Sargassum horneri extract fermented by Lactiplantibacillus pentosus SH803 mediates adipocyte metabolism in 3T3-L1 preadipocytes by regulating oxidative damage and inflammation

Sargassum horneri (S. horneri), a brown seaweed excessively proliferating along Asian coastlines, are damaging marine ecosystems. Thus, this study aimed to enhance nutritional value of S. horneri through lactic acid bacteria fermentation to increase S. horneri utilization as a functional food supplement, and consequently resolve coastal S. horneri accumulation. S. horneri supplemented fermentation was most effective with Lactiplantibacillus pentosus SH803, thus this product (F-SHWE) was used for further in vitro studies. F-SHWE normalized expressions of oxidative stress related genes NF-κB, p53, BAX, cytochrome C, caspase 9, and caspase 3, while non-fermented S. horneri (SHWE) did not, in a H2O2-induced HT-29 cell model. Moreover, in an LPS-induced HT-29 cell model, F-SHWE repaired expressions of inflammation marker genes ZO1, IL1β, IFNγ more effectively than SHWE. For further functional assessment, F-SHWE was also treated in 3T3-L1 adipocytes. As a result, F-SHWE decreased lipid accumulation, along with gene expression of adipogenesis markers PPARγ, C/EBPα, C/EBPβ, aP2, and Lpl; lipogenesis markers Lep, Akt, SREBP1, Acc, Fas; inflammation markers IFN-γ and NF-κB. Notably, gene expression of C/EBPβ, IFN-γ and NF-κB were suppressed only by F-SHWE, suggesting the enhancing effect of fermentation on obesity-related properties. Compositional analysis attributed the protective effects of F-SHWE to acetate, an organic acid significantly higher in F-SHWE than SHWE. Therefore, F-SHWE is a novel potential anti-obesity agent, providing a strategy to reduce excess S. horneri populations along marine ecosystems.

a food additive instead of a standalone food ingredient are in progress.In a previous report, supplementation of S. horneri enhanced lactic acid bacteria (LAB) growth, revealing its potential as a prebiotic supplement 1 .This suggests that fermentation could be utilized to enhance S. horneri biological functions through production of secondary metabolites.LAB are gram-positive, lactic acid-producing organisms, in which possess numerous health benefits.Major strains include probiotics-live microorganisms that confer health benefits to the host when administered in adequate amounts 5 .Among probiotics, strains from the Lactobacillaceae and Bifidobacteriaceae families are involved in fermentation; they regulate microbial growth and the enzymatic conversion of food constituents 6,7 , which in turn produces beneficial end-products, such as organic acids, bacteriocins, and peptides 8 .In a previous report, S. horneri fermentation with Lactiplantibacillus pentosus SN001 increased glycerol levels, in which elevated antihypertensive effects 9 .Therefore, fermentation is a prominent tool for enhancing the value of biomaterials.
Obesity, the state of excess fat accumulation, is a global crisis affecting more than 1 billion people, including 650 million adults, 340 million adolescents, and 39 million children 10 .Obesity-inducing energy overconsumption contributes to symptoms such as adipogenesis, increased fat cell numbers, and adipocyte hypertrophy 11 .Adipogenesis, or adipocyte hyperplasia, is the process of adipocyte precursor cells committing their fate to the adipogenic lineage, gathering nutrients, and transforming into triglyceride-filled mature adipocytes 11 .This determines the lipid storage capacity of adipose tissue; therefore, it is a potential therapeutic target for treating obesity.Furthermore, obesity-associated fat accumulation induces inflammation and hypoxia, which increases oxidative stress 12 .Oxidative stress in turn exacerbates inflammation, leading to obesity-related complications, such as type 2 diabetes, insulin resistance, and infertility 13 .S. horneri and LAB possess antioxidative and antiinflammatory properties, which suggests their potential in attenuating obesity [14][15][16] .Moreover, in previous reports, the antioxidative activities of Sargassum spp.were enhanced by LAB fermentation 10,17 .Therefore, this study aimed to determine the anti-obesity potential of S. horneri and LAB combined through fermentation in vitro.Our study introduces a novel perspective on the utilization of S. horneri as an edible source, which has not been frequently addressed due to its rough texture.Moreover, our study aims to enhance functional properties of S. horneri through fermentation, and ultimately heighten the value of S. horneri as a food product.

