Introduction

Methods

Results

The study presented by Guo et al. [17] evaluated the anti-bacterial efficacy of olive oil polyphenol extract (OOPE) against both Salmonella Typhimurium ATCC 14028 and S. aureus ATCC 13565. The evaluation included the determination of the minimum inhibitory concentration (MIC) and the examination of the survival rate of the bacteria after treatment with OOPE in both sterile normal saline (NS) and Luria-Bertani broth (LB). The MICs of OOPE against S. Typhimurium and S. aureus were 0.625 and 0.625–1.25 mg mL⁻¹, respectively. After treatment with the MIC of OOPE for 3 h and the MIC of OOPE for 5 h, the growth of S. Typhimurium and S. aureus (approximately 10⁸ colony-forming units (CFU) mL⁻¹) was completely suppressed. When S. Typhimurium and S. aureus were exposed to the extract, the physiological functions associated with cell activity were disrupted. This was reflected by a decrease in the intracellular adenosine triphosphate (ATP) concentration, depolarization of the cell membrane, reduced bacterial protein content, and cytoplasmic leakage [17].

Chen et al. [18] investigated the potential value of polyphenolic extract from fresh sorghum stalks as a natural anti-oxidant and anti-bacterial agent. Polyphenols were extracted from sweet sorghum and further identified. The total phenolic content (TPC), polyphenolic content in the extract, and total anti-oxidant activity (AA) were then quantified. The initial TPC of sorghum juice was 2.21 mg g⁻¹, which increased to 5.77 mg g⁻¹ after extraction. The total AA after extraction and purification was 9.4 μM Trolox mL⁻¹. The sweet sorghum polyphenolic extract contained a significant amount of coumarin, vanillin, caffeic acid, coumaric aldehyde, cinnamic acid, and 18 other polyphenols. S. aureus CMCC 26003, L. monocytogenes ATCC 19114, E. coli CMCC 4410 and S. Typhimurium ATCC 14028 were exposed to a 25 mg mL⁻¹ sweet sorghum polyphenolic extract. The extract showed effective inhibition against all four pathogens, although the numbers of apoptotic cells were significantly higher in S. aureus and L. monocytogenes compared to E. coli and S. Typhimurium. The conductivity of the bacterial culture medium increased significantly within 1 h, but hardly changed between 1 and 7 h, suggesting that polyphenols affect the integrity of bacterial membranes and cause leakage of cell electrolytes. In conclusion, sweet sorghum polyphenolic extract has been shown to be rich in phenolic compounds and to exert both, strong anti-oxidant capacity and anti-bacterial activity [18].

Another study also focused on the anti-bacterial and anti-oxidant effects of the polyphenols in sorghum bran [19]. In this work, ionic liquid and three different conventional solvents, namely anhydrous methanol, acidic methanol, and water, were used to determine the TPC, flavonoids, condensed tannins, and anthocyanins. It was found that the TPC expressed as gallic acid equivalents (GAE) was highest in the acidified methanolic extract (AME) at 26.8 mg g⁻¹, although the total phenolic mass fraction expressed as GAE on a dry matter (DM) basis was only 7.4 ± 0.7 mg g⁻¹. The anti-bacterial activity against S. aureus NCDC 109 and E. coli ATCC 5922 was measured by agar well diffusion assay, in contrast to the previous study [18] which used flow cytometry to determine the anti-bacterial efficacy against the selected pathogens. The concentration of all added extracts was kept uniformly at 12 mg mL⁻¹. The only extract that exhibited a clearly visible inhibition zone was the AME against both E. coli and S. aureus. The MIC measurement was then performed to determine the lowest concentration at which a killing effect against the foodborne pathogens was observed. Complete inhibition of S. aureus was observed at a MIC of 1.1 mg mL⁻¹, while E. coli was inhibited only at a concentration of 2.2 mg mL⁻¹ [19]. The authors of both studies [18, 19] concluded that the polyphenolic extracts from sorghum bran and stalks were significantly more effective against Gram-positive than against Gram-negative bacteria, which may be due to the fact that the polyphenols disrupt the integrity of the bacterial cell membrane structure [18, 19].

