The antioxidant activity and metabolomic analysis of the supernatant of Streptococcus alactolyticus strain FGM

Strain-specific probiotics can present antioxidant activity and reduce damage caused by oxidation. Streptococcus alactolyticus strain FGM (S. alactolyticus strain FGM) isolated from the chicken cecum shows potential probiotic properties which have been previously demonstrated. However, the antioxidant properties of S. alactolyticus strain FGM remain unknown. In this view, cell-free supernatant (CFS), intact cells (IC) and intracellular extracts (CFE) of strain FGM and 3 strains of Lactobacillus (LAB) were prepared, and their scavenging capacities against DPPH, hydroxyl radicals and linoleic acid peroxidation inhibitory were compared in this study. The effects of strain FGM cell-free supernatant (FCFS) on NO production, activity of SOD and GSH-Px in RAW264.7 cells and LPS-induced RAW264.7 cells were analyzed. The metabolites in the supernatant were quantitated by N300 Quantitative Metabolome. It was shown that the physicochemical characteristics of CFS to scavenge DPPH, hydroxyl radicals, and linoleic acid peroxidation inhibitory were significantly stronger than that of IC and CFE in the strain FGM (P < 0.05), respectively 87.12% ± 1.62, 45.03% ± 1.27, 15.63% ± 1.34. FCFS had a promotional effect on RAW264.7 cells, and significantly elevated SOD and GSH-Px activities in RAW264.7 cells. 25 μL FCFS significantly promoted the proliferation of RAW264.7 cells induced by LPS, increased the activities of SOD and GSH-PX, and decreased the release of NO. Furthermore, among the differential metabolites of FCFS quantified by N300, 12 metabolites were significantly up-regulated, including lactic acid, indole lactic acid, linoleic acid, pyruvic acid etc., many of which are known with antioxidant properties. In conclusion, FCFS had good antioxidant properties and activity, which can be attributed to metabolites produced from strain FGM fermentation. It was further confirmed that S. alactolyticus strain FGM and its postbiotic have potential probiotic properties and bright application prospects in livestock and poultry breeding.


Strains and cells
S. alactolyticus strain FGM (GenBank accession No. JX435470; China Patent No. 20120141827.5)was derived from the cecum of indigenous chickens, isolated and preserved in the Veterinary Laboratory of Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS.RAW264.7 macrophage cells were obtained from Sunlight Bio-technology (Shanghai, China).The L. acidophilus (BNCC, China, No. 185342), L. reuteri (BNCC, China, No. 192190) and L. casei (BNCC, China, No.134415) were purchased from North Natron link Biotechnology Ltd.

Strains resuscitation and bacteria fractions preparation
S. alactolyticus strain FGM, L. acidophilus, L. reuteri and L. casei stored at − 80 °C were thawed at 37 °C water bath, inoculated into MRS (Huankai, China) nutrient broth and incubated at 37 °C for 24 h.They were then cultured to a second or third passage for subsequent experiments.
Bacterial fractions were prepared according to the method described in the literature 25 .Briefly the bacterial concentration was adjusted to 1 × 10 8 cfu/mL, the supernatant was obtained by centrifugation (4500×g for 10 min at 4 ℃).After passing through a sterile 0.22 μm pore-size filter unit, a CFS was obtained.The cell pellet was washed three times with 0.01 mmol/L phosphate buffer saline (PBS, pH 7.4) and resuspended in PBS.The cell pellet was adjusted to 1 × 10 8 cfu/mL to obtain IC.CFE were obtained from the cell suspensions containing 1 × 10 8 cfu/mL that were subjected to ultrasonic disruption (in ice cold water, ten 5-s strokes and 5-s intervals, 40 min) and centrifuged (12,000×g for 10 min at 4 °C) to remove the cell debris.