Selection of the optimal S. horneri fermentation product and assessment of its antioxidative activity
Combinations of SHWE with bacterial strains were screened to identify the bacterial strains capable of using S. horneri as an energy source (Fig. 1a-e).SHWE supplementation in bacterial growth medium did not inhibit the growth of all four bacterial strains at 48 h, indicating non-toxicity (Fig. 1a-d) (p < 0.05).Moreover, the 48 h growth rates of SH803, SJ422, and SH123 significantly increased through SHWE supplementation (Fig. 1e) (p < 0.05).Specifically, the growth rate of SH803 for 48 h compared to the 0 h in the control medium was 87.98%, which increased to 127.68% with SHWE supplementation.Not only did SHWE supplementation enhance the 48 h growth rate compared to the 0 h by 39.7% in SH803, but also 12.95% in SJ422, and 8.36% in SH123.Based on these results, SHWE was the most effective in improving SH803 proliferation.Therefore, SH803 F-SHWE was used for further assessments.In vitro DPPH radical scavenging activity was measured to determine the antioxidative potential of F-SHWE (Fig. 1f).SHWE and F-SHWE showed high DPPH radical scavenging activities equivalent to 1.2 mM ascorbic acid (p < 0.05).However, the antioxidative activity of the non-supplemented growth media was 4.72%, which was significantly lower than that of SHWE or F-SHWE (89%, p < 0.05).Thus, SHWE enhanced the antioxidative effects, and fermentation did not suppress these properties.

Effects of F-SHWE on H 2 O 2 -induced oxidative stress in HT-29 intestinal epithelial cells
The antioxidative properties of F-SHWE were further evaluated using an oxidative stress-induced cell model.The optimal dosage for in vitro assays was selected based on cell viability measurements using the MTT assay (Fig. 2a-b).SHWE or F-SHWE treatments reduced cell viability to 51.79% at a dose of 10% (p < 0.05).Lower doses of SHWE and F-SHWE (1-2%) maintained cell viability at 90% (p < 0.05).As cell viability reduction > 30% is considered cytotoxic 18 , 2% treatment, the highest dose showing no cell viability reduction, was selected as the optimal dose for HT-29 assays.
H 2 O 2 -induced oxidative stress triggers cell apoptosis.Therefore, the gene expression of H 2 O 2 -induced apoptosis markers was measured using RT-qPCR to determine the reactive oxygen species (ROS)-eliminating effects of F-SHWE (Fig. 2c-h).Compared to the cell positive control treated only with the cell growth medium, H 2 O 2 treatment significantly upregulated the gene expression levels of NF-κB, p53, BAX, cytochrome C, caspase 9, and caspase 3 (p < 0.05).However, NF-κB expression was downregulated 0.4-fold by SHWE compared with the H 2 O 2 -only treated group (p < 0.05) (Fig. 2c).Similarly, F-SHWE suppressed NF-κB expression by 0.9-fold, which was more than twice the inhibitory effect of SHWE (p < 0.05).Furthermore, F-SHWE normalized the gene expression of p53, BAX, cytochrome C, caspase 9, and caspase 3; however, SHWE did not affect these markers (p < 0.05) (Fig. 2d-h).These results suggest that F-SHWE can reduce oxidative stress-induced apoptosis.These effects were minimal with SHWE treatment, suggesting that fermentation enhanced the oxidative stress scavenging abilities.

