Effect of dietary Bacillus subtilis on basic chemical composition, free amino acids, fatty acids and volatile organic compounds in broiler meat

TL;DR

Adding Bacillus subtilis to broiler diets improves meat quality by altering protein, amino acid, fatty acid, and volatile compound profiles.

Contribution

This study reveals how dietary Bacillus subtilis modulates nutrient metabolism to enhance broiler meat quality.

Findings

LBS treatment increased crude protein and flavor amino acids in broiler meat.

BS supplementation reduced saturated fatty acids and increased polyunsaturated fatty acids.

BS increased volatile compounds like hexanal and affected lipid and amino acid metabolism pathways.

Abstract

The aim of this study was to investigate the effect of dietary supplementation with Bacillus subtilis (BS, DSM32324-32325) on the nutritional quality of broiler meat, with emphasis on the content and profile of various chemical components. A total of 144 Arbor Acres male broilers were divided into three dietary treatment groups (CON, control; LBS, 300 mg/kg BS; HBS, 500 mg/kg BS) and fed for 35 days. The results showed that the LBS treatment significantly increased the crude protein content of broiler meat. Dietary BS supplementation increased the content of flavor amino acids (such as serine and alanine) and decreased the content of bitter amino acids such as methionine and isoleucine. Moreover, BS addition reduced the content of saturated fatty acids and increased the content of polyunsaturated fatty acids in broiler meat. Meanwhile, the content of important volatile organic compounds, particularly hexanal, was significantly increased. Finally, tandem mass tags (TMT)-based quantitative proteomics revealed that dietary supplementation with LBS (300 mg/kg BS) favorably affected lipid and amino acid metabolism in broiler meat, and identified key metabolic pathways such as the peroxisome proliferator-activated receptor (PPAR) signaling pathway, amino acid metabolism, and fatty acid degradation. In conclusion, these findings demonstrated that BS supplementation enhanced broiler meat quality by modulating nutrient metabolism pathways.

Figures

Effect of BS on free amino acids in broiler meat. (a) and (b) represent changes in free amino acid content of breast muscle. (c) and (d) represent changes in free amino acid content of thigh muscle. *Ser*: serine, *Ala*: alanine, *Asp*: aspartic acid, *Pro*: proline, *Met*: methionine, *Ile*: isoleucine, *Leu*: leucine, *Gln*: glutamine, *Asn*: asparagine, *Tyr*: tyrosine, *Phe*: phenylalanine. *TAA*: Total amino acid, *EAA*: Essential amino acid, *NEAA*: Nonessential amino acid. ^a-c^ Means different treatments differ at *P* < 0.05. SE: Standard error. *n* = 6. CON: broilers fed with the basa

Effect of BS on fatty acids in broiler meat. (a) and (b) represent changes in fatty acids content of breast muscle. (c) and (d) represent changes in fatty acids content of thigh muscle. SFA: saturated fatty acid, MUFA: monounsaturated fatty acid, PUFA: polyunsaturated fatty acid. ^a, b^ Means different treatments differ at *P* < 0.05. SE: Standard error. *n* = 6. CON: broilers fed with the basal diet; BS: *Bacillus subtilis*; LBS: broilers fed with 300 mg/kg BS; HBS: broilers fed with 500 mg/kg BS.Fig 2 dummy alt text

Effect of BS on volatile organic compounds in broiler meat. (a) and (b) represent changes in some volatile organic compounds content of breast muscle. (c) and (d) represent changes in some volatile organic compounds content of thigh muscle. *n* = 6. CON: broilers fed with the basal diet; BS: *Bacillus subtilis*; LBS: broilers fed with 300 mg/kg BS; HBS: broilers fed with 500 mg/kg BS.Fig 3 dummy alt text

Protein identification and expression profiles in broiler meat. (a) Statistical chart of basic protein identification results; (b) Statistical graph of proteins identified by different subgroups; (c) Box plots of protein expression distribution in samples. *n* = 3.Fig 4 dummy alt text

Screening of differential expressed proteins (DEPs) in broiler meat. (a) Volcano plot of DEPs between CON and LBS groups. Blue and red dots represent down-regulated and up-regulated proteins, respectively, with darker colors indicating more significant protein differences; (b) DEPs clustering heat map. Each row indicates the expression status of each protein in different groups. Different colors were present in the heat map, with red representing highly expressed proteins and blue indicating lowly expressed proteins. *n* = 3.Fig 5 dummy alt textTable 2Top ten up- and down-regulated proteins id

GO enrichment analysis of broiler meat samples from CON and LBS groups. (a) Bar graph of the top30 functional terms in GO enrichment analysis; (b) GO enrichment analysis graph of up-regulated differentially expressed proteins; (c) GO enrichment analysis graph of down-regulated differentially expressed proteins. *n* = 3.Fig 6 dummy alt text

KEGG enrichment analysis of broiler meat samples from CON and LBS groups. (a) Bubble plots of the top 20 pathways in KEGG enrichment analysis; (b) KEGG enrichment analysis graph of up-regulated differentially expressed proteins; (c) KEGG enrichment analysis graph of down-regulated differentially expressed proteins. *n* = 3.Fig 7 dummy alt text

PPI network of differentially expressed proteins between CON and LBS muscle samples. The left figure shows the top 25 connectivity protein interactions network diagram. Circles represent differential proteins, and their size represents the level of connectivity, with larger circles representing higher connectivity and vice versa; the right panel shows a histogram of top 25 connectivity protein expression. *n* = 3.Fig 8 dummy alt text

Full text

Introduction

The overuse of antibiotics has been a threat to human and environmental health as it promotes resistant bacteria strains and exposes the environment including our food sources to residues from these medicines (Chiesa et al., 2017; Li et al., 2015). The use of antibiotics as a supplement in the diet to enhance chicken growth rate and improve productivity isit also damages the quality of chicken meat nutritionally (Cai et al., 2018). The use of probiotics could be an alternative solution in overcoming this problem and replacing antibiotics with safe alternatives. Probiotics have been proven that their bacteria, which are highly resistant to acid and heat conditions and modulate gut microflora population which can enhance feed efficiency and quality of chicken meat (Upadhaya et al., 2019; Yu et al., 2019).

