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International Dairy Journal 21 (2011) 901e906

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International Dairy Journal

journal homepage: www.elsevier .com/locate/ idairyj

Antioxidant and anti-platelet properties of milk from goat, donkeyand cow: An in vitro, ex vivo and in vivo study

Yannis Simos a,*, Apostolos Metsios a, Ioannis Verginadis a, Angela-Gabriella D’Alessandro b,Pasquale Loiudice c, Emilio Jirillo d, Pavlos Charalampidis a, Valantis Kouimanis a,Athina Boulaka a, Giovanni Martemucci b, Spyridon Karkabounas a

a Laboratory of Experimental Physiology, Faculty of Medicine, University of Ioannina, 45110 Ioannina, GreecebDept. PRO.GE.S.A., University of Bari, ItalycAzienda Ospedaliero Universitaria Pisana, Via Paradisa, 2 e Cisanello, 56124 Pisa, Italyd Immunology, Faculty of Medicine, University of Bari, Bari, Italy

a r t i c l e i n f o

Article history:Received 16 December 2010Received in revised form10 May 2011Accepted 27 May 2011

* Corresponding author. Tel.: þ30 26510 07602; faxE-mail address: isimos@cc.uoi.gr (Y. Simos).

0958-6946/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.idairyj.2011.05.007

a b s t r a c t

A study was conducted to evaluate the biological properties of milk from different animals and breedssuch as cows, goats (Prisca, Ionica or Saanen breeds) and donkeys (Martina Franca breed). Methodsincluded in vitro, ex vivo and in vivo experiments to evaluate the activity of milks on platelet aggregationand antioxidant capacity. The in vitro trials results demonstrated the highest total antioxidantcapacity (TAC) in goats’ milk, especially from Prisca breed. Ex vivo trials showed that Prisca goats’ milkinhibits platelet aggregation at lower amounts than milk from other species. Consumption for 40 days of0.6 L day�1 of Prisca goats’ milk significantly increased TAC in healthy volunteers. This study contributesto defining the biological properties of milk from these animals/breeds. Goats’ milk from the autoch-thonous Greek breed (Prisca) provided the best antioxidant capacity and inhibitory properties on plateletaggregation of the milk samples tested.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Milk contains several physiologically functional componentsincluding proteins, vitamins such as vitamin E, and C as well ascarotenoids and flavonoids with antioxidant properties. Therefore,milk with a higher antioxidant capacity will reflect greater oxida-tive stability and provide potentially greater protection for theconsumer from exposure to the oxidative stress that is recognizedas a prominent feature of many acute and chronic diseases (Dalle-Donne, Rossi, Colombo, Giustarini, & Milzani, 2006; Valko et al.,2007).

In recent years, considerable interest has focussed on goats’ anddonkeys’ milk for their nutritional and biofunctional properties(Amati et al., 2010; D’Alessandro, Martemucci, Jirillo, & Leo, 2010;Jirillo et al., 2010; Tafaro et al., 2007). Goats’ milk and deriveddairy products assume importance in the human diet, withparticular relevance in infants for whom goats’ milk represents analternative to cows’ milk (Hanlein, 2001). From a nutritional point

: þ30 26510 07850.

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of view, goats’ milk is different from that of other species(Park, 2006). It is characterized by small sized fatty acid globules(Attaie & Richter, 2000) and a high content of short chain fatty acids(Alonso, Fontecha, Lozada, Fraga, & Juárez, 1999), which improve fatdigestibility and intestinal absorption (Hachelaf et al., 1993)compared with cows’ milk (Chandan, Attaie, & Shahani, 1992).Moreover, the presence of medium-chain fatty acids (caproic,caprilic and capric acid) in goats’ milk has been attributed toa reduction of cholesterol in human tissues by limiting cholesterolstorage and improving its mobilization (Hanlein, 1992). Goats’milkis thought to have a low allergenic potential compared with cowmilk on the basis of characteristics of aS1-casein (Roncada,Gaviraghi, Liberatori, Bini, & Greppi, 2002). In a recent study(Amati et al., 2010), it has been shown that daily use of goats’ milkin the diet of immuno-compromised aged patients acts as a down-regulator of acute inflammation, reducing the exaggerated basalsecretion of interleukin (IL)-8 and IL-6 acute response, and exertinga moderate down-regulation of IL-1b and tumour necrosis factor(TNF)-a production. Casein macropeptides from bovine, ovine andcaprine milk exhibit antithrombotic properties similar to theactivity of g-fibrinogen 400e411 peptide (Manso, Escudero, Alijo, &Lopez-Fandino, 2002; Qian, Jolles, Migliore-Samour, Schoentgen, &

