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USE OF ONION EXTRACT AS DAIRY CATTLE FEED SUPPLEMENT: MONITORING OF PROPYL PROPANE THIOSULFONATE AS MARKER OF ITS EFFECT ON MILK ATTRIBUTES Paloma Abad, Natalia Arroyo-Manzanares, Lidia Gil, and Ana M. García-Campaña J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04395 • Publication Date (Web): 31 Dec 2016 Downloaded from http://pubs.acs.org on January 2, 2017

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Journal of Agricultural and Food Chemistry

USE OF ONION EXTRACT AS DAIRY CATTLE FEED SUPPLEMENT: MONITORING OF PROPYL PROPANE THIOSULFONATE AS MARKER OF ITS EFFECT ON MILK ATTRIBUTES

Paloma Abad †, Natalia Arroyo-Manzanares‡, Lidia Gil † and Ana M. García-Campaña‡*

† DMC Research Center S.L.U., Camino de Jayena nº82, E-18620 Alhendín, Spain ‡ Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Campus Fuentenueva s/n, E-18071 Granada, Spain *Corresponding author, (Tel: +34 958 242 385; Fax: +34 358 243 398; E-mail: [email protected])

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ABSTRACT

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Onion extract is used as feed supplement for dairy cows diet, acting as inhibitor of

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methane production; however its properties could alter sensory attributes of milk. In this

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work, we propose a method to evaluate the influence of this extract on milk properties,

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using propyl propane thiosulfonate (PTSO) as marker. PTSO is extracted using a

6

QuEChERS procedure and monitored by HPLC with UV detection. The method was

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applied to milk samples obtained from 100 dairy cows fed during two months with

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enriched feed. In addition, a milk tasting panel was established to evaluate the PTSO

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residue that should not be exceeded in order to guarantee milk sensory attributes. It was

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established that a value of PTSO lower than 2 mg Kg-1 does not alter milk organoleptic

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properties. This fact makes onion extract an interesting alternative as feed supplement to

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control the methane emissions without any influence on milk attributes.

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Keywords: onion extract, propyl propane thiosulfonate, milk, feed flavors, HPLC-UV

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INTRODUCTION

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Agricultural animal production is a source of gases that has been on the rise in recent

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years, thus becoming a potential ecological risk.1 Global emissions of methane (CH4), a

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potent greenhouse gas, represent 18% of total emissions to the atmosphere, contributing

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to global warming. The main sources of CH4 emissions are natural sources like

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wetlands, oceans, lakes, rivers and anthropoids followed by agricultural sources such as

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manure or enteric fermentation.2,3,4

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Recent studies indicate that changes in rumen ecosystem as a mitigating strategy could

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reduce CH4 emission as well as improve the efficiency of converting plant material into

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milk and meat.5 Ruminants in different production systems have access to different

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types and quantities of feed so the spatial distribution of produced greenhouse gases is

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expected to vary considerably depending on their location. In Europe, most of livestock-

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related CH4 emissions arise from fermentation in the digestive tract of herbivorous

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animals. The main actions for the reduction of ruminant CH4 emissions can be classified

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into three broad categories: addition of rumen modifiers (using specific substances that

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inhibit methanogenesis or biological control directed at reducing methanogens), use of

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diets that decrease H2 production which would be converted to CH4 (feeds, feeding

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management and nutrition), or increase of animal production and efficiency (fewer cows

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are needed to produce the same amount of milk).3

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The reduction of enteric CH4 production in ruminants is of environmental and a

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nutritional interest. The most promising strategy, an important objective for ruminant

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nutritionists, is the development of new feed additives that act as rumen modifiers.6 The

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correct choice of feed ingredients could modify microbial fermentation and hence the

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produced amount of CH4 and volatile fatty acids (VFA) such as propionate, acetate or

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butyrate. VFA generation also affects the amount of CH4 produced, since propionate 3 ACS Paragon Plus Environment


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formation consumes H2, whereas acetate and butyrate formation generate it for

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methanogenesis.7 Thus, a ruminant diet that increases propionate production will

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reduces CH4 generation and consequently atmospheric emissions, whereas a shift in

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favor of acetate and butyrate production will increase ruminant CH4 production.8,9

