Establishment of Fatty Acid Profile and Comparative Analysis of Volatile Substances in Regular and DHA-Biofortified Raw Milk

Abstract

This study aimed to establish fatty acid profiles of regular raw milk and docosahexaenoic acid (DHA)-biofortified raw milk and to compare the volatile substance composition of the two types of raw milk. The fatty acid composition of the two types of raw milk was analyzed by gas chromatography–mass spectrometry (GC). The results revealed the absence of C15:1, C17:1, C18:2, C22:1, and C24:1 in both types of raw milk, while C20:3 and C22:6 were exclusively found in DHA-biofortified raw milk. The fatty acid levels generally followed a pattern of initial increase and subsequent decrease during lactation, with higher concentrations of short- and medium-chain fatty acids being observed in regular raw milk. The C16:0, C18:3, C20:3, and C20:5 contents in the two types of raw milk varied significantly at different lactation stages. The gas chromatography–mass spectrometry (GC-MS) analysis of the volatile substances revealed the presence of aldehydes, ketones, esters, acids, and sulfur-containing compounds. The volatile substance content in the DHA-biofortified raw milk was generally higher than that in the regular raw milk, which was attributed to the elevated levels of unsaturated fatty acids in biofortified DHA raw milk.

1. Introduction

Milk fat is recognized as a highly complex fat containing numerous compounds. It has vital roles in the human body, including supplying and storing energy, sustaining regular cell functions, converting cholesterol into esters, regulating blood lipid levels, and aiding the development of brain nerves [1,2]. Furthermore, milk fat serves as an excellent solvent for natural fat-soluble vitamins (A, D, E, and K) and β-carotene.

Docosahexaenoic acid (DHA) is an essential ω-3 polyunsaturated fatty acid (PUFA), which is almost undetectable in regular raw milk. DHA plays a crucial role in maintaining human health, acting as an important component of the brain and retina [3,4,5,6]. Infants have a high demand for DHA, with dairy products being their primary source of dietary intake. DHA-biofortified milk is one of the main current dietary sources, which is obtained by introducing DHA-rich substances into dairy cattle feeds, thereby promoting DHA accumulation in milk through metabolic processes in dairy cattle [7]. The DHA-biofortified milk produced through the above approach maintains a flavor profile akin to regular milk, devoid of any fishy taste, thereby enhancing consumer acceptance.

DHA is present in four different structures in various substances: free fatty acid (FFA-DHA), triglyceride (TG-DHA), phospholipid (PL-DHA), and ethyl ester (EE-DHA). TG-DHA is predominantly found in DHA from microalgae oil and fish oil [8], while krill oil and krill powder contain PL-DHA, and fish oil products contain EE-DHA after processing and concentration [9]. Bioavailability refers to the absorption and transportation of a nutrient to systemic circulation or physiological activities. The bioavailability of DHA is closely associated with its different forms, with PL-DHA having the highest bioavailability, followed by TG-DHA, FFA-DHA, and EE-DHA. Triglyceride-DHA (TG-DHA) in feed sources, such as microalgae can be converted into phospholipid-DHA (PL-DHA) through animal metabolism, making PL-DHA the most easily absorbed and utilized form by the brain [10], which leads to an increase in DHA content in body organs [11].