Y. Simos et al. / International Dairy Journal 21 (2011) 901e906902

Fiat, 1995a). Lactoferrin isolated from sheep presented similarproperties in reducing platelet aggregation as human lactoferrin(Qian, Jolles, Migliore-Samour, Schoentgen, & Fiat, 1995b). Moreinteresting is the fact that lactoferrin inhibited platelet aggregationin concentrations not far from physiological levels.

Donkeys’ milk is very different from milk of other speciestraditionally used for human feeding such as cows’, goats’ andsheep milks (Alais, 1984), showing a closer similarity to humanmilk (Conte, Calabrò, & Monsù, 2003; Guo et al., 2007). Recentstudies have indicated donkeys’ milk may be a promising food forchildren affected by cows’ milk protein allergy or multiple foodintolerance (Alessandri & Mari, 2007; Carroccio et al., 2000; Iaconoet al., 1992; Tesse, Paglialunga, Braccio, & Armenio, 2009) and in theelderly, because of its ability to up-regulate the immune response(Amati et al., 2010; Tafaro et al., 2007). In particular, the effect ofdonkeys’ milk on immune response resulted in a significantincrease of IL-8 and IL-6 acute response and in up-regulation ofIL-1b and TNF-a production (Amati et al., 2010).

The objective of this study was to evaluate and compare,through in vitro, ex vivo and in vivo experiments the antioxidantactivity and inhibition of platelet aggregation capability of goats’and, donkeys’ milks in comparison with cows’ milk. Milk fromdifferent goat breeds was also considered.

2. Materials and methods

2.1. Reagents and apparatus

Ammonium dichromate, 3-methylbutanol, a-tocopherol, sul-phuric acid and hydrogen peroxide of high purity, adenosinediphosphate (ADP), platelet-activating factor (PAF), g-linolenic acidand linoleic acid were purchased from SigmaeAldrich Co (St. Louis,MO, USA). Total antioxidant capacity (TAC) measurements weremade with a Thermo Scientific MultiSkan Spectrum UV/Vis micro-plate (Thermo Fischer Scientific Inc.Waltham,MA,USA) and cuvettespectrophotometer using disposable polysterene square cuvettes of10 mm optical pathway purchased from Sarstedt Co (Sarstedt AG &Co. Postfach, Nümbrecht, Germany). Platelet aggregation was per-formed in a Chrono-log Corporation Model 500-Ca aggregometer(Chrono-log Corporation, Havertown, PA, USA) using glass cuvettes.Atomic absorption spectroscopy was performed on a Perkin Elmerspectrometer, model 560 (Perkin Elmer Waltham, MA, USA).

2.2. Milk source

The study was performed on milk from different species such asgoat, donkey and cow. Commercial, pasteurized, whole (3.5% fat)cows’ milk was used as the control. As experimental milk, goats’milk was obtained from different breeds from Greece (Prisca, anautochthonous breed) and Italy (Ionica, an autochthonous breed;Saanen, a cosmopolitan breed). Animals of Prisca and Ionica breedswere reared under extensive conditions on natural pasture in thearea of Epirus (Greece, North West) and Apulia (South Italy),respectively, while Saanen goats were reared under semi-extensivesystem (pasture and integrationwith concentrate) according to thecommon breeding system in the area of Apulia. Donkeys’ milk wasobtained from a local Italian breed (Martina Franca) reared underextensive conditions on natural pasture in Apulia. Experimentalmilk samples from goats and donkeys were collected in spring,pasteurized and frozen at �76 �C until analysed.

