Antibiofilm activity of the biosurfactant and organic acids against foodborne pathogens at different temperatures, times of contact, and concentrations

Daiane Carvalho; Rafaela Menezes; Gabriela Zottis Chitolina; Hiran Castagnino Kunert-Filho; Daiane Elisa Wilsmann; Karen Apellanis Borges; Thales Quedi Furian; Carlos Tadeu Pippi Salle; Hamilton Luiz de Souza Moraes; Vladimir Pinheiro do Nascimento

doi:10.1007/s42770-022-00714-4

. 2022 Mar 8;53(2):1051–1064. doi: 10.1007/s42770-022-00714-4

Antibiofilm activity of the biosurfactant and organic acids against foodborne pathogens at different temperatures, times of contact, and concentrations

Daiane Carvalho ¹, Rafaela Menezes ¹, Gabriela Zottis Chitolina ¹, Hiran Castagnino Kunert-Filho ¹, Daiane Elisa Wilsmann ¹, Karen Apellanis Borges ^1,^✉, Thales Quedi Furian ¹, Carlos Tadeu Pippi Salle ¹, Hamilton Luiz de Souza Moraes ¹, Vladimir Pinheiro do Nascimento ¹

PMCID: PMC9151985 PMID: 35260995

Abstract

Biofilm formation has been suggested to play a significant role in the survival of pathogens in food production. Interest in evaluating alternative products of natural origin for disinfectant use has increased. However, there is a lack of information regarding the effects of biosurfactants and organic acids on Salmonella enterica serotype Enteritidis, Escherichia coli, and Campylobacter jejuni biofilms, mainly considering temperatures found in environments of poultry processing, as well as simulating the contact times used for disinfection. The aim of this study was to evaluate the antibiofilm activity of rhamnolipid, malic acid, and citric acid on the adhesion of S. Enteritidis, E. coli, and C. jejuni on polystyrene surfaces at different temperatures (4, 12, and 25 °C), compound concentrations, and times of contact (5 and 10 min), and to analyze the potential use of these compounds to disrupt formed biofilms. All three compounds exhibited antibiofilm activity under all analyzed conditions, both in the prevention and removal of formed biofilms. Contact time was less important than temperature and concentration. The antibiofilm activity of the compounds also varied according to the pathogens involved. In the food industry, compound selection must consider the temperature found in each stage of product processing and the target pathogens to be controlled.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-022-00714-4.

Keywords: Campylobacter jejuni, Citric acid, Escherichia coli, Malic acid, Rhamnolipid, Salmonella Enteritidis.

INTRODUCTION

According to the World Health Organization (WHO), Salmonella spp., Escherichia coli, and Campylobacter spp. are among the most common foodborne pathogens that affect millions of people annually [1]. In the USA and Europe, Salmonella enterica subesp. enterica serotype Enteritidis and Campylobacter spp. are responsible for the majority of foodborne illnesses [2, 3]. In Brazil, the majority of foodborne outbreaks that occurred between 2009 and 2018 were linked to bacterial agents. Of these, approximately 35% were caused by Salmonella spp. or E. coli [4]. Several studies have shown that poultry products are the main source of Salmonella and C. jejuni infections in humans [5]. In addition, some reports suggest that avian pathogenic E. coli (APEC) strains may have zoonotic potential and could be transmitted via chicken meat to humans [6, 7].

The survival and persistence of pathogens in the environment, especially in poultry processing plants, is a significant risk factor that contributes to their spread through the food chain [8]. Biofilm formation by S. Enteritidis, E. coli, and C. jejuni is suggested to play a significant role in the survival of these pathogens in food production and processing environments [9–11]. This represents a risk to consumer health and results in economic losses to the industry [12]. The ability of S. Enteritidis, E. coli, and C. jejuni to adhere to surfaces commonly found in the poultry processing industry, such as stainless steel and plastic, over a wide temperature range of 4 to 42 °C has been previously described [10, 13–20].

Food processing plants are routinely subjected to cleaning and disinfection procedures to promote microbiological control and to prevent bacterial adhesion. However, due to the increasing number of strains resistant to the antimicrobials used, there is a growing concern in the society as a whole, including the scientific community, to identify alternative compounds of natural origin and to evaluate their efficacy against pathogens, mainly due to their safety use [21, 22]. Biosurfactants and organic acids are attractive to the food industry because of their low or no toxicity, high biodegradability, and good performance at extreme temperatures, pH, and salinity [23–25]. They are classified according to their molecular structure into mainly glycolipids, including rhamnolipids. Rhamnolipids are strong surface-active glycolipids with emulsion-forming ability and antimicrobial activity. Pseudomonas aeruginosa is considered the primary rhamnolipid-producing microorganism. However, many other bacterial species have been reported to produce this biosurfactant [26]. Increased interest in the properties of rhamnolipids has led to them becoming a target for production on a commercial scale in personal and home care [27]. However, it still does not have the generally recognized as safe (GRAS) status by the US Food and Drug Administration (FDA), and it is not used in the food industry [28]. Citric and malic acids are organic acids with GRAS status by the FDA [28]. Citric acid (21CFR184.1033) is the primary organic acid present in citrus fruits and has been approved for use in food with no limitations other than current good manufacturing practice [28]. This acid can inhibit bacterial growth through lowering the pH and metal-chelating capabilities. Similarly, malic acid (21CFR582.1069) is normally found in fruits and vegetables and can be used in food in accordance with good manufacturing or feeding practice [28]. Its antibacterial activity is associated with its pH-lowering ability and cytoplasmic damage in bacterial cells [21].

The use of biosurfactants and organic acids against foodborne pathogens has been previously described [29–31]. However, there is still a lack of information concerning the effects of these compounds on S. Enteritidis, E. coli, and C. jejuni biofilms, mainly considering temperatures found in environments of poultry processing, as well as simulating contact times and their concentrations used for disinfection. The aim of this study was to evaluate the antibiofilm activity of rhamnolipid, citric acid, and malic acid on adhesion and biofilm formed by S. Enteritidis, E. coli, and C. jejuni on polystyrene surfaces under different temperatures, times of contact, and concentrations.