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In recent years, essential oils or plant extracts have been widely studied as dairy cows

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feed supplement and they have proven to act on the reduction of CH4 emissions by

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alteration of VFA (acetate, propionate and butyrate) generation. Moreover, it has been

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demonstrated that their use improves feed efficiency and milk production.10,11 As a

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consequence, the interest towards natural products like garlic, onion, cinnamon, yucca,

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anise, oregano or capsicum extracts has increased. These natural alternatives have

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previously shown antimicrobial activity and antioxidant properties and present an

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important effect in a positive alteration of rumen fermentation by VFA

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modification.12,13 Specifically, Allium spp. extracts, including onion extract (OE), have

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shown an important reduction of CH4 emissions by alteration of rumen VFA and this

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fact has been associated with the presence of organosulfur compounds characteristic of

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the alliaceous family.14,15,16

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Despite the beneficial effects of the use OE,17,18 its dosage in cattle feed should be

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controlled in order to preserve organoleptic properties (mainly odor and flavor) of milk

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or its derivative products as yoghurt and cheese. Several studies have demonstrated the

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transmission of onion flavor to milk after the intake indicating that it can be transferred

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to the udder via vascular routes from the digestive system.19,20 Propyl propane

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thiosulfonate (PTSO), an organosulfur compound characteristic of the Allium genus,

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mainly of onion, and responsible of the odor of freshly cut onion,21 has been proposed

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as OE residue marker in milk samples. Previously, in our lab, several analytical

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methods for PTSO analysis in animal feed were developed but none of them for milk or


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its derivative products.22,23 Thus, analytical methods for dairy products quality control

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should be developed.

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As stated above, the presence of PTSO in milk samples could alter its organoleptic

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properties, such as taste and flavor. Consequently, in this paper an analytical method for

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the control of OE residues in milk has been developed and validated. The method

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consisted of a sample treatment based on the extraction of PTSO with a QuEChERS

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(Quick, Easy, Cheap, Effective, Rugged and Safe) procedure. PTSO is then monitored

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in the extract by high-performance liquid chromatography with ultraviolet detection

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(HPLC-UV). The method has been applied for the control of milk samples from 100

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dairy cows that ate feed enriched with OE for two months. In addition, a milk tasting

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panel was carried out in order to determine the maximum content of PTSO residue in

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milk that should not be exceeded to guarantee its quality in relation to preserve its

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organoleptic properties.

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MATERIALS AND METHODS

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Chemicals and reagents

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All reagents were of analytical reagent grade. HPLC-grade solvents methanol (MeOH)

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and acetonitrile (MeCN) were purchased from VWR BDH Prolabo (West Chester,

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Pennsylvania, USA). Perchloric acid (70%), sodium chloride (NaCl) and magnesium

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sulfate (MgSO4) were supplied by Panreac (Madrid, Spain). Formic acid (FA) and

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sodium phosphate monobasic monohydrate were obtained from Sigma Aldrich (St

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Louis, MO, USA). HPLC mobile phases were filtered using a vacuum system with

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Millipore (Milford, MA, USA) filters (nylon, 0.20 µm, 47 mm). Samples were filtered

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through PET Chromafil® (Macherey-Nagel, Germany) filters (polyester, 25 mm, 0.20

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µm). 5 ACS Paragon Plus Environment


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Kits of SampliQ QuEChERS consisting of buffered QuEChERS extraction packet (4 g

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MgSO4, 1 g NaCl, 1 g sodium citrate, 0.5 g disodium hydrogen citrate sesquihydrate)

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and non-buffered QuEChERS extraction packet (4 g MgSO4, 1 g NaCl) were supplied

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by Agilent Tecnologies Inc (Wilmington, DE, USA). C18 and primary secondary amine

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(PSA) sorbents were also supplied by Agilent Tecnologies Inc. and Supelclean LC

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Alumina-N (Al) and SupelTM QuE Z-Sep (Z-Sep) sorbents by Supelco (Bellefonte, PA,

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USA).

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Ultrapure water (18.2 MΩ cm-1, Milli–Q Plus system, Millipore Bedford, MA, USA)

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was used throughout the trials.

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PTSO standard (min 95% of purity) and Garlicon®, an onion commercial extract

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containing PTSO as main active ingredient, were kindly provided by DOMCA S.A.

