Caprine milk fermentation enhances the antithrombotic properties of cheese polar lipids

Journal of Functional Foods 61 (2019) 103507

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Caprine milk fermentation enhances the antithrombotic properties of cheese polar lipids

T

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Ronan Lordana,b, , Aaron Walshc, Fiona Crispiec, Laura Finneganc,d, Martina Demurue, Alexandros Tsouprasa, Paul D. Cotterc,d, Ioannis Zabetakisa,b a

Department of Biological Sciences, University of Limerick, Limerick, Ireland Health Research Institute (HRI), University of Limerick, Limerick, Ireland c Teagasc Food Research Centre, Moorepark, Fermoy, Cork, Ireland d APC Microbiome Ireland, Cork, Ireland e Department of Life and Environmental Sciences, University of Cagliari, via Ospedale 72, 09124 Cagliari, Italy b

A R T I C LE I N FO

A B S T R A C T

Keywords: Polar lipids Fermentation Goat milk Inflammation Antithrombotic Metagenomics

The effect of fermentation on the antithrombotic properties of polar lipids in raw and pasteurised caprine milk was assessed through the production of various cheeses using the same starter culture. The total lipids (TL), total neutral lipids (TNL), and total polar lipids (TPL) were extracted from each milk and cheese and the TPL fatty acid profiles were analysed by GC–MS. It was determined that fermentation influenced the polar lipid fatty acid composition. The milk and cheese polar lipids exhibited potent antithrombotic activities with IC50 values ranging from 79 to 226 µg against platelet-activating factor (PAF) induced platelet aggregation. Finally, shotgun metagenomics determined the species-level microbial composition and functional potential of each milk and cheese. Several microbe-encoded phospholipid biosynthetic genes were identified in the most antithrombotic cheeses. Lactococcus lactis and other microbial species may play a significant role in determining the antithrombotic properties and fatty acid composition of caprine cheese polar lipids.

1. Introduction Cardiovascular disease (CVD) is the leading cause of mortality worldwide (World Health Organization, 2017). Maladaptive diet and lifestyle is responsible for the development of chronic diseases such as CVD, insulin resistance, obesity, and cancer (Mozaffarian, 2016). Previously, dairy products were generally considered a negative component of the diet due to their perceived association with an increased risk of CVD due to their high saturated fatty acid (SFA) and cholesterol content (Lordan, Tsoupras, Mitra, & Zabetakis, 2018). However, an increasing volume of research has demonstrated that dairy products may have a neutral or beneficial impact on cardiovascular health, and do not seem to significantly affect blood cholesterol levels (Lordan, Tsoupras et al., 2018). Indeed, cheese previously was deemed harmful to cardiovascular health due to its high saturated fatty acid (SFA) content, but now research indicates that cheese and other fermented dairy products exert positive effects on cardiovascular health (Guo et al., 2017; Labonté, Couture, Richard, Desroches, & Lamarche, 2013; Lordan & Zabetakis, 2017a; Lordan, Tsoupras et al., 2018; Thorning et al., 2016). Caprine dairy products are popular in the Mediterranean

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Basin and caprine milk is one of the most consumed liquid milks globally (Capurso, Crepaldi, & Capurso, 2018; Lordan & Zabetakis, 2017b). Caprine milk is associated with several health benefits including potential cardiovascular benefits (Haenlein, 2004; Lordan & Zabetakis, 2017b; Lordan, Tsoupras et al., 2018). Consumer trends indicate that the consumption of raw milk and dairy products produced by raw milk have increased in recent years (Verraes et al., 2015), possibly in response to suggestions that heating the milk destroys the nutritional and health benefits of milk (Claeys et al., 2013). While raw milk is associated with more favourable organoleptic properties (Fotou et al., 2011), there is concern that raw milk products may increase the consumer’s risk of exposure of pathogenic organisms such as Listeria monocytogenes, Campylobacter jejuni, Staphylococcus aureus, Escherichia coli, and Salmonella (Claeys et al., 2013). Nevertheless, raw milk artisan cheese products are produced and preferred by consumers due to preference in taste and minimal processing (Haenlein, 2004; Verraes et al., 2015). Chronic diseases such as atherosclerosis and CVD are characterised by low-grade systemic inflammation (Lordan, Tsoupras, & Zabetakis, 2019; Tsoupras, Lordan, & Zabetakis, 2018). Platelet-activating factor

Corresponding author at: Department of Biological Sciences, University of Limerick, Limerick, Ireland. E-mail address: [email protected] (R. Lordan).

https://doi.org/10.1016/j.jff.2019.103507 Received 22 May 2019; Received in revised form 6 August 2019; Accepted 6 August 2019 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.

Journal of Functional Foods 61 (2019) 103507

R. Lordan, et al.

(PAF) is a potent phospholipid proinflammatory mediator implicated in all stages of atheroma development (Tsoupras, Lordan, & Zabetakis, 2018). PAF carries out its proinflammatory and prothrombotic activities through the PAF-receptor (PAF-R). It has been demonstrated that polar lipids from marine and animal origin exert anti-inflammatory and antithrombotic properties against the actions of PAF (Lordan, Tsoupras, & Zabetakis, 2017). In particular, in vitro studies have demonstrated that bovine, caprine, and ovine cheese products possess antithrombotic polar lipids that bind to the PAF-R (Megalemou et al., 2017; Poutzalis et al., 2016; Tsorotioti et al., 2014). It has been suggested that caprine milk, yoghurt, and cheese may possess greater biological activity than bovine or ovine dairy products. However, this has yet to be established (Lordan, Nasopoulou, Tsoupras, & Zabetakis, 2018). Recently, it has been shown that fermentation of ovine milk by specific starter cultures increased the antithrombotic activity of the yoghurts produced (Lordan, Walsh, et al., 2019). It has also been demonstrated that lipids in ovine and caprine meat exhibit antithrombotic activities by inhibiting the PAF pathway (Poutzalis, Lordan, Nasopoulou, & Zabetakis, 2018). In general, milk-derived polar lipids seem to significantly contribute to health and even infant development (Claumarchirant et al., 2016; Rombaut & Dewettinck, 2006). The potential health benefits of milkderived polar lipids warrant further thorough investigation. The aim of this study was to establish if the production of cheeses using raw or pasteurised caprine milk can affect the antithrombotic properties of caprine cheeses. Furthermore, shotgun metagenomics was employed to characterise the species-level microbial composition of the milk and cheeses to determine if the detected species contained genes associated with fatty acid and/or lipid metabolism.