MATERIALS AND METHODS

Bacterial strains

Strains of S. Enteritidis (n = 21), E. coli (n = 30), and C. jejuni (n = 24) were randomly selected from our stock collections for this study. These strains were isolated in Southern Brazil from poultry, food implicated in outbreaks, and poultry slaughterhouses (Table S1). S. Enteritidis and E. coli were kept frozen at − 80 °C in brain heart infusion broth (BHI; Oxoid, UK) supplemented with 15% glycerol (Synth, Brazil). C. jejuni strains were stored at − 80 °C in ultra-high-temperature-processed milk. All strains were previously tested by crystal violet for their ability to produce biofilms at 4 °C (temperature of the handling environment in poultry slaughterhouse), 12 °C (temperature required by the Brazilian sanitary service in cutting rooms of broiler processing plants), and 25 °C (room temperature) [32]. As a positive control for biofilm formation under all conditions tested, a standard strain of S. Typhimurium ATCC 17025 was used.

Inoculum preparation

S. Enteritidis and E. coli strains were reactivated in BHI for 18–24 h at 37 °C. After incubation, the strains were cultured on trypticase soy agar (TSA; Oxoid, UK) plates for 24 h at 37 ºC. One colony of each strain was suspended in 3 mL of trypticase soy broth without glucose (TSB; BD Biosciences, USA) for 18 h at 37 °C. The McFarland standard No. 1 (Probac do Brasil, Brazil) was used as a reference to adjust the turbidity of the bacterial suspension in TSB to a concentration of 3 × 10⁸ CFU/mL. A spectrophotometer SP-22 (Biospectro, Brazil) was used to measure turbidity at 620 nm, which ranged from 0.224 to 0.300. C. jejuni strains were reactivated on blood agar (Kasvi, Brazil) supplemented with 5% defibrinated sheep blood (Newprov, Brazil), followed by incubation under microaerophilic conditions for 48 h at 42 ºC. One colony of each strain was suspended in 3 mL of TSB, and the turbidity of the bacterial suspension was adjusted, as previously described.

Compounds and preparation of solution

Four compounds were selected for this study: (1) biosurfactant rhamnolipid of P. aeruginosa (R90; AGAE Technologies, USA); (2) citric acid (C0759; Sigma-Aldrich, Germany), (3) malic acid (M8304; Sigma-Aldrich, Germany), and (4) disinfectant benzalkonium chloride (Êxodo Científica, Brazil). The disinfectant was used only for the removal of formed biofilms assay. Each compound was evaluated at three concentrations: 1, 3, and 5% (w/v) for the biosurfactant, 2, 5, and 10% (w/v) for organic acids, and 50, 100, and 150 ppm for the disinfectant. All compounds were diluted in sterile distilled water. Concentrations of biosurfactants and organic acids were selected based on previous reports on their use in the food industry [21, 33, 34]. Disinfectant concentrations followed FDA recommendations for surfaces in contact with food [35]. For the organic acids, stock solutions were prepared and the pH was adjusted with HCl, when needed, to reach 3.1–4.8 for citric acid and 3.5–5.1 for malic acid, based on their pKa values.

Prevention of biofilm formation

Techniques used to prevent biofilm formation and remove the formed biofilm were adapted from previous studies [21, 35]. Sterile 96-well flat-bottomed polystyrene microplates (Kasvi, Brazil) were pre-treated with 200 μL of each compound, followed by incubation for 24 h at 4, 12, and 25 °C. For each temperature, one polystyrene microplate was used for each compound concentration, and a microplate treated with distilled water was used as a non-treated control. After incubation, the well contents were removed, and the microplates were washed three times with 230 μL sterile distilled water and were air-dried at room temperature. Subsequently, 200 μL of each bacterial suspension was inoculated into each well in triplicate. Negative control wells containing only TSB without glucose were inoculated in triplicate in each microplate. A positive control (S. Typhimurium ATCC 17025) was inoculated in triplicate in each microplate. The microplates were incubated for 24 h for S. Enteritidis and E. coli and 96 h for C. jejuni at 4, 12, and 25 °C. After incubation, the cell suspension was removed and the wells were washed with distilled water, as previously described. The attached bacteria were fixed with 200 μL of methanol (Nuclear, Brazil) per well for 15 min. The methanol was removed and the microplates were stained with 200 μL of 2% (w/v) Hucker crystal violet (MediQuímica, Brazil) per well for 15 min. The stain was removed slowly, and the plate was washed. The plates were then air-dried at room temperature. The biofilm was resuspended in 200 µL of 33% glacial acetic acid (Nuclear, Brazil) per well. The optical density (OD) of each well was measured at 550 nm using an ELx800 Absorbance Reader (BioTek, USA). The experiment was repeated three times.

Removal of formed biofilms

To evaluate the removal of formed biofilms, 200 µL of each bacterial suspension was inoculated into each well of the sterile 96-well flat-bottomed polystyrene microplates, followed by incubation for 24 h at 4, 12, and 25 °C. Negative control wells containing only TSB without glucose were inoculated in triplicate in each microplate. The cell suspension was removed, and the microplates were washed with 230 μL of sterile distilled water. Then, 200 μL per well of each compound was inoculated at their respective concentrations and maintained for 5 and 10 min of contact, simulating the times commonly used for disinfection in poultry slaughterhouses. For each temperature, a microplate treated with distilled water was used as a non-treated control. The wells were washed with distilled water, as previously described. Subsequently, as mentioned above, the microplates were fixed with methanol, stained with Hucker crystal violet, washed with water, air-dried, resuspended in glacial acetic acid and the OD measured at 550 nm using an ELx800 Absorbance Reader. The experiment was repeated three times.

Measurement of biofilm prevention and removal effects

The ability of each compound to prevent biofilm formation or to remove formed biofilm was assessed by determining the percentage of biomass not formed or removed in relation to the control. The prevention and removal of the biofilm ratio were calculated using the following formula [36]:

\frac{(C - B) - (T - B)}{(C - B)} \times 100 (%)

where B is the mean absorbance per well with no treatment and no biofilm (negative control); C is the mean absorbance per non-treated well with biofilm (positive control, no treatment); and T is the mean absorbance per well for treated wells for each compound evaluated.

For graphic presentation, the strains were grouped according to their results: (1) strains that did not exhibit biofilm prevention/removal (0.00–0.99%); (2) strains that prevented/removed up to 50% of the biofilm (1.00–50.99%); and (3) strains that prevented/removed ≥ 51% of the biofilm (51.00–100%). These graphics are presented in Figures S1 to S21.

Statistical analyses

All statistical analyses were performed using GraphPad Prism software, with a significance level of 5%. Descriptive statistics were used to express biofilm prevention and removal. One-way ANOVA, followed by Tukey’s honestly significant difference (HSD) test, was used to detect differences in biofilm prevention and removal among the treatments, temperatures, times of contact, and concentrations.

