Glycerophospholipid metabolism as a central hub: Sublancin and oregano essential oil differentially reprogram goat meat quality and flavor

Abstract

Sublancin (SUB) and oregano essential oil (OEO) differentially affect goat meat quality. SUB increased carcass weight, cooked meat rate, polyunsaturated fatty acids (PUFA), and antioxidants, reduced L*/b*, and remodeled glycerophospholipid, TCA, and the phospholipase D signaling pathway. Lysophosphatidylcholine reduction correlated with improved color, cooked meat rate, and oxidative stability. OEO improved slaughter performance, reduced pH, L*/b*, cooking loss, and elevated antioxidants via down-regulating glycerolipid/glycerophospholipid metabolism without altering PUFA, indicating antioxidant-mediated preservation. Both reduced certain amino acids without compromising protein quality or flavor. Multi-omics revealed convergent yet pathway-specific mechanisms: SUB integrates energy–lipid metabolism to enhance PUFA and processing traits; OEO acts via antioxidant–lipid crosstalk for stability and water-holding. This provides a mechanistic framework for antibiotic-free ruminant production.

Highlights

• •Sublancin and oregano essential oil improve goat meat quality.
• •Sublancin improves meat quality via TCA and phospholipid pathways.
• •Oregano essential oil improves meat quality by inhibiting glycerolipid synthesis.
• •LPC is a key metabolite hub linking phospholipids to meat traits.
• •Sublancin and oregano essential oil impact broad and pathway-specific mechanisms.

1. Introduction

Goat meat is widely consumed globally, serving as an important source of protein in daily diets, particularly in developing countries, as it is not subject to significant religious or cultural restrictions in terms of production, slaughter, and consumption—aside from the specific requirement for Halal slaughter among Muslim communities (Abhijith et al., 2023). According to the latest 2024 data from the Food and Agriculture Organization of the United Nations (FAO), global goat meat production reached 7.74 million metric tons, making a significant contribution to the increasing global supply of animal protein, as well as to long-term food security and associated health benefits. Male offspring from dairy goat systems also constitute an important component of global goat meat production. In goat meat production, some issues still need to be addressed, including but not limited to: against the backdrop of the prohibition on the use of antibiotics in feed in China, the European Union, and other regions, how to ensure animal health and welfare, and how to enhance meat flavor and improve other meat quality traits.

The overuse of antibiotics leads to issues such as bacterial resistance and drug residues in food, which pose a serious threat to human health and the safety of animal-derived food products (Allen et al., 2014). In response, an increasing number of countries, organizations, or regions have implemented bans on the addition of antibiotics to animal feed (the European Union in 2006, the United States in 2012, Japan in 2008, and China in 2020) (Cao et al., 2025). Antimicrobial peptides, plant essential oils, microalgae (Boukrouh et al., 2025), Azolla pinnata (Soumaya, 2025), Sulla flexuosa (Boukrouh et al., 2023) and other green feed additives possess remarkable advantages including non-resistance, no drug residue, environmental friendliness and sustainability. Nevertheless, their efficacy and action mechanisms vary with different target animal species. Sublancin (SUB)—a kind of antimicrobial peptide extracted from Bacillus subtilis—comprises 37 amino acid residues and features remarkable stability, and it can kill multiple Gram-positive bacteria such as Bacillus cereus, Staphylococcus aureus and Streptococcus pyogenes (Garcia De Gonzalo et al., 2014). Studies have shown that dietary changes can influence goat meat quality and antioxidant activity by altering the rumen or intestinal microbiota (Sun et al., 2022; Uğurlu et al., 2025). Therefore, while exhibiting antibacterial properties, SUB may also exert a notable influence on meat quality and flavor. Although Wang et al. (2025) have demonstrated in poultry studies that Bacillus subtilis can enhance the muscle antioxidant capacity (by increasing the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px)) and improve the meat quality of yellow-feathered broilers. Liu et al. (2022) demonstrated that dietary probiotic supplementation could improve meat quality, remodel muscle fiber type composition, and regulate meat flavor as well as intestinal microbial community in lambs. Oregano essential oil (OEO), an aromatic plant extract, has been shown to possess various biological properties as a dietary supplement, including antioxidant, antifungal, antiviral, and anti-inflammatory effects. Research indicates that its primary active components are phenolic compounds, notably thymol and carvacrol (Ma et al., 2023). Previous studies have demonstrated that OEO can maintain water-holding capacity and meat color of Pingliang Red beef, increase the contents of essential amino acids (EAA), flavor amino acids and total amino acids (TAA), elevate the activities of CAT, GSH-Px and SOD, and thus improve meat quality (He et al., 2023). CAT, SOD and GSH-Px are closely correlated with the antioxidant capacity of the organism (Shirinov et al., 2026). Additional research has demonstrated that dietary supplementation with OEO can reduce the compression strength of mutton while increasing its a* value and unsaturated fatty acid content (UFA). This combination of effects helps prevent lipid oxidation, thereby serving as a viable alternative to monensin in feed to enhance overall meat quality (Garcia-Galicia et al., 2020).

Despite these benefits being established, information on how SUB and OEO influence goat meat quality traits at the metabolic level remains very limited. Meat quality is a multifactorial trait determined by the dynamic interplay of muscle energy metabolism, lipid and protein oxidation, and postmortem biochemical processes—all of which are ultimately regulated by the muscle metabolome (Hwang et al., 2023; Liu, He, et al., 2023). Glycerophospholipids, amino acids and fatty acids serve not only as structural components and metabolic substrates, but also as direct precursors for volatile flavor substances generated from the Maillard reaction and lipid oxidation (Duan et al., 2024). Therefore, characterizing the metabolic alterations induced by SUB and OEO supplementation is essential to understanding how these additives exert their effects on meat quality and flavor. We hypothesized that SUB and OEO affect meat quality and flavor by regulating these metabolites, as well as some shared and specific metabolic pathways. To verify this hypothesis, this study systematically investigated the effects of dietary supplementation with SUB and OEO on goat meat quality, flavor and metabolism for the first time. This work aimed to elucidate the mechanisms underlying meat quality improvement by these two antibiotic alternatives, and provide a theoretical basis for their rational application in ruminant production.

2. Materials and methods

2.1. Animals, experimental design, and sample collection

All animal experiments in this study were strictly conducted in accordance with the Guidance on the operation of the Animals (Scientific Procedures) Act 1986 and associated guidelines, EU Directive 2010/63 for the protection of animals used for scientific purposes, and the NIH Guide for the Care and Use of Laboratory Animals. All experimental animals were purchased from Qingyang Weihe Dairy Products Co., Ltd. (Qingyang, China). The study was approved by the Animal Protection Committee of Gansu Agricultural University (Lanzhou, China) under the approval number GSAU-Eth-AST-2022-001, and was carried out in compliance with the rules and guidelines formulated by this committee. A total of 27 three-month-old male offspring from Saanen dairy goats with similar body weight (16.07 ± 1.54 kg) were randomly divided into three groups based on initial body weight via a completely randomized design (CRD). Each group consisted of nine goats, with each goat serving as a biological replicate. One group served as the control (CON) and was fed a basal diet. The other two groups received, based on the optimal dosages determined by Zhou (2019) and Ren et al. (2019), respectively, 7 g of OEO per goat per day or 2 g of SUB per goat per day. OEO was obtained from Ralco Agricultural Lnc., Marshall, MN, USA. The essential oil was extracted from common oregano by steam distillation at 100 °C. Clinoptilolite powder was employed as an inert carrier to prevent the volatilization of the essential oil. The additive contained 1.3% OEO, with the major active constituents being carvacrol (51.78%; 6.73 g/kg) and thymol (6.93%; 0.90 g/kg). Sublancin was manufactured according to feed additive-Sublancin (NYSL-1004-2022). The antimicrobial peptide sublancin (C₁₆₂H₂₅₄N₅₀OS₅) was formulated using corncob powder as a carrier to achieve a uniform distribution of the peptide, thereby preventing localized over-concentration that could cause adverse effects. The effective content of sublancin in the formulation is 0.1% (1000 mg/kg). Formulation of total mixed rations (TMR) based on nutrient requirements of meat-type sheep and goat (NY/T 816–2021). The dietary ingredients and nutrient concentrations are presented in Table S1. The fatty acid composition of the diet is presented in Table S2. Prior to each feeding, 200 g of the TMR was thoroughly mixed with either OEO or SUB to ensure complete intake of the additives. After which, the remaining TMR was provided. The experimental period comprised of a 15-day adaptation period followed by a 124-day formal trial period. During the experiment, the animals were fed twice daily at 08:00 and 18:00, and were housed in individual pens with free access to feed and water. To ensure that the goats had adequate ad libitum access to feed, approximately 10% of the daily feed was left as orts, and both feed intake and orts were recorded daily. The experiment was conducted in Huanxian County (36°34′N, 107°18′E, altitude approximately 1500 m), Gansu Province, China, which is located in a temperate continental semi-arid climate zone. During the experimental period, the average indoor temperature was 17.0 °C, the average relative humidity was 63%, and natural ventilation was applied. In each group, three goats were excluded due to non-treatment-related reasons (including one goat in the SUB group because of diarrhea). Six goats of comparable body weight from each group were enrolled for further analyses to guarantee the reliability of statistical results. Subsequently, the goats were transported to a commercial slaughterhouse, where they were fasted for 12 h prior to humane slaughter. After slaughter, the head, hooves, skin, and viscera were removed. The kidneys were retained, and the carcass was weighed to obtain the hot carcass weight. The slaughter rate was then calculated by dividing the hot carcass weight by the pre-slaughter live weight. Immediately after slaughter, samples of the longissimus dorsi muscle were collected from the left side of the goat between the 12th and 13th ribs, and excess fat and fascia were trimmed off. The samples were stored at 4 °C, and their meat quality was evaluated. Additionally, approximately 200 g of samples were taken and stored at −80 °C for the determination of amino acids, fatty acids, antioxidant properties, flavor compounds, and metabolomes.

