Fatty acid composition, vitamin A content and oxidative stability of milk in China

ABSTRACT The objective of the present study was to investigate the influence of geographical region and lactation stage on the fatty acid composition, vitamin A content and oxidative stability of milk from Holstein cows and to determine the relationship between milk fat components and milk stability. Fatty acids composition (C10:0, C11:0, C12:0, C13:0, C14:1, C18:0, C18:2 cis-9, trans-11, γ-C18:3, α-C18:3, C20:0, C20:1 and C22:0), vitamin A and oxidative stability were differed between region, but were not affected by stage of lactation. Differences in milk composition could be related to region differences because the use of grass-based forage and altitude condition in four regions. The oxidation stability of milk was closely related to monounsaturated fatty acids (MUFA) and all-trans-retinol. MUFA were negatively correlated with superoxide dismutase (r = −0.207). All-trans-retinol was positively correlated with total antioxidant capacity value (0.208), and negatively correlated with thiobarbituric acid reactive substances (r = −0.265). These findings may be used to assess and improve milk quality of china.


Introduction
Fat is an important component of milk and plays a vital role in human health (Thorsdottir et al. 2004). Milk fat from dairy cows includes 70% saturated fatty acids (SFA), 25% monounsaturated fatty acids (MUFA) and 5% polyunsaturated fatty acids (PUFA), on average (Grummer 1991). The nutritional value of milk can be enhanced by modulating the composition of fatty acids, for example, by reducing the proportion of SFA and increasing the proportion of unsaturated fatty acids (UFA) (Simopoulos 2008). Kromhout et al. (2002) found that the MUFA and PUFA can be especially effective in the prevention and treatment of atherosclerosis and cardiovascular disease. However, raising the UFA content of milk fat may reduce the oxidative stability of milk and increasing the risk of developing spontaneous oxidized flavour (SOF).
Oxidative processes in milk fat, resulting in SOF, reduces the quality of milk and shortens storage life (Juhlin et al. 2010;Rafalowski et al. 2014). Therefore, preventing oxidation of milk is a major challenge confronting the dairy industry. Total antioxidant capacity (T-AOC) can reflect the total antioxidant capacity, which may provide more biologically relevant information than that from measured concentrations of individual antioxidants (Jin et al. 2011). Superoxide dismutase (SOD) is the most important antioxidant enzymes that act as the original protection system against free radicals (Michiels et al. 1994). Assay of Thiobarbituric acid reactive substances (TBARS) measures malondialdehyde (MDA) present in the sample, as well as MDA generated from lipid hydroperoxides by the hydrolytic conditions of the reaction (Trevisan et al. 2001).
Many factors have been found to affect oxidative stability, including the degree of unsaturation and the content of antioxidants such as fat-soluble vitamins and antioxidant enzymes (Havemose et al. 2004). Timmons et al. (2001) found that feeding cows with whole roasted soybeans increased the content of C18:2 and C18:3 in milk fat, which in turn increased the susceptibility to oxidation. Liu et al. (2010) found that infusion with high-linolenic perilla fatty acid emulsion enhanced the content of n-3 PUFAs in milk fat but decreased the oxidative stability of milk fat. The position of double bonds in UFA can affect the oxidative stability of milk. The UFA at the sn-2 position are less susceptible to oxidation than those at positions sn-1 and sn-3 (Rafalowski et al. 2014). Vitamin A is the most abundant of the fat-soluble vitamins (Haug et al. 2007), and occurs mainly as a mixture of retinol, retinal, retinoic acid, retinyl esters and provitamin β-carotenoids. Milk and dairy products provide a significant proportion of the vitamin A consumed (about 15-20%), especially for infants and children (Herrero-Barbudo et al. 2005). Some studies have shown that vitamin A can enhance antioxidation defense systems against oxidative stress (Kleczkowski et al. 2004;Ma et al. 2005).
The composition of milk fat is affected by many exogenous and endogenous factors, including region (Lindmark-Mansson et al. 2003) and lactation stage (Kelsey et al. 2003). Larsen et al. (2010) investigated the influence of regional climatic conditions on milk composition, especially fatty acid composition and content of fat-soluble antioxidants. This study indicates that content of carotenoids, tocopherol, short-chain fatty acid (C4-C14), C18:0 and C 18:3 n-3 are higher in central Sweden than in southern Sweden and that most likely because maize growing is limited to Southern Sweden.
China is the third largest producer of milk in the world. Assessing the composition of milk fat is important for Chinese milk production. The present study aimed to determine the effects of region and lactation period on fatty acid composition, vitamin A content and oxidative stability of milk from the major cow milk-producing regions in China. This information will also provide an indication of the associated factors influencing fatty acid composition, vitamin A and oxidative stability of milk, and we aimed to study the relationships between stability and the composition of fatty acids and vitamin A content.

