Assessment of Oligogalacturonide from Citrus Pectin as a Potential Antibacterial Agent against Foodborne Pathogens Ming-Chang Wu, Hui-chin Li, Po-Hua Wu, Ping-Hsiu Huang, and Yuh-Tai Wang
Abstract: Foodborne diseases are an important public health problem in the world. The bacterial resistance against presently used antibiotics is becoming a public health issue; hence, the discovery of new antimicrobial agents from natural sources attracts a lot of attention. Antibacterial activities of oligogalacturonide from commercial microbial pectic enzyme (CPE) treated citrus pectin, which exhibits antioxidant and antitumor activities, against 4 foodborne pathogens including Salmonella Typhimurium, Staphylococcus aureus, Listeria monocytogenes, and Pseudomonas aeruginosa was assessed. Pectin hydrolysates from CPE hydrolysis exhibited antibacterial activities. However, no antibacterial activity of pectin was observed. Citrus oligogalacturonide from 24-h hydrolysis exhibited bactericidal effect against all selected foodborne pathogens and displayed minimal inhibitory concentration at 37.5 μg/mL for P. aeruginosa, L. monocytogenes, and S. Typhimurium, and at 150.0 μg/mL for S. aureus.
Introduction Foodborne illness resulting from consumption of food contaminated with pathogenic bacteria and/or their toxins is a vital concern to public health. Centers for Disease Control and Prevention (2013) indicated that in 2012 the total of 19531 infections, 4563 hospitalizations, and 68 deaths in the United States were foodborne diseases associated. Therefore, controlling food pathogens will contribute to protect consumers from disease, reduce foodborne disease outbreaks, and assure consumers a safe and wholesome food supply. Food antimicrobials are compounds added to, or present in foods to retard microbial growth or kill microorganisms. Natural antimicrobials are increasingly used to avoid health-related problems, extend shelf-life and minimize deleterious effects of foods induced by microorganisms including off-odor, unpleasant taste, textural problems, or changes in color (L´opez-Malo and others 2000; Nielsen and R´ıos 2000; Feng and Zheng 2007). Several naturally occurring antimicrobial agents have been used to control or prevent the growth of food spoilage bacteria and pathogens for shelf-life extension, including plant volatile oils (Dorman and Deans 2000), nisin (Cleveland and others 2001), lactoferrin and its hydrolysate (Murdock and Matthews 2002), and chitosan (Devlieghere and others 2004). Plant volatile oils are isolated from plant material by distillation methods such as steam or hydrodistillation (Dorman and Deans 2000). Nisin and lactoferrin (and its hydrolysate) are polypeptide-related antimicrobials isolated from lactic acid bacteria and cow’s milk, respectively (Cleveland and others 2001; Murdock and Matthews 2002). Chitosan produced from chitin of crustacean shells by deacetylation has a major draw-
MS 20130693 Submitted 5/23/2013, Accepted 4/30/2014. Authors M-C Wu, Li and Huang are with Dept. of Food Science, Natl. Pingtung Univ. of Science and Technology, Pingtung 91201, Taiwan. Author P-H Wu is with Dept. of Microbiology, Immunology, and Biophamarceuticals, Natl. Chiayi Univ., Nr. 300 Syuefu Rd.,Chiayi City 60004, Taiwan. Author Wang is with Life Science Center, Hsing Wu Univ., Taipei 244, Taiwan. Direct inquiries to author Wang is with (E-mail: [email protected]).
R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12526 Further reproduction without permission is prohibited
back of poor solubility for application (Devlieghere and others 2004; Vel´azquez-del Valle and others 2012). Low molecular weight (Mw) chitosan and nisin are cationic antibacterial agents, which inhibit bacterial growth by interacting with the anionic surface charge of Staphylococcus aureus (Chen and others 2012). Alginate is reported as an auxiliary bacterial membrane of Pseudomonas aeruginosa infections in cystic fibrosis patients and the antibiotic resistance of antimicrobial agents with positive charge (Chan and others 2005). Oligogalacturonide from citrus pectin was reported to express antioxidant activity, lipid oxidation inhibit ability in food emulsion (Huang and others 2011) and showed antitumor activity with membrane permeability on human cancer cells (Huang and others 2012). No antibacterial study of anionic polysaccharide was found, except the results of Men’shikov and others (1997) who reported that pectin exhibited a bactericidal effect on pathogenic and opportunistic microorganisms. Preliminary experiments indicated that oligogalacturonide derived from citrus pectin expressed higher antibacterial activity than citrus pectin. In order to discover new antimicrobial agents from natural sources, the antibacterial activity of the oligogalacturonide from citrus pectin on foodborne pathogens was evaluated.
Materials and Methods Materials Tendril shoots of chayote (TSC; Sechium edule (Jacq.) Swartz) were purchased from a local supermarket. Commercial microbial pectic enzyme Peclyve CP (CPE) from Aspergillus niger containing 133.5 U/mL pectin lyase (PL) activity, 50.6 U/mL pectin methyl esterase (PME) activity, and 22.4 U/mL polygalacturonase activity were purchased from Lallemand Australia Pty. Ltd. (North Adelaide, Australia). Citrus pectins with 60% degree of esterification (DE) and 90% to 93% DE were purchased from Nacalai Tesque (Kyoto, Japan) and Sigma (St. Louis, Mo., U.S.A.), respectively. Nutrient broth was purchased from ST Bio (Taiwan), and penicillin from Biological Industries (Kibbutz Beit Haemek, Israel). Vol. 79, Nr. 8, 2014 r Journal of Food Science M1541
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Keywords: antibacterial activity, citrus pectin, foodborne pathogens, oligogalacturonide.
