025

.

2.2.3. Determination of Antibacterial Rate

CD and Nisin were diluted with LB broth to the designated gradient concentrations, and mixtures were prepared according to the uniform design. For each combination, 100 μL of the mixture was added to a 96-well plate, followed by 100 μL of bacterial suspension at the corresponding concentrations.

Three control groups were established: (i) the growth control group, consisting of 100 μL bacterial suspension and 100 μL LB broth; (ii) the CD control group, consisting of 100 μL CD mixture and 100 μL LB broth; and (iii) the blank control group, consisting of 200 μL LB broth alone. Plates were incubated at 37 °C with shaking at 200 r/min for 24 h. After incubation, absorbance values were measured using a ST-360 microplate reader (Shanghai Kehua Bio-engineering Co., Ltd.; Shanghai, China). The antibacterial rate (Y) was calculated using the following equation:

$$Y\left(\mathrm{\%}\right)={\displaystyle \frac{\left(A_{sz}-A_{kb}\right)-\left(A_{sy}-A_{CD}\right)}{A_{sz}-A_{kb}}}\times 100$$

where Y is the antibacterial rate (%), A_sz is the absorbance of the growth control group, A_kb is the absorbance of the blank control group, A_sy is the absorbance of the experimental group, and A_CD is the absorbance of the CD control group.

2.2.4. Preparation of Braised Chicken Breast

The marination solution was prepared based on meat weight, consisting of 60% water, 2.5% injection salt, and additional ingredients as specified in Table 1. Raw chicken breast was thawed at room temperature, trimmed to remove excess fat, bone fragments, and impurities, and then cut into uniform pieces weighing approximately 50 g with a thickness of about 6 cm. The meat was mixed with the marination solution and processed in a HT-GR50L vacuum tumbler (Zhucheng Hengtai M&T Co., Ltd.; Weifang, China) for 1.5 h, alternating between forward and reverse tumbling for 25 min each with a 5 min interval between cycles, followed marination at 0–4 °C for 24 h. For braising, water and seasonings were measured according to the combination table (Table 1). After boiling the water, spices were added, and the chicken breast was cooked until the core temperature reached 85 °C. The braised chicken breast was then transferred to a sanitized container, covered with plastic wrap, and cooled to approximately 30 °C before being portioned, sealed in sterile resealable bags, labeled, and stored under refrigerated conditions at 0–4 °C.

2.2.5. Analysis of Chicken Breast Indices

• Total viable count (TVC)

TVC was determined according to the Chinese National Standard GB 4789.2-2016 [12].

2.

Coliforms

Coliforms were detected following the Chinese National Standard GB 4789.3-2016 [13].

3.

pH

pH was measured on days 0, 4, 7, 14, 21, and 28. For each measurement, 5.0 g of chicken breast was homogenized with 100 mL of ddH₂O. The pH of the homogenate was measured using a PHB-5 portable pH meter (Hefei Enfan Instrument Equipment Co., Ltd.; Hefei, China).

4.

Color

As the braised chicken breast showed severe deterioration on day 28, color measurements were conducted only on days 0, 4, 7, 14, and 21. The L* (lightness), a* (red-green), and b* (yellow-blue) values were determined on the sample surface using an NH300 portable colorimeter (Guangdong 3nh Technology Co., Ltd.; Guangzhou, China) under controlled conditions (D65 illuminant, 10° observer, 8 mm aperture, in a dark room at 22 ± 1 °C).

5.

Sensory evaluation

Sensory evaluation of braised chicken breast was performed on days 0, 4, 7, 14, and 21 in accordance with the Chinese National standards GB 2726-2016 [14]. Each sample was cut into two uniform cubes and placed on a clean white plate. Twelve trained panelists (six males and six females) with normal sensory acuity participated. Panelists received instructions on standardized evaluation procedures, including gently inhalation when assessing odor, avoiding repeated exposure to strong aromas, and allowing sufficient intervals between assessments. The sensory evaluation criteria are shown in

Table 2. The sensory evaluation criteria for marinated chicken breast.

