Volatile aroma composition of distillates produced from fermented sweet and acid whey

J. Dairy Sci. 102:1–9 https://doi.org/10.3168/jds.2018-14737 © American Dairy Science Association®, 2019.

Volatile aroma composition of distillates produced from fermented sweet and acid whey Derrick Risner, Elizabeth Tomasino,* Paul Hughes,* and Lisbeth Meunier-Goddik Department of Food Science & Technology, Oregon State University, Corvallis 97331

ABSTRACT

as emulsifiers and foaming agents, used within food systems (Ryan and Walsh, 2016). The conversion of sweet whey to these value-added products operates on an economy of scale and is only feasible for large-scale producers, not smaller artisanal creameries, even if such companies pool their whey supplies. Federal and local environmental regulations have eliminated historical small-scale whey disposal methods, such as the dumping of whey through land applications (Kosikowski, 1979; Kettering et al., 2017). These limitations are necessary due to whey’s biochemical oxygen demand of 40 to 60 g/L and chemical oxygen demand of 50 to 80 g/L (Siso, 1996; Dragone et al., 2009). Disposal of whey at a waste treatment facility can be costly for creameries (Dragone et al., 2009) and varies by municipality. Additionally, acid whey processing is problematic at any scale due to differences in both composition and electrostatic protein interactions (Chandrapala et al., 2015). These factors have led artisanal creameries and acid whey producers to seek alternative means of whey processing that are economically and environmentally viable. One avenue includes the conversion of the lactose within whey to ethanol. The fermentation process reduces the biochemical oxygen demand by approximately 75% (Siso, 1996) and it has been illustrated on an artisanal creamery level to be environmentally advantageous (Risner et al., 2018). Research concerning the fermentation of lactose into ethanol has been ongoing for decades (Whittier, 1944; Sansonetti et al., 2009; Tomaszewska and Białończyk, 2016). Kluyveromyces marxianus can convert lactose into glucose and galactose and metabolize these monosaccharides into ethanol via fermentation. Research includes maximization of K. marxianus fermentation efficiency by optimization of fermentation conditions (Diniz et al., 2014; Hadiyanto et al., 2014). Whereas many studies have used K. marxianus as the ethanolproducing organism, others have used Saccharomyces cerevisiae with exogenous enzyme additions (Parashar et al., 2016; Tomaszewska and Białończyk, 2016) or genetic modification (Silva et al., 2010). Ozmihci and Kargi (2007a), Koushki et al. (2012), and Guimarães et al. (2010) compared ethanol production between K.

Lactose within whey can be fermented and distilled to produce a potable distilled spirit. The aim of this study was to determine if acid and sweet whey types can be fermented and distilled using similar processes and to investigate differences in volatile aroma compounds for the 2 distillates. Fermentation and distillation of the 2 whey types progressed in a similar manner, using Kluyveromyces marxianus for the initial fermentation and a glass still fitted with a Vigreux column for the subsequent distillation. Ethanol content of the wash (fermented whey) varied considerably following each fermentation and ranged from 1.2 and 2.0% (wt/wt) with no clear trend between acid and sweet whey samples. Volatile aroma compounds were extracted using headspace solid-phase microextraction and identified via gas chromatography-mass spectrometry. Acid and sweet whey distillates contained unique volatile aromatic compounds, and significant differences in compound peak areas were observed. These differences may have an effect upon the organoleptic qualities of spirits produced from whey; therefore, whey source may be an important factor when fermenting and distilling whey. Key words: artisan spirit, ethanol, headspace solidphase microextraction, gas chromatography-mass spectrometry INTRODUCTION

Whey is broadly categorized into 2 types: sweet and acid whey. Sweet whey is produced from renneted cheese, such as Cheddar and Gouda, whereas acid whey is generally produced from acid-precipitated cheeses, such as cottage cheese and Greek yogurt. Sweet whey is typically centrifuged and clarified to remove curd fragments and lipids before being processed further into protein powders and lactose or specialty products, such Received March 12, 2018. Accepted September 12, 2018. *Corresponding authors: elizabeth.tomasino@​ oregonstate​ .edu and Paul.hughes@​oregonstate​.edu

