Inhibition of Gallic Acid on the Growth and Biofilm Formation of Escherichia coli and Streptococcus mutans Dongyan Shao, Jing Li, Ji Li, Ruihua Tang, Liu Liu, Junling Shi, Qingsheng Huang, and Hui Yang

New strategies for biofilm inhibition are becoming highly necessary because of the concerns to synthetic additives. As gallic acid (GA) is a hydrolysated natural product of tannin in Chinese gall, this research studied the effects of GA on the growth and biofilm formation of bacteria (Escherichia coli [Gram-negative] and Streptococcus mutans [Gram-positive]) under different conditions, such as nutrient levels, temperatures (25 and 37 °C) and incubation times (24 and 48 h). The minimum antimicrobial concentration of GA against the two pathogenic organisms was determined as 8 mg/mL. GA significantly affected the growth curves of both test strains at 25 and 37 °C. The nutrient level, temperature, and treatment time influenced the inhibition activity of GA on both growth and biofim formation of tested pathogens. The inhibition effect of GA on biofilm could be due to other factors in addition to the antibacterial effect. Overall, GA was most effective against cultures incubated at 37 °C for 24 h and at 25 °C for 48 h in various concentrations of nutrients and in vegetable wash waters, which indicated the potential of GA as emergent sources of biofilm control products.

Abstract:

This study revealed that gallic acid (GA) had a great potential in being developed as a naturally sourced, nontoxic, and novel inhibitor of bacteria. It is the first time to investigate the inhibition effects of GA on the growth and biofilm formation of bacteria under different nutrient levels (such as vegetable wash waters that simulate the nutrient-deprived environments of wash step in real food industry), temperatures, and incubation times. The study addresses the potential of phytochemicals as emergent naturally sources of biofilm control products.

Practical Application:

Introduction Biofilm is a concerned source of contamination in food industry sectors including fresh produce, dairy processing, and poultry processing (Lou and others 2013). It is estimated that biofilm contributes to more than 80% of all human infections in foodborne illness (Hancock and others 2010). Biofilm cells are difficult to eradicate because the biofilm protects them from physical and chemical antimicrobial treatments (Costerton and others 1999; Quave and others 2008). Although some antibiotics can effectively destroy biofilms, their use in factories and hospitals accelerates drug resistance (Simoes and others 2009). Therefore, new strategies for inhibiting biofilms are becoming increasingly necessary. Plant extracts and compounds are being explored as natural alternatives to existing synthetic antimicrobials (Lou and others 2013). Chinese gall is a famous traditional Chinese medicine, recognized for its antidiarrheal, anticavity, and antibacterial properties (Chen and others 2009) and gallic acid (GA) is a phenolic acid, the final hydrolysated product of tannin in Chinese gall. GA has MS 20141969 Submitted 11/29/2014, Accepted 4/15/2015. Authors Dongyan Shao, Jing Li, Ji Li, Ruihua Tang, Junling Shi, Qingsheng Huang, and Hui Yang are with Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical Univ., 127 Youyi Xilu, Xi’an, Shaanxi, 710072, China. Author Liu Liu is with College of Food Engineering and Nutritional Science, Shaanxi Normal Univ., 620 Western Changan Street, Xi’an, Shaanxi, 710062, China. Direct inquiries to authors Liu (E-mail: [email protected]) and Shi (E-mail: [email protected]).

R  C 2015 Institute of Food Technologists

doi: 10.1111/1750-3841.12902 Further reproduction without permission is prohibited

