Skip to main content
NCBI home page
Food Science and Biotechnology logoLink to Food Science and Biotechnology
. 2023 Feb 21;32(9):1257–1268. doi: 10.1007/s10068-023-01259-4

Effect of fermentation on antioxidant, antimicrobial, anti-inflammatory, and anti-Helicobacter pylori adhesion activity of Ulmus davidiana var. japonica root bark

Seong-Hyeon Lee 1,#, Jeong-Muk Lim 1,#, Se-Won Lee 1, Tae-Hu Jang 1, Jung-Hee Park 1, Young-Seok Seo 2, Jeong-Ho Lee 3, Kamala-Kannan Seralathan 1, Byung-Taek Oh 1,✉
PMCID: PMC10290026  PMID: 37362805

Abstract

The limited yield of Ulmus davidiana var. japonica root bark (URB) extract is considered an economic loss to the food industry. Improving extraction yield and bioactivity through fermentation increase the industrial usage of URB. The study aims to optimize the fermentation with cellulolytic and pectinolytic bacteria and evaluate the bioactivity and anti-Helicobacter pylori activity of the fermented URB extract. URB fermentation with the Bacillus licheniformis FLa3, isolated from salted seafood (Sardinella zunasi), under optimal conditions (37 °C, pH 6, 10% inoculum dose, and 36 h) improved the extraction yield by 36% compared to the control. The antioxidant and antimicrobial activity of the fermented extract were significantly higher than non-fermented extract. High-performance liquid chromatography results confirmed that the fermentation increased the proportion of bioactive components such as catechin (171.7%), epicatechin (144.3%), quercetin (27.3%), and kaempferol (16.7%). The results confirmed that the fermentation increased both the extraction yield and bioactivity.

Keywords: Ulmus davidiana var. japonica root bark, Fermentation, Hydrolytic activity, Bioactivity, Anti-Helicobacter pylori

Introduction

Plants contain several phytochemicals and mostly these phytochemicals are present in free or soluble conjugated form, whereas some exist in insoluble complex forms within plant cell wall structures such as cellulose, hemicellulose, lignin, pectin, and rod-shaped structural protein. Insoluble phytochemicals present in plant-based food materials are not easily bioavailable to the human system. Improving the yield of phytochemicals in plant-based foods is an efficient and economically viable benefit to the food industry. Fermentation is a well-known traditional processing technique that is used to improve the nutritional quality and health-promoting properties of various plant-based foods. It is a less expensive process than using commercial enzymes. In addition to extending the shelf life of foods and improving their organoleptic properties, fermentation also causes the conversion of macronutrients (carbohydrates and proteins) and the release of antioxidant peptides and phenolic compounds (Leonard et al., 2021). Fermentation with functional microorganisms such as lactic acid bacteria, yeast, and Bacillus spp. converts the chemical composition of the plant materials, thereby, increasing the nutritional value of the foods and providing health benefits (Tamang et al., 2016). Microbial hydrolytic enzymes such as α-amylase, β-glycosidase, cellulase, pectinase, and xylanase have significant roles in improving the nutritional value of fermented food materials (Madeira Junior et al., 2015). In the nutraceutical and pharmaceutical industries, cellulase and pectinase are commonly used for the efficient extraction of various bioactive compounds from natural plants. During fermentation, cellulase and pectinase enhance the elution of some active ingredients by hydrolyzing cellulose and pectin, the main components of the cell wall.

Ulmus davidiana var. japonica (Ulmaceae) is widely distributed in various regions of Asian countries, and the Ulmus davidiana var. japonica root bark (URB) is well-known as “yugeunpi” in Korean traditional medicine. The bioactive components present in the URB extract have antioxidant, anti-arthritic, anti-angiogenic, anti-tumorigenic, and anti-H. pylori activity (So et al., 2019). Catechin isolated from URB extract was reported to inhibit the accumulation of intracellular reactive oxidative species (ROS) in tumor necrosis factor-α (TNF-α)-stimulated normal human dermal fibroblasts (Lee et al., 2020a, b). However, there is no similar study on fermented URB extract. Furthermore, URB is considered a suitable raw material for fermentation because it contains different sugars such as rhamnose, galactose, glucose, fucose, galactosamine, and glucosamine.

Helicobacter pylori is a Gram-negative spiral-shaped bacteria that is designated as a class I carcinogen by the World Health Organization (WHO) and is known to be one of the most infectious microorganisms that commonly infect children. H. pylori is responsible for stomach infections and is susceptible to several types of antibiotics (Graham, 2015). Earlier, H. Pylori infection was almost universally observed in humans, but currently, only half of the world's population is infected. H. pylori eradication rates, which were above 80% in the early 1990s, have now decreased to 60% in some countries inversely with reported rates of antibiotic resistance (Thung et al., 2016). Recently, the range of pathogenic microorganisms resistant to antibiotics is on the rise due to the indiscriminate use of antibiotics. Furthermore, indiscriminate use of antibiotics causes gastrointestinal side effects such as nausea, indigestion, vomiting, bloating, and abdominal pain. These studies indicate the need to develop natural and/or fermented plant-based foods that can replace antibiotics.

With this background, this study was aimed to (i) isolate and identify cellulolytic and pectinolytic bacteria from salted seafood, (ii) optimize URB fermentation conditions with isolated bacteria, (iii) evaluate the bioactive constituents present in the fermented extract, and (iv) assess the fermented extract for antioxidant, antimicrobial, anti-inflammatory, and anti-adhesion activity for H. pylori.