Antibacterial citrus oligogalacturonide . . . Bacteria S. Typhimurium (BCRC10780), S. aureus (BCRC12947), Listeria monocytogenes (BCRC 14845), and P. aeruginosa (BCRC 15803) were used and purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan). Preparation of PME PME was purified from TSC according to the procedure described by Wu and others (2005). Briefly, TSC (50 g) was homogenized with cold distilled water and 0.01 M phosphate buffer (pH 8.0) and filtered. The residues were then homogenized and centrifuged at 14000 × g for 30 min. The supernatant was fractionated by 40% to 70% ammonium sulfate and dialyzed against deionized water to obtain crude TSC PME. Afterward, highly methoxylated cross-linked alcohol-insoluble solid (HM-CL-AIS) chromatography column (2.5 cm × 20.0 cm; flow rate, 30 mL/h; eluent, 0 to 0.3 M NaCl/0.01 M acetate buffer, pH 4.0) was conducted to purify PME. HM-CL-AIS was prepared from the alcohol-insoluble solid from pea pods that was cross-linked with epichlorohydrin and methoxylated in methanolic H2 SO4 . PME activity was analyzed by monitoring the purification.
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Preparation of PL PL was purified from commercial microbial pectic enzyme (CPE) according to the procedure described by Wu and others (2007). An aliquot (0.2 mL) of CPE was applied on the HMCL-AIS column (2.5 cm × 20.0 cm; flow rate, 30 mL/h) washed with 0.01 M acetate buffer (pH 4) and then eluted with the same buffer at 0 to 1 M NaCl gradient. The PL activity of eluant was determined to monitor the purification. Determination of PME activity PME activity was carried out according to the method described by Jiang and others (2002). Protons released in 0.1 M NaCl/0.5% citrus pectin (DE 60%) solution (25 °C) were titrated with 10 mM NaOH to maintain pH 6.5 in an automatic titrator (PHStat Controller PHM-290, Radiometer, Copenhagen, Denmark). One unit of PME activity is defined as 1 μmol of free carboxyl groups formed from pectin per minute.
Table 1–DE and Mw of pectin after commercial microbial pectic enzyme treatment. Treatment time (h) 0 6 12 24
DE (%)
Mw (kDa)
60.0 22.4 17.5 11.6
353.0 1.8 1.7 1.0
followed by incubation (at ambient temperature for 1 h), filtration, and neutralization with dilute phosphoric acid to pH 7.5. The methanol content of solution was analyzed by gas chromatography (Thermo Finnigan Trace GC Ultra, Milan, Italy) that equipped R with flame ionization detector (FID) and Stabilwax fused silica column (Stabilwax [Restek, Pa., U.S.A.], 30 m × 0.25 mm, 0.2μm film thickness). The DE value of pectin was calculated using the following equation: DE(%) = methanolcontent × (31/32) ÷ 16.32 ÷ pectin content ×100%
(1)
Determination of Mw Mw of various pectin samples were measured by size-exclusion chromatography in a high performance liquid chromatographic (HPLC) system by using the method described by Huang and others (2011). Sample (20 μL) was separated on a TSK-Gel G5000 PWXL column (Tosoh, Tokyo, Japan) at 0.6 mL/min and detected using a Hitachi L-2490 refractive index detector (Hitachi, Tokyo, Japan). Data were analyzed using HPLC System Manager (HSM) software, Version 2.0 (Hitachi). The dextran standard series (Sigma) was used as markers for Mw calculation. Disc diffusion assay The freeze-dried pectin hydrolysate was dissolved in double distilled water to a final concentration of 150 μg/mL and filtersterilized through a 0.45 μm Millipore filter for further use. Antibacterial tests were then carried out by disc diffusion (Sahin and others 2003) using 100 μL of suspension containing 108 CFU/mL of each target microorganism spread on nutrient agar, as the inoculated plate. The discs (8 mm in diameter) were placed on the inoculated agar and impregnated with 50 μL of pectin hydrolysate. The inoculated plates were incubated at 37 °C for 24 h. Negative control was prepared using sterilized distilled water, while penicillin (150 μg/mL) and chitosan (1%) were used as positive controls. Antibacterial activity was evaluated by measuring the zone of inhibition against the test organisms.
