The Effect of Adding Different Concentrations of Cows’ Milk on the Antioxidant Properties of Coffee

Polyphenols have been widely studied and considered as a health promoting and disease preventive agents in humans. Several studies investigated the antioxidant properties of polyphenols and their abilities to eliminate free radicals. In this study, the antioxidant activity of coffee in the presence of different types of cows’ milk at different concentrations, 10% or 20%, was investigated. Our results showed that milk could either enhance the scavenging of DPPH or decrease the metal chelating and metal reducing activity of polyphenol. However, more investigations are required to evaluate the mechanisms by which fats in milk can alter the antioxidant activity of coffee.

Oxygen is an essential element for the survival of aerobic organisms. In many chemical and biochemical reactions, oxygen-derived free radicals are produced from oxidation-reduction processes 1 . These free radicals are responsible for oxidative stress that leads to variety of diseases and disorders such as cancer 2 , cardiovascular diseases 3 , neural disorders 4 , and Alzheimer's disease 5 . They are produced under normal physiological conditions and are generated by exogenous chemicals 6 . Reactive oxygen species (ROS) can present in different forms of activated oxygen such as hydrogen peroxide (H 2 O 2 ) or as singlet oxygen 7 . The redox homeostasis of cell is maintained by the antioxidant protection mechanism of the human body. If this mechanism has been unbalanced, the reducing of oxidative damage may be occurred by using antioxidant supplement 6 .
Coffee is an exogenous antioxidant that is widely consumed due to its desirable sensory properties 8 . The proper consumption of coffee is important to health as many negative and positive effects can occur according to the dose. Negative effects of coffee consumption include hypertension, heart diseases, psychiatric disorders and bone diseases. These side effects are mainly attributed to increased caffeine consumption of coffee 9,10 . On the other hand, a regular intake of coffee has shown many positive effects on metabolic, psychoactive and neurological diseases 11 . The positive effects depend on the phenolic and flavonoid compounds such as chlorogenic acids (quinyl esters of hydroxycinnamic acid) 12 , caffeic, ferulic, p-coumaric acids 13 and proanthocyanidins 13,14 . These biological compounds are secondary metabolites from plants 6 and have potent strong antioxidant and radical scavenging activities 15,16 , which help the body to reduce the oxidative stress 17 .
Most of people prefer to drink coffee with additives. Milk is one of the preferable choices of additives because it contains various compounds such as water, fats, sugars, minerals, vitamins, and proteins 18 . When milk is added to coffee, the milk proteins (caseins and whey proteins) can interact with the coffee polyphenols and form a complex, which might affect many properties of coffee particularly the structural, functional, and nutritional ones [19][20][21] . Many studies have investigated the antioxidant capacity of tea after adding different types of milk to it. However, the obtained results are contradictory. Some of them have reported an inhibitory effect, while others have shown no significant effect of adding milk [22][23][24] . Milk fat exists in emulsified form coated by thin layer known as a globule membrane 25 , and its effect on antioxidant activity and bioavailability of coffee polyphenols is unclear until now. Therefore, the objective of this study was to assess the in vitro effect of adding different concentrations of whole, semi-skimmed, and skimmed cow's milk on the antioxidant activity of coffee.

Preparation of samples
Red mug coffee (Nescafé) and different types of long life (Almarai) cow's milk (whole, semiskimmed, and skimmed), the most popular consumed milk brand according to the survey, were purchased from a local supermarket. The types of milk and the percentages used were selected according to a survey performed on 900 participants in Jeddah, Western region of Kingdom of Saudi Arabia.
Coffee brews have been prepared as people in the survey commonly drink it. Each sample was prepared by adding 2g of instant coffee to 200 ml boiling water and then either 20 ml or 40 ml of different types of cow milk was added to have a final concentration of 10% or 20%, respectively.

