Effect of lactation on the distribution of mineral elements in goat milk

The distribution of mineral elements in milk is crucial for their absorption and utilization, however, there has been limited attention given to the status of mineral elements in goat milk. In this study, goat milk was collected at 4 lactation periods (1–3,90,150,240d) and separated into 4 fractions (fat, casein, whey, and aqueous phase). The concentrations of Mg, Ca, Na, K, Zn, Fe, Cu, Mn, Co, Ni, Mo, and Cr in 4 fractions were analyzed using an inductively coupled plasma emission spectrometer (ICP-OES). Our findings reveal that Ca, Zn, Fe, Cu, Mn, and Cr exhibit the highest levels in casein, while Mo demonstrates the highest content in whey. Additionally, Mg, Na, K, and Ni display the highest concentrations in the aqueous phase. Specifically, the contents of Ca, Cu and Fe in casein decrease from 1 to 3 d to 150 d of lactation but increase from 150 d to 240 d of lactation. Furthermore, the content of Mg in the aqueous phase decreases from 1 to 3 d to 90 d of lactation but increases from 90 d to 240 d of lactation. The content of Na and K in the aqueous phase decreases from 1 to 3 d to 150 d of lactation. Notably, the content of Mo in whey increases from 1 to 3 d to 150 d of lactation and decreases from 150 d to 240 d. Our research contributes to the advancement of understanding the bioavailability of mineral elements in goat milk.


INTRODUCTION
Goat milk is replete with lipids, proteins, as well as abundant calcium, phosphorus, magnesium, copper, and other essential elements, making it a valuable natural nutritional supplement (Chen et al., 2023).Globally, there are approximately 1.003 billion goats, of which 203 million are dairy goats, contributing to an annual milk production of 15.26 million tons (Nayik et al., 2022).The growing popularity of goat milk can be attributed to its burgeoning recognition for its diverse biologically active properties.
Mineral elements present in goat milk are essential for human health and development as they play a key role in meeting the nutritional and developmental needs of the body, especially in children (Górska-Warsewicz et al., 2019).These elements include both macro elements such as K, Na, Ca, P, and Mg, as well as minor elements such as Zn, Fe, Cu, Mn, Mo, and B, and trace elements such as Co and Cr (Liu et al., 2019, Astolfi et al., 2020).The presence of these mineral elements in milk influences various milk properties.For instance, mineral elements can impact the stability of milk by enhancing the electrostatic stability of casein micelles.This effect is achieved through the combination of colloidal calcium phosphate clusters and soluble Ca (Sandra et al., 2012).
The mineral elements in goat milk play a crucial role in maintaining milk ion balance, stabilizing its state, and influencing the functional properties of milk proteins (Zamberlin et al., 2012, Siqueiros-Cendón et al., 2014).It is crucial to investigate the presence and distribution of mineral elements in milk to ensure their ability to fulfill their role in promoting normal physiological functions, development, and immunity (Yusha'u et al., 2018).Numerous studies have investigated the mineral elements present in milk from various species.Denholm et al. (2019) utilized inductively coupled plasma mass spectrometry (ICP-MS) to analyze 11 elements in cow