Effects of F-SHWE on LPS-induced inflammatory stress in HT-29 intestinal epithelial cells
F-SHWE was used in an LPS-induced inflammatory cell model to determine its anti-inflammatory properties.The gene expression of inflammatory response-related markers ZO1, IL1β, IFNγ, and COX2 was measured using RT-qPCR (Fig. 3a-d).LPS-induced inflammatory responses impaired gut barrier integrity by significantly reducing the expression of the tight junction protein, ZO1, leading to elevated IL1β, IFNγ, and COX2 gene expression levels (p < 0.05) (Fig. 3a-d).SHWE and F-SHWE repaired ZO1 expression; however, the effects were more pronounced in the F-SHWE treated group than in the SHWE group (p < 0.05) (Fig. 3a).Moreover, F-SHWE normalized the gene expression levels of IL1β, IFNγ, and COX2, downregulating them by at least 0.65-fold (p < 0.05) (Fig. 3b-d).SHWE did not affect IL1β expression; however, it inhibited IFNγ, although the effect was less pronounced than that of F-SHWE treatment (p < 0.05) (Fig. 3b-c).Compared with the LPS-only treated group, SHWE and F-SHWE exhibited a 1.87-and 3.93-fold reduction in IFNγ expression levels (p < 0.05) (Fig. 3c).Therefore, F-SHWE possesses potential anti-inflammatory effects, possibly enhanced through fermentation.www.nature.com/scientificreports/

Effects of fermentation on the SCFA composition in F-SHWE
GC/MS analysis revealed that SH803 fermentation increased the acetate content (Fig. 6a, Table 1).F-SHWE increased acetate content by 20.92 ± 2.89 mM (p < 0.05) compared with SHWE.Propionate, iso-butyrate, and butyrate were non-detected in both SHWE and F-SHWE (Table 1).For further confirmation on whether acetate is the key component of F-SHWE's effects, spearman correlation analysis between gene expression levels of 3T3-L1 markers and acetate levels were carried out.The correlations of adipogenesis markers (PPARγ, C/EBPα, aP2, Lpl), lipogenesis markers (Akt), inflammation markers (NF-κB) were inversely proportional to acetate, indicating that these biomarkers may be involved in the protective effects of F-SHWE in 3T3-L1 cells (p < 0.05) (Fig. 6b).However, the correlation of C/EBPβ, Leptin, Fas, SREBP1, ACC, IFN-γ were proportional to acetate (p < 0.05), indicating the possibility of a different compound contributing to the effects of F-SHWE other than acetate.Overall, these results suggest that compositional change of acetate through fermentation may correlate with the adipocyte-protective effects of F-SHWE in 3T3-L1 cells.