or pasteurised milk and allowed to sit for 18 h at 20 °C. The production methods of the R-log and P-log were identical except for the initial milk used. The curd was drained away from the whey in hanging bags for 18 h at 20 °C and then mixed and placed in log moulds and salt was added (1% w/w of the final product) and stored for 24 h at 20 °C. The logs were then placed at 5 °C for a further 72 h before being removed from the moulds and packaged for transport to the laboratory. The Crottin cheese was produced by placing the same curd, cultures, and salt into a round mould directly from the vats, salt was sprinkled on the outside of the cheese instead of being mixed in as with the other cheeses. The Crottin cheese is then stored in these moulds at 20 °C for 24 h. Following this, the cheese is then placed in fridge at 5 °C for 72 h. The soft cheese was produced using the same composition of cultures and salt concentration, however instead of putting into moulds, the suspension containing both the curd and whey is placed into individual 120 g pots and stored at 20 °C for 24 h, before being placed at 5 °C until used in the laboratory. Samples of both the raw and pasteurised milk used to make each cheese were taken and stored at 4 °C before transportation to the laboratory, which occurred in less than two days. All cheeses and milks were prepared/sampled in triplicate from the same milk bulk tanks on the same day at the production facilities of Inagh Farmhouse cheese Ltd (Inagh, Co. Clare, Ireland). After production, all cheeses and samples of the milks used to make the cheeses were transferred to the laboratory and stored at −20 °C. All cheese lipids were fully extracted within 7 days of delivery to the laboratory. All cheeses described were produced at a pilot scale level at an active cheese production facility in Ireland. The cheese production process is summarised in the production schematic in Fig. 1.

2. Materials and methods

2.3. Lipid extraction

2.1. Materials and chemicals

The total lipids (TL) of all milk and cheese were extracted from 100 g of sample according to the Bligh-Dyer method (Bligh & Dyer, 1959). One-tenth of the samples was stored dry at −20 °C. The TL was then further separated into total neutral lipids (TNL) and total polar lipids (TPL) by counter-current distribution (Galanos & Kapoulas, 1962). All lipid extracts were stored devoid of solvent in sealed vials under nitrogen atmosphere at −20 °C for a maximum of six weeks. All extractions were carried out in triplicate (n = 3).

All organic solvents used in the lipid extraction and isolation process were purchased from Fisher Scientific Ireland Ltd. (Dublin, Ireland). All chemical reagents used for platelet aggregometry and lipid standards for GC–MS were purchased from Sigma-Aldrich (Wicklow, Ireland). All platelet aggregometry consumables were purchased from Labmedics LLP (Abingdon on Thames, U.K.). All GC–MS consumables were purchased from Apex Scientific Ltd. (Kildare, Ireland). The PowerSoil DNA Isolation kit and PowerNad tubes were procured from Cambio (Cambridge, United Kingdom). Lysozyme, mutanolysin and proteinase K were purchased from Sigma-Aldrich (Wicklow, Ireland). The Qubit High Sensitivity DNA assay was purchased from Life Technologies (ThermoFisher Scientific Ltd., Dublin, Ireland).

2.4. GC–MS analysis Fatty acid methyl esters (FAME) of 35 mg of TPL of the milk and cheese samples were prepared using a modified method of Tsoupras, Lordan, Demuru et al. (2018) and Nasopoulou, Stamatakis, Demopoulos, and Zabetakis (2011). In brief, FAME were prepared using a solution of 0.5 M KOH in CH3OH (KOH–CH3OH method) and extracted with n-hexane. A five-point calibration curve was prepared using five solutions of methyl esters of heptadecanoic acid (17:0–50 ppm, 100 ppm, 200 ppm, 400 ppm, 800 ppm) and heneicosanoic acid (21:0—five 500 ppm injections) (Sigma Aldrich, Wicklow, Ireland). Five 1 µL injections of each solution were analysed with a Varian 431-GC equipped with a split/splitless injector and coupled to a Varian 210-MS ion trap mass detector (Agilent Technologies, Palo Alto, CA, USA). The ratio of the mean of heptadecanoic acid (C17:0) to that of the internal standard (C21:0) is used as the y-axis variable, whereas the concentration (ppm) of C17:0 is used as the x-axis variable of the calibration curve. The equation that defined the curve was: y = 0.0041x + 0.12 with an R2 = 0.9969, where the ratio of the area of the analyte peak to that of the internal standard represents the y value for the above equation and the x value represents the analyte concentration of a selected fatty acid in the unknown mixture. Separation of the FAME was performed on an Agilent J&W DB-23 fused silica capillary column (60 m, 0.25 mm, i.d., 0.25 µm; Agilent Technologies Ltd.). The injector was set at 230 °C with a split ratio of 1:20. The carrier gas was high purity helium with a liner flow rate of 1 mL/min.

2.2. Milk processing and cheese production Raw milk was obtained from a bulk tank that contained milk from Saanen, Toggenburg, British Alpine, and Nubian breeds of goat. To make cheeses from raw milk, unhomogenised raw milk was heated to 24 °C before inoculation with freeze dried cultures at a rate of 50 U/ 500 L as per manufacturer’s instructions (Flora Danica®, Chr. Hansen, Cork, Ireland) and rennet at a concentration of 3.06 IMCU/L (International milk clotting units; Kalase®, CSK Food Enrichment C.V., Leeuwarden, The Netherlands). In total, four caprine milk cheeses were produced from the raw milk and pasteurised milk. These were a raw milk log (R-log), a pasteurised milk log (P-log), a soft cheese known as Divine, and a Crottin cheese that is similar to the traditional Crottin de Chavignol. The bacterial culture used (Flora Danica®, Chr. Hansen, Cork, Ireland) contained Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. lactis biovar. diacetylactis, and Leuconostoc spp. To obtain pasteurised milk, raw milk was heated to 72 °C for 15 s before being cooled to 24 °C. To make the cheeses, cultures and rennet were then added as previously described to vats containing either raw 2

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Fig. 1. A schematic detailing the processes used to produce the caprine cheese from raw and pasteurised milk in this study.

(2.5 mg BSA/mL in 0.9% saline). Testing was carried our as described by Tsoupras, Lordan, Demuru et al. (2018). In brief, PAF was added to the cuvettes in order to induce maximum-reversible aggregation (2.6 × 10-8 M, final concentration in the cuvette). Subsequently, polar lipids samples dissolved in BSA saline solution were added to PRP at various concentrations and allowed to incubate for 1 min with stirring prior to the addition of PAF. Then, the amount of PAF that causes maximum-reversible platelet aggregation was added to the cuvettes and a reduction of platelet aggregation was observed and recorded by comparison to the maximum-reversible platelet aggregation. A dosedependent linear relationship was observed. The concentration of polar lipids required to cause 50% inhibition of PAF-induced platelet aggregation was calculated for each sample. This measurement is the IC50 value and denotes the level of platelet aggregation inhibition.