2.2. Meat quality measurements

Muscle pH was measured at 45 min post-slaughter using a portable pH meter (PHS-25/2F, (Beijing Dongnan Yicheng Laboratory Equipment Co., Ltd., Beijing, China)). Subsequently, chromaticity values for redness (a*), yellowness (b*), and lightness (L*) were measured using a colorimeter (CR-10 Konica Minolta, Tokyo, Japan) under D65 illuminant with a 10° observer angle and an 8 mm aperture. Both muscle color and pH were measured three times at random locations. Approximately 30–40 g of muscle tissue was trimmed into rectangular blocks measuring 2 × 2 × 2.5 cm³, weighed (recorded as W₁), sealed in heat-resistant bags under vacuum, and then subjected to cooking loss determination. The bagged samples were immersed in a water bath maintained at 80 °C and removed when the core temperature reached 70 °C. Subsequently, the samples were cooled to room temperature, surface moisture was blotted with filter paper, and the samples were weighed again (recorded as W₂). Cooking loss was calculated as follows: [(W₁–W₂)/W₁] × 100%. Cooked meat rate was calculated as follows: (W₂/W₁) × 100%. Shear force was determined using an Instron universal testing machine (Model 1011, Instron Corp., Canton, MA, USA) fitted with a Warner-Bratzler shear head. The test was conducted in compression mode with a 50 kg load cell. The crosshead speed was set at 100 mm/min. Each meat core was sheared once perpendicular to the direction of the muscle fibers. Muscle samples of the same dimensions were weighed (recorded as W₁), then suspended in plastic containers without contacting the container walls, and stored at 4 °C for 24 h. Afterward, surface moisture was blotted with filter paper, and the samples were weighed again (recorded as W₂). Drip loss was calculated as follows: [(W₁–W₂)/W₁] × 100%. All measurements were performed in triplicate.

2.3. Amino acids and fatty acids profiling

Approximately 0.1 g of the sample was accurately weighed and hydrolyzed with 10 mL of 6 mol/L hydrochloric acid at 110 °C for 22 h. Following cooling, the hydrolysate was made up to 50 mL with water, and a 1 mL aliquot was evaporated to dryness under a nitrogen stream. The residue was reconstituted in 1 mL of water and dried again under nitrogen. It was then redissolved in 1 mL of 0.02 mol/L hydrochloric acid and filtered through a 0.22-μm membrane. Amino acid composition was analyzed using a Hitachi LA8080 amino acid analyzer (Hitachi High-Technologies, Tokyo, Japan). Separation was achieved on a sulfonated cation-exchange resin column. Detection wavelengths were set at 570 nm and 440 nm. A standard H-type amino acid mixture (FUJIFILM Wako Pure Chemical Corporation) was used as the stock solution. For instrument calibration, 1 mL of this stock solution was precisely pipetted and diluted to volume with 0.02 mol/L hydrochloric acid to prepare the working standard solution, resulting in a final concentration of 2 nmol/20 μL for each amino acid. Note on methodological limitations: Under the acid hydrolysis conditions employed (6 mol/L HCl, 110 °C, 22 h), tryptophan is completely destroyed. Moreover, methionine and cysteine are susceptible to oxidation, resulting in their underestimation.

Approximately 1 g of the sample was weighed, and after the addition of an internal standard solution (triundecanoin), hydrolysis was performed. The lipids were then extracted with diethyl ether. To the lipid extract, 8 mL of a 2% sodium hydroxide in methanol solution was added. The mixture was then refluxed at 80 °C in a water bath until the oil droplets disappeared. Then, 7 mL of a 15% boron trifluoride-methanol solution was added, and the mixture was refluxed at 80 °C for an additional 2 min. After cooling to room temperature, 2 mL of n-heptane was added, and the mixture was vortexed for 2 min. Subsequently, a saturated sodium chloride aqueous solution was added, and the mixture was allowed to stand for phase separation. The upper n-heptane extract was collected into a 25 mL test tube. Then, 2–3 g of anhydrous sodium sulfate was added, the mixture was vortexed for 1 min and allowed to stand for 5 min. Finally, the supernatant was transferred to a vial for analysis. Fatty acid methyl esters were analyzed using an Agilent 7890 A gas chromatograph equipped with a flame ionization detector (FID). Separation was performed on a fused-silica capillary column (100 m × 0.25 mm i.d. × 0.2 μm film thickness) coated with a strongly polar stationary phase of poly (dicyanopropylsiloxane). The injector and detector temperatures were 250 °C and 260 °C, respectively. The oven temperature program was: initial 125 °C held for 2 min, increased to 180 °C at 12 °C/min and held for 6 min, then increased to 200 °C at 3.5 °C/min and held for 20 min, and finally increased to 230 °C at 5 °C/min with a final hold of 8 min. Nitrogen was used as the carrier gas. The split ratio was 10:1, and the injection volume was 1.0 μL. Identification of individual fatty acid methyl esters was achieved by comparing the retention times of sample peaks with those of a 28-component fatty acid methyl ester mixed standard and single fatty acid methyl ester standards (ANPEL, China) analyzed under the same chromatographic conditions. Chromatographic peaks were integrated using the GC software. Perform peak integration using GC software, and quantify the content via the internal standard method (expressed as mg/100 g sample).

2.4. Muscle antioxidant status

Muscle samples (15 g) were homogenized with physiological saline at a 1:9 (w/v) ratio using a homogenizer at 5000 r/min for 15 min in an ice-water bath. The activities of T-AOC, CAT, GSH-Px, and SOD, as well as the content of malondialdehyde (MDA), in muscle tissue were determined using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, China) strictly following the manufacturer's instructions. The intra-batch coefficient of variation for all indicators was less than 5%. The limits of detection and units were as follows: T-AOC, 0.125 U/mg protein, CAT, 0.025 U/mg protein; GSH-Px, 0.5 U/mg protein; SOD, 0.2 U/mg protein; and MDA, 0.005 nmol/mg protein. The absorbance was measured using a spectrophotometer (V6000A, Metash Instruments, Shanghai, China), and the corresponding concentration/activity was calculated according to the manufacturer's instructions provided with the assay kit.

2.5. Flavor compound content

The analysis of flavor compounds in muscle tissue was performed by gas chromatography-ion mobility spectrometry (GC-IMS) according to the methods reported by Huang et al. (2025). and Kang et al. (2024). The volatile organic compounds were analyzed using a FlavourSpec® flavor analyzer (G.A.S., Germany). The analysis was performed over a 20 min runtime. The samples were incubated at 60 °C for 15 min with constant agitation at 500 rpm. A 500 μL aliquot of the headspace was injected automatically into the GC-IMS system using an 80 °C syringe. Separation was carried out on an FS-SE-54-CB-1 capillary column (15 m × 0.53 mm i.d.) maintained at 60 °C, with nitrogen as the carrier gas. The IMS detector temperature was set at 45 °C. The retention indices (RI) were determined using n-alkanes (C4–C9) (Sinopharm Chemical Reagent Beijing Co., Ltd., Beijing, China) as external standards. The identification of volatile organic compounds was achieved by matching their retention indices against drift times in the NIST and IMS databases integrated within the software, while relative quantification was conducted according to the intensities of the detected signal peaks.

