The Aroma of Goat Milk: Seasonal Effects and Changes through

Article pubs.acs.org/JAFC

The Aroma of Goat Milk: Seasonal Effects and Changes through Heat Treatment Caroline Siefarth†,‡ and Andrea Buettner*,†,‡ †

Department of Chemistry and Pharmacy, Emil Fischer Centre, Friedrich-Alexander Universität Erlangen-Nürnberg, Schuhstrasse 19, 91052 Erlangen, Germany ‡ Department of Sensory Analytics, Fraunhofer Institute for Process Engineering and Packaging (IVV), Giggenhauser Strasse 35, 85354 Freising, Germany ABSTRACT: Goat milk was characterized and analyzed by human sensory evaluation and gas chromatography/olfactometry (GC/O). Most potent odor-active compounds were determined in (a) raw goat’s milk from two different seasons and (b) heated goat’s milk after different treatment intensities. A trained panel found sensorial differences between winter and summer milks (seasonal effect) and milks from different farms (farm-specific effect). A total of 54 odor-active compounds with flavor dilution (FD) factors ≥8 were detected of which 42 odorants were identified. 4-Ethyloctanoic acid, 3-methylindole (skatol) and one unknown compound (RI 2715) showed highest intensities in all raw milks. With heat treatment, goat-like, stable-like, and (cooked) milk-like odor characteristics decreased while caramel-like or vanilla-like notes increased. In total, 66 odor-active compounds were detected in heated goat milks (FD ≥ 8). To the best of our knowledge, only 16 of the 42 identified odorants were reported before in raw goat’s milk. Additionally, for the first time the presence of 1-benzopyran-2-one (coumarin) could be confirmed in ruminant milk. KEYWORDS: aroma extract dilution analysis (AEDA), aroma profile analysis (APA), fatty acids, mass spectrometry, retention indices, two-dimensional gas chromatography/olfactometry (2D-GC/O)

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content of ketones and terpenes. Skjevdal8 also reported changing flavor intensities of goat’s milk during lactation. Lowest flavor intensities were found at the beginning and toward the end of the lactation cycle. In contrast, high-feeding intensity using concentrates, as it is performed at the beginning of lactation, was reported to increase the overall flavor (intensity) of the milk.8 In view of heat treatment of goat’s milk, Ormiston and Herreid12 investigated the effect of heat sterilization on the flavor of goat milk and described a cooked flavor that was, however, never as intense in goat milk as previously observed in cow milk. However, all these studies lack information on specific odorants underlying the observed sensory effects. Over the past decades, many more details on flavor, flavor variations, and potent odorants in heated cow milk were published. Especially “cooked/heated”, “sulfurous”, and “caramelized” flavors were reported.13,14 As characteristic odorants in cow’s UHT milk, a variety of lactones and acids were found, besides some sweet, sulfurous, and roasty compounds like vanillin (4hydroxy-3-methoxybenzaldehyde, vanilla-like), methional (3methylthiopropanal, cooked potato-like), furaneol (4-hydroxy2,5-dimethyl-3(2H)-furanone, caramel-like), 2-acetyl-1-pyrroline, 2-acetyl-2-thiazoline, and 2-furaldehyde (roasty, popcornlike).15,16 Other studies on odorants in UHT milk additionally identified various pyrazines, alcohols, aldehydes, and ketones

INTRODUCTION Goat milk and its production, quality, and safety are of growing interest worldwide since new markets recently sprang up, for example, with respect to a use of goat milk in infant formulas or in medicinal applications.1 An important quality aspect is the flavor of goat milk and its flavor stability in terms of seasonal variation or after heat application. Various studies have investigated the flavor of goat’s milk cheeses (e.g. refs 2 and 3); a few studies also focused the flavor of pure goat milk.4−6 Generally, the flavor is described as more robust, waxy, and animal-like compared with the plain, milky flavor of cow’s milk.6,7 The intensity of this typical goat milk flavor is reported to be dependent on several factors, for example, breed, season, lactation period, feeding, milk yield, milk fat content, or milk composition.7 Especially C6 to C10 (free) fatty acids, branchedchain fatty acids, cresols, and indole are reported to characterize the flavor of goat milk.5,6,8,9 Thereby, 4-methyloctanoic acid (4meC8) and 4-ethyloctanoic acid (4-etC8) are described as character-impact compounds of goat milk and goat milk cheeses, with 4-etC8 being known for its extremely low odor threshold.2,7,10 However, detailed studies on key odorants in raw goat’s milk and flavor changes during the year or with thermal treatment of the milk can be rarely found in literature. In one of the rare studies, Fedele et al.11 characterized the flavor profile of raw goat milk from three different seasons (winter, spring, summer) based on descriptive profiling and gas chromatographic determination of specific substance classes (alcohols, ketones, esters, and terpenes). Winter milks were characterized by hay-like, green, and cheesy flavors, while summer milks were found to be more fruity, as well as more citrus/mint-like. Seasonal variations were further found in the © 2014 American Chemical Society