Effect of lactation on the distribution of mineral elements in goat milk
Junyu Pan,12* Zhongna Yu,3* Hongning Jiang, 1 Cuiping Shi, 1 Qijing Du, 1 Rongbo Fan, 1 Jun Wang, 1 Latiful Bari, 5 Yongxin Yang,14 and Rongwei Han14 † milk, including Ca, Mg, K, P, and Zn.Liu et al. (2019) determined the concentrations of 11 elements in goat milk, including Fe, Cu, and Zn.Chen et al. (2020) compared the concentrations of 17 elements in goat, cow, buffalo, yak, and camel milk.Astolfi et al. (2020) compared the concentrations of 41 elements, including K, Ca, Na, and Mg in goat milk, donkey milk, and cow milk using ICP-MS.Additionally, prior research has delved into the elemental distribution within milk.Fransson and Lönnerdal (1983a) explored element distribution in human and cow milk, revealing variances in elemental distribution across fat, pellets, particulate matter, whey protein, and low molecular weight (LMW, < 10 kDa) fractions.Yaşar et al. (2013) observed disparities in mineral element distribution between the protein and serum components of human and cow milk.Fantuz et al. (2020) documented significant differences in mineral element content among the fat, casein, whey protein, and aqueous phases.These discrepancies in mineral element distribution across previous studies may correlate with alterations in bioavailability, thereby influencing mineral absorption dynamics (Kibangou et al., 2005, Liu et al., 2022).Moreover, Malacarne et al. (2015) noted substantial changes in colloidal-phase elements in red deer and roe deer milk during the 1-3 mo of lactation.Subsequent investigations have also revealed significant alterations in the mineral profile of donkey milk throughout the lactation period, spanning from 2 to 9 mo (Malacarne et al., 2017).Studies have examined mineral element distribution in human milk, cow milk, and donkey milk, but the distribution in goat milk remains uncertain.
This study focuses on goat milk due to observed species-specific differences in mineral element distribution.Goat milk was collected at 4 lactation stages (Sun et al., 2023a, Sun et al., 2023b), and then separated into 4 fractions, namely fat, casein, whey protein, and aqueous phase.The distribution of 11 elements at different lactation stages was analyzed by inductively coupled plasma optical emission spectrometry (ICP-OES).The results of our study will facilitate research on the bioavailability of elements in milk and the technical characteristics of milk.Furthermore, the findings will provide guidance for the production, processing, and consumption of goat milk.

Animals, Diet, and Sampling
The sampling procedure in this study followed the methodology described by Sun et al. (2023a).Specifically, milk samples were collected from 15 healthy Laoshan goats with the same parity and lactation period.The feed composition used in the study was consistent with that used by Sun et al. (2023a).Samples were collected at 4 different lactation stages, namely 1-3, 90, 150, and 240 d of lactation.A total of 60 raw milk samples were collected in 50 mL sterile sampling bottles and stored at 4°C.The samples were transported to the laboratory under refrigeration within 24 h and subsequently stored at −80°C until analysis.

Sample Preparation
In the current study, whole goat milk was separated into 4 fractions including fat, casein, whey, and aqueous fractions.To obtain the fat fraction, 15 mL of milk samples were centrifuged at 4,000 × g for 30 min at 4°C using a Heal Force Neofuge 18R centrifuge (Heal Force Shanghai, China), and the upper-fat layer was separated.Then the skim milk was subsequently subjected to ultracentrifugation (BC Optima L-80XP Ultracentrifuge, Beckman Coulter, Brea, CA) at 60,000 × g, 2h at 4°C to obtain the lower fraction of casein.After collecting the lower casein precipitate, 4 volumes of acetone at −20°C (GR, Sinopharm Chemical Reagent Co., Ltd.) were then added to the supernatant whey fraction and centrifuged at 9,400 g for 10 min at 4°C (CR22N Refrigerated Centrifuges, HITACHI, Hitachinaka, JP).The resulting samples were divided into 2 components: the lower layer contained the whey protein component, while the upper layer was collected as the aqueous fraction.Finally, the fat fraction, casein fraction, whey protein fraction, aqueous fraction, and raw milk samples were lyophilized and weighed using an Alpha 2-4 LSC plus Laboratory freeze-dryer (Martin Christ, Osterode, Germany).
Accurately weighing 2 g of raw goat milk sample, lyophilized fat fraction, casein fraction, whey protein fraction, and aqueous phase (with an accuracy of 0.001 g) into a conical flask, 10 mL of nitric acid (GR, Sinopharm Chemical Reagent Co., Ltd.) and 2 mL of perchloric acid (GR, Sinopharm Chemical Reagent Co., Ltd.) were sequentially added.Samples were digested on a temperature-controlled hot plate.After digestion at 120°C until the digestion solution was stable and transparent, the temperature was raised to 150°C for 2-3 h, and then to 180°C to observe the color of the digestion solution.If the digestive solution was brown, a small amount of nitric acid was added to the Erlenmeyer flask to continue digestion until the digestive solution became transparent or slightly yellow.After cooling the digestive solution, it was diluted to 50 mL with deionized water, shaken well, and set aside.At the same time, a blank digestion solution was prepared.