The aim of the study by Levy and colleagues [20] was to evaluate the anti-bacterial activity of peanut skin extract (PSE) containing “A-type” procyanidins and grape seed extract (GSE) containing “B-type” procyanidins against foodborne pathogens such as L. monocytogenes LCDC, E. coli O157:H7 1994 beef outbreak isolate, and S. Typhimurium ATCC 14028. GSE had a significantly higher TPC than PSE (96.8 ± 1.04%, GAE, compared to 76.6 ± 7.83%, w/w, GAE). The pour plate method was employed to ascertain the MIC of the two extracts against the selected pathogens. The growth of E. coli O157:H7 was inhibited comparably by PSE (MIC = 51.1 ppm) and GSE (MIC = 47.4 ppm). Significantly higher inhibition of L. monocytogenes (P = 0.0005) and of S. Typhimurium, however, was noted with GSE if compared to PSE. Due to the observed higher growth inhibition with GSE, all subsequent experiments were performed with GSE only. The GSE extract was separated into monomer-rich and oligomer-rich components. The growth patterns of the three pathogens in the presence of total extract, monomer, or oligomer fractions were evaluated individually. The growth of S. Typhimurium was not hindered by any of the extracts. However, in general, the extract with a higher proportion of oligomer components impeded the growth of L. monocytogenes and E. coli O157:H7 when compared to the control. The findings suggest that an extract rich in type B procyanidins, especially with a higher concentration of oligomers, may have enhanced anti-bacterial properties. It is important to emphasize that the current study focused exclusively on growth inhibition. The authors concluded that although the results suggest that GSE is more effective than PSE, the opposite may be true depending on the underlying mechanism considered [20].

Huang et al. [21] investigated whether polyphenol chemistry were suitable for the environmentally friendly synthesis of reproducible and long-lasting surface-enhanced Raman scattering (SERS) tags that could be used for the non-specific detection of bacteria in food. SERS is considered one of the most advanced techniques for bioanalysis. Polyphenol chemistry has enabled the development of a method for the production of SERS tags with biochemical affinity. Metal-phenol networks (MPNs) were used as bio-affinity elements to recognize bacteria. Two foodborne pathogens were selected, namely the Gram-positive S. aureus ATCC 6538 and the Gram-negative E. coli O157: H7 ATCC 700728. These strains were cultivated both in liquid LB and on solid culture media. The results showed that the polyphenolic SERS biosensor was capable of detecting foodborne bacteria to a concentration of 10² CFU mL⁻¹. To evaluate the practicality of the polyphenolic SERS tags in real food samples, an additional study was conducted, and the results indicate that this SERS biosensor could work effectively in dairy and beef samples, with recovery rates ranging from 83.0 to 106.7% for E. coli O157: H7 and from 94.8 to 112.0% for S. aureus, respectively. The authors concluded from their study that polyphenol chemistry can be used for the environmentally friendly synthesis of reproducible and long-lasting SERS tags for the non-specific detection of bacteria in food. In particular, MPNs were shown to have the potential to act as novel artificial bio-recognition elements that target both Gram-positive and Gram-negative bacteria in a non-selective manner [21].

In the study by Appu et al. [22], the hypothesis that novel chitosan-coated cerium oxide nanocomposites (CS/CeO₂ NCs) derived from aqueous extracts of tea polyphenols could be stabilized and reduced by using green chemistry was investigated. The anti-bacterial potential of cerium oxide nanoparticles (CeO₂ NPs) and CS/CeO₂ NCs was tested against the bacterial strains S. aureus CICC 10384 and E. coli CICC 21524 using the disc diffusion method. Additionally, the anti-fungal potential was assessed against Botrytis cinerea in this study. Different concentrations of NPs (25, 50, 75, and 100 mg L⁻¹) were evaluated for their anti-bacterial activity against Gram-positive and Gram-negative bacterial pathogens. At a concentration of 100 mg L⁻¹, the CS/CeO2 NCs exhibited a significant effect on the anti-bacterial and anti-fungal efficacies against E. coli, S. aureus, and B. cinerea, respectively, compared to the bare CeO₂ NPs, with an inhibition zone of 13.5 ± 0.2 mm and 11.7 ± 0.2 mm, respectively. Thus, the CS/CeO₂–NCs were shown to be able to exert both anti-bacterial and anti-fungal effects, but further in vivo studies should address their potential toxicity [22].