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marxianus strains and other species and documented ethanol production efficiencies ranged from 75.9 to 96.6%. These efficiency differences may be due to the use of raw cheese whey (Sansonetti et al., 2009), whey permeate (Diniz et al., 2014), and reconstituted whey cheese powder (Ozmihci and Kargi, 2007b) as the fermentation substrate. Most research has focused on the production of ethanol for biofuel. Bioreactor systems and novel ethanol-separation techniques have been applied to whey processing (Ozmihci and Kargi, 2008; Gabardo et al., 2014; Tomaszewska and Białończyk, 2016). Whey has been demonstrated to be a feasible substrate in batch and continuous bioethanol production processes (Ghaly and El-Taweel, 1997; Gabardo et al., 2014; Hadiyanto et al., 2014). The commercial production of potable ethanol from fermented whey has been documented since 1978 (Ling, 2008). Craft distilleries are beginning to explore the use of whey to produce ethanol, and several commercial examples are readily available including Black Cow and Vermont White vodka (Vermont White, 2016; Black Cow, 2017). Research related to the production of potable distilled whey spirit has not been extensively explored. Volatile compounds within a whey-based spirit have been identified using GC-MS and have shown that whey-based spirits are similar to other distilled beverages and safe for consumption (Dragone et al., 2009). It is not known how distillate composition varies based upon whey source. The lack of research in this area is significant because whey composition can vary depending on style of cheese, suggesting that volatile aroma compounds in the distillates could vary as well. The goal of our research was to determine if a distillate can be produced from acid whey in a manner similar to that of sweet whey and to analyze volatile aroma compounds in acid whey and sweet whey distillates. This information will aid potential producers of wheybased distilled spirits determine which whey type to use as fermentation substrate and thereby promote the development of palatable, consistent products. MATERIALS AND METHODS Materials

Sweet whey, produced during Cheddar cheese production, was obtained from the Arbuthnot Dairy Center (Corvallis, OR) and immediately processed. Acid whey, produced during cottage cheese production, was obtained from Umpqua Dairy Products (Roseburg, OR) and all samples were stored at −20°C until processing. A lactose fermentative yeast strain, Kluyveromyces marxianus Y-1109 from the Saccharomycetaceae famJournal of Dairy Science Vol. 102 No. 1, 2019

ily was obtained from the USDA Agricultural Research Service (ARS) culture collection (Peoria, IL). Yeast peptone dextrose (YPD) and yeast culture-grade agar were purchased from Sunrise Science Products (San Diego, CA). Diammonium phosphate was purchased from Cellar Science (Pittsburg, CA). Sodium chloride was obtained from Fisher Chemical (Geel, Belgium) and alkane series C7-C30 was purchased from Sigma-Aldrich (Darmstadt, Germany). Preparation of Inoculum

Kluyveromyces marxianus was rehydrated in 15 mL of YPD solution (20 g/L of dextrose, 20 g/L of casein peptone, 10 g/L of yeast extract). Inoculum was immediately aseptically plated and streaked for colony separation upon an YPD agar plate (20 g/L of dextrose, 20 g/L of agar, 20 g/L of casein peptone, 10 g/L of yeast extract) and incubated for 48 h at 37°C. Kluyveromyces marxianus was propagated in an approach common to commercial breweries and distilleries (Bellissimi and Richards, 2009). Three K. marxianus colonies were isolated from the YPD agar plate and inoculated into three 1-mL aliquots of YPD. Cultures were grown anaerobically in screw-cap tubes at 37°C at 200 rpm for 24 h. Each inoculum was added to 10 mL of YPD and aerobically grown in test tubes at 37°C at 200 rpm for 48 h. These inocula were added to 100-mL volumes of YPD in 250-mL Erlenmeyer flasks and grown aerobically at 37°C at 200 rpm for 24 h. These 3 inocula were then combined in a single 1,000-mL Erlenmeyer flask and stored at 4°C for no longer than 2 wk. The combined culture was used to inoculate pasteurized whey. The inoculum was replenished as needed by adding 30 mL of the inoculum to 300 mL of YPD in a 1,000-mL Erlenmeyer flask, which was then incubated aerobically at 37°C at 200 rpm for 24 h and stored at 4°C until use. Cheese Whey Fermentation

