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Research| Volume 106, ISSUE 3, P1650-1671, March 2023

Naturally forming benzoic acid orientates perilipin to facilitate glyceride-type polyunsaturated fatty acid degradation via fermentation behavior

Open AccessPublished:January 27, 2023DOI:https://doi.org/10.3168/jds.2022-22381

      ABSTRACT

      Naturally forming benzoic acid in fermented dairy products accumulates in organisms and biomagnifies through collateral transport. The association between benzoic acid agglomeration and susceptible lipid nutrients remains obscure. Horizontal analysis of lipidomic alteration in response to benzoic acid was conducted and the spatially proteomic map was constructed using label-free quantitative proteomics. From synergistic integration of multi-omics in benzoic acid accumulated fermented goat milk model, the biological processes of significant proteins mostly focused on glyceride-type polyunsaturated fatty acids degradation (143.818 ± 0.51 mg/kg to 104.613 ± 0.29 mg/kg). As a physiological barrier shield, perilipin, which is coated on the surface of lipid droplets, protects triacylglycerols from cytosolic lipases, thus preventing triglyceride hydrolysis. The expression of perilipin decreased by 90% compared with the control group, leading to the decrease of triglycerides. Benzoic acid suppressed phosphatidylethanolamines and phosphatidylcholines synthesis by attenuating choline phosphotransferase and ethanolamine phosphotransferase. Less diglyceride generated by the dephosphorylation of phosphatidic acid entered choline phosphotransferase and ethanolamine phosphotransferase-mediated glycerophospholipid metabolisms. Fermentation of goat milk at a low temperature and less incubation time leads to the production of less benzoic acid and mitigation of lipid nutrient loss. The present study delineated the molecular landscape of fermented goat milk containing endogenous benzoic acid and further dissected the trajectory guiding lipid alteration to advance control of benzoic acid residue.

      Key words

      INTRODUCTION

      Goat milk products are gaining popularity due to their lower allergic potentiality and improved fat digestibility. Meta-analyses of prospective cohort studies suggested fermentation modifies mineral bioavailability (
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      Gut microbiota and fermentation-derived branched chain hydroxy acids mediate health benefits of yogurt consumption in obese mice.
      ). Fermented goat milk has been targeted for particular focus since dietary intake has been associated with reduction of weight gain. Cultured dairy products were susceptible to contamination by spoilage microorganisms at various points in the food production and processing continuum (
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      Controlling dairy product spoilage to reduce food loss and waste.
      ). Antimicrobial preservatives were applied to control the growth of pathogenic and spoilage microorganisms. Benzoic acid directly inhibited the growth of various bacteria, yeasts, and fungi in acidic media (
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      A simple and fast method for the inspection of preservatives in cheeses and cream by liquid chromatography-electrospray tandem mass spectrometry.
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      Effect on benzoic acid production of yoghurt culture and the temperatures of storage and milk heat treatment in yoghurts from cow, goat and sheep milk.
      ).
      However, benzoic acid was found in cheese, yogurt, and fermented dairy products. Kurisaki et al. determined that the values of benzoic acid ranged from 1.6 to 40.6 μg/g in cheese (
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      Formation of benzoic acid in cheese.
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      • Gucer L.
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      Status of benzoic acid amount during processing from yoghurt to its by-product drink (Doogh).
      ). During the ripening period of fermented goat milk, benzoic acid was produced by the metabolic activity of fermentative bacteria against hippuric acid (
      • Park S.Y.
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      • Lim S.D.
      Production of benzoic acid as a natural compound in fermented skim milk using commercial cheese starter.
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      WinMLR program for the determination of sorbic and benzoic acids in food samples.
      ). Several researchers have suggested setting the maximum admissible limit of benzoic acid in fermented dairy products at 40.0 mg/kg based on the above reasons (
      • Iammarino M.
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      Survey of benzoic acid in cheeses: Contribution to the estimation of an admissible maximum limit.
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      • Kinik O.
      Benzoic acid formation and its relationship with microbial properties in traditional Turkish cheese varieties.
      ). Benzoic acid biomagnifies in food webs and has profound implications for ecological integrity. Long-term intake of benzoic acid possibly accumulates in the human body and further leads to allergic reactions (rhinitis, hives, and dermatitis) and metabolic abnormalities (
      • Sugihartono V.E.
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      • Shih Y.J.
      • Huang Y.H.
      Photo-persulfate oxidation and mineralization of benzoic acid: Kinetics and optimization under UVC irradiation.
      ). In view of this regulation, the cumulative impacts of endogenous benzoic acid should be evaluated.
      Lipidomics is focused on the large-scale and comprehensive analysis of lipid molecules in various food matrices (
      • Wu B.
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      Mass spectrometry-based lipidomics as a powerful platform in foodomics research.
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      Omics analysis reveals variations among commercial sources of bovine milk fat globule membrane.
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      Hepatic lipidomics analysis reveals the antiobesity and cholesterol-lowering effects of tangeretin in high-fat diet-fed rats.
      ;
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      • Zhao M.
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      • Liu J.
      • Marchioni E.
      Identification and differentiation of wide edible mushrooms based on lipidomics profiling combined with principal component analysis.
      ). Specifically, lipidomic tools based on LC-MS methodology are of great importance for lipid characterization of cow milk (
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      Characterization and comparison of lipids in bovine colostrum and mature milk based on UHPLC-QTOF-MS lipidomics.
      ), goat milk (
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      • Hellgren L.I.
      • Sorensen M.T.
      • Theil P.K.
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      Fatty acid profile of phospholipids and sphingomyelin in milk and regulation of sphingomyelin synthesis of mammary glands in cows receiving increasing levels of crushed sunflower seeds.
      ), and donkey milk (
      • Li M.
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      • Zheng Y.
      • Shao J.
      • Li Q.
      • Kang S.
      • Zhang Z.
      • Yue X.
      • Yang M.
      Quantitative lipidomics reveals alterations in donkey milk lipids according to lactation.
      ). Lipids as the prominent composition of cell membranes or organelles play a multitude of roles in critical biological functions (cellular signaling, energy storage, and membrane structure) and further maintain cell homeostasis (
      • Luque de Castro M.D.
      • Quiles-Zafra R.
      Lipidomics: An omics discipline with a key role in nutrition.
      ). Accurate quantification of lipid molecules is the important foundation and driving force of detailed understanding of the biological role and metabolic regulation of lipids (
      • Köfeler H.C.
      • Ahrends R.
      • Baker E.S.
      • Ekroos K.
      • Han X.
      • Hoffmann N.
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      • Wenk M.R.
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      Recommendations for good practice in MS-based lipidomics.
      ).
      Leveraging the power of multi-omics was to characterize alterations of lipid molecules in fermented goat milk and to elucidate the metabolic pathways and biochemical mechanisms driven by endogenous benzoic acid. An integrated bioinformatics pipeline was applied to elucidate molecular differences between the control group versus benzoic acid accumulation groups and to screen an array of significant lipid molecules. The results of this work will provide guidance for regulating benzoic acid residue through appropriate strategies.

      MATERIALS AND METHODS

      Because no human or animal subjects were used, this analysis did not require approval by an Institutional Animal Care and Use Committee or Institutional Review Board.

      Chemicals and Materials

      Ammonium acetate, hydrochloric acid, potassium ferrocyanide, standard of benzoic acid (99.5%), and zinc acetate were obtained from Merck. HPLC-grade high pure solvents including acetonitrile (ACN), formic acid, isopropanol (IPA), methanol (MeOH), and methyl tert-butyl ether (MTBE) were purchased from Fisher Scientific. Dithiothreitol (DTT; biochemical grade), iodoacetamide (IAA), and trypsin (sequencing grade) were received from Sigma-Aldrich. Ultrapure water with 18.2 MΩ.cm of resistivity was obtained from a Milli-Q water purification system (Millipore).

      Determination of Benzoic Acid

      The determination of benzoic acid content in fermented milk samples was performed in accordance with
      • ISO (International Organization for Standardization)
      ISO 9231:2008-Milk and milk products: Determination of the benzoic and sorbic acid contents.
      . Hydrochloric acid solution (1 mol/L) was prepared by mixing 90 mL of hydrochloric acid with 910 mL of H2O. Zinc acetate solution (219 g/L) was prepared by dissolving 219 g zinc acetate in 1,000 mL of H2O. Potassium ferrocyanide solution (106 g/L) was prepared by dissolving 106 g of potassium ferrocyanide solution in 1,000 mL of H2O. Ammonium acetate (0.02 mol/L) solution was prepared by dissolving 1.54 g of ammonium acetate in 1,000 mL of H2O. An adequate amount of benzoic acid (100 mg) was weighed accurately and diluted with methanol (100 mL) to make a standard stock solution of 1,000 μg/mL. The standard stock solution was stored in a refrigerator (4 ± 1°C) and preserved in darkness.
      Fermented goat milk samples were homogenized by stirring and warming gently to 40°C before sample preparation. The homogenized sample (5 g) was added with 1 mL of hydrochloric acid solution (1 mol/L) and 20 mL of methanol. The mixture was extracted in an ultrasonic bath for 20 min and then added with 1 mL zinc acetate solution (219 g/L) and 1 mL potassium ferrocyanide solution (106 g/L) to precipitate the fats and proteins. Subsequently, 25 mL of methanol was added and let stand for 15 min. Finally, the supernatant liquid was filtered through a 0.45-μm clarification kit (Millipore).
      The concentrations of benzoic acid in samples and the working standard solution were determined by high-performance liquid chromatography and quantified by the external standard method. The sample was loaded on a C18 reversed-phase trap column (250 × 4.6 mm, 5 μm) at 25°C. Gradient elution was carried out using mobile phase A (water with 0.02 mol/L ammonium acetate) and mobile phase B (methanol): 0 min, 75% A; 6.01 min, 50% A; 8.01 min, 30% A; 20.01 min, 75% A; 25 min, 75% A. The flow rate was 1 mL/min and the injection volume was 20 μL. The UV-visible spectrophotometer was set at 225 nm. The concentration of benzoic acid was calculated as Equation 1:
      X = f × (c × V × 1,000)/(m × 1,000),
      [1]


      where X is the concentration of benzoic acid in samples (mg/kg); c is the concentration of standard solution (μg/mL); V is the final volume of sample solution (mL); m is the mass of test sample of final sample solution (g); and f is the dilution multiple.
      The analytical method was validated for limit of detection (LOD), limit of quantitation (LOQ), precision, and recovery according to acceptance criteria recommended by the US Food and Drug Administration (
      FDA Foods Program Regulatory Science Steering Committee
      Guidelines for the Validation of Chemical Methods in Food, Feed, Cosmetics, and Veterinary Products.
      ). The sensitivity of the analytical method was validated by investigating the response of benzoic acid in consecutive dilutions of concentrated working solution until the signal-to-noise reached 3 (for LOD) and 10 (for LOQ). The LOD and LOQ were 0.7 and 2.5 mg/kg, respectively. The accuracy of the analytical method was evaluated through recovery studies. Recovery was obtained at the lowest, intermediate, and highest concentrations of the benzoic acid analytical curve. The current method provided recovery from 91.6% to 100.8%. The analytical method was repeatable and reliable with % relative standard deviation less than 7% (intraday measurements) and 13% (interday experiments).

      Fermented Goat Milk Samples

      Commercial fermented goat milk samples (n = 306) were arbitrarily purchased from 17 stores in Xi'an, Shaanxi Province, China, during February and March 2021. Naturally forming benzoic acid values in 306 samples were determined and the amount of benzoic acid varied between 0.27 and 45.81 mg/kg. A total of 180 raw goat milk samples were obtained from 5 sampling sites in the central Shaanxi plain including Fufeng (n = 32), Fuping (n = 38), Jingyang (n = 33), Sanyuan (n = 40), and Wugong (n = 37) counties. The inclusion criteria of Saanen goats were as follows: homogeneous age (approximately 5 yr), weighing 60 ± 10 kg, and at 50 ± 15 d of lactation. A representative sample of 50 mL was collected from each goat in the early morning. All collected goat milk samples were stored in carbon dioxide ice and transported to the laboratory. Goat milk was heated in a water bath at 95 ± 1°C for 180 min and subsequently cooled to 42 ± 1°C so as to inoculate freeze-dried lactic starter cultures (Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus salivarius ssp. thermophilus, Danisco). The fermentation process was carried out at 42 ± 1°C in a thermostatic water bath and the end point of fermentation was pH at 4.5. The amount of benzoic acid in fermented goat milk samples varied between 0.36 to 41.25 mg/kg. The maximum admissible limit of benzoic acid in fermented dairy products was suggested setting at 40.0 mg/kg (
      • Iammarino M.
      • Di Taranto A.
      • Palermo C.
      • Muscarella M.
      Survey of benzoic acid in cheeses: Contribution to the estimation of an admissible maximum limit.
      ; Amore et al., 2021). Fermented goat milk samples containing benzoic acid with variable concentrations of 0, 5, 10, 20, 30, and 40 mg/kg were collected (5 trials for each concentration gradient, 5 × 6 = 30) for further experimental analysis.

