Salvia miltiorrhiza bunge extracts: a promising source for anti-atopic dermatitis activity.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory condition characterized by the accumulation of reactive oxygen species and the expression of inflammatory factors. Regarding its anti-atopic activity, numerous traditional medicinal materials and secondary metabolic products play pivotal roles in modulating the associated mechanisms. METHODS: This study aimed to utilize Salvia miltiorrhiza Bunge (SMB) as an anti-AD source. In-vitro activity assessments and qualitative and quantitative analyses using UPLC-TQ-MS/MS and HPLC-DAD were conducted in two cultivars ('Dasan' and 'Kosan'). Statistical analysis indicated that the profiles of their secondary metabolites contribute significantly to their pharmacological properties. Consequently, bio-guided fractionation was undertaken to figure out the distinct roles of the secondary metabolites present in SMB. RESULTS: Comparative study of two cultivars indicated that 'Dasan', having higher salvianolic acid A and B, exhibited stronger antioxidant and anti-inflammatory activities. Meanwhile, 'Kosan', containing higher tanshinones, showed higher alleviating activities on anti-AD related genes in mRNA levels. Additionally, performed bio-guided fractionation re-confirmed that the hydrophilic compounds of SMB can prevent AD by inhibiting accumulation of ROS and suppressing inflammatory factors and the lipophilic components can directly inhibit AD. CONCLUSIONS: SMB was revealed as a good source for anti-AD activity. Several bioactive compounds were identified from the UPLC-TQ-MS/MS and different compounds content was linked to biological activities. Characterization of these compounds may be helpful to understand differential role of secondary metabolites from SMB on alleviation of AD.

Peptide-Drug Conjugate with Statistically Designed Transcellular Peptide for Psoriasis-Like Inflammation.

Peptide-drug conjugates (PDCs) are a promising class of drug delivery systems that utilize covalently conjugated carrier peptides with therapeutic agents. PDCs offer several advantages over traditional drug delivery systems including enhanced target engagement, improved bioavailability, and increased cell permeability. However, the development of efficient transcellular peptides capable of effectively transporting drugs across biological barriers remains an unmet need. In this study, physicochemical criteria based on cell-penetrating peptides are employed to design transcellular peptides derived from an antimicrobial peptides library. Among the statistically designed transcellular peptides (SDTs), SDT7 exhibits higher skin permeability, faster kinetics, and improved cell permeability in human keratinocyte cells compared to the control peptide. Subsequently, it is found that 6-Paradol (PAR) exhibits inhibitory activity against phosphodiesterase 4, which can be utilized for an anti-inflammatory PDC. The transcellular PDC (SDT7-conjugated with PAR, named TM5) is evaluated in mouse models of psoriasis, exhibiting superior therapeutic efficacy compared to PAR alone. These findings highlight the potential of transcellular PDCs (TDCs) as a promising approach for the treatment of inflammatory skin disorders.

Anti-inflammatory activities of Levilactobacillus brevis KU15147 in RAW 264.7 cells stimulated with lipopolysaccharide on attenuating NF-κB, AP-1, and MAPK signaling pathways.

Probiotics confer many beneficial effects on several illnesses, ranging from microbial diarrhea to inflammatory diseases. This study was conducted on whether Levilactobacillus brevis KU15147 obtained from kimchi has anti-inflammatory effects in RAW 264.7 cells stimulated with lipopolysaccharide (LPS) and antioxidant potential. L. brevis KU15147 reduced nitric oxide and prostaglandin E2 levels with decreasing the activation of inducible nitric oxide synthase and cyclooxygenase-2 without cell cytotoxicity. In addition, L. brevis KU15147 attenuated proinflammatory cytokine production including tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 in RAW 264.7 cells stimulated with LPS. Additionally, L. brevis KU15147 reduced the activity of nuclear factor-κB, activator protein-1, and mitogen-activated protein kinase signaling pathways. Furthermore, L. brevis KU15147 downregulated the production of reactive oxygen species. Therefore, L. brevis KU15147 was concluded that had an inhibition effect on LPS-induced inflammatory responses and can be used in functional foods to suppress inflammatory diseases. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01318-w.

Phytochemical Investigation of Equisetum arvense and Evaluation of Their Anti-Inflammatory Potential in TNFα/INFγ-Stimulated Keratinocytes.

