Validation of the Methods for the Non-Milk Fat Detection in Artificially Adulterated Milk with Palm Oil

Dairy products are among the most adulterated food products. Due to the current high price of milk fat, it has been replaced by low-cost oils, especially those oils that have the same fatty acid profile as milk fat. This study intends to confirm the lowest level of palm oil added to milk and validate various methods for detecting palm oil in milk, including gas chromatography, reverse-phase high performance liquid chromatography, and Fourier transform infrared. Different amounts of palm oil were prepared in the final liquid milk using five treatments of fresh milk cream and an emulsion of palm oil. The results of this study showed that the values of the saponification number decreased with the increase in the percentages of added palm oil. There was no decrease under the limits of the Egyptian standards until the addition of 50% palm oil. The iodine number is less sensitive than the saponification number in the detection of palm oil. Butyro refractometer reading is unable to detect the palm oil in milk. The fatty acid profile in milk determined by gas chromatography correlated well with the addition of palm oil. Furthermore, there is a positive relationship between the level of added palm oil and the β-sitosterol content as measured by reverse-phase high-performance liquid chromatography. There was no relationship between the behavior of the spectra resulting from Fourier transform infrared spectroscopy and the presence of palm oil.


Introduction
Milk fat is an important compound and plays an essential role in the nutritional and physicochemical properties, sensory, and economics values of milk and dairy products (Kumar et al. 2010). Milk fat content of saturated fatty acids is about 70%, and the content of unsaturated fatty acids is about 30% (Soha et al. 2015;Salem et al. 2019). Fatty acid composition is influenced by the animals' diet, lactation period, individual traits, breed, health status, and weather conditions (Fontecha et al. 2006). The diet of dairy cows plays the most important role (Mishra et al. 2004).
Adulteration of milk fat has always been a serious problem because of the economic advantages of partly replacing highpriced fats with low-priced oils without labeling the product accordingly (Destaillatsa et al. 2006). This also poses a risk to human health and decreases its functional value (Ntakatsane et al. 2013). The fatty acid composition of milk fat has long been used as a criterion to detect adulteration with vegetable oil, mainly because milk fat is characteristic of short-chain fatty acids (Molkentin and Precht 1998). Palm oil is a natural product and has been consumed for many decades. It is used for the manufacture of a wide variety of food products because of its oxidative stability and its plasticity at room temperature (Noor Lida et al. 2002;Mamat et al. 2005;Rosnani et al. 2007). Detection of vegetable oil in milk fat is quite difficult (Gandhi 2014), especially those oils like palm oil that have a nearly identical fatty acid profile to milk fat. Palm oil has a balanced fatty acid composition, with almost equal amounts of saturated and unsaturated fatty acids. The predominant fatty acids in palm oil are palmitic acid C16:0, ranging between 39 and 45%, and oleic acid C18:1n9c, ranging between 37 and 44% (Koushki et al. 2015). The refractive index (RI) is a simple and rapid method and is widely used as a preliminary 1 3 screening method for knowing the quality of milk fat (Sharma et al. 2020).
Bovine milk is indeed among the controversial foods, with cholesterol generally regarded as an undesirable milk component and being a significant external source for regular consumers (German et al. 2009). Cholesterol has also been commonly used to detect mixtures of animal fat in vegetable oils. Usually, the cholesterol content in milk fat is between 204.3 and 382.4 mg/100 g (Precht 2001). Molkentin (2006) reported that the cholesterol content in butter fat was 302.6 mg/100 g. Since milk fat is high in cholesterol, adulteration with foreign oils will be detectable through the cholesterol content. The concentration of cholesterol in milk fat is typically higher, so a quantitative comparison of the chromatograms of the original sample and its admixtures would either show a tendency for the concentration of cholesterol to decline or additional peaks to appear due to the presence of plant sterols derived from palm oil (Marikkar et al. 2016).
Analysis of fat composition by reverse-phase high-performance liquid chromatography (RP-HPLC) for detection of adulteration in oils and fats has been widely used (Marikkar et al. 2016). In recent years, Fourier transform infrared (FTIR) spectroscopy has emerged as a major analytical technique for a wide range of food analyses. It has become an attractive option because of its high speed in analysis and ease of operation (Marikkar et al. 2016). The full potential of infrared spectroscopy has been realized only after the advent of FTIR spectrometers (Sharma et al. 2020). Windarsih and Irnawati (2020) have reported using FTIR spectroscopy combined with chemometrics of partial least square (PLS) for authentication of bovine milk fat from lard oil. Milk adulteration detection techniques need to be very specific and rapid because defrauders have escaped condemnation by claiming less effective than conventional detection techniques (Garcia et al. 2012).
Most published studies considering the problem of milk adulteration have a focus on experimental adulteration, aimed at assessing the sensitivity of analytical methods in detecting the presence of vegetable oils (Abd El-Aziz et al. 2013). Due to the absence of adequate monitoring and the lack of convenient law implementation, fraudsters have escaped (Garcia et al. 2012). The detection of vegetable oil in milk has become a challenge, and this work is designed to validate the methods that are available for the detection of palm oil in liquid milk.