2.6. Metabolomic analysis

The tissue specimens were retrieved from the −80 °C freezer, thawed on ice, and homogenized by grinding under liquid nitrogen, followed by accurate weighing of 20 mg. Add 400 μL of 70% methanol-water internal standard extraction solution, shake at 2500 r/min for 5 min, and let it stand on ice for 15 min. Centrifuge at 4 °C and 12,000 r/min for 10 min, collect 300 μL of the supernatant, and place it in a − 20 °C refrigerator for 30 min. Centrifuge again at 12000 r/min for 3 min under 4 °C, collect 200 μL of the supernatant, and detect it using an UPLC-MS/MS platform. A Waters ACQUITY UPLC HSS T3 C18 column (1.8 μm, 2.1 mm × 100 mm) was used. The mobile phase A was ultrapure water containing 0.1% formic acid, and the mobile phase B was acetonitrile containing 0.1% formic acid. Elution gradient: 95:5 (V/V) water/acetonitrile at 0 min, 80:20 (V/V) at 2.0 min, 40:60 (V/V) at 5.0 min, 1:99 (V/V) at 6.0 min, 1:99 (V/V) at 7.5 min, 95:5 (V/V) at 7.6 min, and 95:5 (V/V) at 10.0 min. The flow rate was 0.4 mL/min. The column temperature was maintained at 40 °C, and the injection volume was 2 μL. The temperature of the electrospray ionization (ESI) source was set to 500 °C, with a mass spectrometry voltage of 5500 V (positive mode) and − 4500 V (negative mode). The ion source gas I (GSI) was set to 55 psi, gas II (GSII) to 60 psi, and curtain gas (CUR) to 25 psi. The parameter for collision-activated dissociation (CAD) was set to high. Qualitative analysis was conducted based on the MetWare Database (MWDB), utilizing the retention time (RT), precursor/product ion pairs, and MS/MS spectral data of the detected compounds. Quality control (QC) samples were prepared by pooling sample extracts and spiking with internal standards of known concentrations. Principal component analysis (PCA) and relative standard deviation (RSD) analysis were performed on QC samples. The favorable results confirmed the stability of the analytical process and the high quality of the data, thereby meeting the testing requirements. Annotation of the identified metabolites was performed with the KEGG Compound database (http://www.kegg.jp/kegg/compound/), followed by mapping to the KEGG pathway database (http://www.kegg.jp/kegg/pathway.html).

2.7. Statistical analysis

Data on meat quality, slaughter performance, amino acids, fatty acids, and muscle antioxidant properties were analyzed by one-way ANOVA using SPSS 27.0, followed by Duncan's post hoc test. Data are expressed as mean ± standard deviation, and a P-value <0.05 was considered statistically significant. Volatile flavor compounds and metabolomics data were analyzed separately against the CON group using Student's t-test. PCA and orthogonal partial least squares-discriminant analysis (OPLS-DA) were subsequently performed. In addition, permutation analysis was conducted to validate the established OPLS-DA model. Compounds with a variable importance in projection (VIP) > 1 and a P-value <0.05 were considered differential flavor substances. Metabolites were considered differentially expressed when variable VIP > 1, FC ≥ 1.5 or ≤ 0.67, and false discovery rate (FDR) < 0.05.

3. Results

3.1. Slaughter performance and meat quality

As shown in Table 1, the L* value of the SUB group was 27.66 ± 1.54, which was significantly lower than that of the CON group (34.09 ± 0.89), with a decrease of 18.86%. The b* value of the SUB group was 7.95 ± 0.48, which was significantly lower than that of the CON group (12.18 ± 0.50), with a decrease of 34.73%. The cooked meat rate of the SUB group was 66.92 ± 0.64, which was significantly higher than that of the CON group (56.82 ± 1.26), with an increase of 17.78%. The carcass weight of the SUB group was 20.32 ± 1.92, which was significantly higher than that of the CON group (17.26 ± 3.08), with an increase of 17.73%. As shown in Table 1, the pH value of the OEO group was 6.28 ± 0.22, which was significantly lower than that of the CON group (6.63 ± 0.25), with a decrease of 5.28%. The L* value of the OEO group was 28.78 ± 1.32, which was significantly lower than that of the CON group (34.09 ± 0.89), with a decrease of 15.58%. The b* value of the OEO group was 8.08 ± 0.66, which was significantly lower than that of the CON group (12.18 ± 0.50), with a decrease of 33.66%. The cooking loss of the OEO group was 24.93 ± 4.60, which was significantly lower than that of the CON group (31.50 ± 3.25), with a decrease of 20.86%. The cooked meat rate of the OEO group was 67.38 ± 2.08, which was significantly higher than that of the CON group (56.82 ± 1.26), with an increase of 18.58%. The carcass weight of the OEO group was 20.92 ± 2.07, which significantly higher than that of the CON group (17.26 ± 3.08), with an increase of 21.21%. The slaughter rate of the OEO group was 50.84 ± 1.26, which was significantly higher than that of the CON group (47.73 ± 3.45), with an increase of 6.52%.

Table 1.

Effects of dietary supplementation with SUB and OEO on meat quality of the longissimus dorsi muscle and slaughter performance in goats.

Item	CON	SUB	OEO	P-value
Meat quality
pH	6.63 ± 0.25a	6.35 ± 0.22ab	6.28 ± 0.22b	0.051
Lightness (L*)	34.09 ± 0.89a	27.66 ± 1.54b	28.78 ± 1.32b	<0.001
Redness (a*)	19.53 ± 0.57ab	19.02 ± 1.15b	20.91 ± 1.77a	0.054
Yellowness (b*)	12.18 ± 0.50a	7.95 ± 0.48b	8.08 ± 0.66b	<0.001
Cooked meat rate (%)	56.82 ± 1.26b	66.92 ± 0.64a	67.38 ± 2.08a	<0.001
Cooking loss (%)	31.50 ± 3.25a	29.00 ± 2.96ab	24.93 ± 4.60b	0.023
Shear force (N)	63.12 ± 27.68	49.32 ± 14.14	45.30 ± 17.54	0.319
Drip loss (%)	1.52 ± 0.37	1.58 ± 0.69	1.99 ± 0.71	0.385
Slaughter performance
Carcass weight (kg)	17.26 ± 3.08b	20.32 ± 1.92a	20.92 ± 2.07a	0.042
Slaughter rate (%)	47.73 ± 3.45b	50.43 ± 1.91ab	50.84 ± 1.26a	0.080

Note: Different lowercase letters within the same row indicate significant differences (P < 0.05).

CON: basic diet group, SUB: sublancin group, OEO: oregano essential oil group.

3.2. Fatty acid profile

As shown in Table 2, the SUB group resulted in a significant increase in C8:0, C10:0, C18:2n6c, C20:3n6, C24:0, and polyunsaturated fatty acids (PUFA) (P < 0.05) compared with the CON and OEO groups.

Table 2.

Effects of dietary supplementation with SUB and OEO on fatty acid profile of the longissimus dorsi muscle in goats.

