Effects of reheating methods on oxidation and digestibility characteristics of precooked braised chicken during in vitro digestion

Abstract

This study investigated the effects of microwave reheating (MW), water boiling reheating (WB), and steaming reheating (ST) on the oxidation and digestibility characteristics of precooked braised chicken (PBC), using a non-reheated control (C). Oxidation markers increased after all reheating treatments; the ST group preserved total sulfhydryl and carbonyl content closest to the C group, indicating minimal oxidation. Following in vitro digestion, reheated samples displayed reduced digestibility as measured by crude protein digestibility, SDS-PAGE profiling and peptide molecular weight distribution. The digestibility of crude protein and the degree of hydrolysis in the ST group (74.40% and 37.39%) were most similar to those of the C group (76.69% and 40.71%). Distribution analysis of digestion products revealed that the ST treatment outperformed the other reheating methods. ST treatment emerged as a viable reheating method for maintaining protein digestibility in precooked meat products.

Graphical abstract

Highlights

• •Steaming preserves protein digestibility better than microwave or boiling.
• •Minimal protein oxidation occurs in steaming (lowest carbonyls, highest sulfhydryl).
• •INFOGEST digestion shows steaming causes protein hydrolysis close to control.
• •Microwave and boiling cause greater oxidation, decreasing protein digestibility.
• •Oxidation of reheated chicken protein inversely correlates with digestibility.

1. Introduction

Meat products played a significant role in the long history of the human diet due to their excellent taste, flavor, and high nutritional value (Klurfeld, 2018; Leroy et al., 2023). Braised chicken is classified as a traditional Chinese stewed meat product. The primary component is the drumstick, which is simmered with sauce and various complementary ingredients. This dish is characterized by its savory flavor profile and enjoys considerable popularity among consumers.

In recent years, the trend of precooked dishes has impacted the entire food industry. Precooked braised chicken (PBC) holds significant commercial potential due to its high protein content and low cost. PBC is produced by tumbling and marinating trimmed chicken thigh meat, then simmering it with broth and seasonings before sealing and sterilizing under high temperature. PBC typically displays a golden appearance and a pronounced aroma, and yields a tender and juicy texture. This product has emerged as one of the representative products in the field of pre-cooked meat dishes.

Although PBC is fully cooked, it typically requires reheating before consumption. Reheating methods vary in temperature, duration and heat transfer mechanism, and these parameters directly influence the extent of protein and lipid oxidation. While most existing studies have focused on the effects of initial cooking (Abdel-Naeem et al., 2021; Ortuño et al., 2021; Soladoye et al., 2017), the underlying oxidative mechanisms are similar for reheating. However, because reheating represents a milder thermal treatment applied to an already cooked product, the extent of oxidation may differ (Arguelo et al., 2016), warranting dedicated investigation. Oxidative modification induces conformational changes in protein tertiary and quaternary structure that compromise their functional properties. Therefore, it is essential to assess how different reheating treatments alter the nutrient composition of heat-treated meat products following in vitro digestion. Currently, techniques for in vitro digestion simulation have advanced significantly. Ideal in vitro digestion models can provide high quality results efficiently. Nevertheless, the majority objective of related research is to investigate various types or different parts of raw meat. There is limited research on precooked meat dishes. The effect of common reheating methods on the digestive properties of cooked meat products and the differences between them remain unclear. Existing literature regarding precooked meat products predominantly focuses on quality changes, leaving a significant gap in this area of research.

The precooked braised chicken (PBC) was sterilized at 115 °C for 55 min, a thermal treatment far more intense than household reheating. This study focuses on the effects of mild reheating on the sterilized ready-to-eat product, which conforms to real consumer usage. We hypothesized that different reheating methods, which differ in heating rate, heat transfer mode, and oxygen exposure, would induce different degrees of protein and lipid oxidation in precooked braised chicken, thereby leading to distinct in vitro digestibility profiles; specifically, we postulated that steaming reheating, characterized by gentle and moist heating, would cause the lowest oxidation level and consequently preserve the highest protein digestibility among the three reheating methods. Therefore, the non-reheated sterilized sample (group C) serves as the most relevant baseline for this research question, whereas a direct comparison with non-sterilized raw product, while informative, is beyond the present scope. In this study, three common household reheating methods, namely microwave reheating (MW), water boiling reheating (WB) and steaming reheating (ST), were used to investigate the changes in protein oxidation, lipid oxidation, digestibility, amino acids and fatty acids profiles of PBC. The relationship between the oxidation and digestive characteristics was further investigated. The purpose of this study is to provide valid information on the changes in digestibility and nutrient composition of PBC by conventional reheating methods.

2. Materials and methods

2.1. Materials and reagents

PBC (already stored at room temperature for 2 months) were provided by Henan Shuanghui Investment and Development Co., Ltd. (Luohe, China). The chickens used in the PBC product were white-feathered broilers sourced from Zhoukou, Henan Province. Each serving of the PBC product has a net weight of at least 200 g, with chicken meat comprising at least 50% and solids accounting for at least 70% of the product. The processing flow is shown in Supplementary Fig. S1. After being sealed in aluminum-foil pouches, the PBC products were sterilized at 115 °C for 55 min to achieve commercial sterility. The product has a shelf life of seven months under ambient storage conditions. The malondialdehyde (MDA) content assay kit, anhydrous ethanol, sodium hydroxide, ethyl acetate and bovine bile salt were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). ColorMixed protein marker, Coomassie Brilliant Blue fast staining solution, protein carbonyl content assay kit, and protein total sulfhydryl content assay kit were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). The reagents for SDS-PAGE analysis were purchased from Beyotime Biotechnology Co., Ltd. (Beijing, China). Pepsin, trypsin, lipase, bowman-birk trypsin inhibitor (BBI), 4-bromophenylboronic acid, serine, o-Phthalaldehyde (OPA) reagent, and electrolyte inorganic salts used in in vitro simulation of digestion were purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). The rest of the chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All chemical reagents were of analytical grade. All solutions were prepared with double-distilled water. Three independent product units (three individual pouches of precooked braised chicken from the same production batch) were used as biological replicates. Each unit was subjected to reheating treatments and all subsequent measurements separately. For each biological replicate, at least three technical replicate measurements were performed for each analytical endpoint.

