Towards understanding the antagonistic activity of phytic acid against common foodborne bacterial pathogens using a general linear model.

The increasing challenge of antibiotic resistance requires not only the discovery of new antibiotics, but also the development of new alternative approaches. Therefore, in the present study, we investigated for the first time the antibacterial potential of phytic acid (myo-inositol hexakisphosphate, IP6), a natural molecule that is 'generally recognized as safe' (FDA classification), against the proliferation of common foodborne bacterial pathogens such as Listeria monocytogenes, Staphylococcus aureus and Salmonella Typhimurium. Interestingly, compared to citric acid, IP6 was found to exhibit significantly greater inhibitory activity (P<0.05) against these pathogenic bacteria. The minimum inhibitory concentration of IP6 varied from 0.488 to 0.97 mg/ml for the Gram-positive bacteria that were tested, and was 0.244 mg/ml for the Gram-negative bacteria. Linear and general models were used to further explore the antibacterial effects of IP6. The developed models were validated using experimental growth data for L. monocytogenes, S. aureus and S. Typhimurium. Overall, the models were able to accurately predict the growth of L. monocytogenes, S. aureus, and S. Typhimuriumin Polymyxin acriflavine lithium chloride ceftazidime aesculin mannitol (PALCAM), Chapman broth, and xylose lysine xeoxycholate (XLD) broth, respectively. Remarkably, the early logarithmic growth phase of S. Typhimurium showed a rapid and severe decrease in a period of less than one hour, illustrating the bactericidal effect of IP6. These results suggest that IP6 is an efficient antibacterial agent and can be used to control the proliferation of foodborne pathogens. It has promising potential for environmentally friendly applications in the food industry, such as for food preservation, food safety, and for prolonging shelf life.

1. Introduction

Food safety is an important public health priority. Each year, around a third of the world population is infected by foodborne pathogens [1, 2]. The highest incidence of foodborne diseases occurs in Africa [1], where more than 91 million people are estimated to fall ill each year, leading to around 137 000 deaths [2]. L. monocytogenes is considered to be one of the most hazardous foodborne bacterial pathogens [3, 4]. It is particularly problematic, as it can form biofilms and survive for long periods of time in a food processing environment [5]. L. monocytogenes causes human listeriosis, a rare disease associated with high rates of hospitalization and mortality [6, 7]. The infection is mainly linked to the consumption of contaminated ready-to-eat foods, such as cheese and other dairy products, processed meats, salads, seafood, and raw eggs [8, 9]. Another bacterium, S. aureus, which belongs to the Gram-positive Micrococcaceae family, is considered to be one of the most common causes of foodborne disease in the world [10]. This bacterium causes gastrointestinal illness by secreting a range of toxins, including staphylococcal enterotoxins [10-12]. S. aureus is also able to form biofilms on food-contact surfaces, which highly increases its stress tolerance and, thus, its persistence in food-related environments [13-16]. Salmonella is also considered to be among the most common foodborne pathogens [17]. It causes Salmonellosis, a disease that is linked to the consumption of contaminated meats, especially poultry products [17]. This disease is responsible for the largest number of hospitalizations and deaths due to foodborne pathogens [18].

In order to prevent the transmission of foodborne disease, the food industry uses a variety of methods, which can be physical (temperature, radiation), chemical (bleach, alcohol, iodine) or chemotherapeutic (antibiotics) [19, 20]. However, several studies have suggested that synthetic sanitizers can have significant side effects, such as bleaching and the formation of toxic compounds [21]. In addition, the growing rate of antimicrobial resistance in foodborne bacterial pathogens is also becoming a major concern for food safety and one of the disquieting threats to global human health [22]. For this reason, the World Health Organization (WHO) recommended that breeders and the food industry stop misusing antibiotics, such as for promoting animal growth and preventing disease in healthy animals, in order to preserve the effectiveness of antibiotics for human medicine [22]. Moreover, the increasing demand for organic food has increased the interest in substituting these chemicals with natural products, which do not damage the host or the environment [23, 24]. Thus, studies of natural compounds with antimicrobial properties are warranted. There is currently much interest in the use of organic acids as ‘environmentally friendly’ sanitizers or preservatives [25], most notably citric acid, acetic acid and lactic acid [26]. Studies have mainly focused on citric acid, which can be used alone or alongside conventional sanitizers and other alternative technologies [27]. It is especially useful for fruit, such as strawberries [27]. However, it has been noted that organic acids, such as formic acid, lactic acid, propionic acid and their salts, often fail to provide the desired result when applied in practice, such as when used for decontaminating animal feed, thus occasioning substantial additional costs for operators (feed producers and farmers) [28]. Therefore, there is currently much interest in research on alternative organic acids that are both cheap and safe to use.

Myo-inositol 1, 2, 3, 4, 5, 6-hexakisphosphate (IP6), commonly known as phytic acid, is a naturally occurring compound that is ‘generally recognized as safe’, according to the U.S. Food and Drug Administration classification [29]. It represents the principal storage form of phosphorus (P) in whole cereals and other edible vegetable seeds, such as legumes and nuts, and may account for 65–85% of the total P in seeds [29]. IP6 is a negatively charged compound that has been considered an anti-nutrient since it acts as a strong chelator of vital minerals like calcium, iron, magnesium, copper, zinc, and potassium, reducing their absorption and bioavailability [30]. However, several recent studies in both humans and animals have demonstrated that this natural molecule acts as a disease-preventing compound [31]. Indeed, IP6 displays a broad range of pharmaceutical properties, including antioxidant [32, 33], neuroprotective [34, 35], anti-inflammatory [36], lipid lowering [37], pathological calcification preventing [38, 39] and anticancer activities [40-42]. In addition, Kim and Rhee [21] described the anti-biofilm effect of IP6 against E. coli O157:H7, especially when combined with NaCl. They suggested that a sanitizer that combines these two naturally occurring antimicrobial agents could be used by food safety managers who encounter thick biofilm formation in food processing environments.

The purpose of the present work is to assess the effectiveness of IP6 against several foodborne bacterial pathogens for the first time. This is achieved by: (i) determining the minimum inhibitory concentration (MIC) as compared to citric acid (CA); (ii) measuring the inhibition diameters of each indicator bacteria growth inhibition or reduction, and (iii) illustrating the mode of action of IP6 for inhibiting pathogen growth.

2. Materials and methods

2.1. Substrates and chemicals

Phytic acid solution [myo-Inositol hexakis (dihydrogen phosphate); IP6] was purchased from Sigma-Aldrich (593648; 50% (w/w) in water). Citric acid (CA), (C6H8O7; CAS: 77-92-9), was purchased from Fluka, Switzerland.

2.2. Bacterial strains, media and culture conditions

The target bacterial strains were obtained from international culture collections (ATCC). They included Gram-positive bacteria: Listeria monocytogenes ATCC 19117 and Staphylococcus aureus ATCC 6538; and Gram-negative bacteria: Salmonella Typhimurium ATCC 14028, Pseudomonas aeruginosa ATCC 49189 and Escherichia coli ATCC 8739. These strains were used as indicator microorganisms for the antibacterial activity assays. L. monocytogenes ATCC 19117 was cultured on Polymyxin Acriflavin Lithium-Chloride Ceftazidime Aesculin Mannitol (PALCAM, LAB M Ltd, U.K) at 37°C for 24 h. S. aureus ATCC 6538 was grown on Chapman medium (Oxoid, Basingstoke, Hampshire, UK) at 37°C for 24 h, and S. Typhimurium ATCC 14028 was cultivated on Xylose Lysine Deoxycholate (XLD, Oxoid) at 37°C for 24 h. For the antagonist tests, the final inoculum concentration used for each indicator bacterium was 106 colony-forming units of bacteria per milliliter (CFU/ml) as used in the method described by Smaoui et al. [24].