Item (mg/100 g)	CON	SUB	OEO	P-value
C8:0	0.27 ± 0.21b	1.23 ± 0.67a	0.33 ± 0.15b	0.050
C10:0	3.33 ± 1.53b	8.00 ± 0.87a	5.00 ± 1.00b	0.007
C12:0	2.00 ± 1.00	3.00 ± 1.00	2.67 ± 1.15	0.533
C13:0	0.70 ± 0.26	1.23 ± 0.68	1.27 ± 0.64	0.434
C14:0	72.33 ± 2.02	89.00 ± 46.17	90.67 ± 30.44	0.750
C14:1n5	6.00 ± 4.00	6.67 ± 4.51	6.33 ± 3.06	0.978
C15:0	16.00 ± 10.15	17.33 ± 3.51	18.67 ± 3.51	0.884
C16:0	619.67 ± 227.18	662.67 ± 258.23	890.67 ± 84.67	0.292
C16:1n7	119.33 ± 21.55	130.33 ± 20.00	154.33 ± 18.18	0.170
C17:0	61.33 ± 14.97	67.67 ± 20.03	67.00 ± 22.91	0.911
C18:0	486.67 ± 230.35	481.66 ± 116.72	557.00 ± 177.81	0.854
C18:1n9t	10.33 ± 7.09	12.67 ± 6.35	11.67 ± 3.21	0.887
C18:1n9c	2560.00 ± 1430.10	3100 ± 2149.51	2526.67 ± 934.04	0.886
C18:2n6t	4.00 ± 1.00	3.00 ± 1.00	3.33 ± 0.58	0.422
C18:2n6c	263.00 ± 32.08b	355.67 ± 53.80a	234.67 ± 17.67b	0.018
C20:0	2.00 ± 1.00	3.73 ± 1.51	1.67 ± 0.58	0.122
C18:3n6c	3.33 ± 1.53	4.33 ± 2.52	3.00 ± 0.00	0.630
C20:1	5.67 ± 2.52	8.00 ± 2.65	6.00 ± 2.65	0.531
C18:3n3	7.33 ± 3.51	9.00 ± 3.61	4.67 ± 0.58	0.264
C21:0	1.47 ± 1.36	1.13 ± 0.81	0.60 ± 0.20	0.544
C20:2	3.33 ± 1.15	3.67 ± 2.08	2.33 ± 0.58	0.524
C20:3n6	4.67 ± 1.15b	6.67 ± 0.58a	4.33 ± 0.58b	0.026
C20:4n6	58.33 ± 11.93	79.00 ± 3.46	61.67 ± 17.21	0.166
C23:0	1.33 ± 0.58	3.67 ± 2.08	1.67 ± 0.58	0.134
C24:0	2.33 ± 0.57b	6.00 ± 2.00a	3.00 ± 0.00b	0.021
C20:5n3	2.00 ± 1.00	3.67 ± 3.79	1.23 ± 0.68	0.462
C24:1n9	2.00 ± 0.00	5.00 ± 2.65	2.00 ± 0.00	0.084
C22:6n3	2.67 ± 0.58	2.60 ± 1.64	0.83 ± 0.28	0.117
SFA 1	1269.43 ± 353.23	1346.33 ± 322.19	1640.33 ± 167.97	0.330
MUFA 2	2703.33 ± 1465.10	3262.67 ± 2175.60	2707.00 ± 951.24	0.889
PUFA 3	348.67 ± 52.44b	467.60 ± 51.70a	316.07 ± 18.43b	0.012

Note: Different lowercase letters within the same row indicate significant differences (P < 0.05).

CON: basic diet group, SUB: sublancin group, OEO: oregano essential oil group.

¹SFA:Saturated fatty acid, (C8:0 + C10:0 + C12:0 + C13:0 + C14:0 + C15:0 + C16:0 + C17:0 + C18:0 + C20:0 + C21:0 + C23:0 + C24:0).

²MUFA: Monounsaturated fatty acids(C14:1n5 + C16:1n7 + C18:1n9t + C18:1n9c + C20:1 + C24:1n9).

³PUFA: Polyunsaturated fatty acids(C18:2n6t + C18:2n6c + C18:3n6c + C18:3n3 + C20:2 + C20:3n6 + C20:4n6 + C20:5n3 + C22:6n3).

3.3. Amino acid profile

As can be seen in Table 3, compared to the CON group, the SUB group exhibited significant reductions in the contents of Ser, Met, Ile, Phe, non-essential amino acids (NEAA), and TAA (P < 0.05) in the longissimus dorsi muscle of goats. Compared to the CON group, the OEO group exhibited significantly lower contents of Ser, Ala, Met, Ile, Tyr and sweet amino acids (SAA) in the longissimus dorsi muscle of goats (P < 0.05).

Table 3.

Effects of dietary supplementation with SUB and OEO on the amino acid profile of the longissimus dorsi muscle in goats.

Item (g/100 g)	CON	SUB	OEO	P-value
Asp	2.01 ± 0.21ab	1.87 ± 0.05b	2.13 ± 0.21a	0.069
Thr	0.97 ± 0.09	0.86 ± 0.08	0.86 ± 0.19	0.275
Ser	0.89 ± 0.06a	0.72 ± 0.05c	0.82 ± 0.05b	<0.001
Glu	3.76 ± 0.19	3.68 ± 0.58	3.64 ± 0.23	0.843
Gly	1.30 ± 0.15	1.24 ± 0.19	1.15 ± 0.25	0.442
Ala	1.48 ± 0.14a	1.32 ± 0.17ab	1.20 ± 0.16b	0.027
Cys	0.21 ± 0.02	0.22 ± 0.03	0.21 ± 0.03	0.872
Val	1.00 ± 0.14	0.94 ± 0.06	0.99 ± 0.13	0.703
Met	0.58 ± 0.04a	0.45 ± 0.05b	0.46 ± 0.08b	0.002
Ile	1.02 ± 0.08a	0.81 ± 0.07b	0.87 ± 0.16b	0.018
Leu	1.53 ± 0.53	1.48 ± 0.13	1.65 ± 0.23	0.666
Tyr	0.76 ± 0.08a	0.65 ± 0.08ab	0.61 ± 0.15b	0.077
Phe	0.96 ± 0.06a	0.83 ± 0.08b	0.88 ± 0.09ab	0.031
Lys	1.89 ± 0.17	1.80 ± 0.18	1.69 ± 0.39	0.411
His	0.75 ± 0.05	0.70 ± 0.11	0.70 ± 0.11	0.650
Arg	1.48 ± 0.22	1.30 ± 0.10	1.33 ± 0.14	0.166
Pro	1.07 ± 0.20	0.87 ± 0.05	0.85 ± 0.40	0.320
FAA 1	11.41 ± 0.55	10.75 ± 0.85	10.70 ± 0.67	0.182
SAA 2	7.60 ± 0.64a	6.82 ± 0.51ab	6.57 ± 0.83b	0.045
EAA 3	8.70 ± 0.80	7.82 ± 0.42	8.01 ± 1.32	0.252
NEAA 4	12.94 ± 0.69a	11.93 ± 0.83b	12.04 ± 0.73ab	0.066
EAA/TAA (%)	40.15 ± 1.89	39.62 ± 2.20	39.78 ± 3.72	0.942
EAA/NEAA (%)	67.24 ± 5.31	65.80 ± 5.81	66.59 ± 10.11	0.945
TAA 5	21.65 ± 1.29a	19.75 ± 0.88b	20.05 ± 1.76ab	0.062

Note: Different lowercase letters within the same row indicate significant differences (P < 0.05).

CON: basic diet group, SUB: sublancin group, OEO: oregano essential oil group.

¹FAA: flavor amino acids (Ala+Asp+Glu + Gly + Phe + Lys).

²SAA: sweet amino acids (Gly + Ser + Thr + Lys + Pro+Ala).

³EAA: essential amino acids (Thr + Val + Met+Ile + Leu + Phe + Lys + Try).

⁴NEAA: non-essential amino acids (Asp+Ser + Glu + Gly + Ala+Cys + Pro+Arg + His).

⁵TAA: total amino acids.

3.4. Oxidative status of muscle

As shown in Table 4, the SUB group exhibited significantly higher activities of T-AOC, CAT, and GSH-Px in the muscle compared to the CON group (P < 0.05). Compared to the CON group, the OEO group demonstrated significantly enhanced activities of CAT and GSH-Px in the muscle (P < 0.05).

Table 4.

Effects of dietary supplementation with SUB and OEO on the antioxidant properties of longissimus dorsi muscle in goats.

Item	CON	SUB	OEO	P-value
T-AOC (U/mg.protein)	1.29 ± 0.12b	1.65 ± 0.18a	1.48 ± 0.21ab	0.008
SOD(U/mg.protein)	10.88 ± 0.69	11.89 ± 1.43	10.67 ± 0.53	0.098
CAT (U/mg.protein)	1.88 ± 0.16c	2.40 ± 0.09a	2.15 ± 0.10b	<0.001
GSH-Px (U/mg.protein)	16.55 ± 1.45c	20.64 ± 1.03a	19.11 ± 0.49b	<0.001
MDA (nmol/mg.protein)	0.47 ± 0.07	0.42 ± 0.02	0.48 ± 0.09	0.338

Note: Different lowercase letters within the same row indicate significant differences (P < 0.05).

CON: basic diet group, SUB: sublancin group, OEO: oregano essential oil group.