The oven temperature was initially programmed to 100 °C for 5 min, followed by a ramp of 3 °C/min until reaching 240 °C, then held isothermally at 240 °C for 10 mins. The transfer line, trap, and manifold temperatures were 250 °C, 200 °C, and 110 °C respectively. The total run time was 61.64 mins. All mass spectra were acquired in the electron impact mode. Scans were performed from 1 to 400 m/z at a rate of 1 scan/sec. FAME were identified using 37-component FAME standards mix (Sigma Aldrich, Wicklow, Ireland) by comparison of the retention times and mass spectra of relative peaks with the aid of a Varian Star Chromatography Workstation Version 6 software (Agilent Technologies Ltd.) and a NIST library (Gaithersburg, MD, USA). 2.5. Human platelet aggregation assay Blood was obtained from healthy human volunteers (n = 12) who provided written informed consent in accordance with the Declaration of Helsinki via venepuncture as described previously by Tsoupras, Lordan, Demuru et al. (2018) and Tsoupras, Zabetakis, and Lordan (2019). All experiments were performed in triplicate and each replicate was repeated using a different donors sample following appropriate control tests of the solvents used on human platelets. In brief, blood was drawn from the median cubital vein or cephalic vein depending on their condition and availability using a 20G needle (Sarstedt Ltd., Wexford Ireland). Blood samples were fasting, and donors were free from all forms of anti-platelet therapy or nonsteroidal anti-inflammatory drugs. Blood was drawn into evacuated sodium citrate monovettes (0.106 mol/L in a 1:10 ratio of citrate to blood; Sarstedt Ltd., Wexford, Ireland), followed by immediate centrifugation at 170g for 18 mins at 24 °C to obtain the supernatant platelet-rich plasma (PRP). A second centrifugation (Eppendorf 5702, Eppendorf Ltd, Hamburg, Germany) at 1500g for 20 mins at 24 °C was carried out to obtain the platelet poor plasma (PPP). The PRP was standardised to 500,000 platelets µL−1 using a Shimadzu UV-1800 spectrophotometer (Kyoto, Japan), before analysis on a Chronolog-490 two channel turbidimetric platelet aggregometer (Havertown, PA, USA), coupled to the accompanying AGGRO/LINK software package. All analyses were carried out within 2.5 h of the initial blood draw and PRP was stored at 24 °C before use. PAF and lipids samples were dissolved in a solution of BSA-saline

2.6. Total DNA extraction from milk and cheese DNA was extracted from 15 mL milk according to the method of Walsh et al. (2016) as follows. Milk was centrifuged at 5444g for 30 min at 4 °C to pellet the microbial cells in the liquid. The cell pellet was resuspended in 200 µL of PowerBead solution from the PowerSoil DNA Isolation kit (Cambio, Cambridge, United Kingdom). The resuspended cells were transferred to a pre-heated (at 60 °C) PowerBead tube (Cambio, Cambridge, United Kingdom). A 90 µL volume of 50 mg/mL lysozyme (Sigma-Aldrich Ltd., Wicklow, Ireland) and 50 µL of 100 U/ mL mutanolysin (Sigma-Aldrich Ltd., Wicklow, Ireland) were added, and the sample was incubated at 60 °C for 15 min. A 28 µL volume of proteinase K (20 mg/mL; Sigma-Aldrich Ltd. Dublin, Ireland) was added, and the sample was incubated at 60 °C for a further 15 min. DNA was then purified from the sample by the standard PowerSoil DNA Isolation kit protocol (Cambio, Cambridge, United Kingdom). For the DNA extraction from the cheeses, a pre-treatment step was included as follows. 5 g of the cheese sample was placed in a stomacher bag with 50 mL of 2% trisodium citrate and homogenized using a masticator mixer (IUL SA, Barcelona, Spain) for 3 min. Then 15 mL of the cheese solution, was placed into sterile Falcon tubes and centrifuged for 30 min at 4500g. After centrifugation, the supernatant was discarded, and the pellet was placed in a 2 mL Eppendorf tube. The pellet was washed 3

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several times with sterile phosphate-buffered saline (PBS) by centrifuging it at 14,500g for 1 min, until the supernatant was completely clear (Bertuzzi et al., 2018). The pellet was then added to PowerBead tubes (Cambio, Cambridge, United Kingdom) and then treated as described above.

Table 1 Content of total lipids (TL), expressed in grams per 100 g of raw (R) and pasteurised (P) caprine milk and cheeses (mean ± SD, n = 3), total polar lipids (TPL) and total neutral lipids (TNL), expressed as percentages of TL in the caprine milk and cheese samples (mean ± SD, n = 3). Sample

2.7. Whole metagenome shotgun sequencing

Milk (R) Milk (P) Cheese Log (R) Cheese Log (P) Soft Cheese (P) Crottin Cheese (R)

Whole-metagenome shotgun DNA was fragmented and adaptors and indices added using the Illumina Nextera XT guide in accordance with manufacturer’s instructions, except that tagmentation time was increased from 5 mins to 7 mins. After indexing, the average fragment size was assessed using an Agilent Bioanalyser High Sensitivity Assay (Agilent Technologies) and quantified using a Qubit High Sensitivity assay (Life Technologies). Subsequently, samples were then pooled equimolarly and the final pool was quantified by quantitative PCR using the Kapa Library Quantification Kit for Illumina (Roche). In accordance with standard Illumina sequencing protocols, the pool was then sequenced on the Illumina MiSeq sequencing platform in the Teagasc sequencing facility, with a 2 × 300 cycle V3 kit.

TL (g/100 g) 1.96 1.65 10.4 11.9 12.2 9.47

± ± ± ± ± ±

TNL (%TL) a

0.22 0.24a 0.77b 0.59c 0.81c 0.14b

92.9 94.3 94.9 95.8 95.7 93.7

± ± ± ± ± ±

TPL (%TL) a

0.5 0.4b 0.1b 0.6c 0.6c 0.8ab

7.10 5.01 3.70 4.05 4.08 6.32

± ± ± ± ± ±

0.2c 1.1ab 0.9a 0.8a 0.9a 0.8bc

a,b,c

Different superscripts indicate significant differences among different cheese and milk samples within the same lipid classes when means are compared using a Tukey’s HSD multiple comparison test (p < 0.05).

were no significant differences in the percentage of TPL present between the raw and pasteurised cheese logs or the soft cheese. However, the Crottin cheese TPL percentage was significantly higher than the other cheeses but not statistically significantly different from either the raw or pasteurised milk.