Received: Revised: Accepted: Published: 11805

August 29, 2014 November 18, 2014 November 18, 2014 November 18, 2014 dx.doi.org/10.1021/jf5040724 | J. Agric. Food Chem. 2014, 62, 11805−11817

Journal of Agricultural and Food Chemistry

Article

good practice, and no specific feeding protocol was established in terms of this study to stay as close as possible to a conventional goat milk aroma and fatty acid composition. Sterilized goat milk was produced by sterilizing raw milk at 121 °C and a pressure of 2 bar for 15 min in a laboratory autoclave (Fedegari Autoklavi Spa, Albuzzano, Italy). Pasteurized and UHT-treated goat milk were purchased from local supermarkets: Leeb Vital Bio-goat milk, pasteurized (Leeb Biomilch GmbH, Schlierbach, Austria), and Andechser Bio-goat milk, UHT-teated (Andechser Molkerei Scheitz GmbH, Andechs, Germany). Preparation of Milk Samples and Fatty Acid Analysis. Sample preparation and gas chromatographic determination of the total fat content and fatty acid profiles were performed according to the DGF standard method C-III 19 (99).18 By means of this DGF method, both free fatty acids and fatty acids bound in mono-, di-, and triglycerides, phospholipids, and sterols can be detected. Isolation of Volatile Organic Compounds (VOCs). Solvent assisted flavor evaporation (SAFE)19 was applied for the isolation of volatiles from fresh goat milk samples and heated goat milks. At room temperature (21 ± 1 °C), 25.0 ± 0.1 mL of goat milk was combined with freshly distilled dichloromethane at a ratio of 2:1 (milk/solvent). The mixture was stirred and distilled, and the organic phase of the distillate was separated, dried, and concentrated as it was reported before by Siefarth et al.20 Characterization and Identification of Goat Milk Odorants. Characterization. Concentrated distillates were characterized by applying them to a high resolution gas chromatography/olfactometry system (HRGC/O, type Trace GC Ultra, Thermo Fisher Scientific, Schwerte, Germany). Application was performed by use of the coldon-column technique (at 40 °C) using the same GC capillaries and settings as described in an earlier study.20 Two panelists screened the distillate for odor-active volatiles. Screening was performed by sniffing the effluent after gas chromatographic separation at the sniffing port and simultaneously describing the perceived odor impressions (GC/ O). Additionally, AEDA was performed on all goat milk samples to screen for the most potent odorants. In detail, an FD series was sniffed via HRGC-O by diluting the concentrated distillates (FD factor 2n, concentrate, FD 20 = FD 1) stepwise with dichloromethane (1:1, v/v). Two microliters of each dilution was analyzed on a DB-FFAP capillary. Identification. Odorants were identified based on the following criteria: odor quality, RIs on two stationary phases, and mass spectra. Linear RIs of the compounds were calculated as described by Van Den Dool and Kratz.21 Identification was performed using a twodimensional gas chromatographic system (2D-HRGC-MS/O), of which the setup and GC conditions were described in an earlier study.20 Mass spectra in the electron impact mode (MS/EI) or chemical ionization mode (MS/CI) were acquired (m/z range 35−249 and 60−299, respectively). In the case of EI mode, mass spectra were generated at ionization energy of 70 eV; in CI mode, methanol was used as the reagent gas at a flow of 2.5 mL min−1. Aroma Profile Analysis (APA). Sensory analyses were performed in a sensory room at 21 ± 1 °C. Goat milk samples (20 mL) were filled into sensory glass beakers (140 mL, J. Weck GmbH u. Co. KG, Wehr, Germany) and closed with a lid. Trained panelists (n = 11−16, male/female, age 23−45) with normal olfactory and gustatory function participated in the APA sessions. They exhibited no known illness at the time of examination. Prior to this study, the assessors were recruited in weekly training sessions for the recognition of about 200 selected odor-active compounds according to their odor qualities by means of an in-house developed flavor language. A preliminary sensory session was performed to evaluate and collect orthonasal (o) and retronasal (r) odor attributes of the different goat milks. Attributes that were detected by more than 50% of the panelists were selected for subsequent sensory evaluations. Panelists were asked to score the perceived intensities (o, r) of the selected attributes on a seven-pointscale from not perceivable (0) to weak (1), medium (2) and intensely perceivable (3), in increments of 0.5. The results for each odor attribute and goat milk sample were averaged and plotted in spiderweb diagrams.