DPPH radical scavenging activity (RSA)
The DPPH RSA of different bacteria fractions was assessed using the method referenced in Lin et al. 26 .Briefly, 500 μL of each sample was mixed with 500 μL of DPPH solution and incubated in the dark for 30 min within a 1.5 mL centrifuge tube.For the blank, 500 µL of absolute ethanol was used, while 500 µL of MRS broth or PBS served as controls.After the incubation period, the absorbance of the mixture was measured at 517 nm and DPPH RSA is expressed as follows.
www.nature.com/scientificreports/Hydroxyl RSA The hydroxyl RSA was measured according to the method described by Zhang 27 .The reaction mixture was prepared by combining the following components in a test tube: 1 mL PBS, 0.5 mL of 1,10-phenanthroline ethanol solution (Solarbio, China), 0.5 mL of FeSO 4 , 0.5 mL of the bacterial fraction being tested, 0.5 mL of H 2 O 2 .This mixture was incubated at 37 °C for 90 min and measured at 536 nm.The data was expressed as a blank.0.5 mL H 2 O 2 was replaced with 0.5 mL distilled water, and the data was expressed as controls, using 0.5 mL distilled water was replaced with 0.5 mL sample, and the data was expressed as sample.The percentage of resistance to hydroxyl radicals was defined as follows.
Linoleic acid peroxidation inhibitory activity 1 mL Linoleic acid (Unsaturated fatty acids) which was prepared of 0.1 mL linoleic acid, 0.2 mL Tween 20 (Solarbio, China) and 19.7 mL deionized water.0.5 mL PBS, 0.2 mL FeSO 4 , 0.2 mL H 2 O 2 and 0.5 mL of different bacterial fractions were mixed and incubated at 37 °C for 12 h.Blank samples contained either PBS or MRS. 2 mL of the reaction solution was mixed with 0.2 mL TCA (Solarbio, China), 2 mL TBA (Solarbio, China) and 0.2 mL BHT (Solarbio, China), and incubated at 100 °C for 30 min before added 2 mL chloroform.The absorbance value of the extracts was measured at 532 nm 26 .The formula is as follows.

Cell viability assay
The CCK-8 assay (ZETA, USA) was used to evaluate the effect of the FCFS on the growth of RAW264.7 cells.Initially, RAW264.7 cells were seeded into 96-well plates and were cultured for 12 h.Subsequently, the medium was refreshed, and different concentrations of FCFS working solutions were added to the cells for another 12 h.0.1 μg/mL LPS as a positive control, while RPMI-1640 complete medium (CCM) as a negative control.After discarding the culture supernatant, each well was incubated with CCM containing 10% CCK-8 at 37 ℃ in the dark for 1 h to 1.5 h.The OD values in each well were measured at 450 nm using a microplate reader.

NO production
The macrophages were seeded into a 6-well plate (1 × 10 6 cfu/mL) for at least 12 h.Subsequently, 1 mL CCM along with various concentrations of the different FCFS were added to each well and the plate was incubated for 24 h.Following incubation, the culture medium at 2000 rpm for 20 min to collected supernatants, and cells supernatant were analyzed with the Griess kit (ThermoFisher, USA) according to the manufacturer's instructions.The NO levels were calculated from the absorbance at 540 nm measured by a microplate reader.

ROS activity
The levels of intracellular oxidative stress were determined using ROS assay kit (Beyotime, China).Briefly, RAW264.7 cells pretreated with FCFS were collected, resuspended in freshly prepared serum-free medium containing 10 μmol/L DCFH-DA and incubated at 37 °C in the dark for 20 min.The cells were then harvested and washed with PBS buffer.The absorbance value was measured the using a fluorescence microplate reader at 488 nm excitation wavelength and 525 nm emission wavelength.
SOD and GSH-Px activity RAW264.7 cells were centrifuged and resuspended to achieve a concentration of 1 × 10 6 cfu/mL, then cultured in 6-well plate for 12 h.Subsequently, 200 μL cell lysis solution was added to each well, which was repeatedly blown to make the cells shed, lyse, and release intracellular proteins, and were centrifuged at 12,000×g for 5 min.The resulting supernatant was collected for the assessment of SOD (Beyotime, China) activity and GSH-Px (Beyotime, China) activity.