2.3. In vitro experiments

A series of in vitro experiments were performed to evaluate totalantioxidant capacity and the levels of zinc (Zn) and cadmium (Cd)

of milk from different species such as cows (commercial whole,pasteurized, 3.5% fat cows’ milk), donkeys and goats. Goats’ milkwas also tested in relation to different breeds (Prisca, Ionica andSaanen goats). Estimation of TAC was performed using the BlueCrO5 assay, according to Charalampidis, Veltsistas, Karkabounas,and Evangelou (2009) and results are expressed as mM ofa-tocopherol. Zinc and cadmium levels in plasma were measuredby atomic absorption spectroscopy (Perkin Elmer Waltham, MA,USA). Samples were diluted with four volumes of deionized water(ddH20) for the zinc measurements. Sensitivity was set at0.018 mg mL�1 of Zn and 0.025 mg mL�1 of Cd for 1% absorption.Results are expressed as ppm for Zn and ppb for Cd. All measure-ments for TAC and atomic spectroscopy were performed intriplicate.

2.4. In vivo experiments

On the basis of the results of the in vitro experiments performedin this study, which have shown the highest TAC for goats’ milkfrom Prisca breed, in vivo experiments were undertaken to evaluatethe effects of consumption of milk or vitamin supplementation onplatelet aggregation and plasma antioxidant capacity.

In the present experiments, 25 healthy volunteers (11 male and14 female), between 23 and 57 years of age (mean age 29 � 10 y)were recruited. The volunteers were divided into two groups, cor-responding to: Group I (Milk Group, 13 volunteers) and Group II(Antioxidants Group, 12 volunteers). The volunteers from Group Iconsumed 0.6 L day�1 of goats’ milk from Prisca breed for 40consecutive days. The volunteers of Group II were supplementeddaily with a mixture of 0.375 g vitamin C in the form of L-ascorbicacid, and 0.125 g of vitamin E in the form of D-a-tocopherol, for thesame period. Blood collection was performed at the beginning andat the end of the experiment.

2.4.1. Volunteer blood donorsThe volunteers followed a stable normal diet, with respect to

energy value, carbohydrates, lipids, proteins, fruits and vegetables,according to individual weight and age. The compliance of volun-teers to their diet was verified every week with a 24 h recall, in aninterview with a certified dietician and weight measurement. Thevolunteers were free of medical problems in their history. More-over, they did not consume any kind of medication or foodsupplements, and did not consume alcoholic drinks during theexperiment. The participation of the volunteers in the study waswithout monetary compensation and in agreement with thehuman rights legislation from the Declaration of Helsinki (1979).

2.4.2. Preparation of plateletsBlood was taken from the basilica vein of the arm from the

volunteers of both groups by free flow with the aid of an appro-priate needle. The blood was placed in test tubes containing citricacid as anticoagulant agent in the proportion 1 mL of anticoagulantsolution to 9 mL of blood. Centrifugation of the blood samples wasperformed at 54 � g for 20 min to obtain the plasma rich in platelet(PRP) fraction. The PRP was placed in a plastic test tube and waskept in a water bath at 37 �C until the termination of the plateletaggregation tests. The test tubes containing the rest of the bloodwere centrifuged again at 645 � g for 20 min to isolate the plasmapoor in platelets (PPP) fraction, which was collected in a plastictube and used for calibration of the aggregometer.

2.4.3. Platelet aggregationAll assays for platelet aggregation and platelet reactions (shape

changes and release reaction) were conducted on the aggreg-ometer. The assessments were conducted in a cuvette, which

Table 2Plasma total antioxidant capacity (TAC) and platelet aggregation (% platelet aggre-gation compared to the control) with adenosine diphosphate (ADP) or platelet-activating factor (PAF) in Group I and Group II volunteers.a

Parameter Group I Group II

D0 D40 D0 D40

TAC (mM a-tocopherol) 27.9 � 3.3a 30.3 � 3.6b 30.7 � 7.7 31.7 � 9.6Platelet aggregation (ADP) 68.4 � 7.8 67.4 � 7.8 60.9 � 4.8 58.9 � 8.2Platelet aggregation (PAF) 72.3 � 6.4a 66.5 � 9.9b 59.3 � 8.1 57.4 � 7.3

a Group I volunteers consumed 0.6 L goats’ milk per day for 40 days; Group IIvolunteers were orally supplemented with 0.375 g vitamin C and 0.125 g of vitaminE per day for 40 days. Blood collection was performed at the beginning (D0) and atthe end of the experiment (D40). Data are presented as mean � SD of threedeterminations. Different superscripts in row denote significantly different,P < 0.05.

