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1 Department of Food Engineering, Istanbul Technical University, 34469 Maslak, Turkey
2 Department of Food Science, University of Wisconsin-Madison, Madison 53706
Corresponding author: Meral Kilic; e-mail: meral.kilic{at}itu.edu.tr.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Key Words: alkylphenol conjugates • ruminant milk
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Conjugation is a detoxification mechanism that enables an animal to solubilize xenobiotics and excrete them in the urine. The main site for conjugation is the liver, but the intestine, kidneys, lungs, and skin also conjugate xenobiotics. The preferred conjugation mechanism for a substrate depends on the species, nature of the compound, the transferase enzyme, and availability of the cosubstrate. Monohydric phenols are commonly conjugated at the hydroxyl group by sulfation, glucuronidation, and to a lesser extent, by acetylation and phosphorylation (Mulder, 1990). Conjugates of p-cresol, 4-ethylphenol, and phenol were identified early in the urine of ruminants (Grant, 1948; Suemitsu et al., 1970; Kao et al., 1979).
Despite extensive studies about the metabolism and urinary excretion of conjugates of phenol (Caldwell, 1980; Mulder, 1982), limited information is available about the identity and quantity of different alkylphenol conjugates present in milk. Understanding of the distribution and quantities of species-related alkylphenols between different conjugation mechanisms could be used to facilitate release of these flavor compounds in dairy products. The present study was undertaken to investigate the amounts of different conjugates of alkyl-phenols present in ruminant milks with a particular emphasis on sheep’s milk using currently available analytical methods.
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MATERIALS AND METHODS |
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Feeding regimens between local farms were reportedly different; sheep from source 3 were on a pasture rotation, including blue grass, white and red clover, and orchardgrass; sheep from source 2 had a more limited supply of pasture without rotation, including red and white clover and orchardgrass. Sheep from source 1 were mostly held in confinement and fed alfalfa hay, but some of the sheep had limited access to pasture, which included kura clover and orchardgrass. All sheep were reportedly fed a supplemental grain mixture at milking in the parlor.
All solvents used were HPLC grade.
Extraction of Conjugated Alkylphenols
Conjugated alkylphenols in skim milk were analyzed using the methods of Zeng (1997) and Lopez and Lindsay (1993) with some modifications. Pasteurized whole milk was centrifuged at 15,000 x g at 4°C for 30 min (Sorvall refrigerated centrifuge, DuPont Inst., Des Plaines, IL) to separate cream and skim milk fractions. Skim milk was then filtered through glass wool to remove residual fat.
Conjugated alkylphenols in skim milk were isolated by elution through an open column of Amberlite XAD-2 beads (Supelco, Bellefonte, PA). The beads (15-g batches) were prepared by immersion in 25 mL of methanol for 10 min and then in 25 mL of double-distilled water for 10 min. Beads were then placed into the column and backwashed with 50 mL of double-distilled water. After removing the water, 2 kg of skim milk was passed through 4 XAD-2 columns at a flow rate of 10 mL/min. Each column was washed with 100 mL of double-distilled water and backwashed with another 50 mL of double-distilled water. After addition of 50 mL of methanol, each column was held for 10 min to allow penetration of the solvent into the beads. Alkylphenols adsorbed by the column were eluted with 100 mL of methanol at a flow rate of 2.5 mL/min. Methanol extracts were concentrated in round-bottom flasks (250 mL) at 40°C using a rotary vacuum evaporator until 3 mL of residue was left. Residues obtained from each elution were collected by washing with 3 mL of distilled water and all fractions were combined in a final extract. Each concentrate obtained was clarified by extraction (3 times) with ether:pentane (3:2) to remove artifacts from the XAD-2 column material. The final extract contained the conjugates from approximately 100 g of skim milk per mL. The extracts were kept at –20°C until analysis.
Hydrolysis of Conjugates
Two milliliters of acetate buffer at pH 5 and 50 µL of o-cresol (10 mg/100 mL of methanol) as an internal standard were added into 500-µL portions of skim milk extracts. Conjugated alkylphenols were released by incubating the extract mixtures with 100 units of acid phosphatase (EC 3.1.3.2) type I from wheat germ or 100 units of arylsulfatase (EC 3.1.6.1) type VIII from abalone entrails containing negligible amounts of ß-D-glucuronidase activity (less than 13 units of ß-D-glucuronidase per 100 units of arylsulfatase) or 1000 units of ß-D-glucuronidase (EC 3.2.1.31) from Escherichia coli (Sigma Chemical Co., St. Louis, MO) at 37°C for 24 h. After incubation, mixtures were cooled to room temperature and acetone was added (1:1) to prevent formation of emulsions and improve the recovery of alkylphenols from the aqueous phase. Free alkylphenols released by each enzyme were then extracted 3 times with ethyl ether (1:1). Ethyl ether extracts were washed with 2 mL of saturated NaCl solution to remove residual aqueous components. Final extracts were dried over excess an-hydrous Na2SO4, and then concentrated under a slow stream of nitrogen to near dryness. Control samples were prepared by including only the milk extract without the enzymes or each enzyme alone to correct for interferences that might occur.