However, altering the diet will not only enhance DHA levels in milk, but also enhance the formation of various fatty acids, ultimately leading to a modified fatty acid composition in milk. The FA composition of milk is not only closely related to the physical properties and sensory of milk but also affects the nutritional quality and functionality of milk and is an important indicator of the health status of cows. Milk FA composition has become an important indicator for evaluating the nutritional value and functional quality of milk. The construction of FA profiles of cow milk is the basis and means for the evaluation of the effects of ration regulation as well as milk quality. In addition, FA profiles can be used to detect non-milk fats in dairy products [12], such as vegetable oil adulteration identification. Therefore, the analysis of FA profiles is of great importance in studies related to cow nutrition and milk quality, and there is a need for in-depth investigation of them [13,14,15]. Gas chromatography is currently the most comprehensive and effective method for fatty acid detection owing to its high sensitivity, rapid analytical speed, and accuracy [16]. The unique flavor of milk is attributed to volatile compounds, mainly organic, such as free fatty acids, alcohols, esters, lactones, aldehydes, ketones, phenols, ethers, sulfur-containing compounds, and terpenoids. These flavor components vary depending on various factors, including feed, seasonal variations, and breeds [17,18]. Therefore, the addition of DHA to dairy cattle feeds alters the composition and content of volatile flavor compounds in milk. Gas chromatography–mass spectrometry (GC–MS) is increasingly used to characterize the flavor of raw milk and dairy products, which facilitates the comparison of volatile substances in regular raw milk and DHA-biofortified raw milk. In this study, we determined the contents and composition of fatty acids and volatile flavor compounds in regular raw milk and DHA-biofortified raw milk. In addition, we determined the fatty acid profile of DHA-biofortified raw milk to establish a scientific basis for the subsequent identification of volatile compounds in DHA-biofortified milk.

2. Materials and Methods

2.1. Milk Sample Collection

Regular raw milk and DHA-biofortified raw milk samples were collected from Holstein cows feeding at Wuchuan pasture, Wuchuan County, Hohhot City, Inner Mongolia. The experiment selected cows with a milk production of 32.4 ± 2.1 kg/d and a body weight of 605 ± 50.9 kg. According to the lactation stage, the cows producing regular milk and DHA-biofortified milk were divided into 3 groups: early lactation (16–100 d), middle lactation (101–200 d), and late lactation (201–300 d). Six cows with close lactation days were selected in each group. All the experimental cows were multiparous cows, and none of the experimental cows developed mastitis, diarrhea, or other clinical signs during the study. The cows were fed a basal diet consisting of 29.29% no-ash neutral washing fiber, 27.01% starch, 18.63% acid detergent fiber, 17.19% crude protein, 5.07% total fat, 3.86% sugar, 0.87% calcium, and 0.47% phosphorus. An amount of 200 g algae powder was added to the feed of cows producing DHA-biofortified milk. The nutritional composition of the algae powder was as follows: the crude protein content was 10.8 g/100 g, the crude fat content was 41.5 g/100 g, the ash content was 14.5%, the water content was 3.76%, and the DHA content was 22 g/100 g. The cows were fed three times a day (7:00, 13:00, and 19:00). They were able to eat freely and drink freely.

The samples of this experiment were collected three times a day (6:00, 12:00, and 18:00). Milk samples of 100 mL were collected in the morning, afternoon, and evening, following a 4:3:3 ratio mix evenly in proportion. After collection, the raw milk samples were stored in a refrigerator maintained at −20 °C in preparation for testing.

2.2. Determination of Fatty Acid Composition and Content

Extraction and derivatization procedure: The raw milk samples were lyophilized to obtain powder, and fat from the milk was extracted according to the standard procedure described in GB 5009.168-2016 [19]. Briefly, 2 mL of toluene was filled with approximately 0.2 g of weighable freeze-dried powder. Afterward, 2.4 mL of an acetyl chloride–methanol (1:9 v/v) solution was added to the samples; then, nitrogen gas was introduced into the test tubes and the mixtures were thoroughly shaken. The mixtures were incubated in a water bath at 80 °C for 2 h. Subsequently, the test tubes were removed from the incubator and allowed to cool to room temperature (25 °C). The reaction solutions were transferred into 15 mL centrifuge tubes and washed two times with 3.6 mL of a sodium carbonate solution. The washing liquid and reaction solution in the centrifuge tubes were thoroughly mixed. The mixture was centrifuged at 4000× g for 5 min, and the supernatant was collected for subsequent analysis.

Gas chromatographic determination procedure: The chromatographic conditions included an injector temperature of 270 °C, a detector temperature of 260 °C, and an SP-2560 capillary column (100 m × 0.25 mm inner diameter, 0.25 μm film thickness; Sigma-Aldrich, St. Louis, MO, USA). The temperature program was as follows: the temperature was initially set at 130 °C for 5 min, then increased at a rate of 4 °C per min until it reached 240 °C and held for 20 min. The injection volume was 1.0 μL, and the carrier gas used was nitrogen with a split ratio of 100:1. The fatty acid methyl ester in the samples was quantified by peak area based on the retention time of the fatty acid methyl ester standard.