Bacillus subtilis is one of the probiotic bacteria belonging to the Bacillus genus which have high resistance against temperature and pH; making it a good candidate to be included in animal feeding (Hong et al., 2005; Sarao and Arora, 2017). The bacteria was found to be a good feed supplement for broilers, (iv)especially in broiler chicken because of their ability to enhance the physiological parameters, metabolic processes, and digestive system health (Sun et al., 2025; Chen et al., 2024). Probiotic that is effective as a feed additive in providing multiple benefits to animals’ health and production. For instance, Park et al. (2020), reported on the beneficial impacts of the same strain when given to young calves before weaning, bacillus subtilis has been found to stimulate both physical growth and metabolic maturation (Antanaitis et al., 2024). In the field of poultry industry, co-administration of B. subtilis with Lactobacillus acidophilus as probiotics increased the immunity status of Cherry Valley ducklings, consequently decreasing inflammation-related tissue damage (Zhang et al., 2024a). In addition, B. subtilis has been shown to have an influence on meat colour properties; it was observed that inoculating pigs with B. subtilis improved the freshness of vacuum-packed raw and cooked pork meats due to inhibition of metmyoglobin formation, thus retaining good appearance for longer time of shelf-life (Zhang et al., 2025b). The most important mechanism of BS supplement on enhancing the tolerance to oxidative stress is related with the activity of GPx and GST (Dong et al., 2023). All in all, results from studies presented above reveal the multiple uses of B. subtilis in livestock feeding.

One potential alternative, not based on antibiotics use, which can be used in order to improve animal welfare is the application of BS–bacillus subtilis DSM32324-32325. The selected microorganism was proved able to change skeletal muscles fibre types (fast-to-slow conversion) as well as increase meat oxidative stability in chicken, ultimately leading to improved meat characteristics (Wang et al., 2024a). Nevertheless, there is little information available about the impact of BS on meat quality and nutrient content in chicken meat. This study aimed at evaluating the impact of BS on several meat quality attributes, including basic nutrients, free amino acids, fatty acid composition and volatile components aiming to provide some guidelines for future application in a broiler production system.

Materials and methods

Animals and feeding

The experiment was conducted on the Experimental Station of Animal Science & Technology College, Taian, Shandong Province,China based on approved guidelines (Wang et al., 2024a). All animal procedures complied with the ethical guidelines of Shandong Agricultural University and were performed following approval from the Animal Care Committee (Approval No. SDAUA-2022-50). Briefly, 144 one-day-old Arbor Acres (AA) male broilers were selected for this study, which lasted for 35 days. The birds were randomly allocated into three experimental groups: CON (basal diet), LBS (basal diet supplemented with 300 mg/kg BS), and HBS (basal diet supplemented with 500 mg/kg BS). The BS (3.2 × 10⁹ CFU/g) additive used in this experiment was purchased from Beijing Chr. Hansen Trading Co., Ltd. (Beijing, China; Product No. DSM32324-32325). All broilers were housed in an environmentally controlled room with ad libitum access to feed and water throughout the experimental period.

Sample collection

On the final day of the experiment, one broiler with a body weight representative of the replicate mean was selected from each replicate per treatment. A total of 18 broilers (n = 6 per treatment) were then euthanized by cervical dislocation. Approximately 10 g of left breast muscle (pectoralis major) and thigh muscle (biceps femoris) were collected within 45 min post-slaughter and stored at -80 °C for subsequent determination of meat nutritional quality-related indices, including moisture, crude protein, crude fat, free amino acids (FAAs), fatty acid composition, and VOCs. Finally, the regulatory effect of BS on the nutritional quality of broiler meat was further investigated using proteomic analysis (n = 3 per treatment).

Determination of basic chemical composition

The contents of moisture, crude protein (CP), and crude fat (CF) in broiler meat were determined according to the Chinese national standards GB5009.3-2016, GB5009.5-2016, and GB5009.6-2016, respectively.

Determination of free amino acids

The types and contents of amino acids in broiler meat were analyzed using a XEVO TQ-S Micro tandem quadrupole mass spectrometry system (Waters, Milford, MA). For sample preparation, 50 mg of tissue was homogenized, and standard solutions of each amino acid were prepared at concentrations of 400, 200, 100, 40, 20, 10, 4, 2, and 1 μmol/L. Chromatographic separation was performed on an ultra-high performance liquid chromatography (UPLC) system using an HSS T3 column (2.1 mm × 150 mm × 1.8 μm; Waters, Milford, MA). The mobile phase consisted of phase A (water with 0.1 % formic acid) and phase B (acetonitrile). The elution gradient was shown in Table S1. The injection volume was set to 5.0 µL, and the flow rate was maintained at 0.5 mL/min. The column temperature was controlled at 50 °C. Finally, quantitative analysis of amino acids was performed by calculating peak areas using MassLynx quantitative software (TargetLynx, Waters, Milford, MA).

Determination of fatty acids

The lipids composition in chicken meat were analyzed by Gas Chromatograph-Mass Spectrometer (GC-MS; Shimadzu TQ8040, Japan). The samples were prepared as per modified methods of the procedure in O’Fallon et al.’s work (2007), namely, the supernatant was filtered using a 0.45 µm membrane (organic), and 2 mL was stored in a freezer (-20 °C) to be analyzed after 7–14 d. GC-MS analytical parameters were: Agilent HP-Innowax capillary column (30 m x 0.25 mm ID, 0.25 µm), carrier gas of high purity He with a constant linear velocity of 1 mL/min; injection port temperature of 260 °C andwith 1 μL of sample injected in the 25:1 split mode.Initial temperature: 50 °C; ramp up to final temp.: +25 °C/min, time at initial temperature: 60 sec; ramp up to final temp.: +4 °C/min, final temp.: 250 °C, time at final temperature: 600 sec.