Preparation of standard solutions
The standard solutions of K, Ca, Na, and Mg, each with an initial concentration of 1000 μg/mL, underwent serial dilution using ultrapure water (Millipore Corp., Billerica, MA).Specifically, the K standard solution was diluted to concentrations of 20 mg/L, 50 mg/L, 100 mg/L, 200 mg/L, and 400 mg/L.Similarly, the Ca standard solution was prepared at concentrations of 25 mg/L, 50 mg/L, 100 mg/L, 250 mg/L, and 500 mg/L.The Na and Mg standard solutions were diluted to concentrations of 5 mg/L, 10 mg/L, 20 mg/L, 50 mg/L, and 100 mg/L.The quantification of K, Ca, Na, and Mg was performed using a standard curve, and the concentration of each element was expressed in μg/g.
For Zn, Fe, Cu, Mn, Co, Mo, Ni, and Cr, standard solutions with an initial concentration of 100 μg/mL were serially diluted using 2.5% nitric acid.Specifically, Zn and Fe standard solutions were diluted to concentrations of 100 μg/L, 200 μg/L, 500 μg/L, 1000 μg/L, and 2000 μg/L.Meanwhile, the Cu, Mn, Co, Mo, Ni, and Cr standard solutions were prepared at concentrations of 20 μg/L, 50 μg/L, 100 μg/L, 200 μg/L, and 500 μg/L.The quantification of Zn, Fe, Cu, Mn, Co, Mo, Ni, and Cr was achieved using a standard curve, and the concentration of each element was expressed in ng/g.

ICP-OES analysis
The concentrations of Mg, Ca, Na, K, Cr, Cu, Fe, Mn, Ni, Zn, Mo, and Co in the mineralization solution were determined by inductively coupled plasma optical emission spectrometer (iCAP 7200 ICP-OES, Thermo Fisher Scientific, Waltham, MA, USA).The instrument operated with the following parameters: radio frequency (RF) generator power of 1,300 W, plasma flow rate of 12.0 L/min, peristaltic pump speed of 45 r/min, fog chamber of high-efficiency cyclonic fog chamber, cleaning time of 30 s, integration time of 0.5 min, frequency of 40.6 MHz, nebulizer flow rate of 0.55 L/min, pump feed volume of 1.5 mL, nebulizer pressure of 0.2 MPa, Auxiliary gas flow rate of 0.5L/min, Repetition number of 3. The instrument stabilizes for approximately 30 min before analysis.The data were collected by the computerized data acquisition system, and a standard curve was generated to quantify the concentrations of the elements in the samples.After analysis, the injection system was washed with 3% dilute nitric acid and deionized water for 5-10 min each.

Methodological validation
The sensitivity and accuracy of the method were evaluated using spike-and-recovery experience of standard mineral elements, determination of detection limit, and quantification limit.The blank samples were analyzed 10 times, and the limit of detection was defined as 3 times the standard deviation of the content value of each element, while the limit of quantification was set at 10 times.The results of the spike recovery, detection limit, and quantification limit by ICP-OES are presented in Table 1.The recovery rates of the 12 elements investigated in this experiment ranged from 91.15% to 107.47%, indicating that the method is accurate and reliable.Furthermore, statistical analysis of the standard curves for each element revealed correlation coefficients above 0.999.

Statistical analysis
The Shapiro-Wilk test was employed to assess the conformity of the data to a normal distribution using SPSS software (version 22.0, SPSS Inc., Chicago, IL).A significance level (P) greater than 0.05 indicates that the data conforms to a normal distribution.Subsequently, a 2-way ANOVA was conducted using SPSS software, followed by confirmation through the Tukey test.Statistical significance was determined with a threshold of P < 0.05.

Changes of Elements Concentration During Lactation
The concentrations of Mg, Ca, Na, K, Cr, Cu, Fe, Mn, Ni, Zn, and Mo in goat milk were determined by ICP-OES.Table 2  except for Co, which were all detected.The concentrations of Mg, Ca, Na, K, Cu, and Fe in goat milk exhibited a decreasing trend during the lactation period 1-3 to 150 d, followed by an increase from 150 to 240 d.On the other hand, the concentrations of Cr, Ni, and Mo showed an increasing trend from 1 to 3 to 150 d of lactation.The concentrations of Mn and Zn increased during the lactation period of 1-3 to 90 d and then decreased from 90 to 240 d.