Milk collection
Milk samples were collected from eight commercial large-scale herd cattle (>300 Holstein cows) dairy farms. These farms were located in four different geographical regions in China (Table 1). The dairy farms were visited in June and July 2014. Farm management and production data were collected by interview and questionnaire. The information of feeding and milk yield is shown in Table S1 as 'Supporting information'. A total of 120 milk samples were used in the present study (Each farm collected 15 milk samples). The milk samples from the four regions were collected at three different lactation periods: early (30-90 d), mid (120-180 d) and late (210-270 d). The mean age at first calving for eight farms was 23-24 months.
Each milk sample was obtained from individual cow throughout the day, including the morning and evening milking. The samples were then stored in a freezer at −20°C for further analysis.

Analysis of fatty acid composition
Milk samples (2 mL) were mixed with 4 mL n-hexane/isopropanol (3:2, v/v) and 2 mL 0.47 M Na 2 SO 4 and slowly agitated for 5 min before centrifugation at 5300g for 10 min. The supernatant and 200 μL of heptadecanoic acid (C17:0) as the internal standard were transmethylated with 2 mL NaOH dissolved in methanol at 50°C for 15 min. The samples were then cooled, 2 mL acetylchloride/methanol (1:10, v/v) were added at 80°C for 30 min. and then 6 mL n-hexane and 3 mL water were added to the mixture. After centrifugation, the upper phase (n-hexane) was separated and analysed for fatty acid methyl esters as previously described (Zhao et al. 2013).
Fatty acid methyl esters were analysed by standard gas chromatography (Model 7890A, Agilent Technologies, Palo Alto, CA, USA) using a SP-2560 column (100 m × 0.25 mm ID; Supelco Inc., Bellefonte, PA, USA) with a flame ionization detector. The initial column oven temperature was 100°C, then raised at 5°C min −1 to 210°C, and held at that temperature for 25 min. The temperature was then increased at 4°C min −1 to 230°C and held there for 2 min. The injector and detector temperatures were maintained at 260°C.

Analysis of vitamin A
Vitamin A, all-trans-retinol contents of milk samples were determined using the procedure described by Chotyakul et al. (2014). 0.25 g ascorbic acid, 50 mL methanol and 5 mL 50% KOH were added to a 5 mL milk sample, then held under bubbling nitrogen for 1 min. After stirring in a water bath set at 80°C for 45 min, then 40°C for 15 min, 50 mL n-hexane and 50 mL water was added. The solution was centrifuged for 5 min at 5000g, the supernatant was filtered, then concentrated to dryness under nitrogen gas at 40°C. The extract was reconstituted in 10 mL acetonitrile/methanol (65:35, v/v) and filtered, and aliquots of 100 μL were injected into an HPLC system (HP1100, Agilent Technologies, Palo Alto, CA, USA) using a diode array detector at 325 nm. The conditions were mobile phase, 65% acetonitrile/35% methanol, flow rate 1 mL min −1 , column: Eclipse Plus C18 (Agilent Technologies, Palo Alto, CA, USA). Samples were quantified using external standard curves.