      Extraction Methods from Fermented Goat Milk Samples

      Lipid extraction involved fermented goat milk samples corresponding to 0, 5, 10, 20, 30, and 40 mg/kg of benzoic acid. Briefly, 3,400 μL of ice-cold H2O (Millipore) and 9,600 μL of methyl tert-butyl ether/methanol (Aladdin) extracting solution (5:1, vol/vol) were added to 600 μL of fermented goat milk samples. A homogeneous mixture containing 20 μL of internal standard (Alabaster) was vortexed for 60 s and sonicated for 10 min in a cold ultrasonic bath (0°C). Then, the sample was centrifuged at 10,000 × g and 4°C for 15 min; the supernatant was transferred and the bottom layer phase was supplemented with 4,000 μL of methyl tert-butyl ether/methanol (5:1, vol/vol). The extraction step was repeated 2 more times. Supernatants collected 3 times were combined, dried under a gentle stream of nitrogen, and reconstituted in 1,000 μL of acetonitrile/isopropanol/H2O (65:30:5, vol/vol/vol). Sample was finally filtered through a 0.22-μm PVDF membrane (Sigma-Aldrich) for UHPLC-Q-Orbitrap analysis.
      Protein extraction involved fermented goat milk samples corresponding to 0 and 40 mg/kg of benzoic acid. SDT lysis buffer (4% SDS, 100 mM Tris-HCl, 1 mM dithiothreitol, pH 7.6) was added to fermented goat milk samples. After boiling in a thermostatic water bath for 10 min, the mixture was centrifuged at 14,000 × g for 40 min at 4°C and protein concentration was quantified using BCA Protein Assay Kit (Thermo Fisher Scientific). Each protein sample was mixed with dithiothreitol (final concentration, 10 mM) and reacted at 56°C for 60 min. Subsequently, iodoacetamide (final concentration, 55 mM) was added and incubated in darkness for 30 min at 25°C. Then, the protein solution was loaded into an ultrafiltration filter (Merck) with a nominal molecular weight cutoff of 10 kDa and centrifuged at 14,000 × g for 20 min at 4°C. Sample was washed twice with UA buffer (8 M urea and 150 mM Tris-HCl, pH 8.0) and then 2 other times with NH4HCO3 buffer and then centrifuged at 7,500 × g for 15 min at 4°C, and the filtrate was discarded. Proteins were digested with trypsin buffer (enzyme: protein = 1:50, wt/wt). Following digestion overnight at 37 ± 2°C, 1% formic acid was added, and the resulting peptides were centrifuged at 14,000 × g for 10 min at 4°C. Finally, these samples were filtered through sterile 0.22 μm pore-size filters (Millipore) for further analysis.

      Lipidomic and Proteomic Profiling by Ultra-High-Pressure Liquid Chromatography Coupled with Quadrupole-Orbitrap High-Resolution Mass Spectrometry Analysis

      Samples were analyzed in triplicate by the Dionex Ultimate 3000 ultra-high-pressure liquid chromatography (UHPLC) system coupled with Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific). The chromatographic separation of lipids was performed on a reverse-phase C18 column (100 × 2.1 mm, 1.7 μm, Thermo Scientific). Mobile phase A (60% acetonitrile with 10 mM ammonium acetate) and mobile phase B (90% isopropanol and 10% acetonitrile with 10 mM ammonium acetate) were used in the positive-ion mode at the following gradient elution: 0 min, 63% A; 6 min, 50% A; 20 min, 15% A; 22 min, 2% A; 29 min, 2% A; 29.1 min, 63% A; 32 min, 63% A. The flow rate was 0.3 mL/min. High-resolution mass spectrometry equipped with heated electrospray ionization (HESI)-II probe was applied for lipid detection. Full scan MS and ddMS2 analyses were adapted with mass resolutions of 70,000 and 35,000 full width at half maximum, respectively. The injection volume was 5 μL and scan range was set at mass-to-charge ratio (m/z) 100 to 1,500. Top 10 ions were selected for fragmentation under the normalized collisional energy of 30 eV. The source parameters were as follows: sheath gas flow rate as 35 arbitrary units (arb), auxiliary gas flow rate as 10 arb, spray voltage as 3.2 kV, and capillary temperature as 320°C.
      Protein extraction sample was loaded on a C18 reversed-phase trap column (Thermo Scientific Acclaim PepMap100, 100 µm × 20 mm, nanoViper C18) and then switched online on a C18 reversed-phase analytical column (Thermo Scientific Easy Column, 75 μm × 150 mm, 3 μm) equilibrated in solvent A (100% water, 0.1% formic acid) and solvent B (80% acetonitrile, 0.1% formic acid): 100 to 45% solvent A for 60 min; 45 to 0% solvent A for 15 min; maintaining 0% solvent A for 15 min; 0 to 95% solvent A for 1 min; 95% solvent A for 9 min. The flow rate was 300 nL/min. The MS data were acquired in Q-Orbitrap at the scan range of m/z 300 to 1,800 with resolving power of 35,000 full width at half maximum. The inference of peptides was from MS/MS spectra, with acetyl (protein N-term) and oxidation (M) modification, accepting 2 maximum missed cleavage and matching the proteome of Capra hircus (UniProt, https://www.uniprot.org/, accessed March 2022). Label-free quantification was achieved by setting the false discovery rate to a value of 1% (
      • Jia W.
      • Zhang R.
      • Liu L.
      • Zhu Z.B.
      • Mo H.Z.
      • Xu M.D.
      • Shi L.
      • Zhang H.
      Proteomics analysis to investigate the impact of diversified thermal processing on meat tenderness in Hengshan goat meat.
      ).

      Data Processing and Multivariate Statistical Analysis

      Raw data were analyzed and processed according to the workflow shown in Figure 1, consisting of the following steps: peak extraction, spectral matching, and peak list generation. The obtained matrix data set was checked for integrity and estimated missing values in MetaboAnalyst 5.0. Missing values were replaced by 1/5 of the minimum positive value of each variable. Data set was transformed by median centering, log-transformation, and Pareto scaling to adjust offset and correct for heteroscedasticity (
      • Ceciliani F.
      • Audano M.
      • Addis M.F.
      • Lecchi C.
      • Ghaffari M.H.
      • Albertini M.
      • Tangorra F.
      • Piccinini R.
      • Caruso D.
      • Mitro N.
      • Bronzo V.
      The untargeted lipidomic profile of quarter milk from dairy cows with subclinical intramammary infection by non-aureus staphylococci.
      ). Normalization result was visualized by examining the distribution curve. Normalized lipidomic data were imported into SIMCA 14.1 (Umetrics) to perform the multivariate statistical elaboration. Unsupervised and supervised dimensionality reduction analysis methods including principal component analysis (PCA) and partial least squares-discrimination analysis (PLS-DA) were implemented. Thereafter, the 200-permutation testing was carried out to exclude overfitting of PLS-DA model. Variable importance in projection (VIP) value is an index for estimating the importance of variables in PLS-DA models to enhance interpretability. Variable importance in projection values of each compound were calculated for discrimination purposes. Lipids and proteins with VIP above a threshold of 1.0 were considered as differentially expressed compounds (
      • Jia W.
      • Wang X.
      • Wu X.X.
      • Shi L.
      Monitoring contamination of perchlorate migrating along the food chain to dairy products poses risks to human health.
      ). The FASTA sequences of significant proteins were blasted against the online Gene Ontology Resource (http://www.geneontology.org/) to retrieve their Gene Ontology (GO) and then mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (https://www.kegg.jp/).
      Figure thumbnail gr1
      Figure 1Schematic illustration of quantitative lipidomic workflow. LC-MS/MS = liquid chromatography-tandem mass spectrometry; RT = retention time; m/z = mass-to-charge ratio; IS = internal standard; a = slope of calibration curves for individual lipid classes; b = intercept of calibration curves for individual lipid classes; slope = calibration curve slope; RF = response factor; PC = phosphatidylcholine; LPC = lysophosphatidylcholine; SM = sphingomyelin.

      Analytical Method Validation

      Accurate quantification of lipid molecules was valuable and possible for investigating lipid metabolism (
      • Zhang R.
      • Zhu Z.B.
      • Jia W.
      Molecular mechanism associated with the use of magnetic fermentation in modulating the dietary lipid composition and nutritional quality of goat milk.
      ). The absolute quantification of lipids was achieved based on the chromatographic peak area, stable isotope-labeled internal standards, and response factor (RF) information (Equation 2). The response factors were defined as the ratio of the slope between stable isotope-labeled internal standards and individual lipid classes (Equation 3). The RF values (deviation below 3%) were obtained from triplicate independent experiments (Table 1). The meaning of the relative abbreviations is as follows: A = chromatographic peak area; C = concentration; IS = internal standard; and Slope = calibration curve slope.
      Table 1Analytical parameters of the established method for the determination of lipids in samples
      Overall recovery = average of 3 concentration levels: 1.0 × LOQ, 2.0 × LOQ, 4.0 × LOQ. LOD = limit of detection; LOQ = limit of quantitation; RSD = relative standard deviation; RF = response factor; Cer = ceramides; DG = diglycerides; LPC = lysophosphatidylcholines; PC = phosphatidylcholines; PE = phosphatidylethanolamines; PI = phosphatidylinositol; SM = sphingomyelins; TG = triglycerides.
      ClassR2Overall recovery (%)LOD (μg/mL)LOQ (μg/mL)Precision (n = 6; RSD, %)RF-value (positive)RF-value (negative)
      IntradayInterday
      Cer0.997393.620.00360.01093.583.803.823.19
      DG0.998486.360.00170.00534.724.845.95
      LPC0.997192.180.00920.02711.294.310.410.83
      PC0.998694.770.00750.02281.422.743.752.65
      PE0.997590.010.00690.02352.172.932.041.28
      PI0.9968102.100.00880.02874.364.447.465.05
      SM0.999498.040.00110.00364.114.681.631.33
      TG0.998897.150.00280.00901.171.918.60
      1 Overall recovery = average of 3 concentration levels: 1.0 × LOQ, 2.0 × LOQ, 4.0 × LOQ. LOD = limit of detection; LOQ = limit of quantitation; RSD = relative standard deviation; RF = response factor; Cer = ceramides; DG = diglycerides; LPC = lysophosphatidylcholines; PC = phosphatidylcholines; PE = phosphatidylethanolamines; PI = phosphatidylinositol; SM = sphingomyelins; TG = triglycerides.
      Clipid = CIS × (Alipid/AIS) × RFlipid,
      [2]


      RFlipid = SlopeIS/Slopelipid.
      [3]


      Method validation was carried out according to acceptance criteria recommended by the US Food and Drug Administration (
      FDA Foods Program Regulatory Science Steering Committee
      Guidelines for the Validation of Chemical Methods in Food, Feed, Cosmetics, and Veterinary Products.
      ). The linearity of the UHPLC-MS/MS method was evaluated by analysis of the regression coefficient measured for each lipid compound. The internal standard was injected in triplicate on each analytical instrument. Excellent coefficient of determination (R2) was from 0.9971 to 0.9994, indicating lipid concentrations could be accurately quantified by chromatographic peak area. The LOD and LOQ were calculated based on the signal-to-noise of 3 and 10 (
      • Yan Y.
      • Chen S.
      • He Y.X.
      • Nie Y.
      • Xu Y.
      Quantitation of pyrazines in Baijiu and during production process by a rapid and sensitive direct injection UPLC-MS/MS approach.
      ). Recoveries of 8 lipid compounds were evaluated by fermented goat milk spiked with 3 fortification levels standards (1.0 LOQ, 2.0 LOQ, and 4.0 LOQ). The analytical procedure was conducted in triplicate at each concentration. Eligible recoveries were in the range of 86.36 to 102.10%. The intraday precisions were verified via determining 6 parallel experiments of spiked samples in the morning, afternoon, and evening of the same day (
      • Jia W.
      • Yang Y.
      • Liu S.X.
      • Shi L.
      Molecular mechanisms of the irradiation-induced accumulation of polyphenols in star anise (Illicium verum Hook. f.).
      ). The interday precisions were measured on 5 consecutive days in 6 replicates. All validation parameters listed in Table 1 illustrate the accuracy of this quantification method.

      Statistical Analysis

      Statistical analysis was performed with GraphPad Prism 9.0 (GraphPad software). Results were presented as the means ± standard deviation. Independent samples t-test was applied to compare means between 2 samples. Statistical significance among the means of 3 or more samples was assessed by one-way ANOVA followed by the least significant difference test. Differences were significant and markedly significant at P < 0.05 and P < 0.01, respectively.