Discussion
ROS, a systemic physiological factor, facilitates adipogenic differentiation in 3T3-L1 pre-adipocytes, and as an outcome of differentiation, mature 3T3-L1 adipocytes end up with higher ROS levels than pre-adipocytes.Furthermore, excessive ROS accumulation induces cell death, such as apoptosis by activating NF-κB and p53 signaling, which triggers an apoptosis-inducing downstream signal of Bax, cytochrome C, caspase 9, and caspase 3 11,19 .In adipocytes, apoptosis worsens obesity by increasing macrophage infiltration into adipose tissue 20 .Therefore, recent studies attempt to attenuate obesity through antioxidative treatments 21,22 .Similarly, in this study, SHWE and F-SHWE both possessed oxygen radical scavenging effects comparable to ascorbic acid, a commercially used antioxidant (Fig. 1f).On treating intestinal epithelial cells, SHWE suppressed apoptosis by reducing NF-κB expression; F-SHWE had a greater suppressive effect than SHWE by downregulating NF-κB and p53 expression, indicating that SHWE and F-SHWE have anti-obesity potential (Fig. 2).
Inflammation is also associated with adipogenesis.Long term consumption of a high-fat diet, a common risk factor for obesity, induces systemic chronic low-grade inflammation 23 .This in turn alters gut microbiota composition and damages intestinal barrier integrity, increasing endotoxin levels in the intestinal lumen and plasma 24,25 .Endotoxins such as LPS intensify the damage of gut barrier proteins (such as ZO1) and induce TLR4 signaling 24 , consequently promoting the production of pro-inflammatory cytokines (such as IL1β), which exacerbate systemic low-grade inflammation and accelerate obesity pathogenesis [26][27][28] .Moreover, LPS can increase the expression of cyclooxygenase-2 (COX2)-a pro-inflammatory enzyme that stimulates prostaglandin production 29,30 .In this study, SHWE fermentation improved the protective effects against LPS-induced intestinal inflammation by increasing tight junction protein and reducing pro-inflammatory cytokine gene expression levels (Fig. 3).Notably, COX2 expression was reduced by F-SHWE treatment.Besides its pro-inflammatory properties, COX2 can inhibit adipogenesis by downregulating PPARγ and C/EBPα expression, thereby suppressing NF-κB signaling cascades, which promote pro-inflammatory cytokine expression (IFN-γ) 31 .While reduced COX2 expression by F-SHWE indicates anti-inflammatory efficacy, there is potential for reduced anti-adipogenic effects as well.Therefore, for the assessment of adipogenesis-related efficacy, further evaluations using an adipocyte differentiation model are necessary.Conventionally, 3T3-L1 murine preadipocytes are used as in vitro adipogenesis models; they enable preadipocyte differentiation pathway-related analysis 32 .Moreover, 3T3-L1 preadipocytes are differentiated by culturing in a growth medium treated with a mixture of IBMX, dexamethasone, and insulin.Dexamethasone-induced C/EBPδ and IBMX-induced C/EBPβ heterodimerize to activate PPARγ and C/EBPα, which are known to promote preadipocyte maturation into functional adipocytes 32,33 .Additionally, PPARγ and C/EBPα regulate the activation of mature adipocyte markers, such as aP2 and LPL 33 .These signaling cascades lead to lipid accumulation in adipocytes, which can be measured using Oil Red O staining 34 .As shown in Fig. 4, SHWE and F-SHWE treatment reduced Oil Red O stainable lipid accumulation in 3T3-L1 cells and the expression of adipogenesis markers (PPARγ, C/EBPα, aP2, and Lpl).These results align with those for the inhibitory effects of SHWE and F-SHWE on lipogenesis-related gene expression (Fig. 5).Lipogenesis is the synthesis of lipids within mature adipocytes, activated by the downstream signaling of Akt, SREBP1, ACC, and Fas 35,36 .Overall, these results indicate that SHWE and F-SHWE have the potential as anti-obesity agents by reducing adipogenesis and lipogenesis.Despite the higher inhibition rate of ROS-mediated apoptosis by F-SHWE than that by SHWE, both these extracts exhibited similar adipogenesis and lipogenesis downregulation.However, F-SHWE was more effective than SHWE in lowering inflammation response marker expressions in adipocytes; inflammatory cytokine IFN-γ and transcription factor NF-κB (Fig. 5f and g).IFN-γ impairs mitochondrial function and fatty acid flux in adipocytes, accelerating inflammation through responses, such as NF-κB regulated pathways 37 .Interestingly, in a previous report, Lactobacillus-fermented Sargassum spp.inhibited NF-κB signaling and increased SCFA productions 38 .SCFA, organic monocarboxylic acids with less than six carbons, reduce systemic inflammation through decreased NF-κB signaling and pro-inflammatory cytokines in obese states 39 .Thus, amounts of major SCFA such as acetate (C2), propionate (C3), and butyrate (C4), were measured to assess the effect of fermentation on its production.As shown in Fig. 6a, acetate concentrations were higher in F-SHWE than SHWE.Other SCFA were non-detected in both SHWE and F-SHWE.Acetate is produced by LAB strains through the subsequent conversion of pyruvate to acetyl phosphate and then to acetate in the presence of acetyl-CoA 40 .In a previous report, acetate reduced lipid accumulation in 3T3-L1 adipocytes through attenuation of fatty acid oxidation 41 .Similarly, in our study, lipid accumulation was reduced in F-SHWE treatment, which had higher concentrations of acetate than that of SHWE (Fig. 4c).Moreover, correlation analysis revealed inverse relationships between acetate concentrations and gene expressions of adipogenesis markers (PPARγ, C/EBPα, aP2, Lpl), lipogenesis markers (Akt), and inflammation markers (NF-κB) (Fig. 6b).This indicates acetate as a potent key contributor to the anti-obesity effects of F-SHWE in 3T3-L1 cells.This study had some limitations that warrant discussion.First, only in vitro experiments were conducted to examine the health benefits of F-SHWE.Oxidation-, inflammation-, and adipocyte metabolism-related effects were notable in adipocytes and intestinal cells treated with F-SHWE.However, these results cannot represent the possible interactions between the intestine and adipocytes on consuming food supplements.To further validate these effects and the related mechanisms of F-SHWE on oral consumption, in vivo studies capable of examining gut-adipose tissue signaling via pathways such as the gut microbiome, adipokines, and inflammatory cytokines are necessary.Second, to confirm whether acetate is the key component responsible for the fermentation-induced aspects of F-SHWE, the effects of F-SHWE and the equivalent amount of acetate should be compared in the same experimental models used in this study.Lastly, metabolites other than acetate may contribute to the fermentation-induced characteristics of F-SHWE.Correlations of C/EBPβ, Lep, Fas, SREBP1, Acc, Fas, IFN-γ gene expression were proportional to acetate, indicating the possibility of different compounds other than acetate also participating in the protective activities of F-SHWE.Other major metabolites produced by LAB-fermentation such as lactic acid could be potential functional compounds in F-SHWE.Lactic acid form acidic conditions in the intestine that result in inhibitory effects on obesity-related pathogenic bacteria 42 .In a previous report, Lactobacillus-fermentation of seaweed species was effective in lactic acid production 43 , indicating the possibility of it as a bioactive component for F-SHWE.Thus, the presence of these molecules may contribute to the effects of F-SHWE on adipocytes.Therefore, additional studies on the effects of F-SHWE oral supplementation in an animal model and on F-SHWE compositional analysis are in progress.
In conclusion, S. horneri fermented by Lactiplantibacillus pentosus SH803 inhibited adipogenesis and lipid accumulation induced by 3T3-L1 preadipocyte differentiation.The bioactive compounds eliminated apoptosisinduced oxidative (ROS) and inflammatory (LPS) stress in intestinal cells, exhibiting promising potential as functional food additives.These antioxidative and anti-inflammatory effects of F-SHWE may have contributed to adipogenesis reduction through PPARγ-mediated signaling and lipogenesis reduction through Akt-mediated Fas signaling.Moreover, F-SHWE inhibited inflammation in adipocytes by suppressing IFNγ and NF-κB expression.Compositional analysis revealed that fermentation increased acetate levels, which may have contributed to the enhanced properties in F-SHWE.Further validation studies are needed using in vivo models and are in progress.Overall, employing S. horneri fermentation as a therapeutic anti-obesity agent is a promising strategy to utilize this common but invasive species of the coastal ecosystem.