3.5. Identification of muscle flavor compounds

Using GC-IMS analysis and subsequent database search, 38 volatile organic compounds were identified. These included nine alcohols, seven esters, six ketones, five aldehydes, four furans, one alkane, and six other compounds (Fig. 1A). PCA revealed that the first two principal components (PC1 and PC2) accounted for a cumulative 60.70% of the total variance (Fig. 1B). PCA showed that the CON group did not separate clearly from the SUB and OEO groups, with considerable overlap observed. This suggests that the overall differences in volatile flavor compounds among the three groups were relatively minor. The fingerprint profile reveals the relative abundance of each volatile organic compound within every sample (Fig. 1C).

Fig. 1.

Volatile organic compounds profile. Categories of volatile organic compounds (A). PCA plot (B). Volatile organic compounds fingerprint (C). CON: basic diet group, SUB: sublancin group, OEO: oregano essential oil group.

Conduct classification analysis of volatile flavor substances and differential analysis of individual compounds. Compounds with a VIP value >1 and P < 0.05 were considered differential flavor compounds. The content of alkanes in the OEO group was significantly lower than that in the CON group (P < 0.05) (Fig. 2A). Compared with the CON group, the relative contents of 3-pentanone, isopentanol, 2-butylfuran, E-3-hexen-1-ol, E-2-hexen-1-ol, and 3-methyl-2-butenal in the SUB group were significantly decreased (P < 0.05) (Fig. 2B). Compared with the CON group, the relative content of methyl acetate in the SUB group was significantly increased (P < 0.05). Compared with the CON group, the relative contents of isopentyl formate, 2,3-butanedione, 1-butene, isobutane, 2-butylfuran, and heptanal in the OEO group were significantly decreased (P < 0.05) (Fig. 2C). In summary, dietary supplementation with SUB or OEO had a limited overall impact on the flavor profile of goat meat, although it altered the content of certain volatile organic compounds.

Fig. 2.

Boxplot of volatile organic compounds. Analysis of volatile organic compound classification (A). SUB group differences in volatile organic compounds (B). OEO group differences in volatile organic compounds (C). * indicates P < 0.05, and ** indicates P < 0.01. CON: basic diet group, SUB: sublancin group, OEO: oregano essential oil group.

3.6. Muscle metabolomics analysis

To further explore the impact of SUB and OEO on muscle metabolism, we identified muscle metabolites by employing a widely-targeted metabolomics strategy. The UPLC-MS/MS platform identified a total of 961 metabolites, including 293 amino acid and its metabolites, 116 organic acid and its derivatives, 99 glycerophospholipids (GP), and 98 fatty acyls (FA), 90 nucleotide and its metabolites, 58 benzene and substituted derivatives, 51 heterocyclic compounds, 47 carbohydrates and its metabolites, 36 alcohol and amines, 16 aldehyde, ketones, esters, 13 hormones and hormone related compounds, 12 bile acids, 12 coenzyme and vitamins, 4 tryptamines, cholines, pigments, 3 glycerolipids (GL), 2 sphingolipids (SP) and 11 others (Fig. 3A). PCA showed that the CON group samples were distinctly separated from the SUB and OEO groups, while there was a partial overlap between the SUB and OEO groups (Fig. 3B, C, F). OPLS-DA further delineated the differences between the CON group and the SUB/OEO groups, with no signs of overfitting in the model (Fig. 3D, E, G, H). These results demonstrate that dietary supplementation with either SUB or OEO significantly altered the metabolite profile in the longissimus dorsi muscle of goats. Differential metabolites were identified based on the criteria of VIP > 1, FC ≥ 1.5 or ≤ 0.67, and FDR < 0.05. Compared to the CON group, the SUB group exhibited a significant increase in 92 metabolites and a significant decrease in 162 metabolites (Fig. 3I, TableS3). Compared to the CON group, the OEO group exhibited a significant increase in 13 metabolites and a significant decrease in 50 metabolites (Fig. 3J, Table S4).

Fig. 3.

The metabolite profile of goat longissimus dorsi muscle. Determine the composition and classification of metabolites (A). PCA analysis diagram of metabolites (B, C, F). OPLS-DA score plot (D, G). OPLS-DA validation plot (E, H). Volcano plot of differential metabolites between CON and SUB groups (I). Volcano plot of differential metabolites between CON and OEO groups(J). CON: basic diet group, SUB: sublancin group, OEO: oregano essential oil group. GP: glycerophospholipids, FA: fatty acyls, GL: Glycerolipids, SP: Sphingolipids.

Refinement of differential metabolites to identify critical metabolites. Fig. 4A and B display the top 20 most differential metabolites in the SUB and OEO groups, respectively. In the SUB group, the levels of compounds GDP-L-fucose, 7α,26-dihydroxy-4-cholesten-3-one, cysteine-glutathione disulfide and succinic anhydride were significantly increased, whereas those of compounds lysophosphatidylcholine (LPC) (18:4/0:0), LPC (16,3/0:0), LPC (18:3/0:0), pyridoxamine and lysophosphatidic acid (LPA) (18,3) were markedly reduced. In the OEO group, the levels of compounds 7α,26-dihydroxy-4-cholesten-3-one, and succinic anhydride were significantly increased, whereas the contents of LPC(O-16:0), maleamic acid, lysophosphatidylethanolamine (LPE) (18,3/0,0), and LPA (18,3) were markedly decreased. Categorization of the top 20 differential metabolites was performed. SUB predominantly altered the metabolism of LPC, organic acids and its derivatives, nucleotides and its metabolites, and phenolic acids (Fig. 4C). OEO primarily altered the metabolism of LPC, LPE and organic acids and its derivatives (Fig. 4D). LPC, LPA, and LPE belong to the class of glycerophospholipids. Phospholipids are mainly present in intramuscular fat. Glycerophospholipids can influence intramuscular fat content, muscle tenderness, flavor, and other attributes by affecting biological processes such as cell membrane structure and function, and cell signaling. Identification of metabolic pathways associated with differential metabolites through KEGG enrichment analysis. In the SUB group, among the 146 metabolic pathways identified for differential metabolites, a total of 7 pathways were significantly enriched after FDR correction (Table S5). The top 10 metabolic pathways are illustrated in Fig. 4E. In the OEO group, among the 45 metabolic pathways identified for differential metabolites, a total of 10 pathways were significantly enriched after FDR correction. The top 10 metabolic pathways are illustrated in Fig. 4F. Key metabolic pathways were determined via KEGG network mapping, and interrupted upstream and downstream signaling cascades were supplemented and identified. The core metabolic pathways in the SUB group include glycerophospholipid metabolism, citrate cycle (TCA cycle), the phospholipase D signaling pathway, and butanoate metabolism (Fig. 4G). In the OEO group, the core metabolic pathways include glycerophospholipid metabolism, glycerolipid metabolism, the phospholipase D signaling pathway and regulation of actin cytoskeleton (Fig. 4H). Notably, the differential metabolites in both the SUB group and the OEO group were commonly enriched in glycerophospholipid metabolism, which was identified as a core metabolic pathway. This suggests that SUB and OEO influence meat quality through both shared and specific metabolic pathways.

Fig. 4.

Key differential metabolites and KEGG enrichment analysis. Top 20 most differential metabolites in the SUB (A) and OEO (B) groups compared to the CON group. Sankey diagram of differential metabolite categories in the SUB (C) and OEO (D) groups compared with the CON group. KEGG enrichment plot for the SUB (E) and OEO (F) groups. KEGG network of the SUB (G) and OEO (H) groups. CON: basic diet group, SUB: sublancin group, OEO: oregano essential oil group. LPA: lysophosphophatidic acid, LPC: lysophosphatidylcholine, LPE: lysophosphatidylethanolamine.

3.7. Integrated analysis of differential metabolites and phenotypic data

Integrated analysis of metabolomic data and phenotypic data was performed to explore the potential mechanisms underlying the changes in muscle phenotypes. As shown in Fig. 5A (Table S7), LPC exhibited positive correlations with L* and b* values, and negative correlations with cooked meat rate, GSH-Px, CAT, and PUFA. As shown in Fig. 5B (Table S8), 7α,26-dihydroxy-4-cholesten-3-one and succinic anhydride were negatively correlated with L* and b* values, and positively correlated with cooked meat rate, CAT, and GSH-PX.

Fig. 5.