Attribute	Score
	7–9	4–<7	1–<4
Tissue condition	Neat cut surface; compact, firm texture; distinct oily sheen	Neat cut surface; compact, firm texture; slight oily sheen	Rough cut surface; loose texture; absence of oily sheen
Texture	Highly elasticity; full recovery after pressure; non-sticky	Moderate elasticity; slow recovery after pressure; slightly sticky	Poor elasticity; severe stickiness
Odor	Characteristic braised chicken aroma; no off-odors	Faint aroma; no off-odors	Absence of aroma; slightly foul odor
Overall acceptability	High acceptability	Moderate acceptability	Low acceptability

.

2.3. Statistical Analysis

Data were analyzed using SPSS 16.0 (SPSS Inc., Chicago, IL, USA) for multiple comparisons and regression analysis, while MATLAB 7.0 and Qtisplot 1.2 for graphical illustration. All experiments were conducted in triplicate, and results are expressed as mean ± standard deviation.

3. Results and Discussion

3.1. Effects of Different Combinations of CD and Nisin on the Inhibition Rates of S. aureus and E. coli

The effects of CD and Nisin concentrations on the inhibition rates of S. aureus and E. coli are shown in Figure 1. Both bacterial suspensions were adjusted to 10⁶ CFU/mL, after which different inhibitor concentrations were applied. The results indicated that increasing concentrations of CD significantly enhanced antibacterial activity. At 0.5%, the inhibition rate reached 70.79% against S. aureus and 58.92% against E. coli. In contrast, concentration below 0.025% exhibited no inhibitory effect on S. aureus, only 1.76% inhibition against E. coli. Similar trends were observed for Nisin. At 0.05%, Nisin inhibited S. aureus by 21.05% but showed no effect on E. coli. Moreover, Nisin concentrations below 0.01% did not inhibit S. aureus. Therefore, the concentration ranges selected for subsequent uniform design experiments were 0.025–0.5% for CD and 0.0025–0.05% for Nisin, to enable a systematic evaluation of their potential synergistic inhibitory effects.

3.2. Effect of Different Combinations of CD and Nisin on Bacteriostasis

3.2.1. Effect of Different Combinations on the Bacteriostasis of S. aureus

The inhibitory effects of seven CD–Nisin combinations on S. aureus at three initial inocula (10⁵, 10⁶, 10⁷ CFU/mL) are shown in Figure 2. It was found that Combinations 1, 2, 4, and 5 consistently achieved inhibition rates >95% across all inoculum levels, whereas Combinations 3, 6, and 7 showed comparatively weaker activity. Notably, even at the lowest inoculum (10⁵ CFU/mL), several combinations containing relatively low preservative levels (e.g., Combination 5: 0.01% Nisin + 0.3% CD) exerted inhibition rates comparable to or higher than those of the positive control (0.05% SDA). This pattern indicates that the antibacterial efficacy is influenced not only by the absolute concentration of each agent, but also by their synergistic interplay, especially under lower bacterial loads. A clear negative correlation was observed between initial bacterial concentration and sensitivity to the preservative combinations. Higher inocula (10⁷ CFU/mL) consistently reduced the inhibition rate, suggesting that dense populations exhibit enhanced tolerance. This tolerance may be explained by mechanisms such as biofilm formation, secretion of resistance-related enzymes, or metabolites [15], and quorum sensing-mediated adaptive responses [16]. The superior performance of low-concentration combinations (e.g., Combination 5) at lower inocula can be explained by a multi-faceted synergistic mechanism. Sub-inhibitory levels of Nisin can perturb the Agr quorum-sensing system, leading to transient virulence down-regulation and biofilm dispersal. Simultaneously, organic acids from CD acidify the extracellular microenvironment, increase membrane fluidity, and thereby enhance the pore-forming efficiency of Nisin. Furthermore, lactic and acetic acids in CD deplete intracellular ATP via proton influx, while low-dose Nisin blocks Lipid II recycling, a dual action that exhausts the cellular energy pool and produces a steeper inhibition slope at lower concentrations than the plateau observed at higher doses. In complex food matrices such as braised chicken breast, high Nisin concentrations may form inactive dimers or bind to phospholipid micelles, reducing the free active fraction. Lower concentrations maintain better molecular dispersion and higher accessibility to membrane targets. At an initial load of 10⁵ CFU/mL, the low-dose combination can interrupt quorum-sensing signaling before it reaches the threshold for virulence amplification, whereas at 10⁷ CFU/mL, extracellular polymeric substances secreted by the dense population may adsorb Nisin, contributing to the observed plateau where further increases in preservative levels yield no additional inhibition. These observations collectively highlight that the antibacterial outcome depends on a delicate balance between inoculum density, preservative concentration, and the synergistic interactions between CD and Nisin, which is essential for designing effective clean-label preservation strategies. Based on the overall inhibition performance and practical additive usage, Combination 5 (0.01% Nisin + 0.3% CD) exhibited the most favorable profile against S. aureus.

