Comparison of Total Antioxidant Capacity of Milk of Crossbred Holstein Friesian and Vechur Cattle during Different Lactation

Milk is a rich source of vitamins, minerals, fats and high quality proteins. The composition of milk has been influenced by a number of genetic and environmental factors such as parity, breed, stage of lactation and climatic conditions (Bernard et al., 2018). The most variable component in milk is fat, which is the primary determinant of its physical and organoleptic qualities (Magan et al., 2021). Milk proteins such as casein, lactoferrin and amino acids in milk like tyrosine, cysteine, tryptophan, lysine, serine and leucine have antioxidant properties (Layman et al., 2018). Antioxidant compounds have the ability to scavenge free radicals and help to relieve stress (Rio et al., 2013). The antioxidants present in milk can be grouped into lipid-soluble and water-soluble antioxidants. Carotenoids, retinol and a-tocopherol are lipid-soluble antioxidants, whereas ascorbic acid is water-soluble antioxidant (Khan et al., 2019). The milk fat globule membrane contains a considerable amount of a -tocopherol, which is regarded as one of the milk’s most potent lipid-soluble antioxidants (Bernard et al., 2018). There are two primary groups of proteins in milk: caseins, which make up 80% of total proteins and come in a variety of forms and serum proteins, all of which contribute significantly to its antioxidant property (Davoodi et al., 2016). Tyrosine and cysteine, Vitamins A and E, carotenoids and enzyme systems including superoxide dismutase, catalase and glutathione peroxidase are some of the sulfur-rich amino acids that contribute to milk and milk products’ antioxidant potential (Gayathri and Renu, 2015). Equol, a polyphenolic metabolite of daidzein, is also present in milk in significant concentrations and research has shown that it has antioxidant properties (Mayo et al., 2019). The antioxidant mechanisms of milk can prevent superoxide radicals (O₂), hydroxyl radicals and peroxide radicals (Akhtar et al., 2018).

Antioxidant properties of milk and its products may improve human health, immunity, nutritional value, prevent or treat health threats, aid in the treatment of diabetes, cancer and cholesterol-related issues and lessen allergic reactions (Manach et al., 2017). A diet rich in antioxidants is crucial for managing the pro- and anti-oxidant balance. It is assumed that milk from the Vechur cattle is healthier than milk from crossbred bovines. In comparison to crossbreed cattle, the Vechur cow has much more fat and solids not fat (SNF) content (Abraham and Gayathri, 2015). Different animal milk and milk products with antioxidant defense systems can be considered to have health-beneficial effects against free radical damage (Stobiecka et al., 2022). Excessive formation of reactive oxygen species and oxygen-free radicals has been linked to oxidative stress, which may lead to chronic diseases such as cancer, cardiovascular diseases and ageing degenerative diseases (Pizzino et al., 2017). The oxidation of membrane phospholipids, proteins and DNA as well as the modification of low-density lipoproteins, can all be exacerbated by oxidative stress (Lopez-Pedrera et al., 2016). Free radicals, in particular reactive oxygen species (ROS) andreactive nitrogen species (RNS), which are produced during typical cellular and metabolic events, are crucial in the aetiology of chronic illnesses (Van-Raamsdonk et al., 2017).  When the natural defense mechanism of body fails to prevent the damage caused by reactive radicals, artificial and natural antioxidants are used to impede the negative consequences of oxidative stress. Artificial antioxidants have been found to be harmful, carcinogenic and hazardous (Uzombah, 2022). The dairy animals possess variable levels of antioxidant across different lactation period. Hence, the objective of the present study was to analyse the antioxidant properties of whole milk of CBHF and Vechur cattle and assess whether the Vechur milk is better in quality as far as antioxidant property is concerned.

The study was conducted in the College of Veterinary and Animal Sciences, Mannuthy, Thrissur, Kerala (Latitude: 10.536; Longitude: 76.265). From the Vechur cattle farm milk samples were collected for the present study. Total antioxidant capacity (TAC) of milk samples of CBHF and Vechur cattle were evaluated across early (5-15 days), mid (90-120 days) and late (>150 days) stages of lactation. A total of 36 milk samples from CBHF (n=6) and Vechur cows (n=6) were used for the study. All of the milk samples were obtained early in the morning and stored at 4oC till processing. The analysis was carried out on the same day of collection of samples.
 