Correlation analysis. Correlation between differential metabolites and phenotypes in the SUB (A) and OEO (B) groups compared with the CON group. Correlation between differential metabolites and differential flavor compounds in the SUB (C) and OEO (D) groups compared with the CON group. (Spearman's correlation coefficient, |r| > 0.6, P < 0.05). Significance is reported as *P < 0.05, **P < 0.01. Red indicates a positive correlation, while blue indicates a negative correlation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

As shown in Fig. 5C (Table S9), GDP-L-fucose, succinic anhydride, 7α,26-dihydroxy-4-cholesten-3-one, and cysteine-glutathione disulfide were negatively correlated with E-2-hexen-ol. GDP-L-fucose, succinic anhydride, and cysteine-glutathione disulfide were negatively correlated with 2-butylfuran. E-2-hexen-1-ol was positively correlated with LPC, LPA (18:0), and pyridoxamine. As shown in Fig. 5D (Table S10), isobutane was negatively correlated with 7α,26-dihydroxy-4-cholesten-3-one and succinic anhydride. Positive correlations were detected between isobutane and partial LPCs, partial LPEs, LPA (18:3), as well as maleamic acid.

4. Discussion

In recent years, researchers have increasingly focused on phytonutrients, probiotics, antimicrobial peptides, and other sustainable alternatives in animal diets(Dong et al., 2023; Long et al., 2026). The aim is to enhance animal growth performance and improve meat quality while minimizing the accumulation of antibiotic residues in animal-derived foods and preventing the development of bacterial resistance. Postmortem hypoxia induces oxidative stress, leading to the accumulation of reactive oxygen species and an accelerated lactate production, ultimately resulting in a decline in muscle pH (Long et al., 2026). A decrease in pH facilitates the degradation of myofibrillar proteins, whereas a relatively high pH may lead to the decomposition of proteins into ammonia (NH₃) and amines during meat storage (Huang et al., 2025). In this study, compared with the CON group, the SUB group showed no significant difference in pH value, while the pH value in the OEO group was significantly decreased. In our previous study, it was found that dietary supplementation with OEO significantly reduced the pH of beef at 30 min and 24 h postmortem (He et al., 2023). Studies have indicated that diets containing essential oils (thymol, eugenol, clove, cashew nut shell liquid, and vanillin) may reduce pH value by slowing the oxidative process, improving proteolysis, and enhance tenderness (Ornaghi et al., 2020). In this study, OEO addition significantly strengthened muscle antioxidant capacity to relieve oxidative stress. Oxidative stress causes intramuscular glycogen consumption and attenuates postmortem glycolysis, contributing to increased muscle pH (Abhijith et al., 2023). Therefore, the improved antioxidant ability induced by OEO may reduce glycogen depletion, explaining the decreased muscle pH in OEO-treated goats. Although decreased, the muscle pH of the OEO group remained within the normal range, with subsequent changes potentially influenced by acid elimination and storage duration. Meat color is generally regarded as a key and readily perceptible sensory attribute that influences consumer purchase decisions. In this study, the SUB group exhibited significant decreases in L*and b* values. Generally speaking, meat color and water-holding capacity are closely related to pH value, with low pH values often associated with pale meat color and reduced water retention in the product (Liu, Liu, et al., 2023). However, in the SUB group, pH values and water-holding capacity did not decrease. These findings indicate that SUB may influence the color of goat meat to some extent, but it does not negatively affect pH value or water-holding capacity. The precise mechanisms underlying these effects remain unclear and warrant further investigation. Furthermore, studies have demonstrated that dietary supplementation with probiotics Bacillus subtilis KT260179 (Zheng et al., 2014) and Bacillus subtilis fmbj (Yang et al., 2016) significantly improved chicken meat color and water-holding capacity. Under normal circumstances, the a∗ value is positively correlated with meat quality, whereas the L∗ and b∗ values show a negative correlation with it (Ruedt et al., 2023). When pH decreases, cellular respiration is inhibited. This leads deoxymyoglobin to compete with mitochondria for oxygen and become oxidized to bright red oxymyoglobin, resulting in an increase in a* value (Xiao et al., 2022). In this study, both the SUB and OEO groups exhibited significant decreases in L* and b* values, with no significant change in a* values. These colorimetric alterations may have contributed to an improvement in meat quality. The increased cooked meat rate and decreased cooking loss further support this finding. OEO is rich in phenolic compounds, such as carvacrol and thymol, and possesses antioxidant properties (Zhang et al., 2015). According to the study by (Garcia-Galicia et al., 2020), OEO significantly improved the color, tenderness, and fat oxidation stability of lamb meat. Previous studies have also shown that dietary OEO supplementation significantly reduced cooking loss in beef but had no significant effect on drip loss (He et al., 2023). The cooked meat rate was also significantly higher in the SUB group than in the CON group. Cooked meat rate reflects the ability of muscle to retain water under cooking conditions (Ma, Song, et al., 2024). The results indicate that dietary supplementation with either SUB or OEO contributes to improved water retention properties of muscle under cooking conditions. As has been widely demonstrated, essential oils and antimicrobial peptides promote animal health and growth (Xiao et al., 2015; Zhang et al., 2023). In the present trial, the increased carcass weight in both the SUB and OEO groups, along with the higher slaughter rate in the OEO group, are consistent with these established findings.

The fatty acid profile of lamb meat plays a pivotal role in determining its nutritional value, flavor, and implications for human health (Zhang, Yang, et al., 2025). The composition of fatty acids in muscle significantly affects meat quality, and in ruminants, it is primarily influenced by diet and ruminal biohydrogenation (Boukrouh et al., 2024). Fatty acids can be classified into saturated and unsaturated fatty acids; excessive intake of saturated fatty acid (SFA) is associated with atherosclerosis, cardiovascular diseases, and metabolic syndrome (Wang et al., 2022). In the present study, the levels of C8:0, C10:0 and C24:0 were significantly higher in the SUB group compared to the CON group. However, analysis of the complete muscle fatty acid profile revealed no significant increase in SFA content. These findings indicate that dietary supplementation with SUB may alter the content of certain individual SFAs, but does not lead to an overall increase in SFAs content in muscle. Unsaturated fatty acids (UFA) generally exert beneficial effects on human health, such as anticancer and hypolipidemic effects, as well as cardiovascular disease prevention (Kong et al., 2023). In the present study, the levels of C18:2n6c, C20:3n6, and total PUFAs were significantly higher in the SUB group compared to the CON group. C18:2n6c (linoleic acid) is an essential fatty acid for humans, which must be obtained from the diet and plays critical roles in various physiological processes, including immune responses and inflammation (Han et al., 2025). Despite the extensive ruminal biohydrogenation process, changes in diet can alter the deposition of PUFAs in the meat of ruminants (Bessa et al., 2015). In general, long-chain fatty acids (including PUFAs and n-3) are more likely to escape ruminal biohydrogenation and subsequently be absorbed and deposited in animal tissues, thereby reducing the risk of cancer and heart disease (El Otmani et al., 2021). The addition of plant essential oils or other antibacterial substances appears to be effective in modulating ruminal biohydrogenation, potentially altering the muscle fatty acid profile (He et al., 2023). Research has found that the addition of essential oils (flaxseed and oregano) to lamb diets increases the concentration of UFAs in lamb meat, particularly that of linolenic acid (Scarpa et al., 2021). Al Rharad et al. (2025) reported that the addition of tannins to the diet of small ruminants directly acts on Butyrivibrio fibrisolvens and inhibits the first step of biohydrogenation, thereby leading to an increase in linoleic acid. This may similarly explain the reason for the increase in C18:2n6c content in the SUB group.