Subsequently, regression analysis was performed using the addition levels of Nisin (x, %) and CD (y, %) as independent variables, with bacteriostasis rates (Z₁, Z₂, Z₃) for S. aureus at 10⁷, 10⁶, and 10⁵ CFU/mL, respectively, as dependent variables. The corresponding regression equations are shown as follows, and the response surface diagrams are shown in Figure 3.

Z₁ = −77.36x + 32.67y + 1464.1x² − 1030.3xy + 346.3y²   (R² = 0.942)

(1)

Z₂ = −79.95x + 23.66y + 1705.8x² − 1307.4xy + 573.0y²   (R² = 0.894)

(2)

Z₃ = −57.35x + 22.08y + 1294.3x² − 1027.8xy + 409y²     (R² = 0.887)

(3)

It was found that when the initial bacterial concentration was high (10⁷ CFU/mL), the combined application of Nisin and CD did not significantly improve inhibition compared with their individual use. Nisin alone showed negligible inhibitory effect on S. aureus at this inoculum level, possibly due to the enhanced preservative tolerance of dense bacterial populations. Overall, inhibition rates at 10⁷ CFU/mL were consistently lower than those at 10⁶ CFU/mL and 10⁵ CFU/mL, indicating that the combined bacteriostatic effect of Nisin and CD was limited under high inoculum conditions. At lower inoculum levels, however, increasing the concentration of CD significantly enhanced inhibition, particularly when Nisin was applied at low levels, indicating a positive synergistic effect. This synergistic effect aligns with previous findings for Nisin combined with other clean-label ingredients such as plant extracts [17], essential oils [18], and organic acids [19], which demonstrated synergistic or additive inhibition of S. aureus in vitro and in food systems. Such combinations often enable stronger microbial control at lower doses through broader and complementary mechanisms of action. In the present study, the effect may reflect their distinct mechanisms of action: Nisin disrupts cell membranes and walls [20], whereas CD modifies the metabolic environment to suppress growth. Moreover, similar to many reported Nisin-based combinations that target the bacterial membrane and cell wall, the Nisin-CD pairing presumably enhances disruptive effects, such as increased permeabilization, leakage of cellular contents, and morphological damage, compared to either agent alone. Notably, synergistic effects were not found between CD and Nisin at high concentrations of S. aureus (10⁷ CFU/mL). However, at 10⁶ CFU/mL and 10⁵ CFU/mL, bacteriostasis reached a complete inhibition plateau of 100% when CD concentrations were sufficiently high. This plateau effect suggests that once bacterial tolerance threshold is exceeded, complete inhibition can be achieved, and further increases in preservative levels confer no additional bacteriostatic benefit [21]. Consequently, in practical applications, maintaining the concentration above this saturation level ensures complete inhibition. Based on the regression equations Z₁, Z₂, and Z₃, the calculated minimum concentrations of CD required for 100% inhibition of S. aureus were 0.5% for 10⁷ CFU/mL, 0.398% for 10⁶ CFU/mL, and 0.271% for 10⁵ CFU/mL. In summary, based on the comprehensive analysis of bacteriostatic effects and optimization of additive usage, Combination 5 (i.e., Nisin 0.01%, CD 0.3%) was identified as the optimal combination.