Equisetum arvense L. (Equisetaceae), widely known as 'horsetail', is a perennial plant found extensively across Asia. Extracts of E. arvense have been used in traditional medicine, particularly for the treatment of inflammatory disorders. This study aimed to determine the phytochemical compounds in E. arvense ethanolic extract and their anti-inflammatory properties. Subsequently, we isolated and identified nine secondary metabolites, including kaempferol 3,7-di-O-ß-D-glucopyranoside (1), icariside B2 (2), (Z)-3-hexenyl ß-D-glucopyranoside (3), luteolin 5-O-ß-D-glucopyranoside (4), 4-O-ß-D-glucopyranosyl caffeic acid (5), clemastanin B (6), 4-O-caffeoylshikimic acid (7), (7S,8S)-threo-7,9,9'-trihydroxy-3,3'-dimethoxy-8-O-4'-neolignan-4-O-ß-D-glucopyranoside (8), and 3-O-caffeoylshikimic acid (9). The chemical structures of the isolated compounds (1-9) were elucidated using HR-ESI-MS data, NMR spectra, and ECD data. Next, the anti-inflammatory effects of the isolates were evaluated in tumor necrosis factor (TNF)α/interferon (IFN)γ-induced HaCaT, a human keratinocyte cell line. Among the isolates, compound 3 showed the highest inhibitory effect on the expression of pro-inflammatory chemokines, followed by compounds 6 and 8. Correspondingly, the preceding isolates inhibited TNFα/IFNγ-induced activation of pro-inflammatory transcription factors, signal transducer and activator of transcription 1, and nuclear factor-κB. Collectively, E. arvense could be employed for the development of prophylactic or therapeutic agents for improving dermatitis.

Fermentation of Inula britannica using Lactobacillus plantarum SY12 increases of epigallocatechin gallate and attenuates toxicity.

This study aimed to increase epigallocatechin gallate (EGCG) levels and attenuate the toxicity in Inulabritannica by fermentation using Lactobacillus plantarum SY12. The optimal medium was composed of 10 g of I. britannica, 4 g of xylose, 5 g of soytone, and 5 g of beef extract. The predicted value of EGCG was 237.327 µg/mL. To investigate damage in HepG2 cell lines by I. britannica extracts (IE) or fermented I. britannica extracts (FIE), cell viability, mitochondria membrane potential, the expression of apoptosis and autophagy genes, and chemical composition were measured. FIE increased cell viability, regulation of the gene expression (decreased p53, p62, p-ERK 1/2, and p-p38; increased CDK2 and CDK4) compared with IE. These results were explained by an increase in 1,3-dicaffeoylquinic acid and a decrease in 1-O-caffeoylquinic acid, 1-O-acetylbritannilactone, and ergolide in FIE. In conclusion, these results indicated that fermentation can mitigate the toxicity in I. britannica.

Protective effect of Tisochrysis lutea on dry eye syndrome via NF-κB inhibition.

Dry eye syndrome (DES) affects the cornea, causes pain and hypersensitivity to light. Although inflammation and endoplasmic reticulum stress are known to be involved, the detailed mechanisms remain unknown. DES is characterized by a decrease in corneal thickness, tear volume, and lacrimal gland size, and damage to corneal cells. Tisochrysis lutea is a microalga that has been shown to reduce immune factors. Therefore, we hypothesized that T. lutea could ameliorate DES. We investigated the role of T. lutea in scopolamine-induced DES in BALB/c mice. Oral administration of T. lutea increased corneal thickness, tear volume, and size of the corneal cells, and reduced damage to the corneal cells. Furthermore, treatment of ARPE-19 human retinal pigmented epithelial cells with T. lutea reduced expression of the inflammatory factor, NF-κB, MAPK, and AKT. T. lutea may be used therapeutically to reduce the symptoms of DES.

Levilactobacillus brevis KU15151 Inhibits Staphylococcus aureus Lipoteichoic Acid-Induced Inflammation in RAW 264.7 Macrophages.

