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Research article
Effect of Lactobacillus Fermentation on the Anti-Inflammatory Potential of Turmeric
1Division of Animal Science, Chonnam National University, Gwangju,,61186, Republic of Korea, 2JNBIO, Jeonnam, 57059, Republic of Korea
Correspondence to:J. Microbiol. Biotechnol. 2019; 29(10): 1561-1569
Published October 28, 2019 https://doi.org/10.4014/jmb.1906.06032
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Introduction
Curcumin (diferuloylmethane), the major bioactive constituent of turmeric with a vibrant yellow appearance, was first identified in 1910 [8]. Many researchers have demonstrated the ability of curcumin to interact with numerous molecular targets in inflammation, including impairing the pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1) production in
Recently, plant-based fermentation by probiotic strains has drawn the attention of both scientific researchers and agricultural industries as a way to improve the nutritional value, flavor, and aroma of food, and for application as a feed additive or adjuvant to enhance the performance of livestock [12]. Probiotics are well known for their beneficial effects on the host; hence, fermentation of plant resources with probiotics might synergistically amplify their beneficial properties. To date, several fermented turmeric products have been developed that not only enhance liver function but also improve palatability while having antioxidant, anti-obesity, and antimicrobial activities [13-16]. However, there are limited studies reporting the immuno-modulatory activity of
Materials and Methods
Cell Culture and Chemicals
Jungnong Bio Inc. (Korea) provided turmeric and
High-Performance Liquid Chromatography Analysis of Lactobacillus -Fermented Turmeric
The curcumin content of the
In Vitro Cytotoxicity Assay
The RAW 264.7 cells were seeded in a 96-well plate at a density of 1 × 105 cells/well in DMEM medium and maintained under conditions mentioned previously until 70% confluency. The cells were treated with various concentrations of
Lipopolysaccharide-Induced Cell Viability Assay
For cell viability assay, RAW 264.7 cells were seeded in a 96-well plate at a density of 1 × 105 cells/well in DMEM medium and maintained under conditions mentioned previously until 70%confluency. The cells were treated with 1 μg/ml of lipopoly-saccharide (LPS;
Nitric Oxide Determination
The nitric oxide (NO) production in LPS-induced RAW 264.7 cells, upon treatment with
2,2-Diphenyl-1-Picrylhydrazyl (DPPH) Radical Scavenging Assay
The
Determination of the Level of Pro-Inflammatory Factors
RAW 264.7 cells (3 × 105 cells/well in 6-well plate) were cultured with 1 μg/ml of LPS and
-
Table 1 . Oligonucleotide primers used in this study.
Genes Oligonucleotide Sequences Annealing Temperature References TNF-α FACTGAACTTCGGGGTGAT 60°C [43] TNF-α RACTTGGTGGTTTGCTACG TLR4 FTTCAGAACTTCAGTGGCTGGATTTA 60°C [43] TLR4 RGTCTCCACAGCCACCAGATTCTC GAPDH FCATGACCACAGTCCATGCCATCAC 60°C [17] GADPH RTGAGGTCCACCACCCTGTTGCTGT
Western Blot Analysis
RAW 264.7 cells (3 × 105 cells/well in 6-well plate) were cultured with 1 μg/ml of LPS and
Statistical Analyses
All values were expressed as mean ± standard deviation from triplicate runs (
Results
Curcumin Content of Unfermented and Lactobacillus -Fermented Turmeric
HPLC analysis revealed that fermentation with
-
Table 2 . HPLC analysis of the curcumin content of
Lactobacillus fermented turmeric.Samples Curcumin content before fermentation, mg/g Curcumin content after fermentation, mg/g L. fermentum -fermented1.855 ± 0.026 2.03 ± 0.012 *** L. plantarum -fermented1.971 ± 0.010 ** L. brevis -fermented1.880 ± 0.013 Results are expressed as mean ± standard deviation of triplicate runs (
n = 3). Paired T-test analysis was performed with asterisks (*) indicating that curcumin contents before and after fermentation differ significantly (**p < 0.01; ***p < 0.001).