The acidic components in each concentrate subsample were neutralized by adding 100 µL of 0.2 M Na2CO3. The pH of the concentrates was adjusted to pH 12 using 50-µL portions of 1 N NaOH and the addition of 100 µL of phosphate buffer at pH 12. Free alkylphenols were derivatized by incubating buffered mixtures with 600 µL of dansyl chloride (5-dimethylamino-napthalene-1-sulfonyl chloride, 5 mg/mL acetone; Aldrich Chemical Co., Milwaukee, WI) at 45°C for 20 min. After cooling to room temperature, excess dansyl chloride in the mixtures was hydrolyzed with 3 drops of 1 N NaOH. Dansylated free alkylphenols were extracted into 500 µL of hexane, and 20-µL aliquots were used for analysis with HPLC. Conjugated alkylphenols in sheep urine were determined using the same procedures except that 100 mL samples were used for isolation of conjugates, and 100 µL of urine extract obtained from Amberlite XAD-2 elution (equivalent to 2.5 g of urine) and 100 µL of internal standard were used in the enzyme incubation mixtures.
HPLC Analysis of Dansylated Free Alkylphenols
Normal phase HPLC was performed using a Lichrosorb Si-60 column (5 µm, 250 x 4 mm i.d.; Alltech Assoc., Inc., Deerfield, IL). The HPLC system included a Beckman 110B solvent delivery module (Beckman Instruments, Inc., Berkeley, CA), and a 20-µL sample loop. The mobile phase was 1.6% acetone in 2,2,4-trimethyl-pentane at a flow rate of 1.5 mL/min. Detection of dansyl derivatives was carried out with a Waters model 420 fluorescence detector (Waters Associates, Inc., Milford, MA) with a gain setting of 8 which was set at an excitation wavelength of 360 nm and an emission wavelength of 425 nm. Chromatograms were recorded using a Spectra Physics model 4200 computing integrator (Spectra Physics, San Jose, CA) with a chart speed of 0.5 cm/ min. Alkylphenols in chromatograms were identified by coincidence of retention times between unknown and authentic compounds.
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RESULTS AND DISCUSSION |
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All of the milk samples contained mostly sulfate conjugates of p- and m-cresols and 3- and 4-ethylphenols with minor amounts of phosphate and glucuronide conjugates (Tables 1
and 2). Lopez and Lindsay (1993) also found that alkylphenols in cows’ and goats’ skim milk were mostly conjugated with sulfate with minor amounts bound as glucuronides. Brewington et al. (1973, 1974) reported glucuronide and sulfate conjugates of p-cresol and 4-ethylphenol in cows’ skim milk, but only indicated estimates of relative abundances of the compounds. Grant (1948) identified 4-ethylphenyl sulfate in goat’s urine. Notably, for the current study, the overall distribution of alkylphenols between sulfate, phosphate, and glucuronide conjugates were similar in all ruminant milk samples even though species differences had previously been reported for conjugation of phenolic compounds in urine (Capel et al., 1972; Williams, 1974; Caldwell, 1980).
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). Moio et al. (1993) and Lopez and Lindsay (1993) found higher amounts of p-cresol in ewes’ and goats’ milk than in cows’ milk. p-Cresol was the major alkylphenol present in all the milks. The major alkylphenol in sheep’s (Bassett et al., 1955; Braden et al., 1967; Martin, 1982) and cows’ (Suemitsu et al., 1970) urine has also been found to be p-cresol. p-Cresol has a characteristic barn-like flavor that blends with the more medicinal notes of m-cresol in milk (Ha and Lindsay, 1991a).
Goats’ milk contained the highest amounts of 3- and 4-ethylphenols among the milk samples analyzed (Table 1). 3-Ethylphenol is a characteristic species-related alkylphenol with a sheep-like and phenolic flavor. 4-Ethylphenol possesses stronger sheep-like flavor character with a particular sheep yard-like aroma (Ha and Lindsay, 1991a). The presence of these compounds (3-and 4-ethylphenols) likely contributes to the strong flavors of goats’ milk. The sheep’s milk sample had lower amounts of 3- and 4-ethylphenols than goats’ milk, and cows’ milk contained slightly higher amounts of 3- and 4-ethylphenols than the sheep’s milk. However, analysis of sheep’s milk from different sources revealed that some variation occurred between different milk samples (Table 2).