2.3. Determination of Volatile Substance Composition and Content

Volatile organic compounds (VOCs) in the raw milk samples were extracted and their contents determined by headspace solid-phase microextraction (HS-SPME) according to the method described by Zhang et al. [20]. Briefly, 5 mL of the raw milk sample, 10 μL of an internal standard (2-methyl-3-heptanone in 50 μg/L methyl alcohol), and 2 g of sodium chloride were transferred into a headspace vial. After equilibration at 60 °C for 15 min, SPME fibers (DVB/CAR/PDMS, 50/30 μm; Supelco, Bellefonte, PA, USA) were inserted in the headspace for 30 min. The SPME fibers were aged at 250 °C for 15 min to remove adsorbed impurities before sample sorption. Finally, the SPME fibers with adsorbed analytes were inserted into the GC port at 250 °C for 5 min to resolve the analytes.

Gas chromatography–mass spectrometry (GC–MS) was performed using an Agilent 7890 GC System (Agilent Technologies, Santa Clara, CA, USA) coupled with an Agilent 7200 Q-TOF (Agilent Technologies, Santa Clara, CA, USA). VOCs were separated on a DB wax capillary column (30 m × 0.25 mm × 0.25 μm; Agilent Technologies, Santa Clara, CA, USA) at a flow rate of 1 mL/min using helium as the carrier gas. The column temperature conditions were as follows: 40 °C for 2 min, increased at a rate of 5 °C per min to 90 °C, and maintained for 10 min; increased at a rate of 5 °C per min to 130 °C and maintained for 3 min; and increased at a rate of 5 °C per min to 230 °C and maintained for 3 min. The inlet and ion source temperatures were 230 °C.

The Unknowns Analysis Tool, MassHunter Quantitative Analysis (B.10.1, Agilent Technologies, Santa Clara, CA, USA), was used for peak selection and deconvolution of the data sets. The mass spectra of VOCs were matched to the reference mass spectra in the NIST17 library, and those with a matching factor above 65 were selected for the subsequent analysis.

2.4. Statistical Analysis

Data were analyzed using IBM SPSS Statistics 27.0 (IBM Corp., Armonk, NY, USA). An analysis of variance was performed to determine significant differences in fatty acid contents between the two types of raw milk. The experiments were repeated more than three times in parallel, and data are presented as means ± standard deviation. Lowercase letters within the same row indicate significant differences (p < 0.05), while uppercase letters indicate highly significant differences (p < 0.01).

3. Results and Discussion

3.1. Fatty Acid Profiles of Regular Raw Milk and DHA-Biofortified Raw Milk

The composition and contents of fatty acids in regular raw milk and DHA-biofortified raw milk from cows at different stages of lactation are presented in

Table 1. Fatty acid profile of regular raw milk and DHA-biofortified raw milk (g/100 g).

