Micro-nano bubble pretreatment differentially regulates gel properties and flavor of lamb meat via fermentation and lipolysis

Abstract

This study explored the effects of micro-nano bubble (MNB) pretreatment combined with Lactiplantibacillus plantarum (Lp) fermentation or lipase (Lip) hydrolysis on lamb meat quality, gel properties, and flavor. SEM revealed that MNB loosened muscle fibers and increased porosity, while FT-IR indicated that MNB alone did not alter protein secondary structure. LF-NMR showed MNB + Lp triggered conversion of immobile to free water, whereas MNB + Lip maintained superior water retention. MNB + Lp achieved the lowest pH, reduced MDA by 71%, raised total free fatty acids to 2.87 g/100 g, and increased volatile aldehydes 7.9-fold; nonanal, (E)-2-nonenal, and (E)-2-decenal were key odor-active compounds, improving flavor. MNB + Lip increased free sulfhydryl and hydrophobic interactions, promoting a uniform gel network with enhanced water holding capacity and springiness of gels. Overall, MNB pretreatment enhanced biological modification in a pathway-dependent manner, enabling differentiated regulation of lamb flavor and processing characteristics, and providing a green and efficient strategy for lamb processing.

Graphical abstract

Highlights

• •MNB loosens muscle fibers and reduces mass transfer resistance.
• •MNB + Lp acidifies, enriching aldehydes while suppressing lipid oxidation.
• •MNB + Lip enhances hydrophobicity & -SH, forming uniform gel with higher WHC.
• •MNB differently regulates lamb: flavor via Lp and gel properties via Lip hydrolysis.

1. Introduction

Lamb meat is favored for its unique flavor and high nutritional value, enjoying broad market prospects. Nevertheless, raw lamb suffers from inherent defects including tough texture, poor water-holding capacity, strong gamey odor, high lipid oxidation susceptibility and unstable heat-induced gel properties (Fu et al., 2025; Xu et al., 2026). These issues impair product quality and shelf life, limiting the high-value development of lamb deep-processing industry.

Traditional physical treatments such as ultrasound, high pressure and vacuum tumbling mainly improve meat texture by disrupting muscle fibers, yet they hardly optimize flavor, oxidation resistance and gel properties simultaneously (Chen et al., 2024; Xu et al., 2025). Microbial fermentation and enzymatic catalysis are promising green strategies for meat modification. Lactiplantibacillus plantarum (Lp) lowers pH, inhibits spoilage bacteria and improves flavor and texture (Jiang et al., 2025). Lipase hydrolyzes intramuscular lipids to release flavor precursors and regulate nutritional traits (Fu et al., 2022; Wei et al., 2023). However, the dense structure of lamb muscle creates high mass transfer resistance, limiting the accessibility of microbes and enzymes to the interior of the meat matrix. Pure biological treatments thus lead to insufficient modification and unstable product quality, restricting their large-scale application.

Micro-nano bubbles (MNBs), defined as gas bubbles smaller than 1 μm, feature large specific surface area, high stability and negatively charged surfaces (Chuenchart et al., 2021; Fan et al., 2023; Zhang et al., 2020). Widely used in fruit and vegetable cleaning, MNBs achieve deep penetration, decontamination and sterilization via their small size and physicochemical properties (Fan et al., 2023; Malahlela et al., 2025). Recent studies have demonstrated the potential of micro- and nano-bubbles in meat and seafood processing: micro-bubble treatment improved the physicochemical properties and textural quality of shrimp (Huang et al., 2025), while nanobubbles were shown to protect myofibrillar proteins and enhance tenderness during mutton cooking (Si et al., 2023). However, systematic studies on combining MNB pretreatment with biological modification for lamb quality regulation remain scarce. MNB can remove residual contaminants, while their nano-scale properties may facilitate mild structural loosening of muscle tissue, potentially enhancing texture and water-holding capacity.

Given this knowledge gap, we hypothesize that MNB pretreatment reduces mass transfer resistance in lamb muscle, thereby enhancing the efficacy of subsequent biological agents. To validate this hypothesis, two representative biological strategies with distinct functional targets were selected: Lp fermentation for flavor improvement (reducing gamey odor and enriching volatile profiles) and lipase hydrolysis for texture modulation (releasing free fatty acids to regulate protein gelation). These pathways are expected to be differentially enhanced by MNB pretreatment for either flavor-oriented or texture-oriented applications.

To our knowledge, this is the first study to employ MNB pretreatment as a physical platform to synergistically enhance subsequent biological modification (Lp fermentation and lipase hydrolysis) for the differentiated regulation of lamb meat quality, gel properties, and flavor. Herein, we systematically investigated the synergistic effects of MNB pretreatment combined with Lp fermentation or lipase hydrolysis on lamb quality, gel properties, water distribution, oxidative stability, and volatile profiles, aiming to clarify the interactions among MNB-induced structural loosening, biological metabolism, protein conformation, lipid oxidation, and flavor formation, and to establish a green and industrially feasible processing technology for differentiated lamb products.

2. Materials and methods

2.1. Materials and reagents

Fresh hind leg meat of Small Tail Han sheep was collected within 2 h post-slaughter from Xinxiang Yuxuan Food Co., Ltd. (Henan, China). The lambs (6-month-old males, live weight 38.9 ± 2.1 kg, carcass weight 18.6 ± 1.3 kg, backfat thickness 0.88 ± 0.11 cm) were raised under intensive conditions with a standard diet and had ad libitum access to water. They were subjected to 24 h feed and 2 h water withdrawal before standard commercial slaughter. All slaughter procedures were performed by qualified personnel in a commercial slaughterhouse in accordance with national regulations on commercial slaughter and animal welfare. This study did not involve live animal experiments; therefore, no animal ethics approval was required. The initial pH and moisture content of the raw meat were 5.8 ± 0.2 and 72.3 ± 0.57%, respectively. Samples of uniform color and free of bruising, obvious fascia, superficial fat, and connective tissue were selected for use.

Lipase (≥100,000 U/g) and freeze-dried Lactiplantibacillus plantarum powder (≥1 × 10⁸ CFU/g, strain HH-LP56) were purchased from Henan Anyue Biotechnology Co., Ltd. and Minsheng Zhongke Jiayi Bioengineering Co., Ltd., respectively. All other reagents were of analytical grade and supplied by Sinopharm Chemical Reagent Co., Ltd.

2.2. Meat sample preparation

Three Small Tail Han sheep were used as three independent biological replicates. Immediately post-slaughter, hind leg meat was collected from each carcass separately. After trimming residual fat and fascia with a sterile scalpel, the meat from each animal was cut into uniform cubes (1.0 cm × 1.0 cm × 1.0 cm), rinsed twice with sterile distilled water, and drained for 5 min at room temperature. From each animal, the cubed meat was randomly allocated to six treatment groups (Control, Lp, Lip, MNB, MNB + Lp, and MNB + Lip), with approximately 250 g of meat from each animal per group. Thus, each treatment group contained three independent biological replicates derived from three individual animals. All pretreatment operations were performed in a sterile laminar flow cabinet to avoid microbial contamination. The whole treatment process consisted of two stages: 1.5 h at 4 °C followed by 1.5 h at 30 °C, with a total treatment duration of 3 h for all groups.

2.2.1. Control treatment (control)

Lamb cubes were immersed in sterile ultrapure water at 4 °C for 1.5 h with gentle shaking every 30 min. After draining, fresh sterile ultrapure water was added, and the samples were continuously soaked at 30 °C for another 1.5 h with regular shaking. Finally, the samples were drained and collected for subsequent analysis.

2.2.2. Lactiplantibacillus plantarum treatment (Lp)

The bacterial suspension was prepared by dissolving 5.0 g of Lp freeze-dried powder and 2.5 g of trehalose in 250 mL of sterile water, yielding a final concentration of 2 × 10⁶ CFU/mL (corresponding to approximately 2 × 10⁶ CFU/g meat after mixing with the samples). Lamb cubes were first soaked in sterile water at 4 °C for 1.5 h, consistent with the first-stage treatment of the Control group. After draining, the samples were mixed with the prepared bacterial suspension and statically fermented at 30 °C for 1.5 h, then stored at 4 °C for subsequent detection. The inoculation concentration and fermentation duration were determined based on literature references and preliminary experiments. This concentration falls within the typical range (10⁶–10⁸ CFU/g) reported for Lactiplantibacillus plantarum fermentation of meat products (Gao et al., 2024; Liang et al., 2026; Velasco et al., 2021). Preliminary experiments comparing three concentrations (10⁶, 10⁷, and 10⁸ CFU/g) showed that the 10⁶ CFU/g level was the most effective, and further fine-tuning within this level identified 2 × 10⁶ CFU/g as the optimal concentration, which achieved the desired acidification within 1.5 h without over-acidification, while longer fermentation (e.g., 3 h) caused excessive acidification and quality deterioration.