Determination of PL Activity PL activity was determined spectrometrically by measuring the increase in absorbance at 235 nm of a reaction mixture containing 0.2 mL of 0.5% pectin (DE 90% to 93%) in 10 mM Tris-HCl buffer (pH 8) plus 0.2 mM CaCl2 and 0.2 mL of enzyme solution at 40 °C (Wu and others 2007). One unit of PL activity is defined as an increase in absorbance of 0.005 per hour at 235 nm under the reaction condition. Microdilution assays Minimum inhibitory concentration (MIC) is defined as the Preparation of pectin hydrolysate lowest concentration of citrus oligogalacturonide to inhibit the Pectin hydrolysate was prepared according to the method de- growth of microorganisms. It was determined by serial dilutions in scribed by Huang and others (2011). Citrus pectin (1%, w/v) and nutrient broth based on a micro-well dilution method according to various pectic enzymes (CPE, PL, and PME) containing solution Zgoda and Porter (2001). Microbial growth was determined by the were incubated at pH 4, 45 °C for 0 to 24 h, heated in a boiling absorbance at 600 nm using Epoch microplate spectrophotometer water bath for 10 min, cooled to room temperature, and then (BioTek, Winooski, Vt., U.SA.) and confirmed by plating sample freeze-dried. from clear wells on nutrient agar. Determination of DE Determination of mechanisms of antibacterial effect DE values of pectin and pectin hydrolysate were determined To elucidate whether the observed antibacterial effect of citrus according to the method described by Jiang and others (2002). oligogalacturonide was bactericidal or bacteriostatic, the mechaPectin sample (10 mg) was added to 10 mL of 0.5 N KOH, nism of antibacterial effect was evaluated according to the method M1542 Journal of Food Science r Vol. 79, Nr. 8, 2014
Antibacterial citrus oligogalacturonide . . . Table 2–Antibacterial activities of penicillin, chitosan and pectin hydrolysates from various reaction time of commercial microbial pectic enzyme against food pathogens using the disc diffusion methoda . Reaction time (h) Bacterial strain
Penicillin
Chitosan
0
6
12
24
S. aureus P. aeruginosa L. monocytogenes S. Typhimurium
9.2 ± 0.1d
± ± ± ±
b
11.6 ± 0.1c 23.3 ± 0.1b 22.8 ± 0.1c 25.0 ± 0.0b
14.0 ± 0.1b 23.5 ± 0.1b 24.8 ± 0.0b 25.3 ± 0.5b
15.1 ± 0.2a 26.0 ± 0.1a 25.1 ± 0.0a 27.1 ± 0.1a
b
15.7 ± 0.1d 18.3 ± 0.1d
0 3.3 4.0 2.6
0.0e 0.0c 0.0e 0.1e
b b b
Zones of growth inhibition (mm) were measured and expressed as mean ± SD, those bearing the same inline alphabets in the same row are not significantly different (P > 0.05). The diameter of the zone of inhibition includes the paper disc (8 mm). The concentrations used for disc diffusion assay were 150 μg/mL for penicillin and pectin hydrolysates from citrus pectin and 1% for chitosan. b No zone of inhibition was observed. a
Statistical analysis Each experiment was carried out in 3 replications. Statistical comparisons were made by one-way analysis of variance followed by Duncan’s multiple range test.
Results and Discussion Antibacterial activity of pectin hydrolysate Preliminary evaluations of the antibacterial activities of the enzymatically hydrolyzed pectins from CPE, PL, and PME against the 4 selected foodborne pathogens were conducted. No inhibition zone was observed in samples treated with pectin hydrolysates from PL and PME, but inhibition was seen with the CPE-treated pectin (data not shown). CPE hydrolysis caused an increase in the antibacterial activity of pectin solution against all selected foodborne pathogens. As shown in Table 1, the Mw of pectin hydrolysates from the CPE treatment ranged from 1.0 kDa (24 h hydrolysate) to 1.8 kDa (6 h hydrolysate), normally less than 10mer; thus, the pectin hydrolysates from CPE-treated citrus pectin in this study was expressed as citrus oligogalacturonide. Antibacterial activity of oligogalacturonide from citrus pectin against foodborne pathogens was qualitatively and quantitatively assessed by the presence of an inhibition zone and the zone diameter using the disc diffusion assay and MIC value using the microdilution assay. It is obvious that citrus oligogalacturonides showed good antibacterial activity against the selected foodborne pathogens and the zones of inhibition ranged from 11.6 to 27.1 mm (Table 2). Men’shikov and others (1997) reported that pectin exhibited a bactericidal effect on pathogenic and opportunistic microorganisms, however no antibacterial activity of pectin was observed in this study. Antibacterial activity of citrus oligogalacturonide increased with increased enzyme reaction time of CPE (Table 2). In other words, digestion of citrus pectin with CPE for 24 h yielded an oligogalacturonide with 1.0 kDa and 11.6% DE, which exhibited the highest antibacterial activity against the selected foodborne pathogens. Huang and others (2012) revealed that the citrus oligogalacturonide was membrane permeable. The pectin hydrolysates from PL (causing a decrease in Mw of pectin) and PME (causing a decrease in DE of pectin) exhibited no antibacterial activity; thus,
Table 3–Antibacterial properties of citrus oligogalacturonide against food pathogens. Bacterial strain S. aureus P. aeruginosa L. monocytogenes S. Typhimurium a
MICa (μg/mL)
Mechanism of antibacterial effect
150.0 37.5 37.5 37.5
b b b b
The lowest concentration of citrus oligogalacturonide from 24-h hydrolysis. The antibacterial effect was classified as bactericidal effect.