Changes of 4 fractions of goat milk during lactation
The content changes in goat milk and its 4 fractions were determined (Table 3).A significant decrease in casein and fat content was observed during the lactation period of 1-3 to 90 d, while no significant changes were observed during 90 to 240 d.Similarly, whey protein content exhibited a significant decrease from 1 to 3 to 150 d, with no significant changes from 150 to 240 d.The content of the aqueous phase in goat milk did not show significant changes throughout the lactation period.

Distribution of elements in goat milk in different fractions
The highest concentrations of Mg, Na, K, and Ni were found in the aqueous phase of goat milk.Comparatively, the concentrations of K, Na, and Ni were higher in the aqueous phase than in casein, whey, and fat fractions.Among the fractions, the aqueous phase had the highest concentration of Mg, followed by whey and casein, while the lowest concentration was observed in the fat fraction.In the case of Ca, Zn, Cr, Cu, Mn, and Fe, significantly higher concentrations were observed in the casein fraction compared with the other fractions.Among the remaining 3 fractions, whey had higher concentrations of Ca compared with fat, followed by the aqueous phase.For Zn and Mn, the concentrations in whey were higher than in the aqueous phase, followed by fat.The concentration of Fe and Cr was higher in fat compared with whey, followed by the aqueous phase.Additionally, the concentration of Cu was higher in the aqueous phase compared with whey, followed by fat.Furthermore, the whey fraction exhibited the highest concentration of Mo, followed by casein and the aqueous phase, while the fat fraction had the lowest concentration.

Changes of element content in 4 fractions during lactation
The changes of element content in each fraction of goat milk during the lactation period are shown in Figure 1 and Table S1.The concentrations of Mg, Na, and Fe in all 4 fractions of goat milk decreased from 1 to 3 to 90 d and increased from 90 to 240 d.In the case of Ca, the content in casein, fat, and whey protein frac-