The effects of FCFS in LPS induced RAW264.7 cells
Cell viability assay RAW264.7 cells (5 × 10 5 cfu/mL) were seeded into 96-well plates and cultured for 12 h.The cells were then treated with 25 μL and 125 μL FCFS and the plates were incubated for additional 12 h.After incubation, 100 μL LPS was added for 24 h, and following two washes with PBS, 100 μL CCM was added, and 10 μL CCK-8 reagent was added to each well.After further incubation 1.5 h, the absorbance was measured at a wavelength of 450 nm to determine cell viability.

NO production
The release of NO from RAW264.7 cells was measured by the quantifying the stable end product of NO oxidation with the Griess reagent, according to a previous report 28 .The cells were pretreated with 25 μL and 125 μL FCFS and the plates incubated for 12 h.Then 0.5 µg/mL LPS was added to stimulate the cells for 24 h.The supernatant was collected and centrifuged at 2000 rpm for 20 min to remove any cellular debris.Each well was added 80 µL supernatant and 80 µL of both Griess reagent I and II.The absorbance was determined at 540 nm using a microplate reader (Supplementary Information).
Hydroxyl RSA (%) = A Sample − A Blank /(AControl − A Blank) × 100%.www.nature.com/scientificreports/SOD and GSH-Px activity RAW264.7 cells were treated with LPS and then harvested for the analysis of antioxidant enzymes.SOD activity and GSH-Px activities were measured using commercial assay kits and the absorbance was measured at 450 nm with a microplate reader, and the corresponding activity was calculated by a formula.

Metabolomics analysis of FCFS
The metabolomics analysis of FCFS was carried out using liquid-chromatography-mass spectrometry (LC-MS) technology, specifically on the ACQUITY UPLC Xevo TQ-S platform, to investigate the metabolomic profiles and their biological functions (data from three groups, 5 biological repeats for each) 29,30 .The MS detection process involved the analysis of blank samples (MRS), quality control samples (QC), and experimental samples.To achieve absolute quantitative results for the metabolites present in the samples, chromatographic data analysis was performed using MassLynx V4.1 software.Subsequent multivariate statistical analyses of the metabolites, including principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), were conducted to identify differences in metabolic patterns of different groups.Volcano map and KEGG pathway database were employed to annotate the distinct metabolites and to highlight the pathways that exhibited differential metabolite enrichment.This metabolomics study was performed in the laboratory of Beijing Novozymes Technology Co.

Statistical analysis
Data were expressed as mean ± SD, One-way ANOVA determined differences between groups with the SPSS 26.0 software and GraphPad Prism 6.0.For all the tests, a two-sided P-value of < 0.05 was considered significant.

DPPH RSA
DPPH RSA has been attributed to the hydrogen donating ability of antioxidants and is commonly employed in antioxidant assays.IC, CFS and CFE from S. alactolyticus strain FGM, L. acidophilus, L. reuteri and L. casei were prepared.The DPPH RSA of different probiotic components is shown in Table 1.The DPPH RSA of VC was 96.87% ± 1.09.The DPPH RSA of CFS, IC and CFE in the strain FGM was 87.12% ± 1.62, 31.05% ± 8.15 and 24.5% ± 8.14, respectively.Notably, the DPPH RSA of CFS in the strain FGM showed no significant differences compared to that of L. acidophilus, L. reuteri and L. casei (P > 0.05).However, the DPPH RSA of IC in the strain FGM was significantly higher than that in L. casei and L. acidophilus.On the other hand, the DPPH RSA of CFE in the strain FGM was notably lower than that in L. acidophilus and L. reuteri, yet significantly higher than L. casei (P < 0.05).

Hydroxyl RSA
Hydroxyl radicals are most reactive entities among the ROS and are known to cause DNA damage and lipid peroxidation 31 .As shown in www.nature.com/scientificreports/ was no difference difference when compared with L. reuteri, and L. casei (P > 0.05).Regarding the hydrolxyl RSA of IC, strain FGM was significantly higher than that of L. reuteri (P < 0.05), noticeably lower than that of L. casei (P < 0.05), and showed no significant difference from L. acidophilus (P > 0.05).For the hydroxyl RSA of CFE, strain FGM was significantly lower than that of L. casei (P < 0.05), but here was no significant difference when compared with L. acidophilus and L. reuteri (P > 0.05).