Determination of volatile organic compounds

Volatile organic compounds (VOCs) were analyzed in raw meat to assess intrinsic lipid oxidation markers and flavor precursors, reflecting BS-induced metabolic changes without thermal interference. The method of Lytou et al. (2018) was adopted for VOC determination, utilizing headspace solid-phase microextraction (HS-SPME) with minor modifications. The sample (3 g) in a headspace vial was stirred together with 10 mL of NaCl solution (25 %) and 10 μL of the internal standard (4-methyl-1-pentanol, final concentration 3200 μg/g) for 2 min. After being held in a water bath at 40 °C for 15 min, an aged SPME fiber (50/30 μm DVB/CAR/PDMS) was exposed to the headspace and extraction was performed at the same temperature for 30 min. Subsequently, the fiber was inserted into the injection port and desorbed in split mode at 250 °C for 5 min before analysis. Separation and analysis were carried out using an HP-5 column (30 m × 0.25 mm × 0.2 μm). The GC-MS conditions were as follows: high-purity helium was used as the carrier gas at a flow rate of 1 mL/min. The oven temperature was initially maintained at 40 °C for 5 min, then raised to 150 °C at 4 °C/min, and finally increased to 250 °C at 30 °C/min and held for 5 min. The interface temperature was set at 280 °C. The mass spectrometer was operated in electron ionization mode with an electron energy of 70 eV, and the scanning range was 29–350 m/z. The MS source and quadrupole temperatures were set at 230 °C and 150 °C, respectively. Compound identification and quantification were performed by matching the acquired data against the reference spectra in the National Institute of Standards and Technology (NIST) database. The calculation was based on the formula C₁ = (S₁ × C₂) / S₂, where C₁ and S₁ represent the concentration and peak area of the target analyte, and C₂ and S₂ are those of the internal standard. All calculated concentrations are presented in units of μg/g.

Tandem mass tags (TMT)-based quantitative proteomics analysis

Given that breast muscles contain higher protein content than thigh muscles and are richer in phospholipid nutrients that contribute to growth and development, and that the results of RT-qPCR and Western blotting in our previous trial indicated that the LBS group generally promoted better meat quality than the HBS group (Wang et al., 2024a), the present experiment compared the CON and LBS groups in breast muscle for proteomic analysis to investigate nutrient metabolism at the proteomic level in chicken meat.

Extraction and digestion of proteins from broiler meat

An appropriate amount of the sample was taken, and the final concentration was adjusted to 1 mM by adding lysis buffer and protease inhibitor. Subsequently, the sample was homogenized and centrifuged to obtain the supernatant. The protein concentration in the supernatant was determined using a BCA kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). After the protein solution was diluted to an appropriate concentration, dithiothreitol (DTT) was added to a final concentration of 5 mM. Iodoacetamide and acetone were added sequentially to the protein sample followed by incubation at -20°C for 4-12h until precipitation of the proteins occurred; this fraction was collected via centrifugation and dissolved into a solution containing 200mM TEAB. Treated samples were digested using enzymes prior to lyophilization and stored at -80°C for further processing.

Peptide labeling

For the labeling reaction, the lyophilized samples were reconstituted in 100 mM TEAB buffer in 1.5 mL Eppendorf tubes and mixed thoroughly. The reconstituted TMT reagent was then added to the sample mixture and incubated at room temperature for 1 h to facilitate the labeling reaction. The reaction was subsequently quenched with 5 % hydroxylamine, after which the samples were lyophilized and stored at -80 °C.

LC-MS/MS analysis

The lyophilized samples were concentrated to a small volume using an Agilent Zorbax Extend-C18 narrow column (2.1 × 150 mm, 5 μm). The samples were then injected at a flow rate of 300 nL/min onto an analytical column (Acclaim PepMap RSLC, 75 μm × 50 cm, RP-C18, Thermo Fisher) for separation. Mobile phase A consisted of ACN-H₂O-FA (99.9:0.1, v/v), while mobile phase B consisted of ACN-H₂O-FA (80:19.9:0.1, v/v). The primary MS mass resolution was set to 45,000. The mass spectral scan was set to a full scan charge-to-mass ratio range of m/z 350–1500, and MS/MS scans were performed on the 20 most intense peaks. All MS/MS profiles were acquired in positive ion mode with the resolution set to 3000.

Data analysis

Data processing for moisture, CP, CF, FAA, fatty acids, and VOCs in this study was performed using SPSS software (version 26.0; IBM Corp., Armonk, NY) and analyzed by comparing means (95 % confidence intervals). The different feed additive concentration treatments were analyzed as dependent variables in a one-way analysis of variance, and differences between treatments were detected using the least significant difference (LSD) post hoc test. Finally, all MS data obtained from TMT-based quantitative proteomics were analyzed using Proteome Discoverer software (version 2.4.1.15; Thermo Fisher Scientific). Differentially expressed proteins (DEPs) between groups were assessed using two parameters: fold change (FC), calculated as log_2_FC = mean (test group) – mean (control group), and P-value obtained by t-test. A fold change > 1.2 and P < 0.05 were used as the criteria for significant upregulation of DEPs, while fold change < 0.83 and P < 0.05 were used as the criteria for significant downregulation. In this experiment, differences were considered statistically significant at P < 0.05.