demonstrated a wide range of antitumorigenic, anti-inflammatory, and antioxidant activities (Yilmaz and Toledo 2004; Liu and others 2006; Kaur and others 2009) and also was reported to possess strong antimicrobial activity (Li and others 2007). The reported results indicated a potential in using GA to replace the synthetic antimicrobials for improved food safety. However, the effect of GA on biofilm of pathogens has not been well studied (Borges and others 2012; Kang and others 2008). Bacterial growth and biofilm development are known to be affected by environmental conditions such as temperature, pH, nutrient level, hydrodynamics, and the presence of specific ions (Di Bonaventura and others 2008; Romeo 2008). It is unknown how GA influences the growth and biofilm formation of pathogens under different treatment times, temperatures, and nutrient levels. In recent years, the increase in the consumption of fresh produce has resulted in more cases of outbreaks of food-borne illnesses, and Escherichia coli (E. coli) is frequently associated with these outbreaks (Patel and others 2011). Produce washing water has been identified as an important source of E. coli or cross-contamination (Hilgren and Salverda 2000; Danyluk and Schaffner 2011). The pathogens often form biofilm communities in their natural setting (Liu and others 2009; Patel and others 2011), especially in low-nutrient environment (Dewanti and Wong 1995). In addition, Streptococcus mutans (S. mutans) is the most studied etiological agent of dental caries in humans and one of the most frequently isolated components of dental plaque biofilm (Al-Ahmad and others 2008). Personal hygiene of workers is considered an important factor that influences transfer of pathogenic bacteria (Olaimat and Holley 2012). S. mutans usually spreads by small droplet through Vol. 80, Nr. 6, 2015 r Journal of Food Science M1299

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Keywords: gallic acid, growth, biofilm, Escherichia coli, Streptococcus mutans

Inhibition of gallic acid . . . Table 1–Antimicrobial activity of different antibacterial agents against Escherichia coli (E. coli) and Streptococcus mutans (S. mutans). Concentrations of inhibitors (mg/mL) Strains

Bacteriostatic agents

Diameter of inhibition zone to E. coli (mm)

Kanamycin Gentamycin Gallic acid Kanamycin Gentamycin Gallic acid

Diameter of inhibition zone to S. mutans (mm)

0

2

4

8

16

n.i ap n.i ap n.i ap n.i ap n.i ap n.i ap

25.8 ± 0.2bq 33.5 ± 0.1br n.i ap 23.8 ± 0.2bq 33.7 ± 0.2br n.i ap

30.5 ± 0.4cq 38.5 ± 0.5cr n.i ap 28.5 ± 0.1cq 35.2 ± 0.3cr n.i ap

38.4 ± 0.3dq 44.7 ± 0.3dr 13.2 ± 0.4bpa 33.4 ± 0.5dq 38.2 ± 0.6dr 12.0 ± 0.2bp

40.5 ± 0.6eq 45.8 ± 0.5er 15.6 ± 0.5cpa 34.5 ± 0.4eq 42.5 ± 0.8er 13.2 ± 0.6cp

Different letters in the same line (a to e) indicate significant difference at P < 0.05. For the same pathogen, different letters between rows (p, q, and r) indicate significant difference at P < 0.05. a Indicate the significant difference (P < 0.05) between the diameter of inhibition zone to E. coli and S. mutans when with the same gallic acid concentration. n.i., no inhibition.

talking or coughing or sneezing. Therefore, it is also a potential contamination source in food-processing environments. The aims of this study were to (1) investigate the minimum inhibitory concentration of GA on the two targeted pathogens Escherichia coli (E. coli) and Streptococcus mutans (S. mutans); (2) study the effect of GA on the growth curves of the tested strains; and (3) investigate the growth and biofilm formation of the tested bacteria in the presence of GA under different nutrient conditions, temperatures, and treatment times.

M: Food Microbiology & Safety

Materials and Methods Experimental design Antimicrobial activities of GA on two pathogens E. coli and S. mutans were investigated with two common commercial antibiotics (kanamycin and gentamycin) as the control, and the minimum inhibitory concentration of GA was determined. Then the growth curves of both test bacteria in the presence of GA were determined at two different temperatures of 25 and 37 °C, which are the common industrial environment and optimal temperature for growth of the tested bacteria, respectively. For studying the effects of GA on the growth and biofilm formation of the tested bacteria under different conditions, different levels of nutrient (Luria-Bertani (LB), 1/8 LB, and 1/20 LB), temperatures (25 and 37 °C) and treatment times (24 and 48 h) were used. For investigating the effects of GA on the tested bacteria in washing step of real food industry, waters from washing cabbage and spinach (nutrient-deprived environment) were prepared and the assays were carried out by the above temperatures and times. Media preparation LB medium was the common medium for growth of the two bacteria and was prepared with 1% tryptone, 0.5% yeast extract, and 0.5% NaCl. It was used in all of the assays except the section about the vegetable wash water. The medium was diluted by using sterile water to 1:7 (v/v) and 1:19 (v/v) to obtain 1/8 LB and 1/20 LB, respectively, to provide the different levels of low-nutrient. The vegetable wash water samples were used to simulate the nutrient-deprived environment of wash step in food industry and prepared with the method as previously described by Liu and others (2009). Fresh cabbage and spinach were purchased from local grocery stores. The produce (250 g) was sliced into (5 × 5) cm2 pieces and washed for 30 min by gentle shaking in a plastic container (33 × 22 × 8 cm3 ) containing 500 mL of deionized distilled water. The wash water was decanted and filtered through a glass wool column in a 50 mL syringe. The filtered water was centrifuged for 10 min at 4000×g at room temperature. M1300 Journal of Food Science r Vol. 80, Nr. 6, 2015