Linoleic acid peroxidation inhibitory activity
Linoleic acid from natural sources has been reported to inhibit lipid peroxidation 32 .As shown in Table 3, the inhibitory activity of the S. alactolyticus FGM strain from CFS, IC and CFE was 15.63% ± 1.34, 5.66% ± 1.89 and 14.47% ± 1.09, respectively.Interestingly, the FCFS exhibited the highest inhibitory activity on Linoleic acid peroxidation and was significantly higher than that of L. casei and L. reuteri (P < 0.05), but there was no significant difference when compared with L. acidophilus (P > 0.05).For the IC form all four strains, there was no significant difference (P > 0.05).Comparing with the CFE of the strains, the FGM strain showed an activity that was significantly higher than L. casei and L. acidophilus (P < 0.05), but there was no significant difference with L. reuteri (P > 0.05).

Cell viability assay
The result of the proliferative capacity of RAW264.7 cells in the FCFS groups and the LPS group are showed in Fig. 1.As the dose of FCFS increased, the cell proliferation rate showed an ascending trend from 5 to 50 μL, but then decreased at 125 μL.The range of 25 μL to 125 μL FCFS, as well as the LPS treatment, significantly promoted cell proliferation in RAW264.7 cells (P < 0.05).

NO production
NO produced during host inflammatory responses is involved in various disorders.The result of No production in RAW264.7 cells stimulated with doses ranging from 5 to 125 μL of FCFS and with 100 μL of LPS are shown in Fig. 2. A clear positive correlation observed between the FCFS dose and cellular NO production, with the group receiving 125 μL FCFS exhibiting significantly higher levels than the control group (P < 0.05).Furthermore, the LPS group showed an exceptionally potent effect in inducing NO production compared to the FCFS groups (P < 0.01).

ROS activity
Excessive ROS can induce oxidative stress and inflammatory responses, serving as one of important indicators of cellular damaged.As shown in Fig. 3, when compared to the control group, the doses of 5 μL, 25 μL, 50 μL, and 125 μL of FCFS did not exhibit a significant effect on the ROS levels in RAW264.7 cells (P > 0.05).However, ROS levels in the FCFS groups were notably lower than those observed in the LPS group (P < 0.05).

SOD and GSH-Px activity
SOD and GSH-Px are both crucial antioxidant enzymes within cells.As presented in Fig. 4, doses of 25 μL and 50 μL of FCFS significantly increased SOD activity compared to the control group (P < 0.05).SOD activity showed an ascending trend with increasing FCFS doses from 5 to 50 μL, followed by a decrease at 125 μL.In contrast, SOD activity in the LPS group was significantly lower compared to control group (P < 0.05).Regarding the GSH-Px activity, 25 μL of FCFS dose was significantly higher than the control group (P < 0.05).However, 50 μL and 125 μL of FCFS doses resulted in significantly lower than the control group (P < 0.05), showing a similar effect with LPS group.

The effects of FCFS in LPS induced RAW264.7 cells
Cell viability assay A cell viability assay was conducted to evaluate the effects of fermented cell-free supernatant (FCFS) on RAW264.7 cells in an LPS-induced oxidative stress model.As depicted in Fig. 5. a dose of 25 μL FCFS significantly promoted cell proliferation in LPS-induced RAW264.7 cells compared to compared to both the 0.5 μg/mL LPS group and CON group (P < 0.05).However, there was no difference with 125 μL CFS (P > 0.05).

NO production
A previous study reported that different strains demonstrate distinct inhibitory effects on LPS-induced NO synthesis 33 .As shown in Fig. 6, both 25 µL and 125 µL doses of FCFS significantly reduced the concentration of NO in LPS-induced RAW264.7 cells compared to 0.5 μg/mL LPS group (P < 0.05).Notably, NO concentration in the group treated with 125 μL FCFS was significantly lower than that in the 25 μL group (P < 0.05).