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(Alhendín, Granada, Spain).

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Individual stock standard solution containing 2 g L-1 of PTSO was prepared in MeOH

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and stored in a dark bottle at -20 ºC. It was stable for at least 3 months. Working

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solutions were prepared by diluting the stock solution in MeOH to the desired

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concentration prior to use.

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Instrumentation and equipment

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The Agilent 1260 Infinity HPLC (Agilent Technologies Inc) system was used for the

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proposed HPLC method, which includes a quaternary pump, an online degasser, an

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autosampler (injection volume from 0.1 – 900 µL, capacity: 100 vials), a column

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thermostat and a diode array detector (DAD). A C18 column Zorbax BONUS RP

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Analytical (150x4.6 mm, 5 µm) from Agilent Tecnologies Inc. was used.

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Additionally, confirmation of the presence of trace amounts of PTSO was carried out

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using an ultra-high performance liquid chromatography (UHPLC) system (Agilent 1290


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Infinity LC, Agilent Technologies Inc) equipped with a binary pump, on line degasser,

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autosampler (5 µL loop) and a column thermostat. Mass-spectrometer measurements

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were performed on a triple quadrupole mass spectrometer API3200 (AB Sciex, Toronto,

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ON, Canada) with electrospray ionization (ESI). A Zorbax Eclipse Plus RRHD (50 x

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2.1 mm, 1.8 µm) chromatographic column from Agilent Tecnologies Inc was used. The

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instrumental data were collected using the Analysts Software version 1.5 with Schedule

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multiple reaction monitoring (MRM)TM Algorithm (AB Sciex).

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A Universal 320R centrifuge (Hettich Zentrifugen, Tuttlingen, Germany) and a vortex-2

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Genie (Scientific Industries, Bohemia, NY, USA) were also used in the sample

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treatment procedure.

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Chromatographic conditions

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HPLC-UV analysis of PTSO

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The determination of PTSO was performed in a C18 column, previously described,

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using a mobile phase consisting of 30 mM aqueous perchloric acid solution (solvent A)

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and MeCN (solvent B), at a flow rate of 0.7 mL min−1. The injection volume was 20 µL

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and the gradient elution program was as follows: 50% B (0-3 min), 50-100% B (3-9

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min), 100% B (9-14 min). The initial conditions were re-established by 2 min of linear

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gradient, followed by equilibration time of 4 min. The total chromatographic run time

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was 20 min and detection wavelength was set at 200 nm (band width = 4 nm).

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UHPLC-MS/MS analysis of PTSO and its derivatives

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The sensitive quantification and confirmation of the presence/absence of PTSO and its

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typical derivatives (s-propylmercaptocysteine, CSSP and s-propyl mercaptoglutathione,

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GSSP) in milk samples was carried out by UHPLC-MS/MS. The analysis was

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performed according to the method previously development in our lab.23 Under these



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conditions PTSO, CSSP and GSSP were determined in 5.5 min, being characterized by

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two precursor-product ion transitions.

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Sample treatment for PTSO extraction

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A 5 ± 0.05 g milk sample was placed into a 50 mL conical bottom screw tube.

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Subsequently, 10 mL of 6.4% of FA in MeCN was added to the tube and vortexed for 1

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min. An extraction packet non-buffered QuEChERS was added and shaken vigorously

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for 1 min and then, the tube was vortexed for 1 min. The sample was centrifuged at

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5000 rpm for 5 min and afterwards an aliquot of the supernatant was filtered through a

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PET Chromafil® syringe filter and transferred to an Agilent CrossLab Vial. Finally it

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was injected into the HPLC.

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Experimental study

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The experiment was carried out at a local farm located in Reus, Tarragona (Spain). A

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total of 200 cows (11-months-old) were selected and isolated from the rest. All cows

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were maintained for 90 days with ad libitum access to feed and water. The feed intake

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was antibiotic-free.

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At the beginning of the experiment, cows were randomly allocated to two groups: 100

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cows were selected as control group (CG) and the other 100 cows were considered as

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treated group (TG). The diet of TG was supplemented with OE containing PTSO. OE

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was introduced gradually in feed. Dosage was increased by 5 g per animal and per day

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during a period of 5 days. During the experiment, the intake per day and per animal was

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established in 25 g of commercial OE (Garlicon®) to avoid rumen alterations derived

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from high concentrations of PTSO.14 The diet of CG had not any additional supplement.