Materials and methods

Plant material and bacterial strains

The URB powder obtained from local farms at Inje-gun, Gangwon-do, South Korea was stored (4 °C) and used in the experiment. Salted seafoods (Sepia esculenta, Loligo beka, Trichiurus lepturus, and Sardinella zunasi), purchased from local markets at Iksan-si, Jeollabuk-do, South Korea, were used for the isolation of beneficial bacteria. Briefly, 0.1 mL of serially diluted food samples were aseptically inoculated onto the surface of MRS or LB agar by spread plate technique. The agar plate was incubated at 25 °C, and the formation of bacterial colonies was observed. Cellulolytic and pectinolytic activity were screened by inoculating the isolates on carboxymethyl cellulose (CMC) agar (CMC 5 g/L, NaNO3 1 g/L, K2HPO4 1 g/L, MgSO4 0.5 g/L, yeast extract 0.5 g/L, and agar 15 g/L) and pectin agar (pectin 10 g/L, NaNO3 2 g/L, KCl 0.5 g/L, MgSO4 0.5 g/L, K2HPO4 1 g/L, tryptone 0.5 g/L, and agar 20 g/L). The plates were incubated at 37 °C for 2 days and observed for clear zone around bacterial colonies after staining with Congo Red solution.

URB fermentation

URB powder (1 g) was added to distilled water and autoclaved at 121 °C for 15 min. The mixture was sufficiently cooled, bacterial suspension (FLa3, FE2, S-1, and S-391 strain, 5 × 1011 CFU/mL in phosphate buffer) was aseptically inoculated, and the final volume was adjusted to 30 mL using sterile water. The flasks were incubated at 37 °C (120 rpm) for 36 h, and the suspension was extracted with 95% ethyl alcohol for 1 h, concentrated using a vacuum rotary evaporator, and freeze-dried using a lyophilizer. The extraction yield was calculated by measuring the weight of the freeze-dried solid material. Experimental variables in the URB fermentation were pH (5–9), inoculum dose (5–15%, v/v), temperature (25–45 °C), and incubation time (0–72 h).

Relative enzymatic activity

Relative cellulase and pectinase activity were evaluated using 3,5-dinitrosalicylic acid (DNS) method (Bhadrecha et al., 2020). 1% CMC solution (in 0.1 M citrate buffer, pH 5.5) and 1% pectin solution (in 0.1 M citrate buffer, pH 5.5) were used as a substrate for enzyme activity assay. Substrate solution (0.4 mL) and the same volume of enzyme solution were mixed at 50 °C for 30 min. Later, 0.6 mL of DNS reagent was added to the reaction mixture and heated at 100 °C for 15 min. The reaction mixture was cooled, and the absorbance was measured at 540 nm using a microplate reader (Tecan infinite M200Pro, Männedorf, Zürich, Switzerland). Calibration curves of glucose and galacturonic acid were used to calculate reducing sugar in the reaction mixture. The relative enzyme activity was calculated by comparing the reducing sugar contents in samples.

Identification of selected strain

The selected strain was identified through partial 16S rRNA sequencing. The genomic DNA was extracted using a DNA extraction kit (QIAGEN, USA) and 16S rRNA was amplified using universal primers 27F and 1492R. Purified amplicons were sequenced using an Automated DNA sequencer (3730XL DNA analyzer, Applied Biosystem, USA) and were compared with the sequence registered in the NCBI database. The phylogenetic tree using the partial 16S rRNA sequences was constructed using the neighbor-joining method with MEGA 7.0 software.

Antioxidant activity

Samples (3.2–10,000 μg/mL) were serially diluted (five times) with ethyl alcohol and used for antioxidant assay. Superoxide dismutase (SOD)-like activity was measured using SOD Assay Kit-WST (Dojindo Laboratory, Japan) according to the manufacturer’s protocol. The H2O2 scavenging activity was measured using a 2,2′-azinobis(3-ethylbenzthiazolin)-6-sulfonic acid (ABTS)-peroxidase system (Muller, 1985). Briefly, 0.1 mL of 10 mM H2O2 and 0.5 mL of citro-phosphate buffer (pH 5.0) were mixed with 0.4 mL of diluted samples and incubated at 37 °C for 5 min. Later, 0.15 mL of 1.25 mM ABTS and 0.15 mL of 1 U/mL peroxidase were added to the mixture, incubated at 37 °C for 10 min, and the absorbance was measured at 405 nm.

Antimicrobial activity against pathogenic bacteria

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of non-fermented and fermented extracts were determined against Helicobacter pylori SS1, Salmonella typhimurium KVCC-BA0000693, and Listeria monocytogenes KVCC-BA0000087. The MIC test was performed by dilution method in a 96-well plate. The extracts were adjusted to various concentrations (5, 7.5, 10, 25, 37.5, 50, 75, 100, 125, 150, 175, 200 mg/mL), the pathogenic bacteria (H. pylori: 1.0 × 108 CFU/mL, S. typhimurium and L. monocytogenes: 2.0 × 108 CFU/mL) were inoculated into the wells, and incubated at 37 °C for 24 h (H. pylori was cultured under a 10% CO2 atmosphere). After incubation, turbidity was evaluated at 600 nm. The concentration at which turbidity did not increase was considered as MIC. The MBC was determined as a concentration at which no colonies were observed (Lee et al., 2020a, b).

