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Inactivation of Enterobacter sakazakii by water-soluble muscadine seed extracts
International Journal of Food Microbiology 129 (2009) 295–299
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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j f o o d m i c r o
Inactivation of Enterobacter sakazakii by water-soluble muscadine seed extracts T.J. Kim a,⁎, J.L. Silva a, W.L. Weng a, W.W. Chen a, M. Corbitt a, Y.S. Jung b, Y.S. Chen c a b c
Department of Food Science, Nutrition and Health Promotion, Mississippi State University, Mississippi State, MS 39762, United States Department of Biochemistry and Molecular Biology, Mississippi State University, Mississippi State, MS 39762, United States Department of Research and Development, BakeMark, Pico Rivera, CA 90660, United States
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Article history: Received 23 October 2008 Received in revised form 2 December 2008 Accepted 10 December 2008 Keywords: Muscadine seed extracts Polar and polyphenol fractions Antimicrobial activity Enterobacter sakazakii Tannic acid
a b s t r a c t Hot and cold water-soluble muscadine (Vitis rotundifolia) seed extracts and their polar and polyphenol fractions from two Muscadine cultivars (‘Ison’, purple and ‘Carlos’, bronze) were investigated for their inhibition of Enterobacter sakazakii. The heat treatment on each seed extract not only increased total phenolics and tannic acid but also enhanced antimicrobial activity against two strains of E. sakazakii. Within 1 h, all seed extracts reduced an initial population (∼ 6 log CFU/mL) of E. sakazakii to a non-detectable level (minimum detection limit, 10 CFU/mL). Regardless of extraction method and cultivar, only the polar fractions which contained malic, tartaric and tannic acids showed antimicrobial activity against two strains of E. sakazakii. The polyphenol fractions which contained gallic acid, catechin, epicatechin, ellagic acid and pigments showed slight inhibition against E. sakazakii. Results showed that water-soluble muscadine seed extracts (pH 3.3–3.78) contained strong antimicrobial inhibitors against E. sakazakii while acidified peptone water (pH 3.3) did not show any antimicrobial activity. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Enterobacter sakazakii is a gram-negative, motile bacterium that is considered an emerging opportunistic pathogen. It causes meningitis and necrotizing enterocolitis in infants with high mortality rates, up to 40–80% and 10–55%, respectively (Kim et al., 2008a). Survivors often develop irreversible neurological disorders (Lai, 2001). Outbreaks of E. sakazakii have been associated with the ingestion of contaminated (less than 1 cell per 100 g) infant formula (Iversen and Forsythe, 2003). Reports that E. sakazakii has also been isolated from infant cereals raises concern about its rapid growth in infant cereals reconstituted with water, milk, or infant formula (Richards et al., 2005). Muscadine grapes (Vitis rotundifolia) are the predominant grape species in the southeastern United States. They are tolerant to hot, humid climate, and Pierce's disease. Muscadines are traditionally used as table grapes, wines, juices, jams and jellies, and U-pick operations (Ector, 2001; Threlfall et al., 2007). Recently, interest in the health benefits of muscadines has increased due to their high phenolics contents. Most phenolics in muscadines are located in the seeds (Pastrana-Bonilla et al., 2003). Gallic acid, catechin and epicatechin are the main phenolics found in muscadine seeds, while ellagic acid and myricetin are the major ones in the skins. Muscadines and their phenolics compounds have excellent antioxidant capacity (Pastrana-Bonilla et al., 2003; Lee and Talcott, 2004), anti-cancer properties (Mertens-Talcott et al., 2006), anti-inflammatory ⁎ Corresponding author. Box 9805, Department of Food Science, Nutrition, and Health Promotion, Mississippi State University, MS 39762, United States. Tel.: +1 662 325 4048; fax: +1 662 325 8728. E-mail address: [email protected] (T.J. Kim). 0168-1605/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2008.12.014
activity (Greenspan et al., 2005) and antimicrobial activity against E. coli O157:H7 (Kim et al., 2008b). It has been reported that high pressure and temperature sterilization (retort or autoclave process) could enhance antioxidant and antimicrobial capacity with liberating low molecular antioxidant and antimicrobial compounds such as ellagic acid from the repeating subunits of high molecular weight polymers (Lee et al., 2003; Ninno et al., 2005). In this study, we prepared muscadine seed extracts and their polar and polyphenol fractions from two cultivars using two different extraction methods, determined their organic acids and phenolics compositions, and then investigated their antimicrobial effects against E. sakazakii. 2. Materials and methods 2.1. Chemicals and reagents Folin–Ciocalteu reagent, sodium carbonate and standards of gallic acid (90% purity), (+)-catechin (95% purity), (−)-epicatechin (90% purity), ellagic acid (95% purity), malic acid and tannic acid were purchased from Sigma (St. Louis, MO). Tartaric acid, acetic acid, hydrochloric acid, sulfuric acid, acetonitrile, methanol, and HPLC grade water were purchased from Fisher Scientific (Pittsburgh, PA). 2.2. Seed extract preparation The defatted ‘Carlos’ (bronze) and ‘Ison’ (purple) muscadine seed powders were provided by the Mississippi State University Truck Crops Branch Experiment Station (Crystal Springs, MS). Both seed powders were kept at −20 °C in the dark until further analysis. To
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Table 1 pH, total phenolics, organic acids and major phenolic compounds of hot and cold water-soluble extracts from Ison and Carlos muscadine seed pH
Total phenolics
Tartaric acid
Malic acid
6.6.a 7.27a 4.17b 4.3bb
1.33b 1.87a 0.97c 1.43b
Tannic acid
Gallic acid
4.3b 7.1a 1.63d 3.63c
0.26ab 0.35a 0.11b 0.20a
Catechin
Epicatechin
Ellagic acid
0.05c 0.10a 0.05bc 0.07b
0.09b 0.17a 0.02c 0.05bc
mg/mL Polar IC IH CC CH
3.37c 3.30c 3.78a 3.69b
1.67c⁎ 2.78a 1.38d 2.08b
Non-polar 0.11a 0.18a 0.07a 0.12a
CC: Carlos cold extract, CH: Carlos hot extract, IC: Ison cold extract, IH; Ison hot extract. ⁎Mean values within (n = 3) column having the same letters are not different (p ≥ 0.05).