Results

Basic chemical composition

The findings in Table 1 indicate that the moisture content in broiler meat ranged from 71.65 % to 75.53 %, but changes in muscle moisture content were not affected by BS supplementation (P > 0.05). The LBS group showed higher CP levels in thigh muscle than in the other treatment groups (P < 0.05). However, BS supplementation did not significantly alter the CF content in the muscles (P > 0.05).Table 1. Effect of BS on basic chemical components in breast and thigh muscles of broilers.Table 1 dummy alt textItems (%)Breast muscleThigh muscleCONLBSHBSSEP-valueCONLBSHBSSEP-valueMoisture72.4073.2673.350.010.7675.1175.5371.650.030.31CP21.0421.8421.110.400.1418.33b19.52a18.91b0.340.02CF1.201.200.800.230.1661.931.731.860.440.90CP: Crude protein. CF: Crude fat.a,bMeans different treatments differ at P < 0.05. SE: Standard error. n = 6. CON: broilers fed with the basal diet; BS: Bacillus subtilis; LBS: broilers fed with 300 mg/kg BS; HBS: broilers fed with 500 mg/kg BS.

Free amino acids

The effect of BS on FAAs in breast muscle is illustrated in Table S2. During the determination, the content of cystine was found to be very low and could be considered negligible; therefore, it was not included in the statistical analysis of this experiment. As shown in Fig. 1a and 1b, compared with the basal diet group, each BS treatment group significantly increased the contents of serine (Ser), alanine (Ala), and non-essential amino acids (NEAAs) in breast muscle, while simultaneously decreasing the levels of methionine (Met), isoleucine (Ile), leucine (Leu), and proline (Pro) (P < 0.05). Relative to the HBS group, the LBS group showed lower levels of phenylalanine (Phe) and Pro (P < 0.05). In addition, following BS supplementation, the levels of glutamine (Gln) and Phe were significantly increased in the LBS and HBS groups, respectively (P < 0.05).Fig. 1. Effect of BS on free amino acids in broiler meat. (a) and (b) represent changes in free amino acid content of breast muscle. (c) and (d) represent changes in free amino acid content of thigh muscle. Ser: serine, Ala: alanine, Asp: aspartic acid, Pro: proline, Met: methionine, Ile: isoleucine, Leu: leucine, Gln: glutamine, Asn: asparagine, Tyr: tyrosine, Phe: phenylalanine. TAA: Total amino acid, EAA: Essential amino acid, NEAA: Nonessential amino acid. ^a-c^ Means different treatments differ at P < 0.05. SE: Standard error. n = 6. CON: broilers fed with the basal diet; BS: Bacillus subtilis; LBS: broilers fed with 300 mg/kg BS; HBS: broilers fed with 500 mg/kg BS.Fig 1 dummy alt text

As illustrated in Fig. 1 (c and d) and detailed in Table S3, we found that dietary supplementation with BS enhanced the levels of Gln, NEAAs, and total FAAs in thigh muscle compared to the basal diet (P < 0.05). It was also observed that the LBS group exhibited increased contents of asparagine (Asn) and aspartic acid (Asp) while simultaneously showing a decreased Pro content (P < 0.05). Moreover, the HBS group showed an increased tyrosine (Tyr) content and a decreased Met content (P < 0.05). Compared with the HBS group, the LBS group had higher levels of Asn and Met, but lower contents of Pro and Tyr (P < 0.05). Previous studies have reported that FAAs can be classified into bitter amino acids (including Ile, Leu, Met, and Pro) and flavor amino acids, such as Asp and sweet amino acids like Ala and Ser (Lorenzo and Franco, 2012). As reported by Yin et al. (2023), serine and alanine function as flavor-enhancing sweet-taste amino acids, whereas methionine and isoleucine impart bitter taste characteristics. Therefore, the observed shifts in FAA composition induced by BS supplementation are consistent with improved meat flavor perception and consumer acceptance (Ma et al., 2020).

Fatty acids

The overall trends in saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) in breast muscle did not change significantly in response to BS supplementation compared with the basal diet group, as presented in Fig. 2a and Table S4 (P > 0.05). However, the LBS group exhibited a significant reduction in the percentage of heptadecanoic acid (C17:0) among SFAs and increased the proportions of γ-linolenic acid (C18:3n6) and eicosapentaenoic acid (EPA, C20:5n3) among PUFAs (Fig. 2b, P < 0.05). Moreover, a higher proportion of C18:3n6 was observed in the LBS group than in the HBS group (Fig. 2b, P < 0.05).Fig. 2. Effect of BS on fatty acids in broiler meat. (a) and (b) represent changes in fatty acids content of breast muscle. (c) and (d) represent changes in fatty acids content of thigh muscle. SFA: saturated fatty acid, MUFA: monounsaturated fatty acid, PUFA: polyunsaturated fatty acid. ^a, b^ Means different treatments differ at P < 0.05. SE: Standard error. n = 6. CON: broilers fed with the basal diet; BS: Bacillus subtilis; LBS: broilers fed with 300 mg/kg BS; HBS: broilers fed with 500 mg/kg BS.Fig 2 dummy alt text

As shown in Fig. 2c, the percentage of total SFA in thigh muscle was significantly decreased following dietary BS supplementation (P < 0.05). Although the proportion of PUFAs showed an increasing trend with rising BS concentration, the differences were not statistically significant (P > 0.05). As detailed in Fig. 2d and Table S5, the percentages of palmitic acid (C16:0) and stearic acid (C18:0) were consistently reduced, while the proportions of trans-oleic acid (C18:1n9t), linoleic acid (C18:2n6c), and γ-linolenic acid (C18:3n6) were significantly increased following dietary BS addition (P < 0.05). Additionally, the HBS group showed a significant increase in the percentage of α-linolenic acid (C18:3n3) (P < 0.05). These data suggest that BS enhanced the content of PUFAs and reduced SFA content in muscle, potentially leading to improved nutritional quality of broiler meat. It has been documented that fatty acid composition significantly affects meat nutritional value, in which reduced SFA and enhanced PUFA levels are desirable traits for human health (Wan et al., 2023).