Meat and meat products serve as a primary source of high-quality protein, supplying EAAs to humans (Battacone et al., 2024). Amino acids are also recognized as flavor precursors, contributing to the formation of specific flavor compounds through the Maillard reaction and Strecker degradation with reducing sugars (Liu et al., 2024). One of the key characteristics of high-quality proteins is the presence of a diverse array of amino acids with balanced proportions among them (Tian et al., 2021). According to the recommendations of the WHO/FAO, the ratio of essential amino acids to total amino acids (EAA/TAA) in muscle tissues is approximately 40%. Proteins with an essential amino acids to non-essential amino acids (EAA/NEAA) ratio of 60% or higher are considered high-quality proteins (Xu et al., 2025). In our study, a decrease in the content of certain amino acids was observed in both the SUB and OEO groups. For example, Ser, Met and Phe were found in the SUB group, while Ser, Ala and SAA were detected in the OEO group. Nevertheless, the ratio of EAA/TAA was close to 40%, and the ratio of EAA/NEAA was above 60% across all groups, with no significant differences observed among the groups. These findings indicate that the meat in question still qualifies as a high-quality protein source. Studies have indicated that dietary supplementation with Bacillus subtilis in yellow-feathered broilers primarily functions during the late growth stage by enhancing intestinal barrier integrity and activating immune responses, rather than by promoting nutrient utilization. This physiological process may lead to energy repartitioning and ultimately result in reduced protein deposition efficiency (Wang, Su, et al., 2025). The reduction in individual amino acid content may be related to the fact that Saanen dairy goats are a milk-producing breed, and the specific mechanism requires further investigation. Collectively, these findings underscore the necessity of considering factors such as animal species, growth stage, and potential trade-offs for meat quality when employing antibiotic alternatives.

After animal slaughter, the synthesis of peroxides from fats and oxygen in muscle via the free radical chain mechanism constitutes a key pathway for the formation of volatile compounds in meat (Liu et al., 2022). Excessive oxidative processes promote the deterioration of lipid and protein components in meat and meat products, leading to a shortened shelf life, increased exudate, and a reduction in nutritional value (Sadeghinejad et al., 2018). In the present trial, compared with the CON group, the SUB group exhibited significantly enhanced activities of T-AOC, CAT, and GSH-Px. T-AOC serves as a comprehensive indicator of the body's antioxidant system functionality, which is attributed to the integrated action of multiple antioxidant enzymes (Xie et al., 2019). GSH-Px acts as an intracellular scavenger of hydrogen peroxide and lipid hydroperoxides, while CAT catalyzes the decomposition of hydrogen peroxide into water and molecular oxygen (Meng et al., 2023). Improving meat quality by modulating the gut microbiota or utilizing probiotics and microbial metabolites has been well established (Wen et al., 2024). Tang et al. (2018). reported that Lactobacillus plantarum exhibits antioxidant potential in vitro. Furthermore, probiotics can elevate the levels of GPX, SOD, and T-AOC, while reducing the MDA content in mice (Li et al., 2019). The enhanced antioxidant capacity observed in the SUB group may be similarly attributed to its regulatory effects on the microbiota. OEO contains phenolic compounds such as carvacrol and thymol, which can scavenge free radicals by donating electrons or hydrogen atoms and chelating metal ions, thereby enhancing the activity of certain enzymes and inhibiting lipid oxidation (Rodriguez-Garcia et al., 2016). In the present study, the activities of muscle CAT and GSH-PX were significantly increased in the OEO group. In previous studies, supplementation with OEO has also been shown to significantly enhance the activities of CAT, peroxidases, and GSH-Px in the longissimus thoracis muscle of Holstein steers (Huang et al., 2025). In the study by Cheng et al. (2017), it was reported that the activities of T-AOC, CAT, and GSH-Px in porcine muscle were relatively higher with OEO supplementation. Studies have shown that dietary polyphenols can be transferred into muscle, thereby improving the oxidative stability of meat and enhancing its fatty acid composition (Fu et al., 2022). These results indicate that OEO contributes to the improvement of the antioxidant properties of meat.

The distinctive flavor profile of lamb meat arises from the synergistic interaction of various volatile organic compounds, including alcohols, ketones, aldehydes, and sulfur-containing heterocyclic compounds, whose overall sensory impact is collectively defined by both their odor thresholds and relative concentrations (Zhang, Song, et al., 2025). In this study, a total of 9 alcohols, 7 esters, 6 ketones, 5 aldehydes, 4 furans, 1 alkane, and 4 other compounds were identified. Research has identified alcohols, aldehydes, and ketones as the major volatile organic compounds in lamb meat. Their low odor thresholds make them key contributors to the overall aroma profile (Liu et al., 2025). The results of this experiment are consistent with this finding. In the PCA analysis, no marked separation was observed among the three groups, which may be attributed to the fact that the muscle samples analyzed were in the raw (uncooked) state. Raw meat exhibits a minimal olfactory-gustatory profile, often characterized by a subtle, blood-like note. In contrast, the complex thermal interactions among flavor precursors such as proteins and lipids in cooked meat subsequently develop its distinctive and intense flavor characteristics (Shi et al., 2024). In addition, as amino acids serve as precursors for flavor formation, the absence of significant differences in flavor-related amino acids among the groups is consistent with the findings of this study. Classification analysis of the detected volatile organic compounds revealed that the alkanes in the OEO group was significantly lower than that in the CON group. Alkane volatile compounds are primarily derived from the cleavage of fatty acid alkoxy groups, and variations in their content may be related to the composition of fatty acids (Liu et al., 2025). Alkanes generally exhibit relatively high odor thresholds, and most possess only a faint odor. Despite this, they can serve as significant intermediates in the formation of heterocyclic compounds. Although there is limited documentation on the odor characteristics of alkanes, some studies suggest they may contribute to certain off-odors (Zhang, Fan, et al., 2024). Individual analysis of volatile compounds revealed that the SUB group showed decreased levels of three alcohols, one ketone, one aldehyde, and one furan, while an increased level of one ester was observed. Analysis revealed that the OEO group exhibited decreased levels of six distinct flavor compounds. Alcohols are generated through the reduction of ketones/aldehydes produced during lipid peroxidation, as well as through the microbial metabolism of proteins and amino acids (Shen et al., 2023). Compared to aldehydes, alcohols have higher odor thresholds. Consequently, they make a limited direct contribution to the characteristic flavor of lamb meat; however, they can exert a synergistic effect on the overall flavor profile (Xu et al., 2025). Elevated levels of alcohols can impart vanilla, woody, and fatty notes to lamb meat. Ketones are generally considered byproducts of lipid oxidation, alkane degradation, and secondary alcohol dehydrogenation. With odor thresholds higher than those of aldehydes, they are known for imparting fruity and creamy notes (Huang et al., 2022). Aldehydes serve as primary odorants in lamb, derived from the degradation of unsaturated fatty acids or the decomposition of alkoxy radicals during lipid oxidation. Characterized by low odor thresholds and an intense aroma, they can impart rancid or other off-odors when their concentration exceeds a certain level (Wang et al., 2021). In summary, although both SUB and OEO treatments reduce the levels of certain flavor compounds in goat muscle, this reduction does not necessarily compromise the overall flavor of the meat due to factors such as the specific odor thresholds of the compounds and synergistic effects among them. On the other hand, the reduction in alkane content observed in the SUB group may help mitigate undesirable odors in lamb meat. In future studies, sensory evaluation can be further incorporated to investigate the effects of SUB and OEO on the flavor characteristics of goat meat.