2.8. Bioinformatics 3.2. GC–MS polar lipid composition

Shotgun metagenomic fastq files were processed as described previously (Walsh et al., 2018). Briefly, raw fastq files were converted to unaligned bam files using SAMtools (Li et al., 2009). Duplicate reads were subsequently removed using Picard Tools (https://github.com/ broadinstitute/picard). Next, low quality reads were removed using the trimBWAstyle.usingBam.pl script from the Bioinformatics Core at UC Davis Genome Centre (https://github.com/genome/genome/blob/ master/lib/perl/Genome/Site/TGI/Hmp/HmpSraProcess/ trimBWAstyle.usingBam.pl). Specifically, reads were trimmed to 200 bp, while all reads with a quality score less than Q30 were discarded. The resulting fastq files were then converted to fasta files using the fq2fa option from IDBA-UD (Peng, Leung, Yiu, & Chin, 2012). Species-level analysis was performed using MetaPhlAn2 (Truong et al., 2015), which measures the abundance of species-specific marker genes in metagenomic reads. Microbial pathway analysis was performed using HUMAnN2 (Abubucker et al., 2012), which measures the abundances of UniRef clusters (Suzek et al., 2015) by aligning sequences against the ChocoPhlAn database.

The data in Table 2 suggests that fermentation and the use of raw or pasteurised milk plays a significant role in augmenting the fatty acid composition of polar lipids of caprine milk in the production of caprine cheeses. Pasteurisation of the raw milk lead to a statistically significant decrease in the percentage of SFA and a significant increase in the percentage of monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) bound to the polar lipids in the TPL fraction of the milk. In particular, there was statistically significant decrease in the percentage of 14:0, 16:0, and 18:0 fatty acids and there was a significant increase in the percentage of 18:1, c9, t12-18:2, 18:3, 22:5ω6, and 22:6ω3 fatty acids bound to the TPL when raw milk was pasteurised. When raw milk was used to produce the R-log there was a significant decrease in the percentage of SFA and a significant increase in the percentage MUFA bound to the TPL. This was also the case for the production of the Crottin cheese (R), but there was a significant decrease in the percentage of PUFA in the TPL of this cheese. Notably, the levels of some fatty acids were significantly increased when raw milk was fermented to R-log, including: 18:1, c6, c9-18:2, t10, c12-18:2, and 22:5ω6. When pasteurised milk was used to produce the P-log, there was a significant decrease in the percentage of PUFA and a non-significant reduction of MUFA and a non-significant increase in the percentage of SFA bound to the TPL. When producing the soft cheese (P), there was a non-significant decrease in the percentage of SFA and PUFA and a non-significant increase in the percentage of MUFA in the TPL. Although, there was a significant increase of 10:0, 12:0, and 14:0 fatty acids in the P-log and the soft cheese (P) bound to the TPL, with an additional increase of 16:0 fatty acids in the P-log TPL.

2.9. Statistical analysis All experimental analyses were completed in triplicate, and the obtained results were expressed as a mean value ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to find the significant statistical differences (p < 0.05). Tukey’s honest significance difference (HSD) test was utilised in order to conduct multiple comparisons of the means at p < 0.05 (SPSS Inc., Chicago, 215 IL, USA). Data visualisation was performed using the ggplot2 package (version 2.2.1) (Wickham, 2016) in R.3.4.2, in addition to hclust2 (https://bitbucket.org/nsegata/hclust2). 3. Results

3.3. Human platelet aggregation assay results

3.1. Lipid extraction

All TL, TNL, and TPL extracts from the caprine milks and cheeses exhibited potent inhibition against PAF-induced platelet aggregation in human PRP (Table 3). Although the IC50 of the TNL and TPL extracts of the pasteurised milk were lower than the raw milk, there were no significant differences between the two milks. These results indicate that in this set of experiments, neither milk was more antithrombotic than the other. Notably, the TL and TPL extracts of the cheeses were considerably more antithrombotic than that of the milks regardless of their milk source. The lowest IC50 determined was the TPL of the pasteurised soft cheese (79.40 µg ± 7.836).

The levels of TL, TNL, and TPL of the milk and cheese samples are presented in Table 1. The TL extract of each cheese was higher than the milks as expected due to the processing of the milk for cheese production, whereby the fat is concentrated. The TL of the raw and pasteurised milk were not significantly different from each other. However, the percentage of TNL and TPL of both the raw and pasteurised milk samples were statistically significantly different from each other, where there was a higher percentage of TPL in the raw milk samples. There 4

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Table 2 Fatty acid profile of total polar lipids (TPL) of milk and each milk and cheese expressed as a percentage (%) of the total fatty acids of each sample (mean ± SD, n = 3). Total saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) are shown as a percentage of total lipid. Fatty acids

R-Milk

P-Milk

P-Log

R-Log

Soft cheese (P)

Crottin (R)

8:0 10:0 11:0 12:0 12:1 c9 13:0 14:0 14:1 c9 15:0 16:0 16:1 c9 17:0 17:1 c9 18:0 18:1ω9 c9 18:2ω7 c9, t11 18:2ω6 c9, t12 18:2ω6 t10, c12 18:2ω9 c6, c9 18:3ω3 c9, c12, c15 19:1 c9 20:0 20:1 c9 20:2ω6 c11, c14 20:3ω9 c5, c8, c11 20:4ω6 c5, c8, c11, c14 20:4ω5 c6, c9, c12, c15 20:5ω3 c5, c8, c11, c14, c17 22:0 22:1 c11 22:4ω6 c7, c10, c13, c16 22:5ω5 c6, c9, c12, c15, c18 22:5ω6 c4, c7, c10, c13, c16 22:6ω3 c4, c7, c10, c13, c16, c19 SFA MUFA PUFA

0.01 ± 0.00a 0.05 ± 0.01a 0.01 ± 0.00b 1.11 ± 0.05ab 0.06 ± 0.04a 0.14 ± 0.01a 6.56 ± 0.36bc 0.24 ± 0.01a 1.31 ± 0.07a 19.86 ± 0.94de 1.19 ± 0.24a 1.08 ± 0.08c 0.78 ± 0.02b 24.58 ± 1.38d 22.37 ± 1.03a 1.56 ± 0.11ab 9.12 ± 0.35ab 0.02 ± 0.00a 0.74 ± 0.07a 0.95 ± 0.05ab 0.58 ± 0.12a 0.22 ± 0.04ab 1.00 ± 0.23a 0.38 ± 0.03a 0.27 ± 0.05b 1.29 ± 0.55a ND ND 0.78 ± 0.11c 0.54 ± 0.17a 1.31 ± 0.90a ND 1.29 ± 0.12bc 0.49 ± 0.05ab 55.72 ± 4.03d 26.76 ± 4.03a 17.41 ± 1.83b