Materials
Fresh cow's milk was obtained from the herd of the Faculty of Agriculture, Alexandria University, and palm oil (shortening, melting point 36-38 °C) was obtained from the market and manufactured by the Wilmar Company, Singapore. It is white in appearance, bland in taste, and stored at ambient temperature. Food stabilizer was from Hamulsion Trce G.C. HAHN and CO. LTD, in the UK. All chemicals used were analytical grade. Cholesterol solution 10 mg/ml in chloroform, β-sitosterol 100 μg/ml in chloroform, and fatty acid methyl ester mixture (Part number: CRM47801, Sigma-Aldrich Co LLC, Merck KGaA, Darmstadt, Germany).

Preparation of Liquid Milk Containing a Mixture of Fats
The whole milk was separated into milk cream (35% milk fat) and skimmed milk. Palm oil emulsion is made by combining palm oil and stabilizer (5%) to get emulsion of 35% fat. Fresh skim milk heated to 65 °C is combined with a milk cream and palm oil emulsion to create milk with 3.5% fat and include fat blends in milk at different levels (milk cream/ palm oil 100:0%, 75:25%, 50:50%, 25:75%, and 0:100%).

Detection of Milk Fat Constants in Artificially Adulterated Milk
The saponification number was determined according to Egyptian standard (ES 51-p5/2005, the iodine number was determined according to ES 51-p4/2005, the peroxide number was determined according to ES 155-p7/2006, and the refractive index was determined according to ISO 1739/2006 using Abbe refractometer model NAR-3 T (ATAGO, Japan)). Buyer Reading and Refractometer index were measured at 40 °C, by ATAGO Digital Butyro-Refractometer (ATAGO, Japan).

Lipid Extraction
A 250-ml centrifuge container was filled with 20 g of each sample of milk, then were added a 20 ml of chloroform and 40 ml of methanol. It was shaken for 2 min, followed by the addition of 20 ml of chloroform and another 30 s of shaking. The mixture was centrifuged at 492-769 × g for 10 min at room temperature. A dry weighing flask was filled with the lowest layer of chloroform after it had been taken off and filtered through coarse filter paper. The chloroform was evaporated, and the extracted lipids were kept at − 18 °C until analysis (Egan et al. 1981).

Fatty Acid Profile Analysis
Preparation of FAMEs Two drops of the residual oil were collected and mixed with 5 ml of benzene (GC grade) and 7 ml of GC solvent (1% H 2 SO 4 in methanol) to prepare the 1 3 fatty acid methyl ester. The mixture was then heated in an oven at 90 °C for 90 min. Two milliliters of distilled water was added, and the mixture was thoroughly shaken until it separated into two layers. The top layer was removed, the surplus moisture was eliminated using sodium sulfate anhydrous, and the filtrate was then put through a Merck Millipore SLGSR33SS Syringe Filter Millex (33 mm, 0.22 μm (EOG)) to prepare it for injection into the GC column (Eder 1995).