Sweet whey (S1, S2, S3, and S4) and acid whey (A1, A2, A3, and A4) were obtained from 2 different regional creameries on 4 different production days for a total of 8 samples (4 samples from each creamery). Each type of whey was processed identically, except frozen acid whey was thawed using a steam-jacketed kettle. Diammonium phosphate was added at a rate of 2.11 g/L before pasteurization as a nitrogen source that aids fermentation. Two liters of whey was batch pasteurized in Erlenmeyer flasks with magnetic stirring at 63°C for 30 min. Each batch of whey was immediately cooled in an ice bath to 37°C and separated into three 500mL replicates for fermentation. All fermentations were conducted in triplicate except S2, which was conducted

FERMENTATION AND DISTILLATION OF SWEET AND ACID WHEY

in duplicate, for a total of 12 acid whey fermentations and 11 sweet whey fermentations. Before inoculation of whey, a cell count of the inoculum was conducted using a hemocytometer (Gizmo Supply Co., Fountain Valley, CA) in the manner described by American Society of Brewing Chemists (ASBC) method (ASBC, 2016). The desired inoculation rate was 2 × 107 cells/mL, which was used to determine the volume of inoculum required for each replicate. The 500-mL aliquots of cooled, pasteurized whey were inoculated immediately and placed in an incubator for aerobic incubation for 120 h at 37°C with no additional agitation. The pH was measured, both immediately after cooling of pasteurized whey and after fermentation of the replicates, using a Corning pH meter 125 (Corning Inc., Corning, NY). Samples of pasteurized whey from each production day (S1, S2, S3, S4 and A1, A2, A3, A4) and samples from each fermented whey replicate (12 samples of fermented acid whey and 11 samples of fermented sweet whey) were stored at −80°C until further analysis. Distillation

Distillation of each fermented whey replicate (a total of 12 acid whey fermentations and 11 sweet whey fermentations) was conducted using a 500-mL laboratory still fitted with a Vigreux column (Glassco, Beckenham, United Kingdom). The laboratory still was filled with 250 mL of fermented whey and nonreactive boiling chips were added. Vapor temperature was kept at or below 95°C at the condenser/column connection point. The ethanol content of each distillate was monitored throughout the distillation. Distillation continued until 99% (vol/vol) of the ethanol was collected from the fermented whey. Collection occurred in this manner to simulate beverage distillation industry standards for ethanol collection (Hughes and Buxton, 2014; Nicol, 2014). The common distillation industry practice of making cuts was not conducted to ensure experimental repeatability. The collected distillate was stored in nonreactive 2-mL, screw-top, amber vials crowned with a solid cap with a polytetrafluoroethylene liner (12 mm) purchased from Supelco (Poznań, Poland) and stored at −10°C until further analysis. Sugar Analysis by HPLC

Immediately before sugar analysis, samples were thawed and fermented whey replicate samples were combined based upon production day. Lactose, glucose, and galactose content of whey and fermented whey was quantified using HPLC using an Agilent HPLC 1100 (Agilent Technologies, Santa Clara, CA) equipped with a refractive index detector 1200 (Agilent Technologies).

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The Agilent HPLC 1100 was fitted with a 300 × 6.5 mm Waters Sugar Pak I column (Waters, Milford, MA) with an external column heater set at 90°C, using HPLC-grade water as the eluent at a flow rate of 0.5 mL/min, and injection volume of 10 µL. The method was derived from AOAC 977.20 (AOAC International, 2016) with the following modifications: 1 g of initial sample was diluted with deionized water for a final weight of 10 g, a sugar test solution was prepared in deionized water on a weight/weight basis. The mobile phase was HPLC-grade water and the addition of galactose. Ethanol Analysis

Ethanol content of the fermented whey was determined by near infrared spectroscopy immediately before distillation using a DMA 4500 M Anton Paar (Anton Paar GmbH, Gaaz, Austria) equipped with an Anton Paar Alcolyzer Beer ME module (Anton Paar GmbH). Sample were prepared as described in ASBC method analysis (ASBC, 2004) with the addition of manufacturer-recommended centrifugation of samples at 1,500 × g for 10 min at room temperature (approximately 20°C) immediately after the filtration step in the method. The ethanol content of the distillate was measured using a hand-held Anton Paar DMA 35 density meter (Anton Paar GmbH), which measures alcohol content based upon the temperature-corrected density of sample. Extraction and Analysis of Volatile Compounds