2.2.3. Lipase treatment (lip)

A total of 1.25 g of lipase (≥100,000 U/g) was dissolved in 250 mL sterile water to prepare the working solution, corresponding to an enzyme dosage of 500 U/g meat. Lamb cubes were pretreated in sterile water at 4 °C for 1.5 h, drained, and incubated with lipase solution at 30 °C for 1.5 h under static conditions. The treated samples were stored at 4 °C. The lipase dosage (500 U/g) and temperature (30 °C) were selected based on supplier recommendations and preliminary experiments. The supplier recommends a usage range of 200–1000 U/g meat for this enzyme in meat systems. Dose screening (200, 500, and 1000 U/g) showed that 500 U/g achieved higher FFA release than 200 U/g, while the difference between 500 and 1000 U/g was marginal; thus, 500 U/g was selected as the optimal cost-effective dosage. Regarding temperature, the supplier indicates an optimal activity range of 30–60 °C; 30 °C was chosen as it falls within this range while minimizing the risk of meat quality deterioration associated with higher temperatures.

2.2.4. Micro-nano bubble treatment (MNB)

MNB water was generated using a bubble generator (ZJC-NM, Shanghai Zhongjing Environmental Protection Technology Co., Ltd., China). The initial pH and DO of MNB water were 7.15 ± 0.03 and 12.83 ± 0.04 mg/L, respectively (vs. 8.44 ± 0.04 mg/L DO for distilled water), and the Zeta potential was −25.63 ± 0.28 mV (Supplementary Methods S1, Fig. S1A–C). The MNB water remained stable over the 1.5 h treatment period, with pH and DO variations of <0.42 and < 0.5 mg/L, respectively (Supplementary Fig. S1A and B). Nanoparticle tracking analysis (NTA) confirmed that the bubbles had a median diameter (D50) of 68 nm, with a size distribution of 60–100 nm (Supplementary Fig. S2, Tables S1 and S2). Lamb cubes were immersed in 250 mL of the freshly prepared MNB water at 4 °C for 1.5 h with gentle shaking every 30 min. After draining, fresh sterile ultrapure water was added, and the samples were soaked at 30 °C for another 1.5 h to maintain a consistent total treatment duration (3 h) across all groups.

2.2.5. MNB combined with Lactiplantibacillus plantarum treatment (MNB + Lp)

Lamb cubes were firstly pretreated with MNB water at 4 °C for 1.5 h under continuous aeration, following the procedure described in 2.2.4. After draining, the samples were mixed with Lp bacterial suspension and statically fermented at 30 °C for 1.5 h, consistent with the fermentation procedure in 2.2.2. The final samples were stored at 4 °C. MNB pretreatment was performed prior to Lp fermentation rather than simultaneously, thereby avoiding direct exposure of Lp cells to MNB water. Accordingly, MNB serves solely to physically modify the meat matrix for enhanced bacterial accessibility and distribution, without affecting bacterial viability.

2.2.6. MNB combined with lipase treatment (MNB + lip)

Lamb samples received the same MNB pretreatment at 4 °C for 1.5 h as described in 2.2.4. After draining, the lipase working solution was added, and the samples were statically incubated at 30 °C for 1.5 h according to the procedure in 2.2.3. All treated samples were stored at 4 °C for subsequent analysis.

2.3. Determination of pH value

After completing the two-stage treatment, 5.0 g of minced meat sample was mixed with 5.0 mL of distilled water and homogenized at 10,000 rpm for 1 min. The pH of the homogenate was measured at room temperature using a pre-calibrated portable pH meter.

2.4. Preparation of heat-induced gels

Myofibrillar protein (MP) was extracted from lamb meat following a modified method (Yu et al., 2025). Minced meat was homogenized with four volumes of 20 mmol/L PBS (pH 7.5) and centrifuged, and the precipitate was rinsed with 0.1 mol/L NaCl. The precipitate was then re-homogenized with six volumes of PBS containing 0.1 mol/L NaCl, followed by filtration and centrifugation to obtain purified MP. The MP suspension was adjusted to 60 mg/mL, and 5 mL was heated sequentially at 40 °C for 30 min and 90 °C for 30 min. The resulting gels were cooled in ice water and stored at 4 °C overnight for subsequent analysis.

2.5. Hydroxyl radical scavenging activity of MNB water

The hydroxyl radical scavenging capacity of MNB water was determined using a commercial assay kit (Nanjing Jiancheng Bioengineering Institute, Cat. No. A018-1-1) based on the colorimetric method. MNB water was freshly prepared as described in 2.2.4. The absorbance was measured at 550 nm, and the scavenging rate was calculated following the manufacturer's instructions.

2.6. Scanning electron microscopy (SEM) observation

Samples (5 × 5 × 1 mm) were fixed in 2.5% glutaraldehyde in 0.2 mol/L PBS (pH 7.2) at 4 °C for 12 h, rinsed with PBS, and dehydrated in graded ethanol series (50–100%) (Wang, Li, et al., 2025). After freeze-drying, specimens were gold-sputtered and examined under a scanning electron microscope (Hitachi S-3400) at 5 kV. Porosity was quantified using ImageJ software (Version 1.53 k, National Institutes of Health, USA). For each treatment group, three SEM images were randomly captured from three independent biological replicates (one image per replicate). After grayscale conversion, pore areas were identified using automatic threshold adjustment (Default algorithm), and porosity was expressed as the percentage of pore area relative to the total image area.

2.7. Low-field nuclear magnetic resonance (LF-NMR) measurement

Water distribution and mobility were determined using an LF-NMR instrument (NMI20-025V-I, Niumag, China) according to the method of Wang et al. Wang, Li, et al., 2025). Uniform meat samples were placed in glass tubes, and transverse relaxation time (T₂) was measured at 25 °C using the Carr-Purcell-Meiboom-Gill pulse sequence. The instrumental parameters were set as follows: spectral width 200 kHz, 90° pulse width 8.52 μs, 180° pulse width 17.04 μs, echo time 0.35 ms, echo number 15,000, scanning times 8, receive gain 20 dB, and digital gain 3.

2.8. Texture profile analysis (TPA)

TPA analysis was performed by the method of Wang et al. (Wang, Li, et al., 2025) with a slight modification. The texture properties of raw lamb and heat-induced gels were determined using a texture analyzer (TA.XTC-18, Bosun, China) equipped with a P/50 cylindrical probe. The test parameters were set as: pre-test speed 2.0 mm/s, post-test speed 2.0 mm/s, strain 50%. The probe was pressed vertically against the muscle fiber direction.

2.9. Fourier transform infrared (FT-IR) spectroscopy

Protein secondary structures of raw and gel samples were analyzed using a Nicolet iZ10 FT-IR spectrometer (Thermo Scientific, UK) following Xu et al. (Xu et al., 2026). Freeze-dried samples were ground to a fine powder, and approximately 2 mg was scanned from 4000 to 400 cm⁻¹ at 4 cm⁻¹ resolution with 32 scans. Baseline correction, Fourier self-deconvolution, second derivative analysis, and Gaussian fitting were performed using OMNIC 8.0 to calculate secondary structure contents.

2.10. Protein intermolecular interaction forces

Four extracting solutions with different dissociation capacities were prepared according to Wang et al. (Wang, Li, et al., 2025): (a) 0.05 mol/L NaCl; (b) 0.6 mol/L NaCl; (c) 0.6 mol/L NaCl +1.5 mol/L urea; (d) 0.6 mol/L NaCl +8 mol/L urea. Briefly, 1.0 g of minced lamb was mixed with 5 mL of each solution, homogenized for 30 s, and shaken at 150 r/min for 2 h at 4 °C. After centrifugation at 10,000 r/min for 15 min at 4 °C, the supernatant protein concentration was determined using a BCA kit. The contributions of different molecular forces were calculated as: ionic bonds = (b) - (a); hydrogen bonds = (c) - (b); hydrophobic interactions = (d) - (c).

2.11. Protein surface hydrophobicity

The surface hydrophobicity of MP was measured according to Zou et al. (Zou et al., 2022) with slight modifications. MP was suspended in 20 mmol/L phosphate buffer (pH 6.0) at 1 mg/mL. Then, 1 mL of MP suspension was mixed with 200 μL of 1 mg/mL bromophenol blue (BPB) solution. The control consisted of 1 mL of the same phosphate buffer (pH 6.0) mixed with 200 μL of BPB solution. The addition of the weakly acidic BPB slightly decreased the pH of the mixtures to approximately 5.8 in both samples and controls. All mixtures were incubated at room temperature for 10 min and then centrifuged at 2000 ×g for 15 min. The BPB binding amount was calculated as follows:

$$\mathrm{BPB}\text{binding}\left(\mathrm{\mu g}\right)=200\times \left({\mathrm{OD}}_{\text{control}}-{\mathrm{OD}}_{\text{sample}}\right)/{\mathrm{OD}}_{\text{control}}$$

2.12. Lipid oxidation (TBARS assay)

Lipid oxidation was evaluated by the TBARS method with slight modifications (Bai et al., 2023). In brief, 2 g of lamb meat was mixed with 25 mL of trichloroacetic acid (TCA) solution, homogenized, shaken thoroughly, and incubated at 50 °C for 30 min. After cooling and filtration, 5 mL of the filtrate was mixed with 5 mL of thiobarbituric acid solution and heated at 90 °C for 30 min. The absorbance was measured at 532 nm. The TBARS value was calculated based on the malondialdehyde standard curve and expressed as mg MDA equivalents per kg of sample.