RESULTS

Prevention of biofilm formation

The effects of citric acid, malic acid, and rhamnolipid in the prevention of biofilm formation by S. Enteritidis, E. coli, and C. jejuni are presented in Tables 1, 2, and 3, respectively. For S. Enteritidis, the antibiofilm activity was affected by the concentration of citric acid at all temperatures, malic acid at 4 ºC, and rhamnolipid at 4 and 12 ºC (p < 0.05). Temperature also influenced the effect of all compounds at all concentrations (p < 0.05). For E. coli, the antibiofilm activity was significantly (p < 0.05) affected by the compound concentration only for rhamnolipid at 4 ºC. Temperature significantly (p < 0.05) increased the effect of citric acid at 5 and 10%, malic acid at 2%, and rhamnolipid at all concentrations for E.coli. For C. jejuni, the antibiofilm activity of organic acids and rhamnolipid was not significantly affected (p > 0.05) by their concentrations, except for rhamnolipid at 25 ºC. The temperature only influenced the effects of malic acid at 2 and 10% (p < 0.05).

Table 1.

Antibiofilm activity of citric acid at 2, 5, and 10% (w/v) on prevention of biofilm formation by Salmonella Enteritidis, Escherichia coli, and Campylobacter jejuni at 4, 12, and 25 ºC

Mean (%) ± standard-deviation
Salmonella Enteritidis
Temperature (ºC)	Concentration (% w/v)
Temperature (ºC)	2	5	10
4	14.62 ± 18.42^b,AB	31.08 ± 27.77^a,A	33.94 ± 28.60^a,AB
12	6.12 ± 15.67^b,B	8.31 ± 16.07^b,B	15.12 ± 15.33^a,B
25	27.40 ± 23.47^b,A	52.78 ± 27.89^a,A	87.67 ± 18.83^a,A
Escherichia coli
Temperature (ºC)	Concentration (% w/v)
Temperature (ºC)	2	5	10
4	22.27 ± 29.07^a,A	25.9 ± 33.30^a,A	33.51 ± 36.36^a,A
12	26.68 ± 34.88^a,AB	29.49 ± 37.27^a,A	33.54 ± 36.39^a,A
25	40.53 ± 26.10^a,B	45.29 ± 27.84^a,B	46.45 ± 28.92^a,B
Campylobacter jejuni
Temperature (ºC)	Concentration (% w/v)
Temperature (ºC)	2	5	10
4	27.33 ± 26.49^a,A	32.43 ± 28.69^a,A	40.22 ± 35.28^a,A
12	11.90 ± 15.66^a,A	24.69 ± 25.33^a,A	32.80 ± 30.96^a,A
25	38.43 ± 31.99^a,A	45.54 ± 28.62^a,A	56.16 ± 32.78^a,A

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Legend:

Different lowercase letters in the same line indicate statistically significant differences (p < 0.05) among different concentrations, with the same temperature

Different capital letters on the same column indicate statistically significant differences (p < 0.05) among different temperatures, with the same concentration

Table 2.

Antibiofilm activity of malic acid at 2, 5, and 10% (w/v) on prevention of biofilm formation by Salmonella Enteritidis, Escherichia coli, and Campylobacter jejuni at 4, 12, and 25 ºC

Mean (%) ± standard deviation
Salmonella Enteritidis
Temperature (ºC)	Concentration (% w/v)
Temperature (ºC)	2	5	10
4	8.47 ± 13.75^B,C	19.47 ± 19.23^ab,B	26.99 ± 26.44^A,B
12	18.56 ± 21.29^a,B	33.83 ± 28.75^a,AB	46.25 ± 29.12^a,AB
25	62.60 ± 35.76^a,A	69.35 ± 31.68^a,A	74.95 ± 28.12^a,A
Escherichia coli
Temperature (ºC)	Concentration (% w/v)
Temperature (ºC)	2	5	10
4	16.27 ± 22.35^a,AB	27.91 ± 29.18^a,A	28.27 ± 34.71^a,A
12	23.12 ± 27.05^a,A	28.26 ± 28.72^a,A	31.83 ± 34.56^a,A
25	7.9 ± 19.15^a,B	16.78 ± 25.55^a,A	14.83 ± 25.70^a,A
Campylobacter jejuni
Temperature (ºC)	Concentration (% w/v)
Temperature (ºC)	2	5	10
4	19.88 ± 22.79^a,B	26.99 ± 30.21^a,A	33.96 ± 34.26^a,B
12	23.23 ± 20.97^a,AB	34.22 ± 9.26^a,A	41.80 ± 32.96^a,AB
25	37.91 ± 27.15^a,A	49.68 ± 28.13^a,A	58.60 ± 30.33^a,A

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Legend:

Different lowercase letters in the same line indicate statistically significant differences (p < 0.05) among different concentrations, with the same temperature

Different capital letters on the same column indicate statistically significant differences (p < 0.05) among different temperatures, with the same concentration

Table 3.

Antibiofilm activity of rhamnolipid at 1, 3, and 5% (w/v) on prevention of biofilm formation by Salmonella Enteritidis, Escherichia coli, and Campylobacter jejuni at 4, 12, and 25 ºC

Mean (%) ± standard deviation
Salmonella Enteritidis
Temperature (ºC)	Concentration (% w/v)
Temperature (ºC)	1	3	5
4	6.57 ± 12.50^b,C	11.85 ± 2.91^b,B	62.99 ± 36.17^a,A
12	0.67 ± 16.72^b,B	10.99 ± 27.39^ab,B	24.80 ± 36.09^a,B
25	54.7 ± 31.39^a,A	58.42 ± 32.20^a,A	76.64 ± 34.98^a,A
Escherichia coli
Temperature (ºC)	Concentration (% w/v)
Temperature (ºC)	1	3	5
4	9.35 ± 19.20^b,B	22.51 ± 25.23^a,B	43.95 ± 28.72^a,AB
12	14.53 ± 26.44^a,B	26.37 ± 35.09^a,B	35.55 ± 42.51^a,B
25	41.06 ± 35.67^a,A	52.75 ± 43.78^a,A	63.94 ± 39.47^a,A
Campylobacter jejuni
Temperature (ºC)	Concentration (% w/v)
Temperature (ºC)	1	3	5
4	37,82 ± 32,93^a,A	40,14 ± 32,92^a,A	43,99 ± 36,53^a,A
12	33,14 ± 29,06^a,A	32,52 ± 28,59^a,A	37,04 ± 31,36^a,A
25	22,61 ± 27,10^b,A	31,19 ± 31,91^b,A	43,29 ± 38,56^a,A