Volatile compounds from each distillate were extracted using headspace solid-phase microextraction (HS-SPME) and analyzed using a Shimadzu GCMS QP2010Ultra (Shimadzu Corporation, Kyoto, Japan) with an AOC-5000plus autosampler (CTC analytics, Zwingen, Switzerland). The GC-MS was equipped with an Rxi-5ms capillary column (Crossbond diphenyl dimethyl polysiloxane, Restek Corporation, Bellefonte, PA) with a length of 15 m, internal diameter of 0.25 mm, and film thickness of 0.25 µm. The GC oven parameters were as follows. The injector temperature was 250°C with a split ratio of 0. The oven temperature was held at 35°C for 10 min, increased to 250°C at a rate of 4°C/min, and then maintained at 250°C for 10 min. Helium was used as a carrier gas with linear velocity flow control set at 55 cm/s and total flow rate of 3.2 mL/min. The total run time was 73.75 min. The GC to MS transfer line was 250°C. The mass spectrometer’s ion source was 200°C and the capillary direct interface was maintained at 250°C. The detector used electron impact ionization (70 eV) in a full-scan mode from 3 Journal of Dairy Science Vol. 102 No. 1, 2019

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to 73 min with a scan range of m/z 33 to 303 at an acquisition rate of 5 spectra/s. Whey distillate was diluted before analysis to 7.0% (vol/vol) ethanol using a saturated NaCl solution (Fisher Chemical) to achieve a total volume of 2 mL contained in 20-mL amber glass, screw-capped vials (22.5 × 75.5 mm; Supelco). All samples were maintained at 7°C until analysis using a stack cooler that was part of the autosampler. Volatile compound extraction occurred using 3 phase Stableflex fiber (50/30 µm DVB/CAR/PDMS, 2 cm, 24 gauge; Supelco). The fiber was conditioned at 250°C for 1 h before analysis. Immediately before extraction, samples were incubated for 10 min at 50°C while the vial was simultaneously agitated at 500 rpm (5 s on, 2 s off). The samples were extracted for 10 min with no further agitation. The fiber was then injected into the GC-MS for 10 min at 250°C followed by conditioning in an NDL heater (CTC Analytics, Zwingen, Switzerland) for 10 min at 250°C. The above method was also used with a Stabilwax column (Crossbond polyethylene glycol, Restek Corporation) with a length of 15 m, internal diameter of 0.25 mm, and film thickness of 0.25 µm to determine compounds that are more suited to polar separation mechanisms. Chromatograms were analyzed using GCMSsolution ver. 4.2 (Shimadzu Corporation). Compound identification was validated using retention index (C7-C30 alkane series, Sigma-Aldrich) and NIST2011 library for fragrance and flavors (NIST, Gaithersburg, MD). Volatile compounds were identified by comparing their mass spectra with the NIST11 spectrum library (Standard Reference Database Program, National Institute of Standards and Technology, Gaithersburg, MD). Linear retention indexes were also calculated from the retention times of n-alkanes (C7–C30) and compared with those from literature. Statistical Analysis

Statistical analysis of the fermented whey pH and ethanol content was conducted using ANOVA single factor analysis and Tukey’s honest significant difference test. Replicate pH readings were converted to H+ concentrations. Ethanol content was not transformed in any manner. Analysis of variance, Tukey’s honest significance difference test, and Welch’s t-test were conducted using Microsoft’s Excel 2016 (Microsoft Corp., Redmond, WA) data analysis add-in. Welch’s t-test was used to determine if differences in the peak areas of sweet and acid whey distillates were statistically significant. Principal component analysis was conducted using XLSTAT version 2014.6.01 (Addinsoft, New York, NY). Journal of Dairy Science Vol. 102 No. 1, 2019