prepare the hot extract with ‘Carlos’ (CH) and ‘Ison’ (IH) muscadine seeds by the autoclave process, the mixture of each seed powder and water (1:5, w/v) was autoclaved (121 °C, 103.4 kPa) for 15 min, mixed in a rotary mixer (Dynal® Inc., NY) at 40 rpm for 1 h at room temperature (∼ 25 °C), and centrifuged at 16,000 ×g for 5 min using an Eppendorf model 5414 microcentrifuge (Eppendorf, Brinkmann Instruments, NY). The supernatant was then filtered through a 0.20 μm syringe filter (Millipore, Bedford, MA). The cold-water extracts from ‘Carlos’ (CC) and ‘Ison’ (IC) muscadine seeds were prepared as described above, except for the autoclave step. 2.3. Total phenolics and pH
2.5. Antimicrobial activity on seed extracts E. sakazakii Fec39 and E. sakazakii MSDH were kindly provided by Dr. Yoshen Chen (Department of Food Science and Technology, Mississippi State University). These strains were clinically isolated by Dr. Ute Römling (Microbiology and Tumorbiology Center, Sweden) (Zogaj et al., 2003) and Mississippi State Department of Health, USA. Stock cultures were maintained at −65 °C in tryptic soy broth containing 10% glycerol. The cultures were thawed and reactivated by subculture in tryptic soy broth. Reactivated cells were stored on tryptic soy agar slant. The strains were separately cultured in tryptic soy broth (Becton Dickinson, Sparks, MD) with shaking at 150 rpm for 16 h at 37 °C (Incubator Shaker, New Brunswick, NJ). After incubation,
The pH of muscadine seed extract was measured using a pH meter (Corning Pinnacle 530; St. Louis, MO) at room temperature (∼25 °C). Total phenolics were determined according to the Folin–Ciocalteu procedure (Waterhouse, 2001). Properly diluted samples were mixed with Folin–Ciocalteu reagent and sodium carbonate and allowed to stand for 2 h. Absorption at 765 nm was measured in a Spectronic genesys 5 UV–vis spectrophotometer (Fisher Scientific, Pittsburgh, PA). Results were expressed as milligrams of gallic acid equivalents per milliliter of seed extract. 2.4. High-performance liquid chromatographic (HPLC) analysis for phenolics and organic acids Reversed-phase HPLC was used to separate and quantify major phenolic compounds and organic acids in muscadine seed extract. Extract samples were mixed with 6 N HCl at 1:10 ratio (v/v) and placed in a water bath at 95 °C for 1 h to separate the free phenolic acids from their conjugated forms (Lee, 2000). After cooling down to room temperature (about 15 min), each supernatant of acid hydrolyzed sample was filtered through a 0.45 μm syringe filter (Millipore, Bedford, MA) and injected into a Gemini C18 column (250 × 4.6 mm, Phenomenex®, Torrance, CA) in an Agilent HPLC 1100 series (Agilent Technologies, Santa Clara, CA) equipped with a diode array detector. The volume of sample injected was 25 μL. Mobile phases consisted of 10% methanol/2% acetic acid in water (solvent A), and 100% acetonitrile (solvent B). A linear gradient for phenolics separation was used as follows: at 0 min, 95% solvent A, 5% solvent B; at 1 min, 90% solvent A, 10% solvent B; at 30 min, 30% solvent A, 70% solvent B; at 31 min, 90% solvent A, 10% solvent B; at 32 min, 95% solvent A, 5% solvent B with 5 min post run. Flow rate was 1 mL/min. Individual phenolics were detected at 260 nm. To separate tartaric, malic and tannic acids, muscadine seed extracts were centrifuged at 16,000 ×g for 5 min, and each supernatant was filtered and injected into a HPLC system as described previously. Mobile phase was 0.01 N H2SO4 with a flow rate of 1 mL/min. Individual organic acids were detected at 215 nm (Kim et al., 2008b). Peaks for phenolic compounds and organic acids were integrated and analyzed using ChemStation software (Agilent Technologies). Individual compounds were identified based on the retention time of the standards and their quantification was made by calibration curves built for each standard of analyzed compounds.
Fig. 1. Antimicrobial effect of hot (CH: Carlos hot extract, IH; Ison hot extract) and cold water-soluble extracts (CC: Carlos cold extract, IC: Ison cold extract) from Ison and Carlos muscadine seed against Enterobacter sakazakii Fec39 (A) and E. sakazakii MSDH (B). CO: acidified peptone water (pH 3.3).