2.2. Reheating process

The samples were assigned to four reheating treatments, C (control): no reheating; MW: microwave reheating at 800 W for 2 min (PM200M3, Midea, China); WB: immersion of the vacuum-packaged PBC in boiling water for 8 min; ST: steam reheating in an electric steamer for 14 min (ZGC322301, Midea, China). The initial central temperature of the samples before reheating was 25.0 ± 0.5 °C. The meat center temperature was monitored using a multiplexed temperature checker (AT4508, Anbai, China), and reheating was terminated immediately upon reaching 72.5 °C at the sample center. The times required to reach 72.5 °C were 2 min for MW, 8 min for WB, and 14 min for ST, reflecting substantial differences in heating rate and total thermal exposure among the three methods.

2.3. Measurement of total sulfhydryl content

Total sulfhydryl content was measured using a commercial assay kit. Briefly, 1 g sample was homogenized in 10 mL of extraction solution on ice (A25, Oulior, China) and centrifuged at 8000 ×g for 10 min (H4-20KR, KeCheng, China). The supernatant was reacted with the kit reagents according to the manufacturer's protocol and incubated at room temperature for 10 min. Absorbance was then recorded at 412 nm using a microplate reader (SPARK, Tecan, China). A standard curve was constructed with reduced glutathione (25 μmol/mL), and total sulfhydryl content was expressed as μmol sulfhydryl per gram of sample. The total sulfhydryl content was calculated using Eq. (1).

$$\text{Total sulfhydryl content}=\mathrm{x}\times \frac{{\mathrm{V}}_{\text{total}}}{\mathrm{W}}$$ (1)

where x is the sample concentration calculated based on the standard curve; V_total is the extracted liquid volume; W is the sample mass.

2.4. Measurement of carbonyl content

Protein carbonyl content was quantified using a commercial assay kit. Briefly, carbonyl groups were reacted with 2,4-dinitrophenylhydrazine (DNPH) to form protein hydrazones, and absorbance was measured at 370 nm. Carbonyl content was expressed as μmol carbonyl per gram of sample.The carbonyl content was calculated using Eq. (2).

$$\text{Carbonyl content}=\frac{\mathrm{\Delta A}}{16\times \mathrm{W}}$$ (2)

where ΔA is the difference between the test tube and the control tube at 370 nm; W is the sample mass.

2.5. Measurement of MDA content

MDA content was determined using a commercial assay kit. Briefly, 1 g of sample was homogenized in 10 mL of extraction solution on ice. The mixture was centrifuged at 8000 ×g and 4 °C for 10 min, and the supernatant was combined with the kit reagents. The mixture was incubated at 95 °C for 30 min, then cooled on ice and centrifuged again at 10,000 ×g for 10 min. Absorbance was measured at 532 nm and 600 nm, and MDA content was expressed as nmol MDA per gram of sample. MDA content is calculated according to Eq. (3).

$$\mathrm{MDA}=\frac{\frac{\mathrm{\Delta A}\times {\mathrm{V}}_{\text{total}}\times {10}^9}{\mathrm{Ɛ}\times \mathrm{d}}}{\frac{\mathrm{W}\times {\mathrm{V}}_{\text{sample}}}{{\mathrm{V}}_{\text{extract}}}}$$ (3)

where ΔA is the difference in absorbance between the supernatant at 532 nm and 600 nm, V_total is the total reaction volume (0.4 mL), Ɛ is the molar absorption coefficient for the MDA (1.55 × 10⁵/L/mol/cm), d is the cuvette pathlength (1 cm), V_extract is extraction volume (10 mL), V_sample is the added sample volume (0.1 mL), W is the sample mass (1 g), and 10⁹ is the conversion factor, where 1 mol = 10⁹ nmol.

2.6. In vitro digestion

In vitro simulated digestion was performed according to the INFOGEST static protocol (Brodkorb et al., 2019), which remains the consensus model for healthy adults (Ménard et al., 2023). The electrolyte stock solutions, simulated saliva fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) were prepared as described in Supplementary Table S1. A pH-test adjustment experiment was conducted before the formal experiment to determine the required volumes of HCl and NaOH.

Oral phase: Five grams of reheated sample were mixed with 4 mL SSF, 975 μL water, and 25 μL 0.3 M CaCl₂ (final CaCl₂: 1.5 mM). The mixture was homogenized at 10,000 rpm for 30 s (twice) and incubated at 37 °C, 180 rpm for 2 min.

Gastric phase: To the oral chyme, 8 mL SGF and 0.8 mL 2 M HCl (pH 3.0) were added, followed by CaCl₂ (0.15 mM final) and pepsin (2000 U/mL final). Water was added to reach 1× SGF concentration. The mixture was incubated at 37 °C, 180 rpm for 2 h.

Intestinal phase: After adding 0.8 mL 2 M NaOH (pH 7.0), 8 mL SIF was added, followed by bile (10 mM final), CaCl₂ (0.6 mM final), pancreatin (100 U/mL final), and lipase (2000 U/mL final). Water was added to reach 1× SIF concentration. The mixture was incubated at 37 °C, 180 rpm for 2 h.

Termination: Protein digestion was stopped by adding BBI (0.05 g/L) and lipid digestion by adding 4-bromophenylboronic acid (1 M in methanol) (Williams et al., 2012). The digest was centrifuged at 4 °C, 10,000 ×g for 20 min. The supernatant was stored at −20 °C for analysis, and the undigested residue was dried at 55 °C to constant weight.

2.7. Protein digestibility

2.7.1. Crude protein digestibility

Undigested residues and pre-digested samples were completely digested in a graphite digester (SH420F, Hanon, China). Crude protein content before and after in vitro digestion was determined using an automated Kjeldahl nitrogen analyzer (K1160, Hanon, China) with a nitrogen-to-protein conversion factor of 6.25. The crude protein content of the undigested residue (W_ad) and of the samples after the reheating treatment (W_bd) was recorded. The crude protein digestibility (DT, %) was calculated using Eq. (4).

$$\mathrm{DT}\left(\%\right)=\frac{{\mathrm{W}}_{\mathrm{bd}}-{\mathrm{W}}_{\mathrm{ad}}}{{\mathrm{W}}_{\mathrm{bd}}}\times 100$$ (4)

2.7.2. Degree of protein hydrolysis

The degree of protein hydrolysis (DH) was determined by quantifying free amino groups before and after in vitro digestion using the o-phthalaldehyde (OPA) method (Wang, Tian, et al., 2023). Three sample sets were prepared for each treatment:

• (1)Total amino groups after complete hydrolysis: 1 g of reheated sample (before digestion) was hydrolyzed with 5 mL of 6 M HCl at 110 °C for 16 h, then neutralized with 5 mL of 6 M NaOH and filtered. The supernatant was diluted appropriately for OPA assay. This value represents the total amino groups from all proteins and peptides.
• (2)Initial free amino groups before digestion: 5 g of reheated sample (before digestion) was mixed with the same volume of enzyme-free digestion working solution (i.e., replacing the enzyme solution with double-distilled water) and immediately filtered. The filtrate was used for OPA assay to determine the initial free amino group concentration.
• (3)Final free amino groups after digestion: The supernatant obtained from the in vitro digestion (Section 2.6) was used directly for OPA assay to determine the free amino group concentration after intestinal digestion.