RESULTS Proximate Analysis

The lactose content and pH of the whey samples are summarized in Table 1. The pH analysis of the pre- and postfermented whey indicate that the sweet whey is acidified during fermentation, which lowers the pH by more than 2 units (approximately 6.8 to 4.3). The acid whey underwent little to no change in pH during fermentation with exception of A2, where the postfermentation pH increased. As expected, the lactose content of each treatment was decreased by the fermentation process. The ethanol contents of all fermented whey samples were not significantly different (α = 0.05) and were within the expected range of 1.5 to 2.5% (wt/wt) (Guimarães et al., 2010; Koushki et al., 2012) with the exception of treatment S3, which was below the expected published range. Glucose and galactose concentrations were below the detectable limit for all samples. Volatile Compounds

The GC-MS analysis revealed distinct differences in the composition of volatiles in acid whey distillate and sweet whey distillate (Table 2). The HS-SPME is a nonexhaustive sampling method, but a comparison of relative compound concentrations can be made between each distillate type based on peak area. This allows for statistical analysis of volatile composition of each distillate. Differences in volatile composition based upon type of whey distillate is illustrated by a principal component analysis (PCA; Figure 1). The variables within the PCA are the compounds identified during the GC-MS analysis. This comparison shows a distinct separation of acid and sweet whey distillates. The distinct separation illustrated in the PCA is due to the presence of volatile compounds unique to each type of fermented whey. Both acid and sweet whey distillates contained compounds unique to their distillate type; however, the majority of identified compounds were present in both distillates. The following compounds were identified in all samples and replicates. Concentrations of ethyl dodecanoate and nonanal were not significantly different between acid and sweet whey distillates. Concentrations of isobutyl alcohol, dodecanoic acid, ethyl hexanoate, and ethyl octanoate were significantly greater in sweet whey distillate, whereas concentrations of farnesol and β-farnesene were significantly greater in acid whey distillate. 2-Heptanone, 2-pentadecanone, 2-tridecanone, and 2-undecanone were identified exclusively in sweet whey

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FERMENTATION AND DISTILLATION OF SWEET AND ACID WHEY

distillate (Table 2); all 5 are part of a homologous series of methyl ketones. Heptyl acetate was identified exclusively in sweet whey distillate. Isobutyl alcohol and 2-nonanone were identified in both whey distillates, but in significantly greater concentrations in sweet whey distillate. Only 1 compound, geraniol, was identified exclusively in acid whey distillate. Farnesol, limonene, and farnesene were identified in both distillates but were of significantly greater concentrations in acid whey distillate. Phenylacetaldehyde was also found in significantly greater concentrations in the acid whey distillate. Sweet whey distillate contained significantly greater concentrations of identified alcohols, acids, esters, and ketones, whereas acid whey distillate contained significantly greater concentrations of identified aldehydes, terpenes, and terpenoids. This demonstrates clear differences in composition of volatile compound between acid and sweet whey distillates. DISCUSSION pH Behavior During Fermentation of Sweet and Acid Whey

The decrease in pH is likely due to the production of organic acids and absorption of basic AA during the fermentation process (Coote and Kirsop, 1976), as K. marxianus has been shown to produce acetic, propionic, succinic, malic, and citric acid (Wilkowska et al., 2015). In contrast, the lower starting pH of the acid whey would likely influence the metabolic products of K. marxianus (Aktaş, et al., 2006). Furthermore, as pH approaches 4.5, whey protein’s isoelectric point (Morand, et al., 2012), additional buffering would occur, possibly limiting the effects of organic acid production upon pH of the fermentate.

Differences in Volatile Aroma Compounds

The significant differences in acid and sweet whey can be roughly based on 2 compositional criteria: the differences in presence of terpenes or terpenoids and microbial metabolism products such as esters, alcohols, aldehydes, and ketones. A larger concentration of free fatty acids could be attributed to differences in processing between the styles of cheese associated with the whey. Cheddar cheese production uses a full-fat milk to form curds, whereas cottage cheese is produced from skim milk. The increased presence of fat in whey produced in cheddar cheese production allows the lipases within bacterial cultures or native to milk more opportunity to hydrolyze triacylglycerides more effectively. This increases the free fatty acids within whey and, subsequently, the esters produced from the fatty acid metabolism by K. marxianus. However, fatty acid peak areas of sweet and acid whey were not found to be significantly different (data not shown). Differences in ester peak areas of the distillates may have come from other conditions affecting the microbial metabolism or other formation pathways, such as transesterification or Fischer esterification. Sweet whey distillate samples contain significantly greater amounts of esters than acid whey distillate (Table 2). The identity of the esters present in each distillate are typically found in alcoholic beverages (Khio et al., 2012). Formation of the esters may be attributed to the fatty acid metabolism by K. marxianus (Lam and Proctor, 2002) as well as other microbial metabolic processes. The esters identified in the whey distillates provide fruity and floral aromas (Flavor and Extract Manufacturers Association, 2017) and are usually desirable in distilled spirits. These compounds provide a positive organoleptic quality to the distillate depending