Distribution of Elements among 4 fractions of goat milk
We observed that Ca, Zn, Cr, Cu, Mn, and Fe have exhibited higher content in the casein fraction compared with the other fractions.Previous studies have reported variations in the distribution of Ca in different milk species.Fransson and Lönnerdal (1983b) found that in bovine milk, the Ca content was highest in casein, followed by whey protein, fat, and the aqueous phase.However, their findings on human milk showed that whey protein had the highest Ca content, followed by the aqueous phase and fat, with the lowest in casein.In donkey milk, Ca content was the highest in the casein fraction, followed by the aqueous phase and whey protein, while it was not detected in the fat fraction (Fantuz et al., 2020).These differences in Ca distribution among milk species can be attributed to species-specific variations.The higher content of Ca in the casein fraction can be attributed to the fact that casein constitutes the majority of the protein in ruminant milk, accounting for approximately 80%, while whey protein constitutes around 20% (Prosser, 2021).The presence of casein micelles, which contain Ca, contributes to the higher Ca content in the casein fraction.The hydrophobic interaction between casein and colloidal calcium phosphate (CCP) facilitates the formation of casein micelles (Gaucheron, 2013).Additionally, a portion of Ca is present in the whey protein fraction.Whey proteins have the ability to bind Ca, with proteins like α-lactalbumin capable of binding one calcium ion per protein molecule (Le Maux et al., 2014).Therefore, it is important to consider the species-specific variations and protein compositions when interpreting the distribution of Ca in different milk fractions.
While our study found that Fe was predominantly distributed in the casein fraction of goat milk, it is important to note that previous research has reported varying results.For instance, in cow milk (Fransson and Lönnerdal, 1983a), it was found that Fe had the highest content in the aqueous phase, followed by whey protein, casein, and the fat fraction.In a study on camel milk, Al-awadi and Srikumar ( 2001) reported that the casein fraction had the lowest Fe content.In goat milk, we observed that Fe was primarily bound to casein, which may be attributed to the high affinity of the casein phosphoserine residue cluster for Fe.However, there appears to be a conflict in the functional properties of casein in mineral metabolism.β-casein and its casein phosphopeptide (CPP) have been shown to promote Fe absorption, whereas α S -casein and its CPP moiety inhibit Fe absorption.Thus, milk and dairy products with varying casein compositions may result in different Fe bioavailability (Kibangou et al., 2005).Fe in the whey fraction primarily binds with lactoferrin, which not only promotes Fe absorption but also possesses important immune properties, such as antibacterial and antiviral effects (Kell et al., 2020).As an iron-free apoprotein, lactoferrin can bind 2 ferric ions, preventing bacteria from utilizing free Fe.Moreover, lactoferrin can bind to the lipopolysaccharide on the bacterial wall, preventing pathogen binding and inducing bacterial cell lysis (Dupont, 2019).It can also transfer ferric iron to ceruloplasmin, thereby preventing the formation of potentially toxic hydroxyl radicals and inhibiting the pathogen utilization of Fe (Kell and Pretorius, 2018).Fe in the fat fraction is mainly present in the milk fat globule membrane, where it binds to xanthine dehydrogenase/xanthine oxidase (XDH/XO).This process requires the participation of clusters involved in the formation of Fe and S to exert its biological activity (Yokoyama andLeimkühler, 2015, Manoni et al., 2021).There are limited studies available on Fe in the aqueous phase, which may be due to its relatively low content in this fraction.Further research is needed to explore the role and distribution of Fe in the aqueous phase of milk.
In our study, we observed that the Cu content in goat milk was higher in casein micelle compared with the whey protein and fat fractions.This finding aligns with previous research in cow milk, where the Cu content in the whey fraction was lower than that in the casein and aqueous phase, but higher than that in the fat fraction.However, in human milk, the Cu content in the whey fraction was found to be higher than that in the casein and aqueous phases (Fransson and Lönnerdal, 1983a).These differences could be attributed to varia- tions in the ratio of caseins to whey proteins in human, cow, and goat milk.Cu in milk binds to the negatively charged phosphoserine residues in casein and plays a role in catalyzing lipid oxidation (Lönnerdal et al., 1982).Aulakh and Stine (1971) conducted equilibrium dialysis experiments to measure Cu binding to various milk proteins, including micellar casein, milk fat globule membrane protein, α-casein, β-casein, and lactoglobulin.They found that Cu had the highest affinity for micellar casein, which can explain the higher content of Cu in the casein fraction.In the whey fraction, Cu binds to ceruloplasmin, and the complex structure of this binding allows Cu to enhance cellular energy production and prevent the formation of oxygen-free radicals.Cu ions bound to ceruloplasmin facilitate electron donation or acquisition, enabling ceruloplasmin to function in Fe metabolism (Harris, 2019).Overall, the distribution of Cu in different milk fractions can be attributed to its binding affinity with specific milk proteins and its role in various metabolic processes.The differences observed in Cu distribution among spe- cies may be influenced by the specific composition and characteristics of their milk proteins.