b
we speculated that the antibacterial activity of citrus oligogalacturonide from CPE (causing a decrease in Mw and DE of pectin) was attributed to the membrane permeability of oligogalacturonide in an appropriate Mw and DE value. The citrus oligogalacturonide displayed different antibacterial activity depending on the bacteria. In general, Gram-negative bacteria are more resistant than Gram-positive bacteria (Marino and others 1999; Nasar-Abbas and Halkman 2004). The mechanism of antimicrobial substances of Gram-positive and Gram-negative bacteria is not well established (Gomes Dos Santos and others 2013; Haney and others 2011; Radulovi´c and others 2013). The antibacterial results of citrus oligogalacturonides in this study revealed a different result. S. aureus (Gram-positive) was found to be more resistant strain than Gram-negative strains tested (Table 2). There is no single antibiotic that provides coverage for all microbes (Benz and others 2004). Therefore, citrus oligogalacturonides can be used as a potential native antibiotic. All of the citrus oligogalacturonides exhibited higher antibacterial activities than penicillin at the same concentration (7.5 μg/disc). Penicillin is preferentially used in the treatment of grampositive bacterial infection as an antibiotic, however penicillin resistance is becoming more common in S. aureus (Lowy 2003), P. aeruginosa (Suginaka and others 1975), Listeria spp. (Wang and others 2012), and Salmonella Typhimurium (Schuman and others 1989). Hence, citrus oligogalacturonides might also be a good antibacterial candidate for pharmaceutical use.
Minimal inhibitory concentration of citrus oligogalacturonide The MIC of citrus oligogalacturonide from 24 h hydrolysis for inhibition of P. aeruginosa, L. monocytogenes, and Salmonella Typhimurium was 37.5 μg/mL, whereas the MIC for S. aureus was 150.0 μg/mL (Table 3). The ethanolic extracts of sumac and avishan-e shirazi inhibited S. aureus at MIC of 0.1% and 0.4%, respectively (Fazeli and others 2007); and lactoferrin and lactoferrin hydrolysate inhibited Vol. 79, Nr. 8, 2014 r Journal of Food Science M1543
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described by Marino and others (1999). The procedure involving the contact of microorganisms to pectin hydrolysates using the disc diffusion assay was described above. Streak plates were prepared by inoculation of 5 loopfuls of the inhibition zones on fresh nutrient agar and incubating the plates at 37°C for 24 h. Samples that showed no growth after re-streaking were considered as demonstrating a bactericidal effect.
Antibacterial citrus oligogalacturonide . . .
M: Food Microbiology & Safety
S. aureus at MIC of 8 and 4 mg/mL, respectively (Murdock and Matthews 2002). The MIC of rosemary essential oil was found to be 2.3 to 3.5 mg/mL for S. aureus (Pintore and others 2002). The MICs of plant essential oils thymol and eugenol for S. aureus were found to be 5 and 8 mg/mL, respectively; and nisin inhibited S. aureus at MIC of 5 mg/mL (Panitee and Vanee 2007). Lactoferrin and lactoferrin hydrolysate inhibited L. monocytogenes at MIC of 2 and 1 mg/mL, respectively (Murdock and Matthews 2002). The MICs of plant essential oils thymol and eugenol for L. monocytogenes were found to be 4 and 11 mg/mL, respectively; and nisin that inhibited L. monocytogenes at MIC of 4 mg/mL (Panitee and Vanee 2007). The essential oil constituents, citral, geraniol, and perillaldehyde, were reported to completely kill Salmonella Typhimurium at 500 μg/mL (Kim and others 1995). The MIC of rosemary essential oil was found to be >4 mg/mL against P. aeruginosa (Pintore and others 2002). In comparison with the other antimicrobials, the MIC of citrus oligogalacturonide against S. aureus, L. monocytogenes, Salmonella Typhimurium, and P. aeruginosa ranged from 37.5 to 150.0 μg/mL, showing that the citrus oligogalacturonide possess stronger antimicrobial activity and may be a suitable antibacterial agent to inhibit pathogen growth in foods. Based on the classifications of antibacterial activity using the disc method followed by streak plate culture, no growth was observed. Hence, the citrus oligogalacturonide from 24 h hydrolysis was found to display a bactericidal effect against the selected pathogens (Table 3). Citrus oligogalacturonide with 1.0 kDa and 11.6% DE was reported to be membrane permeable (Huang and others 2012). The mechanism of antimicrobial substances includes: membrane instability and interfering the intermediary metabolisms or DNA/RNA synthesis/function (Haney and others 2011; Radulovi´c and others 2013). Therefore, the possible antimicrobial pathway of the membrane permeable citrus oligonucleotide is by interfering with intermediary metabolism or DNA/RNA synthesis/function.
Conclusions Recently, bacterial resistance against presently used antibiotics is becoming a public health issue (Kuroda and Caputo 2013). Numerous studies have focused on the discovery of new antimicrobial agents from natural sources or the design and synthesis of new agents. A lot of attention has been paid to the discovery of new antimicrobial agents from natural sources and the design and synthesis of new agents (Yilmaz and Ercisli 2011; Kuroda and Caputo 2013). Citrus oligogalacturonide was reported to exhibit antioxidant activity and antitumor activity with no cytotoxicity (Huang and others 2011, 2012). In this study, the citrus oligogalacturonide was found to exhibited good antibacterial activities and display a bactericidal effect against the selected food pathogens, including S. Typhimurium, S. aureus, L. monocytogenes, and P. aeruginosa. Therefore, citrus oligogalacturonide may have potential to be further developed as an antibacterial agent for the food industry and a novel pharmaceutical compound with cancer prevention and bacterial resistance avoiding benefits.