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Legend:

Different lowercase letters in the same line indicate statistically significant differences (p < 0.05) among different concentrations, with the same temperature

Different capital letters on the same column indicate statistically significant differences (p < 0.05) among different temperatures, with the same concentration

Comparisons were also made between the compounds using equivalent concentrations defined as low, medium, and high at each temperature for this analysis (Table 4). For S. Enteritidis, some significant differences (p < 0.05) were found, where malic acid presented a higher antibiofilm activity than citric acid and rhamnolipid at 12 °C, regardless of the concentration evaluated. At 25 °C, malic acid presented a better result than citric acid at a lower concentration (2%). For E. coli, malic acid presented significantly lower (p < 0.05) antibiofilm activity than citric acid and rhamnolipid at 25 °C, regardless of the concentration evaluated. For C. jejuni, malic acid showed significantly lower (p < 0.05) antibiofilm activity than rhamnolipid at 4 °C.

Table 4.

Antibiofilm activity of citric acid, malic acid, and rhamnolipid at low, medium, and high concentrations on prevention of biofilm formation by Salmonella Enteritidis, Escherichia coli, and Campylobacter jejuni at 4, 12, and 25 ºC

Mean (%) ± standard deviation
Salmonella Enteritidis
Compound	Temperature (ºC) and concentration
	4			12			25
	Low	Medium	High	Low	Medium	High	Low	Medium	High
Citric acid	14.62 ± 18.42^a	31.08 ± 27.77^a	33.94 ± 28.60^b	6.12 ± 15.67^b	8.31 ± 16.07^b	15.12 ± 15.33^b	27.40 ± 23.47^b	52.78 ± 27.89^a	87.67 ± 18.83^a
Malic acid	8.47 ± 13.75^a	19.47 ± 19.23^a	26.99 ± 26.44^b	18.56 ± 21.29^a	33.83 ± 28.75^a	46.25 ± 29.12^a	62.60 ± 35.76^a	69.35 ± 31.68^a	74.95 ± 28.12^a
Rhamnolipid	6.57 ± 12.50^a	11.85 ± 2.9^a	62.99 ± 36.17^a	0.67 ± 16.72^b	10.99 ± 27.39^b	24.80 ± 36.09^b	54.7 ± 31.39^a	58.42 ± 32.20^a	76.64 ± 34.98^a
Escherichia coli
Compound	Temperature (ºC) and concentration
	4			12			25
	Low	Medium	High	Low	Medium	High	Low	Medium	High
Citric acid	22.27 ± 29.07^a	25.9 ± 33.30^a	33.51 ± 36.36^a	26.68 ± 34.88^a	29.49 ± 37.27^a	33.54 ± 36.39^a	40.53 ± 26.10^a	45.29 ± 27.84^a	46.45 ± 28.92^a
Malic acid	16.27 ± 22.35^a	27.91 ± 29.18^a	28.27 ± 34.71^a	23.12 ± 27.05^a	28.26 ± 28.72^a	31.83 ± 34.56^a	7.9 ± 19.15^b	16.78 ± 25.55^b	14.83 ± 25.70^b
Rhamnolipid	9.35 ± 19.20^a	22.51 ± 25.23^a	43.95 ± 28.72^a	14.53 ± 26.44^b	26.37 ± 35.09^a	35.55 ± 42.51^a	41.06 ± 35.67^a	52.75 ± 43.78^a	63.94 ± 39.47^a
Campylobacter jejuni
Compound	Temperature (ºC) and concentration
	4			12			25
	Low	Medium	High	Low	Medium	High	Low	Medium	High
Citric acid	27.33 ± 26.49^ab	32.43 ± 28.69^a	40.22 ± 35.28^a	11.90 ± 15.66^a	24.69 ± 25.33^a	32.80 ± 30.96^a	38.43 ± 31.99^a	45.54 ± 28.62^ab	56.16 ± 32.78^a
Malic acid	19.88 ± 22.79^b	26.99 ± 30.21^a	33.96 ± 34.26^a	23.23 ± 20.97^a	34.22 ± 9.26^a	41.80 ± 32.96^a	37.91 ± 27.15^a	49.68 ± 28.13^a	58.60 ± 30.33^a
Rhamnolipid	37,82 ± 32,93^a	40,14 ± 32,92^a	43,99 ± 36,53^a	33,14 ± 29,06^a	32,52 ± 28,59^a	37,04 ± 31,36^a	22,61 ± 27,10^a	31,19 ± 31,91^b	43,29 ± 38,56^a

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Legend: Different lowercase letters on the same column indicate statistically significant differences (p < 0.05) among different compounds, with the same concentration and temperature

Removal of formed biofilms

The effects of citric acid, malic acid, rhamnolipid, and benzalkonium chloride on the removal of formed biofilms by S. Enteritidis, E. coli, and C. jejuni are presented in Tables 5, 6, 7, and 8, respectively. For S. Enteritidis, the antibiofilm activity was significantly (p < 0.05) affected by the concentration of citric acid at 4 and 12 °C, malic acid at all temperatures, rhamnolipid at 4 ºC, and benzalkonium chloride at all temperatures, regardless of the duration of contact. Temperature influenced (p < 0.05) the effect of citric acid at all concentrations for 5 and 10 min, malic acid at 2% for 5 min, rhamnolipid at 1% for 5 and 10 min, and benzalkonium chloride at 50 ppm for 5 and 10 min. The time of contact significantly (p < 0.05) affects the antibiofilm activity of citric acid at 5 and 10% at 4 °C, and malic acid at 2% at 12 ºC. For E. coli, the antibiofilm activity was influenced (p < 0.05) by compound concentration for citric acid at 4 and 12 ºC, malic acid at 25 ºC, rhamnolipid at 4 ºC, and benzalkonium chloride at 25 ºC, regardless of the time of contact. Temperature significantly (p < 0.05) influenced the results for malic acid at 10% for 5 min and benzalkonium chloride under all conditions. For C. jejuni, the antibiofilm activity was significantly (p < 0.05) affected by the concentration for citric acid at all temperatures, malic acid at 25 ºC, rhamnolipid at 4 and 25 ºC, and benzalkonium chloride at 12 and 25 ºC. Significant (p < 0.05) differences were also observed among temperatures for citric acid at all concentrations, except at 10% for 10 min, as well as for malic acid at 5 and 10% for 5 and 10 min, rhamnolipid at 5% for 10 min, and benzalkonium chloride at all concentrations. The duration of contact did not influence (p > 0.05) the effect of compounds for S. Enteritidis and C. jejuni, regardless of the conditions evaluated.