      RESULTS AND DISCUSSION

      Untargeted Lipidomic Screening Identification

      The workflow for lipidomic data analysis consisted of the following steps: (i) extracted ion chromatograms (EIC). The more ions in the m/z tolerance range, the more likely they belong to the same lipid molecule. The mass spectral space was divided into N subspaces with equal space width (Equation 4). Counter the number of ions in each subspace and the subspace with ion number satisfied Equation 5 will be initialized as a cluster.
      N = (maxm/z − minm/z)/(0.1 × tolerancem/z),
      [4]


      where N = the number of subspace; maxm/z and minm/z = acquired maximal and minimal m/z value for a sample, respectively; and tolerancem/z = selected 0.01 Da as the initial m/z tolerance.
      ni > ni − 1 and ni ≥ ni + 1.
      [5]


      The meaning of relative abbreviations was ni, ni − 1, and ni + 1, the number of ions in the ith, (i − 1)th, and (i + 1)th subspaces, respectively.
      The EIC was constructed based on ions in each cluster. (ii) Raw signal filtering: The noise level was estimated to remove background noise and baseline. (iii) Features detection and peak alignment: Acquired spectra were merged with the R programming language and gap-filling algorithm to fill registered lipids that were undetected (
      • Jia W.
      • Zhang R.
      • Shi L.
      • Zhang F.
      • Chang J.
      • Chu X.G.
      Effects of spices on the formation of biogenic amines during the fermentation of dry fermented mutton sausage.
      ;
      • Vasilopoulou C.G.
      • Sulek K.
      • Brunner A.-D.
      • Meitei N.S.
      • Schweiger-Hufnagel U.
      • Meyer S.W.
      • Barsch A.
      • Mann M.
      • Meier F.
      Trapped ion mobility spectrometry and PASEF enable in-depth lipidomics from minimal sample amounts.
      ). (iv) Peak annotation and normalization: The peak annotation procedure aimed to cluster peaks from the same lipid molecule. Adduct ions including [M + H]+, [M + K]+, [M + Na]+, and [M + NH4]+ were utilized for peak annotation. (v) Lipid identification:
      • Mentana A.
      • Zianni R.
      • Campaniello M.
      • Tomaiuolo M.
      • Chiappinelli A.
      • Iammarino M.
      • Nardelli V.
      Optimizing accelerated solvent extraction combined with liquid chromatography-Orbitrap mass spectrometry for efficient lipid profile characterization of mozzarella cheese.
      introduced an optimizing accelerated solvent extraction procedure for the untargeted lipid profile of mozzarella cheese (
      • Mentana A.
      • Zianni R.
      • Campaniello M.
      • Tomaiuolo M.
      • Chiappinelli A.
      • Iammarino M.
      • Nardelli V.
      Optimizing accelerated solvent extraction combined with liquid chromatography-Orbitrap mass spectrometry for efficient lipid profile characterization of mozzarella cheese.
      ). A total of 13 subclasses including bismethyl phosphatidic acids, ceramides, cholesterol ester, diglycerides, hexosyl ceramides, lysophosphatidylcholines, lysophosphatidylethanolamines, phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, sphingomyelins, triglycerides, and zymosterol ester were identified by using LipidSearch software. The lower lipid-rich CHCl3 phase was collected after passing through the upper MeOH-H2O phase and proteins at the interface of the 2-phase, which may result in contamination. As the operation procedures of the Folch method were labor intensive, time consuming, and complicated, the MTBE method, which accumulated the nonextractable pellet at the bottom and collected the upper lipid-rich laid easier and cleaner, was performed to extract lipids in this study. Lipid categories, carbon chain length, and the content of unsaturated fatty acids were identified by using lipid databases (LIPID MAPS, LipidBlast, and LipidBank) and computational tools (Mascot, Sequest, and Tide). The characteristic structural fragments were applied for further confirmation of lipid molecules structures. According to the measured m/z 732.5537 and a strong product ion m/z 184.0131 (phosphocholine headgroup), PC 32:1 could be confirmed tentatively (Figure 1). Product ions with m/z 497.2361 and m/z 495.2955 were identified as palmitic acid (C16:0) and hexadecenoic acid (C16:1). Previous research has suggested fatty acid anions with higher strength are easy to release from sn-2 position (
      • Yin F.-W.
      • Zhou D.-Y.
      • Zhao Q.
      • Liu Z.-Y.
      • Hu X.-P.
      • Liu Y.-F.
      • Song L.
      • Zhou X.
      • Qin L.
      • Zhu B.-W.
      • Shahidi F.
      Identification of glycerophospholipid molecular species of mussel (Mytilus edulis) lipids by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry.
      ). Therefore, the ion of m/z 731.5537 was identified as PC (16:0/16:1). Accurate characterization of structural isomers was of great prerequisite for deciphering structural composition and investigating biological functions of lipid molecules. Annotation of molecules defined at the structural level required the information of alkyl chain length, degree of unsaturation, and fatty acid composition. In the case of m/z 544.45716 (C31H62O6N1), the structural isomers of TG (6:0/8:0/14:0)+NH4+ and TG (6:0/10:0/12:0)+NH4+ could be assigned on the basis of specific fragments in MS2 spectrum (Figure 2). The discrimination of lipid isomers was achieved by detecting specific diagnostic fragments such as m/z 299.22168 (C17H31O4+) and m/z 383.31558 (C23H43O4+) for TG (6:0/8:0/14:0) and m/z 327.25298 (C19H35O4+) and m/z 355.28428 (C21H39O4+) for TG (6:0/10:0/12:0). (vi) Statistical analysis: Lipidomic data set was subjected to statistical analysis to visualize the overall variations driven by benzoic acid. Stepwise changes and significant distinctions for lipid molecules responsive to 6 different concentrations of benzoic acid was observed (Figure 3A). The separation among sample groups was precise according to the first 2 principal components, accounting for 0.524 and 0.213 of the total variances, respectively. Moreover, PLS-DA score plot (R2X = 0.525, R2Y = 0.208, and Q2 = 0.990) demonstrated lipidomic phenotype stepwise shifts in fermented goat milk samples as the elevated concentration of benzoic acid (Figure 3B). Control samples (with 0 mg/kg benzoic acid) were apparently separated from others in the right half zoom, whereas all the others with different concentrations of benzoic acid were consistently situated on the other half, indicating benzoic acid drives the pronounced alterations of lipidome. The 200-permutation testing was performed to estimate the validity and predictive ability of PLS-DA model. As observed in Figure 3C, R2Y(cum) = (0, 0.310) and Q2(cum) = (0, −0.381) proved the PLS-DA model was robust and without overfitting. With general discriminations identified by the PCA and PLS-DA, specific altered lipids due to changes in different experimental conditions of benzoic acid were determined using multivariate statistics. Lipid molecules were considered significant (Figure 3D) if the statistical parameters fit the criteria that the VIP value (>1.0) and FDR-adjusted P-value (<0.05) (
      • Jia W.
      • Wu X.X.
      • Zhang R.
      • Shi L.
      UHPLC-Q-Orbitrap-based lipidomics reveals molecular mechanism of lipid changes during preservatives treatment of Hengshan goat meat sausages.
      ).
      Figure thumbnail gr2
      Figure 2MS/MS spectra for triglycerides (TG; 6:0/8:0/14:0) + NH4+ and TG (6:0/10:0/12:0) + NH4+. m/z = mass-to-charge ratio.
      Figure thumbnail gr3
      Figure 3Multivariate statistical analysis of lipids in fermented goat milk responsive to different benzoic acid levels (0, 5, 10, 20, 30, and 40 mg/kg). (A) Two-dimensional principal component analysis (PCA) score plot of lipid profiling among all groups (R1X = 0.524 and R2X = 0.213). (B) Partial least squares-discrimination analysis (PLS-DA) score plot of identified lipid in 6 different benzoic acid concentrations (0, 5, 10, 20, 30, and 40 mg/kg) samples (R2X = 0.525, R2Y = 0.208, and Q2 = 0.990). The color and shape of the scatter plot represents the grouping of samples. (C) Permutation test (200 times) based on the corresponding PLS-DA model [R2Y(cum) = (0, 0.310) and Q2(cum) = (0, −0.381)]. (D) Variable importance in projection (VIP) plot of lipid profiling in 6 different benzoic acid concentration (0, 5, 10, 20, 30, and 40 mg/kg) samples calculated by PLS-DA model. 0 ppm = fermented goat milk samples; 5 ppm = fermented goat milk samples with 5 mg/kg benzoic acid; 10 ppm = fermented goat milk samples with 10 mg/kg benzoic acid; 20 ppm = fermented goat milk samples with 20 mg/kg benzoic acid; 30 ppm = fermented goat milk samples with 30 mg/kg benzoic acid; 40 ppm = fermented goat milk samples with 40 mg/kg benzoic acid; QC = quality control.

      Benzoic Acid Drove Differences of Lipid Categories and Contents in Fermented Goat Milk