	16–100 d		101–200 d		201–305 d
Lactation Stage	Native Raw Milk	DHA-Biofortified Raw Milk	Native Raw Milk	DHA-Biofortified Raw Milk	Native Raw Milk	DHA-Biofortified Raw Milk
C4:0	0.72 ± 0.06	0.739 ± 0.024	0.843 ± 0.069 a	0.74 ± 0.064 b	0.855 ± 0.081	0.775 ± 0.156
C6:0	0.225 ± 0.013	0.236 ± 0.01	0.243 ± 0.01 A	0.264 ± 0.011 B	0.236 ± 0.019	0.24 ± 0.027
C8:0	0.139 ± 0.008 A	0.151 ± 0.005 B	0.148 ± 0.007	0.178 ± 0.007	0.146 ± 0.011	0.162 ± 0.02
C10:0	0.335 ± 0.018	0.367 ± 0.026	0.345 ± 0.019	0.454 ± 0.015	0.342 ± 0.023 a	0.405 ± 0.054 b
C11:0	0.042 ± 0.002 a	0.035 ± 0.005 b	0.042 ± 0.004	0.049 ± 0.003	0.044 ± 0.003	0.048 ± 0.002
C12:0	0.39 ± 0.02	0.419 ± 0.039	0.4 ± 0.023 A	0.53 ± 0.018 B	0.4 ± 0.026 a	0.477 ± 0.064 b
C13:0	0.028 ± 0.001 A	0.023 ± 0.003 B	0.027 ± 0.002	0.03 ± 0.002	0.028 ± 0.001	0.029 ± 0.001
C14:0	1.143 ± 0.062	1.134 ± 0.071	1.217 ± 0.05 A	1.343 ± 0.04 B	1.193 ± 0.07	1.237 ± 0.138
C14:1	0.093 ± 0.005 A	0.074 ± 0.008 B	0.101 ± 0.004 a	0.094 ± 0.004 b	0.104 ± 0.006	0.1 ± 0.007
C15:0	0.15 ± 0.008 A	0.118 ± 0.009 B	0.14 ± 0.013	0.14 ± 0.007	0.146 ± 0.008 a	0.132 ± 0.003 b
C15:1	-	-	-	-	-	-
C16:0	3.869 ± 0.198 A	3.38 ± 0.11 B	4.182 ± 0.166 A	3.761 ± 0.142 B	4.11 ± 0.226 a	3.378 ± 0.517 b
C16:1	0.191 ± 0.01	0.179 ± 0.021	0.211 ± 0.006 A	0.175 ± 0.013 B	0.212 ± 0.018 a	0.184 ± 0.016 b
C17:0	0.069 ± 0.003	0.075 ± 0.005	0.07 ± 0.004 A	0.079 ± 0.004 B	0.072 ± 0.006	0.075 ± 0.002
C17:1	-	-	-	-	-	-
C18:0	1.08 ± 0.075	1.158 ± 0.083	1.275 ± 0.032	1.239 ± 0.05	1.26 ± 0.121	1.132 ± 0.125
C18:1T	0.223 ± 0.023	0.247 ± 0.08	0.214 ± 0.01	0.199 ± 0.043	0.22 ± 0.019 A	0.173 ± 0.012 B
C18:1	2.085 ± 0.116	2.107 ± 0.249	2.497 ± 0.064 A	2.117 ± 0.117 B	2.502 ± 0.267 a	2.03 ± 0.214 b
C18:2TT	-	-	-	-	-	-
C18:2	0.312 ± 0.019	0.311 ± 0.011	0.379 ± 0.01	0.388 ± 0.022	0.367 ± 0.018	0.353 ± 0.038
C20:0	0.013 ± 0.001	0.015 ± 0.001	0.016 ± 0.001	0.018 ± 0.001	0.016 ± 0.002	0.017 ± 0.002
C18:3	0.007 ± 0.001	0.004 ± 0.001	0.008 ± 0.001 A	0.004 ± 0.001 B	0.008 ± 0.001 A	0.004 ± 0.001 B
C20:1	0.004 ± 0.001	0.005 ± 0.001	0.004 ± 0.001	0.004 ± 0.001	0.004 ± 0.001	0.01 ± 0.007
C18:3	0.034 ± 0.002	0.032 ± 0.007	0.038 ± 0.008	0.045 ± 0.003	0.04 ± 0.002	0.04 ± 0.005
C21:0	0.025 ± 0.006	0.019 ± 0.001	0.03 ± 0.001	0.023 ± 0.002	0.031 ± 0.002 A	0.024 ± 0.002 B
C20:2	0.003 ± 0.001	0.005 ± 0.001	0.003 ± 0.001	0.005 ± 0.001	0.003 ± 0.001	0.005 ± 0.001
C22:0	0.007 ± 0.001	0.009 ± 0.001	0.008 ± 0.001	0.012 ± 0.001	0.007 ± 0.001 A	0.013 ± 0.001 B
C20:3	0.016 ± 0.001 A	0.009 ± 0.001 B	0.021 ± 0.001 A	0.01 ± 0.001 B	0.021 ± 0.002 A	0.011 ± 0.001B
C22:1	-	-	-	-	-	-
C20:3	-	0.003 ± 0.001	-	0.004 ± 0.001	-	0.004 ± 0.001
C20:4	0.004 ± 0.001	0.005 ± 0.001	0.004 ± 0.001	0.006 ± 0.001	0.005 ± 0.001	0.006 ± 0.001
C23:0	0.021 ± 0.001	0.017 ± 0.003	0.024 ± 0.001 A	0.017 ± 0.001 B	0.024 ± 0.003 A	0.015 ± 0.001 B
C22:2	0.003 ± 0.001	0.003 ± 0.001	0.003 ± 0.001	0.003 ± 0.001	0.003 ± 0.001	0.003 ± 0.001
C24:0	0.005 ± 0.001	0.005 ± 0.001	0.006 ± 0.001	0.007 ± 0.001	0.006 ± 0.001	0.008 ± 0.001
C20:5	0.004 ± 0.001 A	0.011 ± 0.001 B	0.004 ± 0.001 A	0.017 ± 0.002 B	0.004 ± 0.001 A	0.013 ± 0.003 B
C24:1	-	-	-	-	-	-
C22:6		0.084 ± 0.009		0.106 ± 0.005		0.092 ± 0.008
FA	11.24 ± 0.001 A	10.97 ± 0.010 B	12.503 ± 0.001 A	12.061 ± 0.021 B	12.409 ± 0.001 A	11.195 ± 0.023 B
SFA	8.261 ± 0.003 A	7.9 ± 0.008 B	9.088 ± 0.001 A	8.888 ± 0.018 B	8.84 ± 0.001 A	8.167 ± 0.021 B
UFA	2.979 ± 0.001	3.07 ± 0.001	3.415 ± 0.006 A	3.173 ± 0.005 B	3.569 ± 0.007 A	3.028 ± 0.031 B
MUFA	2.596 ± 0.002	2.612 ± 0.004	3.027 ± 0.007 A	2.585 ± 0.001 B	3.042 ± 0.018 A	2.497 ± 0.034 B
PUFA	0.383 ± 0.001 b	0.458 ± 0.010 a	0.388 ± 0.014 B	0.588 ± 0.003 A	0.527 ± 0.001	0.531 ± 0.006
N-6	0.011 ± 0.001	0.009 ± 0.001	0.012 ± 0.001	0.010 ± 0.001	0.013 ± 0.001	0.010 ± 0.001
N-3	0.038 ± 0.002 B	0.127 ± 0.019 A	0.042 ± 0.007 B	0.168 ± 0.015 A	0.044 ± 0.011 B	0.145 ± 0.007 A