and some sulfur containing compounds like dimethyl disulfide and dimethyl trisulfide.17 Different from previous literature reports on goat’s milk, this study aimed to characterize not the entire volatile fraction of the milk or some specific odorants, but the most potent aroma compounds by use of GC/O and AEDA. In detail, the odor profile of raw milks from winter and summer season of different farms were compared with each other, also including a human sensory evaluation by means of aroma profile analysis (APA). Since the composition of milk fat is known to be an important contributor to milks’ odor profiles, also the fatty acid composition of the raw goat milk was investigated. Furthermore, changes in goat milks’ odor profiles as a result of heat-treatment were investigated. In a comparative approach, pasteurized, UHT-treated, and sterilized goat milk were studied by means of analytical and descriptive profiling.

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MATERIALS AND METHODS

Chemicals. Fatty Acid Analysis. The following chemicals with the stated purities were used (supplier in parentheses): ascorbic acid 95% p.a., 1-butanol 95%, formic acid 85%, sodium dihydrogen phosphatedihydrate 95% p.a., tridecanoic acid 99% (Fluka, Steinheim, Germany), Celite 545, potassium hydroxide 98.5% p.a. (Sigma-Aldrich, St. Louis, MO), fatty acids calibration kit (Büchi, Essen, Germany). GC/O Analysis. Dichloromethane p.a. was used as the organic solvent and extracting agent (Th. Geyer GmbH and Co. KG, Renningen, Germany). Anhydrous sodium sulfate was purchased from CHEMSOLUTE (Th. Geyer GmbH and Co. KG, Renningen, Germany). For unequivocal identification, the following reference compounds were used in stated purities: 4-hydroxy-3-methoxybenzaldehyde (vanillin) 99% (ABCR, Karlsruhe, Germany), 2-aminoacetophenone 98%, 1-benzopyran-2-one (coumarin) 99%, δ-decalactone 98%, γ-decalactone 98%, decanoic acid 98%, dimethyl trisulfide 98%, dodecanoic acid 98%, 4-ethyloctanoic acid 98%, 2-furaldehyde (furfural) 99%, hexanoic acid 99.5%, 3-hydroxy-4,5-dimethyl-2(5H)furanone (sotolone) 97%, 3-hydroxy-2-methyl-4-pyrone (maltol) 99%, 2-methoxyphenol (guaiacol) 98%, 2 methylbutanoic acid 98%, 3methylbutanoic acid 99%, 3-methylindole (skatol) 98%, 4-methyloctanoic acid 98%, 3-methylphenol (m-cresol) 99%, 3-methylthiopropanal (methional), (E,E)-nona-2,4-dienal 85%, (E,Z)-nona-2,6-dienal 95%, γ-nonalactone 98%, δ-nonalactone 98%, nonanoic acid 97%, (E)non-2-enal 97%, octanal 99%, octanoic acid 98%, (E)-oct-2-enal 94%, oct-1-en-3-one 50%, phenylacetic acid 99%, phenylethan-2-ol 99%, γundecalactone 98% (Aldrich, Steinheim, Germany), 2-acetyl-1-pyrroline 90%, γ-(Z)-6-dodecenolactone, trans-4,5-epoxy-(E)-dec-2-enal 90% (aromaLAB AG, Freising, Germany), γ-octalactone (EGA Chemie, Steinheim, Germany), 1H-benzo[b]pyrrole (indole) 98.5%, butan-2,3-dione (diacetyl) 99%, butanoic acid 99.5%, (E,E)-deca-2,4dienal 85%, dimethyl disulfide 98%, 4-hydroxy-2,5-dimethyl-3(2H)furanone (furaneol) 99%, pentanoic acid 99% (Fluka, Steinheim, Germany), 3-ethylphenol 98% (Riedel-de-Haen, Seelze, Germany), δdodecalactone 98%, γ-dodecalactone 97%, 3-propylphenol 99%, (E)undec-2-enal 90% (SAFC, Hamburg, Germany). Goat Milk. Two types of raw milk (winter season, summer season) as well as three types of heat-treated goat milk (pasteurized, UHTtreated, sterilized) were analyzed in a comparative approach. In the case of raw milk, the milks were obtained from two different farms during two periods: winter season (“W”, from 15 February to 21 March) and summer season (“S”, from 19 July to 10 August). Farm 1 (W1, S1) had a flock size of 120 goats of the breed “Bunte Deutsche Edelziege”; farm 2 had a flock size of 85 goats of the same breed. According to the farmer’s statements, both herds were fed on a mixture of home-grown pasture and grains (trefoil-grass, mixture of oat, barley, pea, triticale), given as straw/dried fodder and hay (conserved feed). Farm 2 additionally fed rapeseed meal, sugar beet slices, and carrots. At the beginning of the lactation period, both herds were additionally fed on concentrated feed, including rapeseed meal and sugar beet slices. Both farms fed their herds according to their 11806

