Introduction
Helicobacter pylori is a gram-negative bacterium that approximately has 50% global human infection rate (Suerbaum and Michetti, 2002). H. pylori easily survive in the gut’s acidic environment. Thus, it can easily cause various gastrointestinal diseases such as gastritis, peptic ulcers, and stomach cancer (Danesh, 1999). The development of gastric cancer is known to be caused by inflammatory responses produced in gastric epithelial cells against chronic H. pylori infection (Wen and Moss, 2009). The primary causes of inflammatory responses in H. pylori infections are the cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA), which are associated with the progression to gastric cancer (Suriani et al., 2008). CagA is delivered into gastric epithelial cells through a type IV secretion system, and once inside the cell, it can activate the NF-kB pathway, leading to the production of pro-inflammatory cytokines (Jones et al., 2010). VacA induces the production of reactive oxygen species in gastric epithelial cells, which can also result in the production of pro-inflammatory cytokines (Jones et al., 2010). Due to the carcinogenic potential of H. pylori, the World Health Organization (WHO) and International Agency for Research on Cancer (IARC) classified H. pylori as a Group I carcinogen in 1994 (Lochhead and El-Omar, 2007).
Currently, commonly used eradication treatment for H. pylori infection is combination therapy with antibiotics such as ampicillin, amoxicillin, and metronidazole, along with acid-reducing medications (Mahony et al., 1992). However, antibiotic treatments have many health risks. Indeed, 22,292 patients infected with H. pylori who received these treatments, 22% experienced side effects such as a metallic taste, diarrhea, nausea, and vomiting (Nyssen et al., 2021). Therefore, the development and evaluation of safer alternatives to antibiotics are necessary to help patients who are sensitive or allergic to prescription antibiotics (Hafeez et al., 2021).
A possible solution to the aforementioned problem is the utilization of bacteriocin, antibacterial peptides, produced by bacteria (Todorov et al., 2019). Nisin, the most well-known bacteriocin, is an antibacterial peptide produced by generally recognized as safe (GRAS) registered Lactococcus lactis strains and it has been approved as a food preservative through Food and Agriculture Organization (FAO) and WHO (Chalón et al., 2012). Thus, nisin is widely used as a natural food bio-preservative due to the antibacterial effect against a wide range of clinical and food-borne pathogens (Amiri et al., 2021). Recent studies have also suggested that nisin has anti-inflammatory effects by inhibiting the production of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1 β) in rat uterine (Jia et al., 2019). Also, nisin helped wound healing process by down regulating pro-inflammatory cytokines in human keratinocytes (Mouritzen et al., 2019).
However, fermentative production of nisin is complicated and requires additional purification step which increases overall cost of production. This makes nisin relatively more expensive to produce as compared to other antibacterial substances (Elshaghabee et al., 2016). Recently, ongoing research is exploring new ways to use nisin at a lower cost. Previous studies have sought to find substances that can enhance the antibacterial effect of nisin, which is possible to reduce the amount of nisin to help save costs (Kirazli and Tunca, 2022). For example, combination of nisin with essential oils, such as oregano, thyme, or rosemary, have been found to have a synergistic effect, resulting in a reduction in the required dosage of nisin (Bajpai et al., 2012).
Lactic acid (LA), produced by lactic acid bacteria (LAB), is known to effective against a broad range of bacteria to increase the permeability of bacterial membrane (Alakomi et al., 2000). In previous study, nisin and LA can work synergistically to increase the overall antibacterial activity of Escherichia coli in red meat (De Martinez et al., 2002). Also, the combination of nisin and other substances that can enhance the antibacterial effect of nisin can allow for the use of lower concentrations of both agents, which can help to reduce costs (Field et al., 2017).
Yogurt, a type of fermented milk, is a widely consumed fermented dairy product that is popular around the world. During the fermentation process, LAB strains produce LA, which transforms milk into yogurt by coagulation (Jeong et al., 2018). Trials adding nisin to yogurt have not been attempted due to concerns that it may inactivate LAB in yogurt. However, several evidence showed the positive health effects of inactivated LAB, called parabiotics (Kazemi et al., 2021). Parabiotics have shown to have beneficial health effects such as supporting the immune system, improving gut health by supporting the growth of beneficial bacteria, reducing inflammation, and improving digestion (Kazemi et al., 2021). Therefore, exploring the potential health benefits of incorporating nisin into post-fermented yogurt would be a valuable research endeavor.
Consequently, our study aimed to investigate the antibacterial effects of yogurt, which naturally contains LA, and explore the feasibility of supplementing it with nisin. This investigation will assess the potential for incorporating nisin into post-fermented yogurt. We also assessed the antibacterial effect of nisin and LA, as well as their impact on H. pylori bacterial toxins, bacterial membrane damage and inflammatory cytokines in human gastric (AGS) cells infected with H. pylori.
Materials and Methods
Preparation of nisin
A commercial nisin standard (Sigma-Aldrich Chemical, St. Louis, Mo, USA) was used in the present work. A stock nisin solution was prepared by commercial nisin standard into 10 mL of sterile 0.02 N HCl, centrifuging at 5,000×g for 15 min and sterilizing by filtration through 0.22 μm filters. A stock nisin solution was stored at 4℃ until use.
Microorganism culture condition
H. pylori ATCC 43504 strains (obtained from American Type Culture Collection, Manassas, VA, USA) was used as the test organism. H. pylori was grown in Brucella agar (MB Cell, Seoul, Korea) supplemented with 5% fetal bovine serum (FBS; Welgene, Gyeongsan, Korea). A single colony from brucella agar was inoculated in 10 mL of brucella broth supplemented with 5% FBS and incubated at 37℃ with shaking under microaerobic conditions (5%–8% carbon dioxide) until the cell density reached 1×109 CFU/mL.