Cytotoxicity of Lactobacillus -Fermented Turmeric
Upon fermentation, the
-
Fig. 1. Cell viability of murine macrophages, RAW 264.7 cells upon treatment with different concentrations of
Lactobacillus -fermented turmeric (500.00, 250.00, 125.00, 62.50, and 31.25 μg/ml in 0.1% [v/v] DMSO). Untreated RAW 264.7 cells were used as naive control. Results are expressed as means ± standard deviations (n = 3). Tukey’s multiple range analysis was performed with different lowercase letters (a, b, c, and d) demonstrating the significant differences between samples (p < 0.05).
Cell Viability of LPS-Induced RAW 264.7 Cells
In this study, an enhanced protective effect was observed on
-
Fig. 2. Cell viability of murine macrophages, RAW 264.7 cells upon stimulation with lipopolysaccharides (LPS) from
Escherichia coli and treatment with different concentrations ofLactobacillus -fermented turmeric. Non-LPS-induced RAW 264.7 cells were used as a naive control. Results are expressed as means ± standard deviations (n = 3). Tukey’s multiple range analysis was performed with different lowercase letters (a and b) demonstrating the significant differences between samples (p < 0.05).
Nitric Oxide Assay
In the current study, the accumulated nitrite in the culture supernatant was determined via the Griess method as an index for NO produced by the LPS-induced RAW 264.7 cells. As shown in Fig. 3, the production of nitrite was reduced upon treatment with
-
Fig. 3. Effect of
L. fermentum -fermented turmeric (31.25 μg/ml) on nitrite production in LPS-induced RAW 264.7 cells. Results are expressed as means ± standard deviations (n = 3). Paired T-test analysis was performed to determine the group differences. Control group vs. samples (***p < 0.001); untreated group vs. samples (###p < 0.001).
2,2-Diphenyl-1-Picrylhydrazyl (DPPH) Radical Scavenging Activity
In this study, the antioxidant activity of
-
Fig. 4. DPPH radical scavenging activity of
L. fermentum fermented (31.25 μg/ml) and unfermented turmeric (31.25 μg/ml). Results are expressed as means ± standard deviations (n = 3). Positive control consists of 500 μg/ml L-ascorbic acid. Paired T-test analysis was performed to determine the group differences. Negative control group vs. samples (**p < 0.01; ***p < 0.001); Positive control group vs. samples (##p < 0.01; ###p < 0.001).
Gene Expression of Pro-Inflammatory Factors
The gene expressions of TNF-α and TLR4 in LPS-induced RAW 264.7 cells upon treatment with
-
Fig. 5. Effects of
L. fermentum -fermented turmeric (31.25 μg/ml) on the gene expression of (a) tumor necrosis factor-alpha (TNF-α) and (b) Toll-like receptor 4 (TLR4) in LPS-induced RAW 264.7 cells. Results are expressed as means ± standard deviations (n = 3). Paired T-test analysis was performed, with asterisks (*) indicating that the differences between untreated control and samples within the same group are significant (*p < 0.05; **p < 0.01).
Western Blot Analysis
The results obtained from the western blot analysis further justified the anti-inflammatory activity of
-
Fig. 6. Effects of
L. fermentum -fermented turmeric (31.25 μg/ml) on the activation of phosphorylated-JNK (p-JNK) and TNF-α of LPS-induced RAW 264.7 cells. (A ) Protein levels of p-JNK and TNF- α were detected by western blotting using p-JNK and TNF-α specific antibodies, where β-actin was used as an internal control. Data shown are representative of three independent experiments (n = 3). (B ) Relative protein expression of p-JNK and TNF-α was quantified via densitometry analysis. Results are expressed as means ± standard deviations (n = 3). Paired T-test analysis was performed, with asterisks (*) indicating that the differences between control and samples within the same antibody group are significant (**p < 0.01; ***p < 0.001).
