Characterization of Enterococcus faecium mutants resistant to mundticin KS, a class IIa bacteriocin

Youko Sakayori1, Mizuho Muramatsu2, Satoshi Hanada2, Yoichi Kamagata2, Shinichi Kawamoto1 and Jun Shima1

1 National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
2 Research Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan

Correspondence
Jun Shima
shimaj{at}nfri.affrc.go.jp


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The emergence and spread of mutants resistant to bacteriocins would threaten the safety of using bacteriocins as food preservatives. To determine the physiological characteristics of resistant mutants, mutants of Enterococcus faecium resistant to mundticin KS, a class IIa bacteriocin, were isolated. Two types of mutant were found that had different sensitivities to other antimicrobial agents such as nisin (class I) and kanamycin. Both mutants were resistant to mundticin KS even in the absence of Mg2+ ions. The composition of unsaturated fatty acids in the resistant mutants was significantly increased in the presence of mundticin KS. The composition of the phospholipids in the two resistant mutants also differed from those in the wild-type strain. The putative zwitterionic amino-containing phospholipid in both mutants significantly increased, whereas amounts of phosphatidylglycerol and cardiolipin decreased. These changes in membrane structure may influence resistance of enterococci to class IIa and class I bacteriocins.


Abbreviations: ACP, amino-containing phospholipid; CL, cardiolipin; CPA, cyclopropanic acid; PG, phosphatidylglycerol


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacteriocins produced by lactic acid bacteria are generally considered to be safe and therefore their use as natural food preservatives has been widely investigated and discussed (Holzapfel et al., 1995; Jack et al., 1995; Montville & Chen, 1998; Ennahar et al., 2000). The emergence and spread of mutants resistant to bacteriocins would threaten the safety of using bacteriocins as food preservatives. Knowledge of the physiological characteristics of bacteriocin-resistant mutants and the conditions that prevent the emergence of resistant mutants could be used to establish conditions of usage to minimize this threat. Nisin was the first bacteriocin to be isolated and approved for use in foods (Chung et al., 1989), but the emergence of nisin-resistant mutants has been reported (Davies & Adams, 1994; Gravesen et al., 2001). It is believed that treatment with a combination of bacteriocins, such as nisin and a class IIa bacteriocin, would theoretically reduce the incidence of resistance (Song & Richard, 1997; Bouttefroy & Milliere, 2000).

In Listeria monocytogenes, resistance to nisin correlated with changes in membrane fatty acid composition, phospholipid composition and the cell wall, and required divalent cations (Davies & Adams, 1994; Davies et al., 1996; Mazzotta & Montville, 1997; Verheul et al., 1997; Crandall & Montville, 1998; Mantovani & Russell, 2001). Although physiological and molecular biological studies of nisin resistance have been undertaken, there have been few studies of resistance to class IIa bacteriocins (Ennahar et al., 2000; Gravesen et al., 2002). The aim of our study was to isolate mutants resistant to class IIa bacteriocins and to determine the characteristics of these resistant mutants and their resistance mechanisms.

Class II bacteriocins, including subclasses IIa and IIb, are low-molecular-mass, heat-stable, nonlanthionine peptides consisting of 30–60 amino acid residues (Klaenhammer, 1988; Jack et al., 1995; Ennahar et al., 2000). Class IIa bacteriocins contain peptides with the consensus sequence YGNGV near the N terminus (Eguchi et al., 2001; Kawamoto et al., 2002). In general, class IIa bacteriocins are strongly active against strains that are primarily close relatives of their producer strains (Ennahar et al., 2000). Almost all class IIa bacteriocins are active against food-borne bacteria, including Listeria and Clostridium strains (Jack et al., 1995; Eguchi et al., 2001; Kawamoto et al., 2002). Mundticin KS is a 43 amino acid peptide produced by Enterococcus mundtii NFRI 7393 and is active against E. mundtii, Enterococcus faecium, Enterococcus faecalis and L. monocytogenes (Kawamoto et al., 2002). Enterocin SE-K4 is a class IIa bacteriocin that has a YGNGV consensus motif and is active against E. faecium, E. faecalis and L. monocytogenes (Eguchi et al., 2001).

