Effects of Ultraviolet Irradiation on Chemical and Sensory Properties of Goat Milk

Article Outline

• Abstract
• Introduction
• Materials and Methods
  • Goat Milk
  • UV Irradiation
  • Sensory Testing
  • Chemical Analyses
  • Fatty Acid Analysis
  • Statistical Analysis
• Results and Discussion
  • Composition of Goat Milk
  • Sensory Changes
  • Chemical Analyses
• Conclusions
• Acknowledgments
• Supplementary data
• References
• Copyright

Abstract 

Sensory and chemical consequences of treating goat milk using an UV fluid processor were assessed. Milk was exposed to UV for a cumulative exposure time of 18s and targeted UV dose of 15.8±1.6 mJ/cm². A triangle test revealed differences between the odor of raw milk and UV irradiated milk. Oxidation and hydrolytic rancidity was measured by thiobarbituric acid reactive substances and acid degree values (ADV). As UV dose increased, there was an increase in thiobarbituric acid reactive substance values and ADV of the milk samples. A separate set of samples were processed using the fluid processor but with no UV exposure to see if lipase activity and agitation from pumping contributed to the differences in odor. The ADV increased at the same rate as samples exposed to UV; however, sensory studies indicated that the increase of free fatty acids was not enough to cause detectable differences in the odor of milk. Solid phase microextraction and gas chromatography were utilized for the analysis of volatile compounds as a result of UV exposure. There was an increase in the concentration of pentanal, hexanal, and heptanal (relative to raw goat milk) after as little as 1.3 mJ/cm² UV dose. Ultraviolet irradiation at the wavelength 254nm produced changes in the sensory and chemical properties of fluid goat milk.

Key words: goat milk, ultraviolet, solid phase microextraction and gas chromatography, acid degree value

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Introduction 

The market for goat milk products is expanding, and the consumer trend toward fresher and more natural food choices has created a niche market for minimally processed and raw dairy foods (Reed and Grivetti, 2000). Consumer preference for raw dairy products is driven, in part, by perceived superior sensory characteristics that cannot be obtained when the milk is heat-treated (Buchin et al., 1998). Although thermal pasteurization of milk has proven to be effective at reducing pathogenic bacteria and controlling lipase activity, UV irradiation may be an alternative method that would be less costly than traditional pasteurization methods for producers working with smaller volumes of milk.

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 280nm and 700nm, 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 254nm did not report negative sensory data (Capstick, 1946; Burton, 1951; Caserio et al., 1975). Matak et al. (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.

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Materials and Methods 

Goat Milk 

Fresh, raw goat milk was purchased in 18-L quantities from a commercial dairy. Milk was collected from the bulk tank, transported, and stored at ≤4°C in an insulated covered 18.9-L (5-gal) cooler (Igloo Products Corp., Houston, TX) that had been sanitized with 200ppm of hypochlorite solution. Samples were processed within 12h of collection, and all analyses, except fatty acid profile, were concluded within 8h 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],

[1]

Milk (3L) was passed through the processor 12 consecutive times for the targeted exposure time of 18s 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 2s as measured by 2 UVX-25 sensors (UVP Inc., Upland, CA). A separate batch of milk (1.5L) 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 et al. (2004). Total cleaning time was less than 2min, and total processing time (including cleaning) was less than 30min.

An aliquot (100mL) 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 (∼15mL) 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 6h 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 6h 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 532nm (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 et al. (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 1h. 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 500μg/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 (25mL) 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 6h of processing. The solid-phase microextraction fiber was inserted through the septum and positioned approximately 1cm above the milk surface to allow maximum exposure to milk headspace, and exposed for 22min 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×0.32mm, 1.05μm, 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 30s at 35°C, 15°C per min to 180°C, hold 30s, 20°C per min to 260°C, hold 30s.