In goat milk, we found that Zn is mainly distributed in the casein fraction.This is consistent with previous studies, conducted on donkey milk, camel milk, and bovine milk, where Zn content in the casein fractions was higher compared with the other fractions (Al-awadi andSrikumar, 2001, Fantuz et al., 2022).However, in human milk, the whey protein fraction was reported to have the highest Zn content, while the casein fraction had the lowest Zn content, which may be attributed to the lower content of casein in human milk (Fransson and Lönnerdal, 1983b).The binding of Zn to casein is a complex process involving multiple stages.Pomastowski et al. ( 2014) studied the kinetics of Zn binding to casein and identified 2 phases.The initial phase may involve weak binding of Zn ions to polar amino acids such as glutamate and aspartate, as well as casein phosphopeptides.The second phase involves the diffusion of Zn into the inner structure of casein micelles and its association with nonpolar amino acids, such as aliphatic or aromatic groups.Further investigations by Rodzik et al. (2020) using Fourier transform infrared spectroscopy (FT-IR) and Raman spectroscopy revealed that carboxyl aspartate (Asp) and glutamate (Glu) play a crucial role in the formation of the Zn-casein complex.They also demonstrated that β-CN binds to Zn ions through indirect interactions with oxygen ions, while the binding of κ-CN and Zn may be accomplished through weak electrostatic interactions with deprotonated functional groups.Zn in the casein fraction has been shown to regulate the function of glutamate and neurotransmitter receptors, modulate transcription factors, and inhibit tyrosine phosphatase proteins.In the fat fraction, Zn primarily exists in the milk fat globule membrane, where it is associated with alkaline phosphatase (Manoni et al., 2021).The precise role of Zn in the fat fraction and its impact on biological functions warrant further investigation.
In the whey protein fraction, we observed that Mo has a higher content compared with the other 3 fractions.However, there is limited research available on the distribution of Mo in milk, particularly in relation to different fractions.Fantuz et al. (2022) studied the distribution of Mo in donkey milk and found that the Mo content in whey protein was the lowest among the 4 fractions.This difference in findings could be attributed to variations between milk species.Further investigation is required to analyze the form of Mo present in the whey protein fraction and understand its implications.Mo plays a significant role as a cofactor in xanthine oxidase activity on the milk fat globule membrane, participating in various metabolic reactions such as the conversion of hypoxanthine to xanthine and xanthine to uric acid (Nishino and Okamoto, 2015).It has been reported that the xanthine oxidase activity in goat milk is notably lower than that in cow milk, which could be attributed to the lower Mo content in goat milk (Atmani et al., 2004).However, there is a lack of research specifically examining the distribution of Mo element in casein, whey, and aqueous phase.While we observed that most Mo exists in the whey fraction, further studies are needed to elucidate its distribution across the various milk fractions.
We found that the content of Mg, K, and Na in the aqueous phase was the highest, significantly higher than the rest of the fraction.This is consistent with previous findings in bovine and human milk, where Mg was highest in the aqueous phase, followed by the whey protein fraction and casein fraction, and lowest in fat fraction (Fransson and Lönnerdal, 1983b).Similarly, in donkey milk, Mg had the highest content in the aqueous phase, followed by casein, whey protein, and fat.The high content of Mg in the aqueous phase is likely related to the stability of milk (Magan et al., 2022).In contrast to Ca, Mg has a low content in the casein fraction due to its low affinity for inorganic phosphate and citrate in CCP (Philippe et al., 2005).Moreover, a decrease in pH can lead to a reduction in Mg content in casein (Dalgleish and Law, 1989).These factors may contribute to the differences in Mg content in the aqueous phase and casein among different milk species.Mg has the lowest content in the fat fraction, mainly exists on the milk fat globule membrane binds to alkaline phosphatase, and plays a role in activating the enzyme (Linden et al., 1977).However, there is limited research available on Mg in the whey and aqueous phases, and further studies are needed to explore its distribution and functions in these fractions.
Our results show that K and Na in milk mainly exist in the aqueous phase, which has been summarized in a previous review (Gaucheron, 2005).This distribution pattern is also observed in donkey milk, where the majority of K and Na exist in the aqueous phase.The presence of K and Na in the aqueous phase is crucial for maintaining the osmotic balance in both blood and milk, as highlighted by (Stocco et al., 2019).Additionally, it is not uncommon to find small amounts of K and Na in other milk fractions such as casein, whey, and the milk fat globule membrane.Although the reasons for their presence in these fractions are not yet well understood, their distribution in these fractions may be associated with specific physiological functions or interactions with other components of milk (Hogan et al., 2001, Pitkowski et al., 2008).Further investigations are needed to fully elucidate the mechanisms and significance of their distribution in different milk fractions.