Acknowledgment Financial support from the Natl. Science Council of the Republic of China under Grant 98-2313-B-020-013-MY3 is greatly) appreciated.
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Abstract: Foodborne diseases are an important public health problem in the world. The bacterial resistance against presently used antibiotics is becoming a public health issue; hence, the discovery of new antimicrobial agents from natural sources attracts a lot of attention. Antibacterial activities of oligogalacturonide from commercial microbial pectic enzyme (CPE) treated citrus pectin, which exhibits antioxidant and antitumor activities, against 4 foodborne pathogens including Salmonella Typhimurium, Staphylococcus aureus, Listeria monocytogenes, and Pseudomonas aeruginosa was assessed. Pectin hydrolysates from CPE hydrolysis exhibited antibacterial activities. However, no antibacterial activity of pectin was observed. Citrus oligogalacturonide from 24-h hydrolysis exhibited bactericidal effect against all selected foodborne pathogens and displayed minimal inhibitory concentration at 37.5 μg/mL for P. aeruginosa, L. monocytogenes, and S. Typhimurium, and at 150.0 μg/mL for S. aureus.
Introduction Foodborne illness resulting from consumption of food contaminated with pathogenic bacteria and/or their toxins is a vital concern to public health. Centers for Disease Control and Prevention (2013) indicated that in 2012 the total of 19531 infections, 4563 hospitalizations, and 68 deaths in the United States were foodborne diseases associated. Therefore, controlling food pathogens will contribute to protect consumers from disease, reduce foodborne disease outbreaks, and assure consumers a safe and wholesome food supply. Food antimicrobials are compounds added to, or present in foods to retard microbial growth or kill microorganisms. Natural antimicrobials are increasingly used to avoid health-related problems, extend shelf-life and minimize deleterious effects of foods induced by microorganisms including off-odor, unpleasant taste, textural problems, or changes in color (L´opez-Malo and others 2000; Nielsen and R´ıos 2000; Feng and Zheng 2007). Several naturally occurring antimicrobial agents have been used to control or prevent the growth of food spoilage bacteria and pathogens for shelf-life extension, including plant volatile oils (Dorman and Deans 2000), nisin (Cleveland and others 2001), lactoferrin and its hydrolysate (Murdock and Matthews 2002), and chitosan (Devlieghere and others 2004). Plant volatile oils are isolated from plant material by distillation methods such as steam or hydrodistillation (Dorman and Deans 2000). Nisin and lactoferrin (and its hydrolysate) are polypeptide-related antimicrobials isolated from lactic acid bacteria and cow’s milk, respectively (Cleveland and others 2001; Murdock and Matthews 2002). Chitosan produced from chitin of crustacean shells by deacetylation has a major draw-
MS 20130693 Submitted 5/23/2013, Accepted 4/30/2014. Authors M-C Wu, Li and Huang are with Dept. of Food Science, Natl. Pingtung Univ. of Science and Technology, Pingtung 91201, Taiwan. Author P-H Wu is with Dept. of Microbiology, Immunology, and Biophamarceuticals, Natl. Chiayi Univ., Nr. 300 Syuefu Rd.,Chiayi City 60004, Taiwan. Author Wang is with Life Science Center, Hsing Wu Univ., Taipei 244, Taiwan. Direct inquiries to author Wang is with (E-mail: [email protected]).
R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12526 Further reproduction without permission is prohibited
back of poor solubility for application (Devlieghere and others 2004; Vel´azquez-del Valle and others 2012). Low molecular weight (Mw) chitosan and nisin are cationic antibacterial agents, which inhibit bacterial growth by interacting with the anionic surface charge of Staphylococcus aureus (Chen and others 2012). Alginate is reported as an auxiliary bacterial membrane of Pseudomonas aeruginosa infections in cystic fibrosis patients and the antibiotic resistance of antimicrobial agents with positive charge (Chan and others 2005). Oligogalacturonide from citrus pectin was reported to express antioxidant activity, lipid oxidation inhibit ability in food emulsion (Huang and others 2011) and showed antitumor activity with membrane permeability on human cancer cells (Huang and others 2012). No antibacterial study of anionic polysaccharide was found, except the results of Men’shikov and others (1997) who reported that pectin exhibited a bactericidal effect on pathogenic and opportunistic microorganisms. Preliminary experiments indicated that oligogalacturonide derived from citrus pectin expressed higher antibacterial activity than citrus pectin. In order to discover new antimicrobial agents from natural sources, the antibacterial activity of the oligogalacturonide from citrus pectin on foodborne pathogens was evaluated.
Materials and Methods Materials Tendril shoots of chayote (TSC; Sechium edule (Jacq.) Swartz) were purchased from a local supermarket. Commercial microbial pectic enzyme Peclyve CP (CPE) from Aspergillus niger containing 133.5 U/mL pectin lyase (PL) activity, 50.6 U/mL pectin methyl esterase (PME) activity, and 22.4 U/mL polygalacturonase activity were purchased from Lallemand Australia Pty. Ltd. (North Adelaide, Australia). Citrus pectins with 60% degree of esterification (DE) and 90% to 93% DE were purchased from Nacalai Tesque (Kyoto, Japan) and Sigma (St. Louis, Mo., U.S.A.), respectively. Nutrient broth was purchased from ST Bio (Taiwan), and penicillin from Biological Industries (Kibbutz Beit Haemek, Israel). Vol. 79, Nr. 8, 2014 r Journal of Food Science M1541
M: Food Microbiology & Safety
Keywords: antibacterial activity, citrus pectin, foodborne pathogens, oligogalacturonide.