T-AOC, SOD and TBARS assays
T-AOC, SOD and TBARS values of the milk samples were determined as described by Zhao et al. (2013). The milk samples were centrifuged for 30 min at 12000g, then 4% acetic acid was added to the supernatant. This was collected for analysis of the enzyme activity. The kits for determining T-AOC and SOD activity in whey were bought from Nanjing Jiancheng Bioengineering Institute, China. The TBARS level in milk was measured by the QuantiChromTM TBARS Assay kit (DTBA-100) (Bioassay systems, Hayward, CA, USA).

Statistical analysis
The data were analysed using Two-way analysis of variance using Statistical Package for the Social Sciences v. 13.0 (SPSS, Chicago, IL, USA) followed by Duncan's test. Milk samples from cows were defined as a random effect, while region and lactation stages and their interactions were defined as a fixed effect. P value of <.05 was regarded as significant. Correlation analysis was evaluated by Pearson correlation coefficients. 3. Results and discussion

Fatty acid composition
The fatty acid composition of the milk samples is shown in Table 2. Of the 25 types of fatty acid in milk, 12 (C10:0, C11:0, C12:0, C13:0, C14:1, C18:0, CLA, γ-C18:3, α-C18:3, C20:0, C20:1 and C22:0) showed significant differences in content among the four regions. The content of C12:0, C14:0 and C20:2 fatty acids in milk from Hohhot was higher than in milk from other regions. The content of C16:1, C18:0, C18:2, γ-C18:3 and C22:1 was highest in milk from Yinchuan. The Xi'an samples had the highest contents of trans-9 C18:1. The cows' diets differed between farms (Table S1), which can have a great influence on the fatty acid composition of milk ).The content of CLA was higher in milk from Cangzhou city (0.76-0.85 g/ 100 g milk fatty acid) compared with milk from other regions. This was likely related to the use of alfalfa hay rather than maize silage in Cangzhou city. CLA was related to the use of grass-based forage rather than maize silage (Larsen et al. 2010). Environmental conditions such as altitude can also affect fatty acid composition of milk. In the present study, milk from Yinchuan had a higher content of PUFA than other regions ( Table 2). The characteristic fatty acids profile of milk has even been suggested as a biomarker for authentication of its alpine origin (Engel et al. 2007).The differences in composition of fatty acids of milk from highlands and lowlands are likely to be due to different plant species with a different fat composition and/or to the activity of the desaturases of the intestine and of the mammary gland of the cow (Collomb et al. 2002). Our result differs from a previous study which had demonstrated that the stage of lactation had a significant effect on the content of medium-chain fatty acids (C10:1, C12:0, C12:1, C14:1 and C16:0) and long-chain fatty acids (C18:1 and CLA) in Canadian Jersey cows (Kgwatalala et al. 2009). This difference may be attributed to the differences in cattle breeds. There were significant differences of SFA proportion among the four regions studied (P < .05), the content increased during the mid stages of lactation ( Table 2). The SFA proportion of milk from Yinchuan was higher than in milk from other regions. Yang et al. (2013) observed that SFA content was significantly different across geographical regions. The effect of lactation stage on the SFA proportion of milk was also observed by Kgwatalala et al. (2009), who found that the SFA proportion increased from 69.32 g/100 g fatty acids in early lactation to 71.54 g/100 g fatty acids in mid lactation, then decreased to 69.76 g/100 g fatty acids in late lactation. We observed that MUFA and PUFA were significantly affected by region, but not affected by lactation stage. MUFA levels were highest in milk from Cangzhou, while PUFA levels were highest in milk from Yinchuan. Some researchers also found that the proportions of MUFA and PUFA were affected by region (

Vitamin A
Vitamin A is stored in fat globules with its activity being defined in retinol equivalents. In the present study, the all-trans-retinol content did not differ during lactation (Figure 1), but was significantly different among the four regions ( Figure 2). The all-transretinol content was 2.01, 2.28, 2.65 and 1.24 μg/g fresh milk for samples from Cangzhou, Yinchuan, Xi'an and Hohhot, respectively. This result is consistent with the study of Thompson et al. (1964) who reported a wide range of vitamin A content of milk from different geographical locations. This may have been caused by differences in forage type (Noziere et al. 2006).