Blank correction: A reagent blank (double-distilled water instead of sample, processed through the same OPA reaction) was used to correct for background absorbance. Additionally, a blank consisting of digestion fluids (SSF, SGF, SIF) without sample was processed in parallel to account for any free amino groups present in the enzyme solutions.

For the OPA assay, 0.4 mL of appropriately diluted sample or blank was mixed with 3 mL of OPA reagent, incubated for 2 min, and the absorbance was measured at 340 nm. A calibration curve was prepared using serine standards (0–100 μg/mL). The free amino group concentration (expressed as serine equivalent, μmol/mL) was calculated from the standard curve.

The degree of hydrolysis (DH, %) was calculated using Eq. (5):

$$\mathrm{DH}\left(\%\right)=\frac{{\left[{\mathrm{NH}}_2\right]}_{\text{final}}-{\left[{\mathrm{NH}}_2\right]}_{\text{initial}}}{{\left[{\mathrm{NH}}_2\right]}_{\text{total}}-{\left[{\mathrm{NH}}_2\right]}_{\text{initial}}}\times 100$$ (5)

where [NH₂]_initial is the free amino group concentration before digestion, [NH₂]_final is the free amino group concentration after in vitro digestion, and [NH₂]_total is the total amino group concentration after complete acid hydrolysis.

2.8. Particle size

To further characterize the extent of protein digestion, the post-digestion supernatant was added to the sample container of a nanoparticle size analyzer (ZSU3200, Malvern Panalytical, UK), and the procedure was executed in accordance with the operation instructions.

2.9. Free amino acid analysis

Free amino acids in the digestive fluid were quantified using an automated amino acid analyzer (Biochrom30+, Biochrom, UK), referring to the study of Zou et al. (2018) with appropriate modifications. Briefly, 1 mL of digestive fluid was deproteinized by mixing with an equal volume of 5% sulfosalicylic acid and centrifuging at 6000 ×g for 10 min. The supernatant was evaporated to dryness (RE-5000, YIBEIER, China), and the residue was dissolved in 1 mL of 0.2 M sodium citrate buffer. Samples were filtered through a 0.22 μm membrane before injection.

2.10. SDS-PAGE analysis

The digestive fluid of each treatment was combined with an equal volume of SDS-PAGE sample buffer and heated at 95 °C for 10 min to ensure complete protein denaturation. Proteins were separated on 5% stacking and 10% resolving gels. Once polymerized, samples and ColorMixed Protein Marker was loaded, and electrophoresis was carried out at 80 V through the stacking gel, and at 110 V in the resolving gel. Gels were stained with Coomassie Brilliant Blue and destained in double-distilled water until bands were clearly visible. Images were acquired by a gel imaging system (iBright 1500, Thermo Fisher, USA).

2.11. Molecular weight distribution of digestion products

The digested products were analyzed by LC-MS/MS according to the method of Wang, Tian, et al. (2020) with appropriate modifications. The digestive juice of each group was subjected to ultrafiltration (10 kDa) and desalting for instrument testing (Yin et al., 2020). Peptide separation was performed on an EASY-nLC 1200 nanoflow UHPLC system (Thermo Fisher Scientific, USA) equipped with a home-packed C18 column (75 μm × 150 mm, 1.9 μm particles). Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in 84% acetonitrile. The flow rate was 250 nL/min with the following gradient: 4–50% B over 50 min, 50–100% B over 4 min, and held at 100% B for 6 min. Mass spectrometry analysis was performed on a Q-Exactive HF-X mass spectrometer (Thermo Fisher Scientific, USA) coupled to the UHPLC system. Positive electrospray ionization (ESI+) was used with a spray voltage of 2.3 kV and an ion transfer tube temperature of 320 °C. Full MS scans were acquired at a resolution of 120,000 (at m/z 200) over a scan range of m/z 350–1800, with an AGC target of 3e6 and a maximum injection time of 50 ms. The top 20 most intense precursor ions were selected for data-dependent MS²(dd-MS²) acquisition with the following parameters: resolution 30,000 (at m/z 200), AGC target 1e5, maximum injection time 45 ms, normalized collision energy 28%, and isolation window 1.6 m/z. Dynamic exclusion was set to 30 s. The acquired MS data were processed using MaxQuant software (version 1.5.5.1; Max Planck Institute of Biochemistry, Martinsried, Germany) for peptide identification and quantification. Database searching was performed against the UniProt Gallus gallus database. Search parameters were as follows: enzyme = trypsin with up to 2 missed cleavages; precursor mass tolerance = 20 ppm; fragment mass tolerance = 0.1 Da; carbamidomethylation of cysteine as a fixed modification; oxidation of methionine and acetylation of protein N-termini as variable modifications; decoy database pattern = reverse; false discovery rate (FDR) ≤ 0.01 at both the peptide and protein levels.

2.12. Fatty acid profile analysis

Free fatty acids (FFAs) in the post-digestion supernatant were extracted, methylated, and analyzed by gas chromatography (GC). The procedure was adapted from Lian et al. (2023), which originally described lipid extraction from cooked chicken wings, with modifications optimized for liquid digestive samples.

Extraction: One milliliter of the digestive supernatant (obtained from Section 2.6) was transferred to a 15 mL glass tube. 6 mL of methanol/chloroform (1:2, v/v) was added, and the mixture was vortexed vigorously for 2 min. After centrifugation at 3000 ×g for 5 min at 4 °C, the lower organic phase (chloroform layer) was carefully transferred to a new tube. The extraction was repeated once with an additional 3 mL of chloroform, and the combined organic phases were evaporated to dryness under a gentle stream of nitrogen at 40 °C.

Saponification and methylation: The dried lipid residue was reconstituted in 4 mL of 2% sodium hydroxide in methanol and heated at 80 °C for 10 min to saponify the lipids. After cooling to room temperature, 4 mL of 15% boron trifluoride (BF₃) in methanol was added, and the mixture was heated at 80 °C for 2 min to methylate the free fatty acids. The mixture was then cooled, and 2 mL of hexane was added to extract the fatty acid methyl esters (FAMEs). The upper hexane phase was collected, and the extraction was repeated once with another 2 mL of hexane. The combined hexane phases were dried over anhydrous sodium sulfate and concentrated to 0.5 mL under nitrogen.