A,B indicate a significant difference (p < 0.05) and a,b indicate a highly significant difference (p < 0.01) between the two types of raw milk. FA, fatty acid; SFA, saturated fatty acid; UFA, unsaturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; N-6, n-6 PUFA; N-3, n-3 PUFA.

and

Table 2. Proportions of fatty acid contents (%) in regular raw milk and DHA-biofortified raw milk.

	16–100 d		101–200 d		201–305 d
Lactation Stage	Native Raw Milk	DHA-Biofortified Raw Milk	Native Raw Milk	DHA-Biofortified Raw Milk	Native Raw Milk	DHA-Biofortified Raw Milk
SFA	73.5	72.01	72.69	73.69	71.24	72.95
UFA	26.5	27.99	27.31	26.31	28.76	27.05
MUFA	23.1	23.81	24.21	21.43	24.51	22.3
PUFA	3.4	4.18	3.1	4.88	4.25	4.75
n6:n3	28.95	7.09	28.57	5.95	29.55	6.9

. The DHA-biofortified raw milk had a higher DHA content than the regular raw milk, which is consistent with previous research findings. The fat content of the milk from cows fed a DHA-enriched diet was considerably lower than that of the milk without supplementation. The possible reason for this is that DHA has the function of lowering serum triglycerides, the mammary glands need to provide fatty acids through the blood to synthesize milk fat, and the reduction in fatty acids in the blood reduces the milk fat content of milk. Another study attributed the decrease in milk fat percentage mainly to the production of the fatty acids trans 10, cis 12-C18:2, and trans 10-C18:1 in the rumen hydrogenation pathway of polyunsaturated fatty acids from algae that have the ability to strongly inhibit milk fat synthesis [21].