ND 0.03 ± 0.01a 0.01 ± 0.00a 0.62 ± 0.00a 0.04 ± 0.01a 0.09 ± 0.00a 3.96 ± 0.10a 0.14 ± 0.00a 1.17 ± 0.01a 16.80 ± 0.01ab 1.26 ± 0.03a 1.38 ± 1.00d 0.66 ± 0.23ab 16.06 ± 0.32c 33.81 ± 0.45c 2.20 ± 0.14b 11.53 ± 0.06c 0.04 ± 0.01a ND 1.64 ± 0.05c ND 0.44 ± 0.03c 1.09 ± 0.04a 0.46 ± 0.03a 0.32 ± 0.02bc 1.56 ± 0.04a ND ND 0.92 ± 0.56d 0.41 ± 0.01a 0.39 ± 0.04a ND 2.19 ± 0.18e 0.77 ± 0.11c 41.49 ± 0.21ab 37.40 ± 0.37bc 21.12 ± 0.58c

0.01 ± 0.00a 0.61 ± 0.05b 0.04 ± 0.01c 2.19 ± 0.20c 0.16 ± 0.01bc 0.18 ± 0.01a 6.82 ± 0.13c 0.19 ± 0.10a 1.31 ± 0.02a 20.29 ± 0.45e 1.39 ± 0.06a 0.98 ± 0.03bc 0.78 ± 0.01b 13.10 ± 0.51b 31.41 ± 0.77bc 1.52 ± 0.04ab 9.23 ± 0.22ab 0.19 ± 0.05b 0.58 ± 0.01b 1.06 ± 0.06ab 1.28 ± 0.09b 0.22 ± 0.03ab 0.64 ± 0.02a 0.38 ± 0.04a 0.25 ± 0.02b 1.13 ± 0.10a ND ND 0.29 ± 0.00a 0.25 ± 0.03a 0.76 ± 0.17a ND 1.74 ± 0.19cd 0.49 ± 0.10ab 46.04 ± 0.73bc 36.14 ± 0.86b 17.82 ± 0.56b

ND 0.09 ± 0.03a 0.02 ± 0.01b 1.67 ± 0.35bc 0.12 ± 0.03ab 0.16 ± 0.03a 6.93 ± 0.96c 0.29 ± 0.04a 1.31 ± 0.09a 18.73 ± 0.75cd 1.52 ± 0.26a 0.97 ± 0.05bc 0.70 ± 0.05ab 12.98 ± 0.72b 30.04 ± 2.21b 1.67 ± 0.20b 10.64 ± 0.60bc 0.15 ± 0.02b 0.60 ± 0.06b 1.23 ± 0.09b 1.17 ± 0.11b 0.18 ± 0.02a 0.73 ± 0.05a 0.40 ± 0.04a 0.30 ± 0.03b 1.51 ± 0.21a ND ND 0.69 ± 0.11d 0.30 ± 0.03a 0.98 ± 0.17a ND 2.11 ± 0.14de 0.71 ± 0.12bc 43.73 ± 1.94ab 34.88 ± 2.66b 20.22 ± 1.34bc

ND 1.41 ± 0.09c 0.09 ± 0.08cd 1.95 ± 0.24bc 0.09 ± 0.05ab 0.10 ± 0.01a 5.33 ± 0.29b 0.32 ± 0.10a 1.00 ± 0.11a 16.33 ± 0.45a 1.02 ± 0.11a 0.82 ± 0.06ab 0.56 ± 0.03a 10.59 ± 0.31a 38.59 ± 1.43d 1.54 ± 0.14ab 9.83 ± 1.05ab ND 0.51 ± 0.01b 1.14 ± 0.16b 1.34 ± 0.10b 0.41 ± 0.19bc 0.68 ± 0.23a ND 0.39 ± 0.00c 2.41 ± 0.22b 1.77 ± 0.16a ND 0.57 ± 0.15b ND 0.75 ± 0.06a 0.61 ± 0.17a 1.80 ± 0.10cd 0.87 ± 0.01c 38.60 ± 1.58a 42.64 ± 1.08c 20.80 ± 0.79c

1.40 ± 0.18b 5.16 ± 0.24d 0.19 ± 0.10d 3.62 ± 0.34d 0.23 ± 0.05c 0.18 ± 0.08a 7.62 ± 0.57c 0.64 ± 0.06b 1.11 ± 0.25a 18.29 ± 0.05bc 1.61 ± 0.59a 0.65 ± 0.05a 0.56 ± 0.11a 9.20 ± 0.18a 33.42 ± 0.78bc 0.87 ± 0.63a 8.67 ± 0.63a ND ND 0.85 ± 0.04a 0.37 ± 0.13a 0.36 ± 0.06abc 0.69 ± 0.27a ND 0.15 ± 0.05a 0.95 ± 0.24a 0.91 ± 0.11b 0.17 ± 0.04a 0.42 ± 0.03b ND 0.32 ± 0.10a ND 0.91 ± 0.11ab 0.31 ± 0.14a 48.60 ± 1.63c 37.72 ± 0.57bc 13.41 ± 0.55a

a,b,c,d,e Mean values (n = 3), ± standard deviation with different letters in the same row indicating statistical significant differences when means are compared using Fisher’s LSD multiple comparison test (p < 0.05). Abbreviations: ND = non-detectable; MUFA = monounsaturated fatty acids; P = pasteurised; P-log = pasteurised milk log; P-milk = pasteurised milk; PUFA = polyunsaturated fatty acids; R = raw; R-log = Raw milk log; R-milk = raw milk; SFA = saturated fatty acids

2013). To produce each goat cheese, the same starter cultures were used, which included Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. lactis biovar diacetylactis, and Leuconostoc spp.. Notably, several species of the genus Lactococcus were present in significant amounts in each cheese regardless of the starter milk. Additionally, species of the Leuconostoc genus were differentially distributed in each of the cheeses depending on the milk it was produced from; For instance, Leuconostoc mesenteroides was present in the cheeses produced from pasteurised milk (soft cheese and pasteurised log), whereas Leuconostoc pseudomesenteroides was predominant in the cheeses made from raw milk (Crottin cheese and R-log). Both of these strains are present in the starter culture used to produce these cheeses. Both pasteurised cheeses contained Acinetobacter johnsonii and the pasteurised cheese also contained the oleaginous yeast Yarrowia lipolytica, which was also present in both raw cheeses.