Changes in the distribution of elements during lactation
The observed changes in the concentration of Ca in different milk fractions, as well as its impact on Ca distribution, align with previous studies and indicate the importance of total Ca content fluctuations.The decrease in Ca concentration at 1-3 to 150 d and subsequent increase at 150 to 240 d suggests dynamic changes in Ca metabolism during lactation.This trend is consistent with the changes observed in fat, casein, and whey fractions, indicating that total Ca content has a significant influence on Ca distribution.A study by (Malacarne et al., 2017) investigating the changes in Ca and Mg content in casein from donkey milk during lactation also reported a decreasing trend in Ca and Mg contents.However, Petrera et al. (2016) found no significant changes in the Ca and Mg contents of casein from Modenese and Italian Friesian cattle during lactation.These variations could be attributed to factors such as species and lactation period, emphasizing the influence of these factors on Ca dynamics (Singh et al., 2015).In our study, the higher Ca content in casein at 1-3 d of lactation compared with subsequent stages suggests an initial accumulation of Ca in casein.Although the contents of casein and the water phase did not change significantly from 90 to 240 d, the Ca content in both fractions increased significantly.Changes in the ratio of ionized calcium to colloidal calcium can impact the micellar charge, destabilize casein micelles, and reduce the thermal stability of milk, which may be influenced by variations in total Ca concentration (Magan et al., 2022).Overall, the fluctuations in Ca concentration and their impact on casein micelles and milk stability support the notion that total Ca content plays a crucial role in Ca distribution dynamics during lactation.
The significant decrease in Mg concentration at 1-3 to 150 d followed by a significant increase at 150 to 240 d indicates dynamic changes in Mg metabolism during lactation.This trend is consistent with the changes observed in the total Mg concentration and the Mg content in each fraction, suggesting that total Mg concentration drives the changes in Mg distribution.A study by (Donnelly et al., 1984) also reported higher Mg content in casein during late lactation compared with mid-lactation, supporting the finding that Mg content in casein varies throughout lactation.However, studies on bovine milk have shown that Mg in casein does not significantly change during the lactation period of 8 to 21 weeks of lactation (Petrera et al., 2016), indicating potential species-specific differences in Mg dynamics.During the 90 to 240 d period, although the contents of casein and the aqueous phase did not change significantly, the content of Mg combined with them increased.This increase in Mg content, particularly in late lactation, can have negative effects on milk stability.The higher concentration of ionic Mg, accompanied by a decrease in lactose concentration, can impact the thermal stability of milk (Tsioulpas et al., 2007, Magan et al., 2022).Additionally, the increased presence of ionic Mg can lead to enhanced casein destabilization during heating processes (Oh and Deeth, 2017).These findings suggest that the changes in total Mg concentration during lactation can influence the distribution of Mg in different milk fractions, including casein and the aqueous phase, potentially impacting the thermal stability and processing properties of milk.
The observed changes in the total concentration of Zn in goat milk during different lactation stages, with an increase at 1-3 to 90d and a decrease at 90 to 240 d, coincide with the trend observed in the Mg content in the 4 fractions.This suggests a similar pattern of changes in Zn and Mg distribution throughout lactation.Interestingly, at 90 to 240 d, while the fat content did not show significant changes, the Zn content decreased significantly.This finding indicates that factors other than fat content are influencing the decrease in Zn content during this lactation stage.One possible explanation could be the binding of Zn to alkaline phosphatase, as demonstrated in studies on milk fat globule membrane alkaline phosphatase levels in mid-to-late lactation in human milk (Manoni et al., 2021).The binding of Zn to alkaline phosphatase may contribute to the decrease in Zn content observed in the milk fat fraction during the later stages of lactation.
The observed changes in the concentration of Fe in goat milk during different lactation stages, with a decrease at 1-3 to 150 d and an increase at 150 to 240 d, align with the trends observed in the Fe content in the 4 fractions.This suggests a similar pattern of changes in Fe distribution throughout lactation.In the case of casein, although its content did not show significant changes after 1-3 d of lactation, the changes in Fe content combined with casein could potentially impact the absorption of Fe.The interaction between Fe and casein may influence the bioavailability and absorption of Fe in the milk (Kibangou et al., 2005).It is worth noting that the content of lactoferrin, an iron-binding protein, has been reported to decrease with increasing lactation (Villavicencio et al., 2017).However, in our results, while the content of whey protein did not show significant changes, Fe content significantly increased at 150 to 240 d.These findings suggest that other factors may be involved in the dynamics of Fe distribution during lactation, and further research is needed to elucidate the precise mechanisms underlying the distribution and changes of Fe in different milk fractions.
The observed changes in the concentration of Cu in goat milk, with a decrease at 1-3 to 150 d and an increase at 150 to 240 d, align with the trends observed in the Cu content in fat, casein, and aqueous phase fractions.This suggests a coordinated change in Cu distribution throughout lactation.also changed with the Cu concentration.Regarding ceruloplasmin, an enzyme that binds and transports Cu, its concentration has been reported to be higher in early lactation and decreased with prolonged lactation (Hussein et al., 2012).This may contribute to the observed decrease in Cu content in goat milk during the 1-3 to 150 d period.It is worth noting that in our results, although the content of casein decreased at 90 to 240 d, the content of whey protein did not show significant changes.This suggests that factors other than changes in the total protein content may be influencing the Cu content in the milk fractions.Further investigation is needed to understand the specific mechanisms underlying the changes in Cu distribution in different milk fractions during lactation.
The concentration of Na and K in goat milk decreased significantly at 1-3 to 150 d and increased at 150 to 240 d.These changes in Na and K levels can be attributed to various factors, including the health status of the mammary gland and the integrity of tight junctions (TJ).Impairment of the mammary gland health during late lactation can lead to disrupted tight junctions, which are critical for maintaining the barrier function of the mammary epithelium.Studies have shown that such disruptions can influence the concentration of K and Na in the aqueous phase of milk (Ontsouka et al., 2003).In the context of mastitis, an inflammatory condition affecting the mammary gland, there is evidence to suggest that increased Na and chloride (Cl) concentrations in the aqueous phase may result from the disruption of mammary cell tight junctions (Górska-Warsewicz et al., 2019, Stocco et al., 2019).Mastitis-induced inflammation and tissue damage can compromise the integrity of the tight junctions, leading to increased permeability and the leakage of Na and Cl ions into the aqueous phase.It is important to note that the natural progression of lactation involves dynamic alterations in milk composition, and further research specifically focused on the physiological changes occurring during lactation would provide valuable insights into the factors influencing the fluctuations in Na and K levels.

