Effect of bioactive compounds on oxidative and lipolytic stability in cow's milk-based functional drink

Introduction: Lipid peroxidation often occurs in response to oxidative stress and reactive oxygen species cause oxidation of lipids containing (C=C) carbon double bonds (Sies and Jones 2020). Malondialdehyde (MDA), the most common aldehyde with the highest biological activity, is abundantly produced during lipid peroxidation and is commonly used as an indicator of oxidative stress (Barrera et al. 2018). MDA originates from the breakdown of peroxide compounds resulting from the oxidation of polyunsaturated fatty acids (Ayala et al. 2014). Carrots contain natural, nutritious and health-promoting bioactive compounds. The antioxidant properties of β-carotene and other compounds such as phenolic compounds, flavonoids and ascorbic acid in carrots are of great interest (Anjani et al. 2022). The aim of this study was to investigate the effect of different percentages of carrot juice in the preparation of carrot-milk drink. Therefore, during the thirteen-day storage period, quality indicators such as acid number, MDA, antioxidant capacity and bioactive compounds were studied in the drinks to determine the appropriate percentage of carrot juice to inhibit oxidative stress in cow’s milk based functional drink.
Materials and Methods: Carrots and pasteurized cow's milk were obtained from local market. The carrots were washed with clean water, extracted and passed through a filter cloth to separate the carrot pieces. Milk-based drinks were prepared by replacing 0 (only cow milk) 10, 20, 30, 40 and 50% of carrot juice. The samples were filled into sealed glass containers and pasteurized at 70°C for 30 min and stored at 4°C until the experiments. Malondialdehyde and acid number tests were performed on the milk-carrot drink with methanol and isoamyl alcohol (lipolysis index), as well as bioactive compounds such as total phenolic compounds, total carotenoids, ascorbic acid and antioxidant capacity in the drink samples. Malondialdehyde measurement: The thiobarbituric acid method was used to measure malondialdehyde in beverage samples and its amount was reported as µmol MDA L-1 (Fenaille et al. 2001). Acid number measurement (lipolysis index): To determine the total free fatty acids, the method of titration of extracted fat by an alkaline alcoholic solution was used and its amount was expressed in mg NaOH g-1 fat (Evers 2003). Determination of bioactive compounds: 1) Total phenol measurement: Methanol-water solvent (50:50) was used to extract the phenolic compounds of the beverage and the Folin-Ciocalteau method was used by measuring the absorbance of the samples at 765 nm to determine the amount of phenolic compounds. Finally, the amount of phenolic compounds was reported as mg GAE 100 g-1 (Lamuela‐Raventós 2018), 20 Measurement of total carotenoids: Hexane-ethanol solvent (90:10) was used to extract the carotenoid compounds of the beverage and centrifugation (5000 rpm) was used for purification. The amount of total carotenoids (μg g-1) was determined by measuring the absorbance of the sample at 450 nm (Machmudah and Goto 2013), 3) Measurement of ascorbic acid: Ascorbic acid was extracted using a metaphosphoric acid-acetic acid solvent and its amount was determined by measuring the absorbance at 521 nm and the amount of ascorbic acid was reported as mg 100 g-1 (Ruiz et al. 2016) and 4) Measurement of total antioxidant capacity by DPPH test: The antioxidant capacity of the sample was determined by the free radical scavenging ability of the DPPH solution. For this purpose, the absorbance at 517 nm was determined and reported as percentage inhibition (Chen et al. 2015). For statistical analysis, factorial experiments were carried out in a completely randomized design with 3 replications. The effect of carrot juice percentage and storage time on bioactive compounds and oxidative and lipolytic stability of carrot-milk drink was investigated. SAS software (version 9.1, USA) was used for analysis and the least mean squares method (P < 0.05) was used for comparison of means.