Table 5.

Antibiofilm activity of citric acid at 2, 5, and 10% (w/v) on removal of formed biofilm by Salmonella Enteritidis, Escherichia coli, and Campylobacter jejuni at 4, 12, and 25 ºC

Mean (%) ± standard deviation
Salmonella Enteritidis
Temperature (ºC)	Concentration (% w/v) and time of contact
	5 min			10 min
	2	5	10	2	5	10
4	0.47 ± 1.37^b,B	2.98 ± 4.44^b,B	21.21 ± 12.49^a,B	2.03 ± 8.07^b,B	20.36 ± 14.61^a,B	32.08 ± 15.27^a,B
12	0.76 ± 3.04^b,B	12.46 ± 26.20^b,B	18.54 ± 21.02^a,B	3.25 ± 7.85^b,B	2.96 ± 6.9^b,B	22.16 ± 16.28^a,B
25	25.56 ± 35.0^a,A	59.58 ± 33.82^a,A	66.50 ± 33.97^a,A	26.03 ± 37.99^a,A	70.54 ± 29.6^a,A	77.86 ± 24.98^a,A
Escherichia coli
Temperature (ºC)	Concentration (% w/v) and time of contact
	5 min			10 min
	2	5	10	2	5	10
4	18.25 ± 21.68^b,A	34.17 ± 27.94^a,A	49.02 ± 32.71^a,A	24.89 ± 24.06^b,A	40.82 ± 25.58^b,A	60.45 ± 32.71^a,A
12	9.23 ± 17.51^b,A	30.24 ± 33.91^a,A	46.74 ± 36.66^a,A	20.20 ± 30.16^b,A	38.79 ± 34.99^ab,A	52.09 ± 37.48^a,A
25	26.87 ± 29.84^a,A	36.42 ± 32.70^a,A	47.40 ± 36.01^a,A	31.53 ± 31.64^a,A	37.29 ± 32.48^a,A	51.81 ± 38.46^a,A
Campylobacter jejuni
Temperature (ºC)	Concentration (% w/v) and time of contact
	5 min			10 min
	2	5	10	2	5	10
4	20.33 ± 23.56^b,B	29.51 ± 29.34^ab,B	42.90 ± 30.89^a,B	24.52 ± 27.01^b,B	31.67 ± 30.37^b,B	54.32 ± 32.03^a,A
12	31.96 ± 27.94^b,AB	46.92 ± 33.99^ab,AB	58.23 ± 39.17^a,AB	40.52 ± 31.28^a,AB	54.44 ± 38.10^a,AB	58.46 ± 37.93^a,A
25	39.33 ± 27.86^b,A	57.32 ± 27.85^ab,A	67.32 ± 29.02^a,A	52.84 ± 28.55^b,A	62.37 ± 29.56^ab,A	75.06 ± 29.53^a,A

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Legend:

Different lowercase letters in the same line indicate statistically significant differences (p < 0.05) among different concentrations, with the same temperature and duration of contact

Different capital letters on the same column indicate statistically significant differences (p < 0.05) among different temperatures, with the same concentration and duration of contact

Table 6.

Antibiofilm activity of malic acid at 2, 5, and 10% (w/v) on removal of formed biofilm by Salmonella Enteritidis, Escherichia coli, and Campylobacter jejuni at 4, 12, and 25 ºC

Mean (%) ± standard deviation
Salmonella Enteritidis
Temperature (ºC)	Concentration (% w/v) and time of contact
	5 min			10 min
	2	5	10	2	5	10
4	21.89 ± 28.93^b,A	28.63 ± 36.25^ab,A	47.68 ± 38.96^a,A	30.36 ± 34.90^b,A	41.39 ± 39.85^ab,A	65.70 ± 41.80^a,A
12	0.10 ± 0.46^b,B	39.02 ± 39.93^a,A	39.77 ± 28.15^a,A	11.81 ± 16.25^b,A	37.41 ± 37.12^a,A	54.21 ± 29.63^a,A
25	19.99 ± 33.58^b,A	25.49 ± 34.75^ab,A	44.76 ± 39.78^a,A	21.79 ± 37.53^b,A	38.84 ± 39.78^ab,A	44.76 ± 39.78^a,A
Escherichia coli
Temperature (ºC)	Concentration (% w/v) and time of contact
	5 min			10 min
	2	5	10	2	5	10
4	21.28 ± 31.28^a,A	27.44 ± 38.06^a,A	30.68 ± 42.83^a,A	25.13 ± 35.93^a,A	28.44 ± 39.71^a,A	48,77 ± 42.79^a,A
12	19.93 ± 31.64^a,A	25.05 ± 34.15^a,A	35.90 ± 37.46^a,AB	21.23 ± 32.05^a,A	30.19 ± 36.00^a,A	39.69 ± 40.62^a,A
25	21.64 ± 31.14^b,A	38.85 ± 38.14^ab,A	55.59 ± 37.60^a,B	29.30 ± 34.69^a,A	45.51 ± 38.85^a,A	59.62 ± 37.86^a,A
Campylobacter jejuni
Temperature (ºC)	Concentration (% w/v) and time of contact
	5 min			10 min
	2	5	10	2	5	10
4	14.60 ± 18.61^a,A	20.59 ± 23.10^a,B	28.05 ± 26.57^a,B	17.50 ± 20.45^a,A	24.67 ± 25.99^a,B	30.75 ± 26.34^a,B
12	28.84 ± 29.13^a,A	37.11 ± 31.39^a,AB	39.61 ± 34.90^a,AB	35.10 ± 30.41^a,A	38.24 ± 32.71^a,AB	50.03 ± 40.27^a,AB
25	28.97 ± 31.05^b,A	42.45 ± 35.27^ab,A	54.67 ± 37.19^a,A	30.22 ± 31.83^b,A	51.18 ± 36.25^ab,A	57.97 ± 38.72^a,A

Open in a new tab

Legend:

Different lowercase letters in the same line indicate statistically significant differences (p < 0.05) among different concentrations, with the same temperature and duration of contact

Different capital letters on the same column indicate statistically significant differences (p < 0.05) among different temperatures, with the same concentration and duration of contact

Table 7.