In this study, we have characterized mutants resistant to mundticin KS as a model of class IIa bacteriocins. We used E. faecium as the parent strain for deriving resistant mutants because E. faecium, which is closely related to E. mundtii, is highly sensitive to class IIa bacteriocins, including mundticin KS and enterocin SE-K4. The cross-resistance of these mutants to various antimicrobial agents, including six antibiotics and three bacteriocins, was investigated. We also determined the effects of magnesium on mundticin KS resistance and the membrane composition of fatty acids and phospholipids in the mutants.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains, culture conditions and growth media.
E. faecium IFO 13712 (Kawamoto et al., 2002) was used for isolation of mundticin-resistant mutants. E. mundtii NFRI 7393 (Kawamoto et al., 2002) was used for production of mundticin KS. Enterococcus strains were cultivated and maintained in Lactobacilli MRS broth (Difco) at 37 °C without shaking, unless otherwise stated. Growth of E. faecium was monitored by measuring OD600 using an Ultraspec 2100 pro (Amersham Pharmacia).

Chemicals.
Nisin was purchased from Asama Kasei (Japan). Erythromycin, kanamycin and tetracycline were obtained from Nakarai Tesque (Japan). Streptomycin and ampicillin were purchased from Sigma–Aldrich. Hygromycin B was purchased from Wako (Japan). Plates for TLC were purchased from Merck. Phospholipid standards were obtained from Funakoshi (Japan). Other chemicals used in this study were purchased from Nakarai Tesque.

Bacteriocin preparation.
A crude preparation of mundticin KS was incorporated into agar medium for isolation of resistant mutants. To produce mundticin KS, E. mundtii cells were cultivated in MRS medium at 37 °C for 16 h. The culture supernatant was concentrated tenfold by ultrafiltration (Viva Cell System; Sartorius). Purified mundticin KS and enterocin SE-K4 were used in the characterization of the mundticin-resistant mutants. Mundticin KS and enterocin SE-K4 were purified by a previously described method (Eguchi et al., 2001; Kawamoto et al., 2002).

Isolation of mundticin-resistant mutants from E. faecium.
E. faecium IFO13712 was utilized as parent strain to isolate mundticin-resistant mutants. Bacteria were grown for 16 h in MRS medium and portions containing 106–108 cells were plated onto MRS agar containing crude mundticin KS (0·1–2·5 µg ml-1) and cultivated at 37 °C or room temperature.

Assay for sensitivity to antimicrobial substances.
Cells of wild-type or resistant mutants were grown to OD600=1·0 and then washed with MES-buffered saline (0·9 % NaCl, pH 6·5). The cells were resuspended in 50 mM MES buffer (pH 6·5) at a cell density of approximately 5x108 cells ml-1 (OD600=1·0). The cell suspension was divided into 1 ml fractions and antibiotics or bacteriocin were added to the fractions at various concentrations. After addition of enterocin SE-K4, mundticin KS or kanamycin, the cell suspension was incubated at 37 °C for 20 min without shaking. After the addition of nisin, the cell suspension was supplemented with 10 mM MgSO4 and then incubated at 37 °C for 60 min with shaking. After treatment with antibiotic or bacteriocin, cell suspensions were centrifuged, the pellet was resuspended in MES buffer and portions were plated onto MRS agar and incubated at 37 °C for 16 h for determination of c.f.u.

Effect of divalent metal ions on bacteriocin resistance.
Cells of wild-type or resistant mutants were grown in Lactobacilli MRS broth to OD600=1·0. The cells were then washed with MES-buffered saline (pH 6·5) and resuspended at a cell density of approximately 5x108 cells ml-1 (OD600=1·0) in 50 mM MES buffer (pH 6·5) containing either divalent cations (10 mM final concn), divalent cations plus 20 mM EDTA to chelate divalent cations, or MES buffer only. Mundticin KS was added to the cell suspension at a concentration of 3 µg ml-1. The cell suspensions were incubated at 37 °C for 20 min and then plated onto MRS agar and incubated at 37 °C for 16 h for determination of c.f.u.