Preparation of LAB-fermented S. horneri
S. horneri water extracts and fermented products of those were prepared 44 .S. horneri was washed, dried at 50 °C for 24 h, and ground.The resulting powder was mixed with distilled water (1:50 w/v) and autoclaved (121 °C, 15 min) for sterilization.After cooling, cellulase (1:50 v/v, Sigma-Aldrich, MO, USA) was added, incubated (50 °C, pH 4.5) for 48 h, and autoclaved (121 °C, 15 min) for cellulase denaturation and sample sterilization 44 .The resulting mixture was termed S. horneri water extract (SHWE).Subsequently, the minimal broth (peptone and distilled water adjusted to a total volume of 500 mL) was mixed with SHWE and used as bacterial strain culture medium.Thereafter, SH803 culture was centrifuged at 10,800 × g for 3 min (VS-180Cfi, Vision Scientific Co., Daejeon, Korea) and washed twice with phosphate-buffered saline (PBS).Next, the optical density of the harvested bacterial pellets at 600 nm was adjusted to 0.3; the pellets were added to S. horneri culture medium (1:100 v/v) and incubated for 48 h.Sample preparations were done at 0 h and 48 h fermentation and spread on MRS agar plates (Kisan Bio) to assess bacterial growth.SHWE fermented with SH803 was termed F-SHWE.Subsequently, F-SHWE was filtered using 0.45 μm Stericup filters (Merck, Darmstadt, Germany), freeze dried, and kept at − 80 °C until use.