HPLC analysis

The bioactive constituents in the non-fermented and fermented extract of URB were analyzed using an Agilent 1220 Infinity II system (Agilent Technologies, USA) equipped with a Variable Wavelength Detector (VWD) and a ZORBAX Eclipse Plus C18 column (5 μm, 250 × 4.6 mm, Agilent Technologies, USA). Prior to use, the sample and standard were filtered through a 0.2 μm filter. For detection of catechin and epicatechin, the mobile phase consisted of (A) 1% formic acid in water and (B) 1% formic acid in methanol was used according to the following gradient: (0 min) 95% A:5% B, (10 min) 80% A:20% B, (35 min) 70% A:30% B, (45 min) 50% A:50% B. The conditions for HPLC were injection volume of 10 μL (25 °C) at 280 nm. For detection of kaempferol and quercetin, the mobile phase consisted of (A) 0.1% H3PO4 in water and (B) 0.1% H3PO4 in ACN was used according to the following gradient: (0 min) 85% A:15% B, (10 min) 55% A:45% B, (15 min) 45% A:55% B, (20 min) 15% A:85% B. The conditions for HPLC were injection volume of 20 μL (25 °C) at 370 nm.

Cell line studies

AGS cells (human stomach adenocarcinoma cell line, KCLB 21739) were obtained from the Korean Cell Line Bank (KCLB, South Korea). The cell line was cultured using an RPMI 1640 medium containing 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin (P/S) at 37 °C in a 5% CO2 atmosphere condition (humidified incubator). The cells were detached from the cell culture dish using TrypLE Express Enzyme and used for cell viability, anti-adhesion, and anti-inflammatory studies.

Cell viability was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to analyze the cytotoxicity of non-fermented and fermented extract. AGS cells were seeded in a 96-well plate at a density of 1 × 105 cells/mL and incubated for 24 h. The culture medium was removed and the sample diluted using RPMI 1640 medium containing 10% (v/v) FBS and 1% (v/v) P/S was treated on the cell monolayer and incubated at 37 °C for 24 h in a 5% CO2 atmosphere. After incubation, 50 μL of MTT solution (5 mg/mL) was added to each well and incubated for 4 h. The MTT solution was picked off, and the MTT-formazan was dissolved by adding 150 μL of dimethyl sulfoxide. Absorbance was measured at 540 nm using a microplate reader.

Anti-adhesion activity of the non-fermented and fermented extract against H. pylori on AGS cells was evaluated by modified phenol red method (Saavedra et al., 1994). AGS cells were seeded in a 6-well plate at a density of 1 × 105 cells/mL and incubated for 24 h. The H. pylori suspension (MOI 200) and extracts of various concentrations containing RPMI medium supplemented with yeast extract were treated on the cell monolayer and incubated at 37 °C for 6 h in a 5% CO2 atmosphere. Non-adherent H. pylori were washed twice with PBS, and a 2 mL urease reagent (20% urea and 0.012% phenol red in PBS, pH 6.5) was added to evaluate the relative amount of attached H. pylori on AGS cells. After incubation at 37 °C for 1 h, absorbance was measured at 562 nm using a microplate reader.

AGS cells were seeded in a 24-well plate at a density of 1 × 105 cells/mL. The H. pylori suspension (MOI 200) and URB extracts diluted with RPMI medium supplemented with yeast extract were treated on the cell monolayer and incubated at 37 °C for 6 h in a 5% CO2 atmosphere. Determination of nitric oxide (NO) was performed by Griess reagent assay (Saha et al., 2004). Briefly, cell culture supernatant (100 μL) was added to 100 μL of Griess reagent (1% sulfanilamide and 0.1% naphthyl ethylene diamide in 5% phosphoric acid) and reacted at room temperature for 10 min. Absorbance was measured at 540 nm and a calibration curve of sodium nitrite was used to calculate NO concentration. Pro-inflammatory cytokines, such as TNF-α and interleukin 8 (IL-8), were determined by enzyme-linked immunosorbent assay (ELISA) kit (BioLegend, USA) according to the manufacturer’s protocol.

Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was extracted from the AGS cells using TRIzol reagent (QIAGEN, USA) and quantified using a NanoDrop spectrophotometer (NanoDrop Technologies, Montchanin, DE, USA). cDNA was synthesized by reverse transcription using QuantiTect Reverse Transcription Kit (QIAGEN, USA) according to the manufacturer’s instructions. Subsequent PCR amplification was conducted using 2X AmpMaster Taq in thermal cycler using primers.

Western blotting

AGS cells grown with H. pylori in a 6-well plate were rinsed with PBS and then lysed with a radioimmunoprecipitation assay (RIPA) buffer containing a protease inhibitor cocktail at 4 °C for 10 min. The lysates were centrifuged, and the supernatant was collected. Bradford protein assay was performed to measure the protein concentration of cell lysate and an appropriate volume of the lysate was diluted with sample loading buffer. The proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using 10% separating gel combined with 4% stacking gel (30 µg protein/lane) and transferred to a PVDF membrane by electroblotting. The membranes were blocked with 5% skimmed milk in TBST buffer (Tris-buffered saline and 0.1% tween 20) and were incubated overnight at 4 °C with primary antibody for cytotoxin-associated antigen A (CagA), vacuolating cytotoxin A (VacA), and β–actin (Santa Cruz Biotechnology, Dallas, TX, USA). After washing with TBST buffer, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (anti-rabbit or anti-mouse lgG) for 2 h and were visualized using enhanced chemiluminescence (ECL) kit (Thermo Scientific, Waltham, MA, USA) and ChemiDoc imaging system (Bio-Rad, USA).

Statistical analysis

Statistical analysis of mean of triplicated experiments ± standard deviation (SD) was performed using the statistical package, Graph Pad Prism 8.0. Experimental results were analyzed by one-way analysis of variance (ANOVA) and Tukey test. Different letters indicate significant differences between groups (p < 0.05).