Antibacterial citrus oligogalacturonide . . . Bacteria S. Typhimurium (BCRC10780), S. aureus (BCRC12947), Listeria monocytogenes (BCRC 14845), and P. aeruginosa (BCRC 15803) were used and purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan). Preparation of PME PME was purified from TSC according to the procedure described by Wu and others (2005). Briefly, TSC (50 g) was homogenized with cold distilled water and 0.01 M phosphate buffer (pH 8.0) and filtered. The residues were then homogenized and centrifuged at 14000 × g for 30 min. The supernatant was fractionated by 40% to 70% ammonium sulfate and dialyzed against deionized water to obtain crude TSC PME. Afterward, highly methoxylated cross-linked alcohol-insoluble solid (HM-CL-AIS) chromatography column (2.5 cm × 20.0 cm; flow rate, 30 mL/h; eluent, 0 to 0.3 M NaCl/0.01 M acetate buffer, pH 4.0) was conducted to purify PME. HM-CL-AIS was prepared from the alcohol-insoluble solid from pea pods that was cross-linked with epichlorohydrin and methoxylated in methanolic H2 SO4 . PME activity was analyzed by monitoring the purification.
M: Food Microbiology & Safety
Preparation of PL PL was purified from commercial microbial pectic enzyme (CPE) according to the procedure described by Wu and others (2007). An aliquot (0.2 mL) of CPE was applied on the HMCL-AIS column (2.5 cm × 20.0 cm; flow rate, 30 mL/h) washed with 0.01 M acetate buffer (pH 4) and then eluted with the same buffer at 0 to 1 M NaCl gradient. The PL activity of eluant was determined to monitor the purification. Determination of PME activity PME activity was carried out according to the method described by Jiang and others (2002). Protons released in 0.1 M NaCl/0.5% citrus pectin (DE 60%) solution (25 °C) were titrated with 10 mM NaOH to maintain pH 6.5 in an automatic titrator (PHStat Controller PHM-290, Radiometer, Copenhagen, Denmark). One unit of PME activity is defined as 1 μmol of free carboxyl groups formed from pectin per minute.
Table 1–DE and Mw of pectin after commercial microbial pectic enzyme treatment. Treatment time (h) 0 6 12 24
DE (%)
Mw (kDa)
60.0 22.4 17.5 11.6
353.0 1.8 1.7 1.0
followed by incubation (at ambient temperature for 1 h), filtration, and neutralization with dilute phosphoric acid to pH 7.5. The methanol content of solution was analyzed by gas chromatography (Thermo Finnigan Trace GC Ultra, Milan, Italy) that equipped R with flame ionization detector (FID) and Stabilwax fused silica column (Stabilwax [Restek, Pa., U.S.A.], 30 m × 0.25 mm, 0.2μm film thickness). The DE value of pectin was calculated using the following equation: DE(%) = methanolcontent × (31/32) ÷ 16.32 ÷ pectin content ×100%
(1)
Determination of Mw Mw of various pectin samples were measured by size-exclusion chromatography in a high performance liquid chromatographic (HPLC) system by using the method described by Huang and others (2011). Sample (20 μL) was separated on a TSK-Gel G5000 PWXL column (Tosoh, Tokyo, Japan) at 0.6 mL/min and detected using a Hitachi L-2490 refractive index detector (Hitachi, Tokyo, Japan). Data were analyzed using HPLC System Manager (HSM) software, Version 2.0 (Hitachi). The dextran standard series (Sigma) was used as markers for Mw calculation. Disc diffusion assay The freeze-dried pectin hydrolysate was dissolved in double distilled water to a final concentration of 150 μg/mL and filtersterilized through a 0.45 μm Millipore filter for further use. Antibacterial tests were then carried out by disc diffusion (Sahin and others 2003) using 100 μL of suspension containing 108 CFU/mL of each target microorganism spread on nutrient agar, as the inoculated plate. The discs (8 mm in diameter) were placed on the inoculated agar and impregnated with 50 μL of pectin hydrolysate. The inoculated plates were incubated at 37 °C for 24 h. Negative control was prepared using sterilized distilled water, while penicillin (150 μg/mL) and chitosan (1%) were used as positive controls. Antibacterial activity was evaluated by measuring the zone of inhibition against the test organisms.