Determination of total phenolic and flavonoid contents
In a clean test tube, 0.5ml of each coffee sample (with or without milk) was incubated at room temperature for 5 minutes with 5ml deionized water and 5ml Folin-Ciocalteu's reagent (BDH, Poole, England). Then, in a dark place, all tubes were incubated with 1ml of anhydrous sodium carbonate (CDH, New Delhi, India) for 1 hour before the absorbance was measured at 750 nm. A standard curve was prepared from a serial concentration (0 to 1000µg/ml) of Gallic acid (Sigma-Aldrich, Poole, UK) which was prepared in methanol: water (50: 50 v/v) 26,27 .
On the other hand, the total flavonoid content was determined by incubating 250µl of each coffee sample (with or without milk) with 1.25 ml deionized water and 75µl sodium nitrite (Sigma-Aldrich, Poole, UK) for 5 minutes. Then, 150µl of (10%) aluminum chloride (loba chemie PVT.LTD., Mumbai, India), 0.5 ml of (1M) sodium hydroxide (AppliChem Panreac, Missouri, USA) and 275µl of deionized water were added to the previous mixture. After that, the absorbance was read at 510 nm and a standard curve was prepared from a serial dilution (0 to 500µg/ml) of β-Catechin (Sigma-Aldrich, Poole, UK) 28 .

Determination of radical scavenging activity with H 2 O 2 and DPPH assays
In test tubes, 1 ml of sample was incubated for 10 minutes at room temperature with 0.6 ml of (40 mM) hydrogen peroxide (Sigma-Aldrich, Poole, UK). Then, the absorbance was recorded at 230 nm and the radical scavenging activity for H 2 O 2 was calculated by the following equation: inhibition % = [(Absorbance of blank -Absorbance of sample) / Absorbance of blank] × 100 29,30 .
In the DPPH-radical scavenging assay, ethanol (Sigma-Aldrich, Poole, UK) and DPPH reagent (Sigma-Aldrich, Poole, UK) were added to water extract solutions of samples and were incubated in dark place for 1 hour at room temperature. The absorbance was measured at 517 nm and the DPPH radical scavenging activity was calculated using the same equation used to calculate the inhibition percentage of H 2 O 2 31 .

Determination of metal chelating and reducing power activity
To estimate the ferrous ion chelating activity of samples 32 , 5µl of each sample (with or without milk) was incubated for 30 seconds with 50µl of (2mM) ferrous chloride (Sigma-Aldrich, Poole, UK) and 1.5ml distilled water. Then, 100µl of (5 mM) ferrozine (Sigma-Aldrich, Poole, UK) was added to samples and was incubated at room temperature for 10 minutes. The absorbance was measured at 562 nm and the following equation (Chelating Activity % = (Absorbance of control -Absorbance of sample)/Absorbance of control) ×100) was used to estimate the percentage of ferrozine-Fe 2+ complex.
Regarding the reducing power assay 33 , 1ml of sample was mixed with phosphate buffer (0.2M, pH 6.6) (Oxoid, Hampshire, England) and potassium ferricyanide (1%) (Koch-light, Colnbrook Bucks, UK) before they were incubated in 50°C for 30 minutes. Then, trichloroacetic acid (TCA) (10%) (Sigma-Aldrich, Poole, UK) was added to stop reaction and then tubes were centrifuged at 6000 rpm for 10 minutes. The supernatant was mixed with equal amount of distilled water and ferric chloride (1%) (BDH, Poole, England) and the absorbance was immediately measured at 700 nm.

Statistical analysis
GraphPad Prism 7 software was used to analyze the results. Three independent experiments were performed for each assay and the means were assessed using one-way ANOVA test with Bonferroni's test correction. The data were expressed as mean ± SD and results were considered statistically significant when P<0.05.