Results and discussion

Selection and identification of strain for fermentation

Morphologically, 11 different colonies were observed in the LB plates, and 4 strains (S-1, FE2, S-391, and FLa3) were confirmed to have cellulolytic and pectinolytic activity. Each strain was inoculated individually, and fermentation of URB extract was performed to select the efficient bacterial strain. Among the 4 strains, FLa3 increased the extraction yield of URB extract by about 37%, and thus it was used for further studies (Fig. 1(A)). Microbial fermentation is commonly used to solubilize the insoluble phytochemicals present in plant materials. During fermentation, microorganisms generate plant cell wall degrading enzymes and degrade the plant cell wall, thereby increasing the solubility of insoluble complex compounds. Previous studies reported that plant cell wall degrading enzymes such as cellulase and pectinase is mainly used to improve the yield of bioactive phenolic compounds from pistachio green hull (Ghandahari Yazdi et al., 2019) and Syrah grape pomace (Meini et al., 2019). Based on the partial 16S rRNA sequence analysis, the FLa3 strain was identified as Bacillus licheniformis. This was supported by the results of phylogenetic analysis, where the FLa3 and B. licheniformis DSM 13 were in the same cluster with 99.7% 16S rRNA sequence identity (Fig. 1B). A previous study reported high cellulase and pectinase activity of B. licheniformis KIBGE-IB3 (Aslam et al., 2020).

Fig. 1.

Fig. 1

Open in a new tab

Selection of strains and various factors for optimal fermentation. (A) Comparison of relative enzyme activity and extraction yield after fermentation; (B) Phylogenetic tree of Bacillus licheniformis FLa3; (C) Temperature (°C); (D) pH; (E) Inoculum (%, v/v); (F) Fermentation time (h). Different letters indicate significant differences between groups (p < 0.05)

Optimization of the fermentation conditions

Temperature is one of the vital factors that play an important role in enzyme activity. Thus, fermentation was performed at different temperatures (25–45 °C), and the results are represented in Fig. 1(C). Cellulase and pectinase activity were maximum at 37 to 45 °C. Maximum extraction yield (4.9%) was observed at 37 °C and which was 5–12% high when compared to other temperatures. The enzyme activity under different pH (5–9) was evaluated and the results are shown in Fig. 1(D). Maximum cellulase and pectinase activity (100.0%) was observed at pH 6 and minimum activity (23.9% and 31.5%) was observed at pH 5. Furthermore, the initial pH of URB extract also altered the enzyme yield, and it was observed that acidic pH 6 increased the extraction yield by 3–20% compared to other initial pH conditions. The effect of the inoculum dose on the enzyme activity and extraction yield was evaluated and the results are shown in Fig. 1(E). Maximum enzyme activity (100%) and extraction yield (4.8%) were observed at 10% inoculum dose than 5% inoculum dose. The effect of fermentation time on enzyme activity and extraction yield was evaluated and the results are shown in Fig. 1(F). Extraction yield (4.9%) was observed after 36 h of fermentation, and the extraction yield increased by 36.6% compared to the non-fermented group. However, if the fermentation is carried out for more or less than 36 h, both enzyme activity and yield were decreased. Also, both initial bacterial density (10.9 log CFU/mL) and pH 6 were gradually decreased to 5.9 log CFU/mL and pH 5.5 with increasing the fermentation time to 72 h. The decrease in yield after 36 h of incubation could be due to several factors such as decreased bacterial density, instability of the enzymes at acidic pH, and bioconversion of some bioactive constituents that may alter enzyme synthesis. However, this phenomenon needs more study. Enzyme synthesis and activities are highly influenced by environmental conditions such as temperature, pH, microbial density, and incubation time. In addition, since cellulase and pectinase have different enzyme activity at specific temperatures and pH depending on the enzyme-producing organism, each condition should be considered and optimized with enzyme activity. From the results it was observed that the optimum conditions for the FLa3 strain were 37 °C, pH 6, 10% inoculum dose, and incubation time of 36 h (Fig. 1(C)–(F). Our results are consistent with previous studies that reported the optimal condition for cellulase and pectinase production in B. licheniformis. Kim et al. (2008) reported that cellulase produced from B. licheniformis DK42 showed high enzymatic activity at pH 6 and temperature stability at 35–45 °C. B. licheniformis KIBGE-IB3 synthesis pectinase enzyme at pH 7.0 at 37 °C (Aslam et al. 2020). Previous studies have shown that temperature affects the secretion of extracellular enzymes by changing the physical properties of the cell membrane, and pH affects the transport of chemical products and enzymes across the cell membrane (Liang et al., 2009; Sethi et al., 2013). Also, a further increase in inoculum dose is less efficient because high bacterial density increases competition between substrates, nutrients, and bacteria.

Antioxidant and antimicrobial activity

The antioxidant activity of the non-fermented and fermented URB extract is shown in Table 1. The IC50 values of SOD-like activity and hydrogen peroxide scavenging activity of fermented URB extract are about 164.06 and 4246.20 µg/mL (non-fermented URB extract: 302.00 and 4920.40 µg/mL). The antimicrobial activity was evaluated by comparing the MIC and MBC of the URB extracts against pathogenic microorganisms (Table 1). The MIC and MBC of the non-fermented extract were 150 and 175 mg/mL, respectively, for H. pylori, 125 and 175 mg/mL for S. typhimurium, and 100 and 150 mg/mL for L. monocytogenes. However, for fermented extract, it was 100 and 125 mg/mL for H. pylori, 100 and 125 mg/mL for S. typhimurium, and 75 and 125 mg/mL for L. monocytogenes. The antioxidant and antimicrobial activity of the extract has been improved by the bioconversion of B. licheniformis FLa3 through fermentation.