3.2.2. Effect of Different Combinations on the Bacteriostasis of E. coli

The effects of different combinations on the inhibition rates of E. coli are shown in Figure 4. It was found that the inhibition rate generally decreased across all combinations as the initial bacterial concentration increased, indicating that higher concentrations of E. coli exhibit greater tolerance to Nisin and CD. CD contains antibacterial compounds such as organic acids and aldehydes, which inhibit bacterial growth by altering the metabolic environment [22]. In contrast, Nisin is ineffective against Gram-negative bacteria (e.g., E. coli) when used alone, due to its inability to penetrate the outer membrane [23]. However, when combined with other antimicrobial agents, Nisin exhibits synergistic effects through complementary mechanisms, for instance, by disrupting cellular energy metabolism or enhancing the efficacy of other antibacterial substances [24]. Under the same bacterial concentration, significant differences in inhibition were found among different combinations. At 10⁵ CFU/mL, the inhibition rate was 43.73% for Combination 1 and 84.89% for Combination 4, with the latter being significantly higher than the positive control (35.15%). At 10⁶ CFU/mL, Combination 4 achieved an inhibition rate of 34.38%, which also exceeded that of the positive control (20.93%). Even at 10⁷ CFU/mL, Combinations 1, 4, and 5 all outperformed the positive control. Considering overall antibacterial efficacy and additive dosage, Combination 4 demonstrated the best comprehensive performance against E. coli.

Subsequently, regression analysis was performed using the addition levels of Nisin (x, %) and CD (y, %) as independent variables to obtain the following equations:

Z₄ = 13.82x − 3.94y − 234.2x² + 145xy + 52.1y²             (R² = 0.976)

(4)

Z₅ = −1.69x + 0.688y + 18.3x² − 30.9xy + 52.1y²            (R² = 0.943)

(5)

Z₆ = 22.09x − 4.66y − 384.3x² + 246.9y²                        (R² = 0.976)

(6)

where Z₄, Z₅, and Z₆ represent the inhibition rates for E. coli at 10⁷, 10⁶, 10⁵ CFU/mL, respectively (%).

The variation in inhibition rates with respect to Nisin and CD concentrations are shown in Figure 5. It was found that the synergistic antibacterial effect of Nisin and CD on E. coli follows a trend similar to that observed for S. aureus. While Nisin alone exhibited no inhibitory effect on E. coli, the combination led to inhibition rates that were negatively correlated with bacterial concentration. As the initial concentration of E. coli increased, the inhibition rate decreased, indicating greater bacterial tolerance at higher densities. According to the regression equations Z₄, Z₅, and Z₆, when CD was added at 0.5% and Nisin at 0.05%, the calculated inhibition rates were 59.53% at 10⁵ CFU/mL, 12.55% at 10⁶ CFU/mL, and 14.78% at 10⁷ CFU/mL.

Overall, Combination 4 showed the strongest inhibitory effect on E. coli under the tested conditions. These results provide a scientific basis for optimizing food preservative combinations and may contribute to the development of more effective natural preservative combinations.