2.3. Determination of minimum inhibitory concentrations

The minimum inhibitory concentration (MIC) values, representing the lowest concentration of IP6 and CA at which the microorganism did not demonstrate visible growth after incubation, were determined against a panel of five bacteria, as described by Gulluce et al. [43] with minor modifications. The test was performed in sterile 96-well microplates with a final volume of 100 μl per well. A commercial stock solution of IP6 (50% (w/w) in water) and a stock solution of CA at 50% (w/w) in water were used. Then, the corresponding concentrations of IP6 and CA were transferred to each successive well in order to obtain a two-fold serial dilution of the original sample. In fact, each sample was dissolved to a final concentration of 0.078, 0.156, 0.312, 0.625, 1.25, 2.5, 5, 10 and 20 mg/mL and then filtered through 0.22 μm pore-size black polycarbonate filters (Millipore). To each test well 10 μl of cell suspension were added to final inoculum concentration of 106 CFU/ml of bacterium. Positive growth control well consisted of Listeria monocytogenes ATCC 19117, Staphylococcusaureus ATCC 6538 and Salmonella Typhimurium ATCC 14028 respectively growth in PALCAM, Chapman and XLD. Plates were then covered with the sterile plate covers and incubated at 37°C for 24 h. As an indicator of microorganism growth, 25 μl of thiazolyl blue tetrazolium bromide (MTT) indicator solution (0.5 mg/ml) dissolved in sterile water was added to the wells and incubated at 37°C for 30 min. The colourless tetrazolium salt acts as an electron acceptor and was reduced to a red-coloured formazan product by biologically active organisms. Where microbial growth was inhibited, the solution in the well remained clear after incubation with MTT. The determination of MIC values was done in triplicate.

2.4. Agar diffusion method

The antimicrobial activity of IP6 was evaluated by means of agar-well diffusion assays, as described by Valgas et al. [44]. Fifteen milliliters of the molten agar (45°C) were poured into sterile petri dishes (Ø 90 mm). Working cell suspensions were prepared at 106 CFU/mL, and 100 μl was evenly spreaded onto the surface of the agar plates of Luria–Bertani (LB) agar (Oxoid Ltd, UK). Once the plates had been aseptically dried, 06 mm wells were punched into the agar with a sterile Pasteur pipette. IP6 was dissolved in water to a final concentration of 50 mg/ml and then filtered through 0.22 μm pore-size black polycarbonate filters (Millipore). Thus, 50 μl were placed into the wells and the plates were incubated at 37°C for 24 h for bacterial strains. Antibacterial activity was evaluated by measuring the diameter of circular inhibition zones around the well. The un-inoculated media were also tested for inhibitory zones as a control. Tests were performed in triplicate.

2.5. Mode of action of phytic acid

The bacteriostatic or bactericidal mode of action of IP6 was tested using a method described previously by Jiang et al. [45]with some modifications. L. monocytogenes ATCC 19117, S. aureus ATCC 6538 and S. Typhimurium ATCC 14028 were cultivated in 100 mL of PALCAM, Chapman broth and xylose lysine xeoxycholate (XLD) broth, respectively. After 3 h of incubation, the bacterial growth reached the beginning of the exponential phase (about 106 CFU/ml). At this moment, IP6 was added to the cultures at a final concentration of 1×MIC, 2×MIC and 4×MIC. The three indicator strains grown in the absence of IP6 were used as controls. Changes in the turbidity of the cultures were recorded at OD600 nm and the number of CFU/mL was determined by plating the serial decimal dilutions of samples on PALCAM agar for L. monocytogenes ATCC 19117, Champan agar for S. aureus ATCC 6538, and XLD agar for S. Typhimurium ATCC 14028, and then counting the colonies that appeared.

2.6. Statistical analyses

Measurements were carried out in triplicate and repeated three times. A one-way analysis of variance (ANOVA) was run for each parameter using the SPSS 19 statistical package (SPSS Ltd., Woking, UK). Means and standard errors were calculated and a probability level of P<0.05 was used for assessing the statistical significance of the experimental data. Tukey's post hoc test was used to determine whether differences between each of the mean values were significant (P<0.05). Plate count data were converted to logarithms prior to the statistical analyses. Linear mixed models were used, which made certain assumptions about the errors (e.g. constant variance), to compare the CFU values among treatments with different incubation times (measured in h). Mixed models were fitted using SPSS 19 and followed by post hoc contrasts through the origin. The interpretation of the statistical output by analysis of covariance (ANCOVA, SPSS; covariates, time and trial) of a mixed model requires an understanding of how to explain the relationships among the fixed and random effects in terms of the hierarchy levels. The significance or not of all estimates was confirmed by Wald Z.

3. Results and discussion

3.1. Assessment of the antibacterial activity of phytic acid

The antibacterial activity of phytic acid (IP6) was evaluated against Gram-positive (L. monocytogenes ATCC 19117 and S. aureus ATCC 6538) and Gram-negative (S. Typhimurium ATCC 14028, P. aeruginosa ATCC 49189 and E. coli ATCC 8739) foodborne bacterial pathogens. The antagonistic activity was assessed by determining the MIC values in comparison with CA and by measuring the inhibition zones by agar diffusion method.

3.1.1 Determination of MIC values of phytic acid and citric acid

The comparison of the antimicrobial activity of IP6 and CA against both Gram-negative and Gram-positive bacteria, as determined using the MIC values, is illustrated in . We found that the IP6 MIC ranged from 0.244 to 0.976 mg/ml, while the CA MIC ranged from 1.25 to 2.5 mg/ml. For the Gram-positive bacteria, the IP6 MIC and the CA MIC ranged from 0.488 to 0.976 mg/ml and from 1.25 to 2.5 mg/ml, respectively. The lowest MIC values were for S. aureus ATCC 6538. For the Gram-negative bacteria, the IP6 MICs were reduced by at least 50%, while the CA MIC remained the same as that observed for S. aureus ATCC 6538 (1.25 mg/ml). No significant (P> 0.05) difference was observed in terms of the IP6 MIC and CA MIC (0.244 mg/ml and 1.25 mg/ml, respectively) between the three Gram-negative bacteria ().

Minimum inhibitory concentration of IP6 (□) and CA (■) against indicator strains (L. monocytogenes ATCC 19117, S. aureus ATCC 6538, S. Typhimurium ATCC 14028, P. aeruginosa ATCC 49189 and E. coli ATCC 8739.).

Values represent the means of triplicate experiments with comparable results.

These results indicate that IP6 is more efficient than CA against foodborne pathogenic bacteria (Gram-positive and Gram-negative bacteria).

3.1.2. Diameter inhibition assay of phytic acid

The results of the diameter inhibition assays that assessed the antibacterial effects of IP6 are shown in . For all of the tested concentrations, this natural compound had an antagonistic effect on all indicator strains tested. The diameter of the inhibition zones generally increased with higher IP6 concentrations (P <0.05). Furthermore, IP6 showed a strong inhibitory (P<0.05) effect and varying degrees of antibacterial activity against all of the strains tested, and the inhibition zones were in the range of 8.25–26.75 mm. At a concentration of 50 mg/ml, the largest diameters were recorded for S. aureus and S. Typhimurium while L. monocytogenes exhibited the smallest diameter. In general, the inhibition zones evolved exponentially (P<0.05) and, then, not significantly (P>0.05) between 34.16 and 50 mg/ml.