Broth microdilution method
Minimum inhibitory concentration (MIC) values of nisin, LA, and ampicillin against H. pylori was determined using broth microdilution method on 96-well plates. Briefly, each well was inoculated with 100 μL of H. pylori adjusted to 1×106 CFU/mL. Serial 2-fold dilutions of nisin, LA, and ampicillin were treated in each well at final concentrations of 0.01 to 40 μg/mL, 0.04% to 5%, and 0.01 to 50 μg/mL, respectively. The final total volume of each well was 200 μL, and the final bacterial number was adjusted to 5×105 CFU/mL. The MIC value was defined as the lowest concentration of nisin and LA alone or in combination with nisin and LA that showed visible growth inhibition after 24 h incubating at 37℃ under micro-aerobe condition.
Checkerboard assay
A checkerboard assay was carried out to evaluate the synergistic effects of nisin and LA (Zhao et al., 2023). Briefly, the rows on the x-axis of the 96-well plate contained 2-fold dilution of nisin, and the columns on the y-axis contained 2-fold dilution of LA. The final concentration of H. pylori was adjusted to 5×105 CFU/mL for each well and then the plates were incubated at 37℃ under micro-aerobe condition. The synergistic effects of each combination were determined by the fractional inhibitory concentration index (FICI; Shi et al., 2017). The FICI was calculated as following formula: FICI=ΣFIC=FIC (nisin)+FIC (LA), where FIC (nisin)=MIC of nisin in combination/MIC of nisin alone, and FIC (LA)=MIC of LA in combination/MIC of LA alone. The interpretations of ΣFIC values were as follows: Synergy, if ΣFIC≤0.5; no interaction, if ΣFIC>0.5 to <2, and antagonism, if ΣFIC≥2.
Yogurt and supernatant preparation
Yogurt was produced using 12% (w/v) of non-fat milk (Seoul Dairy, Seoul, Korea). The milk was pasteurized at 85℃ for 30 min and then cooled to 24℃. Next, a starter culture (2%, v/v) was added to the milk. The starter culture was prepared by incubating a mixture of 100 mL of non-fat milk (12%, w/v) and 0.34 g of starter culture powder (Samik Dairy and Food, Seoul, Korea) for 5 h. The starter culture contained mixed strains of Lactobacillus acidophilus (35%), Bifidobacterium longum (30%), and Streptococcus thermophilus (35%). The inoculated milk samples were then placed in an incubator at 42℃ until they reached a pH of 4.5–4.6. The supernatants from the yogurt were separated by centrifuging samples (10 g) twice at 4,000×g for 10 min at 4℃. Subsequently, the supernatants were further centrifuged at 10,000×g for 10 min at 4℃. They were then filtered through 0.45-µm syringe filter (Advantec, Tokyo, Japan) and stored at –80℃ until use.
Lactic acid (LA) measurement
To determine the amount of LA in the yogurt, the titratable acidity was measured. At the end of the fermentation process, 10 g of yogurt was mixed with 10 mL of distilled water and titrated to a pH of 8.3 using 0.1 N NaOH. The TA was calculated using the following formula:
Titratable acidity (%) = (mL of NaOH used × 0.009) / (weight of yogurt in grams) × 100 (1)
Here, 0.009 is the conversion factor for LA, which is used to convert the volume of NaOH using into an equivalent amount of LA.
Comparative evaluation of antibacterial effects of yogurt supplemented with nisin and combination of nisin and lactic acid (LA)
To evaluate the antibacterial effect, the agar well diffusion assay was used for both the yogurt supplemented with nisin and the combination of nisin and LA. A 100 μL aliquot of a 1×108 CFU/mL H. pylori suspension was spread over brucella agar with 5% FBS, and then nisin (0, 1.25, 2.5, or 5 μg/mL) or nisin-supplemented yogurt supernatant (0, 1.25, 2.5, or 5 μg/mL) were added to each well on the agar. To further evaluate the effects of nisin, LA and their combination, and ampicillin (positive control), each was added to individual wells in the agar with different concentrations. The clear inhibition zone diameter (mm) was measured after 24 h of incubation at 37℃ under micro-aerobe conditions.
Lactate dehydrogenase (LDH) activity assay
Lactate dehydrogenase (LDH) assay was carried out by investigating the cytotoxicity of H. pylori (Sohn et al., 2020). In brief, AGS cells grown in 96-well plate were treated with different concentration (106, 107, 108, and 109 CFU/mL) of H. pylori and incubated at 37℃ for 24 h. 45 min before the end of incubation, cells were treated with lysis buffer for a positive control. After that, the medium was transferred to a 1.7 mL tube. Centrifugation was done to collect the supernatants. Supernatants 50 μL were transferred to a new 96-well plate and 50 μL of the Cytotox 96® reagent were added. The 96-well plate was incubated at 25℃ for 30 min in the dark. The optical value was measured at 490 nm with spectrophotometer, and the percentage of LDH was calculated as following formula:
LDH release (%) = [(ODsample LDH release / ODmaximum LDH release) × 100] (2)
Cell culture and treatment
AGS cells were maintained in RPMI 1640 medium (Welgene) containing 10% FBS, 1% penicillin /streptomycin (v/v) at 37℃ in humidified atmosphere containing 5% CO2. For the experiment, H. pylori was centrifuged and washed twice with phosphate-buffered saline (PBS). Before cell treatment, H. pylori was resuspended in antibiotic-free RPMI 1640 medium and diluted to the desired concentration.
Cell viability assay
The cell viability was evaluated by the trypan blue dye exclusion assay (Kim et al., 2021). AGS cells were grown in 6-well plates and treated with different concentration of nisin, LA, their combination, or ampicillin, and incubated at 37℃ for 24 h. Viable cell number were counted using a hemocytometer (Hausser Scientific, Horsham, PA, USA) under an optical microscope.