CONCLUSION
In our study, the distribution of 11 mineral elements in different fractions of goat milk during lactation was analyzed using ICP-OES.Our findings revealed that Ca, Zn, Fe, Cu, Mn, and Cr exhibited the highest con-centrations in the casein fraction.Mo was primarily found in the whey fraction.Mg, Na, K, and Ni showed the highest concentrations in the aqueous phase.During lactation, the content of these elements in the measured fractions exhibited changes over time.Specifically, the content of Fe in the fat fraction, as well as Ca, Cu, and Fe in the casein fraction, experienced a decrease during 1-3 to 150 d of lactation and an increase at 150 to 240 d.The content of Mg in the aqueous phase decreased from 1 to 3 to 90 d and then increased from 90 to 240 d.These variations in the element content can be attributed to changes in the levels of substances bound to the respective elements during lactation.Studying the differences in the distribution of mineral elements in goat milk provides valuable insights into the bioavailability of these elements.The variations in the distribution of mineral elements at different lactation stages in goat milk offer the potential for processing goat milk into natural mineral element supplements.Furthermore, it's important to note that the distribution of these elements across lactation stages can also influence the technical properties of goat milk.This knowledge can contribute to further research in this field, focusing on the nutritional properties and potential health benefits associated with consuming goat milk.
Pan et al.: Effect of lactation… tions decreased from 1 to 3 to 150 d and increased from 150 to 240 d, while the content in the aqueous phase decreased from 1 to 3 to 90 d and increased from 90 to 240 d.Regarding K, the content in casein, fat, and whey protein fractions decreased from 1 to 3 to 150 d and increased from 150 to 240 d, whereas the content in the aqueous phase continued to decrease throughout lactation.The content of Cr in casein, fat, and aqueous phase increased from 1 to 3 to 150 d and decreased from 150 to 240 d.In whey protein, the content increased from 1 to 3 to 90 d and decreased from 90 to 240 d.For Cu, the content in casein, fat, and aqueous phase decreased from 1 to 3 to 150 d and increased from 150 to 240 d, while the content in whey protein continued to decrease throughout the lactation.The contents of Zn and Mn in all 4 fractions increased from 1 to 3 to 90 d and decreased from 90 to 240 d.Similarly, the contents of Ni and Mo contents in all 4 fractions increased from 1 to 3 to 150 d and decreased from 150 to 240 d.

Figure 1 .
Figure 1.Distribution of elements in fat, casein, whey protein, and aqueous phase at different stages of lactation.a-d Means the difference of an element in different stages of lactation in same fraction (P < 0.05).α-γ Means the difference in element content in different fractions at the same lactation stage (P < 0.05).

Table 1 .
displays the changes of mineral element content in goat milk at different lactation stages, Pan et al.: Effect of lactation… Spike recovery (%), limit of detection (mg/L) and limit of quantification (mg/L) for 12 elements

Table 2 .
Content of mineral elements expressed as μg/g (and ng/g with *) in goat milk at 3d, 90d, 150d, 240d of lactation Mean values in the same row with different superscripts differ (P < 0.05) for the elements content in different lactation stages.

Table 3 .
Different milk composition in goat milk at different lactation stages a-b Mean values in the same row with different superscripts differ (P < 0.05) for the content of fat, casein, whey protein, and aqueous phase in different lactation stages.
Pan et al.: Effect of lactation…