2.13. Determination of free fatty acids

Lamb meat was mixed with 2 mL of 95% ethanol, 4 mL of water, and 10 mL of 8.3 mol/L HCl, then hydrolyzed at 80 °C for 40 min with shaking every 10 min. After cooling, another 10 mL of 95% ethanol was added, and lipids were extracted three times with 100 mL of petroleum ether-diethyl ether (1:1, v/v). The pooled organic phases were evaporated to dryness under N₂.

The lipids were derivatized with 4 mL of methanolic NaOH (45 °C, 20 min) and 4 mL of BF₃-methanol (45 °C, 20 min). The fatty acid methyl esters (FAMEs) were extracted with 3 mL of n-hexane, and the supernatant was filtered (0.22 μm) for gas chromatograph (7890 A, Agilent, USA) equipped with FID and an HP-88 column (100 m × 0.25 mm × 0.20 μm) with N₂ carrier gas at 10 mL/min, split ratio 20:1, injector 260 °C, detector 250 °C. Oven program: 100 °C (15 min) → 190 °C at 20 °C/min (6 min) → 220 °C at 1 °C/min (7 min).

2.14. Free sulfhydryl content

Free sulfhydryl content was determined using Ellman's method (Zou et al., 2022). Briefly, 3 mL of MP solution (5 mg/mL) was mixed with 20 mL of Tris-glycine buffer (4 mmol/L EDTA, 0.09 mol/L glycine, 0.086 mol/L Tris, pH 8.0). Then 50 μL of Ellman's reagent was added, and the mixture was vortexed and incubated at 25 °C for 1 h. After centrifugation at 10,000 ×g for 10 min, the supernatant absorbance was measured at 412 nm. The free sulfhydryl content was calculated as:

$$\text{Free sulfhydryl content}\left(\text{μmol}/\mathrm{g}\right)=73.53\times \mathrm{A}/\mathrm{\rho },$$

where 73.53 is the constant derived from the molar extinction coefficient, A is the absorbance value, and ρ is the protein concentration (mg/mL).

2.15. Carbonyl determination

Carbonyl content was determined using the 2,4-dinitrophenylhydrazine (DNPH) method (Bai et al., 2023). Briefly, 2.0 g of meat was homogenized in 10 mL of phosphate buffer (20 mM, pH 6.5, 0.6 M NaCl) and centrifuged at 13,000 r/min. The supernatant (0.4 mL, 1 mg protein/mL) was mixed with 0.2 mL of 10 mM DNPH in 2 M HCl and incubated at 30 °C for 1 h in the dark. After adding 1 mL of 40% TCA and standing for 20 min, the mixture was centrifuged for 15 min. The pellet was washed repeatedly with 1 mL of ethyl acetate: ethanol (1: 1) until the supernatant was colorless, with centrifugation at 13,000 r/min for 10 min each time. The final pellet was dissolved in 3 mL of 6 M guanidine hydrochloride, and absorbance was measured at 370 nm. Results are expressed as nmol carbonyl per mg protein.

2.16. Water holding capacity (WHC) of gel

WHC was determined according to the method of Wang et al. (Wang, Li, et al., 2025) with a slight modification. Heat-induced gels prepared in 2.4 section were balanced to room temperature. Approximately 5 g of gel was weighed (m₁) and centrifuged at 10,000 ×g for 15 min at 4 °C. After removing the supernatant and drying the tube surface with filter paper, the residual gel was re-weighed (m₂). The WHC was calculated as:

$$\mathrm{WHC}\left(\%\right)=\left({\mathrm{m}}_2/{\mathrm{m}}_1\right)\times 100$$

where m₁ is the initial gel mass and m₂ is the residual gel mass after removing the released water.

2.17. Electronic nose analysis

Volatile profiles of raw and roasted lamb meat were analyzed using a PEN3 electronic nose (AIRSENSE, Germany) following Xu et al. (Xu et al., 2026) with minor modifications. Briefly, 2.00 g of raw or roasted lamb (180 °C, 15 min) was placed in a 10 mL vial, equilibrated at 60 °C for 20 min, and measured under the following conditions: sampling interval 1.0 s, flush time 60 s, zero calibration 10 s, pre-sampling 5 s, measurement 60 s, and gas flow 300 mL/min.

2.18. GC–MS analysis of volatile compounds

Volatile compounds were analyzed by GC–MS (Agilent 7890B-7000D) with HS-SPME according to Xu et al. (Xu et al., 2026). Samples were placed in 20 mL vials with saturated NaCl and 2-octanol (3 mg/L) as internal standard, equilibrated at 80 °C for 30 min, extracted by SPME fiber at 80 °C for 30 min, and desorbed at 250 °C for 5 min. Separation was performed on an HP-5MS column (30 m × 0.25 mm × 0.25 μm) with helium carrier gas (1.0 mL/min, splitless). Oven program: 50 °C (2 min) → 180 °C at 5 °C/min (5 min) → 250 °C at 10 °C/min (5 min). Mass spectra were acquired in full-scan mode (m/z 40–600). Compounds were identified using the NIST 2020 database and quantified against the internal standard.

Odor activity values (OAV) were calculated as OAV = (C × 1000)/OT, where C is the compound concentration (mg/kg) and OT is the odor threshold (μg/kg); OAV > 1 indicated aroma contribution.

2.19. Quantitative descriptive sensory analysis (QDA)

Sensory evaluation was performed by 12 trained panelists according to ISO 8586:2023. Panelists were recruited from the College of Food Science with prior meat sensory experience, and underwent three training sessions (2 h each) to familiarize with the eight target attributes using reference standards. Panelist consistency was verified by repeated evaluation (RSD < 15%). Evaluations were conducted in individual booths at 22 ± 1 °C under white light, with samples coded and randomized. Eight attributes (aldehyde/fruity aroma, fermentation flavor, fatty aroma, grilled aroma, hardness, juiciness, springiness, and gamey odor) were scored on a 15-cm linear scale (0 = undetected, 15 = extremely strong). Roasted lamb cubes (1.5 cm) were presented in a Latin square order, with water and plain bread rinsing between samples. Detailed training procedures are provided in Supplementary Tables S3 and S4. The sensory evaluation was approved by the Science Ethics Committee of Henan University of Animal Husbandry and Economy (Approval No. HNUAHESER 2512 K0901). All panelists provided written informed consent prior to participation.

2.20. Statistical analysis

All experiments were performed in triplicate, and data were expressed as mean ± SD. One-way ANOVA followed by Tukey's test was applied for group comparisons using Minitab. Mantel test correlation heatmaps were generated via Chiplot (https://www.chiplot.online/). Two separate general linear models (GLMs) were fitted for the MNB + Lp and MNB + Lip systems, with MNB, biological treatment, and their interaction term as fixed factors. The interaction term p-value was used to determine significance (p < 0.05). The interaction coefficient (I) was calculated as I = E_AB − (E_A + E_B − E_C), where E_C, E_A, E_B, E_AB are the group means of the control, single MNB, single biological treatment, and combined treatment, respectively. Effects were classified as synergistic (p < 0.05, coefficient in the beneficial direction), antagonistic (p < 0.05, opposite direction), or additive (p ≥ 0.05). For indicators where lower values are beneficial (e.g., MDA, T₂₃), a positive coefficient indicates antagonism and a negative coefficient indicates synergy. Given n = 3 per group, non-significant interactions should be interpreted with caution.

3. Results and analysis

3.1. pH of meat homogenates

The morphology of the micro-nano bubble water is shown in Fig. 1A, and the pH values of meat homogenates are presented in Fig. 1B. Single MNB treatment significantly reduced pH to 5.95 (p < 0.05), indicating a modest acidifying effect of physical pretreatment alone. The Lip group showed a decrease to 6.06 (p < 0.05), attributable to free fatty acid release via lipase-catalyzed lipid hydrolysis (Pei et al., 2025). Notably, MNB + Lip exhibited an even lower pH (5.83) than Lip alone (p < 0.05), suggesting that MNB-induced muscle loosening (Fig. 2A) likely improved lipase accessibility and distribution by reducing mass-transfer resistance.

Fig. 1.

(A) Appearance of distilled water and micro-nano bubble (MNB) water. (B) pH values of meat homogenates. (C) LF-NMR transverse relaxation time (T₂) spectra of lamb meat.

Fig. 2.

(A) Microstructure of lamb meat. (B) Macroscopic appearance and (C) microstructure of heat-induced myofibrillar protein gels from different treatment groups.

Lp fermentation markedly reduced pH to 5.62 (p < 0.05) due to organic acid accumulation (Jiang et al., 2025), while MNB + Lp achieved the lowest pH (5.42) among all treatments, significantly lower than Lp alone (p < 0.05). This additive acidification reflects that MNB pretreatment remodels the compact muscle structure and likely improves bacterial accessibility, promoting more uniform Lp distribution and in situ acid production. Overall, the enhanced acidification in MNB-combined groups indicates that the “physical loosening + biological metabolism” strategy effectively intensifies biochemical modification of lamb meat, thereby regulating protein conformation, oxidative stability, and subsequent gel and flavor characteristics.

