MATERIALS AND METHODS

Materials: Fresh cow and buffalo butter were obtained from processing milk unit, Faculty of Agriculture, Cairo University, Giza, Egypt. Cow and buffalo butter oil (Anhydrous milk fat) ware prepared by the method of Amer et al.⁸. Fresh butter was melted at 60°C, removing the top oil layer, filtering through glass wool and drying the resulting oil over anhydrous sodium sulphate. The oil then refiltered (under vacuum, Whatman no 41 paper) to obtain clear oil (~99.5% milk fat) and stored at -20°C until used. Coconut oil made in Indonesia, was obtained from the Abou El Houl for Import and Export Co., 815 Port Said St., Ghamra, Hadayk El Kobba, Cairo, Egypt. The sample flushed with nitrogen and stored at -20°C until used. All chemicals used in analytical methods were analytical grade. Solvents (Hexane, chloroform, petroleum ether and ethyl alcohol) were distilled before use. The chemicals were purchased from the following sources: Sigma Chemicals Company and El-Gamhouria Trading Chemicals and Drugs Company, Egypt.

This study was carried out in Food Science Department, Faculty of Agricultural, Ain Shams University, Cairo, Egypt during September, 2017-September, 2018. The different samples of pure and adulterated milk fat were prepared in Dairy production Unit, Faculty of Agricultural, Ain Shams University, Cairo, Egypt. Fatty acids content of the tested samples were determined in National Research Center, Dokki, Giza, Egypt.

METHODS

Analytic methods: The refractive index of tested samples was estimated using a Carl Zeiss Refractometer (JENA, Type 337397, Germany) at 40°C, melting point (MP), acid value, iodine value, saponification number and fatty acids content of the tested samples were determined according to the method described in AOAC⁹.

Statistical analysis: Statistical analysis was performed according to SAS¹⁰ using General Linear Model (GLM) with main effect of treatments. Duncan’s multiple range was used to separate among of three replicates at p<0.05.

Equations of milk fat adulteration were performed according to Microsoft Office Excel¹¹ program for creating values and calculating the slope and intercept.

RESULTS AND DISCUSSION

Physical properties
Refractive index: There are many physical characteristics of the edible fats and oils such as refractive index and melting point, which are played an important role in assessing their quality and palatability, as well as the consumer acceptability of these products. The physical characteristics of fat or oil are dependent on the degree of unsaturation, the carbon chain length, the isomeric fatty acid forms and molecular configuration¹².

As shown in Table 1, no significant difference was observed between the RI value in cow and buffalos milk fat. It is clear from the results that coconut oil showed the lowest (RI) value while cow milk fat had the lowest. The obtained data agreed with Ariponnammal¹³ who observed that, the reflective index of pure milk fat was higher than that of coconut oil.

Table 1:	Refractive index and melting point (°C) of milk fat either pure or adulterated with different ratios from coconut oil
a,b,cMeans with same letter among treatments are not significantly different at p<0.05

Addition of coconut oil to milk fat either cow or buffalo led to decrease the RI value. Also, RI value was decreased with increasing ratio added of coconut oil into milk fat either cow or buffalo. This decrease in RI values with increasing the ratios of coconut oil added could be due to that the coconut oil contained short chain and saturated fatty acids compared with milk fat which may let to increase RI values of treatments. From the obtained data, it could be observed that there were non-significant differences in RI between pure milk fat and adulterated milk fat with coconut oil up to 50%. The refractive index of fats and oils may be correlated with fatty acids content, free fatty acids content, saturation degree and oxidative state^14,15. However, the slope of the change in the refractive index, with the addition of coconut oil, decreases slightly at higher values of the addition. The refractive index of adulterated cow and buffalo milk fat with different levels of coconut oil was within the natural variation of pure milk fat samples. Therefore, this analytical characteristic did not appear to show any significance in detecting adulteration of milk fat with coconut oil.