Table 1. Lactose and ethanol content along with pH of pre- and postfermentation whey samples Prefermentation Sample1

pH

Lactose2 content (%, wt/wt)

A1 A2 A3 A4 S1 S2 S3 S4

4.8 5.2 5.2 4.9 6.8 6.7 6.9 6.7

3.6 4.1 4.4 4.5 4.7 5.1 5.0 5.1

Postfermentation  

Lactose content (%, wt/wt)

pH 4.9 5.53 5.2 4.8 4.2 4.2 4.7 4.2

± ± ± ± ± ± ± ±

0.0 0.2 0.2 0.0 0.1 0.0 0.6 0.0

1.5 <0.5 <0.5 <0.5 0.6 <0.5 1.5 0.8

Ethanol content (%, wt/wt) 1.5 1.7 1.6 1.8 1.6 2.0 1.22 1.9

± ± ± ± ± ± ± ±

0.3 0.1 0.2 0.1 0.2 0.2 0.2 0.1

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A = acid whey, S = sweet whey. Glucose and galactose levels were below detectable limits. 3 Significant difference determined via single factor ANOVA and Tukey’s honestly significant difference test (α = 0.05). 2

Journal of Dairy Science Vol. 102 No. 1, 2019

Journal of Dairy Science Vol. 102 No. 1, 2019

1972 1179 2188 1107 1040 1460

1109 1610 2521 1684 1841 1002

1529 1430 2055 1122 883 1097 2024 1494 1298 1020 1714 1520

1741 1454

Compound

1-Dodecanol 1-Nonanol* 1-Tetradecanol Isobutyl alcohol* Phenylacetaldehyde* Furfural

Nonanal Tetradecanal Dodecanoic acid* Ethyl-9-decenoate Ethyl dodecanoate Ethyl hexanoate*

Ethyl nonanoate * Ethyl octanoate* Ethyl tetradecanoate Heptyl acetate 2-Heptanone* 2-Nonanone* 2-Pentadecanone* 2-Tridecanone* 2-Undecanone* D-Limonene* Farnesol* Geraniol*

α-Farnesene* β-Farnesene*

125037–13–0 18794–84–8

123–29–5 106–32–1 0124–06–01 0112–06–01 110–43–0 821–55–6 2345–28–0 593–08–08 0112–12–09 5989–27–5 4602–84–0 106–24–1

124–19–6 124–25–4 0143–07–07 67233–91–4 106–33–2 123–66–0

112–53–8 0143–08–08 112–72–1 78–83–1 122–78–1 1998–01–01`

CAS

1

Not available Fat, floral, green, oil Not available Apple, bitter, cocoa, wine Berry, geranium, honey, nut, pungent Almond, baked potatoes, bread, burnt, spices Fat, floral, green, lemon Not available Not available Not available Floral, fruit, leaf Apple peel, brandy, fruit gum, overripe fruit, pineapple Floral Apricot, brandy, fat, floral, pineapple Wax Floral, fresh Blue cheese, fruit, green, nut, spice Fragrant, fruit, green, hot milk Green Savory Fresh, green, orange, rose Citrus, mint Oil Geranium, lemon peel, passion fruit, peach, rose Boiled vegetable, floral, wood Not available

FEMA odor descriptors2

2.4E+05 1.6E+05 5.1E+05 3.0E+05 7.2E+05 7.2E+05 5.4E+04 1.8E+06 9.0E+04 0.0E+00 0.0E+00 7.7E+03 0.0E+00 0.0E+00 0.0E+00 3.7E+04 4.3E+05 1.7E+05