T.J. Kim et al. / International Journal of Food Microbiology 129 (2009) 295–299
the bacterial cultures were added in muscadine seed extract to create a population of 6 log CFU/mL. The mixture was incubated at 37 °C, and the viable cells were recorded every 30 min, for up to 120 min. Acidified peptone water (pH 3.3) was used as a control. Viable E. sakazakii was enumerated on tryptic soy agar plates (Becton Dickinson, Sparks, MD) after incubation at 37 °C for 18 to 24 h. After 120-min incubation of E. sakazakii in water-soluble seed extracts, inoculated extract samples (100 μL) were transferred to TSB and then incubated at 37 °C for 24 h to see any recovery of E. sakazakii cells. 2.6. Antimicrobial activity on polar and polyphenol fractions separated from seed extracts Solid-phase extraction (SPE) was performed according to the methods of Nohynek et al. (2006) and Daglia et al. (2007) with minor modifications. The C18 SPE cartridge (SEP-PAK®, Waters, Millipore Corp. Milford, Mass, USA) was pre-washed with methanol (2 mL) and distilled water (2 mL). Five milliliter of water-soluble ‘Ison’ and ‘Carlos’ extracts with or without the autoclave process loaded onto the cartridge. The sample was slowly passed through the cartridge and then eluted with distilled water (0.5 mL) to collect the polar substances (CC1, CH1, IC1 and IH1). The residual water in the cartridge was removed by pushing air through the cartridge. Five milliliters of methanol was loaded into the cartridge to collect the polyphenol substances (CC2, CH2, IC2 and IH2). Methanol in the polyphenol fraction was completely dried on the vacuum drier at 45 °C and reconstituted with mixture (5 mL) of water and dimethyl sulfoxide (Sigma, St. Louis, MO) (1:50 v/v). Antimicrobial activities of each fraction were similarly measured as described above.
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2.7. Statistical design and analysis A completely randomized design with three replications was used for measurement of pH, total phenolics, organic acids and phenolic profiles. A two-way factorial design (treatments × time) with at least three replications in CRD (completely randomized design) were set for measurement of antimicrobial activities. Analysis of variance was performed using mean log microbial population data to determine differences between treatments. Analysis of variance and means were computed using the Statistical Analysis System (SAS, 2001). Fisher's Least Significant Difference test (P ≤ 0.05) was used to determine differences between treatment means. 3. Results 3.1. pH and total phenolics, organic acids, phenolic acids and polyphenol compounds in seed extracts Cold (IC) and hot (IH) extracts from ‘Ison’ differed (P ≤ 0.05) in total phenolics (1.67 and 2.78 mg/mL), malic acid (1.33 and 1.87 mg/mL), tannic acid (4.3 and 7.1 mg/mL), epicatechin (0.05 and 0.1 mg/mL) and ellagic acid (0.09 and 0.17 mg/mL), while cold (CC) and hot (CH) extracts from ‘Carlos’ differed (P ≤ 0.05) in pH values (3.78 and 3.69), total phenolics (1.38 and 2.08 mg/mL), malic acid (0.97 and 1.43 mg/mL), tannic acid (1.63 and 3.63 mg/mL) and gallic acid (0.11 and 0.2 mg/mL) (Table 1). In all extracts, tartaric and malic acids were the major aliphatic organic acids, while tannic acid had the highest concentration among detected phenolic acids (Table 1). Heat treatment generally increased
Fig. 2. Antimicrobial effect of polar (Carlos cold polar, CC1, pH 3.8; Carlos hot polar, CH1, pH 3.7; Ison cold polar, IC1, pH 3.4; Ison hot polar, IH1, pH3.3) and polyphenol fractions (Carlos cold polyphenol, CC2, pH 3.8; Carlos hot polyphenol, CH2, pH 3.7; Ison cold polyphenol, IC2, pH3.4; Ison hot polyphenol, IH2, pH 3.3) from muscadine seed extracts against Enterobacter sakazakii Fec39 (A and C) and E. sakazakii MSDH (B and D).
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concentration of total phenolics, organic acids and phenolic compounds, regardless of cultivar (Table 1). 3.2. Antimicrobial activity of muscadine seed extracts against E. sakazakii The hot ‘Ison’ (IH) and “Carlos” (CH) extracts reduced (P ≤ 0.05) the number of cells of E. sakazakii Fec39 and E. sakazakii MSDH to nearly undetectable levels (∼ 6 log CFU/mL) after 30 min and completely after 60 min (minimum detection limit, 10 CFU/mL). Furthermore, no recovered cells were found in both seed extracts after incubating samples (100 μL) in TSB for 24 h. There were no significant differences (P ≥ 0.05) between IH and CH, or IC and CC on both strains for 30 min. Regardless of strains heat treatment (CC, IC) significantly increased antimicrobial activity of IH and CH in the same time of period (Fig. 1). The acidified control (CO, pH 3.3) did not have any effect on either of strains (Fig. 1). Our results showed that there was not much difference in antimicrobial resistance between the two strains, although they are totally different isolates. 3.3. Antimicrobial activity on polar and polyphenol fractions against E. sakazakii Using SPE (C18), we separated polar (CC1, CH1, IC1 and IH1) and polyphenol fractions (CC2, CH2, IC2 and IH2) from ‘Ison’ and ‘Carlos’ muscadine seed extracts. To determine the antimicrobial effects of each fraction, E. sakazakii were inoculated in individual polar and polyphenol fractions. Regardless of cultivar, all polar fractions of hot extracts reduced E. sakazakii by N5 log CFU/mL (P ≤ 0.05) within 60 min (Fig. 2A and B) while the polyphenol fractions were slightly inhibitory (P ≤ 0.05) to E. sakazakii in the same time period (Fig. 2C and D). This suggests that anti-E. sakazakii activity in seed extracts was mainly due to extracts' polar substances such as tartaric, malic and tannic acids. 