Abutheraa et al. [23] tested the anti-bacterial activities of phenolic extracts from the seed coats of different colored soybeans against foodborne pathogens such as S. Typhimurium ATCC 14028, E. coli O157:H7 GFP-labeled ED 14, and C. jejuni NCTC 11168 in broth cultures and on chicken skin. The phenolic extract from the soybean variety R07-1927 with a black-colored seed coat exhibited the highest TPC at 74.1 ± 2.1 mg chlorogenic acid equivalent/g extract, which was significantly higher than the extracts from the other soybean varieties. The extract from black-colored soybeans resulted in reductions of 2.10 ± 0.08 to 2.20 ± 0.08 log₁₀ CFU mL⁻¹ for both E. coli O157:H7 and C. jejuni after 3 days of incubation in broth culture containing 4 log₁₀ CFU mL⁻¹ of bacteria. Meanwhile, a 6-day incubation resulted in a reduction of S. Typhimurium and E. coli O157:H7 to 2.03 ± 0.05 and 3.3 ± 0.08 log₁₀ CFU mL⁻¹, respectively. The extract also decreased the counts of S. Typhimurium and E. coli O157:H7 attached to chicken skin by 1.39 ± 0.03 and 1.24 ± 0.06 log₁₀ CFU g⁻¹, respectively, after 6-days of incubation. In conclusion, the phenolic extracts from soybean seed coats showed inhibitory effects on pathogenic bacteria both in broth-cultures and on chicken skin, resulting in reductions of approximately 1–3 log₁₀ CFU mL⁻¹ for the three pathogenic bacteria studied [23].

The aim of the study by Villalobos et al. [24] was to determine the anti-oxidant and anti-microbial activities of a phenolic extract from soybean flour against selected foodborne pathogens. The TPC of the aqueous phenolic extract (APE) was 0.859 ± 0.025 mg GAE g⁻¹ DM, and the phenolic compounds in soy flour were mainly identified as isoflavones and phenolic acids, which accounted for 60.5 and 28.3% of the TPC, respectively. The AA of the APE was measured to be 5.226 mg Trolox g⁻¹ DM. Overall, inhibition of 100 and 50% was observed for all pathogenic bacteria tested at concentrations above 1.25 and 0.5 g L⁻¹, respectively. The highest activity was observed against L. monocytogenes CECT 911, B. cereus CECT 131, and Enterococcus faecalis P36. Overall, Gram-positive bacteria were shown to be more sensitive to APE than Gram-negative bacteria, although this did not apply to all strains analyzed. Six yeast strains and four molds that cause fruit decay were also tested, but the sensitivity was generally much lower if compared to pathogenic bacteria. The results indicate that defatted soy flour constitutes an important source of phenolic compounds with remarkable anti-microbial and anti-oxidant activities [24].

Joshi et al. [25] aimed to evaluate the anti-bacterial activity of blueberry proanthocyanidins (PAC) against two isolates of C. sakazakii and to elucidate their mode of action by scanning electron microscopy. Blueberry PAC (5 mg mL⁻¹) was mixed with washed overnight cultures of C. sakazakii and incubated at 37 °C for 30 min, 1, 3, and 6 h. For the two C. sakazakii strains 29,004 and 29,544, a reduction of approximately 1.0 and 1.5 log₁₀ CFU mL⁻¹, respectively, was achieved after 30 min. After 1 h, blueberry PAC resulted in a reduction to undetectable levels for both strains, from initial levels of 8.25 ± 0.12 log₁₀ CFU mL⁻¹ and 8.48 ± 0.03 log₁₀ CFU mL⁻¹, respectively. An interesting observation was made during the scanning electron microscopic examination. Compared to untreated controls, the treated strains showed differences in the morphology of the cell membranes with clumping and blebbing. The authors suggested that the effect of blueberry PAC appeared to be bacteriostatic. However, further in vitro studies in food systems and in vivo animal feeding studies were required before recommendations could be made for the prevention and treatment of C. sakazakii infections [25].