Diameter inhibition zone of IP6 against L. monocytogenes ATCC 19117(□), S. aureus ATCC 6538 (■) and S. Typhimurium ATCC 14028(↘).

Values represent the means of triplicate experiments with comparable results.

Discs containing 50 mg/ml of IP6 showed the largest mean inhibition zone diameter against S. aureus, while the discs containing 0.25 mg/ml showed the smallest diameter. The inhibition zones recorded for IP6 concentrations between 12.5 and 40 mg/ml were 24.5 mm in diameter. As the diameter remained the same (P>0.05) this clearly demonstrates that IP6 at 12.5 mg/ml brought about a significant (P<0.05) reduction in terms of the S. aureus counts. We also found that the inhibitory action of IP6 against S. Typhimurium ATCC 14028 was comparable to that of S. aureus ATCC 6538. Indeed, this inhibition started from a low concentration of IP6 (1.22 mg/ml) and stabilized (P>0.05) at 34.16 mg/ml. However, for L. monocytogenes ATCC 19117, the antagonistic activity of IP6 was only observed from a concentration of 10 mg/ml.

Values with a different letter (a–c) of a same IP6 concentration are significantly different (P< 0.05).

Values with a different letter (A–E) of a same diameter inhibition zone are significantly different (P<0.05)

3.2. Mode of action of phytic acid

3.2.1. Effect of the dose of phytic acid on the growth of L. monocytogenes, S. aureus and S. Typhimurium in vitro using a linear model (ANOVA)

To investigate the effects of varying IP6 dose on L. monocytogenes, S. aureus and S. Typhimurium, the bacterial growth was followed over 24 h and evaluated in comparison with the control culture (without addition of IP6), following the protocol developed by Jiang et al. [45]. IP6 was added to L. monocytogenes ATCC 19117, S. aureus ATCC 6538 and S. Typhimurium ATCC 14028cells after 3 h of incubation, when growth reached the beginning of the exponential phase (cell density of about 106 CFU/ml). shows that when 1×MIC, 2×MIC and 4×MIC of IP6 were added, a significant downward trend (P<0.05) in the viable count was observed and to some extent was dose-dependent. These results indicate that IP6 has a bactericidal activity against L. monocytogenes ATCC 19117, S. aureus ATCC 6538 and S. Typhimurium ATCC 14028. In fact, for L. monocytogenes ATCC 19117 and S. aureus ATCC 6538, a rapid killing action occurred one hour after the addition of 1×MIC, 2×MIC and 4×MIC of IP6 (incubation time of 4 h) with 1.2, 1.45, 4.29 and 4.57 log10 reductions in the density of the cells. The inhibition of growth even persisted 21 h after the addition of IP6. For the control culture, we noted an increase in the number of viable cells cultured in the absence of IP6 at 24 h of incubation (1013 CFU/ml for L. monocytogenes ATCC 19117, 109 CFU/ml for S. aureus ATCC 6538 and 107 CFU/ml for S.Typhimurium ATCC 14028). Interestingly, the early logarithmic growth phase of S. Typhimurium ATCC 14028 resulted in a rapid decrease in a period of less than one hour. It should be noted that this is the first time that it has been shown that IP6 alone can completely prevent the growth of foodborne bacterial pathogens such as L. monocytogenes, S. aureus and S. Typhimurium. This stands in contrast to the study carried out by Bari et al. (2005), which showed that the combination of nisin (50 μg/ml)-IP6 (0.02%) and nisin-pediocin (100 AU/ml)-IP6 caused significant reductions in L. monocytogenes growth, but the total inactivation of the foodborne pathogen was not achieved by either the individual or the combined application of these antimicrobial agents. This could be due to the lower concentration of IP6 used in their study (0.02%) [46]. Taken together, our results indicate that IP6 exerts a dose-dependent bactericidal effect. According to Kim and Rhee [21], this effect might by due to the strong chelating capacity of IP6. Indeed, IP6 contains six reactive phosphate groups, which are responsible for its strong chelating capacity [21, 47].

Influence of the dose of IP6 on the growth of L. monocytogenes ATCC 19117 (a), S. aureus ATCC 6538(b) and S. Typhimurium ATCC 14028 (c) in vitro using a linear model (ANOVA).

Viable cell counts log10 (CFU/ml) in the absence (Control: ♦) and in the presence of 1×MIC (△), 2×MIC (◇) and 4×MIC (▲) of IP6. The time of addition of IP6 (incubation time of 3 h) is indicated by an arrow.

Values represent the means of triplicate experiments with comparable results.

Values with a different letter (a–d) of a same incubation time are significantly different (P< 0.05).

Values with a different letter (A–F) of a same IP6 dose are significantly different (P< 0.05)

3.2.2 Effect of the dose of phytic acid on the growth of L. monocytogenes, S. aureus and S. Typhimurium in vitro using a general linear model (ANCOVA)

Analysis of covariance (ANCOVA) is a general linear model (GLM) that combines ANOVA with linear regression [48]. Descriptive statistics of the mixed model for the time-related survival of L. monocytogenes, S. aureus and S. Typhimurium(in PALCAM, Chapman and XLD broths, respectively) following treatment with various concentrations of IP6 are presented in Tables and . The tests of fixed effects have an ANOVA-style test for each fixed effect in the model. This means that a single overall test can be used to assess the usefulness of a given explanatory variable, without focusing on individual levels. Explanatory variables that do not have a significant fixed effect can be removed and then the mixed effect analysis can be rerun using a simpler model with fewer explanatory variables (see Tables and ). ANCOVA was used to examine the differences in the means of the dependent variables. The independent variables were the six sampling times (0, 1, 2, 3 and 21 h post IP6 addition), and the four treatments (Trial 1: Control samples, Trial 2: 1×MIC, Trial 3: 2×MIC and Trial 4: 4×MIC). As shown in for L. monocytogenes, a significant effect (P<0.05) was found at 0 (P = 0.023), 1 (P = 0.029) and 2 h (P = 0.044). In contrast, for S. aureus, and S. Typhimurium the most significant P values were observed at 0 h (P = 0.031 and P = 0.010, respectively) and 1 hour (P = 0.049 and P = 0.044, respectively). A significant interaction (P<0.05) between the treatments (all trials) and the bacterial growth time was shown for 0, 1 and 2 h for L. monocytogenes and for 0 and 1 hour for S. aureus and S. Typhimurium (. The results shown in Tables and indicate that the first h (0, 1 and 2) are especially important for bacterial growth inhibition. The first 2 h after adding IP6 would appear to be critical for the inhibition of L. monocytogenes, as after this period, no significant inhibition was found. For S. aureus and S. Typhimurium, growth was inhibited one hour after adding IP6.

The covariance parameters are presented in . The intercept variances were estimated as 2.554530, 1.572786 and 4.286251 for L. monocytogenes, S. aureus and S. Typhimurium, respectively (). The null hypothesis for this parameter is a variance of zero, which would indicate that a random effect is not present. This can be assessed using a statistical test called a Wald Z statistic [49-51]. For L. monocytogenes, S. aureus and S. Typhimurium, this test was run and the null hypothesis (Wald Z = 0.000, P = 0.019), (Wald Z = 0.000, P = 0.007) and (Wald Z = 0.000, P = 0.012) was rejected. This suggests that there are important unmeasured explanatory variables that affect the results in a way that appears random because we do not know the values of the missing explanatory variables ().