4. Discussion Several studies have suggested that post-harvest processing can potentially increase antioxidant capacity of processed products by increasing bioactive substances such as phenolics and organic acids. Lee et al. (2003) showed that far-infrared radiation on rice hulls increased total phenolic contents and antioxidant capacity, increasing flavonoids, tannin, or polyphenols from repeating polymers bound in natural plants. Simple heat treatment also increased individual phenolic compounds and the antioxidant activity on extracts from citrus peels (Jeong et al., 2004). The level of water-soluble ellagic acid could be increased by heat-sterilization such as retort sterilization or the ultra-high temperature processing of muscadine seed (Kim et al., 2008b; Ninno et al., 2005). Stojanovic and Silva (2007) showed an increase in phenolic compounds and antioxidants in processed blueberries. In the present study, heat processing of muscadine seeds in water, regardless of cultivar, increased organic acids, total phenolics or individual phenolic compounds. While the acidified control with dimethyl sulfoxide did not show any anti-E. sakazakii effect (Fig. 1A and B), there was enhanced antimicrobial activity in IH1 at the same pH (3.3) (Fig. 2A and B). High content of bioactive compounds in heat-treated seed extracts is likely due to not only the better extraction of organic compounds from the seeds by heat treatment, but also thermal degradation of the repeating subunits of high molecular polymers during heat processing, releasing low molecular polar compounds. The antibacterial action of grapes and their by-products has been previously reported (Jayaprakasha et al., 2003; Özkan et al., 2004; Puupponen-Pimiä et al., 2005; Rhodes et al., 2006). Rhodes et al. (2006) reported that grape seed extract showed a 5-log reduction against Listeria monocytogenes in within 60 min but no reduction
against E. coli and concluded that the unpigmented polymeric phenolics showed antilisterial activity. Jayaprakasha et al. (2003) also reported that Gram-positive bacteria were more sensitive on grape seed extracts than Gram-negative bacteria. In all samples, the polar fraction which contained organic acids and tannic acid (data not shown) showed much stronger antimicrobial activity against E. sakazakii than polyphenol fractions which was bound and eluted with methanol. The latter fraction mainly contained gallic acid, catechin, epicatechin and pigments. This suggests that the major antimicrobial activity in water-soluble seed extracts is derived from organic acids and tannic acid. Nevertheless, the polar fraction was not as active as its original whole extract in inactivating the E. sakazakii. Kim and Beuchat (2005) reported that low initial pH values in apple and strawberry juices play a major role in preventing growth of E. sakazakii at room temperature. However, E. sakazakii can survive below pH 4.0 showing that the predominant acids (citric and malic acids) of strawberry and apple juices did not inactivate the low inoculum numbers (∼ 1.5 log CFU/mL) of E. sakazakii for up to 48 and 168 h, respectively. Edelson-Mammel et al. (2006) also reported that 12 strains of E. sakazakii showed pH resistance at pH 3.5 up to 5 h. Kim et al. (2008b) showed that even though there was a high concentration of tartaric acid in red muscadine seed extracts, tartaric acid alone did not inactivate E. coli O157:H7 as much as the whole seed extracts. This suggests that the low pH associated with weak organic acids cannot explain all the antimicrobial activity of muscadine seed extracts. Ellagitannins and gallotannins (esters of gallic acid and glucose) in muscadine seed extracts are two major groups of hydrolysable tannins, commonly referred to as tannic acid (Akiyama et al., 2001) which could show anti-E. sakazakii activity in muscadine seed extracts and their polar fractions. Tannins have been reported to inhibit a wide range of fungi, yeasts, viruses and foodborne bacteria, including Enterobacter aerogenes, Escherichia coli, Listeria monocytogenes, Salmonella enteritidis, S. paratyphi, Shigella flexneri and Stapylococcus aureus (Chung et al., 1998). Moreover, Akiyama et al. (2001) reported that tannic acid in the concentration range of (0.25–1 mg/ mL) showed a strong inhibition of S. aureus on Mueller-Hinton agar. Thus, high concentration of tannic acid in muscadine seed extracts and their polar fractions could be also responsible for the antimicrobial activity against E. sakazakii. The inhibitory effect of tannic acid to microorganisms may be owed to their strong binding capacity to iron ion that is critical for the survival of most bacteria (Chung et al., 1998) or the formation of complexes with essential proteins or polysaccharides through nonspecific binding (Cowan, 1999). In conclusion, muscadine seed extracts are rich sources of phenolic compounds and organic acids and demonstrate a strong antimicrobial effect against E. sakazakii. The malic, tartaric and tannic acids found in the seed extracts might play a key role in the observed antimicrobial effect. These results suggest that muscadine seed extracts can be used as natural antimicrobial agents against E. sakazakii. Acknowledgement Approved for publication as Journal Article No. J-11501 of the Mississippi Agricultural and Forestry Experiment Station, Mississippi State University. This work was supported in part by the Mississippi Agricultural and Forestry Experiment Station Project Number MIS371272 and by USDA-ARS Grant No. 58-6402-6-075. References Akiyama, H., Fujii, K., Yamasaki, O., Oono, T., Iwatsuki, K., 2001. Antibacterial action of several tannins against Staphylococcus aureus. The Journal of Antimicrobial Chemotherapy 48, 487–491. Chung, K.T., Lu, Z., Chou, M.W., 1998. Mechanism of inhibition of tannic acid and related compounds on the growth of intestinal bacteria. Food and Chemical Toxicology 36, 1053–1060. Cowan, M.M., 1999. Plant products as antimicrobial agents. Clinical Microbiology Reviews 12, 564–582.