Measurement of membrane fatty acids.
Wild-type or resistant mutants were grown in MRS medium with or without 0·2 µg mundticin KS ml-1 to early stationary phase (OD600=1·7). Samples were collected from five cultures incubated separately under each condition. Cellular fatty acids were converted to methyl esters by using anhydrous methanolic HCl. The fatty acid methyl esters were analysed by using a Hitachi M7200A GC/3DQM system equipped with a DB-5ms capillary column (30 mx0·25 mm; J&W Scientific) coated with (5 %-phenyl)-methylpolysiloxane. The column temperature profile was initially maintained at 100 °C for 1 min and then gradually increased to 280 °C at a rate of 10 °C min-1, and finally held at 280 °C for 12 min. In all samples, the measurements with GC/MS were run in triplicate.

Measurement of cellular phospholipids.
The analysis of phospholipids was done as follows. The wild-type or mundticin-KS-resistant mutants were grown in MRS medium with or without 0·2 µg mundticin KS ml-1 to early stationary phase (OD600=1·7). The cells were harvested, washed and then resuspended in 0·9 % NaCl. Total phospholipids were extracted from the cells by the method of Bligh & Dyer (1959). Individual lipids were separated from the total lipid extracts by one- or two-dimensional TLC. Individual lipids on TLC were visualized using the following stains: iodine vapour (general lipid detection), ninhydrin (amino group) and Dittmer–Lester reagents (phosphate group). Major components of phospholipids were visualized by iodine vapour and then extracted from TLC by using chloroform. Phospholipids in the extracts were measured as described by Bartlett et al. (1959).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Isolation of mundticin-resistant mutants from E. faecium
We found two different classes of resistant mutants that appeared on MRS agar medium containing crude mundticin KS. One class appeared on the plates after cultivation for 1 day at 37 °C (MunrF). The other class appeared on the plates after cultivation for 2 or 3 weeks at room temperature (MunrS). To determine the frequencies of appearance of each class of mutants, we counted the number of colonies formed on MRS agar containing mundticin KS. Independent of mundticin KS concentration, both classes of mutants appeared at frequencies of 10-6 to 10-7, similar to the frequencies for one of the kanamycin-resistant mutants (data not shown). The mundticin resistance of both classes of mutants (MunrS and MunrF) remained stable even after further cultivation on mundticin-free medium, repeated at least five times.

First, we did a morphological analysis of the two resistant mutants (MunrS and MunrF) by using phase-contrast microscopy. No significant differences in cell shape, cell size and degree of cell aggregation were observed between wild-type and the two mutants (data not shown). Then, we determined the growth characteristics of wild-type, MunrS and MunrF in MRS liquid medium containing mundticin KS (Fig. 1). The wild-type or the two resistant mutants were inoculated into MRS medium at a concentration of approximately 4·5x105 cells ml-1, incubated at 37 °C and growth was monitored by measuring OD600 every 1·5 h. The growth characteristics of wild-type and Munr mutants were nearly identical in the absence of mundticin KS (Fig. 1a). In contrast, in the presence of 5 µg mundticin KS ml-1 growth of wild-type cells was inhibited (Fig. 1b), whereas growth of the mundticin KS mutants was relatively unaffected.



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Fig. 1. Growth characteristics of wild-type strain (filled circle), and MunrS (open circle) and MunrF (open triangle) mutants in MRS medium (a) and in MRS medium containing 5 µg mundticin KS ml-1 (b).

 
Cross-resistance of Munr mutants to various antimicrobial agents
We measured the sensitivities of the two Munr mutants to various antimicrobial agents, including six antibiotics (streptomycin, ampicillin, kanamycin, erythromycin, tetracycline and hygromycin B) and three bacteriocins (nisin, mundticin KS and enterocin SE-K4). To determine resistance of Munr mutants to these agents, we monitored growth of the mutants on concentration-gradient agar plates containing the antimicrobial agents (Kato et al., 1978). Significant differences in growth between wild-type and Munr mutants were evident on plates containing kanamycin, nisin, enterocin SE-K4 or mundticin KS (data not shown).