Antibiofilm activity of rhamnolipid at 1, 3, and 5% (w/v) on removal of formed biofilm by Salmonella Enteritidis, Escherichia coli, and Campylobacter jejuni at 4, 12, and 25 ºC

Mean (%) ± standard deviation
Salmonella Enteritidis
Temperature (ºC)	Concentration (% w/v) and time of contact
	5 min			10 min
	1	3	5	1	3	5
4	21.00 ± 30.02^b,B	40.74 ± 34.54^ab,A	55.35 ± 33.61^a,A	22.57 ± 31.34^b,B	43.69 ± 34.53^a,A	65.02 ± 32.82^a,A
12	41.48 ± 38.45^a,AB	52.32 ± 40.56^a,A	69.15 ± 34.25^a,A	46.48 ± 38.91^a,A	54.78 ± 38.87^a,A	71.21 ± 30.59^a,A
25	48.19 ± 44.25^a,A	62.14 ± 37.32^a,A	65.60 ± 38.86^a,A	53.87 ± 41.44^a,A	63.98 ± 37.28^a,A	68.88 ± 36.71^a,A
Escherichia coli
Temperature (ºC)	Concentration (% w/v) and time of contact
	5 min			10 min
	1	3	5	1	3	5
4	25.83 ± 27.51^a,A	31.25 ± 31.73^a,A	44.28 ± 31.99^a,A	29.09 ± 30.99^b,A	38.39 ± 31.45^ab,A	53.38 ± 35.68^a,A
12	28.33 ± 28.29^a,A	30.81 ± 29.14^a,A	39.12 ± 37.50^a,A	30.64 ± 29.39^a,A	33.74 ± 3243^a,A	42.67 ± 40.58^a,A
25	27.23 ± 32.36^a,A	38.00 ± 34.74^a,A	48.17 ± 37.79^a,A	30.88 ± 34.83^a,A	42.52 ± 37.57^a,A	52.97 ± 38.47^a,A
Campylobacter jejuni
Temperature (ºC)	Concentration (% w/v) and time of contact
	5 min			10 min
	1	3	5	1	3	5
4	33.93 ± 20.92^b,A	47.91 ± 20.40^ab,A	55.97 ± 25.09^a,A	40.94 ± 18.16^a,A	54.87 ± 23.48^a,A	56.12 ± 23.86^a,B
12	38.82 ± 29.98^a,A	51.74 ± 34.37^a,A	55.99 ± 34.28^a,A	41.28 ± 31.60^a,A	51.46 ± 32.86^a,A	61.98 ± 34.91^a,AB
25	43.79 ± 29.02^b,A	54.87 ± 28.45^ab,A	70.37 ± 28.71^a,A	48.28 ± 29.43^b,A	64.62 ± 30.96^ab,A	78.61 ± 27.42^a,A

Open in a new tab

Legend:

Different lowercase letters in the same line indicate statistically significant differences (p < 0.05) among different concentrations, with the same temperature and duration of contact

Different capital letters on the same column indicate statistically significant differences (p < 0.05) among different temperatures, with the same concentration and duration of contact

Table 8.

Antibiofilm activity of benzalkonium chloride at 50, 100, and 150 ppm on removal of formed biofilm by Salmonella Enteritidis, Escherichia coli, and Campylobacter jejuni at 4, 12, and 25 ºC

Mean (%) ± standard deviation
Salmonella Enteritidis
Temperature (ºC)	Concentration (ppm) and time of contact
	5 min			10 min
	50	100	150	50	100	150
4	4.92 ± 10.76^c,B	34.20 ± 36.91^b,A	61.65 ± 30.18^a,A	15.49 ± 23.10^b,A	37.12 ± 34.58^b,A	75.88 ± 26.86^a,A
12	27.70 ± 27.84^a,A	42.60 ± 38.52^a,A	68.09 ± 33.42^a,A	31.49 ± 33.17^b,A	50.98 ± 39.87^b,A	72.54 ± 31.15^a,A
25	8.75 ± 16.96^b,B	45.90 ± 31.46^a,A	58.68 ± 31.09^a,A	29.17 ± 27.78^b,A	53.04 ± 31.52^a,A	73.53 ± 31.46^a,A
Escherichia coli
Temperature (ºC)	Concentration (ppm) and time of contact
	5 min			10 min
	50	100	150	50	100	150
4	9.50 ± 20.35^a,B	13.76 ± 25.61^a,B	19.98 ± 29.44^a,A	11.73 ± 24.22^a,B	15.99 ± 26.76^a,B	24.05 ± 32.59^a,C
12	25.65 ± 29.62^a,A	31.48 ± 32.40^a,A	40.48 ± 38.03^a,B	27.15 ± 29.43^a,A	36.96 ± 35.27^a,A	46.70 ± 42.00^a,A
25	40.60 ± 29.79^b,A	49.85 ± 31.81^ab,A	60.85 ± 33.00^a,B	41.83 ± 31.52^b,A	54.16 ± 32.83^ab,A	69.62 ± 35.70^a,B
Campylobacter jejuni
Temperature (ºC)	Concentration (ppm) and time of contact
	5 min			10 min
	50	100	150	50	100	150
4	7.63 ± 17.47^a,B	15.32 ± 20.06^a,B	20.21 ± 23.82^a,B	13.85 ± 19.85^a,B	18.75 ± 22.13^a,B	22.14 ± 23.61^a,C
12	12.70 ± 22.01^b,B	23.05 ± 25.07^ab,B	32.68 ± 26.86^a,B	18.74 ± 23.95^b,B	25.60 ± 26.77^ab,B	43.61 ± 34.06^a,B
25	52.97 ± 35.04^a,A	62.15 ± 33.88^a,A	62.15 ± 33.88^a,A	55.09 ± 34.06^b,A	67.75 ± 32.90^ab,A	81.00 ± 31.03^a,A

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Legend:

Different lowercase letters in the same line indicate statistically significant differences (p < 0.05) among different concentrations, with the same temperature and duration of contact

Different capital letters on the same column indicate statistically significant differences (p < 0.05) among different temperatures, with the same concentration and duration of contact

For all experiments, a high standard deviation was observed, which indicates high variability, possibly influenced by the intrinsic characteristics of the selected strains.

DISCUSSION

S. Enteritidis, E. coli, and C. jejuni are linked to outbreaks of foodborne diseases worldwide [1–3]. Food processing plants are an ideal environment for biofilm formation, mainly because of the large amount of nutrients available [37]. To achieve acceptable levels of contamination, chemical disinfectants are routinely used during disinfection programs. However, biofilms are known to provide protection to microorganisms, and attached bacteria are quite different from planktonic cells because of their altered physiological status. Thus, antimicrobial agents should be investigated for biofilm-forming bacteria [21, 38]. Over the past decade, researchers have expended considerable effort in order to find alternatives to improve surface disinfection programs. Nevertheless, there remains a gap in knowledge regarding biofilm prevention and removal [37]. In this context, studies evaluating the mechanisms adopted by these pathogens that allow their survival along the food processing chain are of great importance.