Y. Simos et al. / International Dairy Journal 21 (2011) 901e906 903

contained 450 mL of PRP and 50 mL of each of two diluted plateletagonists (ADP and PAF) in ddH2O.

2.5. Ex vivo experiments

The aims of the ex vivo experiments were the evaluation ofactivity of milk from different origin (cow, donkey and goat breeds),at different doses (2.5, 5.0,10.0 and 20.0 mL) on platelet aggregation.The experiments were carried out on blood collected from thebasilica vein of the arm of 12 healthy volunteers according to thecriteria described above. The same procedure as described abovewas also followed for the preparation of platelet. The control assaywas conducted in a cuvette, which contained 450 mL of PRP and50 mL of each of two diluted platelet agonists (ADP or PAF) inddH2O. Moreover, the experiment assay was also conducted ina cuvette, containing 450 mL PRP and 50 mL of dilutedmilk in ddH2O.Initially, the milk was added into the cuvette, containing 450 mLPRP, and incubated for a few minutes. After that, each of the twoplatelet agonists was added to the cuvette, to observe thepercentage of platelet aggregation. Blood sample analysis wasperformed in triplicate for each of the two platelet agonists.

2.6. Statistical analysis

All statistical procedures were performed using SPSS (SPSS 16.0,SPSS Inc. Chicago, Illinois, USA, 2009). The statistical significancebetween data means was determined by using paired sample t-test.Data are expressed as mean � SD.

3. Results

3.1. In vitro trials

The in vitro assessments of TAC and levels of Zn and Cd in milkfrom the different origins (cow, donkey and goat breeds) arereported in Table 1. Among the various types of milk, milk of Priscagoats showed the highest (P < 0.01) antioxidant capacity (66.7 mM

a-tocopherol). This value of TAC was 2 times higher than in Saanenand Ionica goat breeds (33.6e35.8 mM a-tocopherol, respectively)and in donkeys’ milk (31.2 mM a-tocopherol), and 1.5 fold higherthan in cows’ milk (42.9 mM a-tocopherol).

Analyses of the Cd content showed the highest (p< 0.01) level inmilk of Saanen goats (9.13 ppb) and commercial cows’ milk(8.44 ppb), whereas donkeys’ milk presented the lowest levels(0.60 ppb). Zinc levels were the lowest in donkeys’ and Prisca goats’milk (2.3 and 2.6 ppm, respectively) in comparison with the othermilk (range 3.09e3.90 ppm; 0.05 > P < 0.01). Donkeys’ milk alsohad the lowest Cd/Zn ratio (0.15; P < 0.01).

3.2. In vivo trials

The results of the in vivo trials on the effects of goats’ milk onTAC and platelet aggregation are shown in Table 2. Consumption of

Table 1In vitro total antioxidant capacity (TAC) in plasma and levels of Cd and Zn of milk from d

Component Cows’ milk Goats’ milk from:

Prisca

TAC (mM a-tocopherol) 42.9 � 2.10Ca 66.7 � 2.30A

Cd (ppb) 8.44 � 0.42A 3.33 � 0.25B

Zn (ppm) 3.90 � 0.24A 2.57 � 0.17B

Cd/Zn ratio 2.17 � 0.15Aa 1.29 � 0.11C

a Data are presented as mean � SD of three determinations. Different superscripts inP < 0.05.

goats’ milk (Group I) for 40 days significantly increased plasmaantioxidant capacity (P < 0.05). Conversely, at the end of thesupplementation period with vitamin C and vitamin E, the volun-teers of Group II showed no change in their plasma antioxidantcapacity (P > 0.05).