Table 3 Inhibition of PAF-induced platelet aggregation in human PRP total lipid (TL), total neutral lipids (TNL), and total polar lipids extracts from raw and pasteurised caprine milk and cheeses. This activity is represented by their IC50 (µg) (mean ± SD, n = 3). Sample Milk (R) Milk (P) Cheese Log (R) Cheese Log (P) Soft Cheese (P) Crottin Cheese (R)

TL 519.4 666.1 288.7 210.5 496.0 675.1

TNL ± ± ± ± ± ±

c

59.1 131c 44.2b 32.7b 100c 159c

533.3 519.3 521.7 586.3 649.0 697.3

TPL ± ± ± ± ± ±

a

39.3 58.0a 134a 19.0a 43.7a 137a

225.5 207.4 135.3 121.8 79.40 113.3

± ± ± ± ± ±

13.3c 26.2c 32.4b 19.0b 7.84a 13.7b

a,b,c Mean values (n = 3), ± standard deviation with different letters in the same column indicating statistically significant differences when means are compared using Tukey’s HSD multiple comparison test (p < 0.05).

3.4. Cheese microbial composition

3.5. Gene composition of milk and cheeses

MetaPhlAn2 was used to determine the microbial composition of the samples. The predicted relative abundances of the detected species are presented in the heat map in Fig. 2. The pasteurised milk contained a small proportion of unclassified bacteria and a variety of taxa at low abundances. The raw milk mainly contained species of Pseudomonas and Leuconostoc lactis, which are routinely found in raw milk (Badis, Guetarni, Moussa Boudjema, Henni, & Kihal, 2004; Quigley et al.,

HUMAnN2 (https://bitbucket.org/biobakery/humann2) was employed for functional analysis of the shotgun metagenomic data. Specifically, HUMAnN2 was used to measure the abundances of gene ontology (GO) terms, in addition to level-4 Enzyme Commission (EC) categories related to the synthesis of polar lipids or fatty acids. It is apparent from Fig. 3A that L. lactis has the genetic capacity to biosynthesise polar lipids and fatty acids and seem to have a greater capacity to do so following the fermentation of caprine milk to cheese. 5

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diacylglycerol–glycerol-3-phosphate 2.7.8.5) among others.

3-phosphatidyltransferase

(EC

4. Discussion Previous research has shown that ovine milk fermentation alters the fatty acid composition of polar lipids and that these changes affect their antithrombotic properties. It has also been demonstrated that lactic acid bacteria have the capacity to alter the polar lipid composition of these products, leading to the potential development of antithrombotic functional foods (Lordan, Walsh, et al., 2019). In this study, raw and pasteurised caprine milk was used in the production of various caprine cheeses that all were produced from the same starter culture. The lipid profile, microbial composition, and antithrombotic activity of these cheeses were then assessed. Although all the cheeses were produced using the same starter culture, both raw and pasteurised milk were used and various cheese production techniques were employed, which may have altered the final microbial composition. As discussed in Section 3.4, the bacteria introduced by the starter culture were present in abundance as expected, along with other microbial species common to the dairy industry. It was noted that yeast (Yarrowia lipolytica) was present in the pasteurised cheeses, which is not unusual as it has been reported in milk and cheese previously, and its presence may affect the lipid quality of the cheese products due to its lipolytic and lipid biosynthetic capabilities (Groenewald et al., 2014). Furthermore, these yeasts may beneficially support the growth of probiotics in dairy products (Lourens-Hattingh & Viljoen, 2002). Indeed, it was also noted that all four cheeses contained commonly occurring Lactococcus phage P680, which is an extremely heat-resistant member of the 936 group of L. lactis phages (Atamer et al., 2009). Following the analysis of the microbial composition, functional analysis of the shotgun metagenomic data was carried out using HUMAnN2 to measure the abundances of GO terms, in addition to level-4 EC categories related to the synthesis of polar lipids or fatty acids. In this study, it was determined that L. lactis may play a central role in the fermentation process and the alteration of the polar lipid fatty acid composition due to the abundance of GO terms and level-4 EC categories detected associated with L. lactis following fermentation. In a similar study assessing the effects of ovine milk fermentation on the antithrombic properties of yoghurts, it was demonstrated that fermentation by specific microorganisms such as L. acidophilus increased the antithrombotic properties of the milk TPL. Indeed, several GO terms associated with phospholipid biosynthesis and metabolism were detected in abundance and many of those are in common with those measured in this study (Lordan, Walsh, et al., 2019). While L. lactis seems to be a significant driver of the fermentation process, it is clear that other microbial species have the potential to play a role in polar lipid biosynthesis. Interestingly, GO terms for the phosphatidylglycerol biosynthetic process (GO:0006655) were only detected in the soft cheese and pasteurised cheeses that were linked to the presence of Y. lipolytica; both cheeses exhibited the lowest TPL IC50 values. Other microbial species such as Lactococcus raffinolactis were also associated with several anabolic polar lipid related GO terms including: Phosphatidylserine decarboxylase activity (GO:0004609); phosphatidylethanolamine biosynthetic process (GO:0006646). Furthermore, the presence of Debaryomyces hansenii was associated with several GO terms related to the production of ceramides (GO:0006672), phosphatidylcholine (GO: 006656), and unsaturated fatty acids (GO:0006636). This yeast is routinely found in dairy products where it exhibits proteolytic and lipolytic activities (Quirós et al., 2006). It is clear that these microorganisms have the capacity to alter the polar lipid composition of the milk and dairy products. However, in order to determine whether there is a change in the fatty acid composition in the polar lipids, GC–MS was employed to analyse FAMEs prepared from the polar lipid fractions.

Fig. 2. A heat map showing the 15 most abundant species present in the caprine milk and cheese samples.