Minor amounts and sporadic distributions were obtained for phenol conjugates in all milk samples (Tables 1 and 2). In earlier reports, phenol was thought to be conjugated mostly with glucuronide with lesser amounts as sulfate in the urine (Suemitsu et al., 1970; Williams, 1974; Kao et al., 1979). In this study, however, the glucuronide conjugate of phenol was not detected in cows’ and goats’ milk, and only minor amounts were present in sheep’s milk. Furthermore, the glucuronide conjugates of other phenols were only found at minor amounts compared with sulfate conjugates in all milk samples.
The effect of feeding regimen on alkylphenols in sheep’s milk is illustrated in Table 2. Sulfate conjugates of p- and m-cresols were 3-fold higher in milk from source 3 than in milks from sources 1 and 2. The concentrations of sulfate conjugates of 3- and 4-ethylphenols were about 4 times higher in ewes’ milk from source 2 and 3 than in ewes’ milk from source 1. Ewes from sources 2 and 3 had similar feeding regimens that included mostly pasture, but they also received grain concentrate supplementation at milking. Pasture was also available to some of the sheep in source 1, but only to a limited number of the animals.
The higher concentrations of p- and m-cresols in milk from source 3 than in source 2 could reflect the effect of variety of pasture that was available to the ewes. Formononetin, a constituent of subterranean clover, has been found to be degraded into mostly p-cresol with minor amounts of 4-ethylphenol when administered intraruminally to sheep (Batterham et al., 1965; Braden et al., 1967; Batterham et al., 1971). Isoflavones in subterranean clover, such as biochanin A and genistein, have been shown to be degraded in the sheep rumen to yield 4-ethylphenol as an end product (Batterham et al., 1965; Braden et al., 1967; Batterham et al., 1971). Because a varying distribution of alkylphenols in different conjugate forms was observed between the ewes’ milk from different sources, feed, breed, and individual animal characteristics probably affect the metabolism of alkylphenols. The effect of feed on the flavor of milk has been studied, including that for the cow (Urbach, 1990), the goat (Skjevdal, 1979), and the sheep (Moio et al., 1996). Considerable variation has been observed within and between species, which likely results from the natural variations noted above.
Urine and milk from the same ewe were analyzed for conjugated alkylphenols; the amounts of conjugated alkylphenols in milk were 1.3, 0.8, and 0.5% of the amounts present in the urine for p- and m-cresols, 3-and 4-ethylphenols, and phenol, respectively (Table 3). The same trend as in milk was obtained for the urine for p- and m-cresols and 3- and 4-ethylphenols where sulfate was the major conjugate. Similar amounts of phosphate and glucuronide conjugates were found for each of the alkylphenols. Phosphate and glucuronide conjugates of phenol were present at approximately equal concentrations, but sulfate conjugates of phenol were absent in the urine. Kao et al. (1979) determined that [14C]phenol was excreted in sheep’s urine mainly as glucuronide (49%) and sulfate (32%) conjugates with small amounts associated with phosphate (12%). Lopez and Lindsay (1993) previously reported that sheep’s milk appeared to contain mostly phosphate conjugates of alkylphenols based on hydrolysis studies using crude enzymes. However, these observations were not confirmed in the current study.
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The concentrations of conjugated alkylphenols in milk were found at amounts less than 1.5% of that present in the urine of the sheep (Table 3). Prelusky et al. (1987) reported that less than 0.25% of a dose of deoxynivalenol, a mycotoxin, was excreted into milk of lactating sheep. Minor amounts of fungicides and herbicides were conjugated mostly with sulfate in milk and urine of goat (Paulson et al., 1973; Paulson and Jacobsen, 1974; Chukwudebe et al., 1994).
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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The amounts of alkylphenols varied in ewes’ milk obtained from different local sources, suggesting that the feed, breed, and individual animal affect the metabolism of the alkylphenols. The amounts and distributions of alkylphenols in ewes’ urine were found to be similar to those in milk. Results from this study indicated that species-related alkylphenols were predominantly present as sulfate conjugates in ruminant milks when phenolic compounds or their precursors were normally ingested via feed by the animal. Further studies are needed to determine the factors, such as feeding regimen, that affect the amounts of conjugated alkyl-phenols in milk so that this information could be used to manipulate the amounts of these flavor precursors in milk and dairy products.
Received for publication August 22, 2004. Accepted for publication September 29, 2004.
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