Real-time polymerase chain reaction analysis
The levels of mRNA expression for bacterial toxins (i.e., VacA and CagA) and pro-inflammatory cytokines (IL-6, IL-8, IL-1β, and TNF-α) in AGS cells were evaluated by RT-PCR. For quantification of mRNA expression of H. pylori toxin factors, 100 μL of H. pylori culture was inoculated in 10 mL of brucella broth containing 5% FBS with different concentration of nisin, LA, its combination and ampicillin for 3 h. Then, the bacterial pellet was obtained through centrifugation at 8,000×g for 5 min at 4℃. TRIzol reagent (Life Technologies, Eugene, OR, USA) 500 μL was used to extract RNA and TOPscript RT DryMIX kit (Enzynomics, Daejeon, Korea) was used for cDNA synthesis. The reference gene 16S rRNA expression levels were used to quantify the relative mRNA expression levels. To quantify mRNA expression of pro-inflammatory cytokines, AGS cells were grown in a 6-well plate and treated with 108 CFU/mL of H. pylori in antibiotic-free RPMI medium for 6 h. Afterward, the bacteria-containing medium was withdrawn and rinsed thrice with PBS. Next, the cells were treated with nisin, LA, its combination or ampicillin for 3 h. After incubation, the medium was withdrawn and rinsed thrice with PBS. TRIZOL 500 μL was used to extract RNA, and TOPscript RT DryMIX kit was used for cDNA synthesis. The reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression levels were used to quantify the relative mRNA expression levels. The PCR conditions were as follows: 95℃ for 15 min (denaturation) and 80 cycles of amplification at 95℃ for 20 s, followed by respective annealing temperature and finally held at 4℃. The primer sequences used in current study are shown in Table 1.
Table 1. Primers used in the current study
IL-1β, interleukin-1 beta; IL-6, interleukin 6; IL-8, interleukin 8; TNF-α, tumor necrosis factor-alpha; VacA, vacuolating cytotoxin A; CagA, cytotoxin-associated gene A; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; 16s rRNA, 16s ribosomal ribonucleic acid.
Measurement of DNA and protein leakage
DNA and protein leakage was determined by measuring the absorbance of culture supernatants using spectrophotometer (Kirazli and Tunca, 2022). In brief, nisin (5 μg/mL), LA (0.6%), their combination, or ampicillin (6.25 μg/mL) were added to the 108 CFU/mL of H. pylori suspensions in 1×PBS. The suspensions were incubated at 37℃ with shaking under microaerobic conditions for 3 h. Then, 0.22 μm microporous membrane was used to remove the bacteria. The leakage of DNA and protein was measured using Epoch microplate spectrophotometry at the absorbances of 260 and 280 nm, respectively.
Synergistic effect of nisin and lactic acid (LA) on morphological changes in Helicobacter pylori
The morphology changes of H. pylori were observed by field emission scanning electron microscope (FE-SEM; SU-8010, Hitachi, Tokyo, Japan). H. pylori suspensions were treated with the antibacterial substances at 37℃ for 3 h under micro-aerobe condition (5%–8% carbon dioxide). After incubation, the cells were collected and washed thrice with PBS and were fixed with 2.5% glutaraldehyde overnight at 4℃. After washing with PBS, the cells were dehydrated with 30%, 50%, 70%, 90% ethanol solutions subsequently. After freeze drying, the cells were fixed on the FE-SEM support and sputtered with platinum under vacuum. Then, the morphologies were observed by FE-SEM.
Statistical analysis
Data are presented as mean±SEM. All statistical analyses were performed with SPSS-PASW Ver. 18.0 (SPSS, Chicago, IL, USA). Analysis of variance (ANOVA) was analyzed using one-way ANOVA with Dunnett’s test post hoc test. Differences were considered statistically significant when p-values were <0.05.
Results
Minimum inhibitory concentration (MIC) of nisin and lactic acid (LA), and their synergistic antibacterial effects in combination
The MIC of nisin, LA, and ampicillin alone or in combination against H. pylori was shown in Table 2. The MIC of nisin against H. pylori was determined to be 10 μg/mL and the MIC of LA and ampicillin was determined to be 0.6% and 0.195 μg/mL, respectively. The combined effect of nisin and LA was calculated using checkerboard test (Table 2). The synergistic effect of nisin and LA was observed against H. pylori. After combining nisin and LA, MIC of nisin was 1.25 μg/mL (8 times lower than the MIC when used alone) and MIC of LA was 0.15% (4 times lower than the MIC when used alone). As shown in Table 2, the combined FICI value of nisin and LA was determined to 0.375, indicating synergism (FICI<0.5). The results suggest that combination of nisin and LA could effectively inhibit H. pylori growth and lower the concentration of nisin and LA.
Table 2. MIC of nisin, lactic acid, their combination, or ampicillin and the FICI values of combinations against Helicobacter pylori
MIC, minimum inhibitory concentration; FICI, fractional inhibitory concentration index.
Antibacterial effects of nisin-added yogurt supernatant, and nisin, lactic acid (LA) or its combination against Helicobacter pylori
The yogurt contained 0.61±0.01% LA based on titratable acidity. Agar well diffusion method was performed to determine the combined antibacterial effect of nisin (1.25, 2.5, and 5 μg/mL) and yogurt supernatant (Fig. 1A). The combination of yogurt supernatant with 5 μg/mL of nisin exhibited the highest inhibitory activity against H. pylori, with an inhibition zone diameter of 14.33±0.33 mm. This inhibition zone was significantly greater than that of nisin alone as well as than that of yogurt supernatant alone (Fig. 1A). In fact, nisin alone did not exhibit any inhibition against H. pylori at all concentration. To further investigate the antibacterial effects of nisin, LA, nisin-LA combination, an agar well diffusion assay was conducted (Fig. 1B). Different concentrations of nisin (1.25, 2.5, and 5 μg/mL) were combined with 0.6% LA, and their antibacterial activities were compared with those of nisin and LA alone, as well as with ampicillin. The combination of nisin (5 μg/mL) with LA (0.6%) showed the highest antibacterial activity against H. pylori, with an inhibition zone diameter of 22.33±0.33 mm, which was significantly greater than that of the same concentrations of nisin and LA treated alone, and comparable to that of ampicillin (22.67±0.67 mm).
