Rapid Assay to Evaluate the Total Antioxidant Capacity in Donkey Milk and in more Common Animal Milk for Human Consumption

Rapid Communication

Austin Food Sci. 2016; 1(1): 1003.

Rapid Assay to Evaluate the Total Antioxidant Capacity in Donkey Milk and in more Common Animal Milk for Human Consumption

Beghelli D¹, Lupidi G², Damiano S², Cavallucci C¹, Bistoni O³, De Cosmo A¹ and Polidori P²*

¹School of Biosciences and Veterinary Medicine, University of Camerino, Italy

²School of Pharmacy and Health Product Sciences, University of Camerino, Italy

³Department of Clinical & Experimental Medicine, University of Perugia, Italy

*Corresponding author: Polidori Paolo, School of Pharmacy and Health Product Sciences, University of Camerino, Camerino (MC), Italy

Received: February 22, 2016; Accepted: March 04, 2016; Published: March 10, 2016

Abstract

The milk antioxidants, by preventing lipid peroxidation, maintain milk quality, but they also exert a beneficial effect on the consumer’s health, in particular that of infants. Donkey Milk (DM), for its nutritional, functional and bioactive components, seems to be one of the best substitutes of breast milk when the latter is not available. However, there are few data about its antioxidant properties. In this study, the Total Antioxidant Capacity (TAC) of donkey milk was determined by means of an in micro-plate assay. DM samples were analyzed at the first, third and fifth month of the lactation period (n 6/period), comparing results to those obtained in milk of different dairy species (goat, ewes, cows) and in breast milk using the same assay. The lactation periods did not affect the TAC of DM, whereas significant different values (P<0.001) were observed between species. The breast milk showed the lowest TAC value, followed by its progressive increase in donkey, cow’s, goat’s and ewe’s milk. The rapid test here adopted can be successfully employed for a reliable monitoring of the TAC in DM and, thanks to the constant antioxidant supply, DM can also be sponsored as a valid alternative to infant milk nutrition.

Keywords: Total Antioxidant Capacity (TAC); Donkey milk; Breast milk; Cow milk; Ewe milk; Goat milk

Introduction

Human Milk (HM) besides being the best source of nutrients, also supplies a complex system of defence factors necessary for the health of growing infants [1,2]. The potential of HM to directly affect oxygen-induced tissue injury in the newborn has been demonstrated by experimental studies in animals [2].

The milk antioxidant compounds also exert a beneficial effect on the consumer’s health by giving a potentially greater protection from exposure to the oxidative stress that is recognized as a feature of many acute and chronic diseases [3-5].

Therefore, milk antioxidants, including proteins, carotenoids, flavonoids as well as vitamins such as vitamin E and C, not only carry out important roles in preventing lipid peroxidation which in turn is the underlying cause for generation of hydrolytic off-flavors, but they also could help in reducing the loss of important nutrients and bioactive agents that promote health of offspring or of older consumers [6-8].

When breast milk is not accessible, it is very important that infant nutrition fulfils the right antioxidant requirements to resemble natural feeding as much as possible.

Cow milk is widely employed as a substitute, although it is not routinely fed to human infants as it is, but it needs to be modified into formulas that are more comparable to HM. However, some authors [9] stated that in this adaptation process many factors, including antioxidants, are either absent or poorly represented, as in other artificial feedings. Besides the well known infant formula, in the past few years, Donkey Milk (DM) has gained considerable attention in the Scientific Community due to its nutritional, functional and bioactive components [8].

DM seems to be the best substitute for human milk in infant nutrition [10-13] because it is rich in lactose (the taste of ass’ milk resembles breast milk), lysozyme, -3 and -6 polyunsaturated fatty acids [14]; the ash residue is similar to that of human milk and the protein profile is adequate for the correct development of infant digestive tract [15,16]. In particular, ass’s milk can be consumed by human infants with multiple food allergies or Cow’s Milk Protein Allergy [17-20] and elderly people, because of its ability to up-regulate the immune response [21,22].

Nowadays, what is scarcely known is whether the DM, besides the nutritional characteristics, is as so similar to breast milk to also satisfy the antioxidant requirements of infants? In general, it is particularly difficult to compare the different Total milk Antioxidant Capacity (TAC) values reported in literature because they were obtained by means of diverse analytical methodologies [23] and/or not all the possible substitutes to human milk, produced by the most common dairy animals, were compared for this specific feature [5].

The interest in determining the TAC in milk is increasing since it is able to give an overall picture of the antioxidant potential of this type of food, and also because TAC measurement requires much less work and methodological infrastructure than analyzing the often complex composition of individual antioxidants. This purpose has highlighted the need to develop reliable, easy and fast methods to quantify this property in a basic food such as milk for human development. The luminescence switch-on detection assay, based on an iridium complex, could represent an interesting alternative method, having shown its potential in monitoring proteins and being time and cost effective [24]. However, the method here adopted evaluates the ability of milk samples to contrast with the massive oxidative action of a powerful and physiological oxidant (hypochlorous acid) and has the advantage to be faster than all the other methods cited in literature, though keeping the reliability of the results [25].

Aims of the present work were 1) to evaluate the total antioxidant capacity of donkey milk in different lactation periods and 2) compare the results with data from different dairy animals (cows, ewes and goats) and breast milk, using the same assay.