Oxidative stability of milk fat
The present study found that T-AOC, SOD activity and TBARS values were significantly different among the four regions, but were not affected by the lactation stage (Table 3). The milk samples from Cangzhou were characterized by higher T-AOC activities (6.05-6.46 U/mL fresh milk) than samples from the other three regions. SOD activity was higher in the Yinchuan and Hohhot milk samples than those from Cangzhou and Xi'an   city. Our research has also found that the TBARS values (19.71-21.17 μM MDA equivalents) from the Hohhot samples were higher than the samples from other regions. These could to some extent be attributable to differences in feed composition such as higher shares of Leymus chinenisis in Hohhot. Feeding L. chinenisis could increase C18:1 cis-11 and C18:2 content in milk (Yan et al. 2011). Timmons et al. (2001 demonstrated that cows feeding roasted soybeans could increase concentrations of C18:2 and C18:3 in milk and appearance of SOF in milk. Zhao et al. (2013) reported cows supplement lipids containing longchain fatty acids could increase MDA content in milk. The oxidative stability of milk was closely related to its UFA composition. Significantly, a negative linear correlation was found in milk between SOD activity and MUFA (r = −0.207) ( Table 4). Milk fatty acid profile was important for its oxidation stability. Previous studies have shown that increasing contents of UFA (especially C18:2 and C18:3) in milk will increase the susceptibility of milk to oxidation (Kristensen et al. 2004, Timmons et al. 2001. Liu et al. (2010) found that the relevant antioxidant enzyme activity tended to decrease when PUFA increased in milk fat and the MDA content increased in milk. The all-transretinol content and stability of milk are strongly correlated (Rafalowski et al. 2014). The correlation coefficient between all-transretinol content and T-AOC value in the present study was 0.208 but was negative (r = −0.265) between all-trans-retinol content and TBARS values. Rafalowski et al. (2014) reported that vitamin A and oxidative stability were positively correlated (r = 0.591). Many factors can affect the oxidative stability of milk: PUFA content, antioxidants and the presence of metal ions. Therefore, further investigations should be conducted to explain these data.

Conclusion
This study has demonstrated that geographical location and lactation stage affect the fatty acid composition, vitamin A content and oxidative stability of milk. Some fatty acids such as CLA and C18:3, UFA (including MUFA and PUFA) and vitamin A were found to be affected by region. In contrast, the effect of lactation on fatty acids was minor. The differences in the cow's diet and altitude will influence the milk composition. In addition, the cow's diets also affected the oxidative stability of milk fat. The oxidative stability of milk was closely related to MUFA and vitamin A. These findings may be used to assess and improve milk quality of China.

Disclosure statement
No potential conflict of interest was reported by the authors. Table 3. Distribution of total antioxidant capacity (T-AOC), superoxide diamutase (SOD) and thiobarbituric acid reactive substances (TBARS) values from milk samples from four regions at three stages of lactation (n = 40 for regions and 30 for lactation stages). Notes: Means within a row with different superscript capital letters are significantly different between regions (P < .05), and with lowercase letters are significantly different between lactation stages (P < .05). T-AOC: total antioxidant capacity; SOD: superoxide diamutase; TBARS: thiobarbituric acid reactive substances. Notes: MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; T-AOC: total antioxidant capacity; SOD: superoxide diamutase; TBARS: thiobarbituric acid reactive substances. *Correlation coefficients significant at a level of α = .05. **Correlation coefficients significant at a level of α = .01.