The interaction estimates for the differences in slope between trial 4 (4×MIC) and the other trials are shown in Table 2A for the three strains. It is very important to note that the parameter estimates given in the fixed effects are estimates of mean parameters. The effects of the treatments, the bacterial growth time and their interaction on the inhibition of L. monocytogenes, S. aureus and S. Typhimurium are shown in A significant interaction (P<0.05) was found between all of the treatments and the bacterial growth time. This was highly significant (P<0.001) for the interaction between trial 4 (4×MIC) and time for L. monocytogenes, S. aureus and S. Typhimurium(in PALCAM, Chapman and XLD media, respectively) (). These results show that high concentrations of IP6 coupled with incubation time actively inhibit the studied bacteria. Interestingly, for S. Typhimurium, 1×MIC of IP6 is sufficient for bacterial inhibition.

Table 2

Estimates of covariance parameters in L. monocytogenes ATCC 19117, S. aureus ATCC 6538 and S. Typhimurium ATCC 14028 behavior estimates of treatment (Trial) fixed effects.

Parameter	Estimate	Std. Error	Wald Z	P.	LowerBound	LowerBound
Listeria monocytogenes ATCC19117
Residual	3.065435	1.119339	2.739	0.006	1.498571	6.270570
TRIAL [subject = ID] Variance	0.696690	23726566.4060	0.000	0.007	0.000000	.
Staphylococcus aureus ATCC 6538
Residual	1.887343	.689160	2.739	0.006	.922648	3.860696
TRIAL [subject = ID] Variance	17.194269	7.59250110	0.000	0.019	0.000000	.
Salmonella Typhimurium ATCC 14028
Residual	4.286251	1.565118	2.739	0.006	2.095380	8.767837
TRIAL [subject = ID] Variance	12.54789	257.15489	0.000	0.012	0.000000	.

As presented in , for L. monocytogenes, S. aureus and S. Typhimurium, the intercept variances ((Wald Z = 0.810, P = 0.041), (Wald Z = 0.000, P = 0.031) and (Wald Z = 0.000, P = 0.021)) were found to be greater than zero (). The results shown in Tables show that there are interactions between the trial and incubation time for L. monocytogenes, S. aureus and S. Typhimurium.

4. Conclusion

In this study, we demonstrated for the first time the strong inhibitory effect of phytic acid (IP6) against the proliferation of both Gram-positive and Gram-negative foodborne pathogenic bacteria. This was achieved by determining the MIC values and diameter inhibition assays. Of note, IP6 was found to be especially effective against Gram-negative bacteria and more efficient than citric acid, which is widely used as a natural sanitizer in the food industry to reduce the growth rate of foodborne pathogenic bacteria. The analysis study assessing the mode of action confirms these results and indicates that IP6 exerts a dose-dependent bactericidal effect against L. monocytogenes, S. aureus and S. Typhimurium.

Taken together, our results reinforce suggestions that IP6, a natural and biodegradable GRAS compound, could be used for the development of future environmentally friendly applications in the food industry.

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26 Feb 2020

PONE-D-19-34291

Towards understanding of the antagonistic activity of phytic acid against common foodborne bacterial pathogens using a general linear model

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Reviewer #1: In this study, the authors describe the antagonistic activity of phytic acid against common foodborne bacterial pathogens using a general linear model. The results obtained in this study showed an interesting effect of IP6 against the tested bacteria. Unfortunately, the methods used to perform this work are primitive. However, I recommend adding the following tests in order to improve the quality of this work:

- Studying the kinetic parameters of microbial growth in the presence of IP6 (PLoS ONE 10(2): e0114026. doi: 10.1371/journal. pone.0114026).

- Studying the antimicrobial activity of IP6 in a food model.

- The determination of the mode of action of IP6 on the tested bacteria should be reinforced by others test, like Proton Motive Force (PMF) (to check the effect of IP6 on membrane integrity), Scanning Electron Microscopy (SEM)….( BioMed Research Internationa, Doi: 10.1155/2017/7657190).

- After adding these tests, the title can be changed to: “in vitro and in situ effects of phytic acid on the growth of common foodborne bacterial pathogens”.

In addition, the manuscript writing should be improved:

- Salmonella Typhimurium must be written as Salmonella in the italic form with a capital letter and Typhimurium with the non-italic form with a capital letter.

- Lines no. 75-78: “For this reason, in November 2017, the World…… of antibiotics for human medicine”; the authors talk about the recommendation of WHO published in 2017, but the reference at the end of this sentence was published in 2016 and does not presented these recommendations. So, can the authors check again this reference?

- In materiel and method (2.3. Determination of minimum inhibitory concentrations): Can the authors specify the culture media (name and volume) used for the growth of bacteria in microplates.

- In materiel and method (2.4.Agar diffusion method): the reference Smaoui et al. (2010) (43) does not describe the protocol of the agar diffusion method but they referred to another reference. So, can the authors describe the protocol in detail or put another reference that describes it.

- In materiel and method (2.5. Mode of action of phytic acid): To study the mode of action, authors should not use selective media (PALCAM, CHAMPAN and XLD), because these media contain selective substances that may act in synergic/inhibitory/ antagonistic with the tested compound. So, I recommend using TSB or TSA, TSYEB or TSYEA, Mueller Hinton agar or Mueller Hinton broth…to study the mode of action of IP6.

- The resolution of Figures should be improved.

Reviewer #2: The study presented by Boukhris et al. investigates the antibacterial efficiency of phytic acid against common food associated pathogens.

Since alternative methods to control the all over food quality and consumer safety become more important against the background of antimicrobial resistance and the demand of consumers for healthy, natural foods the subject is of interest to a wide number of readers.

The work was generally well prepared and the manuscript is generally well written and clear to read. Nevertheless, from my point of view the manuscript needs some basical revisions prior to publication.

General comments:

The authors should check the whole document whether all the statements made are sufficiently and accurately referenced and if they are formatted according to the requirements of the journal. The whole documents should be double-spaced (e.g. L221-228). Please uniformize the units (e.g. hours or h) throughout the whole document. Check the text for correct formatting and spaces (e.g. P and P and spaces before and after =)

Please uniformize the row heights and text alignment in all tables.

LL 6-7: Please state which authors contributed equally to this study

L10+12 Tunisie/Tunisia please uniformize

L7: Is the superscript „3“ in formatted in bold?

L 38: Please give the full name of the PALCAM agar as already done for XLD broth

LL 50-51: Please add a reference.

LL60-61: Please add a reference and also check with the sentence in LL 50-51.

L 60: „produces“ as an alternative: leads to / causes.

L 64-65: Please add a reference.

L 73: Could you please check whether this the right reference, since you are referring to a single study and not to review?

LL 73-75: Please add a reference

L 78: Reference 22: The reference doesn’t seem to match. Please cite the original recommendation of the WHO.

LL 83-85: Please add a reference.

LL: 91-93: Please add a reference

LL 98-99: Please add a reference

LL 102-103: Please adapt the output style to the requirements of tthe journal.

LL 110: inhibition diameters: in which test?

LL 113-117: I understand why you are pointing at this, but especially the last sentence confuses me. Wouldn’t it be easier to skip the whole paragraph and just mention IP6 an citric acid in the text/description of the respective method?

Material and Methods:

L120: remove the comma after ATCC 19117

L122: remove the comma after ATCC 49189

L 125: Chapman medium (Oxoid… please complete if mentioned for the first time

L 129 (CFU)/ml

LL 129-130: Please adapt the outputstyle to the requirements of the journal and remove the point after (2014)

LL 135: Please adapt the outputstyle to the requirements of the journal

LL 145-149: It doesn’t come to me easily how exactly the AWDA was performed. Please provide further details

L 152: Please adapt the outputstyle to the requirements of the journal

L 159: CFU per …?