Estimation of total phenolic and flavonoid contents
In this study, a survey was performed on 900 participants that live in Jeddah, Kingdom of Saudi Arabia to assess the most consumed coffee type, the average daily consumption of coffeecups, and the type of additives used commonly when drink coffee. Results from survey revealed that (7%) of the participants prefer to add milk (with either 10% as minimum concentration or 20% as maximum concentration) to coffee. Therefore, this study was interested in studying the effect of adding different milk types with different concentrations on the phenolic compounds bioavailability and the antioxidant activities of coffee. To test all these objectives, many samples were prepared and used in this study [Nescafé red mug coffee, Nescafé red mug coffee with whole milk (10% and 20%), Nescafé red mug coffee with semi-skimmed milk (10% and 20%), and Nescafe red mug coffee with skimmed milk (10% and 20%)].
The total phenolic content was determined with Folin-Ciocalteu assay and was expressed as Gallic acid equivalent whereas; the total flavonoid content was determined with aluminum chloride assay and was expressed as Catechin equivalent. Table (1) represents the total phenolic and flavonoid contents in the samples. Generally, results showed that adding milk to coffee improves the contents of total phenolic and flavonoid compounds especially with the whole milk, either at 10% or 20% concentration.

Estimation of radical scavenging activity
To determine the radical scavenging activity of coffee samples, two assays were performed. The first assay, H 2 O 2 radical scavenging assay, measured the ability of samples to scavenge hydroxyl radical at 320 nm. Hydrogen peroxide is one of the most reactive oxygen species (ROS) that can cross cell membranes rapidly 34 36 . Table 2 represents the results from H 2 O 2 and DPPH radical scavenging assays. The results in Fig 1 showed that adding any type of milk (whole, semi-skimmed, and skimmed) at any concentration, 10% or 20%, had a paradoxical effect on the radical scavenging activity. For H 2 O 2 scavenging activity, adding any type of milk at low concentration (10%) did not show any significant effect on the scavenging activity, whereas; at high concentration (20%), the activity of coffee to scavenge H 2 O 2 reduced significantly when milk is added. In contrast, the addition of milk improves

Estimation of metal chelating and reducing power activity
Two assays were performed to estimate the ability of antioxidants, present in the samples, to act as metal chelating agents and as reducing agents. In ferrous chelating activity assay, the dark color which is formed by the interaction of ferrozine with Fe 2+ is changed into a lighter color in the presence of a chelating agent in the sample 37 . On the other hand, ferric reducing power assay used the Fe 3+ reduction as an indicator of electrondonating activity which is an important mechanism of any phenolic antioxidant 38 . Table 3 represents the results from metal chelating and reducing power assays. Interestingly, the results in Fig 2 showed that adding any type of milk (whole, semi-skimmed, and skimmed) at any concentration, either 10% or 20%, reduces significantly the metal chelating and the metal reducing power activities of the coffee compared to the coffee alone (with no milk added). -All data were represented as Mean ±SD calculated by one-way ANOVA test followed by Bonferroni's test correction.
-P-values were calculated by a comparison made versus the (coffee only) value, which was referring to it as reference.