However, the content of total PUFAs and n-3 PUFA in DHA-biofortified raw milk was significantly higher than that in regular raw milk across all lactation stages, and the n-6/n-3 fatty acid ratio of the DHA-biofortified raw milk was lower than that of the regular raw milk, which is consistent with the findings of a previous study [8]. PUFAs, particularly the n-6 and n-3 series, constitute crucial nutrients in the diet. As a result of the absence of n-3 desaturase in the body, the conversion of these two series of fatty acids into one another is impossible [22], and they must therefore be sourced from the diet. Balancing the ratio of n-6/n-3 is crucial throughout the life process for maintaining internal environment stability, promoting normal growth and development, preserving health, and preventing and treating chronic diseases.

Among the 30 fatty acids identified in regular raw milk, 17 were saturated and 13 were unsaturated fatty acids. In DHA-biofortified raw milk, 32 fatty acids were identified, with 17 being saturated and 15 being unsaturated fatty acids. Notably, C15:1, C17:1, C18:2, C22:1, and C24:1 were absent in both types of raw milk, while C20:3 and C22:6 were exclusively found in DHA-biofortified raw milk. The physiological function of C20:3 has not been extensively studied, but it is uniquely detected in DHA-biofortified raw milk. This may indicate that C20:3 is a distinctive component of DHA raw milk.

The saturated fatty acid content of the regular raw milk decreased gradually as the days of lactation increased, whereas the unsaturated fatty acid content increased. Specifically, the monounsaturated fatty acid content increased gradually, and the polyunsaturated fatty acid content initially decreased and subsequently increased with the progression of lactation duration. The saturated fatty acid content of the DHA-biofortified raw milk initially increased and then decreased with the progression of lactation duration. However, the unsaturated fatty acid content initially decreased and then increased with the progression of lactation duration. The monounsaturated fatty acid content initially decreased and then increased with the progression of lactation duration, whereas the polyunsaturated fatty acid content initially increased and then decreased with the progression of lactation duration. These changes are mainly due to differences in the energy balance of cows in different lactations and changes in fatty acid metabolic activity in the liver, fatty acid tissues, and mammary glands, resulting in differences in the fatty acid composition of milk [23].

No significant differences were observed in the levels of C18:0, C18:2, C18:3, C20:0, C20:2, C20:4, C22:2, and C24:0 between the two types of raw milk at each lactation stage. The levels of C6:0, C8:0, C10:0, C12:0, C17:0, C22:0, and C20:5 in the DHA-biofortified raw milk were higher than those in the regular raw milk at all lactation stages, whereas the levels of C14:1, C16:1, C21:0, and C23:0 in the DHA-biofortified raw milk were lower than those in the regular raw milk at all lactation stages. Additionally, the levels of C16:0, C18:3, C20:3, and C20:5 in the two types of raw milk at different lactation stages varied considerably.

3.2. Comparison of Volatile Organic Compounds in Regular Raw Milk and DHA-Biofortified Raw Milk

The VOCs identified and their contents in the two types of raw milk are shown in

Table 3. Contents of main volatile compounds in regular raw milk and DHA-biofortified raw milk (μg/L).