From the statistical analysis of the obtained data, it could be calculated the ratios of adulteration in the cow and buffalo's milk fat with coconut oil using the following equation:

For cow milk fat:

For buffalo milk fat:

Melting point: Data in Table 1 indicated that, the pure coconut oil showed the lowest melting point among all treatment, this could be due to the lower percentages of long chain fatty acids in coconut oil which led to lower it melting point. On the other side, pure cow's milk fat had the highest melting point compared with other treatments. The results agreed with Celik and Bakirci¹⁶. From the data, it could be noticed that adulteration of milk fat with coconut oil decreased the melting point in both cow and buffalo milk fat. Gradually increasing percentage of added coconut oil into milk fat either cow or buffalo gradually decreased the melting point. Decreasing melting point of pure milk fat blended with coconut oil could be attributed to the decrease in solid fat content of coconut oil as compared to pure milk fat either cow and buffalo. De Martini Soares et al.¹⁷ reported the melting point depends on the solid to liquid phase transition of triacylglycerols which are already present in greater amounts when the blend of coconut oil. According to the data obtained from Table 1, it could be found that, there were significant differences between pure milk fat either cow or buffalo and all adulterated milk fat with 25-75% coconut oil. Therefore, this method may be useful to determine the adulterated the butter fat with different ratios of coconut oil.

From the statistical analysis of the obtained data, it could be calculated the ratios of adulteration the cow and buffalo's milk fat with coconut oil using the following equation:

For cow milk fat:

MP = 35.24-0.11×Y

For buffalos milk fat:

MP = 34.64-0.11×Y

Chemical properties
Acid value: As illustrated in Table 2, it could be noticed that, acid value was significant lower in pure coconut oil than that of milk fat either pure control or adulterated with different ratios of coconut oil. These results agreed with Park et al.¹⁸ who found that the acid value of pure milk fat was 0.48%. Also, Odoom et al.¹⁹ observed that, acid value of collected coconut oil samples were 0.5 mg KOH/g oil (0.05%). Addition of coconut oil to milk fat either cow or buffalo resulted in significant lower acid value being decreased with increasing the ratio added.

Table 2:	Acid value and iodine value (g I2 absorbed\100 g fat) of milk fat either pure or adulterated with different ratios from coconut oil
a,b,cMeans with same letter among treatments are not significantly different at p<0.05

The acid values were slightly higher in cow milk fat than buffalo milk fat at any ratio added of coconut oil. According to Nielsen²⁰, acid values are dependent on different factors such as FFA, acid phosphate and conditions of the storage. Therefore, the acid value cannot be successfully used to detect the blending of cow or buffalo milk fat with coconut oil.

From the statistical analysis of the obtained data, it could be calculated the ratios of adulteration in cow and buffalo's milk fat with coconut oil using the following equation:

For cow milk fat:

AV = 0.45-0.004×Y

For buffalos milk fat:

AV = 0.24-0.0014×Y

Iodine value: Iodine value (IV) is a measure of the extent of fat unsaturation. The value varies with the type and proportion of unsaturated fatty acids present in fat. From the obtained data in Table 2, it could be observed that, the pure coconut oil showed the lowest iodine value among all treatment. On the other side, pure cow's milk fat had the highest iodine value compared with other treatments. The decrease in iodine value of coconut oil could be attributed to the lower unsaturated fatty acid content in coconut oil than pure milk fat either cow or buffalos. These results found to be in agreement with Bolton²¹ who found that, pure milk fat contained about 35% of total saturated acids, most of which consists of oleic acid, the iodine value of milk fat lies around 30. Marina et al.²² stated that, iodine value in coconut oil ranged from 4.47-8.55. The addition of coconut oil to pure milk fat caused a gradual and significant decrease in iodine value of adulterated milk fat. The decrease was proportional to the add level. Iodine value (IV) is a measure of the extent of unsaturation of fat and is a structure index. This value for milk fat ranges from 32-37, which is low in comparison to most other fats²³. Kehar et al.²⁴ also reported that the IV of cow ghee ranged from 27.4-40.5. The average IV of ghee of Deoni cow²⁵ is 27.7 . Many common vegetable oils have high IVs, ground nut 90, sesame 108, linseed 180. Coconut oil is unique in having an IV of only about 9. IV furnishes very clear information on the actual nature of the fat. High values characterize the liquid vegetable fats except the coconut oil and myristica groups and few other fats²¹. Therefore, lower iodine value can be taken as an index of adulteration of ghee with coconut oil.

From the statistical analysis of the obtained data, the ratios of adulteration could be calculated of cow and buffalo's milk fat with coconut oil using the following equation:

For cow milk fat:

IV = 32.8-0.23×Y

For buffalos milk fat:

IV = 32.45-0.24×Y

Where:

Saponification number: Data in Table 3 indicated that, saponification number was significantly affected by adulterating milk fat (cow and buffalo) with different coconut oil ratios. The data showed that coconut oil possessed higher saponification number than that of milk fat either cow or buffalo. Saponification value of buffalo milk fat was slightly lower than that of cow milk fat. These results agreed with Javeed et al.²⁵ and Singhal²³ who reported that, the range of saponification values in cow ghee falls within the lower (222.80) and upper (237.00), respectively. On the other hand, Marina et al.²² observed that saponification value in coconut oil ranged from 250.07-260.67 mg KOH g oil.