Volatile organic compounds

Volatile organic compounds (VOCs) were identified and quantified to characterize the flavor attributes of broiler meat. In total, 24 distinct volatiles were detected and classified—according to their chemical functional groups—into four major categories: alcohols, aldehydes, esters, and miscellaneous compounds. As documented by Sidira et al. (2015), these VOCs arise primarily from oxidative degradation of lipids and proteins, as well as from catabolic processes involving amino acids and other metabolic pathways. As shown in Fig. 3a and 3c, the most abundant organic compounds in broiler meat were alcohols, followed by aldehydes.Fig. 3. Effect of BS on volatile organic compounds in broiler meat. (a) and (b) represent changes in some volatile organic compounds content of breast muscle. (c) and (d) represent changes in some volatile organic compounds content of thigh muscle. n = 6. CON: broilers fed with the basal diet; BS: Bacillus subtilis; LBS: broilers fed with 300 mg/kg BS; HBS: broilers fed with 500 mg/kg BS.Fig 3 dummy alt text

In breast muscle (Fig. 3b and Table S6), all doses of BS supplementation significantly increased the contents of 1-hexanol, 1-nonanol, 1-ethylcyclohexanol, 2,3-octanediol, 2-methylbutyraldehyde, and 3-nonen-5-one, while decreasing the content of benzaldehyde compared to the basal diet (P < 0.05). Additionally, LBS supplementation resulted in higher levels of isohexanol and 1,2-octanediol (P < 0.05). Moreover, (cis)-3-nonen-1-ol content was significantly higher in the HBS group than in the CON group (P < 0.05), whereas the LBS group did not differ significantly from CON (P > 0.05). Relative to the LBS group, the HBS group demonstrated higher contents of 1-nonanol, (cis)-3-nonen-1-ol, benzaldehyde, and 3-nonen-5-one, but lower levels of 1,2-octanediol (P < 0.05).

In thigh muscle (Fig. 3d and Table S7), dietary BS supplementation significantly increased the levels of 1-nonen-1-ol, 3-octanol, 2,3-octanediol, octanal, nonanal, and hexanal (P < 0.05). The LBS group showed higher levels of 1-heptanol, pentanal, and octyl butyrate compared to the other two treatment groups (P < 0.05). Additionally, the HBS group exhibited a marked increase in hexyl octyl ether content (P < 0.05).

TMT proteomics

Protein quality control status

In the TMT proteomics analysis, the CON and LBS groups were compared in this experiment, with three biological replicates per group. Following LC-MS/MS detection and database searching, a total of 348,667 spectra were generated. Among these, 60,592 spectra were identified as valid, corresponding to 24,318 distinct peptides. The obtained peptides matched with 3540 proteins group in total (Fig. 4a), and we could obtain quantification data from 3,356 proteins via spectra (Fig. 4b). We explored protein expression distribution in terms of the number of samples each was expressed in. As shown in Fig. 4c, the read counts were converted into fragments per kilobase per million mapped reads (FPKM), and there was very little variability between replicates of a given treatment, which is also confirmed by the unchanged median values but with evident separation among groups that indicates not only a good repeatability within group but also a marked difference between groups for the proteomic profiles of chicken samples.Fig. 4. Protein identification and expression profiles in broiler meat. (a) Statistical chart of basic protein identification results; (b) Statistical graph of proteins identified by different subgroups; (c) Box plots of protein expression distribution in samples. n = 3.Fig 4 dummy alt text

Differentially expressed protein (DEP) analysis

In order to study the effect of BS on DEPs, we compared the expression profiles of two groups: LBS group with CON group; Screened for proteome changes caused by different treatment conditions. A total of 96 DEPs were obtained, where the distribution is readily apparent from the volcano plot (Fig. 5a). In particular, there were 38 significantly up-regulated and 58 down-regulated proteins. To facilitate the comparison among different groups’ differential expressed proteins (DEPs), We then created the clustered heatmap as shown in Fig. 5b. The top ten up- and down-regulated proteins in broiler meat are listed in Table 2. These proteins are involved in various biological activities, including metabolic processes (ACOX2, AOX1, GANC, GSTK1), cellular functions (NA, NUBP2, SELENOF, FXYD6, XIRP1), and signaling (FBN1).Fig. 5. Screening of differential expressed proteins (DEPs) in broiler meat. (a) Volcano plot of DEPs between CON and LBS groups. Blue and red dots represent down-regulated and up-regulated proteins, respectively, with darker colors indicating more significant protein differences; (b) DEPs clustering heat map. Each row indicates the expression status of each protein in different groups. Different colors were present in the heat map, with red representing highly expressed proteins and blue indicating lowly expressed proteins. n = 3.Fig 5 dummy alt textTable 2Top ten up- and down-regulated proteins identified in proteomics.Table 2 dummy alt textAccessionProtein NameGene NameCoverageMWCalc. pIFCPUpregulated in LBS group vs CON groupA0A3Q2U8M2Aspartate beta-hydroxylaseASPH1919.24.153.9110.026A0A8V0Z3S8Glucosidase alpha, neutral CGANC3495.25.862.2160.001A0A8V0 × 4I0Uncharacterized proteinLOC121108905102.26.541.9860.021A0A8V0ZDI7Fibrillin 1FBN11298.14.981.9020.025Q9DGM5Fast myosin heavy chain isoform 2NA65223.15.871.9000.047E1C5V6Acyl-coenzyme A oxidaseACOX2975.48.001.7700.000Q5ZKV4Cytosolic Fe-S cluster assembly factor NUBP2NUBP2829.36.071.5860.027F1NE68Aldehyde oxidaseAOX19146.56.581.5400.043A0A3Q2U3 × 0Platelet glycoprotein Ib alpha chain-likeLOC101750889276.96.481.5090.013E1BXS6SH3 domain binding kinase family member 2SBK2543.67.251.4860.018Downregulated in LBS group vs CON groupA0A1D5PFR6Selenoprotein FSELENOF2016.44.980.4930.000F1NAY8Mevalonate diphosphate decarboxylaseMVD1368.08.720.5400.046A0A8V0XXP8Scribble planar cell polarity proteinSCRIB13205.45.290.5640.022Q5ZM59FXYD domain-containing ion transport regulatorFXYD62310.24.880.5800.047A0A8V0YQA2Four and a half LIM domains 1FHL15632.48.320.6060.045A0A8V0XTP0Beta-taxilinTXLNB2976.34.930.6580.015A0A8V0XTC7Scribble planar cell polarity proteinSCRIB13208.15.270.6580.014F1NUI2Xin actin binding repeat containing 1XIRP146284.45.810.6640.011F1N9G6Glutathione S-transferase kappaGSTK13625.38.850.6810.048F1P156Crystallin lambda 1CRYL14134.96.790.6920.035FC: Fold change. P<0.05 represents a significant difference between the data. CON: broilers fed with the basal diet; BS: Bacillus subtilis; LBS: broilers fed with 300 mg/kg BS

