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* Human Nutrition and Foods Program, West Virginia University, Morgantown 26506
Department of Food Science and Technology, Virginia Tech, Blacksburg 24061
Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park 16802
Department of Food Science & Technology, Cornell University, New York State Agricultural Experiment Station, Geneva 14456
1 Corresponding author: Kristen.matak{at}mail.wvu.edu
| ABSTRACT |
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Key Words: goat milk ultraviolet solid phase microextraction and gas chromatography acid degree value
| INTRODUCTION |
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Ultraviolet irradiation, a process that does not involve heat to kill microorganisms (Sastry et al., 2000), is effective against numerous microorganisms found in drinking water (Parrotta and Bekdash, 1998) and apple cider (Wright et al., 2000; Hanes et al., 2002; Basaran et al., 2004). Recently, Matak et al. (2005) demonstrated the effectiveness of UV processing for the reduction of the bacterial pathogen Listeria monocytogenes in goat milk. Microbial inactivation from UV light is associated with photochemical changes that take place in proteins and nucleic acids within the cell membrane when UV light is absorbed (Sastry et al., 2000). Mutations occur that disrupt DNA transcription and replication, which ultimately cause death of the microorganism (Miller et al., 1999).
It has been well reported that UV and visible light, with wavelengths between 280 nm and 700 nm, are key factors in the creation of flavor defects and malodors in milk (Bradley, 1980; Cadwallader and Howard, 1998; Borle et al., 2001; Min and Boff, 2002). Historical studies that looked at the potential of UV irradiation for vitamin D enrichment at the specific germicidal wavelength of 254 nm did not report negative sensory data (Capstick et al., 1946; Burton, 1951; Caseiro et al., 1975). Matak and others (2005) demonstrated UV processing as an effective, nonthermal process to attain a greater than 5-log reduction (P < 0.0001) of L. monocytogenes in goat milk. This was accomplished when the flow rate of the milk through the UV processing unit was increased to the point where the milk was exposed to the UV source in a state of turbulent flow. At this flow rate, the milk received a cumulative UV dose of 15.8 ± 1.6 mJ/cm2 (Matak et al., 2005); this current study was conducted to determine if chemical and sensory properties of goat milk were affected by UV processing.
| MATERIALS AND METHODS |
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4°C in an insulated covered 18.9-L (5-gal) cooler (Igloo Products Corp., Houston, TX) that had been sanitized with 200 ppm of hypochlorite solution. Samples were processed within 12 h of collection, and all analyses, except fatty acid profile, were concluded within 8 h of processing. Samples for fatty acid profiles were frozen and maintained at 80°C until analyses were conducted. Separate batches of milk were purchased within a 2-mo period for each of the 3 study replications, and gross composition (total fat, protein, total solids) was evaluated. An infrared analyzer (Infrared Analyzer 115 Vac, Denver Instrument Company, Arvado, CO) was used to measure moisture content and total solids. The modified Babcock procedure was used to measure milkfat (Marshall, 1993). A dye-binding method and commercial assay kit (BioRad protein assay, BioRad, Hercules, CA) was used to measure protein content spectrophotometrically (Spectronic 20 Colorimeter, Bausch & Lomb Inc., Rochester, NY; Bradford, 1976). Fresh, raw goat milk served as the control for all chemical and sensory analyses.
UV Irradiation
Milk samples were exposed to UV irradiation using a commercial UV fluid processor (CiderSure 3500A, FPE Inc., Rochester, NY), the details of which are described elsewhere (Matak, et al., 2005). The UV dose was calculated using equation [1],
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where irradiance was measured as described by Quintero-Ramos et al. (2004), and exposure time was a factor of the flow rate.
Milk (3 L) was passed through the processor 12 consecutive times for the targeted exposure time of 18 s and targeted UV dose of 15.8 ± 1.6 mJ/cm2 (12 pass UV). This UV dose was in the range that is effective at achieving a 5-log reduction in L. monocytogenes in fresh goat milk (Matak et al., 2005). The processor was operated at 75% capacity (a turbulent flow rate of 567 L/h) so that comparisons could be made with findings from previous studies. At this flow rate, the lamps generate approximately 1.3 mJ/cm2 UV dose at exposure times between 1 and 2 s as measured by 2 UVX-25 sensors (UVP Inc., Upland, CA). A separate batch of milk (1.5 L) was passed through the UV apparatus 12 times with the UV lights turned off (12 pass no UV). This was to determine if agitation caused by the act of pumping the milk through the UV apparatus 12 times had an effect on milk properties due to shear and lipase activity.