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0.10 0.01 0.08 0.26 0.11 0.30 0.60 0.22 1.68 0.06 0.64 0.70 0.12 0.03 0.15

± 0.01 (3.8%) ± 0.00 (0.4%) ± 0.01 (3.3%) ± 0.02 (10.4%) ± 0.01 (4.5%) ± 0.02 (11.8%) ± 0.05 (23.9%) ± 0.02 (8.8%) (67.2%) ± 0.01 (1.7%) ± 0.05 (20.3%) (28.0%) ± 0.01 (4.7%) ± 0.00 (1.2%) (6.0%)

W1 0.15 0.01 0.12 0.34 0.16 0.39 0.86 0.34 2.37 0.09 1.09 1.18 0.20 0.01 0.21

± 0.01 (3.8%) ± 0.00 (0.3%) ± 0.01 (3.0%) ± 0.02 (9.0%) ± 0.01 (4.1%) ± 0.01 (10.3%) ± 0.04 (22.5%) ± 0.02 (9.0%) (62.4%) ± 0.01 (2.7%) ± 0.05 (34.4%) (31.1%) ± 0.01 (5.1%) ± 0.00 (0.3%) (5.5%)

W2 0.12 0.01 0.10 0.30 0.13 0.34 0.73 0.28 2.02 0.07 0.86 0.93 0.16 0.02 0.18

(3.8%) (0.3%) (3.1%) (9.6%) (4.2%) (10.9%) (23.1%) (8.9%) (63.9%) (2.2%) (27.6%) (29.8%) (5.0%) (0.6%) (5.6%)

mean 0.09 0.01 0.07 0.24 0.11 0.28 0.60 0.16 1.56 0.04 0.48 0.52 0.10 0.02 0.12

± 0.01 (3.9%) ± 0.00 (0.5%) ± 0.01 (3.1%) ± 0.01 (10.8%) ± 0.01 (4.9%) ± 0.01 (12.5%) ± 0.02 (27.4%) ± 0.01 (7.0%) (70.9%) ± 0.01 (1.8%) ± 0.01 (20.4%) (23.6%) ± 0.01 (4.3%) ± 0.01 (1.0%) (5.5%)

S1 0.09 0.01 0.07 0.24 0.11 0.31 0.72 0.16 1.71 0.05 0.61 0.66 0.12 0.01 0.13

± 0.01 (3.7%) ± 0.00 (0.4%) ± 0.00 (2.8%) ± 0.01 (9.7%) ± 0.00 (4.4%) ± 0.01 (12.2%) ± 0.01 (28.7%) ± 0.00 (6.4%) (68.4%) ± 0.00 (2.1%) ± 0.01 (26.1%) (26.4%) ± 0.01 (4.6%) (0.4%) (5.2%)

S2

summer milk (S) (Φ total FA, 2.4 g/100 g) 0.09 0.01 0.07 0.24 0.11 0.30 0.66 0.16 1.64 0.05 0.55 0.59 0.11 0.02 0.13