Antioxidative activity evaluation using DPPH assay
Investigation of antioxidant activity was measured by the DPPH method, with slight modifications 45 .DPPH (2,2-diphenyl-1-picrylhydrazyl) solutions were prepared by dissolving 0.4 mM DPPH (Sigma-Aldrich) in ethanol (Duksan Chemicals, Incheon, South Korea) until the absorbance at 517 nm was 0.94-0.97.In addition, ascorbic acid solutions were prepared dose-dependently (0.6, 1.2, and 2.4 mM) and used as positive controls.Next, the samples (60 μL) were mixed with 1000 μL DPPH solution and kept in complete darkness for 30 min at room temperature (25 °C).After incubation, absorbance was determined at 517 nm using the Epoch microplate spectrophotometer (BioTek, VT, USA).DPPH radical scavenging activity was calculated using the following equation.

Gene expression measurement using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR)
Gene expressions of biomarkers were measured using RT-qPCR 46 .For antioxidative activity evaluation, cells were seeded at a density of 5 × 10 5 cells/well in 12-well plates.After 24 h incubation, the cells were pre-treated with F-SHWE (1:2 v/v, diluted with RPMI) and incubated for 24 h (37 °C, 5% CO 2 ).To induce oxidative stress, the cells were treated with 100 μM hydrogen peroxide (H 2 O 2, Duksan, Ansan, South Korea) diluted with RPMI and incubated for 24 h (37 °C, 5% CO 2 ).Next, total mRNA was extracted using TRIzol reagent (Life Technologies, CA, USA) following the manufacturer's instructions.The concentration and purity of the extracted RNA were assessed using the NanoDrop spectrophotometer (BioTek, VT, USA) and standardized to a final concentration of 0.1 μg/μL.Following this, cDNA was synthesized using the reverse transcription kit (Thermo-Fisher).The polymerase chain reaction (PCR) cycling conditions were 25 °C for 10 min, 37 °C for 120 min, and 85 °C for 5 min.Subsequently, reverse transcription quantitative real-time PCR (RT-qPCR) was performed using the Bio-Rad CFX96 Real-Time PCR Detection System (Bio-rad, Hercules, CA, USA).Targeted genes were quantified using MG2 x qPCR MasterMix (SYBR green) (MGmed, South Korea).The RT-qPCR cycling conditions were: an initial denaturation cycle at 95 °C for 10 min, followed by 40 cycles of amplification at 95 °C for 15 s, annealing at 55-65 °C for 30 s, and extension at 70 °C for 5 s.Lastly, the mRNA expression levels of each targeted gene were analyzed and normalized to the internal standard gene, GAPDH, using Bio-Rad CFX Maestro (Bio-Rad Laboratories, Hercules, CA, USA).The primer sequences used in this study are listed in Supplementary Table ST For anti-inflammatory activity evaluation, cells were seeded at a density of 5 × 10 5 cells/well in 12-well plates.After 24 h incubation, the cells were pre-treated with F-SHWE (1:2 v/v, diluted with RPMI) and incubated for 24 h (37 °C, 5% CO 2 ).To stimulate inflammatory responses, 1 μg/mL lipopolysaccharide (LPS, Sigma-Aldrich) diluted with RPMI was added and incubated for 24 h (37 °C, 5% CO 2 ).Subsequent procedures were similar to those performed for evaluating antioxidative activity using RT-qPCR.
For evaluation of adipocyte differentiation-related gene expression levels, 3T3-L1 cells were treated with postdifferentiation media as described in Section "Gas Chromatography/Mass Spectrometry (GC/MS) instrumentation and chromatographic condition" and incubated for 6 days.The media was replaced every 2 days.Following this, the total mRNA was extracted using TRIzol reagent (Life) according to the manufacturer's instructions.The RNA, PCR, and RT-qPCR analyses were performed as described in section "Gene expression measurement using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR)".The primer sequences used in this study are listed in Supplementary Table