GC analysis: The FAMEs were analyzed using an Agilent 8890 gas chromatograph equipped with an HP-5 capillary column (30 m × 0.32 mm × 0.25 μm). The injection volume was 0.5 μL, and nitrogen (≥ 99.999%) was used as the carrier gas at a flow rate of 1.2 mL/min. The temperature program was as follows: initial temperature 150 °C; increased to 170 °C at 10 °C/min, hold 0.5 min; increased to 200 °C at 5 °C/min, hold 1 min; increased to 260 °C at 2 °C/min, hold 2 min; then increased to 300 °C at 40 °C/min, hold 2 min. Fatty acids were identified by comparing retention times with authentic standards (Supelco 37 Component FAME Mix), and quantified by area normalization. Results are expressed as relative percentage of total identified fatty acids.

2.13. Statistical analysis

Three independent product units (three individual pouches of precooked braised chicken from the same production batch) were used as biological replicates. Each unit was subjected to reheating treatments and all subsequent measurements separately. For each biological replicate, at least three technical replicate measurements were performed for each analytical endpoint, and the technical replicates were averaged before statistical analysis. Thus, for each treatment, data were obtained from three biological replicates (n = 3). While three biological replicates are conventional for comparative studies in food science, we acknowledge that all three units originated from a single batch; therefore, inter-batch variability was not assessed (see Section 3.9). All measurements were performed on three biological replicates (n = 3 independent product units). Experimental data are expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was performed using SPSS 27.0 (IBM, USA), and differences were considered statistically significant at P < 0.05. GraphPad Prism 10.0.1 (GraphPad, USA) was used for graphical analysis. Correlation analysis was performed using Origin Pro 2021 (OriginLab, USA) with Spearman's rank correlation coefficient.

3. Results and discussion

3.1. Protein and lipid oxidation

3.1.1. Total sulfhydryl content

Total sulfhydryl content is an important index of protein oxidation. Guo et al. (2024) demonstrated that total sulfhydryl determination ranks second only to atomic absorption spectroscopy in sensitivity for assessing protein oxidation levels. As illustrated in Fig. 1a, the C group retained the highest total sulfhydryl content, whereas all reheating treatments induced significant total sulfhydryl loss in the following order: MW > WB > ST. These results indicate that reheating promotes protein denaturation and microstructural disruption, leading to depletion of SH groups. Although all reheating treatments achieved the same final center temperature (72.5 °C), they differed markedly in heating rate and total exposure time (MW: 2 min; WB: 8 min; ST: 14 min). Consequently, the overall thermal intensity can be ranked as MW > WB > ST, which explains why the ST group exhibited the lowest level of protein denaturation and thus the highest residual total sulfhydryl content.

Fig. 1.

The oxidation indexes and digestive characteristics of PBC influenced by different reheating methods. (a) Total sulfhydryl content, (b) Carbonyl content, (c) MDA content, (d) Crude protein digestibility, (e) Degree of protein hydrolysis, (f) Particle size (C: the un-reheated samples, MW: the reheated samples by microwave, WB: the reheated samples by boiling water, ST: the reheated samples by steam, * sign is indicated significance at P < 0.05, ** sign is indicated significance at P < 0.01, *** sign is indicated significance at P < 0.001).

3.1.2. Carbonyl content

The carbonyl content of protein is also recognized as one of the indexes of protein oxidation. As presented in Fig. 1b, the carbonyl content showed a numerical increase in all three reheated groups (MW, WB, and ST) relative to C, but the increase was statistically significant only for the MW group (P < 0.05); the WB and ST groups did not differ significantly from C (P > 0.05). Tavares et al. (2018) reported the same observations in heat-treated fish. This phenomenon can be attributed to the heat treatment process, which leads to the disruption of hydrogen or ionic bonds within protein. Consequently, the NH and NH₂ side chains of amino acids become more accessible, allowing them to interact with reactive oxygen species and subsequently form carbonyl groups. In addition, iron in heme is also released and contributes to the binding of free radicals, which further enhances the carbonylation level of proteins (Zhou et al., 2018). The most significant increase in the MW group (P < 0.05) was attributed to the fact that proteins are susceptible to oxidized lipids (as will be discussed in Section 3.1.3). On the one hand, free radicals generated by fat oxidation can be transferred to proteins; on the other hand, derived aldehydes from lipid oxidation can be covalently bound to proteins (Hu et al., 2017). No significant difference (P > 0.05) was observed between the WB and ST groups, likely reflecting their comparable reheating temperatures. Xia et al. (2021) showed that there is a significant correlation between the level of protein carbonylation and heat treatment temperature. Both protein-oxidation indices exhibited a consistent ranking in terms of oxidation levels following the order MW > WB > ST > C.

3.1.3. MDA content

Fig. 1c represents the changes in MDA content of the PBC after reheating treatments. MDA content increased in all reheated groups compared to the C group, which is consistent with the findings of Cabral et al. (2017). Reheating disrupts the microstructure of meat, facilitating interactions between reactive oxygen species and polyunsaturated fatty acids. This accelerates lipid oxidation and inhibits endogenous antioxidant enzymes. Lipid oxidation increased progressively with both reheating temperature and duration (Sobral et al., 2020). Microwave treatment produced the largest rise in MDA levels; similarly, Arguelo et al. (2016) observed the highest lipid oxidation in microwave-reheated chicken tenderloin. This pronounced effect likely reflects the high energy density of microwaves, which, despite shorter exposure times, intensify oxidative processes. The minimal increase in MDA content observed in the WB group may be attributed to oxygen availability, which is a critical determinant of lipid oxidation in foods containing heme, free iron, or salt. When oxygen supply is restricted, such as under vacuum, cooked meat exhibits lower MDA levels (Ortuño et al., 2021). In the WB treatment, samples were reheated inside their original sealed bags in a boiling water bath, which limited oxygen exposure compared with the ST treatment, in which flowing steam allowed greater oxygen contact. Consequently, the ST group exhibited higher MDA accumulation than the WB group. Furthermore, the pronounced reduction in total sulfhydryl content observed in the MW group (Fig. 1a) can be partly attributed to the propagation of oxidative stress from lipid peroxidation to protein oxidation, as microwave heating induced the highest level of lipid oxidation (Wang, Bao, et al., 2020).