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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j f o o d m i c r o
Inactivation of Enterobacter sakazakii by water-soluble muscadine seed extracts T.J. Kim a,⁎, J.L. Silva a, W.L. Weng a, W.W. Chen a, M. Corbitt a, Y.S. Jung b, Y.S. Chen c a b c
Department of Food Science, Nutrition and Health Promotion, Mississippi State University, Mississippi State, MS 39762, United States Department of Biochemistry and Molecular Biology, Mississippi State University, Mississippi State, MS 39762, United States Department of Research and Development, BakeMark, Pico Rivera, CA 90660, United States
a r t i c l e
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Article history: Received 23 October 2008 Received in revised form 2 December 2008 Accepted 10 December 2008 Keywords: Muscadine seed extracts Polar and polyphenol fractions Antimicrobial activity Enterobacter sakazakii Tannic acid
a b s t r a c t Hot and cold water-soluble muscadine (Vitis rotundifolia) seed extracts and their polar and polyphenol fractions from two Muscadine cultivars (‘Ison’, purple and ‘Carlos’, bronze) were investigated for their inhibition of Enterobacter sakazakii. The heat treatment on each seed extract not only increased total phenolics and tannic acid but also enhanced antimicrobial activity against two strains of E. sakazakii. Within 1 h, all seed extracts reduced an initial population (∼ 6 log CFU/mL) of E. sakazakii to a non-detectable level (minimum detection limit, 10 CFU/mL). Regardless of extraction method and cultivar, only the polar fractions which contained malic, tartaric and tannic acids showed antimicrobial activity against two strains of E. sakazakii. The polyphenol fractions which contained gallic acid, catechin, epicatechin, ellagic acid and pigments showed slight inhibition against E. sakazakii. Results showed that water-soluble muscadine seed extracts (pH 3.3–3.78) contained strong antimicrobial inhibitors against E. sakazakii while acidified peptone water (pH 3.3) did not show any antimicrobial activity. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Enterobacter sakazakii is a gram-negative, motile bacterium that is considered an emerging opportunistic pathogen. It causes meningitis and necrotizing enterocolitis in infants with high mortality rates, up to 40–80% and 10–55%, respectively (Kim et al., 2008a). Survivors often develop irreversible neurological disorders (Lai, 2001). Outbreaks of E. sakazakii have been associated with the ingestion of contaminated (less than 1 cell per 100 g) infant formula (Iversen and Forsythe, 2003). Reports that E. sakazakii has also been isolated from infant cereals raises concern about its rapid growth in infant cereals reconstituted with water, milk, or infant formula (Richards et al., 2005). Muscadine grapes (Vitis rotundifolia) are the predominant grape species in the southeastern United States. They are tolerant to hot, humid climate, and Pierce's disease. Muscadines are traditionally used as table grapes, wines, juices, jams and jellies, and U-pick operations (Ector, 2001; Threlfall et al., 2007). Recently, interest in the health benefits of muscadines has increased due to their high phenolics contents. Most phenolics in muscadines are located in the seeds (Pastrana-Bonilla et al., 2003). Gallic acid, catechin and epicatechin are the main phenolics found in muscadine seeds, while ellagic acid and myricetin are the major ones in the skins. Muscadines and their phenolics compounds have excellent antioxidant capacity (Pastrana-Bonilla et al., 2003; Lee and Talcott, 2004), anti-cancer properties (Mertens-Talcott et al., 2006), anti-inflammatory ⁎ Corresponding author. Box 9805, Department of Food Science, Nutrition, and Health Promotion, Mississippi State University, MS 39762, United States. Tel.: +1 662 325 4048; fax: +1 662 325 8728. E-mail address: [email protected] (T.J. Kim). 0168-1605/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2008.12.014
activity (Greenspan et al., 2005) and antimicrobial activity against E. coli O157:H7 (Kim et al., 2008b). It has been reported that high pressure and temperature sterilization (retort or autoclave process) could enhance antioxidant and antimicrobial capacity with liberating low molecular antioxidant and antimicrobial compounds such as ellagic acid from the repeating subunits of high molecular weight polymers (Lee et al., 2003; Ninno et al., 2005). In this study, we prepared muscadine seed extracts and their polar and polyphenol fractions from two cultivars using two different extraction methods, determined their organic acids and phenolics compositions, and then investigated their antimicrobial effects against E. sakazakii. 2. Materials and methods 2.1. Chemicals and reagents Folin–Ciocalteu reagent, sodium carbonate and standards of gallic acid (90% purity), (+)-catechin (95% purity), (−)-epicatechin (90% purity), ellagic acid (95% purity), malic acid and tannic acid were purchased from Sigma (St. Louis, MO). Tartaric acid, acetic acid, hydrochloric acid, sulfuric acid, acetonitrile, methanol, and HPLC grade water were purchased from Fisher Scientific (Pittsburgh, PA). 2.2. Seed extract preparation The defatted ‘Carlos’ (bronze) and ‘Ison’ (purple) muscadine seed powders were provided by the Mississippi State University Truck Crops Branch Experiment Station (Crystal Springs, MS). Both seed powders were kept at −20 °C in the dark until further analysis. To
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Table 1 pH, total phenolics, organic acids and major phenolic compounds of hot and cold water-soluble extracts from Ison and Carlos muscadine seed pH
Total phenolics
Tartaric acid
Malic acid
6.6.a 7.27a 4.17b 4.3bb
1.33b 1.87a 0.97c 1.43b
Tannic acid
Gallic acid
4.3b 7.1a 1.63d 3.63c
0.26ab 0.35a 0.11b 0.20a
Catechin
Epicatechin
Ellagic acid
0.05c 0.10a 0.05bc 0.07b
0.09b 0.17a 0.02c 0.05bc
mg/mL Polar IC IH CC CH
3.37c 3.30c 3.78a 3.69b
1.67c⁎ 2.78a 1.38d 2.08b
Non-polar 0.11a 0.18a 0.07a 0.12a
CC: Carlos cold extract, CH: Carlos hot extract, IC: Ison cold extract, IH; Ison hot extract. ⁎Mean values within (n = 3) column having the same letters are not different (p ≥ 0.05).