      Integrating information about retention time, accurate mass measurement, and MS/MS fragmentation showed that a total of 189 lipid molecules were structurally identified and their comprehensive information was annotated (Table 2). These lipids were extended into 3 lipid categories, including 137 glycerolipids, 18 glycerophospholipids, and 34 sphingolipids (Figure 4A). These lipid compounds were further classified into 8 subclasses containing 27 ceramides (Cer), 7 diglycerides (DG), 3 lysophosphatidylcholines (LPC), 8 phosphatidylcholines (PC), 6 phosphatidylethanolamines (PE), 1 phosphatidylserine (PS), 7 sphingomyelins (SM), and 130 triglycerides (TG; Figure 4B). As an unsupervised machine-learning algorithm, K-means clustering seeks to group multiple factors of behavioral responses of all individuals into featured clusters, to maximize variations between the groups and minimize variations within the groups (
      • Huang C.
      • Li Y.
      • Wang K.
      • Xi J.
      • Xu Y.
      • Si X.
      • Pei D.
      • Lyu S.
      • Xia G.
      • Wang J.
      • Li P.
      • Ye H.
      • Xing Y.
      • Wang Y.
      • Huang J.
      Analysis of lipidomics profile of Carya cathayensis nuts and lipid dynamic changes during embryonic development.
      ). K-means clusters analysis reflected the content variations of lipid molecules at different concentrations of benzoic acid accumulation and all lipid molecules were distributed into 6 clusters (Figure 4D). Glycerophospholipids were found in all clusters (especially 1, 2, and 6), whereas glycerolipids and sphingolipids were mainly distributed in clusters (3 and 5) and (1, 4, and 6), respectively, reflecting the significance of cluster 1 and 6 (Figure 4C). In cluster 1, 34 lipid molecules (29.41% glycerophospholipids, 5.84% glycerolipids, and 31.43% sphingolipids) were detected, which remained constant in the initial stage and increased rapidly in group C-E. Conversely, in cluster 5, 21 lipid molecules (5.88% glycerophospholipids, 50.36% glycerolipids, and 8.57% sphingolipids) decreased gradually with the increase of benzoic acid concentration. Glycerophospholipids of fermented goat milk samples mainly consist of PC, PE, PS, and LPC. As one of the dominant lipids in interstitial fluids and plasma, the physiological roles of LPC have been elucidated. LPC 16:0, LPC 18:0, and LPC 18:1 were reported to exhibit proinflammatory effects (the expression of cell adhesion molecules and chemokine release) and accelerate the productive rate of reactive oxygen species (
      • Tan S.T.
      • Ramesh T.
      • Toh X.R.
      • Nguyen L.N.
      Emerging roles of lysophospholipids in health and disease.
      ). These LPC species present in fermented milk accumulated as benzoic acid levels rose from 0.244 to 0.405 mg/kg (LPC 16:0), 0.125 to 0.169 mg/kg (LPC 18:0), and 0.349 to 0.549 mg/kg (LPC 18:1), respectively (Table 3). High concentrations of LPC enhanced the expression of cell adhesion molecules resulting in atherogenesis. Excess LPC exacerbated the production of nitric oxide (NO) contributing to enhanced reactive oxygen species production and further leading to cardiovascular disorders (
      • Tseng H.C.
      • Lin C.C.
      • Hsiao L.D.
      • Yang C.
      Lysophosphatidylcholine-induced mitochondrial fission contributes to collagen production in human cardiac fibroblasts.
      ). Regulation of sphingolipid homeostasis is of fundamental significance for multicellular organisms. Sphingolipids including bioactive constituents participated in numerous cellular signaling cascades (
      • Wigger D.
      • Gulbins E.
      • Kleuser B.
      • Schumacher F.
      Monitoring the sphingolipid de novo synthesis by stable-isotope labeling and liquid chromatography-mass spectrometry.
      ). Disturbance of different bioactive sphingolipids severely affected cell function. Quantitative results showed that the concentration of sphingolipids decreased from 15.61 mg/kg in the control group to 12.50 mg/kg in the experimental group. Benzoic acid accumulation affected the beneficial lipid concentration and nutritive value of fermented goat milk.
      Table 2List of 189 lipid molecules detected in fermented goat milk responsive to different benzoic acid levels (0, 5, 10, 20, 30, and 40 mg/kg)
      Compound
      Cer = ceramide; DG = diglyceride; LPC = lysophosphatidylcholine; PC = phosphatidylcholine; PE = phosphatidylethanolamine; PS = phosphatidylserine; SM = sphingomyelin; TG = triglyceride.
      Ionization modeMolecular formulaRT
      RT = retention time.
      (min)
      Experimental mass (m/z)Theoretical mass (m/z)ΔMass (mg/kg)Elemental composition (fragment 1)Theoretical mass (m/z)Elemental composition (fragment 2)Theoretical mass (m/z)
      Cer (d16:0/16:0)M+HC32H66O3N113.12512.5031512.5037−1.18530C16H34NO+256.26349C21H40NO3+354.30027
      Cer (d16:0/18:0)M+HC34H70O3N114.67540.5346540.5350−0.76493C19H32NO+290.24784C26H50NO2+408.38361
      Cer (d16:1/23:0)M+H-H2OC39H76O2N114.79590.5868590.5871−0.49353C16H31O2+255.23186C23H45NO3+383.39400
      Cer (d17:1/16:0)M+HC33H66O3N111.01524.5034524.5037−0.69298C16H30NO+252.23219C17H26NO+260.20089
      Cer (d17:1/16:0)M+H-H2OC33H64O2N110.98506.4929506.4932−0.46885C16H26NO+248.20089C17H30NO+264.23219
      Cer (d18:0/16:0)M+HC34H70O3N112.20540.5353540.53500.58929C16H32NO+254.24784C18H36NO+282.27914
      Cer (d18:1/14:0)M+HC32H64O3N110.40510.4877510.4881−0.66303C14H28N+210.22163C18H32NO2+294.24276
      Cer (d18:1/14:0)M+H-H2OC32H62O2N110.25492.4769492.4775−1.17055C14H27+195.21073C18H36NO2+298.27406
      Cer (d18:1/16:0)M+HC34H68O3N111.40538.5186538.5194−1.37130C16H32NO+254.24784C18H32N+262.25293
      Cer (d18:1/16:0)M+H-H2OC34H66O2N111.45520.5093520.50881.02309C16H33NO+255.25567C18H31NO+277.24002
      Cer (d18:1/22:0)M+HC40H80O3N115.12622.6148622.61332.40522C18H29+245.22638C22H39+303.30463
      Cer (d18:1/22:0)M+H-H2OC40H78O2N115.28604.6025604.6027−0.36631C18H34NO2+296.25841C22H43O+323.33084
      Cer (d18:1/23:0)M+HC41H82O3N115.78636.6291636.62890.25215C18H32NO+278.24784C22H38NO+332.29479
      Cer (d18:1/23:0)M+H-H2OC41H80O2N115.82618.6180618.6184−0.49541C18H31+247.24203C22H47O+327.36214
      Cer (d18:1/24:0)M+HC42H84O3N116.26650.6441650.6446−0.70003C18H35NO+281.27132C24H48N+350.37813
      Cer (d18:1/24:0)M+H-H2OC42H82O2N116.28632.6334632.6340−0.90332C18H34NO+280.26349C24H46N+348.36248
      Cer (d19:1/16:0)M+H-H2OC35H68O2N112.05534.5239534.5245−0.99054C16H33O+241.25259C19H33NO+291.25567
      Cer (d20:0/16:0)M+HC36H74O3N115.73568.5658568.5663−1.00159C16H31N+237.24510C20H43NO2+329.32883
      Cer (d20:0/18:0)M+HC38H78O3N116.84596.5967596.5976−1.46911C18H36NO+282.27914C20H41NO2+327.31318
      Cer (d20:2/18:0+O)M+NH4C38H77O4N221.46625.5871625.5878−1.13568C18H38N+268.29988C20H39NO2+329.29753
      DG (10:0/18:2)M+NH4C31H60O5N19.14526.4469526.44660.53097C10H18O4+202.11996C18H33O+265.25259
      DG (16:0/14:0)M+NH4C33H68O5N112.82558.5089558.5092−0.62751C17H29O2+265.21621C19H33O+277.25259
      DG (16:0/16:0)M+NH4C35H72O5N114.16586.5394586.5405−1.89155C19H35O3+311.25807C24H47O5+415.34180
      DG (16:0/18:2)M+NH4C37H72O5N113.27610.5398610.5405−1.18169C16H28+220.21855C18H30O+262.22912
      DG (18:0/16:0)M+NH4C37H76O5N115.14614.5716614.5718−0.38152C16H29+221.22638C18H31O+263.23694
      DG (18:1/18:1)M+NH4C39H76O5N114.30638.5706638.5718−1.91595C18H33O+265.25259C18H35O2+283.26316
      DG (18:1/18:2)M+NH4C39H74O5N113.37636.5552636.5562−1.46487C22H40O4+368.29210C35H59O3+527.44590
      Hex1Cer (d18:1/16:0)M+HC40H78O8N110.32700.5716700.5722−0.89423C16H33+225.25768C18H32N+262.25293
      Hex1Cer (d18:1/22:0)M+HC46H90O8N113.99784.6641784.6661−2.51250C18H35NO+281.27132C22H44NO+338.34174
      Hex1Cer (d18:1/23:0)M+HC47H92O8N114.54798.6813798.6817−0.58029C18H34NO+280.26349C23H45NO+351.34957
      Hex1Cer (d18:1/24:0)M+HC48H94O8N115.02812.6959812.6974−1.78353C18H36NO+282.27914C24H46NO2+380.35231
      Hex1Cer (m17:0/24:6)M+NH4C47H85O7N227.01789.6386789.63514.38875C17H33NO+267.25567C24H50NO+368.38869
      Hex2Cer (d18:1/16:0)M+HC46H88O13N19.43862.6246862.6250−0.51293C17H36NO2+286.27406C22H40NO2+350.30536
      Hex2Cer (d18:1/23:0)M+HC53H102O13N113.91960.7315960.7346−3.24073C24H47N+349.37030C27H54NO3+440.40982
      LPC (16:0)M+HC24H51O7N1P12.17496.3396496.3398−0.43412C16H32O2+256.23968C19H35O6P+390.21658
      LPC (18:0)M+HC26H55O7N1P13.29524.3709524.3711−0.41663C6H16NO6P+229.07098C18H36O2+284.27098
      LPC (18:1)M+HC26H53O7N1P12.22522.3550522.3554−0.71880C21H33O+301.25259C21H37O4P+384.24240
      PC (16:0/12:0)M+HC36H73O8N1P110.02678.5068678.5068−0.01248C18H37NO7P+410.23022C31H59O5P+542.40946
      PC (16:0/14:0)M+HC38H77O8N1P111.89706.5369706.5381−1.79533C21H37NO5P+414.24039C33H63O5P+570.44076
      PC (16:0/16:0)M+HC40H81O8N1P113.52734.5681734.5694−1.83029C20H38O5P+389.24514C25H48NO8P+521.31121
      PC (16:0/16:1)M+HC40H79O8N1P111.68732.5530732.5538−1.01217C19H31O3+307.22677C23H44NO7P+477.28499
      PC (16:0/18:1)M+HC42H83O8N1P114.69760.5841760.5851−1.34301C18H30O+262.22912C27H48O6P+499.31830
      PC (16:0/18:2)M+HC42H81O8N1P112.27758.5683758.5694−1.48368C20H30O+286.22912C23H38O5P+425.24514
      PC (18:0/18:2)M+HC44H85O8N1P113.37786.5992786.6007−1.95712C17H37O+257.28389C28H52O8P+547.33943
      PC (18:1/18:2)M+HC44H83O8N1P112.11784.5839784.5851−1.55046C18H29+245.22638C23H37O6P+440.23223
      PE (16:0/18:2)M+HC39H75O8N1P110.90716.5227716.52250.32313C19H39O3+315.28937C26H52NO8P+537.34251
      PE (18:0/18:1)M+HC41H81O8N1P113.18746.5673746.5694−2.87780C18H35+251.27333C18H33O+265.25259
      PE (18:0p/18:2)M+HC41H79O7N1P112.92728.5575728.5589−1.91813C18H36O2+284.27098C18H27O+259.20564
      PE (18:1/18:1)M+HC41H79O8N1P111.79744.5532744.5538−0.82260C13H21O6P+304.10703C33H59O7P+598.39929
      PE (18:1/18:2)M+HC41H77O8N1P111.05742.5372742.5381−1.32043C20H34O2+306.25533C27H45O6P+496.29483
      PE (18:1p/18:1)M+HC41H79O7N1P112.55728.5575728.5589−1.92088C18H31O+263.23694C10H18O7P+281.07847
      PS (18:0/18:2)M+HC42H79O10N1P17.72788.5429788.5436−0.00119C18H33O2+281.24751C39H71O4+603.53469
      SM (d14:0/18:1)M+HC37H76O6N2P19.63675.5430675.5436−0.84594C18H36NO2+298.27406C34H64NO3P+565.46183
      SM (d18:1/16:0)M+HC39H80O6N2P111.29703.5751703.57490.35608C16H33NO+255.25567C22H46N2O6P+465.30880
      SM (d18:1/21:0)M+HC44H90O6N2P115.86773.6516773.6531−1.97178C20H43O+299.33084C26H49NO2+407.37578
      SM (d18:1/22:0)M+HC45H92O6N2P116.64787.6696787.66881.12424C18H34N+264.26858C22H45NO+339.34957
      SM (d18:1/23:0)M+HC46H94O6N2P117.93801.6833801.6844−1.38268C23H45NO+351.34957C32H59NO4P+552.41762
      SM (d18:1/24:0)M+HC47H96O6N2P119.28815.6989815.7001−1.36137C24H47NO4P+444.32372C35H66NO5P+611.46731
      SM (d18:1/24:1)M+HC47H94O6N2P116.40813.6822813.6844−2.71908C6H13NO5P+210.05259C20H43NO5P+408.28734
      TG (10:0/10:0/18:1)M+NH4C41H80O6N116.17682.5976682.5980−0.55006C10H21O+157.15869C18H32O3+296.23460
      TG (10:0/14:0/18:1)M+NH4C45H88O6N117.99738.6594738.6606−1.60489C10H18O+154.13522C14H25+193.19508
      TG (10:0/17:1/18:1)M+NH4C48H92O6N118.45778.6902778.6919−2.21458C21H37O+305.28389C36H65O2+529.49791
      TG (10:0/18:1/18:1)M+NaC49H90O6Na118.84797.6616797.6630−1.72438C25H46O4+410.33906C32H61O3+493.46152
      TG (10:0/18:1/18:1)M+NH4C49H94O6N119.03792.7065792.7076−1.36554C27H50O5+454.36528C37H65O5+589.48265
      TG (10:0/18:1/18:2)M+NH4C49H92O6N118.32790.6907790.6919−1.49928C20H35O+291.26824C31H54O5+506.39658
