Preliminary Metabolomic Analysis of Goat Milk from Different Breeds Using Mass Spectrometry

Metabolomics facilitates the identification of numerous metabolites in a sample with mass less than 1 kD. The purpose of this study was to determine the effect of feed on the metabolites in raw goat milk samples obtained from two breeds namely Saanen and Shami that were collected from a farm in Selangor and Negeri Sembilan, Malaysia, respectively. The types of feed given to the goats were recorded. The fat in the samples were removed via centrifugation before analysis using liquid chromatography quadrupole time of flight mass spectrometry (LC-QToF-MS). From the chromatograms obtained the presence of veterinary drug residue, diand tri-peptides, short and long-chain fatty acids, some components from plants, and insecticide residue were detected using HMP, KEGG, LMP, and METLIN databases identification. It is expected that through metabolomics study it may be possible to formulate the feed and determine the breed that give good quality milk. Keywords— Metabolomics, goat milk, LC-QToF-MS, databases.


I. INTRODUCTION
In the last 20 years scientists begin to realise that a simultaneous compound or metabolite identification method would assist in confirming health and physiological status of an organism. Metabolites represent a diverse group of lowmolecular-weight structures including lipids, amino acids, peptides, nucleic acids, organic acids, vitamins, thiols and carbohydrates [1]. Metabolomics aims to detect, identify, and quantify a total population of low molecular weight compounds to gain functional information in a biological system [2].
Milk is a complex biological fluid secreted for neonate nourishment and development. Milk contains water, lipids, carbohydrates, proteins, vitamins, minerals, and smaller metabolites. Changes in chemical composition of milk affect nutritional, safety, and technological properties of the milk [3]. In addition to the normal nutritional composition, goat milk contains other metabolites that have functional properties and may be unique to type of feed and breed [4], [5]. Sundekilde [6] suggested that metabolomics approach can be used to establish biomarkers in bovine milk as a diagnostic tool for determining quality and technological properties. Metabolites as biomarkers for specific biochemical pathways will indicate different physiological status of the organism [2].
In addition, coupling chromatography to mass spectrometry (MS) offers an excellent solution to complex mixture analyses and has been extensively used in metabolomics. Chromatographic separation of metabolites prior to MS analyses has several advantages: i) reduces matrix effects and ionization suppression, ii) separates isomers, iii) provides additional and orthogonal data (i.e. retention time/factor/index) valuable for metabolite annotation, and iv) allows for more accurate quantification of individual metabolites [7].
Nevertheless it has now become relatively routine to comprehensively compare the levels of thousands of metabolite peaks in one sample group to another in an untargeted manner. This approach, called untargeted metabolomics, has the potential to implicate unexpected pathways with a unique phenotype or disease process [8].
Reaves [9] coined out that one of the uses of metabolomics data is to pick out a few interesting compounds for further study. This was the further emphasized by [10], whereby he classified a high quality milk as having low somatic cell count (SCC), low standard plate count (SPC), no human pathogen, and no antibiotic residues. Hence, this preliminary effort aims at profiling the metabolites of goat milk in selected farms in some parts of Peninsular Malaysia.

A. Milk collection and sample preparation
The method of [11] was followed with modification. Goat milk was collected from a farm in Negeri Sembilan and Selangor and the freshly collected milk samples were immediately cooled in ice and appropriate care was observed to prevent any possible cross-contamination. The individually milked samples from different goats (n = 4) were pooled, collected in sterile bottles and transported in an icebox at temperature to the Faculty of Science and Technology, Universiti Sains Islam Malaysia (USIM), then immediately frozen at -20°C until further analysis. Aseptic techniques were applied, wherein all the equipment were pre-sterilized prior to analysis.
Goat milk was thawed and centrifuged at 3,000 x g for 10 min using centrifuge (Novil) at 4°C to remove high molecular weight compounds. The supernatant were then collected and analyses were done on the same day.