SOD, and GSH-Px activity
The oxidative indicators including SOD and GSH-Px were assessed to evaluate the anti-oxidative property of FCFS on oxidative stress in LPS-induced RAW264.7 cells.As shown in Fig. 7, treatment with 25 μL FCFS significantly increased the activities of SOD and GSH-Px in LPS-induced RAW264.7 cells compared to 0.5 μg/mL LPS group (P < 0.05).However, the activities of SOD and GSH-Px in the group treated with 125 μL FCFS were significantly lower than those in the 25 μL FCFS group (P < 0.05).4 and 5.As shown in Fig. 8, the relative content of these metabolites is represented by variations in color intensity on the Heat map.The trend in the changes of differential metabolites can   www.nature.com/scientificreports/be broadly categorized into two zones.In the red zone of the upper half corresponding to the FCFS sample group, the metabolites are predominantly Phenols and Indoles, including 2 Phenylpropanoic Acids, 1 Fatty Acids, 3 Carbohydrates, and 4 Organic Acids, all of which are significantly upregulation.Conversely, the red zone in the lower half highlights the differential metabolites mainly found in the MRS sample group.

PCA and OPLS-DA analysis
In this study, the FCFS group and MRS groups were selected as the research subjects, with the differential metabolites between them analyzed using LC-MC.PCA analysis and OPLS-DA were employed to identify metabolites that contribute to the main differences between the two groups.As indicated in Fig. 9, the first principal component (PC1) was 82% of the variance, while the second principal component (PC2) was 9.89%.The Fig. 9 demonstrates a clear separation trend between the samples of FCFS and MRS Groups, with a cumulative contribution rate of 91.89% from the principal components.This suggests significant differences in metabolic profiles between FCFS and MRS groups.The OPLS-DA model was utilized to further investigate the metabolite differences.Figure 9 shows that the FCFS and MRS groups are completely separated in the model, R2Y represents the interpretation rate of the model, Q2Y is used to evaluate the predictive ability of the PLS-DA model, and the results show that R2Y is 1 and Q2Y is 0.91.R2Y > Q2Y and all close to 1, indicated that the model was well established and had good accuracy, and the differential metabolites could be screened by variable projection importance (VIP) method.

Volcano map and KEGG pathway bubble map analysis
The volcano plot, as depicted in Fig. 10, visualizes the differential metabolites where red dots indicate significantly up-regulated substances, green dots indicate significantly down-regulated substances, and grey dots represent substances that are not significantly different between the two groups.The five most prominent metabolites