Fatty Acid Profile Analysis by Gas Chromatography
Gas chromatographic analysis was carried out using an ACME model 6100 GC (Young LIN Instrument Co., Korea) fitted with a split injector and flame ionization detector. Nitrogen was used as the carrier gas with a flow rate of 0.5 ml/min. The components were separated on a 30-m SP-2380 fusedsilica capillary column with a 0.25-mm i.d. and 0.2-m film thickness (Supelco, Bellefonte, PA), and the detector temperature was set at 260 °C. The injector temperature was set at 220 °C and in split mode (split ratio of 1:50). The column was initially maintained at 140 °C for 5 min, and the temperature was subsequently increased to 240 °C at a rate of 4 °C/min.

Extraction of Unsaponifiable Matter for RP-HPLC Analysis
The samples of fat were dissolved in 10 ml of isopropanol, then added an aliquot of 1 ml to methanolic solution containing 10 mol/l potassium hydroxide (9:1) and refluxed for 30 min. After cooling, 5 ml of deionized water and 10 ml of n-hexane were added, and they were vigorously shaken at 150 rpm for 20 min at 37 °C. To terminate the reaction, the organic layer was separated, rinsed with deionized water, and dried with anhydrous sodium sulfate. The hexane solution was evaporated in fume hood on sand bath, and the leftover material was diluted in 1 ml of methanol prior to HPLC analysis (Borkovcová et al. 2009).

RP-HPLC Analysis
Stock solutions of two different standards (β-sitosterol and cholesterol) in methanol were prepared for standard solutions. Each of the standards was filtered by a 0.22-µm Nylon syringe filter then 10 µl was injected. Samples were prepared and filtered using a 0.22-µm Nylon syringe filter and 10 µl was injected. Samples were eluted by the RP-HPLC Waters 2690 Alliance HPLC system equipped with a Waters 996 photodiode array detector at 205 nm. C18 Xterra: 4.6 × 250-mm, 5-µm column was used. Isocratic elution with a mobile phase of water and methanol (15%:85%) mixture at a flow rate of 1 ml/min was used. The column temperature was set up at Ambient (Borkovcová et al. 2009).

Analysis by FTIR Instrumental
The FTIR spectra of samples (either pure or admixtures) were measured using an FTIR spectrometer. The functional groups present were characterized by a Bruker VERTEX 70v FT-IR Spectrometer connected with platinum ATR model V-100 in the wave number (400-4000 cm −1 ).

Sensory Evaluation
Samples of milk were sensory evaluated by expert staff members according to Bodyfelt et al. (1988), using the scorecard, which assigns 10 points for flavor, 5 points for body and appearance for milk samples.

Statistical Analysis
The observation data were analyzed using IBM SPSS software package version 20.0. (Armonk, NY: IBM Corp). The used tests were F-test (ANOVA) for normally distributed quantitative variables, to compare between more than two groups, and post hoc test (Tukey) for pairwise comparisons. Significance of the obtained results was judged at the 5% level.