Lipid accumulation assessment by Oil Red O Staining
Lipid accumulation was evaluated with Oil Red O staining 47 .Initially, 3T3-L1 cells were seeded at a density of 0.8 × 10 5 cells/well in six-well plates and incubated for 48 h.After incubation, the cell culture media was replaced and incubated for 72 h.Further, cells were co-treated with differentiation medium (DMI; 0.5 mM 3-isobutyl-1-methylanthine [IBMX], 1 μM dexamethasone, 5 μg/mL insulin, 10% FBS, and 1% P/S mixed in DMEM, low glucose) and F-SHWE (1:2 v/v, diluted with differentiation medium) and incubated for 48 h.Subsequently, the cells were treated with post-differentiation media (5 μg/mL insulin, 10% FBS, and 1% P/S mixed in DMEM, low glucose) and incubated for 6 days; the media was replaced every 2 days.After the 6-day incubation, cells were fixed with formaldehyde (10% v/v) for 10 min and washed with isopropanol (60% v/v).Subsequently, 1 mL Oil Red O staining solution (0.5% v/v in isopropanol, Sigma-Aldrich) was added to the cells and incubated at room temperature for 20 min.Finally, the cells were washed with PBS and observed using the Olympus CKX41 inverted phase contrast microscope (Olympus, Tokyo, Japan).Oil Red O-stained area ratio (%) was measured using Image J software (National Institutes of Health, MA, USA).

Figure 1 .
Figure 1.Screening of S. horneri-fermentable bacteria and evaluation of antioxidative properties of the selected product (F-SHWE).(a-d) Bacterial growth of lactic acid bacteria (LAB) strains cultivated in growth media or growth media supplemented with 2% SHWE for 48 h.Results are expressed as mean ± standard error (SE) (n = 3).abc Results in the same series with different lowercase superscript letters are significantly different (p < 0.05).(e) Growth rate of LAB strains cultivated in growth media or growth media supplemented with 2% SHWE for 48 h.Results are expressed as mean ± SE (n = 3) (*p < 0.05, **p < 0.01, ***p < 0.001, compared with the control group) (f) DPPH scavenging activity of SH803 fermented SHWE (F-SHWE).Results are expressed as mean ± SE (n = 3).abcd Results in the same series with different lowercase superscript letters are significantly different (p < 0.05).

Figure 2 .
Figure 2. Effects of F-SHWE on H 2 O 2 -induced oxidative stress in HT-29 cells.(a,b) Viability of HT-29 cells pretreated with different concentrations (0, 1, 2, 10%) of SHWE or F-SHWE for 24 h.(c-h) Effects of SHWE or F-SHWE pre-treatment on the gene expression of apoptosis-related markers in H 2 O 2 -treated HT-29 cells.Results are expressed as mean ± SE (n = 3).abc Results in the same series with different lowercase superscript letters are significantly different (p < 0.05; Cyt C: Cytochrome C, CASP9: Caspase 9, CASP3: Caspase 3).

Figure 3 .
Figure 3. Effects of F-SHWE on LPS-induced stress in HT-29 cells.(a-d) Effects of SHWE or F-SHWE pretreatment on the gene expression of inflammation-related markers in lipopolysaccharide-treated HT-29 cells.Results are expressed as mean ± SE (n = 3).abc Results in the same series with different lowercase superscript letters are significantly different (p < 0.05; IL1B: IL-1β, IFNG: IFN-γ).

Figure 5 .
Figure 5. Effects of F-SHWE pre-treatment on the gene expression levels of lipogenesis and inflammatory cytokines in DMI-activated mature 3T3-L1 adipocytes.(a-e) Expression of adipogenesis-related genes in DMIactivated 3T3-L1 adipocytes pre-treated with SHWE or F-SHWE for 24 h.(f-g) Expression of inflammationrelated genes in DMI-activated 3T3-L1 adipocytes pre-treated with SHWE or F-SHWE for 24 h.Results are expressed as mean ± SE (n = 3).abc Results in the same series with different lowercase superscript letters are significantly different (p < 0.05; Lep: Leptin, IFN-g: IFN-γ).