Multiple passes through the processor were necessary to achieve the desired UV exposure; therefore, after each pass the unit was cleaned and sanitized according to Basaran and others (2004). Total cleaning time was less than 2 min, and total processing time (including cleaning) was less than 30 min.
An aliquot (100 mL) was sampled for chemical analyses after 4, 6, 8 and 12 passes through the processor for subsequent chemical analyses. Samples were collected in 100-mL borosilicate milk dilution bottles (Corning Inc., Corning, NY) and 500-mL HDPE containers (Nalge Nunc Int., Rochester, NY), fitted with lids, and covered with aluminum foil to prevent further light exposure. The calculated UV dose for each of these samples was 5.2, 7.8, 10.4, and 15.6 mJ/cm2, respectively. Sample temperature was maintained (
4°C) by holding samples in an ice bath during sample preparation, between processing passes, and during analytical and sensory preparation.
Sensory Testing
Virginia Tech Institutional Review Board approval was received for the sensory study. Because unpasteurized milk samples were used and UV processing is not yet an approved process for milk, samples were assessed for differences in odor, not taste. Comparisons included fresh, raw goat milk (control) vs. 12 pass UV; 12 pass UV vs. 12 pass no UV; control vs. 12 pass no UV. Each comparison was evaluated using a triangle test, which was conducted once and not replicated. Immediately after processing, samples (
15 mL) were poured into 20-mL semiopaque plastic cups, fitted with plastic lids, assigned a random 3-digit code, and stored and maintained at
4°C until sensory testing was concluded. Sensory tests occurred within 6 h of treatment. All combinations of the 2 samples were presented within each sensory session an equal number of times. Two sets of 3 samples were presented to each panelist, representing a balanced order of presentation. Panelists were instructed to identify the sample that smelled different in each group of 3. There was additional space for comments with instructions to describe any odors associated with the unique sample.
Twenty-four volunteers (
18 yr) were recruited from the Food Science and Technology Department (Virginia Tech) to serve on each panel session. Each panelist contributed 2 observations per testing session for a total of 48 observations per triangle test. Testing was conducted in individual booths in the Food Science and Technology sensory laboratory. Panelists were required to complete a consent form, approved by the Institutional Review Board at Virginia Tech, prior to testing. Each panelist was verbally reminded not to drink the samples but to smell them only.
Chemical Analyses
The extent of oxidation as a result of processing with and without UV irradiation was assessed chemically using 3 different methods: thiobarbituric acid reactive substances (TBARS) test, acid degree values (ADV), and volatile analysis by solid-phase microextraction with gas chromatography. All samples were tested concurrently within 6 h following UV exposure.
The TBARS test measures malondialdehyde and other reactive substances in the sample, and the results are reported as milligrams of sample per liter. Results of this test are based on the color reaction of lipid peroxidation products and thiobarbituric acid; color absorbance was read spectrophotometrically at 532 nm (Spectronic 20 Colorimeter, Bausch & Lomb Inc., Rochester, NY). Samples (control, 4 pass UV, 6 pass UV, 8 pass UV, 12 pass UV, and 12 pass no UV) were tested in duplicate using a modified version of the TBARS test described by van Aardt and others (2005b).
The ADV are classified as a standard method for indication of hydrolytic rancidity resulting from enzymatic activity and used as a measurement of free fatty acids in milkfat recovered from an extraction method and titration. These values, coupled with sensory evaluation, can be used as an indicator of rancid off-flavors in milk. The procedure for measuring ADV is described by Marshall (1993). Samples (control, 4 pass UV, 6 pass UV, 8 pass UV, 12 pass UV, and 12 pass no UV) were tested in duplicate.