3.3. Application of Different Combinations of CD and Nisin in Braised Chicken Breast

3.3.1. Changes in Total Viable Counts (TVC) in Braised Chicken Breast During Storage

Total viable count (TVC) serves as a fundamental microbiological indicator for assessing the hygienic quality and safety of ready-to-eat meat products during storage. For braised chicken breast, controlling microbial growth is essential to extend shelf-life while meeting clean-label consumer preferences. The antimicrobial efficacy of various CD–Nisin combinations was evaluated by monitoring TVC changes in braised chicken breast over 28 days of refrigerated storage, and the results are shown in Figure 6a. Compared with the control group, all combinations exhibited antimicrobial effects. From Day 4 to Day 21, TVC increased across all groups, with detectable microbial growth in all nine combination groups by Day 7. By Day 14, the TVC value was 3.75 log (CFU/g) for the control, 1.49 log (CFU/g) for Combination 2, 3.77 log (CFU/g) for Combination 7, and 2.57 log (CFU/g) for the positive control. These values increased to 5.35, 2.68, 5.00 and 3.97 log (CFU/g), respectively, by Day 21. At day 28, the TVC values reached 6.92 (control), 3.83 (Combination 2), 5.95 (Combination 7), and 4.06 (positive control) log (CFU/g). According to the acceptable limit value (10⁴ CFU/g) and the maximum safe limit (10⁵ CFU/g) specified in Chinese National Standard GB 2726-2016 [14], the TVC of all combinations remained within the acceptable level during the first 7 days of storage. By Day 21, the control exceeded the safety limit, indicating rapid microbial growth without preservatives. Combinations 6 and 7 exceeded the acceptable limit (10⁴ CFU/g) on Day 21, suggesting that low levels of preservatives cannot ensure full shelf-life safety. In summary, Combination 2 showed the strongest inhibition of microbial growth, with a relatively slower increase in TVC and superior efficacy compared with the positive control throughout the 28-day period. Only Combination 2 maintained TVC below the acceptable limit for the entire storage duration. Considering antimicrobial performance and required dosage, Combination 2 is found to be the most suitable combination for application in braised chicken breast.

Subsequently, regression analysis was performed using the TVC [Z₇, log (CFU/g)] of each combination on Day 28 as the dependent variable and the addition levels of Nisin (x, %) and CD (y, %) as independent variables, resulting in the following Equation (7):

Z₇ = 6.305 − 10.16x − 7.78y − 38.8x² + 7.2y²      (R² = 0.981)

(7)

The corresponding contour plot is shown in Figure 6b. According to the regression model, CD at 0.5% and 0.4% resulted in TVCs of 4.215 and 4.345 log (CFU/g), respectively. When Nisin was used alone, 0.05% and 0.04% yielded TVCs of 5.70 log (CFU/g) and 5.83 log (CFU/g), respectively. When CD (0.5%) and Nisin (0.05%) were used in combination, the TVC decreased to 3.61 log (CFU/g), indicating a significant synergistic effect. The regression surface further showed that TVC reached to a peak value when both preservatives were at their lowest levels, reflecting weak inhibition, and lowest at their maximum levels, indicating the strongest effect. Moreover, it was found that when Nisin concentration remained constant, the TVC decreased significantly with increasing CD, confirming its effective antimicrobial activity. Our previous study [25] reported similar results, which showed that CD and Nisin effectively preserved marinated beef, with an optimal ratio of 0.5% CD and 0.05% Nisin, achieving microbial control without compromising sensory quality. Overall, the combination of CD and Nisin effectively reduced TVC in braised chicken breast. Optimizing their ratio allows strong antimicrobial effects at lower concentrations, thereby reducing reliance on conventional chemical preservatives.