Fig. 1. Antibacteral effects of nisin, LA, and nisin-supplemented yogurt against Helicobacter pylori. (A) Agar well diffusion assay was conducted to determine nisin, yogurt supernatant and their combination effects against H. pylori (N: nisin μg/mL; Y: yogurt supernatant) and inhibition zone diameters (mm). (B) Agar well diffusion assay conducted to determine nisin, LA and their combination effects against H. pylori (N: nisin μg/mL; LA: lactic acid %; C: control; Amp: ampicillin μg/mL) and inhibition zone diameters (mm). Values represent means±SEM (n=3). Statistical significance was calculated via one-way ANOVA with Dunnett’s test post hoc test. ** p<0.01 and *** p<0.001 indicate a significant difference compared to the yogurt supernatant only. ### p<0.001 shows a significant difference compared to the nisin 5 μg/mL treated only. †† p<0.01 and ††† p<0.001 indicate a significant difference compared to the control. +++ p<0.001 shows a significant difference compared to the nisin at 5 μg/mL and LA at 0.6%, respectively. ANOVA, analysis of variance.
Cytotoxicity of nisin, lactic acid (LA), combination of nisin and LA, and Helicobacter pylori in human gastric (AGS) cells
Trypan blue dye exclusion assay was used to evaluate the effect of nisin, LA, their combination, or ampicillin of AGS cell viability (Fig. 2A). Treatment of cells with nisin (1.25, 2.5, 5 μg/mL) and ampicillin (3.125, 6.25 μg/mL) for 24 h did not show any significant changes in cell viability compared to the control (p>0.05). Similarly, treatment with LA (0.3%, 0.6%) and combination of nisin and LA group did not significantly affect cell viability (p>0.05). However, treatment with 1.2% of LA for 24 h resulted in a significant decrease in cell viability (p<0.001) compared to the control. Based on these results, further in vitro studies were performed using 5 μg/mL nisin, 0.6% LA, and 6.25 μg/mL ampicillin. AGS cells treated with a concentration of 109 CFU/mL of H. pylori for 24 h showed significantly higher LDH release than control (Fig. 2B). Therefore, a concentration of 108 CFU/mL of H. pylori was used for further in vitro studies.
Fig. 2. Cell viability of nisin, LA, and their combination and cytotoxicity of AGS cells treated with Helicobacter pylori. (A) The percentage of viable cells treated with nisin, LA, their combination, or ampicillin was calculated relative to the untreated control. (B) The cytotoxicity of H. pylori was determined by measuring LDH release after treatment with various concentrations of H. pylori (106, 107, 108, and 109 CFU/mL). Values represents means±SEM (n=3). Statistical significance was calculated via one-way ANOVA with Dunnett’s test post hoc test. *** p<0.001 show a significant difference, compared to the control. LA, lactic acid; AGS, human gastric; LDH, lactate dehydrogenase; ANOVA, analysis of variance.
Reduction of mRNA levels of bacterial toxins in Helicobacter pylori
The results of the mRNA levels of bacterial toxins (i.e., CagA and VacA) in H. pylori are presented in Fig. 3. Treatment of H. pylori with 5 μg/mL of nisin resulted in a decrease in mRNA expression levels of CagA (Fig. 3A) and VacA (Fig. 3B), while LA or ampicillin alone did not affect mRNA levels (p>0.05). However, when nisin and LA were used in combination, the mRNA level of CagA decreased significantly, compared to the control (p<0.001). Similarly, the mRNA level of VacA decreased significantly compared to the control (p<0.05). These results suggest that the combination of nisin and LA effectively reduce bacterial toxins in H. pylori.
Fig. 3. Relative mRNA expression levels of bacterial toxins in Helicobacter pylori following treatment with nisin, LA, and their combination. (A) CagA and (B) VacA mRNA expression levels in H. pylori. H. pylori were treated with nisin, LA, their combination, or ampicillin for 3 h. Values represent means±SEM (n=3). Statistical significance was calculated via one-way ANOVA with Dunnett’s test post hoc test. * p<0.05, and *** p<0.001 indicate a significant difference compared to control. LA, lactic acid; ANOVA, analysis of variance.
Anti-inflammatory effect of nisin and lactic acid (LA) in human gastric (AGS) cells infected with Helicobacter pylori
The mRNA expression levels of pro-inflammatory cytokines, including TNF-α, IL-1β, IL-8, and IL-6, increased in AGS cells upon addition of 108 CFU/mL of H. pylori (Figs. 4A, B, C, and D). Treatment of AGS cells with nisin alone resulted in a decrease in the mRNA expression of these cytokines, while LA alone didn’t show a significant decrease in their mRNA expressions. However, when nisin and LA were used in combination, a significant reduction in the mRNA levels of the pro-inflammatory cytokines was observed, with levels similar to the control without H. pylori infection (p>0.05) for all pro-inflammatory cytokines. In contrast, the ampicillin-treated group showed no such effects. These results suggest that the combination of nisin and LA may have a synergistic effect in downregulating the inflammatory in H. pylori infected AGS cells.
Fig. 4. Anti-inflammatory effect of nisin, LA, and their combination on pro-inflammatory cytokine expression in AGS cells infected with Helicobacter pylori. (A) TNF-α, (B) IL-1β, (C) IL-8, and (D) IL-6 mRNA expression in AGS cells. AGS cells were pretreated with 108 CFU/mL of H. pylori for 6 h and then treated with nisin, LA, their combination, or ampicillin for 3 h. Values represent means±SEM (n=3). Statistical significance was calculated via one-way ANOVA with Dunnett’s test post hoc test. * p<0.05, ** p<0.01 and *** p<0.001 indicate a significant difference compared to control. # p<0.05, ## p<0.01 and ### p<0.001 show a significant difference compared to cells only treated with H. pylori. LA, lactic acid; AGS, human gastric; TNF-α, tumor necrosis factor-alpha; IL-1β, interleukin-1 beta; IL-8, interleukin 8; IL-6, interleukin 6; ANOVA, analysis of variance.
