1. Introduction

The main components of the milk, which contribute to the flavor and other sensory attributes, are proteins and fats. Goat milk has more nutritional qualities that are better for human consumption than cow’s milk, such as less lactose levels, more quantity of vitamins and reduced lipids amount. Other characteristic of goat milk is its distinctive aroma and flavor, which are derived from their lipid fraction and it varies widely based on principal factors such as genetic diversity of breed, feeding practices, season of obtaining milk and lactation stage. Goat milk is different from cow’s milk due to its “goaty” flavor and aroma classified in off-flavor attributes, which are not quite appreciated by consumers. Scientific knowledge on precursors leading to the formation of characteristic goat milk flavor is limited. Mostly the short-chain (C₆, C₈ and C₁₀) fatty acids are held responsible for this undesirable sensorial attribute [1]. Ha and Lindsay reported the triglyceride composition of goat, sheep and cow milks, attributing the presence of minor branched chain compounds in goat milk, including 4-methyl octanoic acid which has an extremely low odor threshold value. Moreover, the goat milk flavor is the result of the lipolytic action of the milk lipoprotein lipase, which in goat milk is largely bound to the fat [2].

Very few publications are available which deal with identification and quantification of volatile compounds present in goat milk [1, 3, 4, 5] and these relate mostly to the feeding systems and their effect on the composition and flavor quality of goat milk. Fedele et al. [3] reported the presence of β-caryophyllene and α-copaene as dominant terpenes in goat milk. Queiroga et al. [6] detected 174 volatile compounds from goat milk, such as phenols, acids, lactones, ketones, alcohols, esters and terpenes. The presence of terpenic compounds is related to the plant species utilized for the feeding systems. Yang et al. [1] identified the compounds responsible for the “goaty” flavor in goat milk as being short fatty acids (C6:0 to C9:0). Flavor of dairy products is a critical parameter affecting consumer acceptance, shelf life, and other attributes [7, 8]. Since goat milk has gained economic importance and is classified as a functional food, it participates in health maintenance and reduces disease. Of late, there is an increase in goat milk production due to its utilization in cheese making. In the Mediterranean and many eastern European countries, it is important to elucidate the aroma composition to help to avoid the development of products with off-flavor in order to increase its consumption [9-11]. Thus, the objective of the present work was to identify the volatile compounds present in goat milk and to acquire an in depth knowledge on key volatile compounds as related to their odor active values contributing to the milk’s overall aroma characteristics.

2. Material and Methods

The goat milk was collected from 20 animals of Saanen breed, which were confined in the Sector of Caprinoculture of Federal University of Paraíba, located in city of Bananeiras. The animals were fed with a feed containing grass Tiffton mixed with a balanced diet.

2.1 Volatiles isolation

The volatile compounds were extracted by using Likens and Nickerson’s [12] apparatus, which uses the simultaneous distillation and extraction technique. The extraction conditions were optimized by Queiroga et al. [6] in which 200 mL of goat milk was diluted with 100 mL of distilled water and extraction was performed with 20 mL of pentane:ether (2:1) for 120 min. The extracts were concentrated to a final volume of 0.3 mL under the flow of nitrogen gas.

2.2 High resolution gas chromatography/mass spectrometry

A combined system of gas chromatograph (Shimadzu GC 17A) coupled with a mass spectrometer (GC/MS-QP5050A) was used. One microliter of the concentrated volatile extract was injected in the column in a splitless mode. Capillary GC investigations were carried out on non-polar capillary column HP-5MS (30 m × 0.25 mm; 0.25 µm). The carrier gas used was helium and column head pressure was maintained at 11.5 psi having a flow rate of 1 mL/min. The oven temperature was programmed: initiation at 30°C for 5 min, increased at 5°C/min to 80°C, maintained at 80°C for 30 min, increased at 5°C/min to 220°C, wherein maintained for 45 min. The temperatures of the injection port and the GC/MS interface were 200°C and 230°C, respectively. The mass spectrometer was operated in the electron ionization mode with an electrical energy of 70 eV and an ion source temperature of 250°C. The mass spectrum was scanned between 30 and 350 atomic mass units at 0.1 sec interval. The identification of compounds was done by using the linear retention index (LRI) values, determined on retention time data obtained by analyzing a series of normal alkanes (C₈-C₂₁). Volatile components were positively identified by matching their LRI values and mass spectra with those of standards, also run under identical chromatographic conditions in the laboratory.

2.3 Quantification of volatile compounds

The volatile compounds were quantified using the analytical curves prepared from the results obtained on chromatographic analysis of aroma compound standards of different classes of organic compounds such as esters, alcohols, ketones, aldehydes and terpenes, also performed under identical analytical conditions as that of the samples.

2.4 Statistical analysis

All chromatographic data were analyzed in triplicate and the results were expressed as mean ± standard deviation values.