Functional analysis of DEPs

To better analyze the functions of the detected DEPs, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed in this experiment. The GO analysis categorized DEPs into three ontologies: biological process (BP), cellular component (CC), and molecular function (MF). A total of 215 GO terms were enriched by the DEPs, of which 140 were significantly enriched, including 19 CC terms, 71 BP terms, and 50 MF terms (Fig. 6a). The top 10 most significantly enriched terms in each ontology are presented. A higher number of up-regulated DEPs were observed in GO terms related to iron-sulfur cluster assembly, peroxisome, mitochondrion, FAD binding, oxidoreductase activity, 2 iron-2 sulfur cluster binding, and amino acid kinase activity (Fig. 6b). Conversely, GO terms enriched with down-regulated DEPs included glutathione metabolic process, ESCRT I complex, cytosol, chaperone complex, A band, Z disc, chaperone binding, glutathione transferase activity, glutathione peroxidase activity, and Hsp90 protein binding (Fig. 6c).Fig. 6GO enrichment analysis of broiler meat samples from CON and LBS groups. (a) Bar graph of the top30 functional terms in GO enrichment analysis; (b) GO enrichment analysis graph of up-regulated differentially expressed proteins; (c) GO enrichment analysis graph of down-regulated differentially expressed proteins. n = 3.Fig 6 dummy alt text

To further identify the signaling pathways associated with DEPs in broiler meat following BS supplementation, KEGG pathway enrichment analysis was conducted using the KEGG database (https://www.genome.jp/kegg/). The results indicated that DEPs were predominantly enriched in metabolic pathways related to various nutrients in meat (Fig. 7), providing evidence that dietary BS supplementation influences metabolic processes in broiler meat. Among the enriched pathways, fatty acid degradation, the peroxisome proliferator-activated receptor (PPAR) signaling pathway, and peroxisome biogenesis were closely associated with lipid metabolism. Additionally, the amino acid metabolism pathways enriched by DEPs included tryptophan, valine, leucine, isoleucine, and lysine metabolism.Fig. 7KEGG enrichment analysis of broiler meat samples from CON and LBS groups. (a) Bubble plots of the top 20 pathways in KEGG enrichment analysis; (b) KEGG enrichment analysis graph of up-regulated differentially expressed proteins; (c) KEGG enrichment analysis graph of down-regulated differentially expressed proteins. n = 3.Fig 7 dummy alt text

Protein-protein interaction (PPI) analysis

Finally, PPI network of the DEPs identified in the BA and BB groups was analyzed in this experiment (Fig. 8). Among the interacting proteins, glutathione peroxidase 1 (GPx1), glutathione S-transferase theta 1 (GSTT1), 3-hydroxybutyrate dehydrogenase 2 (BDH2), fructose-bisphosphatase 2 (FBP2), aldehyde oxidase 1 (AOX1), and lactate dehydrogenase D (LDHD) emerged as key hub proteins critical for stabilizing the network. These proteins are closely associated with multiple nutrient metabolic processes, including glutathione metabolism, amino acid metabolism, fatty acid metabolism, and carbohydrate metabolism.Fig. 8PPI network of differentially expressed proteins between CON and LBS muscle samples. The left figure shows the top 25 connectivity protein interactions network diagram. Circles represent differential proteins, and their size represents the level of connectivity, with larger circles representing higher connectivity and vice versa; the right panel shows a histogram of top 25 connectivity protein expression. n = 3.Fig 8 dummy alt text

Discussion

Effect of BS on basic chemical composition in broiler meat

The basic chemical composition of meat, including moisture, CP, and CF, was determined. The results indicated that dietary BS supplementation had no significant effect on moisture or CF content in the meat. Protein level is a key determinant of broiler meat composition, and the LBS treatment was found to significantly increase CP content. However, a previous study reported that supplementation with Bacillus coagulans ZJU0616 did not significantly affect the chemical composition of broiler meat (Zhou et al., 2010). This discrepancy may be attributed to differences in probiotic strain and rearing duration.

Effect of BS on amino acids in broiler meat

While structural amino acids serve as the building blocks of proteins and play key roles in regulating metabolic pathways, antioxidant systems, and enzymatic processes (Estévez et al., 2020), the present study specifically focused on free amino acids (FAAs), which represent the soluble, non-protein-bound fraction in muscle tissue. Unlike protein-bound amino acids that primarily determine nutritional quality, FAAs are closely related to meat flavor, acting as direct taste-active compounds and volatile flavor precursors (Yang et al., 2020). Although free flavor amino acids influence meat taste, the content of structural essential amino acids plays a decisive role in determining protein quality. A previous study showed that dietary supplementation with Bacillus spp. S62-9 increased the content of free flavor amino acids such as Ser, Ala, Gly, and Arg in broiler breast muscle, which may act as precursors capable of generating subtle flavors and influencing overall meat flavor (Liu et al., 2023). This finding is consistent with the results of the present experiment, in which BS supplementation also significantly increased free Ser and Ala content in breast muscle. The results further revealed that broiler meat contained relatively high levels of three free essential amino acids (threonine, lysine, and Leu) and that HBS supplementation increased free Phe levels in breast muscle. It has been reported that feeding Bacillus licheniformis increases the content of certain essential amino acids in broiler meat, suggesting that BS supplementation may enhance the nutritional value and quality of meat to some extent (Yi et al., 2024). Moreover, we observed that the content of free bitter amino acids such as Met, Pro, and Ile was reduced following BS supplementation. Taken together, these results suggest that dietary BS addition may affect the flavor and nutritional profile of broiler meat primarily by modulating the content of free flavor and bitter amino acids.