To determine resistance to these antimicrobial agents in more detail, we measured numbers of viable cells (c.f.u.) after treating cell suspensions with these antimicrobial reagents. At first, we assessed resistance to class IIa bacteriocins. As shown in Fig. 2(a), the MunrF and MunrS mutants showed higher resistance to mundticin KS compared with the wild-type strain. These mutants were not inhibited even at a mundticin KS concentration of >50 µg ml-1 (data not shown). Both Munr mutants also showed strong resistance to enterocin SE-K4 (Fig. 2b). We next assessed resistance to nisin (Fig. 3a). Because nisin is not highly active against E. faecium, we could not detect differences between wild-type and Munr mutants at low nisin concentrations (<100 µg ml-1). However, at higher nisin concentrations, Munr mutants showed less sensitivity to nisin compared with wild-type. In particular, the MunrF mutant was 10–1000 times more resistant to nisin than wild-type at concentrations of 200–500 µg ml-1. As shown in Fig. 3(b) the MunrF mutant showed sensitivity to kanamycin similar to that of the wild-type strain. However, the MunrS mutant was hypersensitive to >150 µg kanamycin ml-1 Fig. 3(b). These results suggest that Munr mutations cause pleiotropic changes in resistance to different antimicrobial agents.



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Fig. 2. Viabilities of wild-type strain (filled circle), and MunrS (open circle) and MunrF (open triangle) mutants treated with class IIa bacteriocins mundticin KS (a) and enterocin SE-K4 (b) under the conditions described in Methods. After treatment, cells were plated onto MRS medium in duplicate and incubated at 37 °C. Colonies were counted after incubation of plates for 16 h.

 


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Fig. 3. Viabilities of wild-type strain (filled circle), and MunrS (open circle) and MunrF (open triangle) mutants treated with class I bacteriocin nisin (a) and kanamycin (b) under the conditions described in Methods. After treatment, cells were plated onto MRS medium in duplicate and incubated at 37 °C. Colonies were counted after incubation of plates for 16 h.

 
Effects of divalent cations on mundticin resistance of Munr mutants
We determined the effect of divalent cations, which are known to be required for expression of nisin resistance by nisin-resistant mutants, on resistance to mundticin KS. Viabilities of wild-type and Munr mutants after treatment with mundticin KS in the presence or absence of MgSO4 were determined (Fig. 4). The addition of Mg2+ ions significantly reduced the inhibitory activity of mundticin on wild-type cells. The addition of EDTA restored this activity. In contrast, the inhibitory activities on both Munr mutants were not affected by the addition of Mg2+ ions. Other divalent cations, including Ca2+, Mn2+ and Ba2+, had no effect (data not shown). These results suggest that Mg2+ ions protect wild-type cells against mundticin activity and that the effect of Mg2+ ions is suppressed in the Munr mutants.



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Fig. 4. Effect of magnesium on mundticin KS resistance of wild-type strain and Munr mutants. Cells were treated in MES buffer in the absence (+) or presence (-) of 3·0 µg mundticin KS ml-1, 20 mM EDTA and 10 mM MgSO4. After treatment, cells were plated onto MRS medium in duplicate and incubated at 37 °C. Colonies were counted after incubation of plates for 16 h.

 
Composition of fatty acids and phospholipids in Munr mutants
We investigated further the resistance mechanisms of the two Munr mutants by measuring the composition of fatty acids (Table 1) and phospholipids (Table 2, Fig. 5) in the Munr mutants. The fatty acids and phospholipids were extracted from Munr cells grown in the presence or absence of mundticin KS and then compared to those from wild-type cells grown in the absence of mundticin KS.