Discussion
Turmeric has been long used as a food additive and herbal medicine owing to its wide range of biological activity, including anti-inflammatory [9, 10], anti-diabetes [21, 22], and anti-Alzheimer’s disease activities [3, 4]. The wide range of pharmacological benefits is attributed to the presence of curcumin, the major bioactive constituent of turmeric. Curcumin only makes up 2-5% of turmeric [23], hence, probiotics fermentation has been used to improve the curcumin content and pharmacological activities of turmeric. In this study, HPLC analysis revealed that fermentation with
Although the curcumin content has been improved via fermentation, it is of utmost importance to validate the cytotoxicity of the
Subsequently, efforts were made to determine the protective effect of
Pro-inflammatory cytokines are produced by the activated macrophages and are essential to elicit the inflammatory responses. The TLR4, a transmembrane protein on the surface of macrophages, monocytes, and dendritic cells, belongs to the pattern recognition receptor family and is essential for the initiation of the innate immune response [27]. Hence, the presence of LPS is detected by the immune cells, recognized by TLR4 to form a TLR4-myeloid differentiation protein 2 (MD2)-LPS complex, resulting in the activation of intracellular signaling pathway NF-kB and innate immune system to promote inflammation and drive pro-inflammatory cascades [27-29].
As a pro-inflammatory cytokine, TNF-α initiates the pro-inflammatory cascades, recruiting and activating various inflammatory cells for recovery purposes [30, 31]. However, the over-production of TNF-α could lead to chronic inflammation and cell apoptosis [31]. In the present study,
In addition, LPS also induces oxidative stress by stimulating the activated macrophages for the production of NO and ROS. A high level of ROS produced upon LPS stimulation will alter host homeostasis condition, thereby causing oxidative damage and cell apoptosis [32]. Notably,
Previous studies also reported that a high level of NO could induce apoptosis directly or indirectly by rendering many cells susceptible to apoptosis [37, 38]. The data showed that the protective effect of
Interestingly, the protective effect of
The present study clearly demonstrated that
Acknowledgments
This study was supported by the South Jeolla Province (2016 R & D Support Program operated by Jeonnam Technopark).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Related articles in JMB

Article
Research article
J. Microbiol. Biotechnol. 2019; 29(10): 1561-1569
Published online October 28, 2019 https://doi.org/10.4014/jmb.1906.06032
Copyright © The Korean Society for Microbiology and Biotechnology.
Effect of Lactobacillus Fermentation on the Anti-Inflammatory Potential of Turmeric
ChengChung Yong 1, Yonghee Yoon 2, HeeSub Yoo 2 and Sejong Oh 1*
1Division of Animal Science, Chonnam National University, Gwangju,,61186, Republic of Korea, 2JNBIO, Jeonnam, 57059, Republic of Korea
Correspondence to:Sejong Oh
soh@jnu.ac.kr
Abstract
Curcumin, the major bioactive constituent of turmeric, has been reported to have a wide range of pharmacological benefits; however, the low solubility in water has restricted its systemic bioavailability and therapeutic potential. Therefore, in the current study, we aimed to investigate the effect of turmeric fermentation on its curcumin content and anti-inflammatory activity by using several lactic acid bacteria. Fermentation with Lactobacillus fermentum significantly increased the curcumin content by 9.76% while showing no cytotoxicity in RAW 246.7 cells, as compared to the unfermented turmeric, regardless of the concentration of L. fermentum-fermented turmeric. The L. fermentum-fermented turmeric also promoted cells survival; a significantly higher number of viable cells in lipopolysaccharide (LPS)-induced RAW 264.7 cells were observed as compared to those treated with unfermented turmeric. It also displayed promising DPPH scavenging activity (7.88 ± 3.36%) and anti-inflammatory activity by significantly reducing the nitrite level and suppressing the expression of the pro-apoptotic tumor necrosis factor-alpha (TNF-α) and Toll-like receptor-4 (TLR4) in LPS-induced RAW 264.7 cells. Western blot analysis further revealed that the anti-inflammatory activity of the fermented turmeric was exerted through suppression of the c-Jun N-terminal kinase (JNK) signal pathway, but not in unfermented turmeric. Taken together, the results suggested that fermentation with lactic acid bacteria increases the curcumin content of turmeric without increasing its cytotoxicity, while strengthening the specific pharmacological activity, thus, highlighting its potential application as a functional food ingredient.