Determination of PL Activity PL activity was determined spectrometrically by measuring the increase in absorbance at 235 nm of a reaction mixture containing 0.2 mL of 0.5% pectin (DE 90% to 93%) in 10 mM Tris-HCl buffer (pH 8) plus 0.2 mM CaCl2 and 0.2 mL of enzyme solution at 40 °C (Wu and others 2007). One unit of PL activity is defined as an increase in absorbance of 0.005 per hour at 235 nm under the reaction condition. Microdilution assays Minimum inhibitory concentration (MIC) is defined as the Preparation of pectin hydrolysate lowest concentration of citrus oligogalacturonide to inhibit the Pectin hydrolysate was prepared according to the method de- growth of microorganisms. It was determined by serial dilutions in scribed by Huang and others (2011). Citrus pectin (1%, w/v) and nutrient broth based on a micro-well dilution method according to various pectic enzymes (CPE, PL, and PME) containing solution Zgoda and Porter (2001). Microbial growth was determined by the were incubated at pH 4, 45 °C for 0 to 24 h, heated in a boiling absorbance at 600 nm using Epoch microplate spectrophotometer water bath for 10 min, cooled to room temperature, and then (BioTek, Winooski, Vt., U.SA.) and confirmed by plating sample freeze-dried. from clear wells on nutrient agar. Determination of DE Determination of mechanisms of antibacterial effect DE values of pectin and pectin hydrolysate were determined To elucidate whether the observed antibacterial effect of citrus according to the method described by Jiang and others (2002). oligogalacturonide was bactericidal or bacteriostatic, the mechaPectin sample (10 mg) was added to 10 mL of 0.5 N KOH, nism of antibacterial effect was evaluated according to the method M1542 Journal of Food Science r Vol. 79, Nr. 8, 2014
Antibacterial citrus oligogalacturonide . . . Table 2–Antibacterial activities of penicillin, chitosan and pectin hydrolysates from various reaction time of commercial microbial pectic enzyme against food pathogens using the disc diffusion methoda . Reaction time (h) Bacterial strain
Penicillin
Chitosan
0
6
12
24
S. aureus P. aeruginosa L. monocytogenes S. Typhimurium
9.2 ± 0.1d
± ± ± ±
b
11.6 ± 0.1c 23.3 ± 0.1b 22.8 ± 0.1c 25.0 ± 0.0b
14.0 ± 0.1b 23.5 ± 0.1b 24.8 ± 0.0b 25.3 ± 0.5b
15.1 ± 0.2a 26.0 ± 0.1a 25.1 ± 0.0a 27.1 ± 0.1a
b
15.7 ± 0.1d 18.3 ± 0.1d
0 3.3 4.0 2.6
0.0e 0.0c 0.0e 0.1e
b b b
Zones of growth inhibition (mm) were measured and expressed as mean ± SD, those bearing the same inline alphabets in the same row are not significantly different (P > 0.05). The diameter of the zone of inhibition includes the paper disc (8 mm). The concentrations used for disc diffusion assay were 150 μg/mL for penicillin and pectin hydrolysates from citrus pectin and 1% for chitosan. b No zone of inhibition was observed. a
Statistical analysis Each experiment was carried out in 3 replications. Statistical comparisons were made by one-way analysis of variance followed by Duncan’s multiple range test.
Results and Discussion Antibacterial activity of pectin hydrolysate Preliminary evaluations of the antibacterial activities of the enzymatically hydrolyzed pectins from CPE, PL, and PME against the 4 selected foodborne pathogens were conducted. No inhibition zone was observed in samples treated with pectin hydrolysates from PL and PME, but inhibition was seen with the CPE-treated pectin (data not shown). CPE hydrolysis caused an increase in the antibacterial activity of pectin solution against all selected foodborne pathogens. As shown in Table 1, the Mw of pectin hydrolysates from the CPE treatment ranged from 1.0 kDa (24 h hydrolysate) to 1.8 kDa (6 h hydrolysate), normally less than 10mer; thus, the pectin hydrolysates from CPE-treated citrus pectin in this study was expressed as citrus oligogalacturonide. Antibacterial activity of oligogalacturonide from citrus pectin against foodborne pathogens was qualitatively and quantitatively assessed by the presence of an inhibition zone and the zone diameter using the disc diffusion assay and MIC value using the microdilution assay. It is obvious that citrus oligogalacturonides showed good antibacterial activity against the selected foodborne pathogens and the zones of inhibition ranged from 11.6 to 27.1 mm (Table 2). Men’shikov and others (1997) reported that pectin exhibited a bactericidal effect on pathogenic and opportunistic microorganisms, however no antibacterial activity of pectin was observed in this study. Antibacterial activity of citrus oligogalacturonide increased with increased enzyme reaction time of CPE (Table 2). In other words, digestion of citrus pectin with CPE for 24 h yielded an oligogalacturonide with 1.0 kDa and 11.6% DE, which exhibited the highest antibacterial activity against the selected foodborne pathogens. Huang and others (2012) revealed that the citrus oligogalacturonide was membrane permeable. The pectin hydrolysates from PL (causing a decrease in Mw of pectin) and PME (causing a decrease in DE of pectin) exhibited no antibacterial activity; thus,
Table 3–Antibacterial properties of citrus oligogalacturonide against food pathogens. Bacterial strain S. aureus P. aeruginosa L. monocytogenes S. Typhimurium a
MICa (μg/mL)
Mechanism of antibacterial effect
150.0 37.5 37.5 37.5
b b b b
The lowest concentration of citrus oligogalacturonide from 24-h hydrolysis. The antibacterial effect was classified as bactericidal effect.