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Table 1. Cellular fatty acid composition and ratios of different types to the total fatty acids in wild-type and Munr mutants grown with or without mundticin

 

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Table 2. Phospholipid composition of wild-type and Munr mutants

 


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Fig. 5. TLC analysis of phospholipids extracted from wild-type strains and Munr mutants visualized with Dittmer–Lester (a) and ninhydrin (b) reagents. Before visualization, total phospholipids were separated by TLC with the solvent system CHCl3/methanol/acetate (35 : 10 : 4, by vol.). Lanes: 1, CL from bovine heart; 2, PG from egg yolk; 3, PE from porcine liver; 4, phospholipids extracted from wild-type grown in MRS medium; 5, phospholipids extracted from MunrS grown in MRS medium; 6, phospholipids extracted from MunrS grown in MRS plus mundticin KS medium; 7, phospholipids extracted from MunrF grown in MRS medium; 8, phospholipids extracted from MunrF grown in MRS medium plus mundticin KS medium; 9, phospholipids extracted from E. coli DH5{alpha}.

 
The predominant membrane fatty acid in all cultures was an unsaturated fatty acid, C18 : 1 (Table 1), but there were also significant amounts of a saturated fatty acid (C16 : 0) and cyclopropanic acid (CPA-C19 : 0). Although no significant differences in composition of membrane fatty acids were evident between the wild-type and Munr mutants, the amounts of unsaturated fatty acids (C18 : 1 and C16 : 1) were lower in the mutants (approx. 77 %) than in the wild-type (approx. 82 %), whereas the amounts of saturated fatty acids and CPAs were higher. The differences in fatty acid composition were greater in the mutants grown in the presence of mundticin KS; the amounts of unsaturated fatty acids and CPAs were 13 and 15 % higher, respectively, than the wild-type strain. These results indicate that the composition of unsaturated fatty acids correlates with resistance to mundticin KS.

Fig. 5 shows the TLC analysis of phospholipids extracted from wild-type and Munr mutants. Three major components of phospholipids are clearly evident. By comparing the mobilities of the phospholipids on TLC with those of standards and those extracted from Escherichia coli DH5{alpha}, we tentatively identified two of the three as phosphatidylglycerol (PG) and cardiolipin (CL), respectively. The third could not be identified. We designated this third component amino-containing phospholipid (ACP) because the unidentified spot in TLC was reactive with Dittmer–Lester reagents and ninhydrin. We compared phospholipid composition between the wild-type and Munr mutants grown in the presence or absence of mundticin KS. The amounts of PG and CL in both Munr mutants were significantly less than that in the wild-type (Table 2), whereas the amount of ACP was significantly higher. Addition of mundticin KS to the medium did not affect phospholipid composition. These results suggest that the decrease in PG and CL and/or increase in ACP contribute to mundticin KS resistance in Munr mutants.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, we isolated and characterized mundticin-resistant mutants derived from E. faecium. The resistant mutants showed cross-resistance to another class IIa bacteriocin, enterocin SE-K4, and a class I bacteriocin, nisin. The possibilities of cross-resistance between class IIa and class I nisin in Clostridium botulinum and L. monocytogenes have been proposed (Mazzotta & Montville, 1997; Song & Richard, 1997; Crandall & Montville, 1998). As far as we know, our current study is the first report of observed cross-resistance between class I and class IIa bacteriocins in Enterococcus.

In this study, we successfully isolated two types of mundticin-resistant mutants (MunrS and MunrF). Although no genetical analysis of these two mutants was performed, the two mutants apparently have different mutations that determine their mundticin resistance based on their differential sensitivities to a range of antimicrobial agents. MunrS mutants showed hypersensitivity to kanamycin, which is an aminoglycoside antibiotic and inhibits protein translation. The mechanism for this hypersensitivity to kanamycin remains unclear.

Resistance to bacteriocins has been widely studied. Almost all those studies have involved nisin (class I) with L. monocytogenes as the target strain (Mazzotta & Montville, 1997; Verheul et al., 1997; Crandall & Montville, 1998). Recently, resistance to class IIa bacteriocins has been reported. For example, Limonet et al. (2002) reported variations in fatty acid composition of mutants of Leuconostoc and Weissela strains resistant to the class IIa bacteriocin mesenterocin 52A (Mes 52A). They showed that resistant strains contained more saturated fatty acids when cultured with Mes 52A. Vadyvaloo et al. (2002) reported that L. monocytogenes strains resistant to class IIa bacteriocins showed increased levels of desaturated PG.