Results and discussion: Samples with 50% carrot juice on the first day and 10% carrot juice on the thirteenth day had the highest (157.37 µg g-1) and lowest (24.44 µg g-1) carotenoid contents, respectively. The decrease in total carotenoid content during storage can be attributed to the antioxidant and free radical scavenging properties of carrot-milk drink, as carotenoids exhibit antioxidant properties and are capable of scavenging reactive oxygen species at low oxygen concentrations (Ribeiro et al. 2018). Carotenoids have the ability to bind to lipid globules and provide phase and color stability during storage in carrot-milk drink (Sharma et al. 2012). Studies have shown that carrot juice contains significant amounts of vitamins such as thiamine, folic acid, niacin, riboflavin, vitamins A and C (Aubert et al. 2022). During storage, vitamin C decreased in all samples due to degradation and oxidation (Castellom-Estrada et al. 2023). The content of ascorbic acid decreased during storage. The lowest content (2.15 mg 100g-1) on the thirteenth day with 10% carrot juice and the highest content (6.17 mg 100g-1) was observed with 50% carrot juice on the first day. In all samples, the lowest content of total phenols (122.18 mg 100g-1) was on the thirteenth day with 10% carrot juice and the highest content (311.32 mg 100g-1) on the fourth day with 50% carrot juice. The major source of phenolic compounds in carrot-milk drink is carrot juice. The increase in phenolic compounds up to the seventh day can be attributed to the release of phenolic compounds from carrot particles into the drink, but the subsequent decrease is due to oxidation inhibition, antimicrobial properties and preservative role during storage (Talcott and Howard 1999). The lowest antioxidant capacity was found in the sample containing 10% carrot juice on the first day (35.98%) and the highest value was found in the sample containing 50% carrot juice on the thirteenth day (60.62%). Researches has shown that there is a good correlation between DPPH free radical scavenging values, phenolic compounds and vitamin C content on the one hand, and antioxidant capacity and beta-carotene on the other (Castellom-Estrada et al. 2023 and Hashemi et al. 2014). Acid number, as an indicator of lipase enzyme activity, was lower in drinks with 30, 40 and 50% carrot juice than in drinks with 10 and 20% carrot juice, and these changes in acid number were calculated as 0.5-1.2 mg NaOH g-1 in terms of extracted fat. Also, the researchers found that the presence of Pseudomonas spp. in milk leads to the production of microbial lipases at the end of storage. According to the results of the experiments, the decrease in the acid value can be attributed to the inhibitory effect of bioactive compounds from the carrot juice source, especially at higher percentages in the carrot-milk drink. The results of the experiments showed that the MDA content of the drinks changed from 3.5 to 13 µmol L-1 during storage. As a result, the most important pathways for inhibiting and suppressing lipid peroxidation and MDA formation could be the inhibition of free radicals such as hydroxyl (OH·) and peroxyl (LOO·) radicals with the help of bioactive compounds in carrot juice. Aldehydes are the major secondary oxidation products in milk, which generally have a lower taste threshold compared to alcohols and ketones. As a result, when their concentration reaches above the taste threshold, they have a significant effect on the taste of milk. Also, MDA can damage biomolecules such as proteins, phospholipids and other molecules through the formation of covalent and cross-links (Aubourg 1993). Researchers reported that the addition of purple corn anthocyanin to milk resulted in higher resistance to lipid oxidation and MDA formation, and showed lower oxidation of secondary metabolites compared to the control sample (Tian et al. 2022). In lipid oxidation, autoxidation is the most important chain reaction through free radicals. Investigation of Table 1 shows that milk-carrot drinks contain significant amounts of bioactive compounds (polyphenolic compounds, flavonoids, carotenoids, vitamin C) and antioxidant capacity that have the ability to inhibit the peroxidation of unsaturated fatty acids and the formation of MDA (Figures 1 and 2). Lipid peroxidation, in addition to the loss of nutritional value, also leads to the formation of radicals with toxic properties (Figure 3), therefore, the control of oxidative processes in the food industry have a vital importance. Although the oxidation of unsaturated fatty acids has been widely studied, the complex reactions involved in this process, as well as the different pathways and factors affecting them, have caused the oxidation mechanisms to be not yet fully understood (Dominguez et al. 2019). Unsaturated fatty acids are very sensitive to oxidant attack and today, malondialdehyde, 4-hydroxy-2-nonenal, and 2-isoprostane are the main biomarkers for assessing lipid peroxidation, all of which are derived from polyunsaturated fatty acids (Mas-Bargues 2021).
Conclusion: Carrots, as a rich source of carotenoid compounds such as α and β-carotene, different types of phenolic compounds and ascorbic acid, have antioxidant and free radical neutralization properties in carrot-milk drink. The main source of phenolic compounds in carrot-milk drink is carrot juice. During whole storage, changes in antioxidant compounds of carrot-milk drink may be due to inhibition of the formation of harmful compounds such as MDA resulting from the secondary oxidation of unsaturated fatty acids. The most important pathways for inhibiting and suppressing lipid peroxidation and MDA formation can be the inhibition of free radicals such as hydroxyl and peroxyl radicals with the help of bioactive compounds in carrot juice. These compounds can also act as lipolysis inhibitors during storage. Finally, it can be concluded that adding carrot juice with different concentrations to carrot-milk drink, in addition to increasing nutritional value, also plays an important role in the oxidative and lipolytic stability of functional drink.