The supernatant was ensured bacteria-free by filtering through a 0.22-µm nylon filter, and retained for further study.

Bacterial strains and culture conditions The test organisms, E. coli and S. mutans, were supplied by the Food Safety and Hygiene Laboratory (Shaanxi Normal University, Xi’an, China). Both organisms were preserved at −20 °C in LB medium containing 20% (v/v) glycerol. Before testing, each inoculum was prepared and cultivated in LB broth at 37 °C in a shaking incubator at 220 rpm until activated. Antibacterial activity determination The GA, kanamycin and gentamycin (Sigma–Aldrich) solutions were prepared by dissolving the corresponding commercial antimicrobial agents in sterile distilled water to an initial concentration of 16 mg/mL, and then 2-fold serially diluting to 8, 4, and 2 mg/mL. All solutions were filtered through a disposable filter with a pore size of 0.22 µm. Susceptibilities of the two bacteria to GA were determined by the disc diffusion method with minor modifications (Min and others 2008). The two bacteria were serially diluted (10-fold increments) in sterile distilled water until the cell suspension reached 106 CFU/mL. A 100 µL inoculum of the diluted (106 CFU/mL) test organism was spread onto LB agar plates. Discs (dia = 11 mm) saturated with GA solution were aseptically placed on the LB agar media spread with the tested bacteria and then the plates were incubated at 37 °C for 24 h. Negative control discs were separately prepared by saturating with sterile distilled water. For comparison, the sensitivities of each bacterium to commercial kanamycin and gentamycin discs were also evaluated. If an antibacterial agent stopped the bacteria from growing or kills the bacteria, there would be an area around the discs where the bacteria had not grown enough to be visible. This was called a zone of inhibition and if any around the discs (Neycee and others 2012), the diameter was measured by a vernier caliper with the cross method. A stronger antibacterial agent would create a larger zone. Growth curve assay The growth of test bacteria was investigated by the spectrophotometric (microquant microplate spectrophotometer; BioTek Instruments Inc., Winooski, Vt., U.S.A.) method. The overnightgrown cultures were diluted 1:100000 in fresh LB medium or LB medium containing 8 mg/mL of GA, and the cultures were incubated at 25 or 37 °C with shaking at 220 rpm (rotary shaker; Labcon, FSIM-SPO16, U.S.A.). The OD600 (optical density at 600 nm) values were recorded at 2 h interval during the 20-h cell growth period.

Inhibition of gallic acid . . . confidence level by Duncan test, using SPSS software (Ver.17.0; SPSS Inc., Chicago, Ill., U.S.A.).