Models of modes of class IIa bacteriocin activity have been proposed (Ennahar et al., 2000). In brief, the modes of bacteriocins consist of at least three steps: (1) electrostatic interaction between the N terminus of bacteriocin and membrane phospholipids, (2) reorientation and insertion into the membrane, and (3) pore formation. At present, no evidence for a protein receptor for binding of class IIa bacteriocins has been reported (Chen et al., 1997b). In our study, we did not detect significant differences in the mundticin-binding affinity between Munr mutants and wild-type cells, even in the presence of Mg2+ ions (data not shown). An affinity assay suggests that mutation in Munr mutants does not occur in genes that are directly involved in bacteriocin-binding, such as genes for protein receptors.

Recently, Limonet et al. (2002) reported variations in fatty acid composition of mutants of Leuconostoc and Weissela strains resistant to mesenterocin 52A (Mes 52A). We have also found differences in fatty acid (Table 1) and phospholipid contents (Table 2). Based on these observations, the composition of membrane fatty acids appears to affect resistance to class IIa bacteriocins. In particular, an increase in saturated fatty acids seems to correlate with resistance, possibly because saturated fatty acids might change the physical properties of the membrane, making the membrane more rigid.

We also showed that, in the two Munr mutants, the amount of ACP was higher, whereas amounts of PG and CL were lower, compared to amounts in the wild-type strain. We speculate that the chemical structure of ACP might be lysyl PG, because Kocun (1970) reported that lysyl PG is one of the major components of the phospholipids of E. faecalis IoCI, a strain closely related to E. faecium. Conditions favourable to electrostatic interactions in the membrane are important for the binding class IIa bacteriocins to phospholipid vesicles (Chen et al., 1997a). Because ACP could have zwitterionic properties, an increased level of ACP might inhibit binding of bacteriocin by positively charging the membrane. It has been reported that changes in the phospholipid composition occur in nisin-resistant strains of L. monocytogenes Scott A (Verheul et al., 1997). Specific changes include more zwitterionic phosphatidylethanolamine and less anionic PG and CL (Verheul et al., 1997; Crandall & Montville, 1998). Changes in the composition of phospholipids in Munr mutants that we observed are similar to these changes in phospholipids in L. monocytogenes. Thus the level of zwitterionic phospholipids is a critical determinant in the resistance to both class I and class IIa bacteriocins. Vadyvaloo et al. (2002) reported that class-IIa-resistant L. monocytogenes strains showed increased levels of desaturated PG. In our study, we did not determine the composition of fatty acids specifically binding to PG.

The changes in membrane components in the two Munr mutants were similar to those in nisin-resistant strains (Tables 1 and 2). Munr mutants showed cross-resistance to nisin (Fig. 3), suggesting that the resistance mechanisms to class IIa and class I bacteriocins are similar. The relationship between changes in membrane components, such as fatty acids and phospholipids, and the molecular mechanisms of resistance remains unclear for class IIa bacteriocins. At present, we do not know whether a specific gene determines resistance to mundticin KS in Munr mutants. Gene cloning of determinants of mundticin KS resistance might help to clarify the mechanisms of bacteriocin resistance, if a specific resistance gene is present. Cross-resistance between class I and class IIa bacteriocins might be a critical problem when bacteriocins are used as biopreservatives. To overcome this problem, characterization of resistance mechanisms to bacteriocins at the molecular level is crucial.


   ACKNOWLEDGEMENTS
 
We thank Drs A. Ohta, R. Fukuda (University of Tokyo) and A. Nagaro (National Food Research Institute) for the methods of phospholipid analysis. We also thank C. Suzuki (National Food Research Institute) for critical comments on this work. This study was supported partly by a grant-in-aid (MAFF Food Research Project) from the Ministry of Agriculture, Forestry, and Fisheries, Japan.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 23 April 2003; revised 18 June 2003; accepted 1 July 2003.



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