DNA and protein leakage in Helicobacter pylori
The effect of nisin and LA on the leakage of the DNA and protein from H. pylori is shown in Fig. 5. The results showed that the highest levels of DNA and protein leakage were observed in samples treated with 5 μg/mL nisin and 0.6% of LA in combination (Fig. 5A and B). Ampicillin showed similar degree of DNA and protein leakage like nisin-LA combination. However, nisin or LA alone at the same concentration did not show any significant differences in DNA and protein leakage, compared to the control (p>0.05).
Fig. 5. Effect of nisin, LA, and their combination on DNA and protein leakage from Helicobacter pylori. (A) DNA leakage and (B) protein leakage were measured by absorbance at 260 and 280 nm, respectively. H. pylori was cultured for 3 h in the presence of nisin, LA, their combination, or ampicillin. Values represents means±SEM (n=3). Statistical significance was calculated via one-way ANOVA with Dunnett’s test post hoc test. ** p<0.01, *** p<0.001 show a significant difference, compared to the control. # p<0.05 and ## p<0.01 show a significant difference compared to the nisin at 5 μg/mL and LA at 0.6%, respectively. LA, lactic acid; ANOVA, analysis of variance.
Disruption of Helicobacter pylori observed by field emission scanning electron microscope (FE-SEM)
FE-SEM was used to visualize the morphology of H. pylori which was treated with nisin, LA, its combination or ampicillin (Fig. 6). The control group showed intact, smooth, and homogeneous bacterial surfaces (Fig. 6A). Treatment of H. pylori with LA alone caused slight deformation to the bacterial surface (Fig. 6B). Nisin alone resulted in slight membrane damage, with some areas of the bacterial surface appearing rough and uneven (Fig. 6C). However, the nisin and LA combination caused more extensive damage to the bacterial membrane, with significant deformation, and in some cases, visible fragments of the bacteria were observed (Fig. 6D). Ampicillin also resulted in membrane damage with strongly ruptured surfaces (Fig. 6E).
Fig. 6. Effect of nisin, LA, and their combination on the ultrastructure of Helicobacter pylori. The membrane damage (white arrow) of H. pylori in FE-SEM image. (A) Control, (B) nisin (5 μg/mL), (C) LA (0.6%), (D) nisin (5 μg/mL)+LA (0.6%), (E) ampicillin (6.25 μg/mL). The scale bars in the images are 1 µm. LA, lactic acid; FE-SEM, field emission scanning electron microscope.
Discussion
The complete eradication of H. pylori is a challenging task due to its persistent nature in the gut. Antibiotic therapy is the primary approach for H. pylori eradication. However, it may cause dysbiosis in the gut microbiota and modulate the immune system, resulting in unwanted effects like metallic taste, diarrhea, nausea, or vomiting during the treatment process (Çalışkan et al., 2022). Consequently, researchers are seeking safe antibiotic alternatives that are devoid of such side effects (Seal et al., 2013). In this regard, nisin is not only safe but also has a broad antibacterial effect on bacteria (Norouzi et al., 2018). However, due to its complex purifying process and limited supply, nisin is relatively expensive compared to other antibacterial substances (Zhao et al., 2022). Thus, there are several ongoing research regarding development and improvement of nisin production by LAB (Özel et al., 2018).
LA is a metabolite of LAB and is known to be a safe substance. It has been extensively studied for its ability as a permeabilizer to disrupt the outer membrane of bacteria by causing changes in the membrane lipid composition (Gyawali et al., 2011). Yogurt or fermented milk is a fermented dairy product, which possess plenty of LA due to the metabolites of LAB during fermentation. Although yogurt has a plenty of LA, there is no data regarding incorporation of nisin to yogurt due to concerns that nisin could inactivate LAB in fermented food. Although nisin can inactivate LAB in yogurt, recent findings suggest that the inactivated bacteria, known as parabiotics, can still confer beneficial effects in human (Kazemi et al., 2021). Some studies have found that parabiotics have a significant impact on serotonin secretion in the gut (Hara et al., 2018). Additionally, several strains of Lactobacillus parabiotics have demonstrated anti-inflammatory and anti-oxidative effects in in vitro and in vivo experimental models (Chung et al., 2019; Jang et al., 2018). Thus, both live probiotics and inactivated parabiotics can have beneficial effects on human health. Therefore, investigating the potential health benefits of incorporating nisin into post-fermented yogurt would be a valuable research endeavor.
While previous experiments have demonstrated the inhibitory effects of LAB and bacteriocins against H. pylori (El-Adawi et al., 2013; Kim et al., 2003), our study focused on the combination effect of nisin and LA for the growth inhibition of H. pylori. To investigate the possibility of incorporating nisin into yogurt, we evaluated the synergistic antibacterial effect of nisin and LA using checkerboard assay and agar well diffusion assay. MIC of nisin (μg/mL), LA (%) and ampicillin (μg/mL) against H. pylori was 10, 0.6, 0.195, respectively. Previous studies indicated that the MIC of nisin was 0.39–25 μg/mL, and ampicillin was 0.015–0.25 μg/mL and it was similar to our data (Neshani et al., 2019; Weiss et al., 1998). The FICI of combined nisin and LA was 0.375, indicating that nisin and LA have synergistic effect.
The LA content in the yogurt was measured to be 0.6%. When the yogurt supernatant and nisin (5 μg/mL) were combined, the growth of H. pylori was inhibited as observed in the agar well diffusion assay. Among the concentrations of nisin tested (ranging from 1.25 to 5 μg/mL), 5 μg/mL of nisin exhibited better H. pylori growth inhibition effects than the MIC value of nisin (10 μg/mL). When nisin is combined with LA, 5 μg/mL of nisin demonstrated the most pronounced H. pylori growth inhibition effects. Based on these data, only 5 μg/mL of nisin was selected.