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 (1mL) 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 0.5μL, and split ratio 100:1. A Chrompack CP-Sil 88 100 m×0.25mm 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 1min, increased to 100 at 5°C/min, held for 3min, increased to 175°C at 10°C/min, held for 45min, increased to 220°C at 5°C/min and held for 15min. Total runtime was 86.5min. 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, α=0.05, β=0.05, and ρ_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, Tukey's 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.

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Results and Discussion 

Composition of Goat Milk 

Milk composition was typical, averaging 92% moisture and 8% solids for each replication. Fat and protein contents were 4.1% (±0.09) and 2.9% (±0.03), respectively.

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.

Table 1. Triangle test responses for odor differences in UV irradiated raw goat milk (12 pass UV), pumped raw goat milk (12 pass no UV), and untreated fresh goat milk (control)

Chemical Analyses 

In general, the fatty acid profile was not significantly altered by UV treatment (Table 2).There was a change in the conjugated linoleic acids (CLA) after UV processing (P<0.05). Compared with fresh goat milk (control), the 12 pass UV treated milk showed a 52% decrease of C18:2 cis-9, trans-11 and a 1,050% (10-fold) increase of C18:2 trans, trans. Timmons et al. (2001) reported that as concentrations of unsaturated fatty acids in milk increase, particularly of PUFA C18:2 and C18:3, milk becomes more susceptible to oxidation. Kim et al. (2003) reported that volatile compounds were formed in goat milk cheese stored under fluorescent light for 2 d at 30°C. Sensory evaluation confirmed that samples stored under these lighted conditions had more off-flavors than samples stored in the dark. Kim et al. (2003) concluded that the light-induced volatile compounds were formed from singlet oxygen oxidation of oleic acid in the cheese. In this current study, differences in oleic acid concentrations were not detected between UV-treated and non-UV-treated samples; however, sensory differences were detected.

Table 2. Average fatty acid profile of fresh, raw goat milk (control); milk that was pumped through the machine but not exposed to the UV source (no UV); and UV processed milk (UV)¹

The TBARS values increased as UV dose increased in goat milk samples (P<0.05; R²=0.93, 0.97, and 0.95 for replications 1, 2, and 3, respectively). The slope of the line was very similar for each replication; however, when data were combined from the 3 replications, a low R² value, or low predictive power, resulted. This may be attributed to natural variations in the composition of milk, most likely milkfat, between milk collections caused by lactation stage and season. The TBARS values of milk processed with no UV exposure indicated that milk agitation alone did not cause a measurable increase in secondary products of oxidation, such as malondialdehyde (Table 3). The TBARS values ranged from 0.31 (±0.09) mg/L in the control milk to 0.58 (±0.11) mg/L in UV-irradiated milk. These trends are consistent with those reported by van Aardt et al. (2005b) who found that cows’ milk exposed to light for 10h had greater TBARS values (0.92 mg/L) than milk protected from light exposure (0.59 mg/L). The TBARS values reported in this current study are modest when compared with van Aardt et al. (2005b); however, the samples in the previously mentioned study received fluorescent light exposure over the broad visible and UV wavelength spectra, including all the riboflavin excitation wavelengths. Riboflavin absorbs light of specific wavelengths, principally 250, 270, 370, 400, 446, and 570nm when in foods with a neutral pH (Kyte, 1995); it is considered to play a significant role as a sensitizer in the photooxidation process of dairy products. Lee (2002) found that the concentration of riboflavin in milk was directly related to the extent of off-flavors when milk was exposed to fluorescent light; our reported TBARS values may be less than other reported values because the concentration of riboflavin in goat milk is less than cows’ milk (Souci et al., 2000).