      TG (15:0/10:0/16:0)M+NaC44H84O6Na118.53731.6159731.6160−0.15236C18H35O2+283.26316C28H52NaO4+475.37578
      TG (15:0/10:0/16:0)M+NH4C44H88O6N118.27726.6593726.6606−1.82819C14H26+194.20290C20H39O5+359.27920
      TG (15:0/10:0/18:1)M+NaC46H86O6Na118.64757.6310757.6317−0.86780C29H55O3+451.41457C32H60O5+524.44353
      TG (15:0/10:0/18:1)M+NH4C46H90O6N118.74752.6750752.6763−1.62682C23H42O5+398.30268C31H55O3+475.41457
      TG (15:0/10:0/18:2)M+NH4C46H88O6N117.99750.6610750.66060.45231C25H46O5+426.33398C39H70O5+618.52178
      TG (15:0/14:0/16:0)M+NH4C48H96O6N120.29782.7217782.7232−1.95659C28H51O3+435.38327C31H59O6+527.43062
      TG (15:0/14:0/18:1)M+NH4C50H98O6N119.96808.7378808.7389−1.34589C20H36O4+340.26081C31H59O5+511.43570
      TG (15:0/16:0/18:1)M+NH4C52H102O6N120.64836.7695836.7702−0.82994C21H35O2+319.26316C29H52O4+464.38601
      TG (15:0/18:1/18:1)M+NH4C54H104O6N120.64862.7844862.7858−1.64754C22H40O4+368.29211C38H67O5+603.49830
      TG (15:0/18:1/18:2)M+NH4C54H102O6N120.19860.7684860.7702−2.03710C22H37O4+365.26864C29H50O5+478.36528
      TG (15:0/18:2/18:2)M+NH4C54H100O6N119.56858.7533858.7545−1.38395C19H37O5+345.26355C28H52O5+468.38093
      TG (15:0/6:0/16:0)M+NH4C40H80O6N116.94670.5971670.5980−1.38752C22H42O4+370.30776C28H51O4+451.37819
      TG (15:0/6:0/18:1)M+NaC42H78O6Na116.86701.5677701.5691−1.97624C21H33O4+349.23734C29H50O4+462.37036
      TG (15:0/6:0/18:1)M+NH4C42H82O6N116.86696.6135696.6137−0.20452C19H37O3+313.27372C31H55O4+491.40949
      TG (15:0/6:0/18:2)M+NH4C42H80O6N116.13694.5955694.5980−3.60565C21H40O4+356.29211C28H46O4+446.33906
      TG (15:0/8:0/18:1)M+NH4C44H86O6N117.52724.6431724.6450−2.54397C11H21O4+217.14344C21H36O4+352.26081
      TG (16:0/10:0/10:0)M+NH4C39H78O6N116.04656.5811656.5824−1.97610C19H33O+277.25259C26H48O6+456.34454
      TG (16:0/10:0/12:0)M+NH4C41H82O6N117.81684.6131684.6137−0.89900C17H33O5+317.23225C32H63O6+543.46192
      TG (16:0/10:0/14:0)M+NaC43H82O6Na118.05717.5979717.6004−3.36187C11H22+154.17160C23H40O4+380.29211
      TG (16:0/10:0/14:0)M+NH4C43H86O6N118.08712.6442712.6450−1.00537C17H29O+249.22129C33H61O2+489.46661
      TG (16:0/10:0/16:0)M+NaC45H86O6Na118.74745.6303745.6317−1.77228C22H43O5+387.31050C35H66O5+566.49048
      TG (16:0/10:0/16:0)M+NH4C45H90O6N119.24740.6763740.67630.05877C18H34O3+298.25025C27H48O3+420.35980
      TG (16:0/10:0/17:0)M+NH4C46H92O6N119.46754.6911754.6919−1.14281C18H34O+266.26042C30H53O3+461.39892
      TG (16:0/10:0/18:1)M+NaC47H88O6Na118.82771.6462771.6473−1.43650C24H44O5+412.31833C37H65O3+557.49282
      TG (16:0/10:0/18:1)M+NH4C47H92O6N118.98766.6907766.6919−1.54491C11H21O4+217.14344C27H45O4+433.33124
      TG (16:0/10:0/18:2)M+NH4C47H90O6N118.26764.6754764.6763−1.13181C21H33O2+317.24751C32H53O4+501.39384
      TG (16:0/12:0/18:1)M+NH4C49H96O6N119.77794.7225794.7232−0.87385C18H31O+263.23694C28H52O5+468.38093
      TG (16:0/14:0/16:0)M+NH4C49H98O6N120.48796.7384796.7389−0.55661C18H34O3+298.25025C35H66O2+518.50573
      TG (16:0/14:0/18:1)M+NaC51H96O6Na120.56827.7108827.70991.13268C21H39O3+339.28937C30H54O4+478.40166
      TG (16:0/14:0/18:1)M+NH4C51H100O6N120.32822.7534822.7545−1.29987C21H37O+305.28389C35H61O3+529.46152
      TG (16:0/14:0/18:2)M+NH4C51H98O6N119.96820.7374820.7389−1.78555C20H35O+291.26824C39H67O5+615.49830
      TG (16:0/16:0/17:0)M+NH4C52H104O6N121.36838.7845838.7858−1.54208C19H34O2+294.25533C23H40O4+380.29211
      TG (16:0/16:0/18:1)M+NaC53H100O6Na121.08855.7403855.7412−1.09901C22H38O3+350.28155C23H41O5+397.29485
      TG (16:0/16:0/18:1)M+NH4C53H104O6N120.95850.7846850.7858−1.43687C27H46O4+434.33906C38H72O6+624.53234
      TG (16:0/16:0/18:2)M+NaC53H98O6Na120.69853.7243853.7256−1.47292C23H37O3+361.27372C27H47O5+451.34180
      TG (16:0/17:0/18:1)M+NH4C54H106O6N121.13864.7997864.8015−1.99754C20H34O4+338.24516C28H49O4+449.36254
      TG (16:0/18:1/18:1)M+NaC55H102O6Na121.06881.7553881.7569−1.78561C19H37O3+313.27372C29H50O4+462.37036
      TG (16:0/18:1/18:1)M+NH4C55H106O6N121.07876.8002876.8015−1.41249C23H40O4+380.29211C43H76O4+656.57381
      TG (16:0/18:1/18:2)M+NH4C55H104O6N120.55874.7851874.7858−0.85560C18H33+249.25768C28H51O5+467.37310
      TG (16:0/6:0/10:0)M+NH4C35H70O6N114.05600.5194600.5198−0.55197C16H27O+235.20564C30H59O3+467.44587
      TG (16:0/6:0/14:0)M+NH4C39H78O6N117.11656.5817656.5824−0.96480C16H34O2+258.25533C29H57O2+437.43531
      TG (16:0/6:0/16:0)M+NaC41H78O6Na117.08689.5685689.5691−0.82438C15H31O2+243.23186C24H46O5+414.33398
      TG (16:0/6:0/17:0)M+NaC42H80O6Na117.87703.5839703.5847−1.15049C24H43O4+395.31559C34H65O4+537.48774
      TG (16:0/6:0/17:0)M+NH4C42H84O6N117.80698.6288698.6293−0.76073C19H38O4+330.27646C32H61O2+477.46661
      TG (16:0/6:0/18:1)M+NaC43H80O6Na117.29715.5831715.5847−2.26594C25H42O3+390.31285C37H67O2+543.51356
      TG (16:0/6:0/18:1)M+NH4C43H84O6N117.51710.6288710.6293−0.66064C22H38O4+366.27646C35H62O3+530.46935
      TG (16:0/6:0/18:2)M+NH4C43H82O6N116.62708.6127708.6137−1.38506C26H45O3+405.33632C37H68O3+560.51630
      TG (16:0/8:0/10:0)M+NH4C37H74O6N114.92628.5504628.5511−1.12238C16H28O6+316.18804C31H57O5+509.42005
      TG (16:0/8:0/18:1)M+NH4C45H88O6N118.16738.6597738.6606−1.23530C18H36O+268.27607C28H53O4+453.39384
      TG (16:0/8:0/18:2)M+NH4C45H86O6N117.62736.6441736.6450−1.12330C26H45O3+405.33632C33H57O4+517.42514
      TG (17:0/10:0/18:1)M+NH4C48H94O6N119.17780.7074780.7076−0.20426C19H39O2+299.29446C26H43O4+419.31559
      TG (17:0/18:1/18:1)M+NH4C56H108O6N121.20890.8152890.8171−2.12442C21H35O3+335.25807C32H59O5+523.43570
      TG (18:0/10:0/16:0)M+NH4C47H94O6N119.89768.7073768.7076−0.35055C19H38O3+314.28155C27H53O5+457.38875
      TG (18:0/16:0/16:0)M+NH4C53H106O6N121.65852.7997852.8015−2.09717C14H31O+215.23694C26H45O3+405.33632
      TG (18:0/16:0/17:0)M+NH4C54H108O6N121.88866.8164866.8171−0.77233C19H36O5+344.25573C24H45O4+397.33124
      TG (18:0/16:0/18:0)M+NH4C55H110O6N122.13880.8308880.8328−2.18143C16H34O+242.26042C23H44O5+400.31833
      TG (18:0/16:0/18:1)M+NH4C55H108O6N121.60878.8159878.8171−1.37625C20H38O5+358.27318C39H72O2+572.55268
      TG (18:0/17:0/18:1)M+NH4C56H110O6N121.78892.8302892.8328−2.92941C19H33O2+293.24751C20H35O2+307.26316
      TG (18:0/18:1/18:1)M+NH4C57H110O6N121.24904.8293904.8328−3.78024C22H37O3+349.27372C38H71O3+575.53977
      TG (18:1/12:0/18:2)M+NH4C51H96O6N119.19818.7223818.7232−1.10229C22H38O4+366.27646C27H47O4+435.34689
      TG (18:1/14:0/18:1)M+NaC53H98O6Na120.31853.7239853.7256−1.98011C20H37O3+325.27372C34H61O4+533.45644
      TG (18:1/14:0/18:1)M+NH4C53H102O6N120.41848.7694848.7702−0.95370C21H37O2+321.27881C33H65O5+541.48265
      TG (18:1/14:0/18:2)M+NH4C53H100O6N119.87846.7535846.7545−1.25476C21H35O2+319.26316C25H46O6+426.33398
      TG (18:1/18:1/18:1)M+NaC57H104O6Na120.89907.7704907.7725−2.33591C16H31O+239.23694C32H57O4+505.42514
      TG (18:1/18:1/18:2)M+NH4C57H106O6N120.52900.8004900.8015−1.20056C24H39O3+375.28937C39H63O5+601.51904
      TG (4:0/10:0/12:0)M+NH4C29H58O6N111.85516.4252516.4259−1.22664C15H27O+223.20564C27H50O3+422.37545
      TG (4:0/10:0/12:2)M+NH4C29H54O6N17.92512.3943512.3946−0.42247C16H30O+238.22912C28H51O3+435.38327
      TG (4:0/10:0/14:0)M+NH4C31H62O6N111.17544.4566544.4572−1.10472C15H29O2+241.21621C21H40O4+356.29211
      TG (4:0/10:0/15:0)M+NH4C32H64O6N112.17558.4716558.4728−2.25163C16H28O+236.21347C29H53O3+449.39892
      TG (4:0/10:0/16:0)M+NH4C33H66O6N114.23572.4879572.4885−1.06809C14H24O3+240.17200C28H53O2+421.40401
      TG (4:0/10:0/18:1)M+NaC35H64O6Na112.98603.4583603.4595−1.99263C21H35O2+319.26316C30H51O4+475.37819
      TG (4:0/10:0/18:1)M+NH4C35H68O6N112.95598.5025598.5041−2.65407C21H39O3+339.28937C28H47O3+431.35197
      TG (4:0/10:0/18:2)M+NaC35H62O6Na111.99601.4431601.4439−1.28769C19H34O2+294.25533C33H57O5+533.42005
      TG (4:0/10:0/18:2)M+NH4C35H66O6N112.19596.4878596.4885−1.12906C18H35O2+283.26316C34H56O4+528.41731
      TG (4:0/14:0/15:0)M+NH4C36H72O6N114.82614.5351614.5354−0.46778C10H18O5+218.11488C22H43O5+387.31050
      TG (4:0/14:0/16:0)M+NH4C37H74O6N115.27628.5513628.55110.33812C19H33O2+293.24751C31H58O2+462.44313
      TG (4:0/14:0/18:1)M+NaC39H72O6Na115.46659.5209659.5221−1.89451C15H29NaO2+264.20598C21H40NaO5+395.27680
      TG (4:0/14:0/18:1)M+NH4C39H76O6N115.29654.5660654.5667−1.04263C10H19O4+203.12779C24H43O4+394.30776
      TG (4:0/14:0/18:2)M+NH4C39H74O6N114.54652.5505652.5511−0.94318C18H36O4+316.26081C33H55O6+531.40440
      TG (4:0/15:0/16:0)M+NH4C38H76O6N115.95642.5660642.5667−1.04809C10H18O4+202.11996C21H39O5+371.27920
      TG (4:0/15:0/18:1)M+NH4C40H78O6N115.85668.5821668.5824−0.43745C15H29O+225.22129C31H55O3+475.41457
      TG (4:0/15:0/18:2)M+NH4C40H76O6N114.74666.5658666.5667−1.37941C16H30O3+270.21895C29H51O4+463.37819
      TG (4:0/15:0/18:3)M+NH4C40H74O6N113.89664.5498664.5511−1.84707C21H34O4+350.24516C28H51O5+467.37310
      TG (4:0/16:0/18:1)M+NaC41H76O6Na116.62687.5519687.5534−2.22451C10H15NaO4+222.08626C25H43NaO4+430.30536
      TG (4:0/16:0/18:1)M+NH4C41H80O6N116.39682.5982682.59800.24543C17H29+233.22638C29H53O4+465.39384
      TG (4:0/16:0/18:2)M+NH4C41H78O6N115.80680.5814680.5824−1.41713C19H35O3+311.25807C32H55O4+503.40949
      TG (4:0/17:1/18:2)M+NH4C42H78O6N115.02692.5815692.5824−1.18754C25H44O3+392.32850C33H55O4+515.40949
      TG (4:0/18:1/18:2)M+NH4C43H80O6N115.68706.5971706.5980−1.28287C17H33O4+301.23734C29H53O5+481.38875
      TG (4:0/6:0/14:0)M+NH4C27H54O6N18.79488.3940488.3946−1.25814C6H13O3+133.08592C16H29O6+317.19587
      TG (4:0/6:0/15:0)M+NaC28H52O6Na19.47507.3650507.3656−1.19927C25H43O4+407.31559C32H60O4+508.44861
      TG (4:0/6:0/15:0)M+NH4C28H56O6N19.57502.4100502.4102−0.42091C23H44O4+384.32341C34H63O3+519.47717
      TG (4:0/6:0/16:0)M+NaC29H54O6Na110.26521.3806521.3813−1.23992C22H39O5+383.27920C33H62O3+506.46935
      TG (4:0/6:0/16:0)M+NH4C29H58O6N110.24516.4256516.4259−0.57214C6H8O3+128.04680C18H31O6+343.21152
      TG (4:0/6:0/16:1)M+NH4C29H56O6N19.15514.4099514.4102−0.58994C21H38O3+338.28155C34H61O3+517.46152
      TG (4:0/6:0/17:0)M+NH4C30H60O6N110.98530.4416530.44150.18198C21H39O5+371.27920C33H62O4+522.46426
      TG (4:0/6:0/17:1)M+NH4C30H58O6N19.60528.4256528.4259−0.50616C22H38O3+350.28155C32H61O4+509.45644
      TG (4:0/6:0/18:1)M+NaC31H56O6Na110.35547.3962547.3969−1.28695C21H37O2+321.27881C32H63O5+527.46700
      TG (4:0/6:0/18:1)M+NH4C31H60O6N110.43542.4414542.4415−0.22762C17H34O+254.26042C23H43O5+399.31050
      TG (4:0/6:0/18:2)M+NaC31H54O6Na19.24545.3809545.3813−0.65728C21H35O+303.26824C24H39O4+391.28429
      TG (4:0/6:0/18:2)M+NH4C31H58O6N19.14540.4256540.4259−0.48937C17H32O3+284.23460C25H44O4+408.32341
      TG (4:0/6:0/18:3)M+NH4C31H56O6N18.16538.4097538.4102−0.90353C18H32+248.24985C26H47O5+439.34180
      TG (4:0/8:0/10:0)M+NH4C25H50O6N16.87460.3628460.3633−1.03933C7H10O4+158.05736C16H31O6+319.21152
      TG (4:0/8:0/15:0)M+NH4C30H60O6N110.79530.4419530.44150.73812C6H11O4+147.06519C23H42O3+366.31285
      TG (4:0/8:0/18:1)M+NaC33H60O6Na111.89575.4279575.4282−0.60558C16H28NaO+259.20324C24H42O4+394.30776
      TG (4:0/8:0/18:1)M+NH4C33H64O6N111.62570.4723570.4728−0.83697C16H31O3+271.22677C28H50O4+450.37036
      TG (4:0/8:0/18:2)M+NaC33H58O6Na110.62573.4120573.4126−0.97568C14H25NaO2+248.17468C27H45O4+433.33124
      TG (4:0/8:0/18:2)M+NH4C33H62O6N110.52568.4555568.4572−2.84537C20H34O3+322.25025C25H47O5+427.34180
      TG (4:0/8:0/18:3)M+NH4C33H60O6N19.53566.4412566.4415−0.55693C20H35O4+339.25299C26H43O4+419.31559