Results and Discussion

Antibacterial activity of GA Table 1 lists the antimicrobial activities of 0–16 mg/mL GA and standard antibiotics (kanamycin and gentamycin) determined by the disc diffusion method. The control solvent (sterile distilled water) exhibited no antimicrobial activity (the inhibition zone (11.0 mm) equaled the disc diameter). In general, the antimicrobial activity of the various antibacterial agents depended on their dosage and type. The antibacterial activity of GA and both standard antibiotics against E. coli and S. mutans strains was an increasing function of dose. Given the same concentration of GA, kanamycin, and gentamycin, the mean diameters of the inhibition zones increased in the order GA < kanamycin < gentamycin (P < 0.05). Notably, the minimal effective dose of GA was 8 mg/mL, significant higher than those of gentamycin and kanamycin which showed the remarkable antimicrobial activity with the concentration of 2 mg/mL (P < 0.05). It seemed that the antimicrobial activity of the commercial antibiotics exceeded that of GA. However, common food-borne pathogens such as E. coli and Salmonella have been reported to rapidly develop antibiotic resistance (Threlfall and others 2000). As a natural bioactive A1 − A2 material, GA may not cause the drug resistance problems, and GA Percentage of growth/biofilm inhibition (%) = × 100 is not nontoxic. A1 In addition, it was found that with the same effective concen(1) tration of GA (8 and 16 mg/mL), the diameters of inhibition zone where A1 is the OD600 value of the control group and A2 is the to E. coli were significant higher than that of S. mutans, which indicated the higher antibacterial activity of GA to Gram-negative OD600 value of the GA group. All above assays including the growth of bacteria, antimicrobial species than those of Gram positive bacteria. activity, and biofilm formation assays were performed in aerobic Effect of GA on growth curve of E. coli and S. mutans conditions. The effect on growth of E. coli by exposing to 8 mg/mL GA with shaking was determined (Figure 1). It was found that GA Statistical analysis significantly inhibited the growth of E. coli at both temperatures, The biofilm formation assay was performed in five replicates since the OD of E. coli was markedly reduced to lower than 600 and all the other trials were carried out in triplicate, and the 0.2 in the GA culture (P < 0.05), which indicated no obvious average was reported. Significant differences were distinguished growth of E. coli. Especially for 37 °C, the OD of E. coli was 600 by one-way ANOVA and the significance was defined at the 95% 1.2 1.2

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Figure 1–Growth curves of Escherichia coli treated with or without gallic Figure 2–Growth curves of Streptococcus mutans treated with or without acid (8 mg/mL) at temperature of 25 and 37 °C. gallic acid (8 mg/mL) at temperature of 25 and 37 °C. Vol. 80, Nr. 6, 2015 r Journal of Food Science M1301

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Growth and biofilm formation assay The growth and biofilm formation was quantified in regular 96-well microtiter plates as previously described by Liu and others (2009) and Zhang and others (2014) with minor modifications. The overnight-grown cultures were diluted 1:100000 in fresh LB medium or LB medium containing 8 mg/mL of GA, transferred to sterile polystyrene microplates at 200 µL per well and incubated for 24 or 48 h at 25 or 37 °C without shaking. After incubation, the growth of the test bacteria under each set of conditions was quantified by the OD600 of the total biomass. The microplates were then washed five times with 450 µL sterile distilled water. The biofilm were stained with 250 µL of 0.3% crystal violet per well and incubated at room temperature for 45 min. Following incubation, the wells were again washed five times with 450 µL sterile distilled water and allowed to air dry. Then, 200 µL of 95% ethanol was added to each well. To dissolve the crystal violet dye, the well contents were mixed with a multichannel pipettor. The OD600 was then recorded for each well using a microquant microplate spectrophotometer (BioTek Instruments Inc.). The biofilm content was obtained by subtracting the average absorbance of the control wells (containing culture medium only) from each sample well. The averages and standard deviations were calculated from the results of five replicate wells. The percentage of growth/biofilm inhibition by GA was calculated as:

Inhibition of gallic acid . . . temperatures and times. Incubation temperature significantly affected the inhibitory results of GA; in 24 h cultures of E. coli grown at 37 and 25 °C, the GA growth inhibitions were 49.51% and 19.67%, respectively (P < 0.05). At 25 °C, increasing the treatment time to 48 h significantly enhanced the inhibitory effect of GA on E. coli to 36.76% (P < 0.05). However, doubling the treatment time exerted the reverse effect on E. coli grown at 37 °C, which decreased the GA inhibitory effect to 18.31% (P < 0.05). The result may be attributed to the limited growth of E. coli in the 24–48 h incubation time (0.934 in the 48 h culture compared with 0.921 in the 24 h culture) in control sample but meanwhile, the OD600 values in that of the GA group significantly increased from 0.465 to 0.763. When calculated the percentage of inhibition using Eq. (1), the value would be decrease because of the significant increase in the OD600 values of the GA group. Moreover, another possible reason was the depletion of GA, which needs to be further confirmed. Based on the above results, the inhibitory Growth and biofilm formation of E. coli and S. mutans and effect of GA on the growth of E. coli is sensitive to both of the time inhibition effect of GA in different LB media Figure 3A and B show the effects of GA on the growth and and temperature. Similar effects were obtained in the biofilm inbiofilm formation of E. coli cultured at different nutrient levels, hibition experiments. For E. coli cultured at 25 for 48 h and 37 °C