	Regular Raw Milk			DHA-Biofortified Raw Milk
	16–100d	101–200d	201–305d	16–100d	101–200d	201–305d
hexanal	1.07 ± 0.08 a	0.35 ± 0.08 c	0.32 ± 0.01 c	0.51 ± 0.03 b	0.19 ± 0.07 d	0.16 ± 0.04 d
acetone	18.99 ± 1.37 c	26.34 ± 2.51 b	16.05 ± 1.33 d	22.22 ± 1.87b c	46.08 ± 4.19 a	17.37 ± 1.89 c,d
2-butanone	6.48 ± 0.28 b	9.1 ± 0.79 a	5.87 ± 0.08 c	5.49 ± 0.35 c	6.66 ± 0.88 b	5.48 ± 1.29 c
1-octene-3-alcohol	0.27 ± 0.02 c	0.36 ± 0.07 b	0.23 ± 0.03 c	0.46 ± 0.09 a	0.53 ± 0.03 a	0.29 ± 0.09 c
methyl heptanoate	0.16 ± 0.01 c	0.17 ± 0.02 c	0.18 ± 0.01 c	0.38 ± 0.06 a	0.41 ± 0.06 a	0.23 ± 0.03 b
methyl caproate	4.97 ± 1.11 d	7.49 ± 0.2 c,d	7.16 ± 1.28 c,d	12.7 ± 1.15 b	21.69 ± 0.93 a	15.92 ± 1.98 b
methyl butyrate	8.17 ± 0.31 c	5.49 ± 1.02 d	9.14 ± 0.75 c	11.58 ± 0.38 b	15.01 ± 3.05 a	9.88 ± 1.59 c
methyl caprylate	2.86 ± 0.58 e	4.54 ± 0.95 c,d	3.75 ± 0.04 d,e	9.62 ± 0.9 b	15.13 ± 1.21 a	5.67 ± 0.35 c
methyl caprate; methyl decanoate	1.41 ± 0.27 c	1.48 ± 0.24 c	1.53 ± 0.28 c	2.46 ± 0.48 b	3.52 ± 0.13 a	2.67 ± 0.24 b
thanolactone	0.04 ± 0.02 c	0.15 ± 0.02 b	0.16 ± 0.02 b	0.38 ± 0.13 a	0.37 ± 0.08 a	0.17 ± 0.08 b
pentate	0.4 ± 0.13 d	0.52 ± 0.05 c	0.56 ± 0.04 c	1.2 ± 0.13 a	0.88 ± 0.05 b	0.55 ± 0.05 c
hexanoic acid	25.41 ± 1.9 d	38.73 ± 1.7 c	42.62 ± 5.62 c	159.34 ± 18.45 a	148.59 ± 34.18 a	97.75 ± 2.64 b
enanthic acid	0.78 ± 0.15 c	1.19 ± 0.05 b	1.16 ± 0.04 b	2.55 ± 0.41 a	2.24 ± 0.31 a	1.13 ± 0.13 b
octoic acid	25.65 ± 1.07 c	29.34 ± 2.37 c	29.7 ± 1.49 c	99.65 ± 4.37 a	89.44 ± 8.55 a	52.76 ± 5.77 b
pelargonic acid	0.26 ± 0.1 d	0.65 ± 0.03 c	1.32 ± 0.09 b	1.83 ± 0.07 a	1.74 ± 0.21 a	1.03 ± 0.4 b
capric acid	5.25 ± 0.26 e	15.83 ± 0.57 d	15.37 ± 3.41 d	45.42 ± 2.24 b	76.24 ± 2.37 a	26.7 ± 2 c
lauric acid	1.15 ± 0.16 c	1.27 ± 0.12 c	1.44 ± 0.19 c	3.07 ± 0.8 b,c	3.51 ± 0.39 a	3.27 ± 0.63 a
dimethyl sulfoxide	0.29 ± 0.03 a	0.23 ± 0.03 a	0.07 ± 0.02 c	0.15 ± 0.03 b	0.07 ± 0.02 c	0.04 ± 0.02 d
dimethyl sulfone	2.55 ± 0.05 b	2.71 ± 0.05 b	1.73 ± 0.09 c	2.64 ± 0.06 b	3.69 ± 0.43 a	1.95 ± 0.05 c

All the data shown in the table are average ± SD. In the same line, values with different superscripts mean significant difference (p ≤ 0.05), while those with the same letter superscripts mean no significant difference (p > 0.05).

