1. Introduction
Recently, due to the high-quality biomass property of microalgae, it is widely used in bio-energy, pharmaceuticals, food, feed industries, etc. Among all microalgae, Schizochytrium microalgae is rich in nutrients, such as protein, fat, sugar, and other nutrients. Moreover, the lipid content of Schizochytrium microalgae is higher than 70% in comparison with other microalgae. Triglyceride is the main form of Schizochytrium microalgae oil, which took up about 90% of the total lipids [
1]. In addition, the fatty acid (FA) content of Schizochytrium microalgae accounted for 18.3~25% of its dry matter weight. The FA profile of Schizophytrium microalgae is abundant, which is rich in unsaturated fatty acids (UFA) and mainly composed of DHA, docosapentaenoic acid (DPA), and palmitic acid (C16:0) [
2,
3]. A previous study reported that the DHA content of total fatty acids is 35% [
4].
DHA plays a vital role in the brain health, growth, and development of infants [
5]. Jensen (2005) reported the addition of DHA to a pregnant women’s diet could improve the cognitive ability of infants [
6]. Moreover, metabolic abnormalities controlled by the fetal central nervous system were caused by the severe deficiency of DHA for the fetus [
7]. Birch, E.E. (1998) reported that the visual acuity of infants was improved significantly by continuous feeding of DHA-rich foods [
8]. However, the desaturase activity of infants was extremely low and could not satisfy the requirements of DHA synthesis [
9]. Therefore, more and more DHA-enhanced infant formula milk powders, which were directly added with microalgae or fish oil, were produced in China, Japan, and the United States to satisfy the daily nutrient requirement of infants [
7,
10].
However, ruminant milk directly enriched with algal oil may result in a fishy flavor, leading to the unacceptance of consumers. Thus, microalgae had been registered as animal feed additives to increase the content of omega 3 fatty acids in ruminant milk [
11]. Microalgae had been widely studied to increase the UFAs content of cow’s milk in the last two decades. Franklin (1999), Vahmani (2013), and Liu (2020) reported that the content of DHA in cow’s milk increased significantly after dietary microalgae, with the increasing ratio of DHA going from 100% to 760% [
12,
13,
14]. In addition, the increasing trend of DHA content was observed in dairy goat’s milk [
15,
16]. Undoubtedly, ruminant milk fat exists as the triglyceride (TAG) form, which is constituted of three fatty acids and one glycerin [
17]. When the dietary fat enters the rumen, the fatty acid of TAG is hydrolyzed by lipase, and then the free fatty acid was mainly hydrogenated and produced by the microorganism [
18].
The use of Schizochytrium microalgae in dairy goat fodder has rarely been reported. Moreover, the content of DHA in Schizachyrium microalgae is higher compared with other DHA-rich sources, and the direct feeding with Schizachyrium microalgae can reduce production costs in comparison to the addition of microalgae or fish oil in goat’s fodder. Hence, the milk composition, milk fatty acids, and milk sn-2 fatty acids of goat’s milk were detected after the supplement of Schizochytrium microalgae into Shanxi Guanzhong goats’ daily diet in this study, and the results provide theoretical support for producing high-DHA goat’s milk.
2. Materials and Methods
2.1. Materials
The Schizochytrium microalgae powder was provided by Xiamen Huison Biotech Co. Ltd. The tridecylic acid triglyceride (C13:0 TAG) and porcine pancreatic lipase (type II) were purchased from Beijing Manhage Biotechnology company and Sigma Aldrich (St. Louis, MO, USA), respectively. Trichloromethane, diethyl ether, petroleum ether, Sodium cholate hydrate (65%), and potassium hydroxide were bought from Sino Pharm Chemical Reagent (Shanghai, China). Methanol (>99.9% purity) and n-Hexane (≥98% purity, chromatographic grade) were obtained from Aladdin (Beijing, China). Moreover, 37 fatty acid methyl esters (FAMEs) were purchased from ANPEL Laboratory Technologies Inc. (Shanghai, China). All other solvents and reagents were analytical grade (Sino Pharm Chemical Reagent, Shanghai, China).
2.2. Sample Collection and the Detection of Milk Composition
All animal procedures in this experiment were approved by the Animal Care and Use Committee of the Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences (IFST-2019-105), and conducted in keeping with animal welfare and ethics. Specifically, 120 Guanzhong dairy goats, who had been pregnant twice and were in middle lactation stage, were randomly divided into four groups, including the control (C, 0 g/day), low microalgae supplementation (LM, 15 g/day), medium microalgae supplementation (MM, 25 g/day), and high microalgae supplementation (HM, 35 g/day) groups. To ensure the microalgae was adequately eaten, the microalgae powder was mixed thoroughly by a machine. In addition, dairy goats were reared in a specific barn and allowed to freely drink water, and they were milked once a day at three o’clock in the morning. The experiment period lasted 65 days, containing a 15-day adaptation period. Diet formula and fatty acids of microalgae powder are shown in
Table 1 and
Table 2, respectively.
The goat’s milk was obtained at the end of the experiment. Afterward, these milk samples were cooled to 4 °C and transported from Xi’an to Beijing Lab on the same day, and then separated into two parts. One part was stored in the refrigerator at −80 °C for fatty acids and sn-2 fatty acids analysis. The other part was added to a tube and detected by a milk component detector for milk composition at Beijing Dairy Cow Testing Center.