Discussion
Probiotic LAB have been demonstrated to exert positive effects on the anti-oxidative 10 , anti-inflammatory 34 , and anticancer activities 35 .The antioxidant capabilities of probiotics have been evaluated through IC 26 , CFS 10 and CFE or their metabolites in vitro 36 .Previous research has indicated that strains such as L. acidophilus 32 , L. casei 37 and L. reuteri 38 , possess antioxidative properties.Both IC and CFE of S. thermophilus and Bifidobacterium longum 32 , L. casei 39 have demonstrated the capacity to scavengers hydroxyl radicals and inhibit linoleic acid peroxidation.Similarly, the CFS of L. rhamnosus has shown DPPH RSA, which is in line with our findings and recent research indicating.In our study, we assessed the antioxidant properties of the four LAB samples in vitro.We found that the CFS, IC and CFE all exhibited antioxidant capabilities, but the antioxidant properties of the four strains of LAB were found to vary.FCFS showed superior antioxidant activity compared to IC and CFE of the strain FGM, with higher DPPH RSA, hydroxyl RSA and linoleic acid peroxidation inhibitory activity.Overall, compared with other strains, FCFS had the best Linoleic acid peroxidation inhibitory activity, DPPH RSA was higher than that of L. acidophilus and L. reuteri, and Hydroxyl RSA was higher than that of L. casei.
Macrophages play an important role not only in immune responses but also in phagocytosis and tissue repair during damage or transitional inflammatory responses caused by microbial infections 40 .Studies have shown that LPS stimulates macrophages to induce oxidative stress by activating inducible nitric oxide synthase (iNOS).Vitamin C may protect macrophages from LPS-induced oxidative stress by maintaining iNOS activity by increasing the stability of tetrahydrobiopterin 41 .In this study, 5 μL, 25 μL, 50 μL and 125μL FCFS was applied to RAW264.7 cells.It was found that the FCFS promoted cell proliferation and there was no significant effect of each dose on ROS levels in RAW 264.7 cells, indicating that FCFS did not cause intracellular ROS disturbances.However, there was a dose-dependent relationship between the NO production in RAW 264.7 cells and FCFS.Studies have shown that NO is lipid-soluble, it can affect the production of several cytokines and enhance the function of the immune system 42 .SOD and GSH-Px are considered the main antioxidant enzymes for the elimination of ROS in vivo 43 .It was also found that 25 μL and 50 μL FCFS could increase the SOD activity of RAW264.7 cells, but only 25μL FCFS had a significant effect on the GSH-Px activity.When the concentration of FCFS was increased to 125 μL, SOD and GSH-Px activities in RAW 264.7 cells were reduced.Therefore, 25 μL and 125 μL FCFS were used for subsequent experiments.In this study, 0.5 μg/mL LPS treatment at 37 ℃ for 24 h successfully induced a significant increase in cell viability in RAW264.7 cells.They were treated with 25 μL and 125 μL FCFS for 12 h in RAW264.7 cells and then stimulated with LPS for 24 h to evaluate the preventive and protective effects of FCFS on oxidative stress in RAW 264.7 cells.The results showed that 25 μL FCFS could significantly relieve the stress on SOD, GSH-Px activities and NO content in LPS induced RAW 264.7 cells.
To further investigate the potential mechanisms of the antioxidant properties of the strain FGM, we examined the expression profiles of metabolite levels in the FCFS, performing correlation analyses to better understand specific metabolites.Due to the differences in metabolites, and the fact that antioxidant active components occurring differently in each strain, the metabolome has become an effective tool for analyzing metabolite accumulation patterns and has been used for nutritional analysis of many substances 44 .In the quantitative N300 assay performed on the FCFS, a total of 156 metabolites were detected, and 46 metabolites (12 up-regulated and 34 down-regulated) among them were significantly different.Further, the 12 up-regulated metabolites were tartaric acid, phenyl lactic acid, lactic acid, indole lactic acid, linoleic acid, pyruvic acid, maleic acid, homovanillic acid, xylose, fumaric acid, glyceraldehyde, hydroxyphenyl lactic acid which had been produced during the fermentation of the S. alactolyticus strain FGM.Among them, lactic acid, fumaric acid, indole lactic acid, linoleic acid and pyruvic acid (FC value > 4) were the main products.Most of the up-regulated metabolites have been reported with antioxidant properties, such as tartaric acid 45 , phenyl lactic acid 46 , lactic acid 47 , linoleic acid 48 , pyruvic acid 49 , maleic acid 50 , homovanillic acid 37 , xylose 51 , fumaric acid 52 , glyceraldehyde 53 , hydroxyphenyl lactic acid 54 .

Conclusion
In the present study, the CFS, IC and CFE from S. alactolyticus strain FGM were all capable of DPPH RSA, hydroxyl RSA and inhibiting linoleic acid peroxidation.Among these, the FCFS showed then strongest scavenging ability.Beyond its antioxidant physicochemical properties, FCFS was also showed antioxidant activity through the enhancement of SOD and GSH-Px activities, as well as a regulatory effect on NO content in LPS induced RAW 264.7 cells.The antioxidant activity exhibited by FCFS were possibly related to tartaric acid, phenyl lactic acid, lactic acid, linoleic acid, pyruvic acid, maleic acid, homovanillic acid, xylose, fumaric acid, glyceraldehyde and hydroxyphenyl lactic acid, which are secreted by S. alactolyticus strain FGM.6][57] , the circles represent metabolites, in which the green solid circles are labeled as annotated metabolites, the red circles are up-regulated differential metabolites, the blue circles are down-regulated differential metabolites, and the yellow circles contain both up-regulated and down-regulated differential metabolites.