3. Results and Discussion

3.1 Volatile profiles of goat milk

Table 1 lists the volatile compounds identified in goat milk. The data cites the retention indices and the concentration of the identified compounds, which are organized according to their organic classes. It was observed that some compounds have the superscript letter ^a, which signifies that the compound was tentatively identified since there was no pure standard compound available, which could be run under the identical analytical conditions. Thus the identification was considered tentative when it was based mainly on matching an unknown mass spectrum with a spectrum available of NIST (National Institute of Standards and Technology, USA) mass spectral data system or the literature [13, 14]. In a typical chromatogram analyzed for the volatile extracts obtained from goat milk, a total number of 91 components were separated out of which, 63 compounds were positively and 28 tentatively identified. The other constituents could not be identified. Among the identified components in the goat milk of Saanen breed, the largest number of compounds belonged to esters, being 29 compounds, followed by alcohols (13), aldehydes (12), terpenes (11), carboxylic acids (8), ketones (6), lactones (5), aromatics (5) and sulfur compounds (2).

Others authors like Sant’Ana et al. [16], Queiroga et al. [6] and Siefarth and Buettner reported the presence of 19, 174 and 54 compounds, respectively. The main compounds identified in this study, such as hexanol (9481.92 µg/L), pentanoic acid (3040.08 µg/L), 2-pentanone (1651.03 µg/L) were previously reported by these authors. However, in this paper we are reporting the presence of the following compounds in goat milk: Esters (isobutyl acetate, isopropyl butanoate, ethyl tridecanoate, butyl 10-undecenoate, isoamyl cinnamate, benzyl benzoate, butyl dodecanoate, isopropyl tetradecanoate, methyl (Z)-9-hexadecenoate, octadecanol acetate, butyl heptadecanoate), alcohols (2-methyl-1-hexanol, tridecanol, 1,2-dodecanediol, pentadecanol), terpenes (cumene, camphor, β-ionone, β-farnesene, cubenol, α-cadinol, farnesol), ketone (2,3-hexanedione), sulfur compounds (butanethiol, 2-methylthiophene). The prominent sulfur compound found in milk is dimethyl trisulfide that has been reported previously as a flavor compound in both yogurt and cow’s milk [15]. However, in this work on goat’s milk, the presence of 2-methylthiophene (23.46 µg/L) was detected and its presence was reported by Bendall [15] in cow milk and products like yogurt.

The volatile compounds identified which were in higher concentrations in goat milk were hexanol (9481.92 µg/L), pentanoic acid (3040.08 µg/L), 2-pentanone (1651.03 µg/L), methyl 9-octadecenoate (1613.88 µg/L), methyl hexadecanoate (1060.61 µg/L) and 2-pentanol (1683.28 µg/L). Fedele et al. [3] reported the dominant presence of terpenes such as β-caryophyllene and α-copaene. However in this work on goat’s milk, these compounds were not found, although other terpenic compounds such as β-farnesene (80.16 µg/L), α-terpinene (31.28 µg/L), β-ionone (26.39 µg/L), δ-cadinene (23.46 µg/L), α-cadinol (20.53 µg/L) and α-pinene (12.71 µg/L) were found. The presence of terpenes in milk are related to the animal diet, since such compounds can pass from the plants to the milk and can be used as biomarkers in a feeding animal system (Sant’Ana et al., 2019). The goat milk from the breed Saanen grown in the Northeast region of Brazil had more presence of terpenes, which is related to animals feeding with a forage support of Tiffton hay.

LRI_Exp-Linear retention index experimental; LRI_Lit-Linear retention index literature; ^a-Tentatively identified compounds; *-Compounds reported for the first time in this work on goat milk aroma

Table 1: Volatile compounds identified in goat milk along with their characteristic odor notes.

There are various chemical and biochemical routes for generation of aroma compounds. The free fatty acids are formed due to the enzymatic processes involving lipase action which hydrolyzes and produces short chain fatty acids. A series of 2-ketones such as 2-pentanone, 2-pentadecanone, 2-heptanone and 2-hexadecanone have been found in fresh milk, which are produced from β-ketoacid glycerides by hydrolytic and decarboxylation mechanism. In this study a very high concentration of 2-pentanone (1651.03 µg/L) was found in milk. The oxidative flavor of milk fat is originated, primarily from linoleic and linolenic acids and gets substantiated by other polyunsaturated fatty acids. Twelve aldehydes (pentanal, hexanal , (E)-2-hexenal, heptanal, octanal, nonanal, decanal, pentadecanal, hexadecanal, heptadecanal and 9-octadecenal) were detected in goat milk. The main aldehyde concentration was that of (E)-2-hexenal (1231,67 µg/L). However, Sant’Ana et al. [16] could not find any aldehyde when analyzing goat milk volatiles. In general, aldehydes are formed by oxidative processes involving microorganisms and enzymes like lipoxygenase [17].