Indeed, the following GO terms were assigned to L. lactis: biotin carboxylase activity (GO:0004075); biotin-[acetyl-CoA-carboxylase] ligase activity (GO:0004077); lipid metabolic process (GO:0006629); fatty acid biosynthetic process (GO:0006633); glycerophospholipid metabolic process (GO:0006650); lipid biosynthetic process (GO:0008610); phospholipid biosynthetic process (GO:0008654); cardiolipin synthase activity (GO:0008808); cardiolipin biosynthetic process (GO:0032049). Importantly, there were several GOs detected that are especially crucial to the biosynthesis of polar lipids, including: CTP synthase activity (GO:0003883); CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase activity (GO:0008444); CDP-diacylglycerol biosynthetic process (GO:0016024); glycerol-3-phosphate cytidylyltransferase activity (GO:0047348); CDP-glycerol glycerophosphotransferase activity (GO:0047355). The abundances of level-4 EC categories were also measured to characterise the metagenome at the enzyme-level resolution (Fig. 3B). It is clear that the starter cultures, and particularly L. lactis, has the greatest capacity to alter the polar lipid fatty acid composition, which may alter the antithrombotic properties of polar lipids. Several EC categories of interest detected are responsible for the biosynthesis of fatty acids such as acetyl-CoA carboxylase (EC 6.4.1.2), which although expected are present in considerable abundance, with L. lactis being the main source. Other EC categories that are specific to phospholipid biosynthesis were detected, including glycerol-3-phosphate acyltransferase (EC 2.3.1.n3), glycerol-3-phosphate cytidylyltransferase (EC 2.7.7.39), phosphatidate cytidylyltransferase (EC 2.7.7.41), and CDP6

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Fig. 3. (A) The following images depict the abundance of GO terms of interest in the raw milk and the various cheeses according to the corresponding microorganisms associate with the GO term using HUMAnN2 output. (B) The abundance of level-4 EC categories of interest in the raw milk and the various cheeses according to the corresponding bacterium associated with the GO term using HUMAnN2 analysis. The pasteurised milk is not included in either figure due to low abundance of microorganisms present in the pasteurised milk.

compositions were significantly altered when fermented to cheese. Pasteurisation of the raw milk lead to a modest but statistically significant decrease in the percentage of SFA and a significant increase in the percentage of monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) bound to the TPL fraction of the milk. Similar observations have previously been reported in the total lipid fraction of bovine milk (Pestana, Gennari, Monteiro, Lehn, & de Souza, 2015). While it has been reported that pasteurisation only minimally affects the total lipid fatty acid profile, pasteurisation does seem to somewhat effect the SFA content more than the unsaturated fatty acids

Previous research has shown that dairy cultures can alter the general lipid composition of milk by fermentation due to lipolysis and the synthesis of lipids by lactic acid bacteria (Florence et al., 2012; Vieira et al., 2015). Recently, it has been reported that fermentation can also specifically affect the phospholipid fatty acid composition (Ferreiro & Rodríguez-Otero, 2018; Lordan, Walsh, et al., 2019). Furthermore, processing milk by pasteurisation can impact the phospholipid composition of fat globules in the milk (Gallier, Gragson, Cabral, JiménezFlores, & Everett, 2010). In this study, the TPL fatty acid composition modestly differed between raw and pasteurised milk and both milk TPL 7

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Overall, the TPL was the most antithrombotic lipid fraction of the milk and cheeses, which is in accordance with previously reported studies on bovine (Megalemou et al., 2017), ovine (Lordan, Walsh, et al., 2019), and caprine dairy products (Poutzalis et al., 2016). Using raw or pasteurised milk with the exact same starter cultures for the production of each cheese log does not seem to significantly affect the TNL or TPL IC50 values over short fermentation times. The lower IC50 values for the TL in contrast to the TNL the structural differences between these lipids, which determine the level of affinity of PAF-like lipids for binding to the PAF-receptor and thus inhibiting the effects of PAF (Tsoupras, Lordan, & Zabetakis, 2018). Indeed, it is also possible that there is a synergistic effect between these various lipids. These results may indicate that the consumption of the soft cheese log may exert more beneficial antithrombotic effects due to the low IC50 TPL and TL. Furthermore, the classical PAF structure generally contains 16:0 (68%), 18:0 (27%), or 18:1 (4%) at the sn-1 position (Demopoulos, Pinckard, & Hanahan, 1979), with acetic acid esterified to the sn-2 position, and a phosphocholine group at the sn-3 position. In this study, the majority of the fatty acids reported in the polar lipids of these antithrombotic caprine milks and cheeses seem to share similar fatty acid compositions with that of PAF, as the percentage of 16:0, 18:0, and 18:1 is considerably high across all TPL fractions, which has also been demonstrated in a recent assessment of ovine milk products (Lordan, Walsh, et al., 2019). Previous studies of antithrombotic marine polar lipids indicate that phosphatidylethanolamine and phosphatidylcholine are the most bioactive polar lipids against PAF, when bearing 16:0, 18:0, and 18:1 fatty acids in their structures (Lordan et al., 2017). While, the analysis of the goat milk and cheese polar lipids in this study tentatively agrees with previous research that the majority of the bioactive polar lipids bear 16:0, 18:0, and 18:1 fatty acids in their structures, which is most probably responsible for the bioactivity observed, it is important to determine whether these lipid are attached to choline, ethanolamine, or other typical phospholipid headgroups. These phospholipid headgroups most probably do dictate the bioactivity and the bioavailability of the polar lipids. Therefore, further investigation is necessary to determine if there is structural homology between these antithrombotic polar lipids and PAF and whether these effects are demonstratable in a clinical setting. A limitation of this study is the use of intact polar lipids. The majority of polar lipids such as phospholipids are digested by phospholipase A2, which cleaves the ester bond at the sn-2 position that results in free fatty acids and the lyso-forms of the phospholipid, such as phosphatidylcholine. These digestion products are absorbed by enterocytes, which are then transported to the lymphatic and then the circulatory system as chylomicrons, where they are eventually resynthesised as phospholipids or triacylglycerols (Dixon, 2010; Lordan et al., 2017). A proportion of the polar lipids are transferred to high-density lipoproteins (HDL) within 5–6 h of dietary polar lipid ingestion (Tall, Blum, & Grundy, 1983). The HDL can then deliver the polar lipids to their target tissues and organs (Burri, Hoem, Banni, & Berge, 2012; Hussain, 2014). However, approximately 20% of intestinal phospholipids are passively absorbed intact and unaffected by lipases, which are then preferentially incorporated directly into HDL (Lordan et al., 2017; Zierenberg & Grundy, 1982). Furthermore, dietary polar lipids can also be integrated into HDL particles already present in the intestine that can later join the plasma HDL pool (Burri et al., 2012), thus increasing the level of intact polar lipids in circulation that may affect platelet activity. HDL itself may have antithrombotic effects, which has previously been attributed to the polar lipids membrane of the HDL particles (Camont et al., 2013). Therefore, the use of intact dietary polar lipids warrants further intensive investigation for their putative cardioprotective properties. Indeed, simulated gastrointestinal digestion, in vivo studies using enterocytes, and human postprandial studies, are also warranted to fully assess whether the antithrombotic properties of dairy polar lipids survive gastrointestinal digestion in humans, thereby inducing their cardioprotective effects.