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The field trial was extended for 2 months. In this period, the access of CG and TG to

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feed was controlled whereas the access to water was ad libitum.


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The presence of off-flavor depends on the time elapsed between cow consumption and

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milking. Previous studies indicated that onion flavors can persist in milk for longer than

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12 h, moreover, the stronger taste and flavor appears within 2 and 4 h after onion intake

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so milk samples were collected 3 h after eating cattle feed.24

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Once per week, milk samples from both groups were collected, homogenized and

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immediately pasteurized in two different tanks. Milk samples were stored at -20 ºC

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before analysis.

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Procedure for sensory evaluation of real milk samples containing PTSO

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Milk must have a good flavor and an attractive appearance, among other nutritional

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parameters in order to be acceptable to consumers. Organoleptic properties of milk may

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be affected by several external factors, such as feeds and weeds consumed or odors

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inhaled by the cow, internal infections of the cow or microbiological or chemical

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alterations of the milk. Consequently, color, odor and flavor of milk containing OE

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residues were evaluated in order to establish the maximum level of PTSO residue that

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could be present without causing any alteration to the organoleptic properties of milk.

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Sensory evaluation was carried out using a triangle test.

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Triangle test

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The triangle test is a discriminating test consisting of checking whether a difference

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exists between two products. Panelists taste a similar amount of three milk samples at

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the same temperature and they should recognize the differences. In a triangle test, the

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probability of getting the right result is 1/3. This methodology was applied following

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the methodology described in the ISO 4120:2004.25



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Panelist selection

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26 candidates were pre-selected as panelists and 16 of them were chosen for the triangle

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tests. Subsequently, panelists underwent a training session. Training for the formation

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of sensory memory was carried out by direct contact with PTSO (the evaluated

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attributes were odor, taste and flavor). The panel was trained in two sessions of 0.5 h at

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room temperature (20 ºC). The panelists (53% female and 47% male, from Granada,

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Spain) were daily consumers of milk all of them and the ages were between 25 and 60

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years old (average age 37 years old).

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Samples evaluation

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The tests were carried out at DOMCA S.A. laboratories, simulating the conditions

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described in the General Guidance for the design of test rooms (ISO 8589:2007).26 The

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design and analysis (using Thurstonian model) of sensory discrimination tests were

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carried out using XLSTAT software version 2016.1.

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RESULTS AND DISCUSSION

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Influence of the presence of cysteine and glutathione on the PTSO content in milk

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It has been demonstrated that PTSO reacts with cysteine (CYS) and glutathione (GSH),

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both present in a wide range of feed and food, resulting in CSSP and GSSP as products.

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23

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κ-casein), α−lactoalbumin and β−lactoglobuline. Although some of these proteins

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contribute to the presence of small amounts of CYS in milk, it is in its oxidized form,

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namely cystine (C6H12N2O4S2), which does not trigger a reaction with PTSO.27,28 The

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amount of PTSO could also be affected by the presence of GSH (a tripeptide with

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antioxidant properties which contains CYS) which is found in cellular tissues but it is

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expected that PTSO will not react with GSH because it is converted in oxidized

The most abundant proteins in milk are casein (αs1-casein, αs2-casein, βs1-casein and


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glutathione by the reaction with glutathione peroxidase.29,30 This is an important aspect

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to be taken into account when considering this molecule as a marker for OE residues in

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milk. Regardless of this issue, the absence of derivative compounds from PTSO (CSSP

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and GSSP) in milk was tested by applying a previously proposed UHPLC-MS/MS

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method23. As it can be seen in Figure 1, which shows a chromatogram from a cow

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whole milk sample spiked with 20 mg kg-1 of PTSO neither CSSP nor GSSP are

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detected.

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Development of the HPLC-UV method

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HPLC-UV chromatographic method was adapted from a method for PTSO

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determination in feed, previously developed in our laboratory.22 Gradient program, flow

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rate, injection volume and column temperature were re-optimized for PTSO

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determination in milk. All the experiments were performed using whole dairy milk

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samples spiked with 50 mg kg-1 of PTSO.