Nisin and LA are GRAS substances because they have been used in various foods for many years (Müller‐Auffermann et al., 2015). However, to use these two substances together, the safety of their combined effect must be evaluated. As H. pylori is a bacterium that lives in the stomach, we conducted a safety evaluation using the trypan blue dye exclusion assay on AGS cells. Also, since it takes approximately 3 hours for food to be digested in the stomach, the material processing time was set to 3 hours. Live cells exclude trypan blue dye due to intact cell membrane, and blue-stained dye indicates cell death due to cell membrane damage. In previous studies, high concentrations of nisin were reported to possess anticancer properties by inducing apoptosis (Ahmadi et al., 2017). In our study, cell death was not observed in low concentrations of nisin (1.25 to 5 μg/mL), even when used in combination with 0.6% LA. Cell death was only observed in AGS cells treated with 1.2% LA. Based on these results, we selected the concentration of 5 μg/mL of nisin, 0.6% of LA, and 6.25 μg/mL of ampicillin for further in vitro assays.
To determine the optimal concentration of H. pylori to be treated in the cell, an LDH assay was conducted. Cell membrane damage results in LDH release in the culture medium, making it an indicator of damaged cells (Fotakis and Timbrell, 2006). It has been suggested that VacA, which is secreted by H. pylori, regulates pro-apoptotic MMP-9 expression of cells, resulting in apoptosis (Gonciarz et al., 2019). Accordingly, the LDH release increased when AGS cells were treated with 109 CFU/mL of H. pylori. Therefore, the cell density of 108 CFU/mL of H. pylori was selected for further cell culture studies.
H. pylori is known to induce inflammation by modulating pro-inflammatory cytokine production. In previous study, H. pylori treated AGS cells showed an increased mRNA level of pro-inflammatory cytokines (Lamb and Chen, 2013). H. pylori bacterial toxin CagA and VacA are major contributors to the increase in pro-inflammatory cytokines. CagA is transported onto gastric epithelial cells via a type IV secretion system, where it activates the NF-κB pathway, leading to the production of pro-inflammatory cytokines (Jones et al., 2010). Meanwhile, VacA stimulated the production of reactive oxygen species in gastric epithelial cells, which can lead to the production of pro-inflammatory cytokines (Jones et al., 2010). We investigated the effect of nisin and LA on H. pylori infected AGS cells, with a focus on its impact on the mRNA expression levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-8 and TNF-α) and bacterial toxins (CagA and VacA). Our findings demonstrate that the combination of nisin and LA was effective in downregulating the mRNA expression levels of pro-inflammatory cytokines by reducing the mRNA expression levels of bacterial toxins. The bacterial toxins have been shown to play a role in the development of pathogen-associated diseases by leading to inflammation and tissue damage. Previous studies have shown that nisin can downregulate the transcriptional levels of Staphylococcus aureus toxin genes, leading to the reduction of bacterium adhesion and modulation of pro-inflammatory and anti-inflammatory cytokines (Jia et al., 2019; Zhao et al., 2016). Additionally, exposure to nisin could damage the cellular membrane and triggers DNA condensation of S. aureus (Jensen et al., 2020). Our data showed that the combination of nisin and LA is more effective in downregulating the gene expressions of bacterial toxins compared to using them alone. This may be attributed to nisin-LA combination ability to damage the bacterial membrane of H. pylori, allowing nisin to enter the inside the cellular membrane and trigger DNA condensation. The combination of nisin and LA led to the leakage of cytoplasmic contents such as DNA and proteins, indicating membrane damage. Nisin and LA used separately did not show such antibacterial effects. These findings were supported by the results of FE-SEM, which showed nisin alone did not cause significant damage to the bacterial membrane, while the combination of nisin and LA resulted in a notable level of damage.
Incorporating nisin into post-fermented yogurt exhibited antibacterial effect against H. pylori. Combination of nisin and LA was found to have a synergistic antibacterial effect against H. pylori, compared to using them alone. This combination was also effective in downregulating gene expressions of bacterial toxins and inflammatory cytokines in human gastric cells. Based on our data, it is suggested that incorporating yogurt, which naturally contains LA, with nisin could provide a safe approach to inhibit H. pylori infections. This combination shows promise as a potential alternative therapy for H. pylori eradication. Further studies, including animal studies, are necessary to validate our findings and assess the potential of these combinations in clinical settings.
Conflicts of Interest
The authors declare no potential conflicts of interest.
Author Contributions
Conceptualization: Han Seo Gu, Han Sung Gu. Methodology: Han Seo Gu. Investigation: Han Seo Gu, Kwon HC, Kim DH, Hong SJ. Writing - original draft: Han Seo Gu, Han Sung Gu. Writing - review & editing: Han Seo Gu, Kwon HC, Kim DH, Hong SJ, Han Sung Gu.
Ethics Approval
This article does not require IRB/IACUC approval because there are no human and animal participants.
References
- Ahmadi S, Ghollasi M, Hosseini HM. 2017. The apoptotic impact of nisin as a potent bacteriocin on the colon cancer cells. Microb Pathog 111:193-197. https://doi.org/10.1016/j.micpath.2017.08.037
- Alakomi HL, Skytta E, Saarela M, Mattila-Sandholm T, Latva-Kala K, Helander IM. 2000. Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane. Appl Environ Microbiol 66:2001-2005. https://doi.org/10.1128/AEM.66.5.2001-2005.2000
- Amiri S, Moghanjougi ZM, Bari MR, Khaneghah AM. 2021. Natural protective agents and their applications as biopreservatives in the food industry: An overview of current and future applications. Ital J Food Sci 33:55-68. https://doi.org/10.15586/ijfs.v33iSP1.2045
- Bajpai VK, Baek KH, Kang SC. 2012. Control of Salmonella in foods by using essential oils: A review. Food Res Int 45:722-734. https://doi.org/10.1016/j.foodres.2011.04.052
- Caliskan G, French T, Lacalle SE, del Angel M, Steffen J, Heimesaat MM, Dunay IR, Stork O. 2022. Antibiotic-induced gut dysbiosis leads to activation of microglia and impairment of cholinergic gamma oscillations in the hippocampus. Brain Behav Immun 99:203-217. https://doi.org/10.1016/j.bbi.2021.10.007
- Chalon MC, Acuna L, Morero RD, Minahk CJ, Bellomio A. 2012. Membrane-active bacteriocins to control Salmonella in foods: Are they the definite hurdle? Food Res Int 45:735-744. https://doi.org/10.1016/j.foodres.2011.08.024
- Chung IC, OuYang CN, Yuan SN, Lin HC, Huang KY, Wu PS, Liu CY, Tsai KJ, Loi LK, Chen YJ, Chung AK, Ojcius DM, Chang YS, Chen LC. 2019. Pretreatment with a heat-killed probiotic modulates the NLRP3 inflammasome and attenuates colitis-associated colorectal cancer in mice. Nutrients 11:516.