prepare the hot extract with ‘Carlos’ (CH) and ‘Ison’ (IH) muscadine seeds by the autoclave process, the mixture of each seed powder and water (1:5, w/v) was autoclaved (121 °C, 103.4 kPa) for 15 min, mixed in a rotary mixer (Dynal® Inc., NY) at 40 rpm for 1 h at room temperature (∼ 25 °C), and centrifuged at 16,000 ×g for 5 min using an Eppendorf model 5414 microcentrifuge (Eppendorf, Brinkmann Instruments, NY). The supernatant was then filtered through a 0.20 μm syringe filter (Millipore, Bedford, MA). The cold-water extracts from ‘Carlos’ (CC) and ‘Ison’ (IC) muscadine seeds were prepared as described above, except for the autoclave step. 2.3. Total phenolics and pH
2.5. Antimicrobial activity on seed extracts E. sakazakii Fec39 and E. sakazakii MSDH were kindly provided by Dr. Yoshen Chen (Department of Food Science and Technology, Mississippi State University). These strains were clinically isolated by Dr. Ute Römling (Microbiology and Tumorbiology Center, Sweden) (Zogaj et al., 2003) and Mississippi State Department of Health, USA. Stock cultures were maintained at −65 °C in tryptic soy broth containing 10% glycerol. The cultures were thawed and reactivated by subculture in tryptic soy broth. Reactivated cells were stored on tryptic soy agar slant. The strains were separately cultured in tryptic soy broth (Becton Dickinson, Sparks, MD) with shaking at 150 rpm for 16 h at 37 °C (Incubator Shaker, New Brunswick, NJ). After incubation,
The pH of muscadine seed extract was measured using a pH meter (Corning Pinnacle 530; St. Louis, MO) at room temperature (∼25 °C). Total phenolics were determined according to the Folin–Ciocalteu procedure (Waterhouse, 2001). Properly diluted samples were mixed with Folin–Ciocalteu reagent and sodium carbonate and allowed to stand for 2 h. Absorption at 765 nm was measured in a Spectronic genesys 5 UV–vis spectrophotometer (Fisher Scientific, Pittsburgh, PA). Results were expressed as milligrams of gallic acid equivalents per milliliter of seed extract. 2.4. High-performance liquid chromatographic (HPLC) analysis for phenolics and organic acids Reversed-phase HPLC was used to separate and quantify major phenolic compounds and organic acids in muscadine seed extract. Extract samples were mixed with 6 N HCl at 1:10 ratio (v/v) and placed in a water bath at 95 °C for 1 h to separate the free phenolic acids from their conjugated forms (Lee, 2000). After cooling down to room temperature (about 15 min), each supernatant of acid hydrolyzed sample was filtered through a 0.45 μm syringe filter (Millipore, Bedford, MA) and injected into a Gemini C18 column (250 × 4.6 mm, Phenomenex®, Torrance, CA) in an Agilent HPLC 1100 series (Agilent Technologies, Santa Clara, CA) equipped with a diode array detector. The volume of sample injected was 25 μL. Mobile phases consisted of 10% methanol/2% acetic acid in water (solvent A), and 100% acetonitrile (solvent B). A linear gradient for phenolics separation was used as follows: at 0 min, 95% solvent A, 5% solvent B; at 1 min, 90% solvent A, 10% solvent B; at 30 min, 30% solvent A, 70% solvent B; at 31 min, 90% solvent A, 10% solvent B; at 32 min, 95% solvent A, 5% solvent B with 5 min post run. Flow rate was 1 mL/min. Individual phenolics were detected at 260 nm. To separate tartaric, malic and tannic acids, muscadine seed extracts were centrifuged at 16,000 ×g for 5 min, and each supernatant was filtered and injected into a HPLC system as described previously. Mobile phase was 0.01 N H2SO4 with a flow rate of 1 mL/min. Individual organic acids were detected at 215 nm (Kim et al., 2008b). Peaks for phenolic compounds and organic acids were integrated and analyzed using ChemStation software (Agilent Technologies). Individual compounds were identified based on the retention time of the standards and their quantification was made by calibration curves built for each standard of analyzed compounds.
Fig. 1. Antimicrobial effect of hot (CH: Carlos hot extract, IH; Ison hot extract) and cold water-soluble extracts (CC: Carlos cold extract, IC: Ison cold extract) from Ison and Carlos muscadine seed against Enterobacter sakazakii Fec39 (A) and E. sakazakii MSDH (B). CO: acidified peptone water (pH 3.3).