b
we speculated that the antibacterial activity of citrus oligogalacturonide from CPE (causing a decrease in Mw and DE of pectin) was attributed to the membrane permeability of oligogalacturonide in an appropriate Mw and DE value. The citrus oligogalacturonide displayed different antibacterial activity depending on the bacteria. In general, Gram-negative bacteria are more resistant than Gram-positive bacteria (Marino and others 1999; Nasar-Abbas and Halkman 2004). The mechanism of antimicrobial substances of Gram-positive and Gram-negative bacteria is not well established (Gomes Dos Santos and others 2013; Haney and others 2011; Radulovi´c and others 2013). The antibacterial results of citrus oligogalacturonides in this study revealed a different result. S. aureus (Gram-positive) was found to be more resistant strain than Gram-negative strains tested (Table 2). There is no single antibiotic that provides coverage for all microbes (Benz and others 2004). Therefore, citrus oligogalacturonides can be used as a potential native antibiotic. All of the citrus oligogalacturonides exhibited higher antibacterial activities than penicillin at the same concentration (7.5 μg/disc). Penicillin is preferentially used in the treatment of grampositive bacterial infection as an antibiotic, however penicillin resistance is becoming more common in S. aureus (Lowy 2003), P. aeruginosa (Suginaka and others 1975), Listeria spp. (Wang and others 2012), and Salmonella Typhimurium (Schuman and others 1989). Hence, citrus oligogalacturonides might also be a good antibacterial candidate for pharmaceutical use.
Minimal inhibitory concentration of citrus oligogalacturonide The MIC of citrus oligogalacturonide from 24 h hydrolysis for inhibition of P. aeruginosa, L. monocytogenes, and Salmonella Typhimurium was 37.5 μg/mL, whereas the MIC for S. aureus was 150.0 μg/mL (Table 3). The ethanolic extracts of sumac and avishan-e shirazi inhibited S. aureus at MIC of 0.1% and 0.4%, respectively (Fazeli and others 2007); and lactoferrin and lactoferrin hydrolysate inhibited Vol. 79, Nr. 8, 2014 r Journal of Food Science M1543
M: Food Microbiology & Safety
described by Marino and others (1999). The procedure involving the contact of microorganisms to pectin hydrolysates using the disc diffusion assay was described above. Streak plates were prepared by inoculation of 5 loopfuls of the inhibition zones on fresh nutrient agar and incubating the plates at 37°C for 24 h. Samples that showed no growth after re-streaking were considered as demonstrating a bactericidal effect.
Antibacterial citrus oligogalacturonide . . .
M: Food Microbiology & Safety
S. aureus at MIC of 8 and 4 mg/mL, respectively (Murdock and Matthews 2002). The MIC of rosemary essential oil was found to be 2.3 to 3.5 mg/mL for S. aureus (Pintore and others 2002). The MICs of plant essential oils thymol and eugenol for S. aureus were found to be 5 and 8 mg/mL, respectively; and nisin inhibited S. aureus at MIC of 5 mg/mL (Panitee and Vanee 2007). Lactoferrin and lactoferrin hydrolysate inhibited L. monocytogenes at MIC of 2 and 1 mg/mL, respectively (Murdock and Matthews 2002). The MICs of plant essential oils thymol and eugenol for L. monocytogenes were found to be 4 and 11 mg/mL, respectively; and nisin that inhibited L. monocytogenes at MIC of 4 mg/mL (Panitee and Vanee 2007). The essential oil constituents, citral, geraniol, and perillaldehyde, were reported to completely kill Salmonella Typhimurium at 500 μg/mL (Kim and others 1995). The MIC of rosemary essential oil was found to be >4 mg/mL against P. aeruginosa (Pintore and others 2002). In comparison with the other antimicrobials, the MIC of citrus oligogalacturonide against S. aureus, L. monocytogenes, Salmonella Typhimurium, and P. aeruginosa ranged from 37.5 to 150.0 μg/mL, showing that the citrus oligogalacturonide possess stronger antimicrobial activity and may be a suitable antibacterial agent to inhibit pathogen growth in foods. Based on the classifications of antibacterial activity using the disc method followed by streak plate culture, no growth was observed. Hence, the citrus oligogalacturonide from 24 h hydrolysis was found to display a bactericidal effect against the selected pathogens (Table 3). Citrus oligogalacturonide with 1.0 kDa and 11.6% DE was reported to be membrane permeable (Huang and others 2012). The mechanism of antimicrobial substances includes: membrane instability and interfering the intermediary metabolisms or DNA/RNA synthesis/function (Haney and others 2011; Radulovi´c and others 2013). Therefore, the possible antimicrobial pathway of the membrane permeable citrus oligonucleotide is by interfering with intermediary metabolism or DNA/RNA synthesis/function.
Conclusions Recently, bacterial resistance against presently used antibiotics is becoming a public health issue (Kuroda and Caputo 2013). Numerous studies have focused on the discovery of new antimicrobial agents from natural sources or the design and synthesis of new agents. A lot of attention has been paid to the discovery of new antimicrobial agents from natural sources and the design and synthesis of new agents (Yilmaz and Ercisli 2011; Kuroda and Caputo 2013). Citrus oligogalacturonide was reported to exhibit antioxidant activity and antitumor activity with no cytotoxicity (Huang and others 2011, 2012). In this study, the citrus oligogalacturonide was found to exhibited good antibacterial activities and display a bactericidal effect against the selected food pathogens, including S. Typhimurium, S. aureus, L. monocytogenes, and P. aeruginosa. Therefore, citrus oligogalacturonide may have potential to be further developed as an antibacterial agent for the food industry and a novel pharmaceutical compound with cancer prevention and bacterial resistance avoiding benefits.
Acknowledgment Financial support from the Natl. Science Council of the Republic of China under Grant 98-2313-B-020-013-MY3 is greatly) appreciated.
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