The inhibitory effect on platelet aggregation, through either ADPor PAF, was not statistically significant after vitamin C and Esupplementation (Group II). In contrast, in Group I, Prisca goats’milk consumption significantly reduced (P < 0.05) the levels ofplatelet aggregation through the PAF pathway.

3.3. Ex vivo trials

Fig. 1 shows the results of ex vivo trials on evaluation of activityof milk from different origins, according to different doses (2.5, 5.0,10.0 or 20.0 mL), on platelet aggregation using ADP or PAF agonist.Inhibition of platelet aggregation was significantly different withrespect to milk origin. Considering the minimum dose of milk(2.5 mL), the highest (P < 0.01) inhibition of platelet aggregationwas shown by goats’ milk of all the breeds evaluated, when ADP asthe platelet agonist was used (67e71%; Fig. 1A). Using PAF as theplatelet aggregation agonist, at the same dose of milk, the strongerinhibitory capacity was shown by goats’milk from Prisca and Ionicabreeds and cows’milk (71e73%; P < 0.01; Fig. 1B). The volume thatcaused total inhibition of platelet aggregation (100%) was 10 mL forPrisca goats’ milk, using either ADP or PAF (Fig. 1). Milk from theIonica and Saanen goats and cows also showed more or lessa complete inhibitory effect (96e100%) at a dose of 20 mL. Donkeys’milk did not have any inhibitory effect on ex vivo platelet aggre-gation (21e27% inhibition of platelet aggregation at 2.5 mL; 33e44%at 20 mL).

4. Discussion

In vitro trials on milk from different sources show that theantioxidant potential of milk from Prisca goats was higher thanmilk from Ionica and Saanen goats, cows and donkeys. This positive

ifferent origins.a

Donkeys’ milk

Saanen Ionica

35.8 � 2.70BCb 33.6 � 3.20B 31.2 � 2.90B

9.13 � 0.40A 3.04 � 0.15B 0.60 � 0.06C

3.59 � 0.13A 3.09 � 0.49ABa 2.27 � 0.16Bb

2.55 � 0.13Ab 0.78 � 0.05B 0.15 � 0.02D

row differ significantly; upper case letters show P < 0.01, lower case letters show

Fig. 1. Effects of milk from cow ( ), Prisca goats ( ), Saanen goats ( ), Ionica goats ( ) and donkeys ( ) and in increasing doses on ex vivo percentage of platelet aggregationcompared with the control (control ¼ 100%): (A) adenosine diphosphate as platelet agonist; (B) platelet-activating factor as platelet agonist. Blood was collected from 12 healthyvolunteers and trials were performed in triplicate. Data are expressed as means � SD. Different letters indicate the significant differences either among the doses of the milk withinthe same source (A, B, C: P < 0.01; a, b: P < 0.05) or among the milk from different sources within the same dose (D, E, F, G: P < 0.01; d, e: P < 0.05).

Y. Simos et al. / International Dairy Journal 21 (2011) 901e906904

effect shown by Prisca goats’ milk agrees with the increasing trendof plasma TAC found in in vivo trials after its daily consumption for40 days, unlike the daily consumption of vitamins C and E. It couldbe hypothesized that a period of milk consumption longer than40 days could have a more significant effect in terms of antioxidantcapacity. The antioxidant capacity of goats’ milk might be attrib-utable not only to its composition, that is very rich in antioxidantsas reported by some authors (Chilliard, Ferlay, Rouel, & Lamberet,2003; Contarini & Avalli, 2001), but probably to the particularcombination of compounds or their greater bioavailability.

Antioxidant capacity of various types of milk has been deter-mined by different methods such as the oxygen radical absorbancecapacity (ORAC) and trolox equivalent antioxidant capacity (TEAC)assays (Saenz, Elisia, Innis, Friel, & Kitts, 2009; Zulueta et al., 2009).Zulueta et al. (2009) have provided evidence that the majorcontributors of TAC in whole milk are the casein fractions and the

hydrophilic antioxidant compounds, such as vitamin C and uricacid, in the deproteinised milk. Total casein content is similar incows’ milk and goats’ milk but their fraction composition differs toa great extent since the major casein fraction of cows’milk is a aS1-casein and of goats’milk is b1- and b2-caseins (Slacanac et al., 2010).