- Danesh J. 1999. Helicobacter pylori infection and gastric cancer: Systematic review of the epidemiological studies. Aliment Pharmacol Ther 13:851-856. https://doi.org/10.1046/j.1365-2036.1999.00546.x
- De Martinez YB, Ferrer K, Salas EM. 2002. Combined effects of lactic acid and nisin solution in reducing levels of microbiological contamination in red meat carcasses. J Food Prot 65:1780-1783. https://doi.org/10.4315/0362-028X-65.11.1780
- El-Adawi H, El-Sheekh M, Khalil M, El-Deeb N, Hussein M. 2013. Lactic acid bacterial extracts as anti-Helicobacter pylori: A molecular approach. Ir J Med Sci 182:439-452. https://doi.org/10.1007/s11845-013-0909-y
- Elshaghabee FMF, Bockelmann W, Meske D, de Vrese M, Walte HG, Schrezenmeir J, Heller KJ. 2016. Ethanol production by selected intestinal microorganisms and lactic acid bacteria growing under different nutritional conditions. Front Microbiol 7:47.
- Field D, Baghou I, Rea MC, Gardiner GE, Ross RP, Hill C. 2017. Nisin in combination with cinnamaldehyde and EDTA to control growth of Escherichia coli strains of swine origin. Antibiotics 6:35.
- Fotakis G, Timbrell JA. 2006. In vitro cytotoxicity assays: Comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicol Lett 160:171-177. https://doi.org/10.1016/j.toxlet.2005.07.001
- Gonciarz W, Krupa A, Hinc K, Obuchowski M, Moran AP, Gajewski A, Chmiela M. 2019. The effect of Helicobacter pylori infection and different H. pylori components on the proliferation and apoptosis of gastric epithelial cells and fibroblasts. PLOS ONE 14:e0220636.
- Gyawali R, Ibrahim SA, Abu Hasfa SH, Smqadri SQ, Haik Y. 2011. Antimicrobial activity of copper alone and in combination with lactic acid against Escherichia coli O157:H7 in laboratory medium and on the surface of lettuce and tomatoes. J Pathog 2011:650968.
- Hafeez M, Qureshi ZA, Khattak AL, Saeed F, Asghar A, Azam K, Khan MA. 2021. Helicobacter pylori eradication therapy: Still a challenge. Cureus 13:e14872.
- Hara T, Mihara T, Ishibashi M, Kumagai T, Joh T. 2018. Heat-killed Lactobacillus casei subsp. casei 327 promotes colonic serotonin synthesis in mice. J Funct Foods 47:585-589. https://doi.org/10.1016/j.jff.2018.05.050
- Jang HJ, Song MW, Lee NK, Paik HD. 2018. Antioxidant effects of live and heat-killed probiotic Lactobacillus plantarum Ln1 isolated from kimchi. J Food Sci Technol 55:3174-3180. https://doi.org/10.1007/s13197-018-3245-4
- Jensen C, Li H, Vestergaard M, Dalsgaard A, Frees D, Leisner JJ. 2020. Nisin damages the septal membrane and triggers DNA condensation in methicillin-resistant Staphylococcus aureus. Front Microbiol 11:1007.
- Jeong CH, Ryu H, Zhang T, Lee CH, Seo HG, Han SG. 2018. Green tea powder supplementation enhances fermentation and antioxidant activity of set-type yogurt. Food Sci Biotechnol 27:1419-1427. https://doi.org/10.1007/s10068-018-0370-9
- Jia Z, He M, Wang C, Chen A, Zhang X, Xu J, Fu H, Liu B. 2019. Nisin reduces uterine inflammation in rats by modulating concentrations of pro- and anti-inflammatory cytokines. Am J Reprod Immunol 81:e13096.
- Jones KR, Whitmire JM, Merrell DS. 2010. A tale of two toxins: Helicobacter pylori CagA and VacA modulate host pathways that impact disease. Front Microbiol 1:115.
- Kazemi A, Soltani S, Nasri F, Clark CC, Kolahdouz-Mohammadi R. 2021. The effect of probiotics, parabiotics, synbiotics, fermented foods and other microbial forms on immunoglobulin production: A systematic review and meta-analysis of clinical trials. Int J Food Sci Nutr 72:632-649. https://doi.org/10.1080/09637486.2020.1857710
- Kim DH, Jeong CH, Cheng WN, Kwon HC, Kim DH, Seo KH, Choi Y, Han SG. 2021. Effects of kefir on doxorubicin-induced multidrug resistance in human colorectal cancer cells. J Funct Foods 78:104371.
- Kim TS, Hur JW, Yu MA, Cheigh CI, Kim KN, Hwang JK, Pyun YR. 2003. Antagonism of Helicobacter pylori by bacteriocins of lactic acid bacteria. J Food Prot 66:3-12. https://doi.org/10.4315/0362-028X-66.1.3
- Kirazli S, Tunca S. 2022. NISIN and gilaburu (Viburnum opulus L.) combination is a cost-effective way to control foodborne Staphylococcus aureus. Food Control 142:109213.