L 182: measuring the inhibition zones in AWDA?

L 203: indicative strains- indicator strains?

LL 259-262: To my opinion, the data do not prove chelat forming. Perhaps you could rebuilt the sentence as: According to Kimm and Rhee this effect might by due to the strong chelating …. ?

LL 274 The ANCOVA and later on WALD Z statistic was not mentioned in the material and method section.

L285-286: I don’t think, that you have to explain the meaning and interpretation of P.

LL 313-315 Well, yes - but this sentence is confusing.

**********

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Reviewer #1: Yes: Abdelaziz ED-DRA

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18 Mar 2020

Response to Referees: Manuscript ID: PONE-D-19-34291

Title:

“Towards understanding the antagonistic activity of phytic acid against common foodborne bacterial pathogens using a general linear model”

By

Boukhris et al.,

Journal: PLOS ONE

Point-by-point answer to Reviewers comments

First of all, we would like to thank Reviewers for their interesting and constructive comments.

Required modifications are now addressed in all sections of the revised manuscript and are illustrated in red colour.

Answers to the Reviewer 1 Comments

• In this study, the authors describe the antagonistic activity of phytic acid against common foodborne bacterial pathogens using a general linear model. The results obtained in this study showed an interesting effect of IP6 against the tested bacteria.

Dear Reviewer, thank you very much for your kind judgment of our manuscript. We are grateful for the time and energy you expended to improve our work. In the following sections, you will find our responses to each of your points and suggestions.

• Unfortunately, the methods used to perform this work are primitive. However, I recommend adding the following tests in order to improve the quality of this work:

- Studying the kinetic parameters of microbial growth in the presence of IP6 (PLoS ONE 10(2): e0114026. doi: 10.1371/journal. pone.0114026).

- Studying the antimicrobial activity of IP6 in a food model.

- The determination of the mode of action of IP6 on the tested bacteria should be reinforced by others test, like Proton Motive Force (PMF) (to check the effect of IP6 on membrane integrity), Scanning Electron Microscopy (SEM)….( BioMed Research Internationa, Doi: 10.1155/2017/7657190).

- After adding these tests, the title can be changed to: “in vitro and in situ effects of phytic acid on the growth of common foodborne bacterial pathogens”.

Dear Reviewer, thank you very much for these pertinent suggestions. In fact, our study focused on the understanding of the mechanism of natural antimicrobial action of phytic acid (IP6), by the development of predictive mathematical models. To elucidate a better understanding on the phytic acid as an antibacterial agent, linear (ANOVA) and general (ANCOVA) models were mathematically used to model the growth rate of Listeria monocytogenes ATCC 19117, Staphylococcus aureus ATCC 6538 and Salmonella Typhimurium ATCC 14028. Furthermore, the combination of ANOVA analysis and ANCOVA approach was used for linking all data. It should be noted that predictive inactivation models have been developed in liquid laboratory media that can mimic the microbial environment.

In this regard, to the best of our knowledge, no data are available on simultaneously study of inactivation of three food borne pathogens (L. monocytogenes, S. aureus and S. Typhimurium) by predictive mathematical models.

- On the other hand, studying the antimicrobial activity of IP6 in a food model is currently in progress. Indeed, we recently investigated the effects of different concentrations of IP6 on shelf-life, microbiological, physicochemical and sensory qualities of stored raw beef and chicken meat products. The analysis of these results is ongoing and will be the subject of another publication.

- Equally, for a conclusive confirmation of the mode of action of IP6 on the all tested bacteria, an experimental validation by Scanning Electron Microscopy (SEM) is currently in progress due to the unavailability of functional scanning electron microscope in region last year.

• In addition, the manuscript writing should be improved:

- Salmonella Typhimurium must be written as Salmonella in the italic form with a capital letter and Typhimurium with the non-italic form with a capital letter.

Dear Reviewer, according to your remarks; corrections are made in the text.

Please see the revised version

- Lines no. 75-78: “For this reason, in November 2017, the World…… of antibiotics for human medicine”; the authors talk about the recommendation of WHO published in 2017, but the reference at the end of this sentence was published in 2016 and does not presented these recommendations. So, can the authors check again this reference?

Dear Reviewer, you are quite right. According to your remark changes are made as follows:

• For this reason, the World Health Organization (WHO) recommended that breeders and the food industry stop misusing antibiotics, such as for promoting animal growth and preventing disease in healthy animals, in order to preserve the effectiveness of antibiotics for human medicine [22].

The reference

22. Holmes AH, Moore LS, Sundsfjord A, Steinbakk M, Regmi S, Karkey A, et al. Understanding the mechanisms and drivers of antimicrobial resistance. Lancet. 2016;387: 176-187. Was change by:

22. World Health Organization (WHO). Strategic and Technical Advisory Group on Antimicrobial Resistance. Report of the first meeting Geneva, Sept 19–20, 2013; http://www.who.int/drugresistance/stag/amr_stag_meeting report 0913.pdf (accessed July 14, 2015).

Please see the revised version

- In materiel and method (2.3. Determination of minimum inhibitory concentrations): Can the authors specify the culture media (name and volume) used for the growth of bacteria in microplates.

Dear Reviewer, thank you for this interesting observation, as mentioned above, the correction was made and the following paragraphs were added to the section 2.3. Determination of minimum inhibitory concentrations in Materials and Methods part:

The test was performed in sterile 96-well microplates with a final volume of 100 μl per well. A commercial stock solution of IP6 (50% (w/w) in water) and a stock solution of CA at 50% (w/w) in water were used. Then, the corresponding concentrations of IP6 and CA were transferred to each successive well in order to obtain a two-fold serial dilution of the original sample. In fact, each sample was dissolved to a final concentration of 0.078, 0.156, 0.312, 0.625, 1.25, 2.5, 5, 10 and 20 mg/mL and then filtered through 0.22 μm pore-size black polycarbonate filters (Millipore). To each test well 10 μl of cell suspension were added to final inoculum concentration of 106 CFU/ml of bacterium. Positive growth control well consisted of Listeria monocytogenes ATCC 19117, Staphylococcus aureus ATCC 6538 and Salmonella Typhimurium ATCC 14028 respectively growth in PALCAM, Chapman and XLD. Plates were then covered with the sterile plate covers and incubated at 37 °C for 24 h. As an indicator of microorganism growth, 25 μl of thiazolyl blue tetrazolium bromide (MTT) indicator solution (0.5 mg/ml) dissolved in sterile water was added to the wells and incubated at 37 °C for 30 min. The colourless tetrazolium salt acts as an electron acceptor and was reduced to a red-coloured formazan product by biologically active organisms. Where microbial growth was inhibited, the solution in the well remained clear after incubation with MTT. The determination of MIC values was done in triplicate.

Please see the revised version

- In materiel and method (2.4.Agar diffusion method): the reference Smaoui et al. (2010) (43) does not describe the protocol of the agar diffusion method but they referred to another reference. So, can the authors describe the protocol in detail or put another reference that describes it.

Dear Reviewer, thank you for this pertinent remark,

• This reference: [43]. Smaoui S, Elleuch L, Bejar W, Karray-Rebai I, Ayadi I, Jaouadi B et al. Inhibition of fungi and gram-negative bacteria by bacteriocin BacTN635 produced by Lactobacillus plantarum sp. TN635. App Biochem Biotechnol. 2010; 162: 1132-1146 was changed by :

• 44. Valgas C, Souza SMD, Smânia EF, Smânia Jr A. Screening methods to determine antibacterial activity of natural products. Braz. J. Microbiol. 2007; 38: 369-380.