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the bacterial cultures were added in muscadine seed extract to create a population of 6 log CFU/mL. The mixture was incubated at 37 °C, and the viable cells were recorded every 30 min, for up to 120 min. Acidified peptone water (pH 3.3) was used as a control. Viable E. sakazakii was enumerated on tryptic soy agar plates (Becton Dickinson, Sparks, MD) after incubation at 37 °C for 18 to 24 h. After 120-min incubation of E. sakazakii in water-soluble seed extracts, inoculated extract samples (100 μL) were transferred to TSB and then incubated at 37 °C for 24 h to see any recovery of E. sakazakii cells. 2.6. Antimicrobial activity on polar and polyphenol fractions separated from seed extracts Solid-phase extraction (SPE) was performed according to the methods of Nohynek et al. (2006) and Daglia et al. (2007) with minor modifications. The C18 SPE cartridge (SEP-PAK®, Waters, Millipore Corp. Milford, Mass, USA) was pre-washed with methanol (2 mL) and distilled water (2 mL). Five milliliter of water-soluble ‘Ison’ and ‘Carlos’ extracts with or without the autoclave process loaded onto the cartridge. The sample was slowly passed through the cartridge and then eluted with distilled water (0.5 mL) to collect the polar substances (CC1, CH1, IC1 and IH1). The residual water in the cartridge was removed by pushing air through the cartridge. Five milliliters of methanol was loaded into the cartridge to collect the polyphenol substances (CC2, CH2, IC2 and IH2). Methanol in the polyphenol fraction was completely dried on the vacuum drier at 45 °C and reconstituted with mixture (5 mL) of water and dimethyl sulfoxide (Sigma, St. Louis, MO) (1:50 v/v). Antimicrobial activities of each fraction were similarly measured as described above.
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2.7. Statistical design and analysis A completely randomized design with three replications was used for measurement of pH, total phenolics, organic acids and phenolic profiles. A two-way factorial design (treatments × time) with at least three replications in CRD (completely randomized design) were set for measurement of antimicrobial activities. Analysis of variance was performed using mean log microbial population data to determine differences between treatments. Analysis of variance and means were computed using the Statistical Analysis System (SAS, 2001). Fisher's Least Significant Difference test (P ≤ 0.05) was used to determine differences between treatment means. 3. Results 3.1. pH and total phenolics, organic acids, phenolic acids and polyphenol compounds in seed extracts Cold (IC) and hot (IH) extracts from ‘Ison’ differed (P ≤ 0.05) in total phenolics (1.67 and 2.78 mg/mL), malic acid (1.33 and 1.87 mg/mL), tannic acid (4.3 and 7.1 mg/mL), epicatechin (0.05 and 0.1 mg/mL) and ellagic acid (0.09 and 0.17 mg/mL), while cold (CC) and hot (CH) extracts from ‘Carlos’ differed (P ≤ 0.05) in pH values (3.78 and 3.69), total phenolics (1.38 and 2.08 mg/mL), malic acid (0.97 and 1.43 mg/mL), tannic acid (1.63 and 3.63 mg/mL) and gallic acid (0.11 and 0.2 mg/mL) (Table 1). In all extracts, tartaric and malic acids were the major aliphatic organic acids, while tannic acid had the highest concentration among detected phenolic acids (Table 1). Heat treatment generally increased
Fig. 2. Antimicrobial effect of polar (Carlos cold polar, CC1, pH 3.8; Carlos hot polar, CH1, pH 3.7; Ison cold polar, IC1, pH 3.4; Ison hot polar, IH1, pH3.3) and polyphenol fractions (Carlos cold polyphenol, CC2, pH 3.8; Carlos hot polyphenol, CH2, pH 3.7; Ison cold polyphenol, IC2, pH3.4; Ison hot polyphenol, IH2, pH 3.3) from muscadine seed extracts against Enterobacter sakazakii Fec39 (A and C) and E. sakazakii MSDH (B and D).
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concentration of total phenolics, organic acids and phenolic compounds, regardless of cultivar (Table 1). 3.2. Antimicrobial activity of muscadine seed extracts against E. sakazakii The hot ‘Ison’ (IH) and “Carlos” (CH) extracts reduced (P ≤ 0.05) the number of cells of E. sakazakii Fec39 and E. sakazakii MSDH to nearly undetectable levels (∼ 6 log CFU/mL) after 30 min and completely after 60 min (minimum detection limit, 10 CFU/mL). Furthermore, no recovered cells were found in both seed extracts after incubating samples (100 μL) in TSB for 24 h. There were no significant differences (P ≥ 0.05) between IH and CH, or IC and CC on both strains for 30 min. Regardless of strains heat treatment (CC, IC) significantly increased antimicrobial activity of IH and CH in the same time of period (Fig. 1). The acidified control (CO, pH 3.3) did not have any effect on either of strains (Fig. 1). Our results showed that there was not much difference in antimicrobial resistance between the two strains, although they are totally different isolates. 3.3. Antimicrobial activity on polar and polyphenol fractions against E. sakazakii Using SPE (C18), we separated polar (CC1, CH1, IC1 and IH1) and polyphenol fractions (CC2, CH2, IC2 and IH2) from ‘Ison’ and ‘Carlos’ muscadine seed extracts. To determine the antimicrobial effects of each fraction, E. sakazakii were inoculated in individual polar and polyphenol fractions. Regardless of cultivar, all polar fractions of hot extracts reduced E. sakazakii by N5 log CFU/mL (P ≤ 0.05) within 60 min (Fig. 2A and B) while the polyphenol fractions were slightly inhibitory (P ≤ 0.05) to E. sakazakii in the same time period (Fig. 2C and D). This suggests that anti-E. sakazakii activity in seed extracts was mainly due to extracts' polar substances such as tartaric, malic and tannic acids. 