3.2. Microstructure of lamb meat

Microstructural variations are shown in Fig. 2A. Control samples displayed compact, regularly arranged muscle fibers with intact endomysium and minimal inter-fiber gaps. Lp treatment enlarged inter-fiber spaces and loosened muscle tissue via acid-induced activation of endogenous protease (Jiang et al., 2025; Liang et al., 2026). Lip treatment caused obvious fiber breakage and fragmentation, as lipolysis-derived free fatty acids disrupted muscle fiber integrity (Tatiyaborworntham et al., 2022). In the MNB group, the inter-fiber spaces were clearly widened and the endomysium continuity was partially disrupted, whereas the muscle fibers themselves remained intact. Based on the NTA results showing a median bubble diameter of 68 nm (Supplementary Table S2), we infer that nano-sized MNBs may access the muscle tissue and physically loosen the fiber structure. This MNB-induced modification was mild and non-destructive at the tissue level, achieving structural loosening while preserving the overall fiber architecture.

Compared with single treatments, MNB + Lp exhibited obvious muscle fiber contraction and aggregation. MNB-induced loosening likely facilitated uniform Lp distribution, and homogeneous acidification shifted the microenvironment toward the isoelectric point, triggering extensive protein aggregation and fiber bundle contraction. This densification explains the highest hardness of MNB + Lp raw meat (Section 3.4). In contrast, MNB + Lip maintained relatively continuous fibers with uniform fine fissures. MNB reduced localized over-hydrolysis and promoted homogeneous fiber fragmentation, effectively reducing muscle tissue rigidity, consistent with its lowest raw meat hardness.

3.3. Low-field nuclear magnetic resonance (LF-NMR)

Four water populations were identified: T₂₀ (tightly bound), T₂₁ (weakly bound), T₂₂ (immobile), and T₂₃ (free) (Fig. 1C, Table 1). T₂₀ was detected only in Control and Lp groups, with Control showing a stronger signal due to intact myofibrils providing abundant polar binding sites (Zhang et al., 2022). T₂₀ was undetectable in Lip, MNB, MNB + Lp, and MNB + Lip groups, likely due to disruption or shielding of polar binding sites.

Table 1.

Low field-nuclear magnetic resonance and texture parameters of lamb meat.

Sample	Control	Lp	Lip	MNB	MNB + Lp	MNB + Lip
T20 (%)	0.0192 ± 0.0002a	0.0171 ± 0.0005b	ND	ND	ND	ND
T21 (%)	0.0338 ± 0.0003d	0.0326 ± 0.0004d	0.0554 ± 0.0009b	0.0354 ± 0.0007c	0.0549 ± 0.0003b	0.0586 ± 0.0006a
T22 (%)	95.29 ± 0.04a	90.15 ± 0.01b	95.60 ± 0.03a	85.79 ± 0.12d	83.75 ± 0.05e	89.28 ± 0.34c
T23 (%)	4.65 ± 0.04e	9.80 ± 0.01d	4.34 ± 0.03e	14.18 ± 0.12b	16.19 ± 0.05a	10.66 ± 0.34c
Hardness (g)	4743 ± 128d	5880 ± 102b	5438 ± 106c	4515 ± 148d	6551 ± 87a	3844 ± 90e
Adhesiveness (g·sec)	−29.40 ± 1.93b	−23.69 ± 1.41ab	−16.11 ± 1.73a	−31.38 ± 5.47b	−25.09 ± 2.22b	−27.97 ± 0.49b
Resilience (%)	85.88 ± 2.69a	81.94 ± 2.98ab	75.55 ± 1.10b	79.17 ± 2.02ab	80.37 ± 1.97ab	82.70 ± 2.98ab
Springiness (%)	91.52 ± 0.11ab	92.83 ± 0.90a	76.37 ± 2.99c	90.83 ± 0.39ab	88.53 ± 1.75b	89.72 ± 0.55ab
Gumminess	4230 ± 88cd	4995 ± 234b	4562 ± 9c	3933 ± 29d	5737 ± 102a	3376 ± 154e
Chewiness	3872 ± 85c	4638 ± 262b	3530 ± 63c	3572 ± 33c	5078 ± 121a	3030 ± 157d
Cohesion (%)	89.20 ± 0.68a	84.93 ± 2.74a	83.41 ± 1.11a	87.17 ± 2.84a	87.58 ± 1.30a	87.81 ± 1.96a

The data are presented as the mean ± standard deviation. Different superscript letters in each row indicate significant differences (p < 0.05).

ND, not detected.

T₂₂ and T₂₃ were the dominant fractions. Lp treatment decreased T₂₂ and increased T₂₃ (Table 1), indicating fermentation promoted conversion of immobile to free water (Zheng et al., 2025). Single MNB treatment further reduced T₂₂ and increased T₂₃, reflecting mild structural loosening that rearranged water distribution without altering protein conformation (Section 3.5).

The MNB + Lp group exhibited the lowest T₂₂ and highest T₂₃, indicating water expulsion from myofilament lattice to extracellular space. This expulsion likely resulted from acid-induced fiber contraction rather than structural disintegration. The increased free water did not lead to tenderization; instead, compacted fiber architecture was associated with the highest hardness (Section 3.4). This decoupling between water mobility and texture suggests that tenderness is determined by myofibrillar structural integrity, not merely free water content. In contrast, MNB + Lip maintained higher T₂₂ and lower T₂₃ than MNB + Lp, as lipolysis products remodeled water-protein-lipid interactions and enhanced weak water binding, partially offsetting MNB-induced water migration. Overall, MNB + Lp induced thorough water migration and structural densification, while MNB + Lip favored moderate water redistribution and uniform loosening.

3.4. Texture properties

As summarized in Table 1, the MNB + Lp group showed the highest hardness, followed by Lp and Lip, while the MNB + Lip group had the lowest hardness. Consistent with microstructure observations, MNB-induced structural loosening of muscle fibers likely improves transfer and accessibility, enabling more homogeneous Lp fermentation. The uniform near-isoelectric acidification induces protein aggregation and fiber contraction, forming compact and high-hardness muscle structure. This pH-driven structural reorganization is consistent with the established principle that near the isoelectric point, electrostatic repulsion is minimized and hydrophobic interactions dominate, promoting extensive protein aggregation (Qu et al., 2025).

The single Lp group showed elevated hardness due to moderate acid-induced aggregation, while the Lip group exhibited medium hardness. Unlike acid fermentation, lipase hydrolysis disrupted fiber integrity, but free fatty acids partially compensated for tissue softening (Abril et al., 2023). In contrast, MNB reduced the heterogeneous modification of single lipase treatment and achieved uniform fiber loosening across muscle tissue, significantly reducing rigidity and giving the lowest hardness and chewiness. The single MNB group had lower hardness than Control, indicating a mild tenderizing effect of MNB treatment. Overall, raw meat texture was regulated by pH-induced aggregation, lipolysis-mediated disruption, and MNB physical remodeling at the tissue level, which differ fundamentally from the molecular interactions governing heat-induced gel properties (Section 3.11).

3.5. Secondary structure of lamb meat

FT-IR spectra and secondary structure are presented in Fig. 3A and B. FT-IR spectra showed no fundamental changes in functional groups across treatments (Fig. 3A), indicating that MNB, Lp, and Lip did not cause detectable changes in the chemical structure of meat proteins. The characteristic regions were defined as α-helix (1650–1660 cm⁻¹), β-sheet (1600–1640 cm⁻¹), β-turn (1660–1700 cm⁻¹), and random coil (1640–1650 cm⁻¹).

Fig. 3.

(A) FT-IR spectra and (B) secondary structure composition of lamb meat. (C) Surface hydrophobicity (BPB binding capacity), (D) MDA content, (E) hydroxyl radical scavenging rates (HRSR) of MNB water and sterile distilled water, and (F) free sulfhydryl and carbonyl contents of myofibrillar proteins.

Single Lp treatment increased β-sheet with negligible changes in α-helix and random coil (Fig. 3B), consistent with acid-induced β-sheet accumulation (Zheng et al., 2025). Single Lip treatment significantly reduced α-helix and increased β-sheet and random coil (Fig. 3B). Lipolysis-derived free fatty acids altered the microenvironment and disrupted intramolecular hydrogen bonds, promoting conformational disorder (Chen et al., 2023).

In contrast, single MNB treatment resulted in no significant changes in any secondary structure components (α-helix, β-sheet, β-turn, or random coil) compared with the Control (Fig. 3B), indicating that MNB-induced nano-scale physical remodeling did not alter protein conformation. Furthermore, MNB + Lip treatment recovered α-helix content close to the control level and attenuated the increase in random coil, indicating that MNB pretreatment promoted more uniform lipase hydrolysis and alleviated local conformational disorder induced by uneven enzymatic action.

3.6. Intermolecular interaction forces

As shown in Table 2, all treatments increased ionic bond strength relative to Control, and Lip exhibited the highest value. Lipolysis products altered ionic strength and exposed charged protein residues to strengthen electrostatic interactions (Tatiyaborworntham et al., 2022). MNB also showed significantly higher ionic bonds than Control, implying mild nano-perturbation exposed surface charged residues.

Table 2.

Intermolecular interaction forces and free fatty acids of myofibrillar proteins of lamb meat.