Effect of BS on fatty acids in broiler meat

Chicken meat is considered one of the major sources of PUFAs. Apart from free amino acids, intramuscular fatty acids are also regarded as a key indicator of meat quality, particularly eating quality (Zhang et al., 2017). Previous findings have demonstrated that dietary probiotic supplementation can improve meat quality by modulating fatty acid profiles (specifically by reducing SFA and enhancing PUFA contents) in animals such as chickens and pigs (Liu et al., 2017; Tian et al., 2021). It is generally recognized that higher levels of SFAs, especially C16:0 and C18:0, are strongly associated with the development of cardiovascular diseases (Zong et al., 2016). The data obtained in this study suggest that dietary BS supplementation reduced the content of C16:0 and C18:0 in thigh muscle, indicating that BS-treated chicken meat may exert a positive effect on human health. He et al. (2022) reported similar changes in sheep, where inulin and Chinese gallotannin supplementation reduced C14:0 and C16:0 while increasing C18:2n6c content, suggesting that dietary additives can modulate fatty acid composition toward a profile more favorable for cardiovascular health. C18:2n6c, a polyunsaturated fatty acid, has been associated with lowered blood triglyceride and cholesterol concentrations (He et al., 2022). In this study, dietary supplementation with BS led to significantly elevated concentrations of polyunsaturated fatty acids (PUFAs) relative to the control group. Notably, the levels of linoleic acid (C18:2n6c) and both isomers of linolenic acid—C18:3n3 and C18:3n6—were also enhanced, aligning with previous observations reported by Chang et al. (2018). C18:3n3 and C18:3n6 are n-3 and n-6 fatty acids, respectively, and are associated with cardiovascular health (Zhang et al., 2025a). Among these, C18:3n3 serves as a precursor for DHA and EPA, which have beneficial effects on cancer prevention and immune response (Luo et al., 2019). Luo et al. (2019) found that higher muscle C18:3n3 content in pasture-fed lambs coincided with elevated antioxidant enzyme activities (CAT, GPx) and reduced lipid oxidation products (hexanal, nonanal), indicating that n-3 PUFA enrichment can coexist with oxidative stability. Therefore, BS supplementation may improve meat quality by reducing SFA content while increasing MUFA and PUFA levels, thereby enhancing the overall fatty acid composition.

The sensory implications of altered fatty acid composition warrant separate consideration. Phospholipid PUFAs serve as important precursors for meat flavor development during cooking; however, their high unsaturation simultaneously increases susceptibility to oxidative rancidity and quality deterioration (Wood et al., 2004). Tian et al. (2021) further demonstrated that modulation of fatty acid metabolism via probiotic supplementation (specifically through PPARα-induced SCD expression) can maintain intramuscular fat content and promote MUFA synthesis (C16:1 and C18:1), thereby balancing lipid oxidative stability with desirable meat tenderness.

Effect of BS on VOCs in broiler meat

Modification of VOCs through dietary patterns may offer a potential approach to obtaining better-tasting broiler meat for human consumption (Jayasena et al., 2013). To demonstrate the effect of BS on the flavor of broiler meat, VOCs were measured in the muscles of broilers. The results showed that no single VOC class or individual compound dominated the meat flavor profile; instead, multiple alcohols and aldehydes were simultaneously altered by BS treatment, along with limited changes in esters (Fig. 3; Tables S6, S7), indicating that meat flavor results from the interactions among various volatile compounds rather than specific marker substances.

Alcohols can be produced by gluconeogenesis, lipid oxidation, decarboxylation, amino acid dehydrogenation, and aldehyde reduction (Morán et al., 2013). 1-Hexanol, 1-heptanol, and isohexanol accounted for a relatively large proportion of the alcohols detected in breast and thigh muscles. Although BS supplementation resulted in significant differences in their contents, these saturated alcohols were considered to have a lesser effect on meat flavor because they possess relatively higher odor thresholds (e.g., 1-hexanol: 500 μg/kg; 1-heptanol: 425 μg/kg) compared to unsaturated alcohols such as 1-octen-3-ol (1 μg/kg) (Zhao et al., 2024). As an unsaturated alcohol, (cis)-3-nonen-1-ol exhibits a "mushroom" odor (a trait common to meat-derived unsaturated alcohols, as exemplified by 1-octen-3-ol), and has a lower odor threshold than saturated alcohols (Zhao et al., 2024; Wang et al., 2024b). The HBS group was found to noticeably increase (cis)-3-nonen-1-ol content in the breast muscle.

Additionally, important flavor compounds known as carbonyl-containing aldehydes originate from the oxidation of threonine, proline, lysine, and arginine in tissues (Dalle-Donne et al., 2003). Hexanal, which has a "green/grassy" odor, was detected in chicken meat, and its concentration in thigh meat increased significantly after broilers were fed BS. Moreover, hexanal has been recognized as an important odor-active compound in broiler meat and plays a vital role in the production of chicken flavor (Jin et al., 2021; Luo et al., 2022). Octanal has a "fatty/floral" odor, and experimental results showed that BS supplementation also increased octanal content in thigh muscle. Although these aldehydes are much less abundant than hexanal, both may contribute to the overall flavor profile of broiler meat. Benzaldehyde is associated with Strecker degradation of amino acids and has an unpleasant smell that can negatively affect the aroma of broiler meat (Sidira et al., 2015; Yufei et al., 2024). In this experiment, high levels of benzaldehyde were detected in muscles, and BS supplementation inhibited its increase, resulting in a decreasing trend in benzaldehyde content.