      TG (4:0/9:0/18:1)M+NH4C34H66O6N112.41584.4879584.4885−1.03932C18H29O+261.22129C20H31O2+303.23186
      TG (4:0/9:0/18:2)M+NH4C34H64O6N111.48582.4717582.4728−1.90819C18H33O3+297.24242C25H42O4+406.30776
      TG (6:0/10:0/10:0)M+NH4C29H58O6N19.52516.4257516.4259−0.28169C10H110+147.08044C21H36O4+352.26081
      TG (6:0/10:0/12:0)M+NH4C31H62O6N111.36544.4570544.4572−0.30575C13H23O2+211.16926C18H32O5+328.22443
      TG (6:0/10:0/14:0)M+NH4C33H66O6N112.72572.4874572.4885−1.81571C9H18O4+190.11996C19H34O5+342.24008
      TG (6:0/10:0/18:2)M+NaC37H66O6Na113.25629.4745629.4752−1.11120C16H25+217.19508C24H39NaO4+414.27406
      TG (6:0/10:0/18:2)M+NH4C37H70O6N113.15624.5193624.5198−0.71650C18H35O4+315.25299C26H41O4+417.29994
      TG (6:0/10:0/18:3)M+NH4C37H68O6N112.19622.5034622.5041−1.21842C21H35O4+351.25299C26H46O4+422.33906
      TG (6:0/10:0/20:4)M+NH4C39H70O6N112.81648.5185648.5198−2.01454C8H15+111.11683C23H40O4+380.29211
      TG (6:0/14:0/18:1)M+NH4C41H80O6N131.92682.5969682.5980−1.59900C18H37O3+301.27372C22H33O4+361.23734
      TG (6:0/17:1/18:2)M+NH4C44H82O6N116.12720.6125720.6137−1.63398C15H24+204.18725C26H41O4+417.29994
      TG (6:0/18:1/18:2)M+NH4C45H84O6N116.53734.6284734.6293−1.20261C12H19+163.14813C22H37O3+349.27372
      TG (6:0/8:0/10:0)M+NH4C27H54O6N18.44488.3936488.3946−1.98706C10H18O3+186.12505C19H34O3+310.25025
      TG (6:0/8:0/14:0)M+NH4C31H62O6N112.86544.4565544.4572−1.14329C12H22O2+198.16143C16H31O5+303.21660
      TG (8:0/10:0/18:1)M+NH4C39H76O6N115.11654.5654654.5667−1.96385C18H32O2+280.23968C27H47O4+435.34689
      TG (8:0/18:1/18:2)M+NH4C47H88O6N117.48762.6593762.6606−1.77205C16H33O+241.25259C21H33O3+333.24242
      1 Cer = ceramide; DG = diglyceride; LPC = lysophosphatidylcholine; PC = phosphatidylcholine; PE = phosphatidylethanolamine; PS = phosphatidylserine; SM = sphingomyelin; TG = triglyceride.
      2 RT = retention time.
      Figure thumbnail gr4
      Figure 4Lipidomic landscape of fermented goat milk samples with 6 different benzoic acid concentration (0, 5, 10, 20, 30, and 40 mg/kg). (A) Detected lipids are classified by head group. Sphingolipids include Cer and SM. Glycerophospholipids include PC, PE, PS, and LPC. Glycerolipids include DG and TG. Cer = ceramide; SM = sphingomyelin; DG = diacylglycerol; TG = triacylglycerol; PC = phosphatidylcholine; PE = phosphatidylethanolaine; PS = phosphatidylserine; LPC = lysophosphatidylcholine. (B) Distribution of 189 identified lipid molecules. The inner ring presents lipid categories and the outer ring presents lipid subclass. Different lipid categories were individually color-coded. (C) Percentage of glycerophospholipids, glycerolipids, and sphingolipids in 6 clusters. (D) K-means cluster analysis of annotated 189 lipid molecules. Group A = control group; group B = experimental group of 5 mg/kg benzoic acid; group C = experimental group of 10 mg/kg benzoic acid; group D = experimental group of 20 mg/kg benzoic acid; group E = experimental group of 30 mg/kg benzoic acid; group F = experimental group of 40 mg/kg benzoic acid.
      Table 3Dynamic changes of lipid molecule concentrations in fermented goat milk responsive to different benzoic acid levels (0, 5, 10, 20, 30, and 40 mg/kg)
      VIP = variable importance in projection; Cer = ceramide; DG = diglyceride; Hex1cer = hexosylceramide; Hex2cer = dihexosylceramide; LPC = lysophosphatidylcholine; PC = phosphatidylcholine; PE = phosphatidylethanolamine; SM = sphingomyelin; TG = triglyceride.
      ClusterCompoundVIPConcentration (mg/kg)
      0510203040
      1Cer (d18:0/16:0)+H1.0730.046 ± 0.0110.040 ± 0.0170.053 ± 0.0210.077 ± 0.0300.133 ± 0.0290.144 ± 0.025
      1Cer (d18:1/16:0)+H-H2O1.0610.163 ± 0.0060.097 ± 0.0030.098 ± 0.0040.120 ± 0.0050.334 ± 0.0120.819 ± 0.032
      1Cer (d18:1/22:0)+H-H2O0.8090.140 ± 0.0030.054 ± 0.0050.098 ± 0.0060.131 ± 0.0070.158 ± 0.0020.150 ± 0.006
      1Cer (d19:1/16:0)+H-H2O1.5480.111 ± 0.0040.108 ± 0.0210.139 ± 0.0030.164 ± 0.0730.204 ± 0.0210.129 ± 0.051
      1Hex1Cer (d18:1/16:0)+H1.2500.164 ± 0.0022.533 ± 0.0402.743 ± 0.0273.298 ± 0.0923.340 ± 0.0383.045 ± 0.051
      1Hex2Cer (d18:1/16:0)+H1.2120.082 ± 0.0051.352 ± 0.0511.288 ± 0.0511.536 ± 0.0611.527 ± 0.0211.496 ± 0.028
      1LPC (16:0)+H1.6170.244 ± 0.0020.332 ± 0.0290.231 ± 0.0840.625 ± 0.0390.651 ± 0.0070.405 ± 0.036
      1LPC (18:0)+H1.6010.125 ± 0.0030.164 ± 0.0050.115 ± 0.0210.215 ± 0.0080.241 ± 0.0050.169 ± 0.009
      1LPC (18:1)+H1.5750.349 ± 0.0260.436 ± 0.0810.358 ± 0.0390.874 ± 0.0370.875 ± 0.0530.549 ± 0.071
      1PE (18:1/18:2)+H1.1981.233 ± 0.0211.133 ± 0.0131.163 ± 0.0281.294 ± 0.0901.374 ± 0.0161.262 ± 0.053
      1SM (d14:0/18:1)+H1.0100.347 ± 0.0180.325 ± 0.0170.321 ± 0.0100.389 ± 0.0610.360 ± 0.0320.363 ± 0.024
      1SM (d18:1/16:0)+H1.0691.987 ± 0.0221.875 ± 0.0502.100 ± 0.0154.076 ± 0.0744.176 ± 0.0683.630 ± 0.030
      1SM (d18:1/23:0)+H1.1211.013 ± 0.0201.291 ± 0.0490.725 ± 0.0041.095 ± 0.0831.933 ± 0.0852.189 ± 0.058
      1SM (d18:1/24:0)+H1.1380.880 ± 0.0050.957 ± 0.0820.454 ± 0.0020.945 ± 0.0291.623 ± 0.0381.814 ± 0.071
      1TG (15:0/6:0/18:1)+Na0.6693.234 ± 0.0523.044 ± 0.0713.428 ± 0.0133.952 ± 0.0604.040 ± 0.0594.330 ± 0.042
      1TG (4:0/6:0/15:0)+Na0.5112.097 ± 0.0470.892 ± 0.0531.620 ± 0.0213.253 ± 0.0533.162 ± 0.0413.987 ± 0.095
      1TG (4:0/6:0/16:0)+Na1.03425.658 ± 0.08717.280 ± 0.09321.469 ± 0.08424.849 ± 0.07131.742 ± 0.05331.632 ± 0.088
      1TG (4:0/6:0/18:2)+Na0.8172.064 ± 0.0161.849 ± 0.0272.199 ± 0.0224.577 ± 0.0155.308 ± 0.0164.469 ± 0.013
      1TG (4:0/8:0/10:0)+NH41.49717.202 ± 0.05612.149 ± 0.0029.286 ± 0.00214.455 ± 0.00227.181 ± 0.00217.857 ± 0.002
      1TG (4:0/8:0/18:2)+Na1.2125.379 ± 0.0215.391 ± 0.0377.963 ± 0.0329.133 ± 0.02510.334 ± 0.02911.855 ± 0.032
      2Cer (d17:1/16:0)+H-H2O1.5530.047 ± 0.0020.051 ± 0.0030.050 ± 0.0070.055 ± 0.0040.054 ± 0.0050.045 ± 0.006
      2DG (18:1/18:2)+NH41.40317.010 ± 0.06417.546 ± 0.08224.160 ± 0.05923.048 ± 0.04820.657 ± 0.02915.856 ± 0.032
      2PC (16:0/14:0)+H1.3700.803 ± 0.0140.953 ± 0.0151.406 ± 0.0291.251 ± 0.0211.192 ± 0.0270.585 ± 0.038
      2PE (18:0/18:1)+H1.5101.414 ± 0.0211.211 ± 0.0221.638 ± 0.0262.036 ± 0.0291.763 ± 0.0301.083 ± 0.035
      2TG (16:0/16:0/18:1)+Na1.4641.579 ± 0.0191.691 ± 0.0181.833 ± 0.0222.006 ± 0.0301.616 ± 0.0311.677 ± 0.027
      2TG (16:0/18:1/18:1)+Na1.2921.954 ± 0.0271.968 ± 0.0132.218 ± 0.0162.536 ± 0.0191.835 ± 0.0202.052 ± 0.015
      2TG (4:0/8:0/18:2)+NH41.21227.218 ± 0.05323.279 ± 0.07032.778 ± 0.06932.631 ± 0.07527.251 ± 0.09321.117 ± 0.089
      3TG (15:0/18:1/18:1)+NH41.0408.454 ± 0.0414.381 ± 0.0154.604 ± 0.0176.075 ± 0.0223.092 ± 0.0313.549 ± 0.040
      3TG (16:0/16:0/17:0)+NH41.2432.422 ± 0.0171.700 ± 0.0031.103 ± 0.0071.709 ± 0.0050.349 ± 0.0040.472 ± 0.006
      3TG (16:0/16:0/18:1)+NH41.53344.328 ± 0.08627.102 ± 0.03124.995 ± 0.04238.416 ± 0.05321.224 ± 0.08127.863 ± 0.092
      3TG (16:0/18:1/18:1)+NH41.29251.747 ± 0.05128.809 ± 0.01829.855 ± 0.02740.484 ± 0.07124.610 ± 0.09330.933 ± 0.085
      3TG (16:0/18:1/18:2)+NH41.02244.391 ± 0.06922.789 ± 0.07325.321 ± 0.05929.912 ± 0.04020.235 ± 0.03821.376 ± 0.026
      3TG (17:0/18:1/18:1)+NH41.6333.105 ± 0.0231.900 ± 0.0162.210 ± 0.0233.080 ± 0.0191.472 ± 0.0572.359 ± 0.041
      3TG (18:0/16:0/18:1)+NH41.79911.033 ± 0.0455.660 ± 0.0384.941 ± 0.02710.531 ± 0.0413.947 ± 0.0386.956 ± 0.030
      3TG (18:0/17:0/18:1)+NH41.2351.182 ± 0.0120.881 ± 0.0030.663 ± 0.0081.123 ± 0.0020.528 ± 0.0030.544 ± 0.006
      3TG (18:0/18:1/18:1)+NH41.2237.237 ± 0.0263.818 ± 0.0174.372 ± 0.0536.633 ± 0.0704.059 ± 0.0364.989 ± 0.029
      3TG (4:0/15:0/16:0)+NH41.157128.542 ± 0.089107.996 ± 0.082111.854 ± 0.061129.517 ± 0.059141.641 ± 0.06896.970 ± 0.084
      3TG (6:0/10:0/18:3)+NH41.11723.280 ± 0.09116.841 ± 0.08518.681 ± 0.07322.236 ± 0.06919.932 ± 0.07112.264 ± 0.081
      4Cer (d18:1/14:0)+H-H2O1.2120.078 ± 0.0020.159 ± 0.0050.036 ± 0.0010.057 ± 0.0020.027 ± 0.0010.224 ± 0.008
      4Cer (d18:1/24:0)+H0.8970.130 ± 0.0110.083 ± 0.0090.054 ± 0.0070.081 ± 0.0080.114 ± 0.0120.110 ± 0.013
      4Cer (d18:1/24:0)+H-H2O1.2720.091 ± 0.0060.062 ± 0.0020.039 ± 0.0010.054 ± 0.0020.074 ± 0.0030.071 ± 0.004
      4Hex1Cer (d18:1/22:0)+H1.5030.262 ± 0.0134.191 ± 0.0433.191 ± 0.0372.985 ± 0.0213.878 ± 0.0393.457 ± 0.036
      4Hex1Cer (d18:1/23:0)+H1.4340.323 ± 0.0185.286 ± 0.0764.866 ± 0.0535.044 ± 0.0785.564 ± 0.0814.811 ± 0.054
      4Hex2Cer (d18:1/23:0)+H1.4350.153 ± 0.0552.766 ± 0.0451.991 ± 0.0412.360 ± 0.0493.144 ± 0.0312.695 ± 0.029
      4PE (18:1/18:1)+H1.3574.834 ± 0.0434.365 ± 0.0384.259 ± 0.0594.299 ± 0.0765.380 ± 0.0464.569 ± 0.039
      4SM (d18:1/21:0)+H1.2520.203 ± 0.0200.235 ± 0.0270.142 ± 0.0230.167 ± 0.0140.188 ± 0.0100.323 ± 0.014
      4SM (d18:1/22:0)+H1.0891.047 ± 0.0151.137 ± 0.0130.997 ± 0.0200.480 ± 0.0311.136 ± 0.0131.338 ± 0.011
      4TG (4:0/6:0/18:1)+Na1.34314.732 ± 0.09111.140 ± 0.08311.649 ± 0.09711.492 ± 0.08614.806 ± 0.08714.660 ± 0.092
      4TG (4:0/6:0/18:3)+NH41.2190.630 ± 0.0130.466 ± 0.0210.454 ± 0.0130.420 ± 0.0130.514 ± 0.0160.577 ± 0.017
      4TG (6:0/10:0/20:4)+NH41.1102.351 ± 0.0081.341 ± 0.0131.217 ± 0.0211.330 ± 0.0351.634 ± 0.0281.768 ± 0.032
      5Cer (d20:0/18:0)+H2.1670.548 ± 0.0850.411 ± 0.0370.583 ± 0.0130.424 ± 0.0610.427 ± 0.0400.496 ± 0.039
      5PC (18:1/18:2)+H1.1040.868 ± 0.0100.767 ± 0.0090.527 ± 0.0040.459 ± 0.0020.594 ± 0.0260.311 ± 0.015
      5TG (16:0/10:0/16:0)+Na1.1042.268 ± 0.0672.711 ± 0.0741.520 ± 0.0320.943 ± 0.0301.344 ± 0.0421.760 ± 0.057
      6Cer (d16:0/16:0)+H1.0500.469 ± 0.0130.448 ± 0.0130.277 ± 0.0130.270 ± 0.0110.030 ± 0.0110.025 ± 0.018
      6Cer (d16:0/18:0)+H1.4230.511 ± 0.0280.523 ± 0.0210.504 ± 0.0290.446 ± 0.0280.542 ± 0.0220.290 ± 0.031
      6Cer (d16:1/23:0)+H-H2O1.2510.084 ± 0.0120.078 ± 0.0150.076 ± 0.0170.081 ± 0.0200.079 ± 0.0070.055 ± 0.006
      6Cer (d17:1/16:0)+H1.5530.045 ± 0.0010.044 ± 0.0020.044 ± 0.0010.047 ± 0.0020.047 ± 0.0040.038 ± 0.003
      6Cer (d18:1/23:0)+H1.4350.215 ± 0.0160.192 ± 0.0240.179 ± 0.0120.200 ± 0.0180.189 ± 0.0230.122 ± 0.020
      6Cer (d18:1/23:0)+H-H2O1.2590.136 ± 0.0130.125 ± 0.0210.114 ± 0.0150.129 ± 0.0180.124 ± 0.0200.088 ± 0.011
      6Cer (d20:0/16:0)+H1.2810.466 ± 0.0250.357 ± 0.0220.478 ± 0.0210.453 ± 0.0100.340 ± 0.0110.260 ± 0.013
      6DG (10:0/18:2)+NH41.2261.841 ± 0.0172.447 ± 0.0231.909 ± 0.0130.803 ± 0.0120.879 ± 0.0200.889 ± 0.034
      6DG (16:0/18:2)+NH41.17519.655 ± 0.08321.894 ± 0.09824.755 ± 0.09222.200 ± 0.09618.805 ± 0.08814.442 ± 0.095
      6DG (18:1/18:1)+NH41.20515.484 ± 0.09319.021 ± 0.09618.863 ± 0.09411.812 ± 0.0879.838 ± 0.0797.481 ± 0.080
      6PC (16:0/16:1)+H1.1400.516 ± 0.0180.580 ± 0.0190.628 ± 0.0150.543 ± 0.0120.553 ± 0.0170.356 ± 0.013
      6PC (16:0/18:2)+H1.1731.644 ± 0.0711.555 ± 0.0820.976 ± 0.0560.954 ± 0.0361.015 ± 0.0420.427 ± 0.016
      6PC (18:0/18:2)+H1.1711.948 ± 0.0731.873 ± 0.0611.577 ± 0.0691.357 ± 0.0751.609 ± 0.0780.659 ± 0.062
      6TG (18:0/16:0/17:0)+NH41.2650.735 ± 0.0390.686 ± 0.0430.425 ± 0.0360.518 ± 0.0400.264 ± 0.0290.198 ± 0.015
      6TG (18:0/16:0/18:0)+NH41.0840.862 ± 0.0350.895 ± 0.0410.966 ± 0.0330.816 ± 0.0280.730 ± 0.0310.218 ± 0.027
      1 VIP = variable importance in projection; Cer = ceramide; DG = diglyceride; Hex1cer = hexosylceramide; Hex2cer = dihexosylceramide; LPC = lysophosphatidylcholine; PC = phosphatidylcholine; PE = phosphatidylethanolamine; SM = sphingomyelin; TG = triglyceride.