Please see the revised version

- In materiel and method (2.5. Mode of action of phytic acid): To study the mode of action, authors should not use selective media (PALCAM, CHAMPAN and XLD), because these media contain selective substances that may act in synergic/inhibitory/ antagonistic with the tested compound. So, I recommend using TSB or TSA, TSYEB or TSYEA, Mueller Hinton agar or Mueller Hinton broth…to study the mode of action of IP6.

Thank you for this interesting remark. In fact, we have chosen to use only the corresponding selective media (PALCAM, Chapman and XLD) for cultivation of Listeria monocytogenes ATCC 19117, Staphylococcus aureus ATCC 6538 and Salmonella Typhimurium ATCC 14028 to track the exact growth of each bacterium. Equally, we avoided the TSB since it is a Non selective medium.

- The resolution of Figures should be improved.

The Reviewer has perfectly reason for this comment. This comment has been addressed in the revised manuscript.

Please see the revised version

Answers to the Reviewer 2 Comments

The study presented by Boukhris et al. investigates the antibacterial efficiency of phytic acid against common food associated pathogens.

Since alternative methods to control the all over food quality and consumer safety become more important against the background of antimicrobial resistance and the demand of consumers for healthy, natural foods the subject is of interest to a wide number of readers.

The work was generally well prepared and the manuscript is generally well written and clear to read. Nevertheless, from my point of view the manuscript needs some basical revisions prior to publication.

Dear Reviewer, thank you very much for your kind judgment about our paper. We are grateful for the time and energy you expended on our work. In the following sections, you will find our responses to each of your points and suggestions.

General comments:

• The authors should check the whole document whether all the statements made are sufficiently and accurately referenced and if they are formatted according to the requirements of the journal. The whole documents should be double-spaced (e.g. L221-228). Please uniformize the units (e.g. hours or h) throughout the whole document. Check the text for correct formatting and spaces (e.g. P and P and spaces before and after =)

• Please uniformize the row heights and text alignment in all tables.

• LL 6-7: Please state which authors contributed equally to this study

• L7: Is the superscript „3“ in formatted in bold?

• L10+12 Tunisie/Tunisia please uniformize

Dear Reviewer, thank you for these interesting remarks. As suggested, corrections have been addressed in the revised manuscript. There is no equal contribution for the authors to this study.

Please see the revised version

• L 38: Please give the full name of the PALCAM agar as already done for XLD broth

Dear reviewer, as recommended, the full name of the PALCAM agar has been introduced

Please see the revised version

• LL 50-51: Please add a reference.

Dear Reviewer, as recommended, two references were introduced in L50-51

1. Havelaar AH, Kirk MD, Torgerson PR, Gibb HJ, Hald T, Lake RJ, et al. World Health Organization global estimates and regional comparisons of the burden of foodborne disease in 2010. PLoS Med. 2015; 12.

2. Bhaskar SV. Foodborne diseases—disease burden. In Food Safety in the 21st Century. Academic Press; 2017. pp. 1-10.

Please see the revised version

• LL60-61: Please add a reference and also check with the sentence in LL 50-51.

Dear Reviewer, as recommended, the following reference was introduced in L60-61

10. Morach M, Käppeli N, Hochreutener M, Johler S, Julmi J, Stephan R, et al. Microarray based genetic profiling of Staphylococcus aureus isolated from abattoir byproducts of pork origin. PLoS One. 2019;14(9).

Please see the revised version

• L 60: „produces“ as an alternative: leads to / causes.

Dear Reviewer, as suggested, correction was made.

Please see the revised version

• L 64-65: Please add a reference.

Dear Reviewer, as recommended, the following reference was introduced in L64-65

17. Nilsson OR, Kari L, Steele-Mortimer O. Foodborne infection of mice with Salmonella Typhimurium. PLoS One. 2019;14(8).

Please see the revised version

• L 73: Could you please check whether this the right reference, since you are referring to a single study and not to review?

Dear Reviewer, thank you for this pertinent remark,

• This reference: 21. Kim NH, Rhee MS. Phytic acid and sodium chloride show marked synergistic bactericidal effects against nonadapted and acid-adapted Escherichia coli O157: H7 strains, Appl Environ Microbiol. 2016;82:1040-1049, was changed by :

• 21. Parish ME, Beuchat LR, Suslow TV, Harris LJ, Garrett EH, Farber JN, Busta FF. Methods to reduce/eliminate pathogens from fresh and fresh‐cut produce. Compr. Rev. Food Sci. Food Saf. 2003;2: 161-173.

Please see the revised version

• LL 73-75: Please add a reference

Dear Reviewer, as recommended, the following reference was introduced in L64-65

22. World Health Organization (WHO). Strategic and Technical Advisory Group on Antimicrobial Resistance. Report of the first meeting Geneva, Sept 19–20, 2013; http://www.who.int/drugresistance/stag/amr_stag_meeting report 0913.pdf (accessed July 14, 2015).

Please see the revised version

• L 78: Reference 22: The reference doesn’t seem to match. Please cite the original recommendation of the WHO.

Dear Reviewer, as recommended, the following reference was introduced in L64-65

22. World Health Organization (WHO). Strategic and Technical Advisory Group on Antimicrobial Resistance. Report of the first meeting Geneva, Sept 19–20, 2013; http://www.who.int/drugresistance/stag/amr_stag_meeting report 0913.pdf (accessed July 14, 2015).

Please see the revised version

• LL 83-85: Please add a reference.

Dear Reviewer, as recommended, the following reference was introduced in L64-65

27. Lin CM, Sheu SR, Hsu SC, Tsai YH. Determination of bactericidal efficacy of essential oil extracted from orange peel on the food contact surfaces. Food Control. 2010; 21(12):1710-1715.

Please see the revised version

• LL: 91-93: Please add a reference

Dear Reviewer, as recommended, the following reference was introduced in L64-65

29. Schlemmer U, Frolich W, Prieto RM, Grases F. Phytate in foods and significance for humans: food sources, intake, processing, bioavailability, protective role and analysis. Mol Nutr Food Res. 2009;53:S330-S375.

Please see the revised version

• LL 98-99: Please add a reference

Dear Reviewer, as recommended, the following reference was introduced in L64-65

31. Bhowmik A, Ojha D, Goswami D, Das R, Chandra NS, Chatterjee TK, et al. Inositol hexa phosphoric acid (phytic acid), a nutraceuticals, attenuates iron-induced oxidative stress and alleviates liver injury in iron overloaded mice. 2017. Biomed Phramacother. 2017;87: 443-450.

Please see the revised version

• LL 102-103: Please adapt the output style to the requirements of the journal.

Dear Reviewer, thank you for this remark. As suggested, correction was made.

Please see the revised version

• LL 110: inhibition diameters: in which test?

Dear Reviewer, thank you for your pertinent remark. The sentence ‘This is achieved by: (i) determining the minimum inhibitory concentration (MIC) as compared to citric acid (CA); (ii) measuring the inhibition diameters, and (iii) illustrating the mode of action of IP6 for inhibiting pathogen growth., was replaced by:

This is achieved by: (i) determining the minimum inhibitory concentration (MIC) as compared to citric acid (CA); (ii) measuring the inhibition diameters of each indicator bacteria growth inhibition or reduction, and (iii) illustrating the mode of action of IP6 for inhibiting pathogen growth.