Solid-phase microextraction with gas chromatography is an extraction and analytical technique used to detect concentrations of volatile flavor compounds in foods and beverages. The solid-phase microextraction fibers (75-µm carboxen-polydimethyl siloxane), manual holder assemblies, Viton septum, caps, micro stirring bars, and 40-mL glass bottles were purchased from Supelco Inc. (Bellefonte, PA). Prior to use, fibers were conditioned in a gas chromatography injection port at 280°C for 1 h. Volatile compounds associated with light oxidation (hexanal, heptanal, pentanal, and methyl sulfide) were purchased from Sigma Chemical (St. Louis, MO). Identification was made for each day of analysis by comparing gas chromatography retention times (RT) of stock solutions containing each compound at concentrations of 100 and 500 µg/mL (Marsili, 1999; van Aardt et al., 2001, 2005a). Concentrations of volatile compounds were not quantified; therefore, peak areas of identified compounds were compared relative to fresh, raw goat milk (control).
Volatile compounds were assessed in the control, 12 pass UV, and 12 pass no UV samples to consider differences as related to sensory evaluation outcomes. Milk (25 mL) from each treatment (control, 12 pass UV, and 12 pass no UV) was transferred into 40-mL glass bottles covered with aluminum foil. Bottles were fitted with Teflon-coated septa and held at
4°C until analyses were conducted.
Volatile compound analysis was completed in triplicate for each treatment within 6 h of processing. The solid-phase microextraction fiber was inserted through the septum and positioned approximately 1 cm above the milk surface to allow maximum exposure to milk headspace, and exposed for 22 min at 45°C with magnetic stirring. The loaded solid-phase microextraction fiber unit was placed into the injector port of a Hewlett Packard gas chromatograph (model 5890 Series II Plus, Hewlett Packard, Avondale, PA) equipped with Chem-Station (Agilent Technologies, Palo Alto, CA; van Aardt et al., 2001). Volatile compounds were separated using a 30 m x 0.32 mm, 1.05 µm, Rtx-S capillary column (Restek Corp., Bellefonte, PA), and helium as the carrier gas with flow rates of 1.8 mL/min. Injector temperature was 280°C, and flame ionization detector temperature was 300°C. Temperature program was 30 s at 35°C, 15°C per min to 180°C, hold 30 s, 20°C per min to 260°C, hold 30 s.
Fatty Acid Analysis
Fatty acid analyses were conducted in duplicate on fresh, raw goat milk (control), 12 pass UV, and 12 pass no UV samples to indicate if profiles among treatments changed as a result of UV processing. This test was completed for one replication of the study because the natural variations in milk composition between collections could create differences in fatty acid profile, potentially masking the effect of the process.
Milk samples were frozen and maintained at 80°C until analyses were conducted. The procedure for extraction and methylation was as follows: milk was thawed, warmed, and gently mixed to provide a uniform sample. Milk (1 mL) was weighed into a 50-mL extraction tube. Lipid was extracted using a modified Folch procedure (Folch et al., 1957). Lipid residue was weighed after drying at 40 to 45°C under a stream of nitrogen. Fatty acids were transesterified to methyl esters with 0.5 N NaOH in methanol and 14% BF3 (Park and Goins 1994). Undecenoic acid (Sigma-Aldrich Corp., St. Louis, MO) was added prior to methylation as an internal standard.
Chromatographic analysis was conducted on prepared samples. All samples were analyzed in duplicate on a 6890N gas chromatograph with a 7683 autoinjector, split/splitless capillary injector and flame ionization detector (Agilent Technologies). Ultrapure H2 was used as the carrier gas with gas velocity set at 30 cm/sec, flow rate at 1.5 mL/min, injection volume 0.5 µL, and split ratio 100:1. A Chrompack CP-Sil 88 100 m x 0.25 mm id capillary column (Varian Inc., Palo Alto, CA) was used to separate fatty acids and methyl esters. Temperature program for separations began at 70°C, held for 1 min, increased to 100 at 5°C/min, held for 3 min, increased to 175°C at 10°C/min, held for 45 min, increased to 220°C at 5°C/min and held for 15 min. Total runtime was 86.5 min. Temperatures for injector and detector were 250 and 300°C, respectively. A customized mixture of pure methyl ester standards as described by Loor and Herbein (2003) was used to identify peaks and determine individual response factors. Data were integrated and quantified using ChemStation (Agilent Technologies).