It has been demonstrated that the reduction of biofilm cells by chemical disinfectants is concentration- and contact-time-dependent, and the antibiofilm activity usually increases with higher disinfectant concentrations and longer periods of contact time [37]. Previous studies have shown that some compounds require up to 10 min of surface contact time to be effective [39, 40]. In addition, the determination of product concentration is a critical point, since there are limitations surrounding the maximum concentrations of certain active agents on surfaces in contact with food. To evaluate the effect of contact duration on the antibiofilm activity for biofilm removal, two contact times of 5 and 10 min were evaluated in the present study for all compounds in order to simulate the procedures before the start of operations, called pre-operational, and during operation, called operational, as determined by Brazilian legislation [32, 41]. The results showed significant differences in some specific cases, which were not sufficient to generalize the potential effect of time on antibiofilm activity. In general, contact time did not influence the results of this study.

To evaluate the influence of compound concentrations, all concentrations tested in this study were determined based on previous reports of their use in the food industry or on FDA recommendations [21, 33–35]. The results showed significant differences in compound concentrations for both the prevention and removal of biofilms. However, these differences were not equally distributed and seemed to be more frequently found in S. Enteritidis, followed by E. coli and C. jejuni at lower temperatures such as 12 °C. Compound concentration did not influence the prevention of biofilm formation by C. jejuni, regardless of the temperature or compound. However, in the removal experiments, the concentration was significant in terms of antibiofilm activity.

Further analyses are needed to evaluate whether higher compound concentrations and longer contact duration up to 60 min would result in an increase in antibiofilm activity. The absence of a strong relationship between concentration and, in particular, contact time for the tested compounds was surprising, since previous studies have reported that a longer exposure to higher concentrations of the compound would increase its antibiofilm capacity [39, 40, 42]. However, a previous study with S. Heidelberg did not identify significant differences in the antimicrobial activity of electrochemically activated water, an alternative bactericidal compound, between the contact times employed of 5 and 10 min [43]. These results are important because during work shifts in a poultry slaughterhouse, the disinfection process must be efficient, with product concentration prepared according to international regulatory organizations in a short period of time.

The biofilm formation process is complex and can be influenced by several factors, including pathogen species, surface characteristics, availability of nutrients, and environmental conditions, such as relative humidity, pH, and temperature [44, 45]. Despite being an important factor, most studies have assessed the ability of these species to adhere under ideal conditions of laboratory cell culture, such as at 37 °C for S. Enteritidis and E. coli, and 42 or 37 °C for C. jejuni, optimal growth temperatures for these pathogens [46–48], limiting the extrapolation of results. These species have the ability to adhere to abiotic surfaces at lower temperatures, such as 4 and 12 °C [13, 15, 49]. Traditionally, lower temperatures are used for food maintenance to prevent microorganism growth [50, 51]. However, a decrease in the temperature of the treatment is usually followed by a decrease in the efficiency of disinfectant compounds [37]. In this context, the present study evaluated three temperatures commonly encountered in poultry slaughterhouses: 4 °C (temperature of the handling environment of poultry slaughterhouse), 12 °C (temperature required by the Brazilian sanitary service in cutting rooms of broiler processing plants), and 25 °C (room temperature) [32].

All compounds tested presented varying degrees of antibiofilm activity to prevent or remove biofilms at all temperatures. In some cases, the temperature significantly influenced the action of the tested compounds under specific conditions. The influence of temperature on biofilm prevention seemed to be more important for S. Enteritidis and E. coli than for C. jejuni, regardless of the compound involved. When the temperature influenced the results, it was observed that, in most cases, increased antibiofilm activity was observed at 25 ºC.

The influence of temperature on biofilm removal was less evident. A decrease in antibiofilm activity was observed at lower temperatures, especially among organic acids. Previous studies have presented variable data regarding the antibiofilm and antimicrobial activity of organic acids at lower temperatures. Borges et al. [52] demonstrated the good performance of gallic and ferulic acids at room temperature, resulting in a greater than 70% reduction in biofilm formation by E. coli, Staphylococcus aureus, P. aeruginosa, and Listeria monocytogenes. In contrast, Koyuncu et al. [53] demonstrated a lower bactericidal effect of formic acid at 5 °C than at 15 °C against Salmonella enterica Infantis and Salmonella enterica Typhimurium. These variations probably indicate that both antibiofilm and antimicrobial effects of organic acids at low temperatures are related to the intrinsic characteristics of the active agents [37, 54]. However, further analyses are required to confirm this hypothesis.

In addition to the experimental conditions, such as treatment temperature, contact duration, and concentration of compounds, the specific characteristics of microorganisms can also influence antibiofilm activity. Once the active agents of compounds bind to specific target sites of microorganisms, their efficacy depends on the specific characteristics of the bacterial species [37]. In addition to species differences, it is also important to consider the inter-strain variability that exists within the same species [37, 40]. Several studies have shown important variations in phenotypic profiles, including biofilm formation ability, related to strains intrinsic characteristics [13, 55, 56]. Differences in gene repertoires among isolates from the same species have been previously reported [57–60]. These variations are probably responsible for their varied responses to disinfectants [37]. It is important to note that biofilms are generally not composed of a single bacterial species, but of bacterial communities with several species that interact both intra- and interspecies [61, 62]. These interspecific interactions lead to cooperation to provide coexistence and benefits to all species present, and enhancing certain properties, including increased tolerance against antimicrobial agents [61]. Thus, the study of multi-species biofilms is needed.

In recent years, organic acids have been used as alternative and natural compounds to reduce food contamination and spoilage by bacteria, especially because of their low cost and effectiveness [63]. A possible disadvantage of organic acids is that solutions of acids can be caustic and toxic at high concentrations. However, typical-use dilutions are considered non-toxic and non-irritating [64]. The beneficial properties of organic acids have been previously demonstrated. Citric acid presents low toxicity and has been widely used worldwide as a flavor enhancer, pH regulator, firming agent, and pharmaceutical reagent [24]. Its antibiofilm and antimicrobial effects have been shown against Yersinia, Listeria, and Shigella species [65–67]. Malic acid is also a safety reagent that has been used in foodstuffs, metals, textiles, pharmaceuticals, water treatment, and bioremediation industries [24]. Previous studies have shown that malic acid is one of the strongest antimicrobial acids against several pathogens [68]. In addition to their bactericidal effect on planktonic cells, their antibiofilm properties for biofilm prevention and removal were demonstrated in the present study. Both organic acids displayed antibiofilm activity in terms of biofilm formation prevention and removal of the three pathogens at all temperatures and compound concentrations.