Determination of total antioxidant activity
 
The ferric reducing antioxidant power assay (FRAP) was used as a direct method for measuring the total antioxidant capacity. At low pH, ferric 2,4,6-tripyridyl-s-triazine [Fe (III)-TPTZ] complex gets reduced to ferrous 2,4,6-tripyridyl-s-triazine [Fe (II)-TPTZ] complex, which has an intense blue colour, which can be monitored by measuring the change in absorption at 593 nm. Working FRAP reagent was prepared by using 300 mM acetate buffer, pH 3.6 (3.1 g sodium acetate trihydrate and16 mL glacial acetic acid); 10 mM TPTZ (2,4,6-tris (2-pyridyl)-s-triazine), in 40 mM HCl; and 20 mM FeCl₃•6H₂O in the ratio of 10:1:1. Milk samples (100 ml) at room temperature were mixed with 3ml of FRAP reagent and kept in dark for 10 minutes. The samples were then centrifuged at 8000 rpm for 5 minutes and supernatant solution was taken. The absorbance of the supernatant solution was measured using spectrophotometer at wavelength 593 nm. Aqueous solutions of FeSO₄•7H₂O (100-3000 mM) were used as standard. The data is shown as FRAP values [mM ml^-1 Fe (II)].
 
Statistical analysis
 
Data were expressed as means±standard error. For statistical comparisons between periods of lactation, the results were subjected to one-way ANOVA using Statistical Package for the Social Sciences (SPSS). Significant differences (P <0.01) between CBHF and Vechur cattle at each stage of lactation were analyzed by Independent t-test (Gerald, 2018).

The present study shows that, milk samples from CBHF and Vechur cattle during the early stages of lactation had significantly higher total antioxidant capacity than milk samples from later stages of lactation (Table 1). This could be because of the high fat content in the early phases, which lead to higher readings. a-Tocopherol is largely present in milk fat globule membrane and it is considered as the most effective lipid-soluble antioxidant present in milk of dairy animals (Bernard et al., 2018). Chen et al., 2000) found that there was a link between milk fat content and antioxidant activity. In both CBHF and Vechur cattle, TAC values of early and mid-lactation (Fig 1) and early and late lactation (Fig 2) varied significantly. However, the TAC values of mid and late lactation milk in both CBHF and Vechur cattle, did not differ significantly (Fig 3).








       
The total antioxidant content of Vechur milk was higher than the total antioxidant capacity of CBHF milk throughout lactation. The TAC values of Vechur milk decreased as lactation progressed, but the net value remained significantly greater than the milk of CBHF.  Vechur milk is distinguished from crossbred cow milk by its superior digestion, buffering capacity and therapeutic properties (Rajeev, 2007). Vechur milk had shown to have significant potential for decreasing blood cholesterol levels in numerous nutritional and medical studies (Chinnamma et al., 2015). The milk of the Vechur cow contains higher beta casein A₂ than A₁. The levels of SNF, protein and lactose in Vechur cattle were stable throughout the seasons, suggesting the breed’s adaptability and climatic resilience in terms of the primary milk ingredients (Anisha et al., 2012). Along with the above mentioned previous studies, the higher TAC value of Vechur milk observed in the present study is indicative of its better medicinal value than the CBHF milk.

Throughout lactation, the total antioxidant capacity of milk samples of CBHF and Vechur Cattle was examined in this study. Antioxidant levels were shown to fluctuate throughout different phases of lactation, with early lactation showed higher TAC values than later stages of lacation. In addition, when compared to CBHF milk, Vechur cattle milk had a higher total antioxidant capacity, indicating that Vechur cattle milk has a higher medicinal value.

The authors are grateful to Kerala Veterinary and Animal Sciences University for providing necessary facilities needed for carrying out the research.

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