Sample	Control	Lp	Lip	MNB	MNB + Lp	MNB + Lip
Intermolecular interaction forces (mg/mL)
Ionic bonds	197.90 ± 5.10c	322.17 ± 4.27b	426.43 ± 5.57a	336.80 ± 10.22b	326.47 ± 12.91b	321.80 ± 7.94b
Hydrogen bonds	463.96 ± 3.84a	432.45 ± 11.89b	408.22 ± 5.83c	474.63 ± 5.36a	458.90 ± 3.64a	376.37 ± 10.58d
Hydrophobic interactions	312.92 ± 3.59d	324.43 ± 10.66cd	378.80 ± 7.52b	370.98 ± 8.68b	332.61 ± 1.50c	514.02 ± 2.68a
Free fatty acids (g/100 g)
C14:0	0.0226 ± 0.0001f	0.0565 ± 0.0004b	0.0310 ± 0.0003e	0.0339 ± 0.0003d	0.0676 ± 0.0002a	0.0443 ± 0.0002c
C15:0	0.0036 ± 0.0002d	0.0079 ± 0.0003b	0.0048 ± 0.0003cd	0.0060 ± 0.0010c	0.0100 ± 0.0002a	0.0085 ± 0.0005b
C16:0	0.2889 ± 0.0007f	0.5923 ± 0.0009b	0.3492 ± 0.0006e	0.3650 ± 0.0003d	0.7116 ± 0.0003a	0.4875 ± 0.0006c
C16:1	0.0256 ± 0.0002d	0.0520 ± 0.0004b	0.0367 ± 0.0003c	0.0364 ± 0.0002c	0.0620 ± 0.0003a	0.0527 ± 0.0004b
C17:0	0.0118 ± 0.0002e	0.0249 ± 0.0004b	0.0143 ± 0.0002d	0.0159 ± 0.0002c	0.0320 ± 0.0002a	0.0243 ± 0.0003b
C18:0	0.1850 ± 0.0003f	0.3649 ± 0.0002b	0.1989 ± 0.0003e	0.2176 ± 0.0003d	0.4521 ± 0.0005a	0.3118 ± 0.0003c
C18:1n9c	0.4952 ± 0.0014f	1.0262 ± 0.0007b	0.6649 ± 0.0004d	0.6410 ± 0.0012e	1.2387 ± 0.0010a	0.9592 ± 0.0007c
C18:2n6c	0.1091 ± 0.0005e	0.1520 ± 0.0006c	0.0974 ± 0.0010f	0.1143 ± 0.0011d	0.2080 ± 0.0007a	0.1739 ± 0.0009b
C20:3n6	0.0056 ± 0.0056c	0.0034 ± 0.0034d	ND	0.0069 ± 0.0069b	0.0083 ± 0.0083a	0.0062 ± 0.0062bc
C20:4n6	0.0399 ± 0.0002c	0.0470 ± 0.0005b	0.0364 ± 0.0004d	0.0399 ± 0.0002c	0.0618 ± 0.0003a	0.0464 ± 0.0004b
C24:1	0.0129 ± 0.0004d	0.0174 ± 0.0003c	0.0119 ± 0.0005d	0.0120 ± 0.0003d	0.0233 ± 0.0005a	0.0190 ± 0.0005b
TFFA	1.2002 ± 0.0027f	2.3446 ± 0.0036b	1.4454 ± 0.0023e	1.4890 ± 0.0033d	2.8754 ± 0.0033a	2.1336 ± 0.0020c

The data are presented as the mean ± standard deviation. Different superscript letters in each line indicate significant differences (p < 0.05).

TFFA, Total free fatty acids; ND, not detected.

Lip and MNB + Lip significantly reduced hydrogen bond content compared with Control, and MNB + Lip reached the minimum (Table 2), owing to hydrogen bond disruption by lipolysis-derived free fatty acids (Chen et al., 2023). No significant difference was found between MNB and Control, suggesting mild physical perturbation by MNB barely disrupted hydrogen bonds, differing from hydrogen bond breakage induced by ultrasonic cavitation (Liu et al., 2022). Lp treatment also caused a modest but significant decrease in hydrogen bond content compared with Control.

For hydrophobic interactions, MNB + Lip exhibited the highest level (Table 2), which facilitates stable gel network construction. MNB enables uniform lipolysis and reduces localized over-hydrolysis, thereby promoting extensive hydrophobic cross-linking. This result agrees with previous work reporting that lipid oxidation products strengthen gel via hydrophobic interactions among myofibrillar proteins (Feng et al., 2024). Single Lp exerted negligible influence on hydrophobic interactions, consistent with its limited capacity to trigger protein unfolding.

3.7. Surface hydrophobicity of myofibrillar proteins

Surface hydrophobicity, reflecting protein unfolding and hydrophobic residue exposure, was measured by BPB binding (Fig. 3C). Binding capacity followed the order: MNB > MNB + Lip > Lp > MNB + Lp > Control > Lip. The highest hydrophobicity of single MNB group likely arose from nano-induced mild structural loosening and exposure of buried hydrophobic residues (Section 3.2). MNB + Lip showed marginally lower hydrophobicity, as free fatty acids partially shielded exposed hydrophobic sites.

Lp exhibited higher hydrophobicity than MNB + Lp. Stronger acidification in MNB + Lp (pH 5.42 vs 5.62) approached the isoelectric point, inducing aggregation and re-burial of hydrophobic group (You et al., 2025). Control displayed low hydrophobicity due to compact native conformation. Lip obtained the minimum BPB binding, which cannot be fully explained by FFA shielding: MNB + Lp contained higher TFFA (2.872 vs 1.443 g/100 g, Section 3.9) yet possessed higher hydrophobicity. Instead, severe oxidative aggregation dominated in Lip; the highest carbonyl level (Section 3.10) was associated with extensive cross-linking and embedded hydrophobic residues within aggregates (Jiang et al., 2021; Zhu et al., 2024). Notably, BPB-determined surface hydrophobicity and intermolecular hydrophobic interactions (Table 2) characterize surface accessibility and inter-protein association separately, explaining low BPB binding accompanied by strong hydrophobic interactions in Lip. Overall, MNB combined treatments yielded moderate hydrophobicity through regulated protein unfolding, which differed from severe oxidation-driven aggregation in Lip.

3.8. Lipid oxidation (malondialdehyde content) of lamb meat

Malondialdehyde (MDA) content varied significantly among treatments (Fig. 3D). The Control group exhibited the highest MDA content, indicating spontaneous lipid oxidation occurred in untreated lamb meat during soaking (Li et al., 2025). The Lip group showed the second highest MDA content, as lipase hydrolysis released unsaturated fatty acids acting as oxidation substrates (Tatiyaborworntham et al., 2022), while the Lp group exhibited markedly lower MDA content due to antioxidant metabolites generated during fermentation (Luo et al., 2024). Single MNB treatment further reduced MDA content below the Lp group, and the combined treatments (MNB + Lp and MNB + Lip) showed the lowest MDA contents with no significant difference between them, suggesting an effective inhibition of advanced lipid oxidation. Nevertheless, interaction analysis indicated that the interaction between MNB and Lp for MDA was antagonistic, with the combined reduction being smaller than the additive expectation (Supplementary Table S5).

The effective inhibition of MDA formation likely arises from MNB's hydroxyl radical scavenging activity (Fig. 3E), which interrupts lipid oxidation chain reactions, and MNB-induced structural loosening that regulates oxygen diffusion within meat matrix (Zheng et al., 2023). Additionally, by improving mass transfer uniformity, MNB likely facilitates the antioxidant action of biological modifiers. The lower MDA in MNB + Lp reflects controlled oxidation toward aldehyde accumulation rather than terminal MDA formation (Section 3.12.2).

3.9. Free fatty acids of lamb meat

The total free fatty acids (TFFA) content followed the order: MNB + Lp > Lp > MNB + Lip > MNB > Lip > Control (Table 2). Single Lip treatment increased TFFA by 20.4% compared with Control (p < 0.05), indicating exogenous lipase retained partial activity (Pei et al., 2025), consistent with the pH reduction in Section 3.1. Single MNB treatment showed a 24.1% increase relative to Control, indicating that nano-scale modification alone exerted only a limited lipolytic effect. Lp fermentation markedly elevated TFFA by 95.43% versus Control, attributable to sustained microbial lipolytic activity (Pei et al., 2025).

MNB + Lp exhibited the highest TFFA, 22.6% higher than Lp alone and 139.6% higher than Control. This synergistic enhancement is attributed to MNB-induced muscle loosening (Section 3.2) facilitating uniform Lp distribution and in-situ lipolysis. MNB + Lip also showed significantly higher TFFA than Lip alone (p < 0.05), suggesting improved lipase accessibility and distribution via MNB. This increase was also consistent with the synergistic interaction between MNB and Lip for TFFA (Supplementary Table S6). However, MNB + Lip remained lower than Lp alone, suggesting that exogenous lipase, even with MNB assistance, was less efficient than the sustained enzymatic activity of viable Lp. This is consistent with the finding that microbial lipases from starter cultures often play a more persistent role in lipid hydrolysis than single exogenous enzyme addition (Pei et al., 2025).