      Benzoic Acid Accumulation Perturbed Glycerophospholipid Metabolism

      To image the protein expression patterns in different concentrations of benzoic acid accumulation groups, the control group and 40 mg/kg group were selected for proteomic analysis. A total of 27 proteins significantly upregulated [fold change (FC) >1.50 and P < 0.05] and 12 proteins significantly downregulated (FC <0.67 and P < 0.05) in the 40 mg/kg group compared with control group (Table 4). From synergistic integration of multi-omics in benzoic acid accumulated fermented goat milk model, the biological processes of significant proteins mostly focused on glycerophospholipid metabolism and triglycerides degradation. Total circulating glycerophospholipids analysis revealed the decrease in phosphatidylcholine and phosphatidylethanolamine. Specifically, 14 lipid molecules, including 6 phosphatidylethanolamines and 8 phosphatidylcholines, were figured to be inextricably linked to glycerophospholipid metabolism (Figure 5). Phosphatidylcholine was formed by the transformation of choline via cytidine diphosphate choline (CDP-choline) branches of the Kennedy pathway (
      • Ecker J.
      • Liebisch G.
      Application of stable isotopes to investigate the metabolism of fatty acids, glycerophospholipid and sphingolipid species.
      ). The precursor choline was catalyzed by choline kinase (CK), CTP: phosphocholine cytidylyltransferase (CT), and choline phosphotransferase (CPT). Ethanolamine kinase phosphorylated ethanolamine to generate phosphoethanolamine followed by condensing with CTP to yield CDP-ethanolamine. Choline phosphotransferase-ethanolamine was converted to phosphatidylethanolamine by ethanolamine phosphotransferase (EPT) (
      • Cassilly C.D.
      • Reynolds T.B.
      PS, It’s complicated: The roles of phosphatidylserine and phosphatidylethanolamine in the pathogenesis of Candida albicans and other microbial pathogens.
      ). To verify the results obtained from lipidomic analysis, the protein expression levels of CPT (P = 0.3294) and EPT (P = 0.2279) were examined, which were key enzymes participating in glycerophospholipid metabolism. The level of diglyceride appeared to regulate its conversion to phosphatidylcholine, phosphatidylethanolamine, or triglyceride. Diglyceride generated by the dephosphorylation of phosphatidic acid fueled the synthesis of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine (
      • Hermansson M.
      • Hokynar K.
      • Somerharju P.
      Mechanisms of glycerophospholipid homeostasis in mammalian cells.
      ). When triglyceride synthesis was boosted by overexpression of diacylglycerol acyltransferase, the synthesis of phosphatidylcholine and phosphatidylethanolamine was markedly reduced, probably due to the shortage of diglyceride.
      Table 4Differentially abundant proteins of fermented goat milk from the comparisons with different benzoic acid amounts (0 and 40 mg/kg)
      Gene nameProtein nameProtein IDLengthMass (Da)Fold changeVIP
      VIP = variable importance in projection.
      40/0 mg/kgP-value
      AGPAlpha-1-acid glycoproteinA0A452E2F620223,1313.16230.00131.2721
      AHSGAlpha-2-HS-glycoproteinA0A452FH7835938,1850.45190.04131.0632
      ALAAlpha-lactalbuminB2YKX612213,8580.33700.03211.0473
      APOA1Apolipoprotein A1A0A452FI1426530,3220.24730.34081.1471
      B2MBeta-2-microglobulinA0A452DY3712414,00611.96100.00011.0974
      BTN1A1Butyrophilin subfamily 1 member A1A3EY5252659,2660.33520.03401.0485
      CATHL2Cathelicidin-2P8201817619,84632.49203.66E-071.3649
      CPT1Cholinephosphotransferase 1P1789839344,8290.32940.03211.0254
      Crisp3Cysteine-rich secretory protein 3D6PX6224427,3260.23750.01531.0487
      CSN1S1Alpha-S1-caseinP1862621424,2902.90628.71E-081.5286
      CSN1S2Alpha-S2-caseinP3304922326,3891.83220.01441.0702
      CSN2Beta-caseinP3304822224,8650.34650.04421.0385
      CSN3Kappa-caseinP0267019221,4412.78121.40E-081.5410
      DBIDiazepam binding inhibitor, acyl-CoA binding proteinA0A452EQX68710,0402.88241.76E-071.5224
      EPT1Ethanolaminephosphotransferase 1P2214039144,5600.22790.01451.0700
      FAM217AFamily with sequence similarity 217 member AA0A452FSL447252,7362.32850.04511.0888
      GLYCAM1Glycosylation-dependent cell adhesion molecule 1A0A452EGX615216,7622.60830.00081.2013
      JCHAINJoining chain of multimeric IgA and IgMA0A452G7G815818,0342.96278.26E-081.5290
      LALBAAlpha-lactalbuminA5JSS814216,25513.72501.02E-061.0561
      LGBBeta-lactoglobulinP0275618019,9763.20711.23E-061.4998
      LOC100860781Serum amyloid A proteinA0A452ECD312814,2782.38160.00371.2082
      LOC100861164AntiluteolysinQ6UZ4719522,3546.97250.02331.0712
      LOC102169231Cathelicidin-1A0A452FQC015517,6063.04719.90E-081.5275
      LOC102171351Short palate, lung and nasal epithelium carcinoma-associated protein 2BA0A452DYG716117,3612.73997.35E-091.5445
      LOC102180465Mammaglobin-AA0A452F4499410,6592.56520.00081.2990
      LOC102182127ClusterinA0A452E2Y043950,89315.55401.02E-051.0280
      LOC106503728Serum amyloid A proteinA0A452EX5313014,6171.66910.01451.0701
      LPOLactoperoxidaseA0A452E9Y671280,3661.72630.01441.0736
      map28MAP28 proteinQ9XSQ815817,7452.36890.01131.0977
      OPNOsteopontinA9YUB727730,8833.20711.23E-061.4998
      PIGRPolymeric immunoglobulin receptorA0A452ED2974781,4772.76031.61E-081.5403
      PLCH2Phosphoinositide phospholipase CA0A452DMZ81,116123,6520.34850.02081.0423
      PLIN1PerilipinA0A452FW8451655,1140.10620.00141.3527
      PLIN2PerilipinA0A452EIE637841,3060.43310.03401.0443
      PLIN3PerilipinA0A452FUU542746,2990.33700.03201.0473
      SDC2SyndecanA0A452ES9319721,9132.56330.05761.0881
      TET2Methylcytosine dioxygenase TETA0A452FBQ62,009223,8001.58450.01511.0692
      TLN2Talin 2A0A452FIW22,390255,1362.34720.00171.2552
      WWC3WWC family member 3A0A452DLQ61,087122,5203.08710.00161.2616
      1 VIP = variable importance in projection.
      Figure thumbnail gr5
      Figure 5Pathways of glycerophospholipid biosynthesis. Lipids are indicated in blue, and the enzymes catalyzing the respective reactions are indicated in green. CDS = CDP-diacylglycerol synthase; CDP-choline = cytidine diphosphate choline; CK = choline kinase; CPT = choline phosphotransferase; CT = CTP: phosphocholine cytidylyltransferase; DG = diglyceride; EPT = ethanolamine phosphotransferase; ET, CTP = phosphoethanolamine cytidylyltransferase; EK = ethanolamine kinase; LPA = lysophosphatidic acid; LPAAT = lysophosphatidic acid acyltransferase; PA = phosphatidic acid; PC = phosphatidylcholine; PE = phosphatidylethanolamine; PEMT = phosphatidylethanolamine N-methyltransferase; PI = phosphatidylinositol; PIS = phosphatidylinositol synthase; PS = phosphatidylserine; PSS = phosphatidylserine synthase; TG = triglyceride.