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The mobile phase consisted of 30 mM aqueous perchloric acid solution (A) and MeCN

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(B) and the final gradient program was as follows: 50% B (0-3 min), 50-100% B (3-9

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min), 100% B (9-14 min). The initial conditions were re-established by 2 min of linear

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gradient, followed by an equilibration time of 4 min. The total run time was 20 min.

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Different injection volumes (5, 10, 20 and 30 µL) were evaluated in order to select the

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optimum for obtaining the highest signal-to-noise ratio (S/N) and satisfactory peak

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efficiency. It was observed that peaks were not symmetrical from 30 µL, so a volume of

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20 µL was chosen. Results are shown in table S1. Column temperature was studied

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between 20 °C and 40 °C and an optimum of 25 ºC was selected as a compromise

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between retention time and column shelf life, since no significance differences were

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observed among all tested temperatures (see table S2). Finally, flow rate was evaluated



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from 0.6 to 1.2 mL min-1 and 0.7 mL min-1 was selected (see table S3) as a compromise

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between signal intensity and run time.

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Optimization of the sample treatment

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Sample treatment based on QuEChERS procedure has already been developed for the

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extraction of several food contaminants like antibiotics31, mycotoxins32 or pesticides33

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in milk but to the best of our knowledge, this is the first time that a QuEChERS

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procedure is used for the extraction of PTSO in Allium spp. The optimization was

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carried out using 5 g of whole dairy milk (ultra-high temperature, UHT) spiked at 50

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mg kg-1 with PTSO. Different extraction solvents were evaluated: MeOH, ethyl acetate,

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acetone, and MeCN (10 mL in all cases). Using MeOH, no protein precipitation was

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observed, with ethyl acetate no PTSO was detected and recovery values for acetone and

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MeCN were 19.8 and 76.2 % respectively. The best results in terms of recovery values

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were obtained with MeCN so it was selected for the rest of experiments.

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Thus, the extraction/partitioning process of the QuEChERS procedure was optimized

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for the analysis of PTSO in milk by using different solvents and Agilent SampliQ

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QuEChERS extraction kits: (a) 10 mL of MeCN, (b) 10 mL of MeCN + non-buffered

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QuEChERS kit, (c) 8 mL of H2O + 10 mL of MeCN + non-buffered QuEChERS kit,

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(d) 10 mL of MeCN + buffered QuEChERS kit, (e) 8 mL of H2O + 10 mL of MeCN +

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buffered QuEChERS kit, and (f) 8 mL of 30 mM NaH2PO4, buffer pH 7.1 + 10 mL of

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MeCN + buffered QuEChERS kit. The best recovery percentages and lower losses of

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analyte were obtained for the option (b) (see table S4).

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The second step of the QuEChERS procedure, based on a dispersive solid-phase

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extraction (dSPE) for cleaning up, was also evaluated and different commercial sorbents

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(C18, Al, Z-Sep and PSA) were tested. However, this step did not show any

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improvement on the analytical signal intensity so it was discarded. 12 ACS Paragon Plus Environment

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Once the sample treatment was selected, the amount of each salt (MgSO4 and NaCl) in

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the extraction kit and the influence of the presence of acid (FA) in the extracting solvent

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(10 mL of MeCN) were optimized by a multivariable approach to improve the PTSO

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recovery, using an experimental design and taking into account possible interactions

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among the chosen variables. A central composite design (23+ star, faced centered) with

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three spaced central points (17 runs) was used to generate the response surface, using

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the recovery percentage of PTSO in the extraction process as analytical response. These

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variables were studied in the following ranges: amount of NaCl (0-2 g), amount of

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MgSO4 (0-5 g) and percentage of FA in MeCN (0-10%).

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A Pareto chart (Figure 2a) was obtained from the screening experimental design

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showing that some of the studied variables and/or the interactions between them have a

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significant effect (positive or negative) on the response when their value changed inside

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the selected experimental domain. The length of each bar is proportional to the value of

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the corresponding effect (main effects or interactions between factors) statistically

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estimated by STATGRAPHICS. The vertical line shows the limit of decision to

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consider the significance of the factors and or of the interactions between them (based

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on the standardized effect = estimated effect/standard error; p-value 0.05 at 95% of

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confidence level).