3.2. Protein digestibility

Crude protein digestibility and protein hydrolysis degree both serve as indicators of overall degree of protein digestion. Fig. 1d and Fig. 1e illustrate the effects of reheating methods on the in vitro digestibility of PBC. The digestibility of chicken proteins was reduced after reheating treatment, as demonstrated by both crude protein digestibility (DT) and degree of hydrolysis (DH). One-way ANOVA followed by Tukey's post-hoc test revealed that all pairwise differences among the four groups (C, ST, WB, and MW) were statistically significant for both DT and DH (P < 0.05 for each comparison), with the order C > ST > WB > MW in both indices. Such a decline is likely a general consequence of excessive thermal processing in meat products. Li et al. (2017) and Clifford (1930) similarly reported decreased digestibility in meat products subjected to prolonged high-temperature cooking. Reheating precooked meat constitutes a secondary thermal treatment of an already cooked product, typically representing a category of overcooking.

Meanwhile, Wang, Wu, et al. (2023) reported that protein digestion indices were negatively correlated with protein oxidation, and these findings mirror the trends of protein oxidation indices (total sulfhydryl content and carbonyl content) in the present study. This phenomenon may be attributed to two aspects, on the one hand, excessive heating induces aggregation of myofibrillar proteins in meat, thereby reducing the binding sites for digestive enzymes (Zhou et al., 2019); on the other hand, reheating treatment promotes protein oxidation, which in turn alters amino acid residues near enzyme recognition siteand consequently modifies protein digestibility (Yu et al., 2022). The three reheating methods differ in heating rate and heat-transfer mode, which consequently affect the extent of protein denaturation and water migration. Microwave reheating, characterized by rapid internal heating, may promote protein oxidation and aggregation, thereby reducing protease-accessible binding sites. The moderate heat transfer rate and uniform heating during boiling water reheating also cause partial denaturation of proteins. Although steam reheating requires a longer treatment time, it delivers heat more gradually in an approximately 100 °C moist environment. The sustained, gentle thermal input under high humidity favors progressive structural relaxation rather than extensive cross-linking, thereby limiting the formation of insoluble aggregates and preserving enzyme-accessible regions. These differences in heating kinetics and moisture exposure provide a plausible explanation for the relatively preserved digestibility observed in the ST treated samples.

Furthermore, it is noteworthy that the digestibility of crude protein and the extent of protein hydrolysis exhibited similar trends, the variability in the degree of protein hydrolysis was greater than that observed in the digestibility of crude protein. Additional factors may influence protein hydrolysis during digestion. Luo et al. (2018) reported that trace elements (excluding iron) exerted a pronounced effect on the degree of hydrolysis. Ozvural and Bornhorst (2018) found that changes in cooking conditions altered water migration, pH, and structural properties during digestion, thereby further affecting the functional properties of the protein.

3.3. Particle size

Smaller particle size is associated with more efficient protein hydrolysis and digestion. As shown in Fig. 1f, reheating increased the z-average particle size of the digested juice in the order MW > WB > ST > C. Accordingly, the control group achieved the highest digestibility, whereas the MW treatment exhibited the lowest. These findings mirror our earlier digestibility results. Cao et al. (2024) likewise reported that among four heating methods applied to yellow-feathered chicken, WB group yielded smaller particle size than MW group. The most prevalent view in numerous studies to date is that excessive thermal exposure promotes protein oxidation, disrupting spatial structure and thereby impairing the activity of digestive enzymes (Cao et al., 2024; Zhao et al., 2019).

3.4. SDS-PAGE analysis

Fig. 2 shows that most digestion products distributed below 63 kDa. Different reheating treatments had significant effects on the intensity of bands corresponding to actin, creatine kinase (Zhao et al., 2021), tropomyosin β chain, troponin T, tropomyosin α chain, and myosin light chains (MLC1 and MLC2). All reheating treatments exhibited reduced digestibility compared with group C, with band intensities ordered as MW ≈ WB > ST > C for proteins in bands <48 kDa. Gawat et al. (2024) observed a similar pattern in microwave and vacuum-steamed mutton, where microwave-treated samples retained more undigested or partially digested peptides on SDS-PAGE. By contrast, Kaur et al. (2014) reported that raw beef showed better digestibility on SDS-PAGE with increasing intensity of cooking conditions and concluded that beef proteins are more sensitive to protein hydrolysis after protein denaturation.

Fig. 2.

The SDS-PAGE on the protein profile of PBC influenced by different reheating methods. (C: the un-reheated samples, MW: the reheated samples by microwave, WB: the reheated samples by boiling water, ST: the reheated samples by steam).

These discrepancies suggest that the extent of protein oxidation critically modulates digestion. Moderate oxidation during heating can alter spatial structure and increase permeability, thereby exposing additional hydrolysis sites and enhancing enzyme access. However, excessive oxidative modification induces cross-linking and aggregation, which diminishes hydrolase binding and reduces overall digestibility (Bhat et al., 2019; Lee et al., 2023; Mitra et al., 2022). As a cooked product, PBC undergoes substantial protein oxidation upon reheating, explaining the observed decline in digestion; notably, trends in oxidation and digestibility were closely aligned in this study. Actin, known for its resistance to complete hydrolysis (Zhao et al., 2021), showed no significant differences in the MW and WB treatments, whereas ST treatment reduced actin band intensity relative to group C. Salt concentration further influenced digestion: increasing salt concentration inhibits protein digestibility within a certain range (Choi & Chin, 2024). The flowing steam in ST promoted partial salt loss, resulting in lower residual salt and improved digestibility compared with other reheating methods. Finally, the emergence of a distinct band below 11 kDa range in the reheating groups, in contrast to the group C, suggests that reheating may modify specific enzyme-binding sites in PBC, thereby impeding the extent of peptide hydrolysis.