Ford et al. [26] investigated the potential use of polyphenols from brown seaweeds as anti-bacterial agents in animal feed. Two phlorotannin extracts from the brown seaweeds Ascophyllum nodosum and Fucus serratus were extracted and analyzed for their bactericidal activity against E. coli O157:H7 EDL 933, Salmonella agona (food isolate from AFBI collection), and Streptococcus suis DSM 9682. A. nodosum proved to be significantly more effective than F. serratus with MICs between 1.56 and 0.78 mg mL⁻¹, while F. serratus reached 3.13 mg mL⁻¹ for all pathogens tested. The two extracts proved to be effective in eliminating three foodborne pathogens without adversely affecting the intestinal cells of the pig. The authors suggested that further research was needed to purify and identify the specific biologically active molecules within respective compounds [26].

In the study by Sánchez-Gutiérrez et al. [27], the researchers compared for the first time the bioactivity of phenolic compounds with nutraceutical potential from olive leaves, which are a by-product of olive oil production. The comparison involved the use of microwave-assisted extraction (MAE) at short duration and low temperatures, along with environmentally friendly solvents such as glycerol, ethanol, and water. The TPC, the AA, the phenolic profile of the extracts and the MIC were determined. The extracts showed high values of TPC (up to 76.1 mg GAE g⁻¹ DM) and AA (up to 78 mg Trolox g⁻¹ DM). Using high performance liquid chromatography (HPLC) analysis, oleuropein was found to be the predominant compound in all extracts, whereas six additional phenolic compounds were identified. These included a simple phenol (hydroxy-tyrosol), four flavonoids (luteolin, luteolin-7-O-glucoside, apigenin, and apigenin-7-O-glucoside), and a cinnamic acid derivative (verbascoside). The anti-bacterial activity was tested against five foodborne pathogens, namely L. monocytogenes CECT4032, S. Typhimurium CECT 704, E. coli CECT 8295, S. aureus CECT 5193, and Y. enterocolitica CECT 754. The MIC values ranged from 2.5 to 60 mg mL⁻¹. Thus, olive leaf waste products could be shown to be rich in phenolic compounds with anti-oxidant and anti-bacterial properties [27].

Shimamura et al. [28] evaluated the inhibitory effects of 14 food additives derived from polyphenolic samples on staphylococcal enterotoxin A (SEA) production and biofilm formation by S. aureus C-29. Western blot analysis revealed that Tannic acid AL (TA), Purephenon 50 W (PP), and Polyphenon 70A (POP) at 0.25 mg mL⁻¹, as well as Gravinol®-N (GN), Blackcurrant polyphenol AC10 (BP), and Resveratrol-P5 (RT) at 1.0 mg mL⁻¹ led to a significant reduction in SEA production by S. aureus. Despite unsuccessful suppression attempts by PP and POP, TA, GN, BP, and RT effectively inhibited the expression of the sea gene in S. aureus, leading to the conclusion that TA, GN, BP, and RT are able to inhibit the production of SEA. Each of the six samples demonstrated a significant inhibition of biofilm formation at a concentration that did not affect S. aureus growth. The outcome of this study shows that low concentrations of polyphenol food additives without direct anti-bacterial effectiveness, could have an inhibitory effect on toxin production and biofilm formation by S. aureus [28].

Calvo et al. [29] investigated the anti-bacterial and anti-biofilm activities of polyphenolic extracts against foodborne pathogens. In this context, the authors characterized two hydro-organic extracts, namely LE050 and PG050, with a high polyphenol content, obtained from the shrub Cytisus scoparius, as potential anti-pathogenic candidates. A total of 24 different polyphenols were detected in the extracts, of which only 20 were present in both. The LE050 extract had a greater diversity of polyphenols, whereas the PG050 extract contained higher relative amounts of phenolic acids. The most common polyphenols identified in the extracts were flavonoids. The in vitro anti-bacterial efficacy of these extracts (by measuring the MIC and the half maximal inhibitory concentration (IC50)) was evaluated against both Gram-positive and Gram-negative pathogenic bacteria, with a particular emphasis on those associated with food contamination. The anti-bacterial efficacy varied significantly among the tested extracts, with LE050 exhibiting the most substantial inhibitory values, especially in terms of IC50 percentages. LE050 showed IC50 values between 0.625 and 3.27% v/v, while PG050 displayed values between 4.87 and 20% v/v. Nevertheless, both extracts demonstrated anti-bacterial effects with remarkable activity against the most common foodborne pathogens, namely S. enterica CECT 554, L. monocytogenes CECT 4032, and Y. enterocolitica. A pattern of action was observed, with Gram-negative bacteria reacting more sensitively to the extracts than Gram-positive bacteria. This suggests that the anti-bacterial activity could be directly linked to the composition of the bacterial wall. S. aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853 and L. monocytogenes CECT 4032, which are among the most common biofilm-forming bacteria, were selected for the biofilm inhibition tests, and the minimum biofilm inhibitory concentrations were determined. No complete eradication of the biofilm was observed for S. aureus and P. aeruginosa with the PG050 extract. However, 20% of the extract was sufficient to completely disperse the biofilm of L. monocytogenes. The LE050 extract proved to be quite effective in inhibiting biofilm formation by all three pathogens at concentrations ranging from 0.625 to 2.5% v/v. Due to their specific composition, both the LE050 extract and the PG050 exhibited pronounced anti-bacterial and anti-biofilm forming activities [29].