Materials and Methods

Sampling and preparation of milk samples

Donkey milk: Eighteen individual milk samples were manually collected from mammary gland in a semi-extensive herd (pasture and integration with concentrate) of the Southern Italy (Ponte Cagnano, Salerno) from 18 pluriparous jennies of different breeds (Ragusana, Amiantina and mixed breeds), age (5-18 years) and lactation period (at one, three or five months; six ass/lactation period), so that the early, middle and late lactation periods were investigated in the same feeding and seasonal conditions. Samples were collected in spring (April 2014) and transferred into a ‘mobile’ refrigerator to laboratory at 4oC where they were frozen as individual samples at -20°C until analysis (within the first month of storage). Further three different 50 ml bulk milk samples of eight pluriparous donkeys (Martina Franca breed, Az. Agricola Cambiotti, Gualdo Tadino, Perugia, Italy), were collected in two different days and treated as afore mentioned. The TAC values of the latter samples were compared to those obtained in the milk samples of the dairy animals considered in the present study.

Cow milk: Three bulk milk samples were collected in an intensive herd of 130 Frisona cows (primiparous and multiparous together) situated in the Central Italy (S. Eraclio, Foligno), by three different tanks where daily production is stored (pasteurized) at 4°C until it is withdrawn by local dairy transformers. Refrigerated milk samples (milked 3 hours before) were collected in spring (April 2014) and transferred into a mobile refrigerator to laboratory at 4°C where the same were stored at -20°C individually or by combining two samples in turn to form three pools until analysis (within the first month by time of collection), for a total of six samples.

Ewe and Goat milk: Three individual milk samples were manually collected from mammary gland in a semi-extensive herd (pasture and integration with concentrate) of central Italy (S. Maria Rossa, Perugia), by three different ewes (Sardinian, pluriparous animals) or goats (Umbrian local breed, pluriparous animals) at the middle of their lactation periods (April 2014). Samples were treated as referred for cow’s samples.

Breast milk: Three individual milk samples were collected at the S. Maria della Misericordia Hospital (Perugia, Italy) in spring (April 2014) from three voluntary women within their first week of lactation. All the milk donors provided written informed consent in accordance with the declaration of Helsinki. The study was approved by the local Ethics Committee (CEAS Umbria).

Milk samples were withdraw (with a mechanical breast pump) from the mothers into sterile flasks for their hospitalized babies and an aliquot (10 ml)/each was kindly given for this purpose. Samples were transferred to laboratory at 4°C into a ‘mobile’ refrigerator and stored at -20°C until analysis (within the first month by collection).

TAC assay and milk procedure

The TAC assay was performed using the Oxy Adsorbent Test (Diacron International, Grosseto, Italy) and a spectrophotometric plate reader (FLUO star Omega, Multi-mode microplate reader BMG Labtech, Ortenberg, Germany) at 546 nm wavelength.

This test, normally used on a serum or plasma matrix, was adapted to a microplate assay by Brambilla et al. [26]; whereas, Bianchi et al. [25] reported its use in the evaluation of milk antioxidant activity. Each milk sample was tested in triplicate. Briefly, the TAC assay evaluates the ability of samples to contrast with the massive oxidative action of a Hypochlorous acid (HClO) solution and TAC values are expressed in μ mol neutralized HClO/ml of sample. Bilirubin, uric acid, vitamins C and E, albumin and in general, the macromolecular complexes (e.g. as glycoproteins) that act as shock absorber against free radicals, help to buffer the oxidizing action of hypochlorous acid. HClO was selected among other oxidant agents because it is not only a powerful but also a physiological oxidant. As soon as the ‘free’ HClO reacts with a correctly buffered chromogenic substrate (N,N-diethylparaphenylendiamine), a colored complex develops. The optical density generated by the colored complex is directly proportional to the concentration of HClO and indirectly related to the antioxidant capacity.

In the present study the micro-plate assay has been slightly modified. In particular, the optical densities at the beginning of the assay (absorbance blank: A blank 0) were subtracted to the values obtained after the incubation period (10 minutes) and the suggested sample dilutions (1:100in distilled water) for blood samples were reduced to 1:75, for whole milk samples.

Statistical analysis

The data were analyses using the GLM procedure of SPSS® 13 (Chicago, IL, SPSS Inc 2004). An ANOVA model, with the dairy animal milk/breast milk as fixed variable, was used. For the DM samples collected in three different lactation periods, the sampling time was also included as a fixed effect. Data were reported as least squares means and Standard Error (SEM). Differences were considered to be significant when P≤0.05.

Results and Discussion

The TAC of DM did not significantly vary during the different lactation periods evaluated (Table 1) and these results could be an important index of good nutritional quality of donkey’s milk. Milk TAC values obtained from different dairy animals and breast milk (Table 2). Goat’s and ewe’s milk showed significantly higher TAC values compared to that from donkey, cow’s and human milk, being the latter significantly lower.

Citation: Beghelli D, Lupidi G, Damiano S, Cavallucci C, Bistoni O and De Cosmo A. Rapid Assay to Evaluate the Total Antioxidant Capacity in Donkey Milk and in more Common Animal Milk for Human Consumption. Austin Food Sci. 2016; 1(1): 1003.