(3.8%) (0.4%) (2.9%) (10.2%) (4.6%) (12.3%) (28.0%) (6.7%) (69.0%) (2.0%) (23.1%) (25.1%) (4.5%) (0.7%) (5.2%)

mean b c b c c e e e d e c e

b b b b

W2/S2

b c b b b b c c

W1/S1

b b

b b

b c b b b c c c

Wmean/Smean

seasonal effects

b e

e e

b c b c b d b b

b d

b e

b c b d c b c c

S1/S2

farm-specific effects W1/W2

level of significance

Mean values and relative amounts (percentage of total fatty acids) of individual fatty acids in milk (g/100 g of milk). SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids. bNot significant. cp < 0.05. dp < 0.01. ep < 0.001.

a

C4:0 butyric C6:0 caproic C8:0 caprylic C10:0 capric C12:0 lauric C14:0 myristic C16:0 palmitic C18:0 stearic C4:0−18:0 total SFA C16:1 palmitoleic C18:1 oleic C16:1−18:1 total MUFA C18:2 linoleic C18:3 linolenic C18:2−18:3 total PUFA

fatty acid

winter milk (W) (Φ total FA, 3.2 g/100 g)

Table 1. Main Fatty Acid Composition of Raw Goat Milk from Winter (W) and Summer (S) Seasons and Different Farms (1,2)a

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Figure 1. Orthonasal (A) and retronasal (B) odor profiles of goat milk: raw milk profiles from summer (S) and winter (W) season. Statistical Analysis. For statistical analyses, Student’s t test (Welch-test) or Mann−Whitney U-test was performed using OriginPro 9.1 (OriginLab Co., Northampton, MA). The level of statistical significance was set at 5%.

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Sensory Characterization of the Raw Goat Milks by Means of APA. Orthonasal (o) and retronasal (r) odor profiles of the raw goat milk samples were described by the following eight attributes: milk-like, fatty, goat-like, metallic, stable-like, vanilla-like, sweaty, and hay-like (Figure 1). Those attributes were detected by more than 50% of the trained panelists in one or more of the different raw milks. Orthonasally, the strongest odor attributes were milk-like and fatty (weak to medium intense; mean values 1.3) followed by weak perception of the attribute goat-like (mean value 0.5). All other attributes were just slightly perceivable with mean values below 0.5. Retronasally, goat-like and fatty were the strongest perceived odor attributes (medium intense; mean values 1.6 and 1.5). The attributes milk-like, sweaty, and metallic were evaluated with weak intensities (mean values 1.0, 0.8, and 0.6), and stable/vanilla-like odors were just slightly perceived (mean values 0.5 units). Retronasally, odor differences became more clear with respect to the attribute goat-like (mean intensities S1,S2: 1.9 and 1.4), but also sweaty was perceived more intense in S1 (mean intensities S1,S2: 1.1 and 0.5) and milk-like in S2 (mean intensities S1 and S2, 0.8 and 1.3). Metallic and stablelike were ranked slightly higher in S1. Thus, the raw milk odor profiles of one season were found to vary from one farm to another.

RESULTS

Fatty Acids Composition of Raw Milks. The fatty acid composition of goat’s milk was investigated in a comparative approach of winter versus summer milk and thus during different lactation stages (Table 1). Overall, the fatty acid percentage composition of summer and winter milk was quite similar to each other as the statistical comparison of Wmean and Smean shows. Thus, average seasonal effects on fatty acid composition of goat’s milk were found to be low. Significances were only detected in a few cases (C6:0, C14:0, C16:0, C18:0, p < 0.05). Generally, winter milk contained a higher percentage of total MUFAs (monounsaturated fatty acids) while summer milk contained a higher percentage of total SFAs (saturated fatty acids). PUFA (polyunsaturated fatty acid) contents were quite similar between summer and winter milks. However, differences in fatty acid composition were more significant by direct comparison of (a) winter/summer milks from the same farm (W1/S1, W2/S2, seasonal effect) or (b) winter/summer milks from the same season but different farms/herds (W1/W2, S1/ S2, farm-specific effect) (Table 1). In detail, most variations were detected in case of the fatty acids C10:0, C14:0, C16:0, C18:0, C16:1, C18:1, C18:3, p < 0.01. Thus, farmer’s seasonal changes in the goat’s diet (types of pasture, grains) as well as farm-specific differences between two farms/herds of the same breed (e.g., due to feeding or animal-specific differences) showed an effect on percentage fatty acid composition. 11808