Xu and colleagues [30] tested the anti-oxidant, anti-bacterial, and anti-biofilm activities of polyphenols derived from muscadine grape Vitis rotundifolia Michx. (Noble and Carlos) pomace against selected foodborne pathogens. The research also focused on the time-killing curves of the polyphenols and explored possible synergies with synthetic antibiotics including ampicillin and streptomycin. The TPC of the skin and seed extracts varied from 35.9 ± 2.5 to 58.2 ± 3.5 mg GAE g⁻¹ DM. The skin of Noble grape contained mainly anthocyanins as the main phenolic compounds, while the skin of the Carlos grape was characterized by ellagic acid and its conjugates. In the seeds of Noble and Carlos grapes, the main phenolic components were flavan-3-ols and condensed tannins. The AA of the individual polyphenols ranged from 5 to 11.1 mmol Trolox g⁻¹, with ellagic acid and its conjugates having the highest AA per unit. The polyphenolic extracts demonstrated a broad spectrum of anti-bacterial activities against the S. aureus isolates ATCC 35548, ATCC 12600-U, and ATCC 29247 with MICs of 74–367 mg L⁻¹ for skin and 67–173 mg L⁻¹ for seed, while they exhibited only minimal to no anti-bacterial activity against the Gram-negative bacteria S. Typhimurium, Shigella sonnei ATCC 25931, and E. coli O157:H7 (204P). The inhibition by the seed polyphenols occurred in a concentration-dependent manner, while the inhibition by the skin polyphenols showed a negative dependence on the concentration. Although Carlos grape skin was used at a higher polyphenolic concentration than Carlos grape seed, its inhibition was significantly weaker. The authors concluded that the anti-bacterial activity of the phenolic compounds in the extracts did not only depend on the concentration but also on the specific type of polyphenol. Noble skin and seed extracts caused a decrease in S. aureus cell viability of 4.7 and 4.9 log₁₀ CFU mL⁻¹ within 6 h of lysis at 4-fold MIC, while they inhibited biofilm formation at a 0.5-fold MIC and eradicated biofilms at a 16-fold MIC. These findings suggest that grape polyphenols can inhibit the formation of bacterial biofilms at a sub-MIC without causing bacterial cell death. However, a high concentration of phenolic compounds was required to eliminate pre-formed biofilms. Polyphenols from muscadine grapes also showed synergistic effects with the synthetic antibiotics ampicillin and streptomycin, causing a maximum decrease in cell viability of 6.2 log₁₀ CFU mL⁻¹ at sub-inhibitory concentrations. Muscadine phenolics have therefore been shown to exert a strong anti-bacterial effect, to inhibit the formation of biofilms, and to act synergistically with synthetic antibiotics [30].