B. Instrumentation
Untargeted metabolomics profile of three goat milk samples (1 = Saanen species, 2 = Shami species) were carried out using LC-QToF-MS (LCMS iFunnel Q-TOF) at Agro Biotechnology Institute (ABI), MOSTI, MARDI, Serdang. Specifications include column type: RRHD Zorbax Eclipse Plus C18; 1.8µ x 2.1mm x 100mm with temperature of 23°C, injection volume of 1.00 µL, flow rate of 0.300 mL/min, acquisition mode applied a minimum range of 100m/z to a maximum of 1700m/z. An Agilent G6550A Accurate Mass QTOF was used to obtain the MS data. The mobile phases used were highly purified water + 0.1% formic acid (A) and acetonitrile+0.1% formic acid (B). The flow rate was set up at 0.6 mL/min with 30 min total run time. The prepared samples were placed into the LCMS autosampler. The injection volume of sample was 10 L. The samples were run at gradient 5% B (0-0.5 min); 30% B (0.5-13 min); and 95% B (13-22.0 min). Analysis was performed in positive ion mode with the following settings:-capillary voltage: 3500 V; nozzle voltage: 1000 V; fragmentor voltage: 175 V; nebulizer pressure (N): 35 psi; drying gas: 5L/min at 350°C, and sheath gas: 11 L/min at 350°C. The mass range was at 100-1700 m/z.

C. Database Annotation
For compound identification, several databases were referred to including KEGG, HMDB, LMP, and METLIN that were all from Mass Hunter Qualitative analysis software, Agilent Technologies.

III. RESULTS AND DISCUSSIONS
Metabolite levels, although present at low concentrations, can provide information of biochemical status in response to environments or genetic manipulation. These components can have a profound impact on the development and maintenance of metabolic, immunological and physiological processes [2], [12]. Figures 1 and 2 show the extracted compound chromatogram (ECC) of goat milk from both Saanen and Shami breeds at the end of the analysis period (30 min), respectively. The ECC profiles of goat milk were distinct with dissimilar chromatogram patterns suggestive of different breeds. Reference [2] specified that metabolomic analysis could detect changes in the metabolites as a result of genetic influence or feed given [13], [14].
The distinctive feed given to both breeds was barli sprout to Saanen, and indigofera spp. and palm leaves to Shami breeds. The other feed given were quite similar at any other goat farms namely napier leaves and brans. Table I listed out the known compounds detected, from the smallest to the highest mass present in milk from Saanen (only). However, the overall mass spectrometry analysis identified compounds with masses ranging from 0.099 kD to 19.6 kD and 0.098 kD to 18.8 kD for Saanen (22117 compounds) and Shami (20679 compounds) species, respectively. The ranges above than 1 kD indicate the poor sample preparation for metabolomic analysis. The metabolites were grouped into several categories namely plant secondary metabolites, drugs, naturally occurring metabolites, environmental contaminants, microbial metabolism in diverse environments including microbial secondary metabolites, and 'others' for easy classification purposes (Table II). Some microbial metabolites were noticed due to both long cold storage and the milking management practices of the milk samples obtained as supported by [15] and [16], correspondingly. Other compounds detected were drug metabolites such as pinacidil, spiramycin 3, deschlorobenzoyl indomethacin, methotrimeprazine sulfoxide and environmental contaminants (trichlorfon). Trichlorfon is an organophasphate which has adverse effects on health [17], [18].
The list of 20 highest concentrations of known compounds detected present in milk from Saanen and Shami are shown in Table II. It was observed that although the milk samples were from different breeds, some similarities existed. Such compounds include asp-phe-arg, maltose, pantothenic acid, 3-butyrl propionic acid, GPEtn(18:1(9Z)/0:0), and, neu5Ac alpha2-6Galbeta1-4Glcbeta-Sp., all of which were categorized as naturally occurring metabolites.
In addition, [19] indicated that valine and glycine were specific to goat milk, talose and malic acid to cow milk, and hydroxyglutaric acid to pasteurized samples via a gas chromatography-mass spectrometry (GCMS). It was noted that LCMS detected amino acids in the form of di-and tripeptides compared to GCMS that can identify individual amino acids [19] envisaging the beneficial effects towards health. For lipids, by-products of metabolism of polyunsaturated fatty acids (PUFA) generated hydroxy fatty acids, oxo fatty acids, conjugated fatty acids, and partially saturated trans-fatty acids as intermediates [20].

IV. CONCLUSIONS
Preliminary study of metabolite profiling of milk by mass spectrometry is able to generate the high quality data needed for further qualitative analysis. Goat milk has compounds related to health benefits in the form of conjugates.