Additionally, esters, ketones, acids, olefins, and ethers were detected in broiler meat. According to Cui et al. (2023), ketones and other volatile classes were not identified as key aroma contributors (OAV ≥ 1) in multispecies meat comparisons, whereas aldehydes, sulfur compounds, and alcohols were the predominant contributors to meat aroma.

Effect of BS on chicken tissues analyzed by TMT proteomics

In the present study, proteomic analysis was performed on breast muscle samples from the CON and LBS groups of broilers. A total of 96 DEPs were identified, including 38 up-regulated and 58 down-regulated proteins. Through GO and KEGG enrichment analyses, DEPs associated with lipid and amino acid metabolism (such as AOX1, ACOX2, CPT2, ACAT1, CCBL1, BCKDHA, HYKK, and PECR) were identified. These proteins are involved in fatty acid degradation, lipid metabolism, various amino acid metabolic pathways, and peroxisomal metabolic processes.

The DEPs detected in broiler meat were enriched in numerous pathways associated with nutrient metabolism. Among these, the PPAR signaling pathway and fatty acid degradation pathway are known to coordinately regulate lipid metabolism, and a close relationship exists between them (Zhang et al., 2024b). The relevance of these two pathways is supported by their roles in lipid metabolism and adipocyte differentiation, as demonstrated by Guo et al. (2020) in ducks and Li et al. (2023) in chickens. It has been suggested that the PPAR signaling pathway is involved in the regulation of adipocyte development and intramuscular fat (IMF) accumulation (Zhang et al., 2022). Moreover, IMF is characterized by a rich content and composition of PUFAs (Li et al., 2022). The results of the present experiment showed that BS supplementation increased PUFA content, providing nutritional evidence that BS can enhance meat quality. The PPAR signaling pathway improves livestock and poultry meat quality by regulating the expression of genes related to lipid metabolism, including those involved in fatty acid synthesis, oxidation, transport, and deposition (Su et al., 2024).

Several key proteins related to lipid metabolism were identified among the DEPs, including acetyl-coenzyme A acetyltransferase 1 (ACAT1), acyl-coenzyme A oxidase 2 (ACOX2), and carnitine palmitoyltransferase 2 (CPT2). ACAT1 is an essential enzyme downstream of the PPAR pathway that maintains cellular cholesterol homeostasis and participates in lipid oxidation by converting excess free cholesterol to cholesteryl esters (CE), the storage form of cholesterol (Kang and Shim, 2021; Zhang et al., 2024b). ACAT1 is also involved in mitochondrial lipid processing for energy production and catalyzes the final step of ketolysis (Ahlawat et al., 2023). ACOX2 was identified among the up-regulated proteins and plays an essential role in the peroxisomal degradation of long-chain branched fatty acids and bile acid intermediates (Zhang et al., 2024b). Furthermore, ACOX2 is enriched in lipid metabolism pathways and participates in fatty acid metabolic processes (Lim et al., 2015). CPT2 induces fatty acid β-oxidation and is involved in lipolysis (Lee et al., 2015; Cui et al., 2018). Our findings suggest that the PPAR pathway regulates lipid metabolism by inducing the expression of downstream genes such as CPT2, thereby contributing to various quality indicators of broiler meat, this observation aligns with the results reported by Su et al. (2024).

A number of DEPs critically associated with amino acid metabolic pathways were additionally detected. AOX1 participates in the degradation pathway of valine, leucine, and isoleucine and promotes the metabolism of leucine and valine, generating products such as acetyl coenzyme A and succinyl coenzyme A that enter the mitochondria and participate in the TCA cycle (Hu et al., 2021). BS supplementation induced changes in lipid and amino acid metabolism as well as in the TCA cycle, thereby affecting ATP synthesis. These data suggest that dietary BS supplementation exerts beneficial effects on biometabolic processes and energy balance in animals, contributing to the maintenance of good meat quality. Branched-chain keto acid dehydrogenase E1 subunit alpha (BCKDHA) is a key regulatory enzyme exclusively involved in the oxidative decarboxylation of branched-chain keto acids—the immediate metabolites of branched-chain amino acids (BCAAs) (Biswas et al., 2019). It has been shown that PPARα can regulate leucine catabolism by controlling the expression of BCKDHA (Huang et al., 2023). In addition, research indicates that feed additives can influence the metabolic pathways of amino acids and fatty acids in chickens, largely through their ability to boost endogenous antioxidant defenses (Wassie et al., 2022). While this study indicates that dietary supplementation with BS influences amino acid and fatty acid metabolism while enhancing the antioxidant potential of broiler meat, the interrelationships among these three effects remain unclear and merit deeper exploration.

Conclusion

The current data suggest that dietary BS supplementation can modulate the nutritional quality of broiler meat through multiple metabolic pathways. This is mainly manifested through significant alterations in the profiles of amino acids, fatty acids, and volatile organic compounds (VOCs) present in broiler meat. BS supplementation markedly elevated the levels of flavor-associated and essential amino acids, whereas the opposite trend was observed for bitter amino acids. Additionally, dietary BS supplementation resulted in a marked reduction in SFA content, while PUFA content was significantly increased. Furthermore, the contents of important VOCs such as hexanal and octanal were significantly elevated. Drawing on experimental findings related to meat quality, antioxidant activity, and nutritional composition, the LBS group exhibited the best overall performance. Finally, proteomic analysis identified the key DEPs and pathways through which 300 mg/kg BS affects broiler meat quality. These DEPs were predominantly associated with fatty acid and amino acid metabolic pathways, offering insights into the mechanistic basis of BS-mediated regulation of broiler meat quality.

CRediT authorship contribution statement

Hairong Wang: Writing – original draft, Investigation. Jiqiang Li: Investigation. Rongrong Liang: Methodology. Yunge Liu: Software. Zhigang Song: Writing – review & editing, Visualization, Validation, Resources. Johan Buyse: Resources. Lixian Zhu: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition. Huixin Zuo: Writing – review & editing.

Disclosures

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.