3.3.2. Effects of Different Combinations of CD and Nisin on S. aureus in Braised Chicken Breast

S. aureus is a major Gram-positive foodborne pathogen frequently associated with poultry meat contamination and a leading cause of food poisoning outbreaks [26]. Effective control of S. aureus in ready-to-eat meat products is therefore crucial for food safety. The efficacy of different CD and Nisin combinations in inhibiting the growth of S. aureus was assessed in braised chicken breast during 28 days of refrigerated storage, assessed against the microbiological limits set by the Chinese National Standard [27]. The changes in S. aureus counts under different combinations are shown in Figure 7a. Compared with the control group, all combinations exhibited inhibitory effects on S. aureus. During storage, S. aureus counts in all samples showed an overall increasing trend. S. aureus was not detected in any group on Day 0. By Day 4, growth was observed in the control, Combinations 5, 6, 7, and the positive control. All groups showed detectable S. aureus by Day 7. On day 14, the control group showed the highest count at 2.53 log (CFU/g), while Combination 1 showed the lowest at 1.06 log (CFU/g). By Day 21, the control reached 3.25 log (CFU/g), and Combination 2 showed the lowest count at 1.49 log (CFU/g). At the end of storage (Day 28), the control group count peaked at 4.77 log (CFU/g), whereas Combination 1 demonstrated the strongest inhibition, maintaining the lowest count at 2.28 log (CFU/g). According to GB 29921-2013 [27], all combinations maintained S. aureus counts within the acceptable level during the first 14 days of storage. By Day 21, the control group exceeded the maximum limit [2–3 log (CFU/g)], indicating rapid growth and increased spoilage risk between Days 14 and 21. In summary, Combinations 6 and 7 exhibited the weakest inhibition, with counts approaching the maximum limit by day 21. Combination 1 demonstrated the strongest suppression of S. aureus growth, maintaining the slowest increase in counts, and its value at Day 28 [2.28 log (CFU/g)] remained below the maximum limit. Considering both antimicrobial efficacy and additive usage, Combination 5 was identified as the most suitable combination for application in braised chicken breast.

Subsequently, regression analysis was performed using the addition levels of CD (x, %) and Nisin (y, %) as independent variables and the S. aureus count at day 28 [Z₈, log (CFU/g)] as the dependent variable. The resulting regression equation is as follows (R2 = 0.972):

Z₈ = 4.7794 − 19.531y − 6.40x − 2.97x² − 20.834y²     (R² = 0.972)

(8)

The response surface plot for S. aureus count as a function of CD and Nisin addition is shown in Figure 7b. The analysis indicates that under constant Nisin levels, increasing the CD concentration significantly reduced S. aureus counts, suggesting that acidic metabolites from CD fermentation disrupt cell membrane integrity and interfere with energy metabolism [28]. Conversely, with fixed CD levels, higher Nisin concentrations also led to lower bacterial counts, attributable to its pore-forming mechanism via Lipid II binding [29]. The combination likely exhibits complementary and synergistic actions: CD weakens the cell wall or membrane barrier, facilitating Nisin’s targeting and pore formation, leading to rapid cell death. This synergistic effect allows for effective inhibition at lower individual concentrations, supporting the potential of this clean-label combination to reduce reliance on traditional chemical preservatives.

3.3.3. Effects of Different Combinations of CD and Nisin on E. coli in Braised Chicken Breast

As a common indicator of fecal contamination and a potential foodborne pathogen, the control of E. coli is critical for ensuring the safety of ready-to-eat poultry products. To develop effective clean-label preservation strategies, the efficacy of different CD and Nisin combinations in inhibiting E. coli growth was investigated throughout refrigerated storage (Figure 7c). It was found that all combinations except the control group exhibited inhibitory effects on E. coli. Notably, Combinations 1 and 4 completely suppressed growth during the first seven days. Over time, E. coli counts increased in all groups except Combination 4. By Day 14, the control group and Combination 7 exceeded the safety limit, and by Day 21, the control group, Combinations 3, 6, and 7 also surpassed threshold. In contrast, Combination 4 maintained E. coli counts at 0 CFU/g throughout the 28-day storage. In summary, Combination 4 demonstrated the most effective inhibition, completely suppressing E. coli in braised chicken breast stored at 0–4 °C for 28 days.

Subsequently, regression analysis was performed using E. coli counts on Day 28 as the dependent variable and the addition levels of CD and Nisin as independent variables, yielding the following regression Equation (8):

Z₉ = 4.06 − 38.7x − 4.74y + 1007.8x² − 56.3xy − 2.9y²   (R² = 0.993)

(9)

where Z₈ represents log (CFU/g) of E. coli, x is the Nisin concentration (%), and y is the CD concentration (%).