3.5. Molecular weight distribution of digestion products

Proteins were digested to generate small molecular peptides and free amino acids. Small molecular peptides have higher bioavailability than large molecular peptides because they are more easily absorbed by the body through the intestinal wall. In addition, molecular peptides <1000 Da have certain antioxidant properties, and the higher the content of small molecular peptides in the digested products, the higher the nutritive value can be demonstrated (Wang et al., 2021). As displayed in Fig. 3, the molecular weight distribution can be divided into four intervals, namely <500 Da, 500–1000 Da, 1000–1500 Da and > 1500 Da. The figure shows that the digestive products were mainly concentrated in the two ranges of <500 Da and 500–1000 Da. The relative content of small molecular weight peptides in the C (96.36%) and ST (96.56%) groups was much higher than those in the MW (92.18%) and WB (90.53%) groups. Although the number of peptide chains in this interval in the MW (1434) and WB (1276) groups was not significantly different from that in the C (1285) group (P > 0.05), it was far less than that in the ST (1757) group. In the two intervals of 1000–1500 Da and > 1500 Da, there was no significant difference between the all groups. These results correspond to the previous results of SDS-PAGE, in which more large peptides were hydrolyzed into small peptides in groups C and ST. Wang et al. (2018) similarly reported that the peptide content of each digested product identified was positively correlated with its in vitro digestibility under the same enzyme conditions of treatment. The reduction in small peptide content of the MW and WB groups might be due to the fact that the higher degree of oxidative denaturation of their proteins in these groups (Yin et al., 2020), which was consistent with the trend of changes in oxidative indexes as we discussed in this paper. The ST group showed better performance in the creation of low molecular weight (< 1000 Da) digestive products compared to the C group. That might indirectly indicate that protein oxidation is not the only factor that can affect the degree of digestion. We hypothesize that the ST reheating treatment, which involves a relatively slow heating rate, uniform heat diffusion, and a high-humidity environment (approximately 100  °C saturated steam), may preserve the native-like conformational flexibility of proteins better than the other methods. This could expose or retain specific enzyme-binding sites while minimizing heat-induced aggregation and cross-linking, thereby facilitating the generation of peptides within the <1000 Da range. In contrast, microwave reheating (rapid volumetric heating) and water boiling (direct convective heat transfer in liquid) may cause more extensive protein denaturation and oxidation, leading to a lower yield of small peptides. Thus, both the temperature profile and the heat transfer mechanism appear to contribute to the observed differences.

Fig. 3.

The peptide distribution in digestion product of PBC influenced by different reheating methods. (a) The count of peptide chains of each range, (b) The relative percentage of peptide chains of each range (C: the un-reheated samples, MW: the reheated samples by microwave, WB: the reheated samples by boiling water, ST: the reheated samples by steam, * sign is indicated significance at P < 0.05, ** sign is indicated significance at P < 0.01, *** sign is indicated significance at P < 0.001).

3.6. Visualization of the correlation

Fig. 4 shows correlations among protein oxidation, lipid oxidation and digestion characteristics. In the upper right of the plot, the gap between the long and short axes of the ellipse indicates the absolute magnitude of the correlation coefficient, and the color of the ellipse is used to represent the positivity or negativity of the correlation coefficients. The corresponding value of the corresponding index could be found in the lower left corner. As can be seen, the content of total sulfhydryl content showed a significant positive correlation with crude protein digestibility and hydrolysis degree (r = 0.74 and 0.78, P < 0.01), and showed a significant negative correlation with particle size (r = −0.90, P < 0.001). Meanwhile, there was a negative correlation between total sulfhydryl content and carbonyl content (r = −0.64, P < 0.05). Carbonyl groups exhibited an opposite trend in the correlation results with digestion property parameters compared to total sulfhydryl content. This result conforms to the majority of current research (Ma et al., 2021; Sun et al., 2011). Moreover, the total sulfhydryl content was negatively correlated with the proportion of peptides >1000 Da (P < 0.05), suggesting that oxidative modification of proteins may be associated with reduced digestibility (Jiang et al., 2021; Liu et al., 2021). Other co-varying factors, such as heat induced structural denaturation, protein aggregation, water redistribution and matrix effects, may also contribute to the observed shift in peptide size distribution and the reduction in overall digestibility. In terms of lipid oxidation, the content of MDA showed a negative correlation with the degree of protein hydrolysis (r = −0.61, P < 0.05), and a positive correlation with the particle size (r = 0.71, P < 0.01). Whilst, it showed a significant positive correlation with the number of peptides in the <1500 Da range (P < 0.05).

Fig. 4.

Correlation analysis between protein oxidation, lipid oxidation and various digestion property parameters. A: total sulfhydryl content; B: carbonyl content, C: MDA content, D: DT, E: DH, F: particle size, G: percentage of peptides in the <500 Da range, H: percentage of peptides in the 500–1000 Da range, I: percentage of peptides in the 1000–1500 Da range, J: percentage of peptides in the >1500 Da range, K: number of peptides in the <500 Da range, L: number of peptides in the 500–1000 Da range, M: number of peptides in the 1000–1500 Da range, N: number of peptides in the >1500 Da range (* Correlation is significant at P < 0.05, ** Correlation is significant at P < 0.01, *** Correlation is significant at P < 0.001).

In contrast, Öztürk-Kerimoglu et al. (2019) reported that there was no correlation between lipid oxidation degree and in vitro digestibility in heat-treated fermented sausages. The reason for this phenomenon may be due to the following points. First, matrix differences. Fermented sausages contain curing salts (nitrite/nitrate), which act as radical scavengers and can inhibit lipid-mediated protein carbonylation. Nitrite reacts with lipid hydroperoxides and secondary oxidation products, thereby uncoupling the MDA-digestibility relationship. In our precooked braised chicken, no curing agents are added, making proteins more susceptible to aldehyde-induced cross-linking and aggregation. Second, oxidation timing and intensity. In fermented sausages, lipid oxidation occurs gradually during the ripening period (weeks to months), allowing endogenous antioxidant systems or matrix adaptations to mitigate the impact of oxidative damage on protein digestibility. In contrast, our reheating treatment imposes a brief (2–14 min) but intense thermal insult on an already cooked product, promoting simultaneous and concentrated oxidative damage that may overwhelm any adaptive responses. Third, aldehyde-protein adduction chemistry. MDA and other secondary lipid oxidation products can covalently modify nucleophilic amino acid residues (lysine, arginine, histidine) via Schiff base formation or Michael addition, generating intra- and intermolecular cross-links that reduce protease accessibility. This mechanism is well-documented in cooked meat systems (Ma et al., 2021; Zhou et al., 2018) but may be less pronounced in fermented sausages, where lower pH (typically 4.5–5.0) and extensive proteolysis during ripening alter the reaction environment and may preferentially degrade aldehyde-modified proteins.

Therefore, while lipid oxidation is not the sole determinant of protein digestibility, its impact becomes significant when oxidation occurs rapidly and in the absence of curing salts—conditions that characterize the reheating of precooked meat products. This discrepancy highlights the importance of considering product-specific factors (formulation, processing history, oxidation kinetics) when interpreting correlations between lipid oxidation and protein digestibility.