3.2 Key volatile aroma compounds in goat milk

Table 2 lists the key volatile compounds which had odor activity values (OAV) higher than 1 and hence considered to contribute to the overall aroma in the goat milk matrix. The volatile compounds hexanol and (E)-2-hexenal are known to be associated with the formation of rancid odor while 2-pentanone contributes to a strong fatty odor. Methyl 9-octadecenoate and isopropyl tetradecanoate are known to be the principal compounds responsible for the odor of goat milk. According to OAV’s the most important compounds contributing to goat milk aroma were decanal (88), (E)-2-hexenal (72), hexanol (47), octanal (40), ethyl hexanoate (35), pentanal (34), nonanal (30), 2-pentanone (24), heptanal (23), methyl hexanoate (20), γ-dodecalactone (11), 2-heptanone (10), heptanol (9), ethyl octanoate (9), 2-pentadecanone (9), β-ionone (8), α-pinene (5), 2-methylthiophene (5), octadecanal (5). In the sensorial analysis of goat milk, Sant’Ana et al. [16] reported the principal odor notes that characterized goat milk were “buttery” and “herbaceous”. In this work hexanol, heptanol and α-pinene were detected which have high OAV’s and their odor notes are characterized as “herbal”. Other compounds with odor notes similar to “buttery” found in this work, and which also possessed a high OAV’s were aldehydes like heptanal, decanal, octanal and nonanal that have odor notes classified as “fatty” and “waxy”.

Yang et al. [1] reported that “goaty” aroma in goat milk is due to presence of straight-chain fatty acids like C6:0 to C9:0 and some branched-chain C9:0 and C10:0 and that its participation in aroma depend mainly on their concentrations. In this study on aroma profile of goat milk, the compounds which had OAV>1 were octanoic acid (C8:0) (3), pentanoic acid (C5:0) (3), heptanoic acid (C7:0) (1) characterizing with odor notes of “cheesy”, “fatty”, “rancid” and “milky”. In other publications, hexanoic acid (C6:0) is described as having pungent, goaty, sweaty, and blue cheese flavor notes; octanoic acid has waxy, soapy, goaty, musty, rancid, and fruity notes; and decanoic acid (C10:0) is described as soapy, bitter, goaty and rancid notes.

In this study, lactones which could contribute to the characteristic aroma of goat milk were γ-dodecalactone (11), δ-dodecalactone (4), δ-hexadecalactone (1). For goat milk, Sienfart and Buettner [18] reported the presence of γ-octalactone, γ-nonalactone, δ-nonalactone, δ-decalactone, γ-undecalactone and γ-dodecalactone as potent odor participants in goat milk aroma. Bendall, [15] also described these compounds as responsible for fruity and sweet characteristics in cow milk aroma. According to Chilliard [2], the compounds which contribute to milk flavor formation could be classified according to their origin: compounds originated from animal metabolism and/or feed forage, compounds produced by chemical reactions, enzymatic activity or from microbial flora before its processing, compounds generated by thermal treatment or the ones developed during storage. Although interpreting the characteristic aroma of foods is an extremely complex matter, the initial step is to identify the odor-impact compounds and then to evaluate the concentration of the major compounds present in the matrix.

^aButtery et al. [19]; ^bBurdock [20]; ^cPino and Quijano [21]; ^dCzerny et al. [22]; ^eSaberi et al. [23]; ^fPuvipirom and Chaiseri [24]; ^gKaragul-Yuceer et al. [25]; ^gOng and Acree [26]; ⁱWagner et al. [27].

Table 2: Key volatile compounds (OAV>1) present in goat milk along with their characteristic odor notes.

Sieffart and Buettner [18] reported that six compounds were associated with goat-like odor characteristics accompanied by the attributes stable-like, fecal, or leather-like, and these were saturated acids like nonanoic acid, decanoic acid, dodecanoic acid. Although these compounds were found in the volatile profile of goat milk analyzed in this study, but they characterized for lower aroma impact as these compounds had their OAV’s less than 1. Sieffart and Buettner [18] classified 3-methylbutanoic acid as a potent odorant in goat milk with notes as “sweety” and “cheesy”; in this study the same compound was found having OAV of 4, and hence corroborates with authors previously published work.

4. Conclusion

This work reports the presence of main volatile compounds identified in milk obtained from Saanen breed of goat, grown in the northeast region of Brazil. A total of 91 volatile compounds were identified and a majority of them belonged to esters, alcohols, aldehydes and terpenes. The work also reports the most promising odorous compounds viz. decanal (88), (E)-2-hexenal (72), hexanol (47), octanal (40), ethyl hexanoate (35), pentanal (34), nonanal (30), 2-pentanone (24), heptanal (23), methyl hexanoate (20), γ-dodecalactone (11), 2-heptanone (10), heptanol (9), ethyl octanoate (9), 2-pentadecanone (9), β-ionone (8), α-pinene (5), 2-methylthiophene (5), octadecanal (5), which could be responsible for goat milk aroma and its over-all flavor.

Acknowledgements

Authors (MTSLN) gratefully acknowledges the financial support in the form of a post-doc fellowship received from CAPES (Ministry of Education, Brazil).

Notes

The authors declare that there is no conflict of interest.

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