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Table 2. Characterization and Identification of Potent Odorants (FD ≥ 8) in Raw Goat Milk from Summer and Winter Season Using Gas Chromatography/Olfactometry (GC/O) FD-factord in raw goat milk RI on no. 1 2 3 4 5 6 7 8 9 10/11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

odorant

a

butan-2,3-dione (diacetyl) dimethyl disulfideb oct-1-en-3-one (E)-oct-2-enal (Z)-non-4-enalb 3-methylthiopropanal (methional) (E)-non-2-enal (E,Z)-nona-2,6-dienal butanoic acid 2-/3-methylbutanoic acid (E,E)-nona-2,4-dienal pentanoic acidb (E,E)-deca-2,4-dienal hexanoic acid phenylethan-2-olb γ-octalactone trans-4,5-epoxy-(E)-dec-2-enal γ-nonalactone octanoic acid δ-nonalactone 4-methyloctanoic acid (4-meC8) unknown 3-methylphenol (m-cresol) γ-decalactone nonanoic acid 3-ethylphenol δ-decalactone 3-hydroxy-4,5-dimethyl-2(5H)-furanone (sotolone)b 4-ethyloctanoic acid (4-etC8) 2-aminoacetophenon 3-propylphenol γ-undecalactone decanoic acid unknown unknown γ-dodecalactone γ-(Z)-6-dodecenolactone δ-dodecalactone 1-benzopyran-2-one (coumarin) 1H-benzo[b]pyrrole (indole) unknown dodecanoic acid 3-methylindole (skatol) unknown unknown phenylacetic acid 4-hydroxy-3-methoxybenzaldehyde (vanillin) unknown unknown unknown unknown unknown unknown

c

odor quality

DB-FFAP

buttery sweaty, fatty mushroom-like green, fatty, plastic-like green, fatty cooked potato-like fatty, green fatty, cucumber-like cheesy, sweaty sweaty, cheesy fatty, green sweaty, cheesy fatty, green urine-like, pungent flowery, fruity, sweet coconut-like metallic peach-like, woodruff-like medicinal, fatty, musty sweet, peach-like musty, urine-, stable-like fruity, soapy, sweet leather-like, medicinal peach-like fatty, fecal, goaty fatty, leather-like, medicinal coconut-like spicy, maggi-like

996−1004 1138−1144 1294−1297 1414−1419 1429−1433 1445−1449 1512−1526 1576−1580 1614−1618 1652−1659 1689−1691 1715−1727 1797−1799 1829−1834 1885−1904 1904−1908 1983−1996 2010−2017 2037−2046 2063−2066 2074−2082 2097−2103 2089−2099 2130−2132 2147−2160 2151−2167 2178−2188 2189−2197

goaty, stable-like flowery, peach-like, soapy musty, medicinal, leather-like peach-like fatty, goaty, leather-like fatty, pungent, plastic-like sweaty, goaty soapy, fatty, flowery flowery flowery, daisy-like cinnamon-like, sweet fecal, goaty flowery, soapy soapy, goaty, fecal fecal, stable-like rubber-like, balloon-like fruity, flowery, pleasant honey-like vanilla-like spicy, meat-like canola-like, metallic, green flowery spicy, urine-like flowery, soapy facial cream-like, oil paint-like

2190−2202 2208−2214 2235−2244 2247−2255 2253−2264 2331−2341 2345−2353 2368−2381 2395−2405 2421−2430 2437−2445 2440−2458 2456−2465 2468−2477 2478−2482 2507−2512 2530−2535 2543−2551 2549−2557 2565−2567 2712−2716 2755−2759 2760−2764 2832−2835 >2900

DB-5 761−768 975−981 1060−1066 1096−1099 908−909 1156−1166 1148−1151 826−833 866−868 1210−1215 912−914 1303−1314 1020−1022 1103−1113 1251−1260 1371−1375 1351−1360 1262−1275 1333−1336 1228−1242 1082−1091 1459−1468 1271−1288 1166−1171 1487−1497 1079−1082 1318−1323 1290−1295 1564−1574 1368−1388

1676−1682 1664−1674 1713−1718 1433−1446 1306−1309 1566−1585 1379−1383 1789−1791 1785−1799 1246−1268 1392−1410

1471−1474

summer milk

winter milk

S1

S2

W1

W2

8 e 16 8 16 8 8 e 64 64 16 16