In the following study by Nazareth et al. [31], polyphenols from an unexplored unripe and ripe fruit of Carissa spinarum were analyzed for their anti-oxidant and anti-quorum sensing activities. In addition, the inhibition of violacein, the anti-biofilm and the effect on the motility of foodborne pathogens were investigated. Phytochemical analysis revealed a TPC and a total flavonoid content of 273.2 mg GAE 100 g⁻¹ and 453.8 mg quercetin equivalent per 100 g fresh weight in ripe fruits, respectively. The main phenolic acids identified in both stages of the fruit were syringic acid and resveratrol, with the resveratrol content being significantly higher in ripe fruit than in unripe ones. Chlorogenic acid and epicatechin were found in higher concentrations in unripe fruit. Both fruit stages showed highly pronounced anti-oxidant potential with an IC50 of 4.69 mg mL⁻¹ for the ripe fruit and 8.61 mg mL⁻¹ for the unripe fruit when analyzed for free radical scavenging activity using the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay. The MIC of the extract from ripe and unripe fruit was 5 and 9 mg mL⁻¹, respectively. Anti-quorum sensing activity was higher in the extract from ripe fruit at 1.8 mg mL⁻¹ than in the extract from unripe fruit and inhibited the violacein production of Chromobacterium violaceum CV026 CECT 5999 by 78.65%. In addition, at 1.2 mg mL⁻¹, it inhibited the swimming motility and biofilm formation of P. aeruginosa KR 476388 by 66.25% and of Y. enterocolitica KT 266804 by 59.36%. The analysis showed that the bioactive compounds from the fruits had considerable anti-oxidant potential and successfully inhibited the bacterial quorum sensing system [31].

Freitas et al. [32] investigated the anti-oxidant and anti-bacterial activities of extracted isoflavones and phenolics from soy flour in an in vitro simulated gastrointestinal digestion process followed by ex vivo colonic fermentation. For this purpose, the polyphenols and their bioavailability in aqueous soya bean flour extract were identified and quantified. The predominant isoflavones were genistin, daidzin, glycitin, and malonylgenistin. In addition to the isoflavones, 16 different phenolic compounds were identified in the aqueous extract of which vanillic acid, hesperidin, syringic acid, gallic acid, and caffeic acid were the most abundant ones. The isoflavone content in the aqueous extract (pre-digestion) was 138.9 mg 100 g⁻¹ DM. Reductions of 5, 19, and 29% were observed after digestion in the salivary, gastric, and intestinal tracts, respectively. During colonic fermentation, the isoflavone content was measured at 62.9, 47.7, and 29.1 mg 100 g⁻¹ DM after 4, 24, and 48 h of fermentation, respectively. The TPC content was initially 73.7 mg 100 g⁻¹ DM and decreased to 40.9 mg 100 g⁻¹ DM after intestinal digestion. Following oral digestion, a lower release of phenolic compounds (i.e., of 27.4%) was observed, followed by an increase in the content of phenolic compounds after the digestion in the gastric and intestinal tract (i.e., 49.6 and 55.9%, respectively). None of these phenolic compounds were found in detectable concentrations after colonic digestion. The extract inhibited lipid peroxidation and thus, showed the ability to inhibit oxidative degradation. The AA was 207.67 µmol Trolox g⁻¹. The aqueous extract was able to inhibit the growth of strains of different species and genera, such as Gram-positive S. aureus ATCC 14458 and coagulase-negative Staphylococcus saprophyticus KT95505 as well as Gram-negative Acinetobacter genomospecies 3 ATCC 17922, Aeromonas hydrophila ATC7966, S. enterica ATCC 12325, V. parahaemolyticus ATCC 17802, E. coli ATCC 43895, Pseudomonas fluorescens ATCC 13525, among others. The MICs against all pathogens tested ranged from 12.5 to 150 mg mL⁻¹. The cytotoxicity of the aqueous soybean meal extract was evaluated by in vitro assays against vital cells. For this purpose, healthy mouse bone marrow cells (5.0 × 10⁵ cells mL⁻¹) and fibroblasts (1.5 × 10⁵ cells mL⁻¹) were treated with soybean meal extract at concentrations ranging from 125 to 0.97 mg mL⁻¹, and cell viability was assessed after 24 h. The extract did not show any cytotoxicity to the cells tested. In conclusion, the aqueous extract exhibited anti-oxidant activity that was enhanced after in vitro digestion in the gastrointestinal tract, inhibited lipid peroxidation, showed anti-bacterial activity against both Gram-positive and Gram-negative foodborne pathogens, and exhibited no cytotoxicity to healthy mouse cells [32].

Discussion

Ethics statement

Not applicable (literature survey).

Funding sources

No financial support was received for this study.

Authors' contributions

CBM conceived and designed the survey, wrote the paper. SM edited the paper. SB provided critical advice in design of the survey, edited the paper. MMH supervised the survey, co-wrote the paper.

Conflict of interests

SB and MMH are Editorial Board members, therefore they did not take part in the review process in any capacity and the submission was handled by a different member of the editorial board.