of bovine milk according to the data of Pestana et al. (2015). A notable limitation of milk treatment is that pasteurisation and minimal thermal treatment provoke an increase of free fatty acids due to disruption of the milk fat globule membrane (MFGM) and lipolysis (Pereira, Martins, & Vicente, 2008). It is possible that the observed decrease of the percentage of SFA bound to the TPL in this study is due to these minor effects of pasteurisation. However, the observed percentage increase of MUFA and PUFA bound to the TPL may be due to the observed reduction of SFA rather than an actual absolute increase of these fatty acids. Although identical bacterial cultures were used to produce each of the cheeses, there were significant differences in the fatty acid composition of the TPL between the various cheeses. This is potentially due to the slight differences in the manufacturing process and the type of starter milk used during the cheese production. When raw milk was used to produce the R-log there was a significant decrease in the percentage of SFA and a significant increase in the percentage MUFA bound to the TPL. This was also the case for the production of the Crottin cheese (R), but there was a significant decrease in the percentage of PUFA in the TPL of this cheese. This increase in unsaturated fatty acids in the TPL when raw milk was fermented suggests that the reduction in the percentage of the SFA bound to the TPL may be due to the microbial utilisation of the SFA for various metabolic processes including the biosynthesis of unsaturated fatty acids. However, the same patterns of fatty acid distribution in the TPL were not evident in the production of the Crottin cheese (R). Instead there was a significant increase in the overall percentage of MUFA in the TPL of these cheeses. Notably, 18:1 accounted for the most significant percentage increase of TPL fatty acids, whereas other MUFA and in general the overall percentage of PUFA in the TPL decreased. Although the initial bacterial starter cultures used were the same for each cheese, it is clear from the microbial composition analysis that the slightly differing production methods lead to different microbial compositions, which seems to play a role in the reported differences between the lipid profiles of the TPL of both raw cheeses produced. When pasteurised milk was used to produce the P-log, there was a significant decrease in the percentage of PUFA and a non-significant reduction of MUFA and a non-significant increase in the percentage of SFA bound to the TPL. When producing the soft cheese (P), there was a non-significant decrease in the percentage of SFA and PUFA and a nonsignificant increase in the percentage of MUFA bound to the TPL. Considering the same cultures and production method were used, the reduction in the percentage of PUFA in the P-log TPL and the increase in the R-log TPL suggests that the use of raw milk or pasteurised milk influences the polar lipid fatty acid composition. Indeed, this study has demonstrated that the variances in polar lipid fatty acid composition observed in the various cheese products is due to the use of pasteurised and raw milks and the effect of the different cheese production techniques used on the fermentation process. The changes observed in the TPL fatty acid composition as milk was fermented and used to make cheeses, seems to have a significant effect on the antithrombotic properties of the caprine dairy polar lipids. Previous research demonstrated that as caprine milk was fermented to yoghurt and then to cheese, the antithrombotic properties increased (Poutzalis et al., 2016). It was hypothesised that the fermentation process may affect these activities (Lordan & Zabetakis, 2017a). In this study, all of the TL, TNL, and TPL extracts from the caprine milks and cheeses exhibited potent inhibition against PAF-induced platelet aggregation in human PRP as demonstrated in Table 3. Notably, the lowest IC50 value was from the soft cheese TPL may be attributable to the fact that this was the only cheese produced that did not drain the whey during production. Draining the whey in cheese production can lead to a loss of almost 20% of the phospholipid content in normal cheese production (Ferreiro & Rodríguez-Otero, 2018), thus potentially affecting the antithrombotic properties of the cheeses produced. However, further research is warranted. 8

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Finally, another limitation of this study is the length of time allowed for fermentation. Longer fermentation periods may induce more significant effects on the overall lipid profile, thus affecting the antithrombotic properties. Therefore, further studies are required to find the optimum starter milk, starter culture, and fermentation parameters to develop further highly antithrombotic and functional foods.

authors reviewed and approved the final manuscript.

5. Conclusions

References

This study demonstrates that the use of raw or pasteurised starter milks and processing techniques can minimally alter the polar lipid fatty acid composition of caprine cheeses. However, fermentation seems to play a key role in augmenting the milk polar lipid fatty acid composition and the improvement of the antithrombotic properties of dairy products during cheese production. There was no significant difference between the antithrombotic activities of raw or pasteurised milk polar lipids; however, their polar lipid fatty acid composition was different, and the respective cheeses produced also had varying polar lipid fatty acid compositions. Notably, the soft cheese TPL had a low IC50 value, thus a very strong antithrombotic effect. As this was the only cheese to include the whey, it may be the case that some bioactive lipids are lost in the whey during typical cheese production that does not include the whey, leading to lower antithrombotic properties in those cheeses. Further research is required to confirm these observations. The metagenomic data indicates that although the same starter culture were used in the production of each cheese, the starter milk used and the slight differences between the production methods significantly alters the microbial composition of the final cheeses. HUMAnN2 analysis confirmed that the microbial populations in the milks and cheeses had the capacity to alter the phospholipid and sphingolipid composition of the milks and cheeses. Therefore, future research should aim to optimise milk processing and fermentation parameters to create potent and functional antithrombotic dairy products that may benefit cardiovascular health.

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Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2019.103507.

Ethics statement Experiments were performed in accordance with the Declaration of Helsinki. Ethical approval was granted by the University of Limerick and informed consent was obtained for experimentation with human subjects. Supplementary data https://www.ebi.ac.uk/ena/data/view/PRJEB30079. Acknowledgements The authors would like to thank all the volunteers for their donations and Elaine Ahern for her phlebotomy assistance and advice. The authors acknowledge the financial support of both Enterprise Ireland (Grant reference: IP/2017/0596-Y) and the Department of Biological Sciences, University of Limerick, Ireland. The authors would like to extend their sincere gratitude to Siobhan, Brian, and team at Inagh Farmhouse Cheese Ltd, Inagh, Co. Clare for their support and advice on this project. Declaration of Competing Interest The authors declare no conflict of interest. The grant providers had no role in the design of the study, in the collection, analyses or interpretation of the data; in the writing of the manuscript; nor in the decision to publish the results. R.L and I.Z designed the study and received the funding. RL performed the experiments. A.T and M.D supported the analysis. A.M.W., F.C, L.F, P.D.C., and R.L conducted the metagenomic and bioinformatic analyses. R.L and A.M.W wrote the manuscript. All 9

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