Table 1.

Antioxidant and antimicrobial activity of non-fermented and fermented URB extract

Antioxidant activity
Sample SOD-like (IC50, μg/mL) H2O2 scavenging (IC50, μg/mL)
Non-fermented 302.00 ± 1.08 4920.40 ± 1.17
Fermented 164.06 ± 1.53 4246.20 ± 1.17
L-ascorbic acid 103.87 ± 1.25 96.07 ± 1.19
Antimicrobial activity
Pathogenic bacteria Sample MIC (mg/mL) MBC (mg/mL)
H. pylori Non-fermented 150 175
Fermented 100 125
S. typhimurium Non-fermented 125 175
Fermented 100 125
L. monocytogenes Non-fermented 100 150
Fermented 75 125
Open in a new tab

Comparison of bioactive constituents in URB extracts

Bioactive constituents in both fermented and raw URB extract were analyzed to confirm the changes in the contents of catechin, epicatechin, quercetin, and kaempferol, and the results are shown in Fig. 2. As the fermentation of URB using B. licheniformis FLa3 progressed, the contents of catechin and epicatechin increased by 171.7% and 144.3% (from 1.59 and 0.88 mg/g to 4.32 and 2.15 mg/g). Also, the contents of quercetin and kaempferol, which are common polyphenols present in most plants, were increased by 27.3% (from 0.11 to 0.14 mg/g) and 16.7% (from 0.06 to 0.07 mg/g), respectively (Table 2). Acosta-Estrada et al. (2014) reported an increase in kaempferol content in cabbage leaves through fermentation with Lactobacillus plantarum and Lactobacillus collinoides. Also, Phanerocheate chrysosporium increased the total polyphenol content (3.5 times) of apple waste extract through fermentation (Ajila et al., 2011). The increase in solubility of phytochemicals contributed to the high antioxidant and antimicrobial activity of the fermented extract (Table 1). Catechins with high antioxidant activity were present in several plant sources such as grape seeds and pine bark, and showed high antimicrobial activity against various pathogenic bacteria, regardless of Gram stain-based bacterial differentiation (Ma et al., 2019). Epicatechin belongs to a group of flavonoids with antioxidant, antimicrobial, anti-inflammatory, anti-tumor, and cardioprotective activity (Calabriso et al., 2016). Quercetin and kaempferol are polyphenolic compounds present in various edible plants and medicinal plants, thus being a common element in a healthy diet. With potent free-radical scavenging, cardioprotective, hypotensive, anti-diabetic, and anti-tumor activity, quercetin has been reported to possess anti-H. pylori activity by inhibiting the virulence factor VacA (Butkovic et al., 2004; Russo et al., 2012). A study on the H. pylori growth inhibitory activity of kaempferol and epicatechin reported that both polyphenol compounds inhibited the growth of H. pylori 26695 and H. pylori 43504 strains, and epicatechin showed a dose-dependent protective role on AGS cells from H. pylori infection (Escandón et al., 2016).

Fig. 2.

Fig. 2

Open in a new tab

HPLC chromatogram of non-fermented and fermented URB extract. (A) Catechin and epicatechin; (B) Quercetin and kaempferol

Table 2.

The contents of catechin, epicatechin, quercetin, and kaempferol in the non-fermented and fermented URB extract

Sample Contents (mg/g)
Catechin Epicatechin Quercetin Kaempferol
Non-fermented 1.59 ± 0.01 0.88 ± 0.01 0.11 ± 0.00 0.06 ± 0.00
Fermented 4.32 ± 0.02 2.15 ± 0.01 0.14 ± 0.00 0.07 ± 0.00
Open in a new tab

Effect of URB extracts on the cytotoxicity, anti-adhesion, and anti-inflammatory activity

Cytotoxicity of non-fermented and fermented URB extract on AGS cells is shown in Fig. 3(A). Treatment with non-fermented and fermented URB extract at a high concentration range showed cytotoxicity. However, each extract below 500 μg/mL had no effect on the cell viability of AGS cells. The extracts effectively inhibited the adhesion activity of H. pylori. Compared to the H. pylori-infected group (100%), adhesion activity was 65% and 50% in the 500 μg/mL non-fermented and fermented URB extract-treated groups, respectively, (Fig. 3(B)). H. pylori has excellent survival, proliferation, and disease-causing ability due to its unique delivery mechanism and sophisticated virulence factors (Suerbaum and Michetti, 2002). Adhesion to gastric epithelial cells and colonization in the gastric mucosa are some of the prerequisites for gastric disorder pathogenesis (Rieder et al., 2005). The URB extract effectively inhibited the attachment of H. pylori to AGS cells, which was further enhanced through fermentation (Fig. 3(A)–(B)). According to previous studies, the herbal extract of Ixeris chinensis was reported to inhibit the adhesion activity of H. pylori (Lu et al., 2018).

Fig. 3.

Fig. 3

Open in a new tab

Cytotoxicity, anti-adhesion, and anti-inflammatory activity of non-fermented and fermented URB extract in AGS cells. (A) Cell viability; (B) Adhesion activity of H. pylori (MOI 200); (C) NO production; (D) TNF-α secretion; (E) IL-8 secretion. Different letters indicate significant differences between groups (p < 0.05)

The effects of non-fermented and fermented URB extract on the inflammation in AGS cells by H. pylori were investigated (Fig. 3(C)–(E)). Treatment with non-fermented and fermented URB extracts effectively inhibited NO production induced by H. pylori in a concentration-dependent manner. Compared to the H. pylori-infected group (100%), NO production was decreased to 69% and 57% in the 500 μg/mL non-fermented and fermented URB extract-treated groups (Fig. 3(C)). Compared with the H. pylori-infected group (100%), in the 500 μg/mL non-fermented and fermented URB extract-treated groups, TNF-α was 78% and 74% and IL-8 was 75% and 65% (Fig. 3(D)–(E)).