Physicochemical Analysis of Fluid Milk Containing Milk Fat and Palm Oil at Different Levels
As observed in Table 1, there was a considerable difference between the studied milk cream mixtures regarding saponification number, iodine number, and refractometer (p ≤ 0.05). Saponification number (SN) of the studied milk mixtures ranged from 198.6 in pure palm oil to 224.4 in pure milk fat (MF), meaning that the saponification number decreases as the percentage of palm oil (PO) in the milk fat mixture increases (p ≤ 0.05). These results for milk fat were in the normal range reported by Özkanlı and Kaya (2007) and Samet-Bali et al. (2009). Also, our findings agreed with Hamed et al. (2019), who suggested that the increase of added vegetable oils to buffalo butter reduced the saponification number and concluded that the saponification number is outside the normal range of pure butter in mixtures containing more than 50% of vegetable oils. Furthermore, Hamed et al. (2019) concluded that if the added level of vegetable oils is lower than 25%, saponification numbers cannot be successfully used in the detection of adulteration in buffalo butter. The saponification number in cow milk fat should be in the range of 211.7-243.3 mg KOH/g oil (Egyptian standard (ES) 154-P5/2005), the SN of the sample (25% PO of its fat) was 216.3 mg KOH/g oil, and this is in the normal range recommended by ES. Therefore, the SN could not be used to detect replacement milk fat by palm oil less than 50%.
According to Table 1, the iodine number (IN) ranged from 32.94 in pure milk fat to 50.90 in pure palm oil. Our results indicated that the IN increased by increasing the palm oil (p ≤ 0.05) level in milk, and there is a direct relationship (p ≤ 0.05) between the percentage of palm oil in the milk fat mixture and the iodine number. These findings agree with Zaidul et al. (2007), who assumed that iodine number increased with the presence of a high amount of unsaturated fatty acids, especially oleic and linoleic acids in the fat. The same results were recorded by Singhal (1980) and Abd El-Aziz et al. (2013). In addition, Kumar et al. (2010) suggested that the iodine number was out of the normal range by the addition of vegetable oils to butter in a ratio of 50% or more. The IN should be in the range of 26.4-43.1 of milk fat (ES 154-P5/2005). In this study, the IN of a sample (50% MF/50% PO) was 40.07. This meant the IN could not be used to detect replacing milk fat with palm oil at a level of 50% or less.
The refractive index (RI) of the fat extracted from milk samples in this study ranged from 1.452 in 100% milk fat to 1.460 in 100% palm oil (Table 1). The RI should be in the range of 1.4522-1.4543 of milk fat (ES 154-p 5/2005). The RI of the sample (75:25 MF: PO) was 1.4549. This is out of the normal range of pure milk fat, but the results are not significant with pure milk fat (p ≥ 0.05). These findings agree with those of Dehanzadeh et al. (2018), who concluded that there is a direct relationship between the amount of palm oil in milk fat and the refractive index. BR measurement is normally obtained at 40 °C (Gandhi and Lal 2017), when the milk fat sample is clear and transparent. This characteristic may be used to identify adulteration of milk fat with vegetable oils and fats (39-40), except palm oil (38-39) and coconut oil, since the typical values for the Butyro refractometer reading of milk fat (40-45) and vegetable oils and fats (over 50) are so far away from one another (over 50).
The BR range for animal body fats is 44 to 51 (Kumar et al. 2005). According to Gandhi and Lal (2017), the BR readings for pure cow ghee ranged from 41.10 to 42.20, whereas that for pure buffalo ghee ranged from 40.20 to 41.20. Palm olein and sheep body fat added individually could not be detected even at 15% levels in pooled cow and buffalo ghee samples. Mixture of palm olein and sheep body fat was detectable only at 9 + 21 (30) % levels. Butyro-refractometer reading of fat extracted from different samples of fluid milk had the highest reading in 100% milk fat (43.33) compared to the lowest value in 100% palm oil (41.07). By using butyric reading, it was not possible to find any significant difference between the fluid milk samples containing different levels of palm oil, except for 100% palm oil (p ≥ 0.05).

Fatty Acid Profile Analysis by Gas Chromatographic
In GC separation, several types of fatty acids were detected in the samples. Each peak was detected and quantified by comparing the obtained chromatogram with the standard chromatogram of fatty acids, and the value of each peak was expressed in fatty acid percent of total fatty acids in the sample (Table 2; Fig. 1). According to the fatty acid profile analysis by GC (Table 2), a sample of 100% MF had the highest values of capric acid (C10:0), lauric acid (C12:0), myristic acid (C14:0), and stearic acid (C18:0) (3.10, 3.79, 12.16, and 13.54, respectively of total fatty acids). All these corresponding values were the lowest in the 100% PO sample. In addition, the 100% MF sample exhibited the greatest value of saturated fatty acid (SFA) composition (68.31) compared to the lowest value in the 100% PO sample (49.88).
On the other hand, the 100% PO sample had the highest composition of palmitic acid (C16:0), oleic acid (C18:1n9c), and linoleic acid (LA; C18:2n6c), 45.40, 39.92, and 9.55, respectively, of total fatty acids (Table 2). Unsaturated fatty acids (USFA) and USFA/SFA had the highest values in the 100% PO sample, 49.92 and 1.00, respectively (Table 2).  The milk samples containing different levels of palm oil were easily detected by GC, as, for example, the reduction in myristic acid (C14:0) was at a level of 24% in a milk sample containing 25% palm oil of total fat. Results of the current study agree with Elaaser (2017), who found that milk fat samples from local Cairo markets had an apparent increment in the palmitic (C16:0), and a decrease or absence in some other fatty acids, as C4:0, C6:0, C8:0, and C10:0, or fatty acids that were found in low content, as C12:0 and C14:0, and concluded that this sample characterized shortening palm oil. Furthermore, Calvo et al. (2007) discovered that the fatty acid profile of the cheese fat was significantly changed by replacing milk fat with vegetable oils in many processed kinds of cheese on the Egyptian market.