Inflammation is a host defense response to harmful agents, such as pathogenic invasion, and is necessary for health. Excessive inflammation may result in the development of inflammatory disorders. Levilactobacillus brevis KU15151 has been reported to exhibit probiotic characteristics and antioxidant activities, but the effect of this strain on inflammatory responses has not been determined. The present study aimed to investigate the anti-inflammatory potential of L. brevis KU15151 in Staphylococcus aureus lipoteichoic acid (aLTA)-induced RAW264.7 macrophages. Treatment with L. brevis KU15151 reduced the production of nitric oxide and prostaglandin E2 by suppressing the expression of inducible nitric oxide synthase and cyclooxygenase-2. Additionally, the production of proinflammatory cytokines including tumor necrosis factor-α, interleukin (IL)-6, and IL-1ß, decreased after treatment with L. brevis KU15151 in aLTA-stimulated RAW 264.7 cells. Furthermore, this strain alleviated the activation of nuclear factor-κB and mitogen-activated protein kinase signaling pathways. Moreover, the generation of reactive oxygen species was downregulated by treatment with L. brevis KU15151. These results demonstrate that L. brevis KU15151 possesses an inhibitory effect against aLTA-mediated inflammation and may be employed as a functional probiotic for preventing inflammatory disorders.

Optimization of an Industrial Medium and Culture Conditions for Probiotic Weissella cibaria JW15 Biomass Using the Plackett-Burman Design and Response Surface Methodology.

The objective of this study was to optimize industrial-grade media for improving the biomass production of Weissella cibaria JW15 (JW15) using a statistical approach. Eleven variables comprising three carbon sources (glucose, fructose, and sucrose), three nitrogen sources (protease peptone, yeast extract, and soy peptone), and five mineral sources (K2HPO4, potassium citrate, L-cysteine phosphate, MgSO4, and MnSO4) were screened by using the Plackett-Burman design. Consequently, glucose, sucrose, and soy peptone were used as significant variables in response surface methodology (RSM). The composition of the optimal medium (OM) was 22.35 g/l glucose, 15.57 g/l sucrose, and 10.05 g/l soy peptone, 2.0 g/l K2HPO4, 5.0 g/l sodium acetate, 0.1 g/l MgSO4·7H2O, 0.05 g/l MnSO4·H2O, and 1.0 g/l Tween 80. The OM significantly improved the biomass production of JW15 over an established commercial medium (MRS). After fermenting OM, the dry cell weight of JW15 was 4.89 g/l, which was comparable to the predicted value (4.77 g/l), and 1.67 times higher than that of the MRS medium (3.02 g/l). Correspondingly, JW15 showed a rapid and increased production of lactic and acetic acid in the OM. To perform a scale-up validation, batch fermentation was executed in a 5-l bioreactor at 37°C with or without a pH control at 6.0 ± 0.1. The biomass production of JW15 significantly improved (1.98 times higher) under the pH control, and the cost of OM was reduced by two-thirds compared to that in the MRS medium. In conclusion, OM may be utilized for mass producing JW15 for industrial use.

Anti-adipogenic Effects of the Probiotic Lactiplantibacillus plantarum KU15117 on 3T3-L1 Adipocytes.

In this study, we investigated the probiotic properties and anti-obesity effects of bacterial strains isolated from homemade kimchi. Lactiplantibacillus plantarum KU15117 was isolated using lactobacilli selective medium. L. plantarum KU15117 did not produce ß-glucuronidase and showed high tolerance to artificial gastric juice and bile salt, acceptable resistance to antibiotics, and high adhesion ability to HT-29 cells. The anti-adipogenic activity of L. plantarum KU15117 at 109 CFU/well was confirmed by the reduction of oil red O staining and intracellular triglyceride level. Additionally, the expression levels of fatty acid synthase, CCAAT/enhance-binding protein-α, and peroxisome proliferator-activated receptor-γ, which are associated with the early stage of adipocyte differentiation, were significantly lower in the probiotic-treated group than in the control group. These results suggest that L. plantarum KU15117 has probiotic properties and anti-obesity effects and could be used as a prophylactic probiotics.

Chrysanthemum indicum suppresses adipogenesis by inhibiting mitotic clonal expansion in 3T3-L1 preadipocytes.