For individual FFAs, C16:0, C18:0, and C18:1n9c accounted for over 75% of TFFA across all groups. MNB + Lp showed the greatest increases, with oleic acid (C18:1n9c) reaching 1.2387 g/100 g (2.5-fold of Control), a key precursor for volatile aldehydes. C20:4n6 was also highest in MNB + Lp, suggesting microbial metabolism facilitated the release of membrane phospholipid-bound fatty acids. C18:2n6c, a major precursor for hexanal and (E)-2-nonenal (Wang et al., 2022), was also highest in MNB + Lp, aligning with the elevated aldehyde profile in this group (Section 3.12.2). Overall, MNB + Lp synergistically enhanced TFFA accumulation through improved microbial accessibility and metabolic activity, enriching unsaturated fatty acid precursors for thermal flavor formation.

3.10. Protein oxidation of lamb meat

Free sulfhydryl (-SH) reflects protein integrity, while carbonyl indicates oxidative side-chain damage (Bai et al., 2023). As shown in Fig. 3F, MNB + Lip exhibited the highest -SH and lowest carbonyl, followed by MNB and MNB + Lp. MNB may scavenge ROS to alleviate protein oxidation and preserve structural integrity. Additionally, by promoting uniform distribution of lipase, MNB reduces localized over-oxidation in MNB + Lip.

Notably, Lip had the highest carbonyl yet its -SH was not the lowest, indicating carbonylation and disulfide formation do not always coincide. In Lip, oxidative modification preferentially targeted amino acid side chains (e.g., lysine, arginine) rather than cysteine thiols, consistent with the residue-specific attack patterns of lipid-derived radicals (Jiang et al., 2021; Zhu et al., 2024). Thus, -SH loss is not the sole indicator of oxidative damage; carbonyl better reflects the overall extent of protein oxidation. Groups with higher -SH (MNB + Lip, MNB, MNB + Lp) all exhibited lower carbonyl, suggesting that MNB-assisted modification may effectively inhibit protein oxidation. The high TFFA in MNB + Lp did not translate into severe oxidation, as MNB scavenging and Lp's reducing microenvironment intercepted oxidative propagation, decoupling FFA accumulation from protein oxidation. Interaction analysis indicated an additive effect for protein carbonyl in MNB + Lp (Supplementary Table S5).

This combined effect likely involves two interrelated aspects: hydroxyl radical scavenging by MNBs (Fig. 3E), and MNB-facilitated uniform distribution of biological agents enabling effective action of Lp-derived antioxidants. Additionally, the loosened muscle structure may have altered the physicochemical microenvironment of the meat matrix, potentially affecting the accessibility of oxygen to lipid substrates (Zheng et al., 2023).

3.11. Gel properties of lamb meat

3.11.1. Macroscopic appearance of gels

The macroscopic appearance of heat-induced myofibrillar protein gels differed markedly among treatment groups (Fig. 2B). The Lip and MNB + Lip groups showed slight shrinkage. The MNB group exhibited slight swelling, a relatively loose structure, and poor integrity. The Lp and MNB + Lp groups had the larger gel volumes, with a plump and swollen appearance, presenting a soft and fluffy macroscopic state, which contrasted sharply with the compactness of the other groups. These contrasting appearances arise from different gelation mechanisms: acid-induced protein pre-aggregation in Lp-treated groups versus lipolysis-facilitated hydrophobic cross-linking in Lip-treated groups during heating.

3.11.2. Microstructure of gels (SEM)

The microstructure of gels is shown in Fig. 2C. The Lip group exhibited a dense, uniform gel network with small, evenly distributed pores and thick pore walls, attributable to hydrophobic cross-linking driven by lipolysis-released free fatty acids (Feng et al., 2024; Xia et al., 2019). MNB + Lip showed an even more uniform and compact network, as MNB pretreatment reduced uneven lipase distribution and localized over-modification, promoting ordered protein aggregation.

In contrast, the MNB group exhibited a rough surface, uneven pores, and network breaks, indicating that MNB treatment alone does not favor ordered gel formation. The Lp group displayed a highly porous network with large pores, consistent with acid-induced gel loosening, as also observed in Lactiplantibacillus plantarum-fermented surimi gels where fermentation induced a structural transition toward a β-sheet-rich conformation and formed a loose, porous network (Wang, Liu, et al., 2025). MNB + Lp exhibited a moderately refined pore structure and better network density than Lp alone, suggesting that MNB pretreatment reduced excessive gel loosening. Quantitative porosity analysis confirmed that the Lp group had the highest porosity, while the MNB + Lip group had the lowest (Table 3).

Table 3.

The porosity, texture parameters and water holding capacity (WHC) of lamb meat gels.

Sample	Control	Lp	Lip	MNB	MNB + Lp	MNB + Lip
Porosity (%)	18.00 ± 0.42e	33.53 ± 0.84a	21.84 ± 1.57d	25.73 ± 0.67c	28.25 ± 0.45b	12.78 ± 0.27f
Hardness (g)	210.93 ± 2.54c	236.50 ± 8.80b	313.09 ± 11.39a	153.28 ± 5.05d	255.68 ± 10.77b	240.30 ± 3.37b
Adhesiveness (g·sec)	/	−9.62 ± 0.32cd	−7.58 ± 1.32bc	−12.23 ± 0.49d	−5.94 ± 1.29ab	−3.87 ± 0.50a
Resilience (%)	31.29 ± 0.03c	31.35 ± 1.54c	43.36 ± 1.02a	25.91 ± 0.69d	26.69 ± 1.75d	35.95 ± 0.44b
Springiness (%)	86.03 ± 0.24b	80.34 ± 0.23c	93.13 ± 0.71a	87.34 ± 3.08b	92.88 ± 0.85a	89.65 ± 0.29ab
Gumminess	169.32 ± 2.23d	183.40 ± 10.24cd	275.78 ± 7.81a	126.70 ± 3.38e	204.57 ± 11.42bc	207.83 ± 4.38b
Chewiness	145.66 ± 1.51c	147.33 ± 7.98c	256.83 ± 7.61a	110.72 ± 6.57d	190.05 ± 11.73b	186.31 ± 4.22b
Cohesion (%)	80.27 ± 0.09ab	77.61 ± 4.89b	88.11 ± 1.45a	82.67 ± 0.59ab	80.10 ± 5.37ab	86.48 ± 0.61a
WHC (%)	56.51 ± 0.23c	54.93 ± 0.22d	61.92 ± 0.51a	53.01 ± 0.07e	53.64 ± 0.13de	59.34 ± 1.01b

The data are presented as the mean ± standard deviation. Different superscript letters in each row indicate significant differences (p < 0.05).

3.11.3. Texture and water holding capacity of gels

Gel texture and WHC are shown in Table 3. The Lip group exhibited the highest gel hardness and WHC, indicating that moderate lipid oxidation promotes hydrophobic interactions and disulfide cross-linking, enhancing gel strength and water retention.

Zheng et al. (Zheng et al., 2024) reported that mild oxidation maximizes gel hardness and WHC via enhanced hydrophobic interactions. Jiang et al. (Jiang et al., 2021) showed that moderate lipid oxidation increased gel WHC from 55.2% to 65.1%.

The MNB + Lip group showed slightly lower hardness and WHC than the Lip group but possessed significantly improved springiness and resilience. MNB-modulated homogeneous lipolysis leads to fine and uniform gel networks, avoiding the overly rigid structure of single lipase-treated gels and optimizing gel elasticity. Lipid degradation and oxidation products have been reported to promote the formation of disulfide bonds and hydrophobic interactions, which benefit gel properties (Zheng et al., 2024).

Notably, the opposite texture trends between raw meat and gels in lipolysis-modified groups are reasonable. Raw meat texture depends on intact fiber bundles, easily disrupted by lipolysis and MNB loosening, reducing hardness. Gel texture depends on thermal aggregation of myofibrillar proteins: free fatty acids and mild lipid oxidation promote protein unfolding and hydrophobic cross-linking during heating, forming a dense, high-strength gel network. Thus, the combination of tender raw meat and high-performance gels in the MNB + Lip and Lip groups reflects the dual regulatory effects of lipolysis. Interaction analysis indicated an additive effect for gel hardness and a synergistic effect for WHC in MNB + Lip (Supplementary Table S6).

3.11.4. Protein secondary structure of gels

The secondary structure of heat-induced gels is shown in Fig. 4A. Lip and MNB + Lip gels exhibited low α-helix and high random coil contents, consistent with changes in raw meat proteins. Lipolysis and mild lipid oxidation disrupted hydrogen bonds, irreversibly destroying α-helix and inducing disorder. This trend is well documented (Zhu et al., 2024). The transition from helix to disordered structure promotes protein unfolding and cross-linking, facilitating dense gel formation and improved WHC. Accordingly, gel strength of lipolysis-modified samples depends mainly on hydrophobic interactions and β-sheet stacking rather than α-helix (Jiang et al., 2021). MNB mainly improves the spatial uniformity of lipolysis and does not alter the intrinsic thermal transformation mechanism of myofibrillar proteins.

Fig. 4.

(A) Secondary structure composition of heat-induced myofibrillar protein gels. (B, C) PCA score plots of electronic nose data for raw and roasted lamb.