3.7. Free amino acid analysis

Amino acids, as essential protein constituents, are nutrients that need to be ingested for humans. They not only influence meat flavor but also contribute significantly to product texture (Dashdorj et al., 2015; Zou et al., 2021). Fig. 5 presents the profile of free amino acids in the digestive fluid following in vitro digestion of PBC subjected to different reheating treatments. As shown in Fig. 5a, all reheated groups released greater amounts of free amino acids than the non-reheated group C. This enhancement likely reflects heat treatment promoted the hydrolysis of proteins and peptide chains, resulting in higher levels of free amino acids after digestion (Rotola-Pukkila et al., 2015).

Fig. 5.

Free amino acids profiles in final digestive juice of PBC influenced by different reheating methods. (a) The content of various free amino acids, (b) The ratio of EAA to TAA and NEAA. Notes: C: the un-reheated samples, MW: the reheated samples by microwave, WB: the reheated samples by boiling water, ST: the reheated samples by steam. * indicates significant differences at a level of P < 0.05; ** denotes significant differences at a level of P < 0.01; *** indicates significant differences at a level of P < 0.001.

The essential amino acid (EAA) / non-essential amino acid (NEAA) is a key indicator of protein quality, ranging from 0.6 to 0.9, with increasing values indicating better protein quality. In this study, EAA dominated the overall amino acid (TAA), and the ratio of EAA to NEAA was greater than 0.9 in all groups (Figure  5b). The EAA/NEAA ratios were 0.98 (C), 1.01 (MW), 1.02 (WB), and 1.03 (ST). One-way ANOVA showed a significant overall difference among the four groups (P < 0.05). Post-hoc comparisons revealed that each of the three reheated groups (MW, WB, ST) had significantly higher EAA/NEAA ratios than the non-reheated control C (P < 0.05), whereas no significant differences were detected among the three reheating methods themselves (P > 0.05). This observation may be attributed to the fact that EAA could be more extensively released from cooked meat proteins than NEAA during in vitro digestion. Cooked meat products such as beef, turkey, chicken and pork had EAA/NEAA ratios of 1.68, 2.13, 2.24 and 2.87 after digestion, respectively (Hernández-Olivas et al., 2022). Considering the goal of enhancing the bioavailability of amino acids by achieving elevated levels of EAA/NEAA, ST treatment emerges as a superior method for reheating.

3.8. Free fatty acid analysis

The fatty acid profiles of the digestive fluids after in vitro digestion are presented in Table 1. All samples originated from the same production batch, and the reheating treatments (MW, WB, ST) did not alter the total fat mass; therefore, differences in the relative proportions of fatty acids primarily reflect oxidation during reheating and subsequent differential release during digestion.

Table 1.

Effect of different reheating methods on FFAs.

Fatty acids	FA content (% total FA)
	C	MW	WB	ST
C4:0	0.09 ± 0.02a	0.08 ± 0.01ab	0.06 ± 0.05bc	0.05 ± 0.01c
C6:0	2.25 ± 0.31a	2.19 ± 0.15a	2.15 ± 0.87a	2.07 ± 0.14a
C8:0	12.31 ± 1.59a	12.21 ± 0.02a	10.08 ± 2.11a	12.03 ± 1.27a
C10:0	0.73 ± 0.05a	0.73 ± 0.06a	0.80 ± 0.17a	0.76 ± 0.09a
C11:0	0.07 ± 0.10a	0.81 ± 0.03a	0.72 ± 0.08a	0.77 ± 0.13a
C12:0	1.07 ± 0.06a	1.29 ± 0.03a	1.27 ± 0.19a	1.38 ± 0.24a
C14:1	0.99 ± 0.08b	1.24 ± 0.04ab	1.18 ± 0.13ab	1.31 ± 0.20a
C15:0	0.63 ± 0.16a	0.62 ± 0.05a	0.59 ± 0.10a	0.59 ± 0.11a
C16:0	27.52 ± 0.79a	30.66 ± 0.59a	25.55 ± 3.15ab	18.39 ± 7.14b
C16:1	25.99 ± 1.97a	24.64 ± 0.41a	29.14 ± 2.42a	31.24 ± 7.24a
C17:1	0.13 ± 0.01b	0.13 ± 0.00b	0.15 ± 0.03ab	0.17 ± 0.02a
C18:1n9c	13.40 ± 0.92a	13.22 ± 0.28a	14.24 ± 2.49a	14.84 ± 4.98a
C18:3n6	1.23 ± 0.47a	1.41 ± 0.30a	8.42 ± 6.48a	8.59 ± 6.76a
C20:0	7.87 ± 1.32a	2.54 ± 1.29b	0.49 ± 0.18b	1.96 ± 1.98b
C21:0	1.51 ± 1.28b	4.18 ± 0.43a	2.77 ± 0.45ab	3.55 ± 1.28a
C24:0	NDb	NDb	0.41 ± 0.37ab	0.97 ± 0.82a
C24:1	1.72 ± 0.25a	1.46 ± 0.32ab	1.19 ± 0.32bc	0.84 ± 0.13c
C22:6n3 (DHA)	1.89 ± 0.98a	2.58 ± 0.11a	0.82 ± 0.13b	0.48 ± 0.19b
SFA	54.64 ± 1.18a	55.31 ± 0.04a	44.87 ± 5.97b	42.53 ± 5.73b
MUFA	42.23 ± 2.59a	40.70 ± 0.23a	45.90 ± 1.73a	48.34 ± 12.04a
PUFA	3.12 ± 1.41a	3.99 ± 0.19a	9.23 ± 6.37a	9.07 ± 6.59a
UFA	45.36 ± 1.18b	44.69 ± 0.04b	55.13 ± 5.97a	57.47 ± 5.73a
N-6 PUFA	1.23 ± 0.47a	1.41 ± 0.30a	8.42 ± 6.48a	8.59 ± 6.76a
N-3 PUFA	1.89 ± 0.98a	2.58 ± 0.11a	0.82 ± 0.13b	0.48 ± 0.19b
Ratio of N-6/N-3	0.71 ± 0.24b	0.55 ± 0.13b	11.23 ± 8.86ab	22.17 ± 18.32a

Note: C: the un-reheated samples, MW: the reheated samples by microwave, WB: the reheated samples by boiling water, ST: the reheated samples by steam.

SFA: C4:0, C6:0, C8:0, C10:0, C11:0, C12:0, C15:0, C16:0, C20:0, C21:0, C24:0. UFA: C14:1, C16:1, C17:1, C18:1n9c, C18:3n6, C24:1, C22:6n3 (DHA). MUFA: C14:1, C16:1, C17:1, C18:1n9c, C24:1. PUFA: C18:3n6, C22:6n3. N-6 PUFA: C18:3n6. N-3 PUFA: C22:6n3 (DHA).

Means in the same row with different letters differ significantly (P < 0.05). ND is represented as undetected.