Table 3:	Saponification number (mg KOH \g fat) and peroxide value of milk fat either pure or adulterated with different ratios from coconut oil
a,b,cMeans with same letter among treatments are not significantly different at p<0.05

Addition of coconut oil to pure milk fat either cow or buffalo led to significant increase of saponification number in mixture of fat. However, saponification value was gradually increased by increasing the ratio of added coconut oil to milk fat either cow or buffalo. The increase in saponification number of blends pure milk fat coconut oil could be correlated to the low molecular weight of coconut oil fatty acids. Saponification value (SV) gives an indication of average molecular weight of fatty acids present in the fat. Since coconut oil may be containing a high proportion of fatty acids of low molecular weight compared with pure milk fat either cow or buffalo. Also, coconut oil was exceptional in having an SV of 243-262, due to its high content of lauric (12:0) and myristic (14:0) acids^23,26. From the data in Table 3, it could be observed that, adulterating the pure milk fat with different coconut oil caused a significant effect on saponification. Therefore, saponification number could be used for detecting adulteration of milk fat with different ratios coconut oil.

From the statistical analysis of the obtained data, the ratios of adulteration the cow and buffalo's milk fat with coconut oil could be calculated using the following equation:

For cow milk fat:

SN = 228.8+0.24×Y

For buffalos milk fat:

SN = 226.3+0.26×Y

Peroxide value: It is clear from the data in Table 3 that, peroxide value was lower in coconut oil than that of pure milk fat either cow or buffalo milk fat. These results confirmed with Celik and Bakirci¹⁶ who found that, peroxide value of the milk fat recorded 1.3 mEq Kg and Marina et al.²², who observed that, coconut oil samples characterized with low peroxide value (0.21-0.57 mequiv oxygen kg). Adulteration of pure fat samples either cow or buffalo milk fat with coconut oil caused a significant decrease in peroxide value of the mix. This is due to high saturated fatty acids content of coconut oil compared to buffalo or cow milk fat. Many research reported that, coconut oil addition to other vegetable oils improves their oxidative stability indicating that coconut oil can be used as a natural antioxidant through the blending process.

From the statistical analysis of the obtained data, the ratios of adulteration the cow and buffalo's milk fat with coconut oil could be calculated using the following equation:

For cow milk fat:

PV = 1.19-0.010×Y

For buffalos milk fat:

PV = 1.75 - 0.015×Y

Fatty acids profile: Fatty acid profile of cow and buffalo milk fat either pure or adulterated with different ratios from coconut oil are presented in Table 4 and 5. The obtained data showed that, in coconut oil butyric, heptadecenoic Cis 10, linoleic (trans), linolenic (cis), linolenic and docosanoic fatty acids were not found, while caprilic, lauric, myristic and oleic fatty acids were the most abundant. Percentage of caprilic, capric, lauric, myristic and oleic fatty acids in coconut oil were higher than that in pure fat milk either cow or buffalo. While other fatty acids (butyric, caproic, tridecylic, tetradecenoic, pantadecylic, pentadecanoic ME, palmitic, palmitoleic, heptadecanoic, heptadecanoic Cis 10, stearic, oleic, linolenic (cis), linolenic, arachidic, eicosenoic and docosanoic) in pure coconut oil were lower than that in pure cow and buffalo milk fat. The results are agreement with the results obtained by Lopez et al.²⁷ who reported that, milk fat is characterized by short-chain (C4-C8, 8.3%), medium-chain (C10-C12, 6.6%) and long-chain (C14-C18, 81.9%) length fatty acids. Moreover, milk fat is a relatively high saturated fat about 65% saturated fatty acids (mainly C16:0, C18:0 and C14:0) and about 35% unsaturated fatty acids (mainly C18:1). Shin et al.²⁸ also found that the fatty acids composition of butter fat contained 3.9% butyric acid, 2.9% caproic acid, 2.1% caprylic acid, 4.4% capric acid, 6.2% lauric acid, 14.0% myristic acid, 6.2% lauric acid, 14.0% myristic acid, 29.5% palmitic acid, 2.0% palmitoleic acid, 11.0% stearic acid, 17.9% oleic acid, 1.0% linoleic acid and 0.5 % linolenic acid.