14 60,0004 2,200–16,0007 1008 2,0009 13 8506 1477 8008 1,4009 0.9–3,0007 5–2004 1,44010 ND 54,0007 104 204 40–754

9.6E+04 3.1E+05

3.5E+05 4.6E+03 1.0E+04 1.7E+06 9.4E+04 2.3E+05

0.01–0.053 504 ND5 8,000–20,0006 47 3,000–23,0004

ND ND

Acid whey distillate peak area

Odor threshold (µg/L)

5.6E+04 1.4E+05

5.2E+04 9.5E+05 4.5E+04 0.0E+00 0.0E+00 1.4E+04 0.0E+00 0.0E+00 0.0E+00 5.0E+04 2.4E+05 1.6E+05

1.4E+05 1.1E+05 2.0E+05 2.3E+05 4.4E+05 3.7E+05

2.8E+05 1.1E+04 2.2E+04 6.2E+05 4.0E+04 3.3E+05

SD (n = 12)

7.7E+03 1.9E+05

3.8E+05 1.2E+07 7.1E+04 2.4E+04 1.8E+05 6.0E+05 2.0E+05 6.6E+05 1.1E+06 4.4E+03 2.3E+05 0.0E+00

2.4E+05 1.9E+05 8.5E+05 2.8E+06 5.6E+05 1.6E+06

5.0E+05 8.2E+04 2.3E+04 3.9E+06 1.8E+04 2.9E+05

Sweet whey distillate peak area

2.5E+04 5.0E+04

1.7E+05 5.0E+06 6.1E+04 4.8E+04 4.7E+04 2.0E+05 7.6E+04 1.3E+05 2.7E+05 1.5E+04 8.0E+04 0.0E+00

8.3E+04 1.2E+05 3.3E+05 7.5E+05 1.9E+05 5.4E+05

5.1E+05 8.3E+04 3.3E+04 1.6E+06 2.6E+04 1.5E+05

SD (n = 11)

2

Chemical Abstract Service (CAS) registry number used as unique chemical identifier. Odor descriptors were taken from the Flavor and Extracts Manufacturers Association (FEMA) flavor ingredient library (Flavor and Extract Manufacturers Association, 2017). 3 (Ruth, 1986). 4 (Leffingwell and Associates, 1998a). 5 ND = no data found. 6 (Börjesson et al., 1996). 7 (Molimard and Spinnler, 1996). 8 (Tao and Zhang, 2010). 9 (Leffingwell and Associates, 1998b). 10 (Ho, 2013). *Significant differences determined using Welch’s t-test (α = 0.05).

1

Kovats number

Table 2. Average peak area, odor descriptors and thresholds of volatile compounds detected in sweet and acid whey distillate

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FERMENTATION AND DISTILLATION OF SWEET AND ACID WHEY

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Figure 1. Separation of whey distillates by volatile compounds using principal component analysis. Vectors for aroma compound variables are in (A) and scores for whey distillates (A = acid, S = sweet) are in (B). 1 = phenylacetaldehyde; 2 = cis-β-farnesene; 3 = α-farnesene; 4 = d-limonene; 5 = farnesol; 6 = geraniol; 7 = ethyl dodecanoate; 8 = ethyl tetradecanoate; 9 = tetradecanal; 10 = dodecanoic acid; 11 = heptyl acetate; 12 = 1-nonanol; 13 = 2-pentadecanone; 14 = isobutyl alcohol; 15 = 2-tridecanone; 16 = 2-nonanone; 17 = 2-heptanone; 18 = ethyl nonanoate; 19 = ethyl octanoate; 20 = 2-undecanone; 21 = ethyl 9-decenoate; 22 = ethyl hexanoate; 23 = 1-tetradecanol; 24 = furfural; 25 = 1-dodecanol; 26 = nonanal.