Herbs have been of interest to treat diseases, including obesity, owing to their various bioactive constituents that exhibit therapeutic and prophylactic properties. The present study examined the anti-adipogenic effects and mechanisms of Chrysanthemum indicum aqueous extract (CAE) in 3T3-L1 preadipocytes. CAE comprises 1,3-dicaffeoylquinic acid, chlorogenic acid, kaempferol-3-O-glucoside, caffeic acid, and apigenin, which were corresponded with previous reports. CAE inhibited the accumulation of lipid droplets and significantly alleviated the expression of lipogenesis- and adipogenesis-associated biomarkers. Treatment with CAE inhibited the mitotic clonal expansion (MCE), corroborated by cell cycle arrest at the G0 /G1 phase, and mitigated the expression of cell cycle progression-associated proteins and in addition to phosphorylation of MCE-promoting transcription factors. Moreover, CAE downregulated the activation of Akt and extracellular signal-regulated kinase 1/2 signaling pathways. In summary, CAE facilitates adipogenic inhibition during the early phase of differentiation, especially MCE, and its phenolic compounds can contribute to its anti-obesogenic properties. PRACTICAL APPLICATIONS: Chrysanthemum indicum has been mainly used as traditional herbal tea and drinks. Chrysanthemum indicum aqueous extract (CAE) inhibits adipogenesis by suppressing mitotic clonal expansion during the early phase of differentiation in 3T3-L1 preadipocytes. 1,3-Dicaffeoylquinic acid, chlorogenic acid, kaempferol-3-O-glucoside, caffeic acid, and apigenin were detected in CAE. Based on these findings, CAE can be used as nutraceutical agents for prevention and treatment of obesity.

Inula britannica Inhibits Adipogenesis of 3T3-L1 Preadipocytes via Modulation of Mitotic Clonal Expansion Involving ERK 1/2 and Akt Signaling Pathways.

The flower of Inula britannica contains various phenolic compounds with prophylactic properties. This study aimed to determine the anti-adipogenic effect of an I. britannica flower aqueous extract (IAE) and its underlying mechanisms in the 3T3-L1 preadipocytes and to identify the phenolic compounds in the extract. Treatment with IAE inhibited the adipogenesis by showing a dose-dependent suppressed intracellular lipid accumulation and mitigated expression levels of lipogenesis- and adipogenesis-associated biomarkers including transcription factors. IAE exerted an anti-adipogenic effect through the modulation of the early phases of adipogenesis including mitotic clonal expansion (MCE). Treatment with IAE inhibited MCE by arresting the cell cycle at the G0/G1 phase and suppressing the activation of MCE-related transcription factors. Furthermore, IAE inhibited adipogenesis by regulating the extracellular signal-regulated kinase 1/2 and Akt signaling pathways. Protocatechuic acid, chlorogenic acid, kaempferol-3-O-glucoside, and 6-methoxyluteolin, which are reported to exhibit anti-adipogenic properties, were detected in IAE. Therefore, modulation of early phases of adipogenesis, especially MCE, is a key mechanism underlying the anti-adipogenic activity of IAE. In summary, the anti-obesity effects of IAE can be attributed to its phenolic compounds, and hence, IAE can be used for the development of anti-obesity products.

Inula britannica fermented with probiotic Weissella cibaria D30 exhibited anti-inflammatory effect and increased viability in RAW 264.7 cells.

The objective of this study was to increase the bioavailability of Inula britannica (IB) through fermentation with probiotic Weissella cibaria D30, and to evaluate the chemical composition, viability, and anti-inflammatory effect of fermented I. britannica (FIB). IB was fermented with W. cibaria D30 at 37 °C for 24 h. FIB increased total phenolic content and decreased total flavonoid content of IB. 1-O-acetylbritannilactone and ergolide production, which are associated with the viability, increased from 1.38 to 4.13 µg/mg, and decreased from 5.24 to 0.94 µg/mg, in the control and FIB, respectively. In addition, the cell viability of RAW264.7 cells increased when pretreated with 400 µg/mL FIB. FIB inhibited the production of nitric oxide and proinflammatory cytokines by inhibiting NF-κB and MAPKs pathways. Therefore, FIB with W. cibaria D30 reduced the toxicity and increased the anti-inflammatory properties. These results indicate that FIB is a potential beneficial bioactive agent for functional foods.

Immune-stimulating Effect of Lactobacillus plantarum Ln1 Isolated from the Traditional Korean Fermented Food, Kimchi.

This study aimed to determine the immune-stimulating effects of heat-killed Lactobacillus plantarum Ln1 (HK-Ln1) through the production of nitric oxide (NO) and pro-inflammatory cytokine achieved by inducing NF-κB and mitogen-activated protein kinase (MAPK)-signaling pathways in macrophages. HK-Ln1 showed higher NO and cytokine production compared t°Control (nonstimulated lipopolysaccharide); in addition, the expression of inducible nitric oxide synthase (iNOS) was induced through HK-Ln1treatment. The phosphorylation of IκB-α and p65 increased following treatment by HK-Ln1, which implicates IκB-α degradation and the translocation of p65 to nucleus. In addition, the phosphorylation of MAPKs, ERK 1/2, JNK, and p38 was induced following HK-Ln1 treatment.