reduced by more than 5-fold (0.199 compared with 1.086 in the GA-free culture) by GA after 14 h culture. Similar results were obtained for the Gram-positive species S. mutans (Figure 2). Overall, GA showed significant inhibition effect on both strains at both temperatures, particularly at 37 °C (P < 0.05). Several mechanisms have been proposed for the antimicrobial effect of various antibacterial agents, such as destabilization of cytoplasmic and plasma membranes, inhibition of extracellular microbial enzymes and metabolisms, and deprivation of an essential growth substrate (Ikigai and others 1993; Puupponen-Pimi¨a and others 2005). Although the inhibition mechanism of GA to E. coli and S. mutans growth remains unknown, earlier studies about the fore body of GA have provided some hints. Chung and others (1998) reported that tannic acid inhibited the growth of intestinal bacteria because of the strong iron binding capacity.

1.0

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GA/LB

Control/1/8LB

GA/1/8LB

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GA/1/20LB

Figure 3–The inhibition of gallic acid (GA, 8 mg/mL) on growth (A) and biofilm formation (B) of E. coli in LB, 1/8LB, and 1/20LB broth media at different temperatures for different times (∗ indicates significant difference between control and GA treated group at P < 0.05 level).

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GA/1/20LB

GA inhibits biofilms by the factors additional to the antibacterial effect. Similar results were reported on the polyphenol extract of tea (Zhang and others 2014). The biofilm mode of growth protects bacterial cells from cell stressors such as desiccation, predators, and antibiotics (Bendaoud and others 2011). Therefore, extremely low nutrition combined with the hostile effects of GA may stimulate biofilm production as a survival strategy. These results are valuable for assessing the pathogenicity of E. coli under low-nutrient conditions. The Gram-positive S. mutans strain (Figure 4A and B) yielded some different results from E. coli. The growth and biofilm formation of this strain were markedly reduced under low nutrient and temperature conditions, but the inhibition activity of GA was overall lower than for the Gram-negative organism E. coli. This result is consistent with the reports by Borges and others (2012), where the activity of GA was evaluated on the prevention and control of biofilms formed by E. coli, Pseudomonas aeruginosa, S. aureus, and L. monocytogenes. It is likely that the viability of S. mutans exceeds that of E. coli (the OD600 values were consistently

Figure 4–The inhibition of gallic acid (GA, 8 mg/mL) on growth (A) and biofilm formation (B) of S. mutans in LB, 1/8LB, and 1/20LB broth media at different temperatures for different times (∗ indicates significant difference between control and GA treated group at P < 0.05 level).

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Conditions B Vol. 80, Nr. 6, 2015 r Journal of Food Science M1303

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for 24 and 48 h, GA exerted a significant inhibitory effect on the biofilm formation (P < 0.05), and the highest inhibition (60.47%) was observed at 37 °C for 24 h culture. Doubling the treatment time at 25 °C dramatically increased the inhibitory effect of GA from 16.29% to 44.35% (P < 0.05). When LB medium was diluted to 1/8 LB in sterile water, the consequent reduction of nutrients significantly reduced the OD600 values for growth and biofilm formation of E. coli (P < 0.05). However, the inhibition activities of GA on the growth and biofilm formation were similar to that in LB medium. In 1/20 LB medium, the growth and biofilm formation of E. coli were significantly inhibited regardless of GA presence. And the inhibition activity (41.46%–50.77%) of GA on the growth of the pathogen was significantly higher in this medium than in the LB (18.31%–49.51%) and 1/8 LB (16.28%–48.77%) media (P < 0.05). However, under most of the tested conditions, GA exerted less inhibitory effect on biofilm formation in 1/20 LB medium (28.78%–36.05%) than in the other two media (16.29%–60.47% for LB and 22.28%–59.70% for 1/8 LB medium, respectively) (P < 0.05). This indicates that

Inhibition of gallic acid . . . higher in S. mutans than in E. coli, regardless of cultivation conditions, suggesting that S. mutans is more resistant to GA. At 37 °C, GA significantly inhibited the growth of S. mutans in all the tested media (P < 0.05) and the inhibition activities were reduced at the longer incubation time. However, at 25 °C, GA exhibited a dramatic inhibitory effect only in a nutrient-rich environment treated for 48 h (P < 0.05). Similarly, GA significantly inhibited biofilm formation only in high-nutrient LB medium, regardless of cultivation conditions (37.11%–48.32%) (P < 0.05). In the nutrient-poor environments (1/8 and 1/20 LB media), GA exerted no significant effects on biofilm formation under any cultivation condition (P > 0.05). As hypothesized for E. coli, the nutrient deprived environment stimulated biofilm production as a protective strategy (Hol´a and others 2006).