- Lamb A, Chen LF. 2013. Role of the Helicobacter pylori-Induced inflammatory response in the development of gastric cancer. J Cell Biochem 114:491-497. https://doi.org/10.1002/jcb.24389
- Lochhead P, El-Omar EM. 2007. Helicobacter pylori infection and gastric cancer. Best Pract Res Clin Gastroenterol 21:281-297. https://doi.org/10.1016/j.bpg.2007.02.002
- Mahony MJ, Wyatt JI, Littlewood JM. 1992. Management and response to treatment of Helicobacter pylori gastritis. Arch Dis Child 67:940-943. https://doi.org/10.1136/adc.67.7.940
- Mouritzen MV, Andrea A, Qvist K, Poulsen SS, Jenssen H. 2019. Immunomodulatory potential of Nisin A with application in wound healing. Wound Repair Regen 27:650-660. https://doi.org/10.1111/wrr.12743
- Muller-Auffermann K, Grijalva F, Jacob F, Hutzler M. 2015. Nisin and its usage in breweries: A review and discussion. J Inst Brew 121:309-319. https://doi.org/10.1002/jib.233
- Neshani A, Zare H, Akbari Eidgahi MR, Hooshyar Chichaklu A, Movaqar A, Ghazvini K. 2019. Review of antimicrobial peptides with anti-Helicobacter pylori activity. Helicobacter 24:e12555.
- Norouzi Z, Salimi A, Halabian R, Fahimi H. 2018. Nisin, a potent bacteriocin and anti-bacterial peptide, attenuates expression of metastatic genes in colorectal cancer cell lines. Microb Pathog 123:183-189. https://doi.org/10.1016/j.micpath.2018.07.006
- Nyssen OP, Perez-Aisa A, Tepes B, Castro-Fernandez M, Kupcinskas J, Jonaitis L, Bujanda L, Lucendo A, Jurecic NB, Perez-Lasala J, Shvets O, Fadeenko G, Huguet JM, Kikec Z, Bordin D, Voynovan I, Leja M, Machado JC, Areia M, Fernandez-Salazar L, Rodrigo L, Alekseenko S, Barrio J, Ortuno J, Perona M, Vologzhanina L, Romero PM, Zaytsev O, Rokkas T, Georgopoulos S, Pellicano R, Buzas GM, Modolell I, Rodriguez BJG, Simsek I, Simsek C, Lafuente MR, Ilchishina T, Camarero JG, Dominguez-Cajal M, Ntouli V, Dekhnich NN, Phull P, Nunez O, Lerang F, Venerito M, Heluwaert F, Tonkic A, Caldas M, Puig I, Megraud F, O'Morain C, Gisbert JP, Hp-EuReg Investigators. 2021. Adverse event profile during the treatment of Helicobacter pylori: A real-world experience of 22,000 patients from the European registry on H. pylori management (Hp-EuReg). Am J Gastroenterol 116:1220-1229.
- Ozel B, Simsek O, Akcelik M, Saris PE. 2018. Innovative approaches to nisin production. Appl Microbiol Biotechnol 102:6299-6307. https://doi.org/10.1007/s00253-018-9098-y
- Seal BS, Lillehoj HS, Donovan DM, Gay CG. 2013. Alternatives to antibiotics: A symposium on the challenges and solutions for animal production. Anim Health Res Rev 14:78-87. https://doi.org/10.1017/S1466252313000030
- Shi C, Zhang X, Zhao X, Meng R, Liu Z, Chen X, Guo N. 2017. Synergistic interactions of nisin in combination with cinnamaldehyde against Staphylococcus aureus in pasteurized milk. Food Control 71:10-16. https://doi.org/10.1016/j.foodcont.2016.06.020
- Sohn H, Chang YH, Yune JH, Jeong CH, Shin DM, Kwon HC, Kim DH, Hong SW, Hwang H, Jeong JY, Han SG. 2020. Probiotic properties of Lactiplantibacillus plantarum LB5 isolated from kimchi based on nitrate reducing capability. Foods 9:1777.
- Suerbaum S, Michetti P. 2002. Helicobacter pylori infection. N Engl J Med 347:1175-1186. https://doi.org/10.1056/NEJMra020542
- Suriani R, Colozza M, Cardesi E, Mazzucco D, Marino M, Grosso S, Sanseverinati S, Venturini I, Borghi A, Zeneroli ML. 2008. CagA and VacA Helicobacter pylori antibodies in gastric cancer. Can J Gastroenterol 22:255-258. https://doi.org/10.1155/2008/521724
- Todorov SD, de Melo Franco BDG, Tagg JR. 2019. Bacteriocins of Gram-positive bacteria having activity spectra extending beyond closely-related species. Benef Microbes 10:315-328. https://doi.org/10.3920/BM2018.0126
- Weiss K, Laverdiere M, Restieri C. 1998. Comparison of activity of 10 antibiotics against clinical strains of Helicobacter pylori by three different techniques. Can J Gastroenterol 12:181-185. https://doi.org/10.1155/1998/765795
- Wen S, Moss SF. 2009. Helicobacter pylori virulence factors in gastric carcinogenesis. Cancer Lett 282:1-8. https://doi.org/10.1016/j.canlet.2008.11.016
- Zhao G, Kempen PJ, Shetty R, Gu L, Zhao S, Jensen PR, Solem C. 2022. Harnessing cross-resistance-Sustainable nisin production from low-value food side streams using a Lactococcus lactis mutant with higher nisin-resistance obtained after prolonged chlorhexidine exposure. Bioresour Technol 348:126776.
- Zhao G, Kempen PJ, Zheng T, Jakobsen TH, Zhao S, Gu L, Solem C, Jensen PR. 2023. Synergistic bactericidal effect of nisin and phytic acid against Escherichia coli O157:H7. Food Control 144:109324.
- Zhao X, Meng R, Shi C, Liu Z, Huang Y, Zhao Z, Guo N, Yu L. 2016. Analysis of the gene expression profile of Staphylococcus aureus treated with nisin. Food Control 59:499-506. https://doi.org/10.1016/j.foodcont.2015.06.018