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In this case, two main significant effects were observed: NaCl and MgSO4 amounts (B

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and C). Both of them have a positive effect on the response. There is also a significant

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interaction between NaCl amount and FA percentage (AB). This interaction causes a

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positive effect on PTSO recovery. Another interaction, with negative effect in this case,

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is the observed between NaCl and MgSO4 amount (BC).

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Based on the information obtained from Pareto chart, the three selected variables

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(amount of NaCl and MgSO4 and FA percentage) were simultaneously optimized by a



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response surface design. Optimum conditions were obtained (Figure 2b): NaCl = 1.9 g,

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MgSO4= 2.5 g and FA in MeCN = 6.4%. The p-value of the lack-of-fit test was 0.089

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and the determination coefficient (r2) was 84.1%, proving the suitability of this design.

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To verify the selected optimum values for the studied variables, several milk samples

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(n=5) were treated with the proposed method. A mean value of 97.5 ± 1.1% was

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obtained for PTSO recovery.

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Finally, the extraction volume (MeCN with 6.4 % FA) was evaluated in the range of 2.5

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to 10 mL and a volume of 10 mL was chosen as optimum since a decrease of the

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volume amount involved lower recovery percentages (see table S5). Sample size was

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also evaluated between 1 and 10 g, selecting 5 g in order to obtain better signal intensity

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and therefore lower limits of detection (LOD) (see table S6).

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Characterization of the method

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In order to check the suitability of the proposed method, linear dynamic range, limits of

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detection (LOD) and limits of quantification (LOQ) were evaluated for milk samples

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from different ruminants: pasteurized dairy milk, UHT dairy milk (whole, semi-

297

skimmed and skimmed), UHT semi-skimmed goat milk and UHT semi-skimmed sheep

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milk. In addition, studies of precision and recovery for PTSO content in each matrix

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were carried out. Figure 3 shows a chromatogram of a whole milk sample spiked with

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50 mg Kg-1 of PTSO.

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Calibration curves and performance characteristics

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Matrix-matched calibration curves were established by spiking milk blank samples at

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six different concentration levels of PTSO (ranging from 5 to 500 mg kg−1 for

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pasteurized dairy cow milk, UHT semi-skimmed goat milk and UHT semi-skimmed

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sheep milk and from 10 to 500 mg kg−1 for whole, semi-skimmed and skimmed dairy

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cow milk). Each level was prepared in duplicate and injected in triplicate. Statistical 14 ACS Paragon Plus Environment

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parameters were calculated by least-square regression. Obtained values for LOD (3xS/N

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ratio) and LOQ (10xS/N ratio), linear dynamic range and linearity are shown in Table 1.

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Precision study

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The precision of the whole method was evaluated in terms of repeatability (intraday

311

precision) and intermediate precision (interday precision). Repeatability was assessed

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by applying the whole procedure to six types of milk samples on the same day

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(experimental replicates) spiked at three different concentration levels of PTSO: 10, 300

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and 500 mg kg-1 and each sample was injected in triplicate (instrumental

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replicates). Intermediate precision was evaluated with a similar procedure, but in this

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case, five samples of each concentration level were analyzed five different days. The

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results, expressed as relative standard deviation (RSD) of peak areas, are shown in

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Table 2. Satisfactory precision was obtained, with RSDs lower than 10% in all cases.

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Recovery study

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Three milk samples were spiked at three different concentration levels of PTSO,

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submitted to the QuEChERS procedure and injected per triplicate. Recoveries were

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calculated as 100 × [(signal of a spiked sample / signal of spiked extract]; additionally

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in all cases blank samples were previously analyzed and none of them gave a positive

324

result for PTSO. Results are shown in Table 3, demonstrating the trueness of the

325

proposed method (all of the recovery were higher than 82%).

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Analysis of real milk samples

327

The validated method was applied for the analysis of milk samples from CG and TG.