The rhamnolipid of P. aeruginosa (PC Code 110029) is a glycolipid and represents one of the most promising biosurfactants, and the US Environmental Protection Agency (US EPA) has approved its use in agricultural crops [69, 70]. No adverse effects on humans or the environment caused by rhamnolipids have been described [69]. Rhamnolipids usually affect cell surface compounds, change cell surface properties, and damage bacterial cells [71]. Similar to organic acids, here, rhamnolipids presented satisfactory prevention and removal of biofilms formed by the three pathogens.

Benzalkonium chloride is an organic salt that is classified as a quaternary ammonium compound. It is among the most common active ingredients in disinfectant products, mainly because of its broad-spectrum antimicrobial properties against several microorganisms [72]. According to the US EPA, benzalkonium chloride is a skin and eye irritant [73]. Previous studies have shown that this compound is ineffective in preventing surface biofilm formation and has lower biocide performance [74, 75]. Therefore, in the present study, this compound was tested only for the removal of formed biofilms. As expected, benzalkonium chloride displayed antibiofilm properties against the three pathogens at higher temperatures.

Significant differences (p < 0.05) were observed among the compounds at the corresponding concentrations for each species. Malic acid had a greater (p < 0.05) effect than citric acid and rhamnolipid in the prevention of biofilm formation by S. Enteritidis at 12 ºC. In contrast, for the prevention of biofilms by E. coli, malic acid showed lower (p < 0.05) antibiofilm activity than citric acid and rhamnolipid at 25 ºC. For C. jejuni, no significant differences (p > 0.05) were observed among the three products. Regarding the removal of formed biofilms, significant differences (p < 0.05) were mostly found for S. Enteritidis. For these pathogens, citric acid presented a lower (p < 0.05) removal power than rhamnolipid and benzalkonium chloride at 4 °C after 5 min of contact. In general, at 12 °C, organic acid presented reduced (p < 0.05) antibiofilm activity compared to rhamnolipid and benzalkonium chloride at lower and intermediate concentrations, regardless of the duration of contact. At 25 °C, the differences were less evident. For E. coli, significant differences were found only at 4 °C, and in most cases, benzalkonium chloride presented higher antibiofilm activity, especially when compared to citric acid. For C. jejuni, at 4 °C, rhamnolipid revealed significantly (p < 0.05) better results than malic acid and benzalkonium chloride at lower concentrations, regardless of the time of contact. At intermediate and high concentrations, this product presented higher antibiofilm activity than all other products for both 5 and 10 min of contact. At 12 °C, benzalkonium chloride presented significantly (p < 0.05) lower antibiofilm activity than rhamnolipid at lower concentrations and lower than that of citric acid and rhamnolipid at intermediate concentrations. At 25 °C, no significant (p > 0.05) differences were found among the compounds.

The most effective way to prevent biofilm formation is to disinfect regularly before bacterial adhesion starts [75]. However, the attachment of microorganisms to abiotic surfaces occurs rapidly and usually takes only a few hours [76]. If preventing the formation of biofilms is difficult, the process of removing already formed structures is even more so. The main challenge in biofilm removal with chemical compounds is the failure of an agent to penetrate the full depth of the biofilm structure and to reach bacterial cells [73]. Despite these difficulties, the ineffective action of compounds could be a result of their inappropriate use under specific conditions [37]. In this context, the choice of the product to be used must take into consideration the specific characteristics of food processing plants, the average temperature of processing rooms and the target pathogens to be controlled.

All compounds exhibited antibiofilm activity under all conditions analyzed. The contact duration for 5 and 10 min seemed to be less important than temperature and concentration. The antibiofilm activity of the compounds also varied according to the specific pathogen. Our results provide evidence that these products may be an alternative to the traditional compounds. However, future studies should be carried out to assess their antibiofilm activity on other surfaces, such as polyethylene and stainless steel, and in the presence of organic matter.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 258 kb)^{(258.1KB, docx)}

Authors' contributions

D.C., C.T.P.S., H.L.S.M., and V.P.N. conceived and designed the experiments. D.C., R.M., G.Z.C., H.C.K.F., and D.E.W. performed the experiments. D.C., K.A.B., T.Q.F. analyzed the data, wrote and prepared the manuscript text. All authors critically reviewed and approved the manuscript.

Funding

This work was supported by the National Council for Scientific and Technological Development (CNPq) through the concession of a scholarship to Daiane Carvalho.

Availability of data and material

Not applicable.

Code availability

Not applicable.

Declarations

Ethics approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

All authors give their consent for the publication of this manuscript.

Conflicts of interest/Competing interests

The authors declare that there is no conflict of interest.

Footnotes

Responsible Editor: Luiz Henrique Rosa

Publisher's note

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Associated Data

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Supplementary Materials

Supplementary file1 (DOCX 258 kb)^{(258.1KB, docx)}

Data Availability Statement

Not applicable.

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PERMALINK

Antibiofilm activity of the biosurfactant and organic acids against foodborne pathogens at different temperatures, times of contact, and concentrations

Daiane Carvalho

Rafaela Menezes

Gabriela Zottis Chitolina

Hiran Castagnino Kunert-Filho

Daiane Elisa Wilsmann

Karen Apellanis Borges

Thales Quedi Furian

Carlos Tadeu Pippi Salle

Hamilton Luiz de Souza Moraes

Vladimir Pinheiro do Nascimento

Abstract

Supplementary Information

INTRODUCTION

MATERIALS AND METHODS

Bacterial strains

Inoculum preparation

Compounds and preparation of solution

Prevention of biofilm formation

Removal of formed biofilms

Measurement of biofilm prevention and removal effects

Statistical analyses

RESULTS

Prevention of biofilm formation

Table 1.

Table 2.

Table 3.

Table 4.

Removal of formed biofilms

Table 5.

Table 6.

Table 7.

Table 8.

DISCUSSION

Supplementary Information

Authors' contributions

Funding

Availability of data and material

Code availability

Declarations

Ethics approval

Consent to participate

Consent for publication

Conflicts of interest/Competing interests

Footnotes

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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