Among saturated fatty acids (SFA), C16:0 (palmitic acid) was the predominant species. Its relative content was significantly higher in the C (27.52%) and MW (30.66%) groups than in the ST (18.39%) group (P < 0.05). C20:0 was markedly lower in WB (0.49%) and ST (1.96%) compared to C (7.87%) and MW (2.54%) (P < 0.05). C24:0 was detected only in WB (0.41%) and ST (0.97%), while it was undetectable in C and MW. The total SFA content followed the order MW (55.31%) > C (54.64%) > WB (44.87%) > ST (42.53%), with significant differences between the two groups with higher oxidation (C and MW) and the two groups with lower oxidation (WB and ST) (P < 0.05). This pattern is consistent with the MDA results (Fig. 1c), where MW exhibited the highest lipid oxidation and ST the lowest. More extensive oxidation likely leads to the relative enrichment of SFA due to the preferential degradation of unsaturated fatty acids.

For monounsaturated fatty acids (MUFA), C16:1 was the most abundant. No significant differences were observed among the four groups for total MUFA or total polyunsaturated fatty acids (PUFA) (P > 0.05), as indicated by the shared superscript letters in Table 1. However, individual PUFA species showed notable changes. The n-3 PUFA DHA (C22:6n3) was significantly higher in C (1.89%) and MW (2.58%) than in WB (0.82%) and ST (0.48%) (P < 0.05). The n-6 PUFA (C18:3n6) content was numerically but not significantly higher in WB (8.42%) and ST (8.59%) compared to C (1.23%) and MW (1.41%) (P > 0.05 due to large standard deviations). Consequently, the n-6/n-3 ratio varied widely among treatments, from 0.55 (MW) to 22.17 (ST), although the differences were not statistically significant owing to high variability. These results suggest that the release and/or degradation of specific PUFA during digestion are differentially affected by the reheating method, but the overall unsaturated fatty acid pool remains largely unchanged across treatments.

In summary, the degree of lipid oxidation induced by different reheating methods correlates with the relative SFA content in the digesta, with higher oxidation leading to greater SFA enrichment. However, the total MUFA and PUFA proportions were not significantly altered, likely because of counteracting effects of oxidation and differential enzymatic release.

3.9. Study limitations

The following limitations should be considered when interpreting the findings of this study. First, regarding product source and batch variability. This study used a single commercially produced PBC from Henan Shuanghui Investment and Development Co., Ltd. Although three independent product units from the same production batch were used as biological replicates, all units originated from the same batch. Consequently, inter-batch variability (e.g., differences in raw material quality, seasoning formulation, or sterilization conditions) was not assessed. Future studies should include products from multiple production batches to evaluate the robustness of the findings. Second, regarding brand and formulation differences. The conclusions drawn from this study are specific to the particular brand and formulation tested. Different manufacturers may use distinct formulations (e.g., varying levels of salt, sugar, spices, or food additives) or different processing parameters (e.g., sterilization temperature and time), all of which could influence the extent of protein and lipid oxidation during reheating and, consequently, the in vitro digestibility. Caution should therefore be exercised when extrapolating our conclusions to other precooked braised chicken products or to precooked meat products in general. Third, regarding the in vitro digestion model. This study employed the static INFOGEST protocol, which is a standardized and widely accepted model for simulating gastrointestinal digestion. However, static models have inherent limitations: they do not capture dynamic physiological processes such as gradual pH changes, continuous enzyme and bile salt secretion, peristaltic mixing, or the progressive absorption of digestion products. Dynamic in vitro digestion models (e.g., DIDGI®, TIM-1, or the dynamic gastric model) offer more physiologically relevant predictions of in vivo digestion by simulating these processes, but they are more resource-intensive and require specialized equipment. Our static model results should therefore be considered an initial screening of the effects of reheating methods on digestibility, and validation using dynamic digestion models is warranted in future research. Fourth, regarding extrapolation to in vivo conditions. While in vitro digestion models provide valuable insights into the fate of food components under simulated gastrointestinal conditions, they cannot fully replicate the complexity of the human digestive system, including the gut microbiota, immune responses, and individual physiological variations. Consequently, extrapolation of our findings to in vivo nutrient absorption should be made with caution, and further studies using animal models or human trials are needed to confirm the nutritional implications. Fifth, regarding the absence of a non-sterilized raw control. All samples used in this study were sterilized (115 °C, 55 min) prior to reheating treatments. Therefore, we cannot distinguish the individual contributions of sterilization versus reheating to the observed protein and lipid oxidation, nor to the reduction in digestibility. The non-reheated sterilized sample (group C) served as a practical baseline reflecting the no reheating consumer scenario, but it does not represent the unprocessed raw meat. Future studies should include a non-sterilized control (e.g., raw or simply cooked chicken without commercial sterilization) to fully decouple the effects of sterilization and reheating.

4. Conclusion

This study investigated the effects of different reheating methods on oxidation levels, digestive characteristics, and partial nutrient contents of PBC, and evaluated the correlations between oxidative and digestive indices. The results showed that the reheating treatment would cause overcooking on the original cooked meat products, which was manifested by the deepening of the oxidization degree, and then led to the decrease of digestive characteristics. The ST treatment exhibited the minimal degree of oxidation and the highest degree of digestion within the reheating groups. These findings provide useful guidance for selecting reheating methods for precooked meat dishes in household settings. Nevertheless, given the inherent limitations of static in vitro digestion models, further validation using in vivo studies (e.g., animal models or human trials) is necessary to confirm the nutritional implications of our findings under physiological conditions. However, the mechanisms by which various reheating treatments affect the physicochemical properties of these products remain poorly understood. Similarly, the impact of reheating on nutritional components, such as amino acids and fatty acids, has yet to be clarified and necessitates further investigation.

CRediT authorship contribution statement

Junguang Li: Writing – review & editing, Methodology, Conceptualization. Hewei Shi: Writing – review & editing, Methodology. Yaxin Bai: Formal analysis. Yu Wang: Writing – original draft, Validation, Investigation. Lichuang Cao: Writing – review & editing, Validation. Sihao Liu: Validation, Supervision. Dianbo Zhao: Validation, Supervision. Shaohua Meng: Formal analysis. Yanhong Bai: Supervision, Project administration, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the supplementary data related to this article.

Supplementary Fig. S1.

Processing flowchart of precooked braised chicken.

Supplementary Table S1

Volumes of electrolyte stock solutions of digestion fluids for a volume of 400 mL diluted with water (1.25× concentrations).

Data availability

Data will be made available on request.