Investigating the antimicrobial and antibiofilm effects of cinnamaldehyde against Campylobacter spp. using cell surface characteristics.

Campylobacter species are known as biofilm-forming bacteria in food systems. The aim of this study was to evaluate the antimicrobial and antibiofilm effects of cinnamaldehyde against Campylobacter jejuni and Campylobacter coli isolated from chicken meat. The biofilm-forming C. jejuni and C. coli strains from chicken meat were investigated using minimum inhibitory concentration (MIC) and Campylobacter spp. characteristics. The MIC value was 31.25 µg/mL for the Campylobacter strains tested. Cinnamaldehyde had an inhibition and degradation effect on Campylobacter biofilms at concentrations > 15.63 µg/mL. Campylobacter strains treated with 15.63 µg/mL CA exhibited significantly decreased autoaggregation, motility, exopolysaccharide production, and soluble protein. In addition, Campylobacter biofilms formed on stainless steel were degraded following cinnamaldehyde treatment, as determined by scanning electron microscopy. Taken together, these results suggest that cinnamaldehyde constitutes a potential natural preservative against Campylobacter and a nontoxic biofilm remover that could be applied to control food poisoning in the poultry manufacturing-related food industry. PRACTICAL APPLICATION: Cinnamaldehyde was able to effectively remove the biofilm of Campylobacter in the small crack of stainless steel. Cinnamaldehyde has a potential to replace the synthetic antimicrobial and/or antibiofilm agent as well as has a positive influence on consumer concern for the food safety issues of the poultry industries.

Anti-Inflammatory Potential of Probiotic Strain Weissella cibaria JW15 Isolated from Kimchi through Regulation of NF-κB and MAPKs Pathways in LPS-Induced RAW 264.7 Cells.

Probiotics are known to provide the host with immune-modulatory effects and are therefore of remarkable interest for therapeutic and prophylactic applications against various disorders, including inflammatory diseases. Weissella cibaria JW15 (JW15) has been reported to possess probiotic and antioxidant properties. However, the effect of JW15 on inflammatory responses has not yet been reported. Therefore, the objective of the current study was to evaluate the anti-inflammatory potential of JW15 against lipopolysaccharide (LPS) stimulation. The production of pro-inflammatory factors and the cellular signaling pathways following treatment with heat-killed JW15 was examined in LPS-induced RAW 264.7 cells. Treatment with heat-killed JW15 decreased nitric oxide and prostaglandin E2 production via downregulation of the inducible nitric oxide synthase and cyclooxygenase-2. In addition, treatment with heat-killed JW15 suppressed the expression of pro-inflammatory cytokines, interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α. The anti-inflammatory properties of treating with heat-killed JW15 were associated with mitogen-activated protein kinase signaling pathwaymediated suppression of nuclear factor-κB. These results indicated that JW15 possesses antiinflammatory potential and provide a molecular basis regarding the development of functional probiotic products.

Antagonistic and antioxidant effect of probiotic Weissella cibaria JW15.

The objective of this study was to evaluate the functional properties of Weissella cibaria JW15 (JW15) by investigating its antagonistic and antioxidant activities. Lactobacillus rhamnosus GG (LGG) was used for comparison as a reference strain. JW15 inhibited the growth of pathogenic bacteria (Listeria monocytogenes, Salmonella Typhimurium, S. Enteritidis, and Escherichia coli). Compared to LGG, JW15 showed rapid organic acid production, with the amounts of lactic and acetic acids being lower and higher, respectively. In addition, JW15 significantly inhibited intestinal epithelial adherence in the tested pathogens. JW15 exhibited antioxidant effects by scavenging radicals including DPPH, ABTS, and hydroxyl radicals, and inhibiting lipid peroxidation. JW15 exhibited significant antagonistic and antioxidant activities compared to LGG in the tested assay (p < 0.05). The results suggested that JW15 might possess a potential for amelioration of disorders induced by pathogenic bacteria or oxidative stress.

Antioxidant and immunostimulatory effect of potential probiotic Lactobacillus paraplantarum SC61 isolated from Korean traditional fermented food, jangajji.