Keywords: Turmeric, curcumin, Lactobacillus, RAW 264.7 macrophage cells, anti-inflammatory
Introduction
Curcumin (diferuloylmethane), the major bioactive constituent of turmeric with a vibrant yellow appearance, was first identified in 1910 [8]. Many researchers have demonstrated the ability of curcumin to interact with numerous molecular targets in inflammation, including impairing the pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1) production in
Recently, plant-based fermentation by probiotic strains has drawn the attention of both scientific researchers and agricultural industries as a way to improve the nutritional value, flavor, and aroma of food, and for application as a feed additive or adjuvant to enhance the performance of livestock [12]. Probiotics are well known for their beneficial effects on the host; hence, fermentation of plant resources with probiotics might synergistically amplify their beneficial properties. To date, several fermented turmeric products have been developed that not only enhance liver function but also improve palatability while having antioxidant, anti-obesity, and antimicrobial activities [13-16]. However, there are limited studies reporting the immuno-modulatory activity of
Materials and Methods
Cell Culture and Chemicals
Jungnong Bio Inc. (Korea) provided turmeric and
High-Performance Liquid Chromatography Analysis of Lactobacillus -Fermented Turmeric
The curcumin content of the
In Vitro Cytotoxicity Assay
The RAW 264.7 cells were seeded in a 96-well plate at a density of 1 × 105 cells/well in DMEM medium and maintained under conditions mentioned previously until 70% confluency. The cells were treated with various concentrations of
Lipopolysaccharide-Induced Cell Viability Assay
For cell viability assay, RAW 264.7 cells were seeded in a 96-well plate at a density of 1 × 105 cells/well in DMEM medium and maintained under conditions mentioned previously until 70%confluency. The cells were treated with 1 μg/ml of lipopoly-saccharide (LPS;
Nitric Oxide Determination
The nitric oxide (NO) production in LPS-induced RAW 264.7 cells, upon treatment with
2,2-Diphenyl-1-Picrylhydrazyl (DPPH) Radical Scavenging Assay
The
Determination of the Level of Pro-Inflammatory Factors
RAW 264.7 cells (3 × 105 cells/well in 6-well plate) were cultured with 1 μg/ml of LPS and
-
Table 1 . Oligonucleotide primers used in this study..
Genes Oligonucleotide Sequences Annealing Temperature References TNF-α FACTGAACTTCGGGGTGAT 60°C [43] TNF-α RACTTGGTGGTTTGCTACG TLR4 FTTCAGAACTTCAGTGGCTGGATTTA 60°C [43] TLR4 RGTCTCCACAGCCACCAGATTCTC GAPDH FCATGACCACAGTCCATGCCATCAC 60°C [17] GADPH RTGAGGTCCACCACCCTGTTGCTGT
Western Blot Analysis
RAW 264.7 cells (3 × 105 cells/well in 6-well plate) were cultured with 1 μg/ml of LPS and
Statistical Analyses
All values were expressed as mean ± standard deviation from triplicate runs (
Results
Curcumin Content of Unfermented and Lactobacillus -Fermented Turmeric
HPLC analysis revealed that fermentation with
-
Table 2 . HPLC analysis of the curcumin content of
Lactobacillus fermented turmeric..Samples Curcumin content before fermentation, mg/g Curcumin content after fermentation, mg/g L. fermentum -fermented1.855 ± 0.026 2.03 ± 0.012 *** L. plantarum -fermented1.971 ± 0.010 ** L. brevis -fermented1.880 ± 0.013 Results are expressed as mean ± standard deviation of triplicate runs (
n = 3). Paired T-test analysis was performed with asterisks (*) indicating that curcumin contents before and after fermentation differ significantly (**p < 0.01; ***p < 0.001)..