DISCUSSION
Coffee is one of the most popular beverages in the world. Many of the studies indicated that adding some additives to coffee can alter the bioactive compounds present in it, and therefore, may affect the biological activity of coffee as an important antioxidant. In many cases, coffee is consumed with milk. Milk protein may form interaction with coffee polyphenols, therefore, the present study aimed to investigate the total phenol and flavonoid contents and the antioxidant activity of bioactive components when different types of milk (whole, semi-skimmed, and skimmed) at both concentration (10% and 20%) were added to coffee. Our results indicated that adding milk to coffee increased the total phenol and flavonoid contents. This might be explained by the presence of other compounds in milk that can act as an antioxidant such as lactoferrin, ascorbic acid, tocopherols and tocotrienols 39 .
Hydrophobic interaction and hydrogen bond are the major reactions that derive polyphenol-protein complex formation. Several factors can influence the extent of these binding forces such as structure of polyphenols and proteins as well as some physical conditions particularly (temperature, pH, and ionic strength) 40 . In our study, our results revealed that at 10% milk concentration, there was no significant difference in hydrogen peroxide scavenging activity between the coffee with and without milk. Interestingly, adding milk at higher concentration (20%) decreased the H 2 O 2 scavenging activity for coffee especially with skimmed milk. Some flavonoids have a high affinity to bind to proteins 40 . This affinity makes many phenol groups of flavonoids occupied and thus H 2 O 2 scavenging do not occur properly. The formation of polyphenol-protein complex can result in protein unfolding. In the presence of lipids, which are classified as Amphiphilic molecules, hydrophobic lipid phase interacts with hydrophobic part of the protein, which has been exposed to the outer phase due to denaturation. This prevents the association between hydrophobic sites of polyphenols with the aromatic groups and aliphatic side chains of hydrophobic amino acids 41 . However, those polyphenols when bind with the polar head groups of lipid can probably make phenol groups somehow in a position that allows phenols to scavenge H 2 O 2 , while polyphenol-protein complex without lipid presence require the involvement of many hydrogen and hydrophobic bonds in addition to the high affinity of flavonoids toward protein binding 40 . These findings may explain why whole milk sample has H 2 O 2 scavenging activity higher than semi-skimmed and skimmed milks. Three different types of milk were tested (whole, semiskimmed, and skimmed) at two concentrations (10% and 20%). A & B) represents the H 2 O 2 and DPPH radical scavenging activity of samples at 10% milk concentration, whereas; C & D) represents the H 2 O 2 and DPPH radical scavenging activity of samples at 20% milk concentration. Results revealed that the addition of milk at any concentration or type decreases the H 2 O 2 radical scavenging activity of the coffee, whereas; increases the DPPH radical scavenging activity. The values expressed as mean±SD (n=3). Comparisons of means were made using one-way ANOVA followed by a correction with Bonferroni's test (*P<0.05,** P≤0.01, and *** P≤0.001).

Fig. 2.
Metal chelating and reducing power activity of red mug coffee with and without various milk types and concentrations. Three different types of milk were tested (whole, semi-skimmed, and skimmed) at two concentrations (10% and 20%). A & B) represents the metal chelating and reducing power activity of samples at 10% milk concentration, whereas; C & D) represents the metal chelating and reducing power activity of samples at 20% milk concentration. Results revealed that the addition of milk at any concentration or type decreases the metal chelating and reducing power activity of the coffee. The values expressed as mean±SD (n=3). Comparisons of means were made using oneway ANOVA followed by a correction with Bonferroni's test (*P<0.05,** P≤0.01, and *** P≤0.001).
On other hand, in the present study, results from DPPH radical scavenging activity experiments are in contrast with that of H 2 O 2 for all tested samples. In addition to the probabilities that mentioned above about the difference of phenols, some amino acids with aromatic and bulky side groups are considered as effective radical scavengers because they can act as H-donors due to the presence of special groups in their side chains. For example, the phenolic group in tyrosine, indolic group in tryptophan, imidazole group in histidine, and sulfur hydrogen 42 in methionine and cysteine. Moreover, several studies refer to the correlation between the high concentration of flavonoids and polyphenols with the high DPPH scavenging activity 43,44 . In our study, the polyphenol and flavonoid contents were higher in all coffee with milk samples compared to the plain coffee sample.
In general, many studies, reported that the polyphenols interaction with proteins, not only affects the scavenging activity but also affects the chelating and metal reducing activity of coffee 18,45 . The hydrophobic stacking of the aromatic groups of the protein and polyphenols, or the interaction of the -OH groups of the polyphenols with the protein chain can resulted in the formation of aggregates 40 . Our results showed that the ferrous chelating activity increased when coffee is not mixed with milk. This might be due to the presence of free -OH groups of polyphenols or might be related to the binding of polyphenols to the milk caseins by covalent and non-covalent interactions either in a multi-site or in a multidentate interaction 24 . Our results are in agreement with other similar studies 22,46 that used FRAP assay to assess the ferrous chelating activity.
In conclusion, the results of the current study revealed that milk might either enhance the scavenging of DPPH or decrease the chelating and metal reducing activity of polyphenol. However, more investigations are required to evaluate the mechanisms by which fats in milk can alter the antioxidant properties of coffee.