In contrast, Lp and MNB + Lp gels maintained significantly higher α-helix contents than Lip-treated groups. Mild acidification by Lp caused only reversible structural disorder in raw meat, and heating promoted refolding to recover α-helix. Studies on pH-dependent gelation of yak myofibrillar proteins have shown that at pH values near the isoelectric point (approximately 5.0), the intermolecular interactions (including hydrophobic interactions, hydrogen bonds, and disulfide bonds) are minimized, resulting in the lowest gel hardness and water-holding capacity (Qu et al., 2025). Acidic pH-shifting causes irreversible myosin conformational change, leading to rapid aggregation and loss of gel-forming ability (Sun et al., 2019). The slightly increased β-sheet in MNB + Lp was attributed to MNB-mediated physical modification promoting ordered peptide stacking. In summary, lipolytic modification induced irreversible helix disruption, whereas acid fermentation led to reversible, thermally recoverable structural changes, indicating distinct mechanisms for protein thermal transformation.

MNB + Lp and MNB + Lip exhibited opposite gelation behaviors. MNB + Lp formed a loose, porous network with the highest porosity and the lowest WHC via pH-driven isoelectric aggregation. Although the absolute WHC of MNB + Lp was the lowest among the gel samples, interaction analysis indicated a synergistic effect on WHC (Supplementary Table S5), i.e., the WHC of MNB + Lp was higher than the additive expectation from MNB and Lp alone.

In contrast, MNB + Lip produced a dense, uniform gel with the lowest porosity and a higher WHC than MNB + Lp, although slightly lower than that of Lip alone, through hydrophobic interactions and disulfide cross-linking (Table 2) (Feng et al., 2024; Zheng et al., 2024). Thus, MNB + Lp suits flavor-oriented products, whereas MNB + Lip targets texture-oriented applications.

3.12. Volatile flavor profile of lamb meat

3.12.1. Electronic nose flavor profile analysis

Principal component analysis (PCA) was performed to characterize flavor differences among raw and roasted lamb samples. For raw lamb, the PCA model explained 83.9% of the total variance (PC1 + PC2) with clear group separation (Fig. 4B). The Control group was associated with alkane and aromatic sensors, while Lp and Lip groups showed positive correlations with alcohols, representing distinct fermentation- and lipolysis-dominated flavor pathways, respectively. The single MNB group was located in a distinct region from the Control, indicating that MNB pretreatment alone altered the volatile profile of raw lamb. This change may be related to MNB-induced structural changes and water redistribution, although the underlying mechanisms require further verification. The MNB + Lp and MNB + Lip groups showed distinct flavor separation from their corresponding single biological treatment groups, suggesting that MNB pretreatment influenced the flavor profile.

For roasted lamb, PC1 and PC2 explained 87.2% of the variance (Fig. 4C). The Control group was associated with W1W, W2W, W1S, and W2S sensors (sulfides, nitrogen oxides, aldehydes), indicating off-flavor characteristics. The Lip group was associated with W3S (long-chain alkanes), while Lp and MNB + Lip groups aligned with W1C, W3C, and W5C (fermentative aromatics). The MNB + Lp group formed a distinct region separated from all others, suggesting that MNB pretreatment broadened the flavor profiles of modified lamb.

3.12.2. GC–MS analysis of volatile flavor compounds

GC–MS identified distinct flavor profiles across treatments. The MNB + Lp group had the highest total aldehyde content, followed by MNB + Lip, while Control and single Lp had lower levels (Table 4). Notably, MDA and total aldehydes showed opposite trends: Control had the highest MDA but the lowest aldehydes, whereas MNB + Lp had low MDA but the highest flavor-active aldehydes. This paradox arises because MDA and volatile aldehydes represent terminal versus intermediate products of lipid oxidation. As reviewed by Wu et al. (Wu et al., 2024), lipid oxidation in muscle foods proceeds through a cascade of reactions in which primary oxidation products can either accumulate or undergo further degradation to secondary and terminal products such as MDA. In Control and Lip groups, unchecked auto-oxidation depletes aldehydes and accumulates MDA. In MNB + Lp, however, radical scavenging by MNB and the reducing microenvironment of Lp limit oxidation at the aldehyde stage, enabling aldehyde accumulation while suppressing MDA formation. MNB + Lp accumulated high OAV (>1) aldehydes, including hexanal, nonanal, and (E)-2-nonenal, with (E,E)-2,4-decadienal uniquely detected in this group, indicating that MNB-promoted uniform fermentation facilitates continuous accumulation of desirable unsaturated aldehydes while limiting over-oxidation to MDA.

Table 4.

Key flavor compounds in GCMS of raw lamb meat.

Compound	Odor threshold (μg/kg)	Control (mg/kg)	Lp (mg/kg)	Lip (mg/kg)	MNB (mg/kg)	MNB + Lp (mg/kg)	MNB + Lip (mg/kg)
Hexanal	4.5	ND	ND	ND	0.025 ± 0.004b (OAV > 1)	0.108 ± 0.003a (OAV > 1)	ND
Nonanal	1	0.171 ± 0.005a (OAV > 1)	0.106 ± 0.004b (OAV > 1)	0.263 ± 0.003c (OAV > 1)	0.254 ± 0.004c (OAV > 1)	0.937 ± 0.032d (OAV > 1)	0.487 ± 0.010e (OAV > 1)
(E)-2-Nonenal	0.1	0.018 ± 0.004c (OAV > 1)	0.015 ± 0.002cd (OAV > 1)	0.007 ± 0.002d (OAV > 1)	0.035 ± 0.004b (OAV > 1)	0.269 ± 0.004a (OAV > 1)	0.033 ± 0.004b (OAV > 1)
Decanal	0.1	0.005 ± 0.001a (OAV > 1)	0.004 ± 0.001a (OAV > 1)	0.012 ± 0.002b (OAV > 1)	0.011 ± 0.001bc (OAV > 1)	0.028 ± 0.002bc (OAV > 1)	0.026 ± 0.006c (OAV > 1)
(E)-2-Decenal	0.3	0.021 ± 0.002d (OAV > 1)	0.014 ± 0.002d (OAV > 1)	0.017 ± 0.003d (OAV > 1)	0.045 ± 0.004c (OAV > 1)	0.262 ± 0.006a (OAV > 1)	0.123 ± 0.001b (OAV > 1)
2-Undecenal	5	0.024 ± 0.004d (OAV > 1)	0.015 ± 0.002de (OAV > 1)	0.012 ± 0.002e (OAV > 1)	0.048 ± 0.003c (OAV > 1)	0.219 ± 0.005a (OAV > 1)	0.188 ± 0.004b (OAV > 1)
(E, E)-2,4-Decadienal	0.3	ND	ND	ND	ND	0.044 ± 0.004 (OAV > 1)	ND
(E)-3-Hexen-1-ol	70	ND	ND	ND	ND	1.112 ± 0.009 (OAV > 1)	ND
α-Terpineol	300	0.018 ± 0.003a	0.017 ± 0.002b	0.034 ± 0.003b	0.019 ± 0.002c	0.049 ± 0.003c	0.035 ± 0.004c
Benzaldehyde	350	ND	ND	ND	0.011 ± 0.002b	ND	0.027 ± 0.002a
Methyl octanoate	200	0.018 ± 0.001c	0.012 ± 0.002d	0.081 ± 0.002b	0.017 ± 0.002cd	0.085 ± 0.003b	0.153 ± 0.003a
Methyl nonanoate	15	0.005 ± 0.001c	0.002 ± 0.001c	0.017 ± 0.001b (OAV > 1)	0.006 ± 0.002c	0.023 ± 0.001a (OAV > 1)	0.019 ± 0.001b (OAV > 1)
Methyl decanoate	70	0.027 ± 0.004d	0.008 ± 0.001e	0.355 ± 0.007b (OAV > 1)	0.032 ± 0.002d (OAV > 1)	0.117 ± 0.002c	0.937 ± 0.004a (OAV > 1)
Methyl tetradecanoate	4.5	0.008 ± 0.002d	0.004 ± 0.001d	0.482 ± 0.004a (OAV > 1)	0.008 ± 0.001d	0.016 ± 0.001c (OAV > 1)	0.096 ± 0.001b (OAV > 1)
Total aldehyde	–	0.266 ± 0.011d	0.170 ± 0.005d	0.434 ± 0.012c	0.456 ± 0.012c	2.107 ± 0.101a	1.135 ± 0.007b
Total ester	–	0.175 ± 0.012d	0.075 ± 0.005e	3.052 ± 0.022b	0.205 ± 0.006d	0.509 ± 0.010c	4.567 ± 0.018a

The data are presented as the mean ± standard deviation. Different superscript letters in each raw indicate significant differences (p < 0.05).

OAV > 1 indicates a potential contribution to the overall aroma. Values without OAV marking have OAV < 1.

ND = not detected.

Notably, the increased aldehydes reflect accumulation of specific odor-active compounds rather than simply higher total volatiles. Nonanal (fatty, citrus, green; threshold 1 μg/kg), (E)-2-nonenal (fatty, cucumber, green; threshold 0.1 μg/kg), and (E)-2-decenal (waxy, fatty, orange, floral; threshold 0.3 μg/kg) are recognized key aroma contributors to roasted lamb. Their OAVs in MNB + Lp group, although the highest, remained within ranges contributing pleasant fatty, fruity, and green notes rather than off-flavors. This is supported by QDA results (Fig. 5A), where MNB + Lp received the highest “aldehyde/fruity” and lowest “gamey” scores, suggesting that aldehyde accumulation translated to improved sensory perception.

Fig. 5.