Statistical Analysis
The data for each triangle test were analyzed by the number of correct responses vs. the total number of responses. Because 2 sets of 3 samples were presented to each panelist, responses were considered correct only when the panelist was able to identify the odd sample for both presentations. Parameters were defined at n = 24,
= 0.05, ß = 0.05, and
d = 50%; the critical number of correct responses for significance was 13 out of 24 (Meilgaard et al., 1999). One test was administered per repetition, and tests were not replicated.
One-way ANOVA, Tukeys honestly significant difference, and linear regression were used to analyze the data. A P-value of < 0.05 was considered to be significant. Data were analyzed using Jmp In (Version 4.04, SAS Institute, Cary, NC) software.
| RESULTS AND DISCUSSION |
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Sensory Changes
Fresh, high quality milk has a delicate odor and flavor, so minor variations in chemical composition could render it unacceptable by consumers (Walstra and Jenness, 1984). The odor of fresh, raw goat milk (control) was different (P < 0.05) from goat milk that had been exposed to UV light (12 pass UV) but not different from milk that was passed through the processor 12 times without being exposed to UV (12 pass no UV; Table 1
). Based on these comparisons, it is evident that the UV exposure did cause changes in the milk that affected the milk odor. Panelists did not describe any off-odors for the control or 12 pass no UV samples; however, some panelists described the odors from the 12 pass UV samples as manure, stinky, barnyard, and goaty, suggesting that the changes were not positive.
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Acid degree values were used as an indicator of hydrolytic rancidity. Initial ADV were less than the values after 6 passes and approximately 7.8 mJ/cm2 UV dose (P < 0.05). The ADV increased as a result of turbulence (Table 3
) regardless of UV exposure. The final ADV for 12 pass UV and 12 pass no UV samples were different than the corresponding control samples as determined by Tukeys honestly significant difference (P < 0.05).
Historically, an ADV of >1.0 mEq/100 g in cow milk is indicative of rancid off-flavors. The greatest ADV in this study was 0.51 ± 0.05 mEq/100 g for milk treated with approximately 15.6 mJ/cm2 UV dose (P < 0.05). According to the Standard Methods for Evaluation of Dairy Product (Marshall, 1993), an ADV of <1.0 mEq/100 g suggests that a sensory change should not be evident. Duncan and Christen (1991) evaluated the relationship between ADV and rancid off-flavors. They found that short-chained fatty acids (C4 to C8) did not enter the fat phase recovered by the ADV procedure in quantities comparable with medium- (C10 to C16) or long-chain (C18:0 to C18:1) fatty acids. Their results implied that the ADV procedure did not measure the fatty acids responsible for rancid flavor (C4 to C12) at the same rate as the longer chained fatty acids (Duncan and Christen, 1991). Relative to cow milk, goat milk has a greater amount of short-chain fatty acids; therefore, there may have been limitations of the ADV analysis to measure a true hydrolytic rancidity score. Regardless, the results of this study show that the ADV values were not great enough to imply rancidity in fresh goat milk and milk that had been subjected to agitation only; this was consistent with triangle test results.
The temperature of milk is very important during agitation because lipase activity is greatest between 37 and 40°C and least at cold storage temperatures below 5°C (Deeth and Fitz-Gerald, 1995). The processing temperature in this study was maintained at or below 4°C to minimize lipase activity. The turbulent flow induced by the pumping of the milk through the CiderSure 3500 UV processor was not enough to cause perceivable changes in the odor of the milk in the first 6 h after processing. No shelf life study was conducted to see if changes in odor developed after time.
Gas chromatograms of headspace volatile compounds in fresh, raw goat milk (control) and milk that had been treated with approximate UV doses of 7.8 and 15.6 mJ/cm2 are shown in Figure 1
. The chromatogram of fresh milk exhibited a large peak after a RT of
2.92 min and then a much smaller peak after
3.15 min. These peaks were consistent for each treatment, and there were no statistical differences in peak area for each treatment. The formation of other volatile peaks was displayed on the chromatogram after a UV dose of 7.8 mJ/cm2. Pentanal, hexanal, and heptanal were detected after exposure to UV, but methyl sulfide was not.
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| CONCLUSIONS |
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| ACKNOWLEDGEMENTS |
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Received for publication October 3, 2006. Accepted for publication March 12, 2007.
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