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All samples were prepared following the proposed sample treatment and analyzed in

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triplicate by the HPLC-UV method. None of them gave a positive result of PTSO levels

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above the LODs. To confirm the absence of PTSO, a more sensitive and confirmatory



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UHPLC-MS/MS method previously developed in our lab was applied to all samples

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(LOQ=250 µg Kg-1).23 As it was expected, negative results were obtained from samples

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belonging to CG. However, in all samples from TG the presence of PTSO was detected

334

and confirmed. Thus, a sensory evaluation was carried out in order to confirm that the

335

detected amount of PTSO did not affect to organoleptic properties of the milk samples.

336

Sensory evaluation of real milk samples containing PTSO

337

Three different tests were performed in three consecutive days. Test 1 and test 2 were

338

performed in order to establish a sensorial limit for PTSO in milk samples whereas test

339

3 was performed to check organoleptic properties in real milk samples. In all cases, 100

340

g of milk were offered to the panelists at a temperature of 20 ±1 °C in a transparent

341

plastic vessel coded with a two-digit number. The first test consisted of free-PTSO milk

342

samples versus milk samples containing 10 mg kg-1 of PTSO. Test samples for each

343

panelist were randomly selected by XLSTAT program. Milk samples containing PTSO

344

were correctly identified by 88% of panelists and therefore it can be said that this

345

concentration of PTSO modifies milk organoleptic properties (p-value < 0.0001). The

346

second test consisted of the comparison of milk samples containing 2 mg kg-1 of PTSO

347

versus free-PTSO milk samples. In this case, 87.5% of panelist did not find any

348

significant differences among the tested samples showing that a concentration of 2 mg

349

kg-1 of PTSO does not alter organoleptic properties of milk (p-value = 0.661). All

350

samples were prepared with pasteurized dairy milk.

351

Finally, the test 3 was carried out with real milk samples from TG at the end of the

352

experiment (results shown in table S7). In this case, samples from CG were faced to

353

samples from TG. None of the panelists found significant differences among the tested

354

samples (p-value= 0.661).


Page 17 of 30


355

Hence, although small traces of PTSO were found in milk samples from TG by

356

UHPLC-MS/MS, the identified concentration does not alter organoleptic properties of

357

milk and Garlicon® can therefore be used as supplement feed at doses of 25 g per day.

358

This positive result allows us to draw a strategy to reduce CH4 emissions without

359

compromising milk quality. Moreover, the proposed analytical method is suitable for

360

controlling the presence of residues of PTSO in milk as marker of OE, at concentrations

361

that could alter organoleptic properties of milk (above 2 mg kg-1).

362

ABBREVIATIONS USED

363

CH4, methane; VFA, volatile fatty acids; PTSO, propyl propane thiosulfonate; HPLC,

364

high performance liquid chromatography; UV, ultraviolet detection system; CYS,

365

cysteine; GSH, glutathione; CSSP, s-propyl mercaptocysteine; GSSP, s-propyl

366

mercaptoglutathione; UHT, ultra-high temperature.

367

SUPPORTING INFORMATION DESCRIPTION

368

Table S1. Effect of The Injection Volume on Area Values and S/N Ratio for the

369

Proposed HPLC-UV.

370

Table S2. Effect of Column Temperature on Peak High and Retention Time for

371

the Proposed HPLC-UV Method.

372

Table S3. Effect of Flow Rate on Peak Area and Width and Retention Time for

373

the Proposed HPLC-UV Method.

374

Table S4. Influence of QuEChERS Procedure on PTSO Recovery Values for the

375

Proposed HPLC-UV Method.

376

Table S5. Effect of the Extraction Volume of MeCN (6.4 % FA) on PTSO

377

Recovery Values for the Proposed HPLC-UV Method.

378

Table S6.

379

Proposed HPLC-UV Method.

380

Table S7a. Samples Assigned to Each Judge to Perform the Triangular Test.

Effect of Sample Size on PTSO Extraction Efficiency Using the

Table S7b. Results of Sensory Analysis of Real Milk Sample. 17 ACS Paragon Plus Environment


381

Page 18 of 30

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URL: 20 ACS Paragon Plus Environment

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http://www.iso.org/iso/home/store/catalogue_tc/catalogue_detail.htm?csnumber=33495. (Friday, October 21, 2016). 26

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Page 22 of 30

NOTE: The authors are thankful for the financial support from DOMCA S.A., the Campus of International Excellence – BioTic Granada (Project- mP_BS_2, CAPRILACT) and the research group FQM-302 (University of Granada, Spain).