Determination of the Sterol Content of the Fat Blenders in Milk at the Levels by RP-HPLC
RP-HPLC results for of determination the cholesterol levels (Table 3) showed that there was a pronounced decrease in cholesterol content as the palm oil percentage increased in the milk (392.76 µg/ml in 100% MF and 56.97 µg/ml in 100% PO), while β-sitosterol concentrations were much higher as the palm oil percentage increased in the milk (3.06 µg/ml in 100% PO and 0.10 µg/ml in 100% MF).
Determination of cholesterol in animal fats and oils is an important topic in the food industry since high amounts of cholesterol in foods are closely related to cardiovascular disease risks. Cholesterol has also been commonly used to detect mixtures of animal oils in vegetable oils (Khorsandmanesh et al. 2020).
The presence of phytosterols in milk fat could be the most important indicators for the detection of the presence of vegetable oil in milk fat (Clemente and Cahoon 2009). Therefore, the sterol profile of oils can identify the origin of the oils much more than their fatty acid compositions (Khorsandmanesh et al. 2020). These findings were close enough to those of Elaaser (2017), who studied three doubtful milk fat samples purchased from the local market.
Also, Hamed et al. (2019) found that the cholesterol content decreased from 278.34 to 117.5 mg/100 g fat, while β-sitosterol in buffalo butter increased from 0.10 mg/100 g fat in the buffalo butter to reach 39.65 mg/100 g fat when palm oil was replaced 75% of total milk fat.
Moreover, Contarini et al. (2002) mentioned that pure butter contains only cholesterol, except traces of isomer 7-cholesterol, which is usually less amount than 1% but could not contain other sterols. Also, Nurseitova et al. (2021) noticed that a large replacement of milk fat by vegetable oils changed the sterol profiles completely, as they observed that six sour cream samples contained more than 95% cholesterol (pure butter) while one contained only 2.1%, which could be regarded as adulterated. Furthermore, our results correspond well with the work of Khorsandmanesh et al. (2020), who studied samples containing 1, 2, 5, 10, 20, and 50% of palm oil of total fat and suggested that among all sterols, β-sitosterol at a level of 5% in all the samples could be a good indicator for the detection of milk fat adulteration.

FTIR Instrumental Analysis of Artificial Adulterated Milk with Palm Oil
Different levels of percentages at different wavelengths of absorbance for each functional group were recorded for the milk fat samples (Table 4; Fig. 2). The absorbing and relative intensities of wavenumbers differ slightly. However, after careful observation, some variations could be reported regarding the stretching of > CH 2 of acyl chains as asymmetric (63% in 100% PO and 72% in 100% MF mixtures), the stretching of > CH 2 of acyl chains (76% in 100% MF and 82% in 100% PO mixtures), C-O-C stretching (68% in 100% MF and 82% in 100% PO mixtures), and PO stretching (symmetric) of > PO 2 polyphosphate phospholipid (80% in 100% MF and 87% in 100% PO mixtures).
Fourier transform infrared (FTIR) spectroscopy has wide applications in food analysis, including milk, butter, cheese, fat, and oil (Du et al. 2019;Saputra et al. 2018). FTIR spectroscopy offers food manufacturers rapid quantitative quality control tools and the means of verifying the identity, authenticity, and purity of the raw materials and ingredients that are used in the food industry (Rutkowska et al. 2015). Table 4 illustrates the FTIR spectra of milk containing different palm oil levels (0%, 25%, 50%, 75%, and 100% of total fat in the sample). The FTIR spectra of milk samples 1 3 in the current study were in the range of 3012-725 cm −1 . The spectra of milk fat were dominated by typical peaks assigned to functional groups. These findings agree with Hamed et al. (2019), who revealed that FTIR spectral bands of butter gradually increased with increasing the addition level of palm oil, as FTIR spectral bands of buffalo butter were lower in absorption in the regions of 3000-2800 nm than those of vegetable oils. The maximum absorption of bands for buffalo butter was at 2925 and 2855, which shifted to 2928 and 2857 nm for vegetable oils, respectively. Also, these results correspond with Cuibus et al. (2017), who studied the ATR-FTIR spectra of seven different butter samples spiked with 1-44% palm oil and found that the FTIR spectral was in the range of 3873-690 cm −1 . The large variety of functional groups makes the overall butter spectra very complex, and not easy to understand, and it might be difficult to identify small variations within the spectra due to adulterant traces. The absorption peaks correspond to different and specific wavenumber ranges, which were close enough to those reported by other authors (Karoui et al. 2005;Rodriguez-Saona et al. 2006;Subramanian et al. 2011;Gori et al. 2012), as they found 3873-3000 cm −1 for O-H stretching modes of water absorbing, the CH stretching vibrations in fatty acids (3000-2800 cm −1 ), the stretching of C = O bonds in acids and esters (1750-1650 cm −1 ), and C = O stretching of acids (1200-800 cm −1 ). So, the FTIR spectra could not observe significant differences among all samples, and this instrument could not be validated as a method to detect the adulteration of milk fat.