upon concentration. It should be noted that distillate produced from sweet whey contained greater levels of esters, which could lead to a spirit with a stronger fruity and floral character. Differences in ketones between acid and sweet whey distillates may also be explained by the increase in free fatty acid concentration of the whey. Ketones formed in whey distillates are likely to be due to β-oxidation of free fatty acids (Cao et al., 2014). These free fatty acids are oxidized to produce β-ketoacids, which in turn are oxidized to produce alkan-2-ones; however, most of these methyl ketones were not found in acid whey distillates, suggesting another possible path for ketone formation. 2-Heptanone is described as having a distinct blue cheese aroma and a detection threshold in water as low as 0.9 µg/L (Molimard and Spinnler, 1996). All ketones identified except 2-undecanone may have a negative effect upon distillate organoleptic quality based their aroma descriptions in Table 2. In the context of this work the presence of 2-heptanone is considered to be highly undesirable due to its low-level odor threshold and distinct blue cheese aroma. A major difference between sweet and acid whey distillates is due to terpene and terpenoid content. Geraniol was only present in acid whey distillates and

demonstrated greater peak areas than the other terpenes and terpenoids. These differences are likely due to the source of milk. Minor constituents of milk can vary depending on the diet of the cattle and geographic location (Turbes et al., 2016; Borge et al., 2016). Terpenes and terpenoids are organic compounds found widely in plants, and cattle feed is a significant source of these compounds in milk and milk products (Lejonklev et al., 2013). Microbial activity is also known to alter terpene and terpenoid content in many food products, and fermentation may also alter the content in the whey (Borge et al., 2016). d-Limonene and geraniol can imbue distillates with citrus, floral, and fruit aromas, whereas farnesol and α-farnesene may negatively affect the distillates’ organoleptic qualities due to their oil and boiled vegetable aromas (Flavor and Extract Manufacturers Association, 2017). It should be noted that the odor detection limits of several of the identified terpenes and terpenoids were below 50 µg/L, concentrations that may affect the organoleptic qualities of the distillate. Due to challenges associated with obtaining whey from 2 different cheese processes, the acid whey was frozen before delivery whereas the sweet whey was processed fresh. It is standard practice to freeze samples when storing for aroma analysis by GC-MS, as this Journal of Dairy Science Vol. 102 No. 1, 2019

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causes any potential aromatic reactions to stop (Jervis et al., 2015; Izawa et al., 2015). Although we could not find any research specifically into potential aroma or aroma precursor changes in whey due to freezing, Sattin et al. (2016) used HS-SPME–GC-MS on frozen and fresh whey samples and found no relationship for aroma composition based on fresh versus frozen whey. This suggests that it is the further treatment of the whey and not the frozen versus fresh storage that is responsible for aroma composition. Composition of whey distillates will depend on the production processes used to produce whey (or the different cheeses). These process variations could present a challenge to producers of potable whey distillate who desire to produce a consistent, palatable product. Especially if multiple small-scale cheese companies decide to pool whey sources for processing within a single facility. Volatile compounds, which may be under the odor threshold initially, may be concentrated above their odor threshold by the distillation process. This could produce an undesirable and inconsistent product. Clarification, centrifugation, and filtration would likely be used in a commercial process to refine the lactose stream before fermentation and distillation. In addition, commercial distillers would blend distillates from multiple batches to minimize batch-to-batch variability. In our study, complete distillates were collected. Distillers typically use cuts, which mean they only keep the desired portions of the distillate based upon organoleptic qualities (Piggot, 2009; Hughes and Buxton, 2014). The use of a commercially scaled still and cuts would enable greater separation of volatiles, which allows for the production of a potable spirit with fewer perceivable aromatic compounds other than ethanol. Another commercial approach for the removal of low levels of flavor-active compounds is to filter the spirit through activated carbon (Piggot, 2009). The aroma compounds identified in our study contain volatile aroma compounds that would be present in distillates from cheese whey without commercial distilling processing and treatments, which could be described as a worst-case scenario due to the many diverse aroma compounds present. Commercial distillers would likely use methods to control and remove many of the volatile aromas detected in our study. The differences in volatile composition of each distillate type identified in our study indicates that whey to ethanol processors may want to employ one or more of the methods described above to ensure consistency from batch to batch. ACKNOWLEDGMENTS

We thank Robin Frojen at the Arbuthnot Dairy Center (Corvallis, OR) and Greg Admire at Umpqua Dairy Journal of Dairy Science Vol. 102 No. 1, 2019

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