4. Discussion Several studies have suggested that post-harvest processing can potentially increase antioxidant capacity of processed products by increasing bioactive substances such as phenolics and organic acids. Lee et al. (2003) showed that far-infrared radiation on rice hulls increased total phenolic contents and antioxidant capacity, increasing flavonoids, tannin, or polyphenols from repeating polymers bound in natural plants. Simple heat treatment also increased individual phenolic compounds and the antioxidant activity on extracts from citrus peels (Jeong et al., 2004). The level of water-soluble ellagic acid could be increased by heat-sterilization such as retort sterilization or the ultra-high temperature processing of muscadine seed (Kim et al., 2008b; Ninno et al., 2005). Stojanovic and Silva (2007) showed an increase in phenolic compounds and antioxidants in processed blueberries. In the present study, heat processing of muscadine seeds in water, regardless of cultivar, increased organic acids, total phenolics or individual phenolic compounds. While the acidified control with dimethyl sulfoxide did not show any anti-E. sakazakii effect (Fig. 1A and B), there was enhanced antimicrobial activity in IH1 at the same pH (3.3) (Fig. 2A and B). High content of bioactive compounds in heat-treated seed extracts is likely due to not only the better extraction of organic compounds from the seeds by heat treatment, but also thermal degradation of the repeating subunits of high molecular polymers during heat processing, releasing low molecular polar compounds. The antibacterial action of grapes and their by-products has been previously reported (Jayaprakasha et al., 2003; Özkan et al., 2004; Puupponen-Pimiä et al., 2005; Rhodes et al., 2006). Rhodes et al. (2006) reported that grape seed extract showed a 5-log reduction against Listeria monocytogenes in within 60 min but no reduction
against E. coli and concluded that the unpigmented polymeric phenolics showed antilisterial activity. Jayaprakasha et al. (2003) also reported that Gram-positive bacteria were more sensitive on grape seed extracts than Gram-negative bacteria. In all samples, the polar fraction which contained organic acids and tannic acid (data not shown) showed much stronger antimicrobial activity against E. sakazakii than polyphenol fractions which was bound and eluted with methanol. The latter fraction mainly contained gallic acid, catechin, epicatechin and pigments. This suggests that the major antimicrobial activity in water-soluble seed extracts is derived from organic acids and tannic acid. Nevertheless, the polar fraction was not as active as its original whole extract in inactivating the E. sakazakii. Kim and Beuchat (2005) reported that low initial pH values in apple and strawberry juices play a major role in preventing growth of E. sakazakii at room temperature. However, E. sakazakii can survive below pH 4.0 showing that the predominant acids (citric and malic acids) of strawberry and apple juices did not inactivate the low inoculum numbers (∼ 1.5 log CFU/mL) of E. sakazakii for up to 48 and 168 h, respectively. Edelson-Mammel et al. (2006) also reported that 12 strains of E. sakazakii showed pH resistance at pH 3.5 up to 5 h. Kim et al. (2008b) showed that even though there was a high concentration of tartaric acid in red muscadine seed extracts, tartaric acid alone did not inactivate E. coli O157:H7 as much as the whole seed extracts. This suggests that the low pH associated with weak organic acids cannot explain all the antimicrobial activity of muscadine seed extracts. Ellagitannins and gallotannins (esters of gallic acid and glucose) in muscadine seed extracts are two major groups of hydrolysable tannins, commonly referred to as tannic acid (Akiyama et al., 2001) which could show anti-E. sakazakii activity in muscadine seed extracts and their polar fractions. Tannins have been reported to inhibit a wide range of fungi, yeasts, viruses and foodborne bacteria, including Enterobacter aerogenes, Escherichia coli, Listeria monocytogenes, Salmonella enteritidis, S. paratyphi, Shigella flexneri and Stapylococcus aureus (Chung et al., 1998). Moreover, Akiyama et al. (2001) reported that tannic acid in the concentration range of (0.25–1 mg/ mL) showed a strong inhibition of S. aureus on Mueller-Hinton agar. Thus, high concentration of tannic acid in muscadine seed extracts and their polar fractions could be also responsible for the antimicrobial activity against E. sakazakii. The inhibitory effect of tannic acid to microorganisms may be owed to their strong binding capacity to iron ion that is critical for the survival of most bacteria (Chung et al., 1998) or the formation of complexes with essential proteins or polysaccharides through nonspecific binding (Cowan, 1999). In conclusion, muscadine seed extracts are rich sources of phenolic compounds and organic acids and demonstrate a strong antimicrobial effect against E. sakazakii. The malic, tartaric and tannic acids found in the seed extracts might play a key role in the observed antimicrobial effect. These results suggest that muscadine seed extracts can be used as natural antimicrobial agents against E. sakazakii. Acknowledgement Approved for publication as Journal Article No. J-11501 of the Mississippi Agricultural and Forestry Experiment Station, Mississippi State University. This work was supported in part by the Mississippi Agricultural and Forestry Experiment Station Project Number MIS371272 and by USDA-ARS Grant No. 58-6402-6-075. References Akiyama, H., Fujii, K., Yamasaki, O., Oono, T., Iwatsuki, K., 2001. Antibacterial action of several tannins against Staphylococcus aureus. The Journal of Antimicrobial Chemotherapy 48, 487–491. Chung, K.T., Lu, Z., Chou, M.W., 1998. Mechanism of inhibition of tannic acid and related compounds on the growth of intestinal bacteria. Food and Chemical Toxicology 36, 1053–1060. Cowan, M.M., 1999. Plant products as antimicrobial agents. Clinical Microbiology Reviews 12, 564–582.
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