Table 3. Thiobarbituric acid-reactive substances (TBARS) values (mean±SD) and acid degree values (ADV; mean±SD) of fresh, raw goat milk in response to increasing doses of UV light

Approximate UV dose (mJ/cm2)	Number of passes	TBARS (mg/L)	ADV (mEq/100g)
	Mean±SD
0.0	01	0.31a±0.09	0.1a±0.05
0.0	122	0.34a±0.11	0.36b±0.15
5.2	4	0.37a±0.11	0.29a±0.05
7.8	6	0.44a,b±0.10	0.36b±0.12
10.4	8	0.50b±0.12	0.31a,b±0.11
15.6	123	0.58b±0.11	0.40b±0.09

There was no increase in TBARS values in nonUV-treated samples, which implies that UV light exposure was a significant factor for increasing production of oxidation byproducts.

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 3s) 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 Tukey's honestly significant difference (P<0.05).

Historically, an ADV of >1.0 mEq/100g in cow milk is indicative of rancid off-flavors. The greatest ADV in this study was 0.51±0.05 mEq/100g 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/100g 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, 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 6h 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 ∼2.92min and then a much smaller peak after ∼3.15min. 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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• Figure 1. 

  The production of headspace volatile compounds was detected by solid-phase microextraction and gas chromatography of A) fresh, raw goat milk, B) goat milk exposed to a UV dose of approximately 7.8 mJ/cm², and C) goat milk exposed to a UV dose of approximately 15.6 mJ/cm². 1=pentanal; 2=hexanal; and 3=heptanal. Compounds identified by retention time to known standards.

The relative peak area (area counts) for volatile compounds identified by gas chromatographic RT in fresh and treated goat milk samples are contained in Table 4. Retention times of known compounds were pentanal (RT 4.46), hexanal (RT 6.09), and heptanal (RT 8.35). There was an increase in peak areas of pentanal, hexanal, and heptanal after 7.8 and 15.6 mJ/cm² UV dose, respectively (P<0.05). A preliminary study indicated that these compounds were formed in goat milk exposed to as little as 1.3 mJ/cm² UV dose. Lee (2002) found that milk stored at 4°C under fluorescent light displayed pentanal and hexanal formation before 2h and heptanal formation in fewer than 4h. Marsili (1999) reported that the reaction kinetics of pentanal and hexanal were different and offered that these differences were due to the availability of substrates. Lee (2002) also reported that fat content had an effect on the formation of these volatile compounds; as fat content increased from 0.5 to 1.0, 2.0, and 3.4%, there was an increase in formation of the volatile compounds (Lee, 2002). The peak areas for each compound in photosensitized whole milk (8h under fluorescent light at 4°C) were consistent with those reported in this current study (Lee, 2002). These compounds were not present in the samples that received zero light exposure.

Table 4. Mean relative peak area (area counts)±SD (1×10⁴) of headspace volatile compounds in goat milk formed as a result of UV irradiation

			UV dose (mJ/cm2)3
RT1	Volatile compound2	Fresh goat milk	0	7.8	15.6
4.46	Pentanal	ND4	ND	0.91a±0.21	1.76b±0.38
6.09	Hexanal	ND	ND	2.03a±0.22	3.07b±0.27
8.35	Heptanal	ND	ND	0.60a±0.07	1.38b±0.32

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Conclusions 

The pursuit to find alternative processing technologies to replace or augment traditional thermal methods should begin with an assessment of safety parameters. However, when developing novel technologies, sensory properties and consumer acceptance must also be given serious consideration. Even though we have previously shown that UV treatment is effective for the reduction of the bacterial pathogen Listeria monocytogenes in goat milk, the present study indicated that UV irradiation at the wavelength 254nm had aromatic and chemical consequences.

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Acknowledgments 

This work was funded by USDA-CSREES #2001-5110-11363. Resources were also provided by The Virginia Tech Dairy Foods Research Group. We would like to thank Phil Hartman of FPE Inc. for the time and effort put into the maintenance of the CiderSure 3500. Thanks to Wendy Wark and Joseph Herbein (Dairy Forage Labs, Virginia Tech) for their work on the fatty acid analyses.

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Supplementary data 

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