In our study, milk from Prisca goats, as well as Ionica goats,showed a higher anti-platelet effect than that of Saanen goats andof donkeys. It could be attributed to different milk characteristicsdue to genotype and/or rearing system and/or diet composition ofthe animals. In the present study, Prisca and Ionica goats werereared under extensive breeding conditions on pasture. The milkfeeding system can have an important role on nutritional quality ofmilk, sincemilk from pasture-fed animals contains high amounts ofpolyunsaturated fatty acids, particularly 18:2 and 18:3 (Chilliardet al., 2003; Palmquist, 1988; Shingfield, Chilliard, Toivonen,Kairenius, & Givens, 2008). In milk from pasture-fed cows milk,

Y. Simos et al. / International Dairy Journal 21 (2011) 901e906 905

Schroeder, Gagliostro, Bargo, Delahoy, and Muller (2004) alsoreported that fatty acid content is highly unsaturated (average70e90%), with a large amount of linoleic and linolenic acids.Pizzoferrato et al. (2007) correlated the degree of antioxidantprotection (DAP) with quality of goat milk. Specifically, DAP valuewas higher when pasture herbage was dominant in animal feed, asis likely to have occurred in our study for Prisca and Ionica goats’milk. Moreover we can speculate that the lower response of theSaanen goats’ milk in comparison with Prisca and Ionica breeds,besides the genetic difference, could be linked to the differentfeeding system of the animals, constituted by pasture and supple-mentation of concentrate. In fact, Pizzoferrato et al. (2007) showedthat in grazing goats, the supplementation of feeding with barley,chickpeas, corn or beans grain, tended to reduce the levels of a-tocopherol and DAP in milk. Kondyli, Katsiari, and Voutsinas (2007)have found that goats’ milk of the native Greek breed reared onpasture contains a high quantity of vitamins such as vitamin C.

Boosting the human body’s defence mechanism with adequateamounts of natural antioxidants provided by diet can boostimmunity and reduce the chance of disease. When the body hasinsufficient antioxidants, oxidative stress increases and this canlead to significant damage and disease (Dalle-Donne et al., 2006).Many mainstream health and nutrition organizations world-widerecommend daily consumption of dairy products for optimalhealth (Bishop-MacDonald, 2005). In this context, the presentstudy contributes to identifying some bioactive components of milkfor human health.

5. Conclusions

This study evaluates the biological properties of milk fromdifferent sources such as goat (Prisca, Ionica or Saanen breeds) anddonkey (Martina Franca breed) in comparison with cows’ milk, inthree experimental models (in vitro, ex vivo and in vivo). Theresults showed that the species from which the milk is sourcedaffects the biological characteristics of milk in terms of TAC andanti-platelet effect. Milk from Prisca goats had the highest in vitroTAC in comparison with the other goat breeds (Ionica and Saanen),cows’ milk and donkeys’ milk. This higher antioxidant capacity ofPrisca goats’ milk was confirmed in an in vivo trial where partici-pants consumed the milk for 40 days, in comparison with theconsumption of a mixture of vitamin C and E, suggesting a betterbioavailability of antioxidant compounds of milk compared withvitamin integration in the diet. Donkeys’milk had a low in vitro TACand did not have any inhibitory effect on ex vivo platelet aggre-gation. The ex vivo trial showed that milk of Prisca and Ionica goatshad the highest inhibition of platelet aggregation with theminimum dose of milk, using either ADP or PAF agonists. Thesefindings lead us to hypothesize the positive influence of naturalpasture on total antioxidant capacity and inhibitory properties onplatelet aggregation of goats’ milk. Further studies are needed tostrengthen the knowledge on factors (feeding, season, etc.,) andmechanisms involved in the biological properties of goat milk.

Acknowledgements

The research was supported by INTERREG III/A Greece-Italy(Project Code I 2101030) Paper N. 31.

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