Given the close relationship between muscle metabolites and meat quality, muscle nutrient composition, and flavor characteristics (Liu et al., 2023), a comprehensive analysis of the muscle metabolite profile will contribute to a better understanding of the underlying mechanisms. PCA and OPLS-DA analyses demonstrated overall differences in metabolites between the SUB and OEO groups compared with the CON group, and the models showed no signs of overfitting. The number of metabolites that decreased in both the SUB and OEO groups exceeded that of those that increased. The compounds that decreased in the SUB group were predominantly glycerophospholipids. The metabolic pathway of glycerophospholipid metabolism was also identified through KEGG enrichment analysis. Phospholipids are the main components of intramuscular fat (IMF). Glycerophospholipids can exert significant effects on intramuscular fat deposition, meat juiciness, flavor, and tenderness by influencing cell membrane structure and function, cell signal transduction, and other processes (Ma, Yang, et al., 2024). Zhang, Cai, et al. (2025) demonstrated that the superior meat quality of porcine semimembranosus, psoas major, and semitendinosus compared to longissimus thoracis is primarily attributed to more active carnitine metabolism and higher phospholipid content. In this experiment, the reduction in the content of certain flavor compounds in the SUB group may be related to this. Furthermore, studies indicate that the differential expression of lipid metabolites in meat during storage at 4 °C is predominantly driven by the oxidation of glycerophospholipids and fatty acyl groups, ultimately leading to an increase in PUFA content (Hwang et al., 2023). This mechanism may account for the increased PUFA content observed in the SUB group. Fatty acid degradation produces acetyl-CoA, which is closely associated with fat utilization and oxidation, while butyrate can regulate energy and lipid metabolism. In the SUB group, the citrate cycle (TCA cycle), phospholipase D signaling pathway, and butanoate metabolism were also identified as core metabolic pathways by the KEGG network. Previous studies have also indicated that these pathways are closely associated with meat quality and flavor (Zhao et al., 2025). The TCA cycle serves as a central energy-yielding pathway in animal energy metabolism and provides precursors for various metabolic processes. Elevated levels of metabolites within this pathway may enhance ATP production in the TCA cycle, thereby optimizing muscle energy status and improving meat quality (Zeng & Shen, 2025). The primary functions of phospholipases include remodeling glycerophospholipids and serving as products of second messengers, whereas specific phospholipase D can hydrolyze phosphatidylcholine to generate phosphoric acid (Panda et al., 2018). Butyrate can provide energy to meet basal metabolic requirements for organisms, serve as a substrate involved in lipid synthesis, and act as a signaling molecule recognized by cell surface G protein-coupled receptors to activate downstream pathways, thereby regulating lipid metabolism (Kimura et al., 2019). Butyric acid and its derivatives are important components contributing to the characteristic flavor of meat products. For instance, methyl butyrate exhibits a fruity aroma, and together these compounds shape the layered aroma profile of meat (Duensing et al., 2024). Collectively, SUB modulates the quality and flavor of goat meat via regulating lipid and energy metabolism pathways, such as glycerophospholipid metabolism, citrate cycle (TCA cycle), phospholipase D signaling pathway, and butanoate metabolism. In the OEO group, the differential metabolites were primarily enriched in lipid metabolism-related pathways such as glycerophospholipid metabolism, regulation of actin cytoskeleton, the phospholipase D signaling pathway, and glycerolipid metabolism. Lipid molecules serve as the primary constituents of IMF, and a high IMF content plays a critical role in the development of meat flavor (Zhang et al., 2022). KEGG enrichment analysis revealed that the aforementioned lipid-related metabolic pathways were downregulated, which may explain the reduced content of certain flavor compounds in the OEO group. Furthermore, alterations in these pathways may also be associated with changes in meat quality phenotypes. For example, the regulation of actin cytoskeleton pathway can affect myofibril structure, muscle shear force, water-holding capacity, and meat color, among others (Chai et al., 2022). Although both were commonly enriched in the glycerophospholipid metabolism pathway, the resulting phenotypic changes were not entirely identical. In addition to the aforementioned core metabolic pathways, the cancer-related metabolic pathways, including choline metabolism in cancer in the SUB group, as well as choline metabolism in cancer and pathways in cancer in the OEO group, were all significantly downregulated. It is indicated that SUB and OEO may suppress these unfavorable pathways, thereby contributing to the maintenance of meat quality and its nutritional benefits. These findings are consistent with those reported by Long et al. (2026) who investigated the effects of allicin supplementation in the diet of Guizhou black goats. These biological pathways provide effective information for revealing the effects of SUB and OEO on goat meat quality, but further validation is still required in subsequent studies.

Correlation analysis revealed that LPC was positively correlated with L∗ and b∗, and negatively correlated with cooked meat rate, GSH-PX, CAT and PUFA. Wang, Qi, et al. (2025) similarly found that phospholipids and triglycerides were positively correlated with L* and a* values, suggesting that lipid molecules may influence meat color. A portion of PUFAs are not present as free fatty acids but are predominantly incorporated into glycerophospholipids (Zhang, Chen, et al., 2024). These pathways may indirectly influence IMF deposition (Zhao et al., 2025). Additionally, studies have demonstrated that the muscles of Tan sheep in the higher IMF group exhibited significantly greater levels of both saturated and monounsaturated fatty acids compared to those in the lower IMF group (Zhang et al., 2022). Correlation analysis further revealed that LPC, LPE, pyridoxamine, and maleamic acid exhibited significantly positive correlations with E-2-hexen-1-ol and 2-butylfuran, highlighting their importance and complexity as flavor precursors. These findings highlight the complexity of the effects of lipids on the fatty acid composition, color, and flavor characteristics of meat, and further investigations combined with lipidomics may be warranted in subsequent research. Current research on antimicrobial peptides and other related products has mostly focused on aspects such as promoting animal growth and regulating the gastrointestinal microbiota of animals; therefore, this paper does not conduct an extensive comparison with similar literature. Nevertheless, the ultimate goal of promoting animal growth and health is always to produce high-quality animal-derived products.

This study provides the first integrated evidence that dietary supplementation with SUB and OEO—two structurally and functionally distinct antibiotic alternatives—differentially remodel the muscle metabolome and phenotypic traits of goat meat through convergent yet pathway-specific mechanisms.

SUB supplementation enhances carcass weight, cooked meat rate, and PUFA deposition, while improving muscle color and antioxidant capacity. Mechanistically, SUB modulates lipid metabolism pathways including glycerophospholipid metabolism and the phospholipase D signaling pathway, as well as energy metabolism pathways consisting of the citrate cycle (TCA cycle) and butanoate metabolism, thereby synergistically enhancing nutritional value, oxidative stability and processing quality. The reduction in specific LPC species and their negative correlation with cooked meat rate and antioxidant enzymes suggest that phospholipid remodeling is a central hub linking SUB treatment to meat quality improvement.

OEO supplementation improves slaughter performance, meat color, cooked meat rate, and antioxidant status. These phenotypic benefits are associated with the down-regulation of glycerolipid metabolism, the phospholipase D signaling pathway, regulation of actin cytoskeleton, and glycerophospholipid metabolism, reflecting reduced lipid oxidation and membrane turnover under improved redox homeostasis. Unlike SUB, OEO does not significantly alter muscle PUFA content, indicating that its primary mode of action is antioxidant-mediated quality preservation rather than nutritional modulation.

Notably, both additives reduced the content of certain amino acids (e.g., Ser, Met, Ile). However, the EAA/TAA and EAA/NEAA ratios remained within the range of high-quality protein sources, and the overall flavor profile was not substantially compromised—although reductions in specific volatile compounds (e.g., E-2-hexen-1-ol, 2-butylfuran, isobutane) were observed. These changes may reflect shifts in phospholipid-derived flavor precursors rather than generalized suppression of amino acid metabolism.

In recent years, research on the regulation of animal product quality by gastrointestinal microbiota has emerged as a research hotspot. As novel antibiotic alternatives, both SUB and OEO exhibit antibacterial properties. The rumen, functioning as a fermentation chamber in ruminants, exerts a crucial impact on animal growth and animal product quality. However, this study did not involve data related to rumen microecology, which may be regarded as one of its limitations. In future research, a comprehensive investigation can be conducted to explore the combined effects of SUB and OEO on rumen microbiota, meat quality, lipid metabolism, and flavor profiles.

5. Conclusion

In conclusion, SUB and OEO represent effective but mechanistically differentiated antibiotic alternatives for improving goat meat quality. SUB acts primarily through energy–lipid metabolic integration to enhance PUFA content and processing traits, whereas OEO functions via antioxidant–lipid pathway crosstalk to improve oxidative stability and water-holding capacity. These findings provide a mechanistic framework for the rational selection and potential combination of these additives in ruminant production systems. Future studies integrating rumen metagenomics, host transcriptomics, and sensory evaluation are warranted to further validate the proposed pathways and translate these metabolic signatures into practical quality biomarkers.

CRediT authorship contribution statement

Ruixin Yang: Writing – review & editing, Writing – original draft, Formal analysis, Data curation, Conceptualization. Lei Xu: Resources. Xiao Zhang: Investigation. Beibei Guo: Resources. Wenliang Tao: Resources. Yue Ma: Visualization. Yongliang Huang: Methodology. Wangjing Liu: Writing – review & editing, Funding acquisition, Conceptualization. Zhaomin Lei: Writing – review & editing, Supervision, Project administration, Funding acquisition.

Funding

This work was supported by the Science and Technology Support Project for Modern Cold and Arid Agriculture Seed Industry Breakthrough (grant no. ZYGG-2025-15); National Natural Science Foundation of China (grant no.32402789; 32260846); Agriculture Research System of China (grant no. CARS-38); Discipline Team Project of Gansu Agricultural University (grant no.GAU-XKTD-2022-22); Gansu Provincial University Postgraduate Innovation Star Project (grant no. 2026CXZX-785).

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 1

Supplementary Table of Diet Composition, Nutrient Levels and Fatty Acid Profiles.

Supplementary material 2

Metabolomics and Correlation Analysis – Supplementary Table.

Data availability

Data will be made available on request.