(A) QDA radar plot of roasted lamb meat across eight sensory attributes. (B) Mantel test-based correlation analysis between physicochemical parameters and major aldehydes (OAV > 1). “Key aldehydes” is the sum of concentrations of all aldehydes with OAV > 1.

3.13. Quantitative descriptive analysis (QDA)

Sensory evaluation (Fig. 5A) revealed distinct profiles among the six groups. MNB + Lp scored highest in aldehyde/fruity aroma and lowest in gamey odor, suggesting that MNB-assisted fermentation enhances fruity notes while suppressing mutton off-flavor. Its fermentation flavor score was also the highest, reflecting the characteristic sensory imprint of Lp metabolism. MNB + Lp additionally exhibited the highest hardness, consistent with acid-induced protein aggregation.

MNB + Lip showed the highest juiciness and springiness, with moderate hardness and prominent fatty/grilled note, indicating a tenderized texture with good water retention. Single treatments (Lp, Lip, MNB) displayed intermediate profiles. The Control group scored highest in fatty and gamey notes, and lowest in aldehyde/fruity and fermentation flavor. Overall, QDA results were consistent with instrumental analyses (TPA, GC–MS, SEM), suggesting that the combined MNB and biological treatments achieved distinct quality improvements.

The HS-SPME-GC–MS method used in this study primarily detects medium-to-high volatility compounds. Branched-chain fatty acids (e.g., 4-methyloctanoic and 4-ethyloctanoic acid) and skatole, which are key contributors to lamb gamey odor, were not quantified in this study because derivatization was not performed and they respond poorly on non-polar columns. Thus, our flavor conclusions are based on aldehydes, esters, and QDA results. Future targeted analysis of BCFA using derivatization combined with GC × GC–MS is recommended.

3.14. Correlation and interaction analysis

Correlation analysis revealed close associations among physicochemical properties, protein oxidation, protein structure, texture, and flavor of lamb (Fig. 5B). MDA was positively correlated with protein carbonyl content, suggesting the lipid-to-protein oxidative cascade (Gao et al., 2024), and also with immobile water (T₂₂), suggesting lipid-protein oxidation remodels water-binding sites and redistributes water mobility, as supported by LF-NMR studies on oxidized myofibrillar proteins (Zhang et al., 2022). Gel hardness and chewiness were positively correlated with WHC, with moderate protein carbonylation enhancing this relationship via hydrophobic cross-linking in the gel network.

Raw meat β-sheet content was negatively associated with hydrogen bond strength, while α-helix content showed a negative correlation with gel chewiness. Moderate protein unfolding, hydrophobic group exposure, and mild carbonylation facilitated dense, elastic gel formation, whereas excessive hydrogen bonding and highly ordered α-helices inhibited heat-induced protein aggregation (Zhang et al., 2022).

Major flavor aldehydes with OAV > 1 (including (E)-2-decenal, (E)-2-nonenal, 2-undecenal, and decanal) were analyzed, with their total concentration defined as “Key aldehydes”. Both individual and total key aldehydes correlated significantly with physicochemical indicators: (E)-2-nonenal correlated with immobile water (T₂₂), and total key aldehydes correlated with raw meat chewiness.

TFFA was significantly negatively correlated with pH (p < 0.01), suggesting the acidifying effect of lipolysis and organic acid accumulation (Tatiyaborworntham et al., 2022). Notably, TFFA also exhibited a negative correlation with protein carbonyl content (p < 0.05), indicating that the abundant unsaturated FFA in MNB + Lp did not trigger severe protein oxidation, owing to the combined antioxidant effects of MNB and Lp metabolites (Zheng et al., 2023). The significant correlation between key aldehydes and raw meat chewiness supported the linkage between volatile flavor profiles and textural properties, indicating that flavor formation is closely related to protein structural changes and water redistribution during processing.

Beyond these statistical associations, the enhanced efficacy of MNB-combined treatments may be attributable to reduced mass transfer resistance. SEM revealed widened inter-fiber spaces and loosened endomysium (Fig. 2A), which may shorten diffusion pathways, while LF-NMR indicated increased water mobility (Table 1), suggesting enhanced diffusivity. Functionally, the greater pH reduction in MNB + Lp and higher TFFA accumulation in MNB + Lip indicated a potential link that MNB facilitated the accessibility and uniform action of biological agents. Collectively, these lines of evidence are consistent with the hypothesis that MNB pretreatment reduces mass transfer resistance in lamb muscle, providing a mechanistic link between physical restructuring and the observed synergistic biochemical outcomes.

It should be noted that the proposed mechanism of MNB-reduced mass transfer resistance is currently supported by multiple lines of indirect evidence. SEM showed widened inter-fiber spaces, LF-NMR revealed increased free water (T₂₃: 4.65% → 14.18%), and functional outcomes (lower pH in MNB + Lp, higher TFFA in MNB + Lip) further corroborate this inference. Direct visualization of bacterial or enzyme penetration was not performed. Therefore, the proposed improvement in accessibility and distribution is based on indirect evidence from SEM, LF-NMR, and functional outcomes. Future studies using fluorescent labeling are warranted to provide direct evidence.

Interaction effect analysis (Supplementary Tables S5 and S6) revealed that MNB + Lp had significant interactions for TFFA, porosity, gel hardness, WHC, and T₂₃; however, the direction of these interactions was indicator-dependent. In particular, MDA showed an antagonistic interaction (p < 0.05, interaction coefficient + 0.33), indicating that the combined reduction in MDA was smaller than the additive expectation, despite the lowest absolute MDA value being observed in the combined groups. MNB + Lip showed significant interactions for TFFA, MDA, porosity, WHC, T₂₃, and protein carbonyl (p < 0.05); however, the direction of these interactions was indicator-dependent. For MDA, protein carbonyl, TFFA, WHC, porosity, and T₂₃, the interaction was synergistic, whereas gamey odor showed an antagonistic interaction. These results indicate pathway-dependent effects of MNB pretreatment. For MNB + Lp, synergistic interactions were observed for TFFA, porosity, gel hardness, WHC, and T₂₃, whereas MDA showed an antagonistic interaction; pH, protein carbonyl, and gamey odor were additive. For MNB + Lip, synergistic interactions were observed for TFFA, MDA, porosity, WHC, T₂₃, and protein carbonyl, whereas gamey odor showed an antagonistic interaction; pH and gel hardness were additive (Supplementary Tables S5 and S6). This quantitative interaction analysis further supports our core innovation hypothesis that MNB pretreatment acts as a physical platform to amplify the modification efficiency of subsequent biological agents, with synergistic effects on several key quality indicators, while other indicators showed additive or antagonistic interactions.

3.15. Microbiological safety

Microbiological safety was assessed by total viable count (TVC), lactic acid bacteria (LAB), and total volatile basic nitrogen (TVB-N) (Table S7). LAB counts in the non-Lp groups were below the detection limit (<3.00 log CFU/g), whereas the Lp and MNB + Lp groups reached 7.04 and 7.81 log CFU/g, respectively. Their TVC values (7.08 and 7.84 log CFU/g) were close to the corresponding LAB counts, indicating that the high TVC mainly originated from the inoculated Lactiplantibacillus plantarum rather than spoilage bacteria. All TVB-N values (4.34–9.19 mg/100 g) were well below the national limit for fresh lamb (15 mg/100 g), confirming that the 3-h treatment did not cause protein spoilage. Detailed methods and data are provided in Supplementary Methods S2, S3 and Table S7.

4. Conclusion

This study demonstrates that MNB pretreatment synergistically enhances Lp fermentation and lipase hydrolysis on lamb meat through distinct physical-biochemical pathways. MNB-induced structural loosening creates micropores and reduces mass transfer resistance without altering protein conformation, which likely improves the accessibility and uniform action of subsequent biological agents.

MNB + Lp induces homogeneous acidification toward the isoelectric point, triggering protein aggregation and fiber contraction that expel immobile water and increase hardness, while achieving a 71% MDA reduction and a 7.9-fold increase in key volatile aldehydes (nonanal, (E)-2-nonenal, and (E)-2-decenal) through sustained microbial lipolysis coupled with antioxidant protection. In contrast, MNB + Lip promotes uniform hydrolysis, releasing free fatty acids that enhance hydrophobic interactions and disulfide cross-linking during heating, forming a compact gel network with superior water-holding capacity and springiness.

Collectively, this work proposes a novel physical synergistic strategy based on micro-nano bubbles, which innovatively realizes differentiated targeted modification of lamb via two distinct biological pathways, providing new experimental evidence for the combined application of MNB technology and biological processing in mutton deep processing.

CRediT authorship contribution statement

Meijuan Xu: Writing – original draft, Methodology, Investigation, Conceptualization. Xinfeng Yue: Software, Methodology, Formal analysis, Data curation. Yuxian Liu: Software, Methodology, Data curation. Fuyi Li: Resources, Methodology, Conceptualization. Yating Han: Software, Methodology, Data curation. Xi Han: Software, Methodology, Investigation. Zeyu Peng: Supervision, Formal analysis, Conceptualization. Jian Zou: Writing – review & editing, Funding acquisition, Conceptualization.

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

Supplementary material

Characterization of micro-nano bubble water and its effects on the physicochemical, microbiological, and sensory quality of lamb

Data availability

Data will be made available on request.