Table 4:	Fatty acids content of cow milk fat either pure or adulterated with different ratios from coconut oil
TSC: Total short chain, TLC: Total long chain, USFA: Unsaturated fatty acids, SFA: Unsaturated fatty acids, a,b,cMeans with same letter among treatments are not significantly different at p<0.05. ND: Not detected

Table 5:	Fatty acids content of buffalo milk fat either pure or adulterated with different ratios from coconut oil
TSC: Total short chain, TLC: Total long chain, USFA: Unsaturated fatty acids, SFA: Unsaturated fatty acids a,b,cMeans with same letter among treatments are not significantly different at p<0.05. ND: Not detected

From the data it could be noticed that, pure milk fat either cow or buffalo was characterized by lower content of short-chain (TSC) fatty acids than that of coconut oil. There were non-significant differences in content of TLC fatty acids between coconut oil and pure milk fat either cow or buffalo. Coconut oil also contains lower USFA (%) than pure milk fat (cow and buffalo). On the other hand, the content of SFA in coconut oil was higher compared to pure cow or buffalo milk fat. Such results have been reported by other researchers as well Krishna et al.²⁹, who stated that, coconut oil contains 92% of saturated fatty acids, most of them (about 70%) are lower chain saturated fatty acids known as medium chain fatty acids (MCFAs). Therefore, adulteration of pure milk fat either cow or buffalo with different ratios of coconut oil caused considerable changes in certain fatty acids; i.e., butyric, caprilic, lauric, palmatic, stearic and oleic acids, the change was increased with increasing percentages of coconut oil (Table 4, 5). In particular, with increasing levels of coconut oil, butyric, palmetic and oleic acids were gradually decreased, while caprilic, lauric and myristic acids gradually increased. Moreover, TSC and SFA gradually increased while USFA gradually decreased with the increasing of coconut oil levels in milk fat. The obtained results agree with Farag et al.³⁰, who found that adulteration of cow and buffalo milk fat with various levels of lard or margarine caused significant changes in certain fatty acids, i.e., 22:0, 18:1, 18:0 and 16:0. Fox et al.³¹ found that the addition of vegetable fat will, in effect, dilute the concentrations of the short-chain acids by an amount equal to the concentration of the added vegetable fat. Sharma and Singhal³² found that the fatty acid composition of ghee containing hydrogenated vegetable oil was significantly different from that of control ghee containing no additional fats.

From the data in Table 4 and 5 it could be noticed that, pure milk fat either cow or buffalo was characterized by lower percentage of TSC/TLC and higher percentage of USFA/SFA compared with pure coconut oil. Adulteration of pure milk fat, either cow or buffalo with various levels of coconut oil caused increase in TSC/TLC% and decrease the percentage of USFA/SFA. This may be due to the addition of coconut oil in milk fat increase the concentrations of the TSC and SFA fatty acids while dilute the concentrations of the TLC and USFA fatty acids.

From the statistical analysis of the obtained data, the ratios of adulteration the cow and buffalo's milk fat with coconut oil could be calculated using the following equation:

For cow milk fat:

TSC = 5.59+0.03×Y

For buffalos milk fat:

TSC = 4.83+0.04×Y

For cow milk fat:

USFA = 31-0.21×Y

For buffalos milk fat:

USFA = 33.6-0.25×Y

For cow milk fat:

SFA = 66.2+0.25×Y

For buffalos milk fat:

SFA = 63.5+0.28×Y

Where:

For cow milk fat:

TSC/TLC (%) = 6+0.03×Y

For buffalos milk fat:

TSC/TLC (%) = 5.2+0.04×Y

CONCLUSION

Finally, it could be concluded that, some physical and chemical methods such as determination of melting point, iodine value, saponification number and percentage of TSC, saturated and unsaturated fatty acids may be useful to determine the adulteration of butter fat with different ratios of coconut oil.

SIGNIFICANCE STATEMENT

This study discovers the importance of using some physical and chemical methods such as determination of melting point, iodine value, saponification number and percentage of TSC, saturated and unsaturated fatty acids to determine the adulteration of milk fat with different ratios of coconut oil. From the obtained data, it could be calculated the ratios of adulteration in the cow and buffalo's milk fat with coconut oil by using different equations (to the best of researchers' knowledge, no researchers used such additives in previous studies) depended on the different of some physical and chemical properties between milk fat and coconut oil. This study will help the researchers and food analysts to detect the adulteration of milk fat with coconut oil by using some equations; therefore, this may be help to solve the serious problem in the food industry.