Please see the revised version

• LL 113-117: I understand why you are pointing at this, but especially the last sentence confuses me. Wouldn’t it be easier to skip the whole paragraph and just mention IP6 and citric acid in the text/description of the respective method?

Dear Reviewer, you are quite right, the correction was made as proposed, and the sentence’ All of the other chemicals used in this study were commercially available in analytical grade’ was deleted

Material and Methods:

• L120: remove the comma after ATCC 19117

• L122: remove the comma after ATCC 49189

Dear Reviewer, the correction was made as proposed.

Please see the revised version.

• L 125: Chapman medium (Oxoid… please complete if mentioned for the first time

Dear Reviewer, the correction was made as proposed: …Chapman medium (Oxoid, Basingstoke, Hampshire, UK)…

Please see the revised version.

• L 129 (CFU)/ml

Dear Reviewer, the correction was made as proposed: ….106 colony-forming units of bacteria per milliliter (CFU/ml)…

Please see the revised version.

• LL 129-130: Please adapt the output style to the requirements of the journal and remove the point after (2014)

Dear Reviewer, the correction was made as proposed.

Please see the revised version.

• LL 135: Please adapt the output style to the requirements of the journal

Dear Reviewer, the correction was made as proposed.

Please see the revised version.

• LL 145-149: It doesn’t come to me easily how exactly the AWDA was performed. Please provide further details

Dear Reviewer, thank you for this pertinent comment. The following paragraph was added to the ‘2.4. Agar diffusion method’ section in the Materials and Methods part of the revised Manuscript. According to reviewer Comment, we insert this part:

2.4. Agar diffusion method

The antimicrobial activity of IP6 was evaluated by means of agar-well diffusion assays, as described by Valgas et al. [43]. Fifteen milliliters of the molten agar (45 °C) were poured into sterile petri dishes (Ø 90 mm). Working cell suspensions were prepared at 106 CFU/mL, and 100 μl was evenly spreaded onto the surface of the agar plates of Luria–Bertani (LB) agar (Oxoid Ltd, UK). Once the plates had been aseptically dried, 06 mm wells were punched into the agar with a sterile Pasteur pipette. IP6 was dissolved in water to a final concentration of 50 mg/ml and then filtered through 0.22 μm pore-size black polycarbonate filters (Millipore). Thus, 50 μl were placed into the wells and the plates were incubated at 37 °C for 24 h for bacterial strains. Antibacterial activity was evaluated by measuring the diameter of circular inhibition zones around the well. The un-inoculated media were also tested for inhibitory zones as a control. Tests were performed in triplicate.

Please see the revised version.

• L 152: Please adapt the output style to the requirements of the journal

Dear Reviewer, the correction was made as proposed.

Please see the revised version.

• L 159: CFU per …?

Dear Reviewer, the correction was made as proposed.

Please see the revised version.

• L 182: measuring the inhibition zones in AWDA?

Dear Reviewer, you are quite right, corrections are introduced.

The antagonistic activity was assessed by determining the MIC values in comparison with CA and by measuring the inhibition zones by agar diffusion method.

Please see the revised version.

• L 203: indicative strains- indicator strains?

Dear Reviewer, the correction was made as proposed.

Please see the revised version.

• LL 259-262: To my opinion, the data do not prove chelat forming. Perhaps you could rebuilt the sentence as: According to Kimm and Rhee this effect might by due to the strong chelating …. ?

Dear Reviewer, the correction was made as proposed.

Please see the revised version.

• LL 274 The ANCOVA and later on WALD Z statistic was not mentioned in the material and method section.

Dear Reviewer, correction was made according to your remark

• The following paragraph was revised in the Materials and Methods part in the 2.6. Statistical analyses section. According to Reviewer comment we insert this part:

…. Mixed models were fitted using SPSS 19 and followed by post hoc contrasts through the origin. The interpretation of the statistical output by Analysis of Covariance (ANCOVA, SPSS; covariates, time and trial) of a mixed model requires an understanding of how to explain the relationships among the fixed and random effects in terms of the hierarchy levels. The significance or not of all estimates was confirmed by Wald Z.

Please see the revised version.

• L285-286: I don’t think, that you have to explain the meaning and interpretation of P.

Dear Reviewer, Thank you for the pertinent remark. In fact, an upper decision limit is obtained using adjusted P-value Also, in our study; the test of homogeneity of variances gives P-value 0.05 that supports homogeneity of variances Equally, as indicated in the ‘Statistical analyses’ section in Materials and Methods part, the probability level of P<0.05 was used for assessing the statistical significance of the experimental data, and Tukey's post hoc test was used to determine whether differences between each of the mean values were significant (P<0.05).

• LL 313-315 Well, yes - but this sentence is confusing.

Dear Reviewer, you are quite right, this sentence was removed from the text

Please see the revised version.

Submitted filename: Response to reviewers.doc

Click here for additional data file.

24 Mar 2020

Towards understanding the antagonistic activity of phytic acid against common foodborne bacterial pathogens using a general linear model

PONE-D-19-34291R1

Dear Dr. Hichem Chouayekh,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

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Academic Editor

PLOS ONE

2 Apr 2020

PONE-D-19-34291R1

Towards understanding the antagonistic activity of phytic acid against common foodborne bacterial pathogens using a general linear model

Dear Dr. Chouayekh:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Table 1

L. monocytogenes ATCC 19117, S. aureus ATCC 6538 and S. Typhimurium ATCC 14028 behavior estimates of treatment (Trial) fixed effects.