This study aimed to isolate and demonstrate their antioxidant and immunostimulatory activities of potential probiotics. The isolated strains, S. Pum19, SC28, and SC61 showed potential probiotic properties including stability in artificial gastric and bile conditions, non-production of ß-glucuronidase, suitable antibiotic susceptibility, and attachment to intestinal cells. S. Pum19, SC28, and SC61 strains were identified as Leuconostoc citreum, Pediococcus pentosaceus, and Lactobacillus paraplantarum, respectively. Of the 3 potential probiotic LAB strains, intact cells of L. paraplantarum SC61 showed higher antioxidant activity, including DPPH radical scavenging, ß-carotene bleaching inhibition, reducing power, superoxide anion scavenging, and ABTS radical scavenging activity. In addition, L. paraplantarum SC61 produced the most nitric oxide production and its mRNA expression level for iNOS, IL-1ß, IL-6, and TNF-α were superior to those of L. rhamnosus GG. Therefore, L. paraplantarum SC61 was demonstrated to exhibit antioxidant and immunostimulatory activity and to have potential use as a probiotic product.

Application of stabilizer improves stability of nanosuspended branched-chain amino acids and anti-inflammatory effect in LPS-induced RAW 264.7 cells.

This study investigated the use of polyglyceryl esters (PGE) as stabilizer in improving stability and anti-inflammatory activity of nanosuspended branched-chain amino acids (BCAAs). BCAAs nanosuspended with stabilizer (BS) exhibited improved stability at concentration of 5% saturation level during storage as compared to BCAAs nanosuspended with aqueous solution (BA). Additionally, anti-inflammatory activity of BS was found to be greater than that of BA. Nitric oxide scavenging activity was found to be dose-dependent, with activity of BS in sodium nitroprusside system being significantly higher than that of BA (p < 0.05) at 2.5-20 mg/mL. BS also possesses greater inhibitory activity on production of pro-inflammatory factors including inducible nitric oxide synthase, cyclooxygenase-2 (COX-2), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells through suppressed phosphorylation of p65 subunit of NF-κB at 0.5, 2, 8 mg/mL. These results suggest that PGE used as stabilizer improves solubility and biological activity of nanosuspended BCAAs.

Evaluation of the Quality of Yogurt Using Ginseng Extract Powder and Probiotic Lactobacillus plantarum NK181.

The objective of this study was to evaluate the composition, pH, titratable activity, microbial properties, and antioxidant effect of yogurt using ginseng extract powder (GEP), Lactobacillus plantarum NK181, and Streptococcus thermophilus as the starter culture. Different concentration of GEP (0%, 0.5%, 1%, 1.5%, and 2% (w/v)) were used in the yogurt. During yogurt fermentation, pH was decreased; however, titratable acidity and viable cell counts were increased. The addition of GEP to yogurt led to a decrease in moisture content and an increase in the fat, ash, and total solids content. The antioxidant effect using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging, ß-carotene bleaching, and ferric reducing antioxidant power (FRAP) assay gradually increased with added GEP. Overall, yogurt fermented with 1% GEP was acceptable in terms of cell viability and antioxidant effect. These results might provide information regarding development of ginseng dairy products with enhanced antioxidant activities and probiotic properties.

Benzoic Acid Production with Respect to Starter Culture and Incubation Temperature during Yogurt Fermentation using Response Surface Methodology.

Benzoic acid is occasionally used as a raw material supplement in food products and is sometimes generated during the fermentation process. In this study, the production of naturally occurring yogurt preservatives was investigated for various starter cultures and incubation temperatures, and considered food regulations. Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus plantarum, Bifidobacterium longum, Bifidobacterium lactis, Bifidobacterium bifidum, Bifidobacterium infantis, and Bifidobacterium breve were used as yogurt starter cultures in commercial starters. Among these strains, L. rhamnosus and L. paracasei showed the highest production of benzoic acid. Therefore, the use of L. rhamnosus, L. paracasei, S. thermophilus, and different incubation temperatures were examined to optimize benzoic acid production. Response surface methodology (RSM) based on a central composite design was performed for various incubation temperatures (35-44℃) and starter culture inoculum ratios (0-0.04%) in a commercial range of dairy fermentation processes. The optimum conditions were 0.04% L. rhamnosus, 0.01% L. paracasei, 0.02% S. thermophilus, and 38.12℃, and the predicted and estimated concentrations of benzoic acid were 13.31 and 13.94 mg/kg, respectively. These conditions maximized naturally occurring benzoic acid production during the yogurt fermentation process, and the observed production levels satisfied regulatory guidelines for benzoic acid in dairy products.