Effect of URB extracts on gene expression of proinflammatory cytokines and translocation of CagA and VacA to AGS cell

RT-PCR was used to compare the mRNA levels of TNF-α and IL-8 on AGS cells infected with H. pylori. The results of gene expression analysis of proinflammatory cytokines shown in Fig. 4 support the results of TNF- α and IL-8 production. Treatment of non-fermented and fermented URB extract significantly suppressed H. pylori-induced TNF-α and IL-8 gene expression. The fermented URB extract exhibited higher anti-inflammatory activity such as decreased gene expression and lower production of pro-inflammatory cytokines than the non-fermented URB extract. A previous study reported that kaempferol reduced H. pylori-induced increases in mRNA levels of TNF-α and IL-8 (Yeon et al., 2019). The fermented extract contains higher kaempferol compared to non-fermented extract and it was responsible for decreased gene expression and lower production of TNF-α and IL-8.

Fig. 4.

Fig. 4

Open in a new tab

Effect of non-fermented and fermented extract on the mRNA expression level in AGS cells infected with H. pylori and translocation of CagA and VacA to AGS cells from H. pylori. (A)–(B) Relative levels of TNF-α and IL-8 normalized to GAPDH; (C) Expression of CagA and VacA analyzed by western blotting; (D)–(E) Relative intensity of CagA and VacA normalized to β-actin. Different letters indicate significant differences between groups (p < 0.05)

The effects of non-fermented and fermented URB extract on the translocation of CagA and VacA from H. pylori to AGS cells were measured using Western blotting and the results are represented in Fig. 4(C)–(E). Treatment with non-fermented URB extract effectively inhibited CagA and VacA translocation in a concentration-dependent manner. Also, a significant decrease in the CagA and VacA translocation was observed after fermentation. The bioactive compounds contained in the extract inhibited the translocation of toxic proteins by inhibiting H. pylori adhesion to AGS cells. CagA is translocated to the host cell through the cag PAI-encoded type IV secretion system (T4SS), and the translocated CagA is phosphorylated inside the host cell to induce morphological cell changes. Unphosphorylated CagA can affect tight junctions in cells, cell polarity, cell proliferation and differentiation, cell scattering, induction of inflammation, and cell elongation. Severe atrophic gastritis and high levels of gastric mucosal inflammation are highly correlated with CagA-positive H. pylori infection (Ferreira Junior et al., 2015). VacA is secreted through the Type V secretion system (T5SS) of H. pylori and VacA complex enters the host cell by endocytosis (Jain et al., 2011). Internalized VacA accumulates and induces apoptosis, thereby promoting the destruction of epithelial cell tight junctions and suppressing the proliferation effect of T cells recruited to the site of infection causing inhibition of T cells activation (Djekic and Muller, 2016). T cells with downregulated activity result in upregulation of pro-inflammatory cytokines such as IL-8 and TNF-a through activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) (Kalali et al., 2014). Hence, the internalization of CagA and VacA causes the expression of inflammatory and proinflammatory cytokines in gastric epithelial cells. Our results confirmed that the translocation of CagA and VacA proteins expressed in H. pylori to AGS cells was suppressed by the fermented extract (Fig. 4(C)–(E)). Various plant-derived extracts exhibiting anti-H. pylori activity (Ngnameko et al., 2019), and kaempferol present in the plant products was reported to inhibit the translocation of CagA and VacA (Yeon et al., 2019).

In summary, B. licheniformis FLa3 isolated from salted seafood (Sardinella zunasi) was used to increase the yield of phytochemicals present in the URB extract. The strain FLa3 has excellent cellulolytic and pectinolytic activity. The optimized fermentation conditions were 37 °C, pH 6, 10% inoculum dose, and 36 h incubation time. The level of bioactive compounds such as catechin (2.71 times), epicatechin (2.44 times), quercetin (1.27 times), and kaempferol (1.17 times) was increased in fermented extract compared to non-fermented extract. The fermented extract inhibited the adhesion of H. pylori to AGS cells and the induction of inflammation and reduced the translocation of CagA and VacA to AGS cells from H. pylori. These results demonstrated the benefits of the fermentation system using cellulolytic and pectinolytic strains and improved anti-H. pylori activity of the fermented extract. Accordingly, fermented extracts can be used as functional foods that can replace antibiotics.

Acknowledgements

This work was supported by a grant from the Honam National Institute of Biological Resources (HNIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (HNIBR202102121).

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Seong-Hyeon Lee and Jeong-Muk Lim are co-first authors.

Contributor Information

Seong-Hyeon Lee, Email: dltjdgus7712@naver.com.

Jeong-Muk Lim, Email: limjm1227@kakao.com.

Se-Won Lee, Email: dltpdnjs1029@naver.com.

Tae-Hu Jang, Email: xogn960@naver.com.

Jung-Hee Park, Email: junghee.park@jbnu.ac.kr.

Young-Seok Seo, Email: dudtjr3610@naver.com.

Jeong-Ho Lee, Email: wooju1119@naver.com.

Kamala-Kannan Seralathan, Email: kannan@jbnu.ac.kr.

Byung-Taek Oh, Email: btoh@jbnu.ac.kr.