Parameter	Estimate	Std. Error	df	T	Sig.	LowerBound	LowerBound
Listeria monocytogenes ATCC19117
Intercept	12.195532	1.836295	15.000	6.641	0.000 (***)	8.281562	16.109502
Hour 0 (time of IP6 addition)	-6.105627	2.596913	15.000	-2.351	0.023 (*)	-11.640817	-0.570438
Hour 1 (post IP6 addition)	-5.254537	2.596913	15.000	-2.023	0.029 (*)	-10.789727	0.280652
Hour 2(post IP6 addition)	-2.815143	2.596913	15.000	-1.084	0.044 (*)	-8.350332	2.720047
Hour 3(post IP6 addition)	-2.103050	2.596913	15.000	-0.810	0.131 (ns)	-7.638239	3.432139
Hour 21(post IP6 addition)	0a	0	.	.	.	.	.
Trial	-2.959106	0.553664	15.000	-5.345	0.069 (ns)	-4.139213	-1.779000
Hour 0 × Trial	2.959106	0.782999	15.000	3.779	0.002 (**)	1.290184	4.628029
Hour 1 × Trial	2.443571	0.782999	15.000	3.121	0.017 (*)	0.774649	4.112493
Hour 2 × Trial	0.984288	0.782999	15.000	1.257	0.027 (*)	-0.684635	2.653210
Hour 3 × Trial	0.781291	0.782999	15.000	0.998	0.134 (ns)	-0.887631	2.450214
Hour 21 × Trial	0a	0	.	.	.	.	.
Staphylococcus aureus ATCC 6538
Intercept	9.402978	1.440860	15.000	6.526	0.000 (***)	6.331858	12.474098
Hour 0	-3.028230	2.037683	15.000	-1.486	0.031 (*)	-7.371449	1.314990
Hour 1	-0.928906	2.037683	15.000	-0.456	0.049 (*)	-5.272125	3.414314
Hour 2	0.995682	2.037683	15.000	0.489	0.249 (ns)	-3.347537	5.338902
Hour 3	1.181910	2.037683	15.000	0.580	0.611(ns)	-3.161310	5.525129
Hour 21	0a	0	.	.	.	.	.
Trial	-2.267845	1.058362	528.35	-2.143	0.075 (ns)	-4.346960	-0.188731
Hour 0 × Trial	2.267845	1.496750	528.35	1.515	0.013 (*)	-0.672467	5.208157
Hour 1× Trial	1.043681	1.496750	528.35	0.697	0.039 (*)	-1.896631	3.983994
Hour 2 × Trial	0.067139	1.496750	528.35	0.045	0.254 (ns)	-2.873173	3.007451
Hour 3 × Trial	-0.063827	1.496750	528.35	-0.043	0.566 (ns)	-3.004139	2.876485
Hour 21 × Trial	0a	0	.	.	.	.	.
Salmonella Typhimurium ATCC 14028
Intercept	6.683287	2.171377	15.000	3.078	0.008 (**)	2.055107	11.311466
Hour 0	-0.802473	3.070790	15.000	-0.261	0.010 (*)	-7.347707	5.742761
Hour 1	-1.983238	3.070790	15.000	-0.646	0.044 (*)	-8.528472	4.561996
Hour 2	-2.442463	3.070790	15.000	-0.795	0.065 (ns)	-8.987697	4.102771
Hour 3	-2.425511	3.070790	15.000	-0.790	0.249 (ns)	-8.970746	4.119723
Hour 21	0a	0	.	.	.	.	.
Trial	-1.670822	8.797076	488978	-0.190	0.104 (ns)	-18.912811	15.571168
Hour 0 × Trial	1.670822	12.44094	488978	0.134	0.016 (*)	-22.713034	26.054677
Hour 1× Trial	0.495809	12.44094	488978	0.040	0.033 (*)	-23.888046	24.879665
Hour 2 × Trial	0.610616	12.44094	488978	0.049	0.226 (ns)	-23.773240	24.994471
Hour 3 × Trial	0.606378	12.44094	488978	0.049	0.346 (ns)	-23.777478	24.990233
Hour 21 × Trial	0a	0	.	.	.	.	.

a:This parameter is set to zero because it is redundant.

Std. Error: standard error; df: The degrees of freedom; t: the Student t-statistic; Sig.: the p-value (associated with the correlation).ns: P>0.05;

*P<0.05;

**P<0.01;

***P<0.001.

Table 3

L. monocytogenes ATCC 19117, S. aureus ATCC 6538 and S. Typhimurium ATCC 14028 behavior estimates of incubation time (h) fixed effects.

Parameter	Estimate	Std. Error	df	T	Sig.	LowerBound	LowerBound
Listeria monocytogenes ATCC19117
Intercept	3,909020	0,957314	14,666	4,083	0.014 (*)	1,864494	5,953546
Trial 1	3,415564	1,218892	12,745	2,802	0.277 (ns)	0,776946	6,054182
Trial 2	3,015635	1,218892	12,745	2,474	0.122 (ns)	0,377017	5,654253
Trial 3	2,494664	1,218892	12,745	2,047	0.130 (ns)	-0,143954	5,133282
Trial 4	0,172542	1,218892	12,745	,142	0.910 (ns)	-2,466076	2,811161
Hour	-0,636441	0,342916	2,829	-1,856	0.022 (*)	-1,766075	0,493193
Trial 1 ×Hour	0,485649	0,127775	12,745	3,801	0.021 (**)	0,209046	0,762251
Trial 2 ×Hour	0,053229	0,127775	12,745	,417	0.008 (**)	-0,223374	0,329831
Trial 3 ×Hour	-0,098872	0,127775	12,745	-,774	0.006 (**)	-0,375474	0,177731
Trial 4 ×Hour	-0,008869	0,127775	12,745	-,069	0.000 (***)	-0,285471	0,267733
Staphylococcus aureus ATCC 6538
Intercept	3,330309	1,001639	14,862	3,325	0.004 (**)	1,193637	5,466980
Trial 1	4,751291	1,299773	12,677	3,655	0.071 (ns)	1,936014	7,566569
Trial 2	3,670235	1,299773	12,677	2,824	0.093 (ns)	,854958	6,485513
Trial 3	2,967218	1,299773	12,677	2,283	0.122 (ns)	,151940	5,782495
Trial 4	0,077913	1,299773	12,677	0,060	0.966 (ns)	-2,737364	2,893191
Hour	-0,541623	0,307282	1,927	-1,763	0.049 (*)	-1,913269	,830023
Trial 1 ×Hour	0,267524	0,136253	12,677	1,963	0.022 (*)	-,027597	,562645
Trial 2 ×Hour	0,007960	0,136253	12,677	0,058	0.009 (**)	-,287161	,303082
Trial 3 ×Hour	-0,125313	0,136253	12,677	-0,920	0.001 (**)	-,420434	,169808
Trial 4 ×Hour	-0,004005	0,136253	12,677	-0,029	0.000(***)	-,299126	,291116
Salmonella Typhimurium ATCC 14028
Intercept	4.968089	0.753077	12.138	6.597	0,000 (***)	3.329336	6.606842
Trial 1	3.683758	0.883156	11.714	4.171	0,001 (**)	1.754296	5.613219
Trial 2	3.90459×10−15	0.883156	11.714	0.000	0.214 (ns)	-1.929461	1.929461
Trial 3	3.94568×10−15	0.883156	11.714	0.000	0.354 (ns)	-1.929461	1.929461
Trial 4	3.94568×10−15	0.883156	11.714	0.000	0.555 (ns)	-1.929461	1.929461
Hour	-2.148155	0.927415	3.026	-2.316	0,031 (*)	-5.085513	.789204
Trial 1 ×Hour	0.238283	0.092580	11.714	2.574	0,0011(*)	.036020	.440545
Trial 2 ×Hour	-2.7329×10−16	0.092580	11.714	0.000	0.000 (***)	-.202263	.202263
Trial 3 ×Hour	-2.8090×10−15	0.092580	11.714	0.000	0.000 (***)	-.202263	.202263
Trial 4 ×Hour	-2.8090×10−16	0.092580	11.714	0.000	0.000 (***)	-.202263	.202263

Std. Error: standard error; df: The degrees of freedom; t: the Student t-statistic; Sig.: the p-value (associated with the correlation).ns: P>0.05;

*P<0.05;

**P<0.01;

***P<0.001.

Trial 1: Control (No IP6 added); Trial 2: 1×MIC. Trial 3: 2×MIC. Trial 4: 4×MIC.

Table 4

Estimates of covariance parameters in L. monocytogenes ATCC 19117. S. aureus ATCC 6538 and S. Typhimurium ATCC 14028 behavior estimates of incubation time (h) fixed effects.

Parameter	Estimate	Std. Error	Wald Z	P	LowerBound	LowerBound
Listeria monocytogenes ATCC19117
Residual	2.450288	0.935779	2.618	0.009	1.159147	5.179597
HOUR [subject = ID] Variance	0.085107	0.105096	0.810	0.041	0.007566	0.957389
Staphylococcus aureus ATCC 6538
Residual	3.501211	1.107180	3.162	0.002	1.883843	6.507165
HOUR [subject = ID] Variance	32.26787	11.51787	0.000	0.031	0.000000	.
Salmonella TyphimuriumATCC 14028
Residual	1,325082	0,547535	2,420	0,016	0,589548	2,978285
TRIAL [subject = ID] Variance	3,123839	2,784617	1,122	0,021	0,544403	17,924910