. The flavor of raw milk is complex and is determined by a variety of volatile compounds with aroma activity, which predominantly include aldehydes, ketones, esters, acids, and sulfur-containing compounds. The sensory characteristics of raw milk largely depend on the relative balance of flavor compounds from fats, proteins, or carbohydrates [9]. One of the primary mechanisms for generating flavor elements is through the hydrolysis or oxidation of fatty acids in milk fat, thereby making milk fat a key source of raw milk aroma.

Lipid oxidation products, such as aldehydes and ketones, can impart a rancid and “oxidized” flavor to dairy products [24]. The oxidation process initially targets the polyunsaturated phospholipids within the milk lipid globule membrane then progresses to triacylglycerols, where unsaturated fatty acids undergo self-oxidation to form aldehydes. Linoleic and linolenic acids are further oxidized to generate propionaldehyde and hexanal. Hexanal was identified in both types of raw milk, with a higher concentration being observed in the regular raw milk than that in the DHA-biofortified raw milk, which could be due to the lower linolenic acid C18:3 content in DHA-biofortified raw milk than in regular raw milk. Acetone and 2-butanone are typically present in small quantities in raw milk, primarily originating from the metabolic processes of dairy cows. Acetone offers a fruity aroma, while the sensory profile of 2-butanone resembles that of acetone [25].

1-Octene-3-ol is generated through the degradation of unsaturated fatty acids. The concentration of 1-octene-3-ol in the DHA-biofortified raw milk was higher than that in regular raw milk, which is consistent with the results of fatty acid levels. The total content of unsaturated fatty acids in the DHA-biofortified raw milk was higher than that in regular raw milk.

Esters are the principal constituents of neutral volatile compounds found in various types of animal milk. Esters identified in both types of raw milk contribute to the fruity and sweet flavor profile of the milk. The content of esters in the DHA-biofortified raw milk exceeded that in the regular raw milk due to variations in the feeding regimen of the two groups of dairy cattle.

Acids are generated through metabolic processes, such as lipolysis and microbial fermentation. Unsaturated fatty acids undergo oxidation to form aldehydes, which are further oxidized into acids. The concentration of unsaturated fatty acids in DHA-biofortified raw milk has been shown to be higher than that in regular raw milk [26].

Sulfur compounds are unique substances found in raw milk, with dimethyl sulfide, carbon disulfide, dimethyl carbon disulfide, and dimethyl sulfone being the main compounds [27]. Dimethyl sulfone, a major sulfur-containing compound, is recognized as a key odorant in various types of milk, and constitutes 25% of the volatile compounds in cow, goat, and sheep milk [28]. The aroma of dimethyl sulfone is described as resembling that of hot milk, leather, and cow sweat. However, its impact on milk flavor is a subject of discussion due to the lack of sufficient data regarding its content in milk, which varies considerably depending on dietary conditions. Sulfur compounds in milk primarily originate from the diet, although they can also be produced through the binding of cysteine and methionine within proteins, with β-lactoglobulin playing a crucial role in this process [18]. Experimental findings suggest that DHA- biofortified raw milk contains higher levels of dimethyl sulfone than regular raw milk, which is potentially attributed to the influence of supplementing dairy cow diets with DHA on the protein-binding mechanisms in these animals.

4. Conclusions

This study investigated the fatty acid composition and content in regular raw milk and DHA-biofortified raw milk. The results revealed that the total contents of unsaturated fatty acids, PUFA, and DHA were higher in DHA-biofortified raw milk than in regular raw milk. The levels of C16:0, C18:3, C20:3, and C20:5 between the two types of raw milk were significantly different at each lactation stage. A comparison of the volatile compound composition between the two types of raw milk revealed differences that were attributed to the altered fatty acid profiles of raw milk. This study provided a preliminary comparison of the fatty acid composition and volatiles of DHA-biofortified raw milk and regular raw milk, laying the groundwork for the rapid identification and quality control of DHA-biofortified cow’s milk in production. However, this study was limited to milk samples from different lactation periods, and the sample size was restricted. Future research should involve a broader comparison of milk samples from various breeds and regions to further refine the establishment of fatty acid profiles for DHA-biofortified raw milk.