Growth and biofilm formation of E. coli and S. mutans and the inhibition effect of GA in different wash waters In low-nutrient environments, bacteria easily form biofilm that seriously contaminates the vegetables. E. coli grew poorly in cabbage wash water, as evidenced by the low OD600 values (Figure 5A). However, GA exhibited significant and similar

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M: Food Microbiology & Safety

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inhibition activity on the growth of E. coli, regardless of cultivation conditions. Doubling the incubation time from 24 to 48 h significantly increased the amount of biofilm for cultures at 25 and 37 °C (Figure 5B). GA showed relatively higher inhibition activity on the formation of biofilm in 48 h culture at 25 °C (37.72%), and in 24 h culture at 37 °C (34.23%) than in other cultivation conditions. Spinach wash water was more conducive to microbial growth than cabbage wash water because of its higher nutrient value. The OD600 values of both growth and biofilm formation were much higher in spinach than in cabbage wash water (P < 0.05). Similarly to cabbage wash water, GA exerted its highest inhibition effect on both of the growth and biofilm formation of E. coli in 48 h culture at 25 °C, and in 24 h culture at 37 °C. Figure 6A and B show the inhibition activity of GA on the growth and biofilm formation of S. mutans incubated in wash water under different conditions. Like E. coli, S. mutans grew less and formed significantly less biofilm in cabbage wash water than in spinach wash water (P < 0.05). The growth and biofilm formation of S. mutans were significantly inhibited by GA in cabbage wash water under most sets of conditions with the values of 17.34%–23.39% and 22.77%–28.67%, respectively (P < 0.05).

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B Figure 5–The inhibition of gallic acid (GA, 8 mg/mL) on growth (A) and biofilm formation (B) of E. coli in cabbage and spinach wash water at different temperatures for different times (∗ indicates significant difference between control and GA treated group at P < 0.05 level).

M1304 Journal of Food Science r Vol. 80, Nr. 6, 2015

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Figure 6–The inhibition of gallic acid (GA, 8 mg/mL) on growth (A) and biofilm formation (B) of S. mutans in cabbage and spinach wash water at different temperatures for different times (∗ indicates significant difference between control and GA treated group at P < 0.05 level).

The exception was GA showed no significant inhibitory effect on the biofilm formation at 25 °C for 24 h (P > 0.05). In spinach wash water, GA exhibited the greatest inhibition activity on the growth (39.01%) and biofilm formation (60.23%) of 48 h cultures at 25 °C. Overall, the inhibition activity of GA on biofilm formation was higher in spinach wash than in cabbage wash water.

Conclusions The minimum antimicrobial concentration of GA was found to be 8 mg/mL showing higher antibacterial activity to Gram negative specie (E. coli) than that of Gram positive bacteria (S. mutans). The growth of tested pathogens was more sensitive to environmental conditions than biofilm formation. The inhibition effect of GA on biofilm could be due to other factors in additional to the antibacterial effect. All tested three factors, nutrient level, treatment temperature and incubation time, had effect on the inhibition activity of GA. The highest inhibition of GA was observed in 24 h cultures incubated at 37 °C, and in 48 h cultures incubated at 25 °C in serial LB media or in vegetable wash waters. Further investigation is needed to elucidate the mechanisms of GA exerts its inhibition activity under different conditions.

Acknowledgments The work was supported by the Fundamental Research Funds for the Central Universities (GK201002023 and 3102014JKY15011). There is no conflict of interest.

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Vol. 80, Nr. 6, 2015 r Journal of Food Science M1305

M: Food Microbiology & Safety

Inhibition of gallic acid . . .

Inhibition of Gallic Acid on the Growth and Biofilm Formation of Escherichia coli and Streptococcus mutans.

New strategies for biofilm inhibition are becoming highly necessary because of the concerns to synthetic additives. As gallic acid (GA) is a hydrolysa...
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