Cytotoxicity of Lactobacillus -Fermented Turmeric
Upon fermentation, the
-
Figure 1. Cell viability of murine macrophages, RAW 264.7 cells upon treatment with different concentrations of
Lactobacillus -fermented turmeric (500.00, 250.00, 125.00, 62.50, and 31.25 μg/ml in 0.1% [v/v] DMSO). Untreated RAW 264.7 cells were used as naive control. Results are expressed as means ± standard deviations (n = 3). Tukey’s multiple range analysis was performed with different lowercase letters (a, b, c, and d) demonstrating the significant differences between samples (p < 0.05).
Cell Viability of LPS-Induced RAW 264.7 Cells
In this study, an enhanced protective effect was observed on
-
Figure 2. Cell viability of murine macrophages, RAW 264.7 cells upon stimulation with lipopolysaccharides (LPS) from
Escherichia coli and treatment with different concentrations ofLactobacillus -fermented turmeric. Non-LPS-induced RAW 264.7 cells were used as a naive control. Results are expressed as means ± standard deviations (n = 3). Tukey’s multiple range analysis was performed with different lowercase letters (a and b) demonstrating the significant differences between samples (p < 0.05).
Nitric Oxide Assay
In the current study, the accumulated nitrite in the culture supernatant was determined via the Griess method as an index for NO produced by the LPS-induced RAW 264.7 cells. As shown in Fig. 3, the production of nitrite was reduced upon treatment with
-
Figure 3. Effect of
L. fermentum -fermented turmeric (31.25 μg/ml) on nitrite production in LPS-induced RAW 264.7 cells. Results are expressed as means ± standard deviations (n = 3). Paired T-test analysis was performed to determine the group differences. Control group vs. samples (***p < 0.001); untreated group vs. samples (###p < 0.001).
2,2-Diphenyl-1-Picrylhydrazyl (DPPH) Radical Scavenging Activity
In this study, the antioxidant activity of
-
Figure 4. DPPH radical scavenging activity of
L. fermentum fermented (31.25 μg/ml) and unfermented turmeric (31.25 μg/ml). Results are expressed as means ± standard deviations (n = 3). Positive control consists of 500 μg/ml L-ascorbic acid. Paired T-test analysis was performed to determine the group differences. Negative control group vs. samples (**p < 0.01; ***p < 0.001); Positive control group vs. samples (##p < 0.01; ###p < 0.001).
Gene Expression of Pro-Inflammatory Factors
The gene expressions of TNF-α and TLR4 in LPS-induced RAW 264.7 cells upon treatment with
-
Figure 5. Effects of
L. fermentum -fermented turmeric (31.25 μg/ml) on the gene expression of (a) tumor necrosis factor-alpha (TNF-α) and (b) Toll-like receptor 4 (TLR4) in LPS-induced RAW 264.7 cells. Results are expressed as means ± standard deviations (n = 3). Paired T-test analysis was performed, with asterisks (*) indicating that the differences between untreated control and samples within the same group are significant (*p < 0.05; **p < 0.01).