References

  1. Acosta-Estrada BA, Gutierrez-Uribe JA, Serna-Saldivar SO. Bound phenolics in foods, a review. Food Chemistry. 2014;152:46–55. doi: 10.1016/j.foodchem.2013.11.093. [DOI] [PubMed] [Google Scholar]
  2. Ajila CM, Brar SK, Verma M, Tyagi RD, Valéro JR. Solid-state fermentation of apple pomace using Phanerocheate chrysosporium-liberation and extraction of phenolic antioxidants. Food Chemistry. 2011;126:1071–1080. doi: 10.1016/j.foodchem.2010.11.129. [DOI] [Google Scholar]
  3. Aslam F, Ansari A, Aman A, Baloch G, Nisar G, Baloch AH, Rehman HU. Production of commercially important enzymes from Bacillus licheniformis KIBGE-IB3 using date fruit wastes as substrate. Journal of Genetic Engineering and Biotechnology. 2020;18:46. doi: 10.1186/s43141-020-00060-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bhadrecha P, Bala M, Khasa YP, Arshi A, Singh J, Kumar M. Hippophae rhamnoides L. rhizobacteria exhibit diversified cellulase and pectinase activities. Physiology and Molecular Biology of Plants. 2020;26:1075–1085. doi: 10.1007/s12298-020-00778-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Butkovic V, Klasinc L, Bors W. Kinetic study of flavonoid reactions with stable radicals. Journal of Agricultural and Food Chemistry. 2004;52:2816–2820. doi: 10.1021/jf049880h. [DOI] [PubMed] [Google Scholar]
  6. Calabriso N, Scoditti E, Massaro M, Pellegrino M, Storelli C, Ingrosso I, Giovinazzo G, Carluccio MA. Multiple anti-inflammatory and anti-atherosclerotic properties of red wine polyphenolic extracts: Differential role of hydroxycinnamic acids, flavonols and stilbenes on endothelial inflammatory gene expression. European Journal of Nutrition. 2016;55:477–489. doi: 10.1007/s00394-015-0865-6. [DOI] [PubMed] [Google Scholar]
  7. Djekic A, Muller A. The immunomodulator VacA promotes immune tolerance and persistent Helicobacter pylori infection through its activities on T-cells and antigen-presenting cells. Toxins (basel) 2016;8:187. doi: 10.3390/toxins8060187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Escandón RA, del Campo M, López-Solis R, Obreque-Slier E, Toledo H. Antibacterial effect of kaempferol and (-)-epicatechin on Helicobacter pylori. European Food Research and Technology. 2016;242:1495–1502. doi: 10.1007/s00217-016-2650-z. [DOI] [Google Scholar]
  9. Ferreira Junior M, Batista SA, Vidigal PV, Cordeiro AA, Oliveira FM, Prata LO, Diniz AE, Barral CM, Barbuto RC, Gomes AD, Araujo ID, Queiroz DM, Caliari MV. Infection with CagA-positive Helicobacter pylori strain containing three EPIYA C phosphorylation sites is associated with more severe gastric lesions in experimentally infected mongolian gerbils (Meriones unguiculatus) European Journal of Histochemistry. 2015;59:2489. doi: 10.4081/ejh.2015.2489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ghandahari Yazdi AP, Barzegar M, Sahari MA, Ahmadi GH. Optimization of the enzyme-assisted aqueous extraction of phenolic compounds from pistachio green hull. Food Science and Nutrition. 2019;7:356–366. doi: 10.1002/fsn3.900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Graham DY. Helicobacter pylori update: Gastric cancer, reliable therapy, and possible benefits. Gastroenterology. 2015;148:719–31 e3. doi: 10.1053/j.gastro.2015.01.040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jain P, Luo ZQ, Blanke SR. Helicobacter pylori vacuolating cytotoxin A (VacA) engages the mitochondrial fission machinery to induce host cell death. Proceedings of the National Academy of Sciences. 2011;108:16032–16037. doi: 10.1073/pnas.1105175108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kalali B, Mejias-Luque R, Javaheri A, Gerhard MH. pylori virulence factors: influence on immune system and pathology. Mediators of Inflammation. 2014;2014:426309. doi: 10.1155/2014/426309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kim MJ, Lim SJ, Kang DK. Isolation of a Bacillus licheniformis DK42 producing cellulase and xylanase, and properties of the enzymes. Journal of Animal Science and Technology. 2008;50:429–436. doi: 10.5187/JAST.2008.50.3.429. [DOI] [Google Scholar]
  15. Lee SW, Lim JM, Mohan H, Seralathan KK, Park YJ, Lee JH, Oh BT. Enhanced bioactivity of Zanthoxylum schinifolium fermented extract: anti-inflammatory, anti-bacterial, and anti-melanogenic activity. Journal of Bioscience and Bioengineering. 2020;129:638–645. doi: 10.1016/j.jbiosc.2019.12.003. [DOI] [PubMed] [Google Scholar]
  16. Lee S, Yu JS, Phung HM, Lee JG, Kim KH, Kang KS. Potential anti-skin aging effect of (-)-catechin isolated from the root bark of Ulmus davidiana var. japonica in tumor necrosis factor-alpha-stimulated normal human dermal fibroblasts. Antioxidants. 2020;9:981. doi: 10.3390/antiox9100981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Leonard W, Zhang P, Ying D, Fang Z. Lignanamides: sources, biosynthesis and potential health benefits: A minireview. Critical Reviews in Food Science and Nutrition. 2021;61:1404–1414. doi: 10.1080/10408398.2020.1759025. [DOI] [PubMed] [Google Scholar]