Sensory Evaluation of Artificial Adulterated Milk with Palm Oil
Sensory evaluations of artificially adulterated milk with palm oil samples were carried out by a group of 15 students and postgraduate students at the Faculty of Agriculture, Alexandria University, according to the scorecard suggested by Bodyfelt et al. (1988), who gave flavor 10 points, body 5 points, and appearance 5 points. There was no significant difference regarding sensory traits (Table 5), such as the appearance and flavor of artificially adulterated milk with different levels of palm oil and the control of normal milk (p = 0.103 and 0.224, respectively), while a significant difference was recorded regarding the body (p = 0.048).
The color and appearance of artificial adulterated milk with palm oil and normal milk ranged from 4.70 (in 100% MF) to 3.67 (in 100% PO). The study of Abd-ElGhany et al. (2020), who investigated the color analysis of fresh cream substitution of red palm oil, revealed that lightness exhibited a decreasing trend with the increase of the percentage of red palm oil replacement in milk.
In the current study, the flavor scores of artificially adulterated milk with palm oil decreased gradually but not significantly as the palm oil percentage increased (from 9.00 in 100% MF to 7.40 in 100% PO). These findings agree with Abd-ElGhany et al. (2020), who declared that the incorporation of red palm oil affected the flavor of the resultant ice milk. They revealed low scores for both mouth feel and flavor for iced milk with red palm oil at different levels, which could be attributed to the slight flavor of red palm oil, which was not commonly accepted by some panelists. In addition, Corradini   (Table 5), but there were no significant differences between normal milk sample (100% MF) and all other samples containing 25, 50, and 75% PO of total milk fat. The only significant difference was between natural milk (100% MF) and a sample containing 100% PO. This discovery should pique the interest of government officials in detecting palm oil in milk.

Conclusion
The results presented herein indicated that the values of saponification value, iodine number, and refractive index could be used as indicators of replacing milk fat with vegetable oil by adding more than 25%. Gas chromatography can be a reliable approach to identifying vegetable oil in dairy products. The concentration of some fatty acids could be used as an indicator of milk fat adulteration and associated with the level of adding foreign fats to milk fat. It could be used to detect the level of adulteration by adding 10% milk fat. Also, the determination of cholesterol and β-sitosterol content by RP-HPLC could be used for the detection of the addition of vegetable oil to milk fat. The spectrum behavior produced by FTIR spectroscopy in adulterated samples is almost the same, so this technique could not be used to detect the vegetable oil in milk fat. Our recommendations are to use GC and HPLC methods for detecting the presence of non-milk fats or oils in milk and dairy products.
Author Contribution All the authors contributed to the study's conception and design. Laboratory experimentation was carried out by Ph.D. student Marwa El-Nabawy under the supervision of Sameh Awad and Amal Ibrahim. The original draft was done by Marwa El-Nabawy. Writing-review and editing were done by Sameh Awad. The authors read and approved the final manuscript.
Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Data Availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations
Ethics Approval and Consent to Participate Not applicable.
Competing Interests Marwa El-Nabawy declares no competing interests. Sameh Awad declares no competing interests. Amel Ibrahim declares no competing interests.
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