The changes in E. coli counts with varying addition levels of CD and Nisin are shown in Figure 7d. According to Equation (8), CD alone at 0.5% and 0.4% reduced E. coli counts to 0.965 and 1.700 log (CFU/g), respectively. In contrast, Nisin alone at 0.05% and 0.04% resulted in counts of 4.64 and 4.12 log (CFU/g), respectively, similar to the control group [4.01 log (CFU/g)], confirming its limited effect on E. coli. However, when CD (0.5%) and Nisin (0.05%) were used in combination, the E. coli counts dropped to 0.142 log (CFU/g), suggesting a significant synergistic effect.

As shown in Figure 5, E. coli counts declined significantly with higher CD concentrations under constant Nisin levels. Similar results were also found with higher Nisin addition levels under constant CD levels. The lowest E. coli counts were observed when both additives were applied at maximum levels. Within the examined range, the inhibitory effect improved consistently with increasing concentrations of CD and Nisin. Overall, these results demonstrate that the combination of CD and Nisin effectively overcomes Nisin’s inability to inhibit Gram-negative bacteria, providing enhanced preservation in braised chicken breast.

3.3.4. Effects of CD and Nisin on the Color of Braised Chicken Breast

Color is a key quality attribute of meat products, directly influencing consumer acceptance and purchase decisions. In braised chicken breast, color stability during storage is particularly important, as discoloration can be an early indicator of spoilage or quality deterioration. The effects of CD and Nisin on the L*, a*, and b* values of braised chicken breast are shown in Figure 8a–c. From Day 7 to Day 21, L* values, which reflect brightness, declined across all groups, with the control group showing the greatest decrease. Combinations 1 and 4 exhibited lower overall L* values, possibly due to the yellowish color of CD. On Day 21, L* values for Combinations 1, 4, and 5 were 77.53, 79.20, and 80.25, respectively, indicating that the preservatives mitigate L* loss, with Combination 5 demonstrated the most stable brightness. Both a* and b* values generally decreased during storage, with no significant differences, likely reflecting natural variability in chicken breast color. Overall, Combination 5 demonstrated the best color preservation.

3.3.5. Effects of CD and Nisin on the pH of Braised Chicken Breast

The pH of braised chicken breast is a critical indicator of its freshness and microbiological stability, as it reflects biochemical changes and microbial activity during storage. During storage, pH fluctuations mainly result from organic acids (e.g., lactic acid) produced by microbial glycogenolysis and amines generated from protein catabolism [30]. The effect of partially replacing Nisin with CD on the pH of chicken breast is shown in Figure 8d. It was found that, on Day 28, the control group exhibited a pH of 6.33, compared with 6.42 in Combination 5. All combination groups, except the control, followed a trend of initial pH decline followed by a gradual increase. From Day 7 to Day 14, pH generally decreased, with Combination 3 reaching the lowest pH value on Day 7. After Day 7, the control group showed a significantly greater pH increase than the treated groups (p < 0.05), indicating more rapid spoilage. In contrast, Combination 3 showed the smallest pH increase, suggesting that the combined use of CD and Nisin effectively preserved chicken breast quality. The pH stabilization observed in braised chicken breast treated with the CD and Nisin blend aligns with findings from previous studies on clean-label preservatives. For instance, vinegar (acetic acid) has been shown to effectively lower meat pH and inhibit microbial growth, thereby delaying spoilage during refrigerated storage [31]. Similarly, protein hydrolysates from kidney beans were reported to reduce pH increase in chicken meat by suppressing bacterial metabolic activity, which otherwise leads to ammonia production and pH elevation [32]. In the present study, the combination of CD and Nisin not only mitigated pH fluctuations but also demonstrated a synergistic effect in maintaining meat quality over 28 days of storage. These results collectively suggest that clean-label antimicrobial blends, such as CD–Nisin, can function as effective pH buffers and microbial growth inhibitors, contributing to extended shelf life without the use of synthetic preservatives.

3.3.6. Sensory Evaluation

Sensory attributes, including tissue condition, texture, odor, and overall acceptability, are critical determinants of product quality and consumer preference, especially for clean-label meat products. The sensory evaluation results for braised chicken breast at different storage intervals are shown in

Table 3. The sensory evaluation results.