  18. Liang Y, Feng Z, Yesuf J, Blackburn JW. Optimization of growth medium and enzyme assay conditions for crude cellulases produced by a novel thermophilic and cellulolytic bacterium, Anoxybacillus sp. 527. Applied Biochemistry and Biotechnology. 2009;160:1841–1852. doi: 10.1007/s12010-009-8677-x. [DOI] [PubMed] [Google Scholar]
  19. Lu MC, Chiu HF, Lin CP, Shen YC, Venkatakrishnan K, Wang CK. Anti-Helicobacter pylori effect of various extracts of ixeris chinensis on inflammatory markers in human gastric epithelial AGS cells. Journal of Herbal Medicine. 2018;11:60–70. doi: 10.1016/j.hermed.2017.08.002. [DOI] [Google Scholar]
  20. Ma Y, Ding S, Fei Y, Liu G, Jang H, Fang J. Antimicrobial activity of anthocyanins and catechins against foodborne pathogens Escherichia coli and Salmonella. Food Control. 2019;106:106712. doi: 10.1016/j.foodcont.2019.106712. [DOI] [Google Scholar]
  21. Madeira Junior JV, Teixeira CB, Macedo GA. Biotransformation and bioconversion of phenolic compounds obtainment: An overview. Critical Reviews in Biotechnology. 2015;35:75–81. doi: 10.3109/07388551.2013.803020. [DOI] [PubMed] [Google Scholar]
  22. Meini MR, Cabezudo I, Boschetti CE, Romanini D. Recovery of phenolic antioxidants from syrah grape pomace through the optimization of an enzymatic extraction process. Food Chemistry. 2019;283:257–264. doi: 10.1016/j.foodchem.2019.01.037. [DOI] [PubMed] [Google Scholar]
  23. Muller HE. Detection of hydrogen peroxide produced by microorganisms on an ABTS peroxidase medium. Zentralbl Bakteriol Mikrobiol Hyg a. 1985;259:151–154. doi: 10.1016/s0176-6724(85)80045-6. [DOI] [PubMed] [Google Scholar]
  24. Ngnameko CR, Njayou FN, Fowora M, Nemg FBS, Fewou PM, Smith SI. Inhibitory effect of medicinal plants from cameroon on the growth and adhesion of Helicobacter pylori. European Journal of Integrative Medicine. 2019;30:100957. doi: 10.1016/j.eujim.2019.100957. [DOI] [Google Scholar]
  25. Rieder G, Fischer W, Haas R. Interaction of Helicobacter pylori with host cells: Function of secreted and translocated molecules. Current Opinion in Microbiology. 2005;8:67–73. doi: 10.1016/j.mib.2004.12.004. [DOI] [PubMed] [Google Scholar]
  26. Russo M, Spagnuolo C, Tedesco I, Bilotto S, Russo GL. The flavonoid quercetin in disease prevention and therapy: Facts and fancies. Biochemical Pharmacology. 2012;83:6–15. doi: 10.1016/j.bcp.2011.08.010. [DOI] [PubMed] [Google Scholar]
  27. Saavedra JM, Bauman N, Perman J, Yolken R, Oung I. Feeding of Bifidobacterium bifidum and Streptococcus thermophilus to infants in hospital for prevention of diarrhoea and shedding of rotavirus. The Lancet. 1994;344:1046–1049. doi: 10.1016/S0140-6736(94)91708-6. [DOI] [PubMed] [Google Scholar]
  28. Saha K, Lajis NH, Israf DA, Hamzah AS, Khozirah S, Khamis S, Syahida A. Evaluation of antioxidant and nitric oxide inhibitory activities of selected malaysian medicinal plants. Journal of Ethnopharmacology. 2004;92:263–267. doi: 10.1016/j.jep.2004.03.007. [DOI] [PubMed] [Google Scholar]
  29. Sethi S, Datta A, Gupta BL, Gupta S. Optimization of cellulase production from bacteria isolated from soil. ISRN Biotechnology. 2013;2013:985685. doi: 10.5402/2013/985685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. So HM, Yu JS, Khan Z, Subedi L, Ko YJ, Lee IK, Park WS, Chung SJ, Ahn MJ, Kim SY, Kim KH. Chemical constituents of the root bark of Ulmus davidiana var. japonica and their potential biological activities. Bioorganic Chemistry. 2019;91:103145. doi: 10.1016/j.bioorg.2019.103145. [DOI] [PubMed] [Google Scholar]
  31. Suerbaum S, Michetti P. Helicobacter pylori infection. The New England Journal of Medicine. 2002;347:1175–1186. doi: 10.1056/NEJMra020542. [DOI] [PubMed] [Google Scholar]
  32. Tamang JP, Watanabe K, Holzapfel WH. Review: diversity of microorganisms in global fermented foods and beverages. Frontiers in Microbiology. 2016;7:377. doi: 10.3389/fmicb.2016.00377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Thung I, Aramin H, Vavinskaya V, Gupta S, Park JY, Crowe SE, Valasek MA. Review article: The global emergence of Helicobacter pylori antibiotic resistance. Aliment. Pharmacology and Therapeutics. 2016;43:514–533. doi: 10.1111/apt.13497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yeon MJ, Lee MH, Kim DH, Yang JY, Woo HJ, Kwon HJ, Moon C, Kim SH, Kim JB. Anti-inflammatory effects of kaempferol on Helicobacter pylori-induced inflammation. Bioscience, Biotechnology, and Biochemistry. 2019;83:166–173. doi: 10.1080/09168451.2018.1528140. [DOI] [PubMed] [Google Scholar]

Articles from Food Science and Biotechnology are provided here courtesy of Springer

RESOURCES