2.3. Determination of Fatty Acids Profiles
The detection method for fatty acid (FA) was referred to in a previous study and modified slightly [
19]. To be specific, 250 μL goat milk, 300 μL internal standard solution (C13:0 TAG, 3,1 mg/mL), 2 mL methanol, 2 mL HCL/methanol (3N), and 1 mL
n-hexane were mixed and vortexed in screw glass test tube. In the next step, the tube was tightly capped and then heated for 1 h at 100 °C in the water bath. After that, the tube was cooled to room temperature, 2 mL water was pipetted into it, and it was vortexed for 30 s. Finally, this tube was centrifuged at 1200×
g for 5 min, and the upper phase (
n-hexane) was transferred and filtered through a 0.22-µm filter into the vials for the gas chromatography (GC) test.
The FAMEs sample was analyzed by GC with a hydrogen flame ionization detector (Agilent 8890 B) and a capillary column (DB-23 60 m × 0.25 mm × 0.25 μm; Sigma-Aldrich). Both the injector and detector temperatures were 250 °C. Nitrogen was used as the carrier gas with a flow rate of 0.8 mL/min and the split ratio was 1:20. The program of the column oven temperature was as follows: the initial temperature was 50 °C and kept for 1 min, and then the column oven temperature increased to 175 °C with a rate of 20 °C/min. Afterward, the column oven was heated to 230 °C at a rate of 1.3 °C/min and maintained for 5 min. The FAMEs were identified by the retention times comparison between the sample peaks and those of known FAME standards.
Quantification of DHA was performed using the following formulas and data are expressed in mg of fatty acid per 100 g of goat milk:
where X
DHA: quantity of DHA expressed as mg per 100 g goat milk; FDHA: response factor; A
DHA: peak area of DHA in the sample chromatogram; A
C13: peak area of C13:0 internal standard in the sample chromatogram; M
C13: mass of C13:0 internal standard added to the sample solution, in mg; 1.0059: conversion coefficient of triglyceride to methyl of C13:0; M: mass of test sample, in mg; 0.9590: the coefficient of DHA fatty acid methyl ester converted into fatty acid; 1000: the conversion coefficient between grams and milligrams; ρ
FAMEs-DHA: the concentration of DHA in the standard mixture of 37 FAMEs; A
FAMEs-C13: peak area of C13:0 in the 37 FAMEs; A
FAMEs-DHA: peak area of DHA in the 37 FAMEs; ρ
FAMEs-C13: the concentration of C13:0 in the standard mixture of 37 FAMEs.
2.4. Detection for Sn-2 Fatty Acids
The milk fat was extracted following a modified version of the Folch method [
20]. Briefly, 3 mL goat milk, 6 mL methanol, 12 mL trichloromethane, and 6 mL water were added to the tube, and then the mixed solution was vortexed thoroughly and centrifuged at 3500 rpm for 15 min. In the next step, the bottom solution was extracted and dried by nitrogen gas.
The detection method for sn-2 fatty acids included the preparation of sn-2 MAG and methylation, and it was referred to the previous study reported by Qi (2018) and Sahin (2005) [
21,
22]. Specifically, 200 μL
n-hexane, 2 mL TRIS buffer (pH 8.0), 0.5 mL bile salts (0.05%), 2 mL calcium chloride (2.2%), and 20 mg pancreatic lipase (porcine pancreatic lipase type II) were pipetted into the tube which contained 25 mg milk fat. The mixture was hydrolyzed by shaking in a water bath (37 °C) for 40 min. After the hydrolysis, 1 mL HCl solution (6 M) and diethyl ether were added in sequence into the mixture solution. In the next step, the mixing solution was vortexed for 2 min and then centrifuged subsequently at 4000 rpm for 5 min. After that, the supernatant was transferred into a new tube and separated on a silica gel plate with a developing solvent that comprised
n-hexane, diethyl ether, and acetic acid (50:50:1,
v/
v/
v). Finally, the band corresponding to sn-2 MAG was isolated and extracted twice with 5 mL diethyl ether. The diethyl ether blended with sn-2 MAG was removed by nitrogen gas, and then it was methylated and detected by the GC method mentioned in 2.3 of this study.
2.5. Statistical Analysis
The percentage of fatty acid and sn-2 fatty acid is expressed as mean and pooled standard errors (SEM), and they were subjected to one-way analysis of variance using SPSS software (Version 24, SPSS, Chicago, IL, USA). The normality and homogeneity of variance were assessed by Shapiro–Wilk and Levene’s test, respectively. Bonferroni and Dunnett’s T posthoc tests were used to identify the difference when variance did or did not satisfy the condition of homogeneity.
5. Conclusions
In conclusion, the content of goat milk protein, SCC, production, and dry matter intake was not affected by the dietary Schizochytrium microalgae. Moreover, the milk fat content increased in LM goat’s milk compared with the control goat’s milk, and the lactose content showed a dropping trend in MM goat’s milk. The absolute concentration of DHA increased significantly in the experimental goat’s milk, with 29.485, 32.351, and 24.817 mg/100 g raw milk in the LM, MM, and HM goat milk, respectively. In addition, the sn-2 DHA content rose in MM and HM goat’s milk in comparison with the control goat’s milk. However, the proportion of sn-2 DHA decreased in LM goat’s milk. In addition, the content of major UFAs showed an increasing variation, except for the proportion of C18:1n9c which decreased in the test goat’s milk. The fatty acid profiles of goat ‘s milk and Schizochytrium microalgae was compared, and it was worth noting that C22:0 and C24:1 were only detected in the test goat’s milk. Therefore, the profile and content of the fatty acid and sn-2 fatty acid of goat’s milk were significantly affected by dietary Schizochytrium microalgae, especially in DHA. This result would promote the production of high DHA goat milk in practical production.