Combination	Sensory Score
	Day 0	Day 7	Day 14	Day 21
Negative control	17.34 ± 0.59 Ad	15.56 ± 0.61 Bc	13.09 ± 0.59 Cb	11.69 ± 0.59 Db
1	19.26 ± 0.45 Ab	18.23 ± 0.59 Ba	15.63 ± 0.59 Ca	13.63 ± 0.59 Da
2	18.85 ± 0.98 Ac	18.89 ± 0.52 Aa	15.12 ± 0.52 Ba	13.23 ± 0.12 Ca
3	20.23 ± 0.45 Aa	18.56 ± 0.58 Ba	15.54 ± 0.57 Ca	13.62 ± 0.12 Da
4	20.37 ± 0.34 Aa	18.13 ± 0.51 Ba	15.55 ± 0.19 Ca	13.94 ± 0.11 Da
5	19.86 ± 0.67 Aa	18.36 ± 0.12 Ba	15.35 ± 0.08 Ca	13.95 ± 0.13 Da
6	19.12 ± 0.78 Ab	18.76 ± 0.55 Ba	15.07 ± 0.16 Ca	10.21 ± 0.47 Dc
7	19.98 ± 0.52 Aa	18.49 ± 0.53 Ba	15.13 ± 0.17 Ca	10.55 ± 0.12 Dc
Positive control	19.46 ± 0.51 Ab	17.52 ± 0.56 Bb	15.63 ± 0.48 Ca	11.63 ± 0.48 Db

Combination 1: Nisin 0.05% + CD 0.4%; Combination 2: Nisin 0.04% + CD 0.2%; Combination 3: Nisin 0.03% + CD 0.05%; Combination 4: Nisin 0.02% + CD 0.5%; Combination 5: Nisin 0.01% + CD 0.005%; Combination 6: Nisin 0.005% + CD 0.1%; Combination 7: Nisin 0.0025% + CD 0.025%; Negative control: Nisin 0% + CD 0%; Positive control: SDA 0.05%. Values are presented as mean ± standard deviation (n = 12). For the same day (within a column), values with different lowercase letters (a–d) are significantly different among combinations (p < 0.05, Duncan’s multiple range test). For the same combination (within a row), values with different uppercase letters (A–D) are significantly different across storage days (p < 0.05, Duncan’s multiple range test).

. At Day 0, significant sensory score differences were not found among various combinations, indicating that varying levels of CD and Nisin did not affect sensory quality. By Day 7, the control group showed a significant score decrease compared to other combination groups (p < 0.05), while significant differences were not found among the combination groups. These results suggest that the combination of CD and Nisin within the tested range did not negatively influence texture, flavor, and overall acceptability of chicken breast. Over time, sensory scores decreased across all groups, with the control group exhibiting the most rapid decline. Similar effects have been reported for other clean-label ingredients such as date palm seed extract [33] and olive leaf extract [34], which preserve sensory quality and extend shelf-life in poultry meat through antioxidative and antimicrobial mechanisms. This correlation between sustained sensory scores and the inhibition of oxidative and microbial spoilage in the present study further reinforces that clean-label ingredient systems (e.g., the CD–Nisin combination) can effectively delay sensory deterioration without compromising consumer acceptance.

4. Conclusions

This study demonstrated a significant synergistic antibacterial property of CD combined with Nisin against S. aureus and E. coli. The combination of 0.02% Nisin and 0.5% CD proved to be the optimal choice for preserving braised chicken breast, due to its balanced and superior performance in ensuring microbial safety while maintaining key quality parameters, including pH, color, and sensory attributes. Therefore, this combination of Nisin and CD is recommended as a promising clean-label preservation strategy for meat products. By extending shelf life and maintaining quality without synthetic preservatives, this approach is anticipated to address both consumer demand for natural ingredients and industry needs for safe, stable meat products. It should be noted that this study was conducted under constant refrigeration (0–4 °C). Future research is warranted to evaluate the combination’s stability under dynamic temperature scenarios and explore its application in other meat matrices to develop more robust natural preservation systems.