      Benzoic Acid Accumulation Induced Glyceride-Type Polyunsaturated Fatty Acid Degradation

      Multifarious fatty acids were esterified to triglycerides situated on the stereospecific numbering positions 1, 2, or 3. Long-chain polyunsaturated fatty acids (LCPUFA) with chain lengths longer than C18 to C20 were regarded as the key elements to make bone maintenance and joint health (
      • Abshirini M.
      • Ilesanmi-Oyelere B.L.
      • Kruger M.C.
      Potential modulatory mechanisms of action by long-chain polyunsaturated fatty acids on bone cell and chondrocyte metabolism.
      ). The existing evidence from in vitro and in vivo research has suggested consumption of n-3 LCPUFA C18:3 (α-linolenic acid, ALA) and n-6 LCPUFA C18:2 (linoleic acid, LA) and C20:4 (arachidonic acid, ARA) has substantiated a close relationship with bone homeostasis (
      • Wang Y.
      • Zhang T.
      • Liu R.
      • Chang M.
      • Wei W.
      • Jin Q.
      • Wang X.
      Reviews of medium- and long-chain triglyceride with respect to nutritional benefits and digestion and absorption behavior.
      ;
      • Zheng J.
      • Yang J.
      • Yang X.
      • Cao Z.
      • Cai S.
      • Wang B.
      • Ye J.
      • Fu M.
      • Zheng W.
      • Rao S.
      • Du D.
      • Liao Y.
      • Jiang X.
      • Xu F.
      Transcriptome and miRNA sequencing analyses reveal the regulatory mechanism of alpha-linolenic acid biosynthesis in Paeonia rockii.
      ). Endogenous capacity of LCPUFA synthesis was limited in mammals because of the lack of ωx desaturases and elongases, and dietary intake of LCPUFA was crucial to satisfy essential requirements (
      • Levental K.R.
      • Surma M.A.
      • Skinkle A.D.
      • Lorent J.H.
      • Zhou Y.
      • Klose C.
      • J. T.Chang J.F.
      • Hancock
      • Levental I.
      Omega-3 polyunsaturated fatty acids direct differentiation of the membrane phenotype in mesenchymal stem cells to potentiate osteogenesis.
      ). Nutrient deficiency potentially induced the perturbation of gene transcription and cellular signaling processes. Compared with the control group, the concentration of glyceride-type polyunsaturated fatty acids was significantly (P < 0.05) decreased from 143.818 ± 0.51 mg/kg to 104.613 ± 0.29 mg/kg driven by benzoic acid. Benzoic acid facilitated attenuation of perilipin associated with glycerolipid metabolism resulting in triglycerides degradation (Figure 6). In adipocytes, 3 neutral lipases were involved in triglycerides breakdown. Adipose triglyceride lipase (ATGL; encoded by PNPLA2) was the main enzyme responsible for triglycerides hydrolysis to diglycerides (
      • Morigny P.
      • Boucher J.
      • Arner P.
      • Langin D.
      Lipid and glucose metabolism in white adipocytes: Pathways, dysfunction and therapeutics.
      ). The second enzyme, hormone-sensitive lipase (HSL; encoded by LIPE), hydrolyzed diglycerides, although it also displayed triglycerides hydrolysis activity. The third enzyme, monoglyceride lipase, catalyzed the hydrolysis of monoglyceride to glycerol and a fatty acid (
      • Kintscher U.
      • Foryst-Ludwig A.
      • Haemmerle G.
      • Zechner R.
      The role of adipose triglyceride lipase and cytosolic lipolysis in cardiac function and heart failure.
      ). The ATGL was located on lipid droplets and CGI58 (encoded by ABHD5) was essential for the activation of full ATGL in stimulated lipolytic states. Perilipin was a major protein of the adipocyte lipid droplet (
      • Kulminskaya N.
      • Oberer M.
      Protein-protein interactions regulate the activity of adipose triglyceride lipase in intracellular lipolysis.
      ). The perilipin protein molecule has 2 states: nonphosphorylated and phosphorylated, which are regulated by PKA (
      • Sletten A.
      • Seline A.
      • Rudd A.
      • Logsdon M.
      • Listenberger L.L.
      Surface features of the lipid droplet mediate perilipin 2 localization.
      ). In basal conditions, perilipin as a physiological barrier shield protects stored triacylglycerols from cytosolic lipases, thus preventing the hydrolysis of triglycerides by lipase (
      • Sztalryd C.
      • Brasaemle D.L.
      The perilipin family of lipid droplet proteins: Gatekeepers of intracellular lipolysis.
      ). As shown in Table 4, the expression of perilipin decreased significantly (P < 0.05). Furthermore, lipolysis was inhibited through activation of cAMP degrading enzyme phosphodiesterase 3B (PDE3B). The inhibition of cAMP synthesis was mediated by the activation of G protein-coupled receptors (GPCR) coupled to Gαi (
      • Brasaemle D.L.
      • Subramanian V.
      • Garcia A.
      • Marcinkiewicz A.
      • Rothenberg A.
      Perilipin A and the control of triacylglycerol metabolism.
      ). Benzoic acid attenuated perilipin expression and facilitated glyceride-type polyunsaturated fatty acids degradation.
      Figure thumbnail gr6
      Figure 6Pathways of triacylglycerol biosynthesis. Perilipin, which is coated on the surface of lipid droplets, protects triacylglycerols from cytosolic lipases, thus preventing triglycerides hydrolysis. Furthermore, lipolysis was inhibited through activation of cAMP degrading enzyme phosphodiesterase 3B (PDE3B). The inhibition of cAMP synthesis was mediated by the activation of G protein-coupled receptors (GPCR) coupled to Gαi. DG = diglycerides; MG = monoglycerides; TG = triglycerides.

      Mitigation Strategies to Regulate Benzoic Acid Residue

      An optimal fermentation method of goat milk to regulate benzoic acid formation was investigated. The amount of naturally forming benzoic acid was influenced by fermented temperature and incubation time. Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus salivarius ssp. thermophilus were inoculated into goat milk and incubated at 37, 40, and 42°C for 12 h at 2-h intervals. As shown in Figure 7A, the content of benzoic acid constantly increased at a constant temperature from 2 to 12 h. Benzoic acid accumulated as increasing temperatures within the same time and the production reached 34.88 mg/kg (42°C, 12 h). For example, the concentration of TG (16:0/18:1/18:2; Figure 7B), showed an upward trend from 12.25 mg/kg (42°C, 12 h) to 37.09 mg/kg (37°C, 12 h). The concentration of TG (16:0/18:1/18:2) was higher in fermented goat milk with 37°C fermentation after 8 h incubation than 12 h, achieving a value of 22.13 mg/kg. Fermentation of goat milk at a low temperature and less incubation time leads to the production of less benzoic acid and mitigation of lipid nutrients loss.
      Figure thumbnail gr7
      Figure 7(A) The amount of benzoic acid in fermented goat milk incubated at 37, 40, and 42°C for 12 h at 2-h intervals. (B) The concentration of triglycerides (TG; 16:0/18:1/18:2) in fermented goat milk incubated at 37, 40, and 42°C for 12 h at 2-h intervals. Three samples were collected from each site, and values are the mean ± SD. Lowercase letters (a–e) indicate a significant difference (P < 0.05).
      The findings of this study identified numerous lipids and proteins alterations induced by benzoic acid in fermented goat milk. A possible relationship between benzoic acid accumulation and glycerolipid metabolism was revealed. Perilipin coated on the surface of lipid droplets protects triacylglycerols from cytosolic lipases, thus preventing triglyceride hydrolysis by lipase (
      • Sztalryd C.
      • Brasaemle D.L.
      The perilipin family of lipid droplet proteins: Gatekeepers of intracellular lipolysis.
      ). Benzoic acid accumulation decreased the expression of perilipin and facilitated the degradation of glyceride-type polyunsaturated fatty acids. In addition, the formation of benzoic acid with different fermentation temperatures (37, 40, and 42°C) and incubation time (8, 10, and 12 h) was evaluated. The results demonstrated that 37°C fermentation for 8 h most likely contributes more to the mitigation of lipid nutrients loss. This study not only investigated the effect of naturally forming benzoic acid on lipids and proteins variations in fermented goat milk but also found a potential relationship between benzoic acid and glycerolipid metabolism. The findings of this study provide a benchmark for the food industry to select fermentation conditions and provide the rationale for regulating benzoic acid residue and mitigating lipid nutrients loss.

      CONCLUSIONS

      This study investigated the dynamic changes of lipids and proteins in fermented goat milk with different concentrations of benzoic acid accumulation (0.0 to 40.0 mg/kg). A total of 189 lipid molecules, including 137 glycerolipids, 18 glycerophospholipids, and 34 sphingolipids were structurally identified, and 39 differentially expressed proteins (27 upregulated and 12 downregulated) were mostly focused on glycerophospholipid metabolism and triglyceride degradation. Benzoic acid facilitated attenuation of perilipin associated with glycerolipid metabolism, resulting in glyceride-type polyunsaturated fatty acids degradation. Fermentation of goat milk at a low temperature and less incubation time leads to the production of less benzoic acid and mitigation of lipid nutrients loss. These preliminary findings deciphered mechanisms of lipid nutrients loss induced by benzoic acid accumulation in fermented goat milk and provided a theoretical foundation for developing mitigation strategies to regulate benzoic acid residue.

      ACKNOWLEDGMENTS

      The research was financially supported by the National Natural Science Foundation of China (no. 32272401; Beijing), Xianyang Science and Technology Plan Project (no. 2021ZDYF-NY-0025; Xianyang, China), Xi'an Science and Technology Plan Project (no. 21NYYF0056; Xi'an, China), the Department of Science and Technology of Shaanxi Province (no. 2022NY-004; Xi'an, China), and the Innovation Capability Support Program of Shaanxi (no. 2021KJXX-37; Xi'an, China). The authors have not stated any conflicts of interest.

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