Western Blot Analysis
The results obtained from the western blot analysis further justified the anti-inflammatory activity of
-
Figure 6. Effects of
L. fermentum -fermented turmeric (31.25 μg/ml) on the activation of phosphorylated-JNK (p-JNK) and TNF-α of LPS-induced RAW 264.7 cells. (A ) Protein levels of p-JNK and TNF- α were detected by western blotting using p-JNK and TNF-α specific antibodies, where β-actin was used as an internal control. Data shown are representative of three independent experiments (n = 3). (B ) Relative protein expression of p-JNK and TNF-α was quantified via densitometry analysis. Results are expressed as means ± standard deviations (n = 3). Paired T-test analysis was performed, with asterisks (*) indicating that the differences between control and samples within the same antibody group are significant (**p < 0.01; ***p < 0.001).
Discussion
Turmeric has been long used as a food additive and herbal medicine owing to its wide range of biological activity, including anti-inflammatory [9, 10], anti-diabetes [21, 22], and anti-Alzheimer’s disease activities [3, 4]. The wide range of pharmacological benefits is attributed to the presence of curcumin, the major bioactive constituent of turmeric. Curcumin only makes up 2-5% of turmeric [23], hence, probiotics fermentation has been used to improve the curcumin content and pharmacological activities of turmeric. In this study, HPLC analysis revealed that fermentation with
Although the curcumin content has been improved via fermentation, it is of utmost importance to validate the cytotoxicity of the
Subsequently, efforts were made to determine the protective effect of
Pro-inflammatory cytokines are produced by the activated macrophages and are essential to elicit the inflammatory responses. The TLR4, a transmembrane protein on the surface of macrophages, monocytes, and dendritic cells, belongs to the pattern recognition receptor family and is essential for the initiation of the innate immune response [27]. Hence, the presence of LPS is detected by the immune cells, recognized by TLR4 to form a TLR4-myeloid differentiation protein 2 (MD2)-LPS complex, resulting in the activation of intracellular signaling pathway NF-kB and innate immune system to promote inflammation and drive pro-inflammatory cascades [27-29].
As a pro-inflammatory cytokine, TNF-α initiates the pro-inflammatory cascades, recruiting and activating various inflammatory cells for recovery purposes [30, 31]. However, the over-production of TNF-α could lead to chronic inflammation and cell apoptosis [31]. In the present study,
In addition, LPS also induces oxidative stress by stimulating the activated macrophages for the production of NO and ROS. A high level of ROS produced upon LPS stimulation will alter host homeostasis condition, thereby causing oxidative damage and cell apoptosis [32]. Notably,
Previous studies also reported that a high level of NO could induce apoptosis directly or indirectly by rendering many cells susceptible to apoptosis [37, 38]. The data showed that the protective effect of
Interestingly, the protective effect of
The present study clearly demonstrated that
Acknowledgments
This study was supported by the South Jeolla Province (2016 R & D Support Program operated by Jeonnam Technopark).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.

Fig 2.

Fig 3.

Fig 4.

Fig 5.

Fig 6.

-
Table 1 . Oligonucleotide primers used in this study..
Genes Oligonucleotide Sequences Annealing Temperature References TNF-α FACTGAACTTCGGGGTGAT 60°C [43] TNF-α RACTTGGTGGTTTGCTACG TLR4 FTTCAGAACTTCAGTGGCTGGATTTA 60°C [43] TLR4 RGTCTCCACAGCCACCAGATTCTC GAPDH FCATGACCACAGTCCATGCCATCAC 60°C [17] GADPH RTGAGGTCCACCACCCTGTTGCTGT
-
Table 2 . HPLC analysis of the curcumin content of
Lactobacillus fermented turmeric..Samples Curcumin content before fermentation, mg/g Curcumin content after fermentation, mg/g L. fermentum -fermented1.855 ± 0.026 2.03 ± 0.012 *** L. plantarum -fermented1.971 ± 0.010 ** L. brevis -fermented1.880 ± 0.013 Results are expressed as mean ± standard deviation of triplicate runs (
n = 3). Paired T-test analysis was performed with asterisks (*) indicating that curcumin contents before and after fermentation differ significantly (**p < 0.01; ***p < 0.001)..
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