Page 23 of 30


FIGURE CAPTIONS

Figure 1. Chromatogram of a milk sample spiked with 20 mg Kg-1 of PTSO extracted with the optimized extraction procedure and analyzed by UHPLC-MS/MS.

Figure 2. (a) Pareto chart showing the effects of the studied variables on the recovery percentage. Vertical line shows the limit of decision to consider the significance of the factors (based on the effect = estimated effect/standard error, P-value = 0.05 at 95% of confidence); (b) Response surface plot showing the effect of FA concentration on MeCN and NaCl and MgSO4 on PTSO recovery.

Figure 3. Chromatograms from (a) a blank whole milk sample and (b) a whole milk sample spiked with PTSO at 10 mg kg-1(b).



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TABLES

Table 1. Statistical and Performance Characteristics of the Proposed HPLC-UV Method. LOD

LOQ

(mg kg-1)

(mg kg-1)

Linear dynamic R2

Matrix range (mg kg−1) A

6.0-500

0.9957

1.8

6.0

B

6.3-500

0.9987

1.9

6.3

C

6.0-500

0.9997

1.8

6.0

D

4.7-500

0.9996

1.4

4.7

E

2.7-500

0.9995

0.8

2.7

F

3.7-500

0.9994

1.1

3.7

A: UHT skimmed dairy milk; B: UHT semi-skimmed dairy milk; C: UHT whole dairy milk; D: Pasteurized whole dairy milk; E: UHT semi-skimmed goat milk; F: UHT semi-skimmed sheep milk.


Page 25 of 30


Table 2. Precision Study (% RSD of Peak Areas) of the Proposed HPLC-UV Method. Repeatability

Intermediate precision

(n = 9)

(n = 15)

Matrix/Level

Level 1

Level 2

Level 3

Level 1

Level 2

Level 3

A

3.01

0.97

2.56

7.94

1.57

3.23

B

8.61

1.83

1.22

8.66

2.08

4.46

C

4.45

0.82

1.67

6.57

1.52

1.29

D

5.43

0.68

0.26

6.82

1.50

1.61

E

4.97

1.10

0.39

7.29

3.63

0.98

F

5.28

3.22

1.03

5.78

1.73

1.01

Level 1: 10 mg kg−1; level 2: 300 mg kg−1; level 3: 500 mg kg−1 A: UHT skimmed dairy milk; B: UHT semi-skimmed dairy milk; C: UHT whole dairy milk; D: Pasteurized whole dairy milk; E: UHT semi-skimmed goat milk; F: UHT semi-skimmed sheep milk.



Page 26 of 30

Table 3. Recovery Study of the HPLC-UV Proposed Method. Recovery (n = 9) Matrix/Level

Level 1

Level 2

Level 3

A

82.5 (8.8)

93.1 (1.8)

92.5 (1.2)

B

82.4 (4.5)

92.2 (0.8)

95.7 (1.7)

C

84.0 (5.4)

92.0 (0.7)

96.3 (0.3)

D

84.3 (5.3)

93.3 (3.2)

91.9 (1.0)

E

82.5 (8.8)

93.1 (1.8)

92.5 (1.2)

F

89.4 (5.0)

96.5 (1.1)

97.5 (0.4)

Level 1: 10 mg kg−1; level 2: 300 mg kg−1; level 3: 500 mg kg−1 A: UHT skimmed dairy milk; B: UHT semi-skimmed dairy milk; C: UHT whole dairy milk; D: Pasteurized whole dairy milk; E: UHT semi-skimmed goat milk; F: UHT semi-skimmed sheep milk.


Page 27 of 30


FIGURE GRAPHICS Figure 1

PTSO 2.3e6 2.2e6 2.1e6 2.0e6 1.9e6 1.8e6 1.7e6 1.6e6 1.5e6 1.4e6

Intensity, cps

1.3e6 1.2e6 1.1e6 1.0e6 9.0e5 8.0e5 7.0e5 6.0e5 5.0e5 4.0e5 3.0e5 2.0e5 1.0e5 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

Time, min

27

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Figure 2


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Figure 3



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