Figure 1 .
Figure 1.Effects of different concentrations of FCFS on the proliferation of RAW264.7 cells.Effects of different concentrations of FCFS on the proliferation of RAW264.7 cells.In the figure, groups that share the same lowercase letter are not significantly different (P > 0.05), whereas groups with different letters are significantly different (P < 0.05).

Figure 2 .
Figure 2. Effects of different concentrations of FCFS on production of NO of RAW264.7 cells.In the figure, groups that share the same lowercase letter are not significantly different (P > 0.05), whereas groups with different letters are significantly different (P < 0.05).

Figure 3 .
Figure 3.The effects of different concentrations of FCFS on ROS level of RAW264.7 cells.In the figure, groups that share the same lowercase letter are not significantly different (P > 0.05), whereas groups with different letters are significantly different (P < 0.05).

Figure 4 .
Figure 4.The effects of different concentrations of FCFS on SOD (a) and GSH-Px activity of RAW264.7 cells (b).In the figure, groups that share the same lowercase letter are not significantly different (P > 0.05), whereas groups with different letters are significantly different (P < 0.05).

Figure 5 .
Figure 5. Effects of different concentrations of FCFS on the proliferation of RAW264.7 cells induced by LPS.In the figure, groups that share the same lowercase letter are not significantly different (P > 0.05), whereas groups with different letters are significantly different (P < 0.05).

Figure 6 .
Figure 6.Effects of different concentrations of FCFS on production of NO of RAW264.7 cells induced by LPS.In the figure, groups that share the same lowercase letter are not significantly different (P > 0.05), whereas groups with different letters are significantly different (P < 0.05).

Figure 7 .
Figure 7.The effects of different concentrations of FCFS on SOD (a) and GSH-Px activity of RAW264.7 cells induced by LPS (b).In the figure, groups that share the same lowercase letter are not significantly different (P > 0.05), whereas groups with different letters are significantly different (P < 0.05).

Figure 8 .
Figure 8.Heat map of FCFS and MRS differential metabolites, the heatmap was created in "clusterProfler" R software package, version 3.4.3.The longitudinal is the clustering of FCFS and MRS, and the transverse is the clustering of metabolites, with shorter cluster branches representing higher similarity.

Figure 9 .
Figure 9. PCA plots (a) and OPLS-DA plots (b) of FCFS and MRS Differential metabolites.The abscissa PC1 and ordinate PC2 in the PCA plot represent the scores of the principal components at levels 1 and 2, respectively.The abscissa in the OPLS-DA plot is the score of the sample on the first principal component.The ordinate is the score of the sample on the second principal component.R2Y represents the explanatory rate of the model, and Q2Y is used to evaluate the predictive ability of the PLS-DA model.

Figure 10 .
Figure 10.Volcano plot of FCFS and MRS Differential metabolites.The horizontal axis represents the fold change of the metabolites in different groups (log2FC), and the vertical axis represents the significance level of the difference (− log10(P-value)).

Figure 11 .
Figure 11.Bubble map of the effects of KEGG metabolic pathways in FCFS and MRS.In the KEGG pathway map[55][56][57] , the circles represent metabolites, in which the green solid circles are labeled as annotated metabolites, the red circles are up-regulated differential metabolites, the blue circles are down-regulated differential metabolites, and the yellow circles contain both up-regulated and down-regulated differential metabolites. 100%.

Table 2
, the hydroxyl RSA of VC was52.22%±1.39.The hydroxyl RSA of CFS, IC, and CFE in the strain S. alactolyticus FGM was 45.03% ± 1.27, 34.14% ± 2.05 and 8.48% ± 5.16, respectively.Compared with L. acidophilus, strain FGM exhibited a significantly lower level of hydroxyl RSA in CFS (P < 0.05), but there Table1.Total DPPH radical scavenging activity assays.In each column, values followed by different letters are statistically different according to the ANOVA test with the same small letter mean no significant difference (P > 0.05), while with different small letters means significant difference (P < 0.05).The same as below.

Table 3 .
Total Linoleic acid peroxidation inhibitory activity assays.

Table 5 .
Metabolites with significant differences in FCFS.