The effect of nanoemulsified methionine and cysteine on the in vitro expression of casein in bovine mammary epithelial cells

Objective Dairy cattle nutrient requirement systems acknowledge amino acid (AAs) requirements in aggregate as metabolizable protein (MP) and assume fixed efficiencies of MP used for milk protein. Regulation of mammary protein synthesis may be associated with AA input and milk protein output. The aim of this study was to evaluate the effect of nanoemulsified methionine and cysteine on the in-vitro expression of milk protein (casein) in bovine mammary epithelial cells (MAC-T cells). Methods Methionine and cysteine were nonionized using Lipoid S 75 by high-speed homogenizer. The nanoemulsified AA particle size and polydispersity index were determined by dynamic light scattering correlation spectroscopy using a high-performance particle sizer instrument. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to determine the cytotoxicity effect of AAs with and without nanoionization at various concentrations (100 to 500 μg/mL) in mammary epithelial cells. MAC-T cells were subjected to 100% of free AA and nanoemulsified AA concentration in Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/F12) for the analysis of milk protein (casein) expression by the quantitative reverse transcription polymerase chain reaction method. Results The AA-treated cells showed that cell viability tended to decrease (80%) in proportion to the concentration before nanogenesis, but cell viability increased as much as 90% after nanogenesis. The analysis of the expression of genetic markers related to milk protein indicated that; αs2-casein increased 2-fold, κ-casein increased 5-fold, and the amount of unchanged β-casein expression was nearly doubled in the nanoemulsified methionine-treated group when compared with the free-nanoemulsified methionine-supplemented group. On the contrary, the non-emulsified cysteine-administered group showed higher expression of genetic markers related to milk protein αs2-casein, κ-casein, and β-casein, but all the genetic markers related to milk protein decreased significantly after nanoemulsification. Conclusion Detailed knowledge of factors, such nanogenesis of methionine, associated with increasing cysteine and decreasing production of genetic markers related to milk protein (casein) will help guide future recommendations to producers for maximizing milk yield with a high level of milk protein casein.


INTRODUCTION
Dairy cattle nutrient requirement systems consider amino acids (AA) requirements to be essential in aggregate as metabolizable protein (MP) and assume fixed efficiencies of MP used for milk protein. They are used to determine the amount of absorbed AAs or nitrogen (N) required to support a preferred level of milk production with milk protein [1]. The AA com position affects the synthesis of milk protein. The exact amount of limited AA in dairy cow diets is unknown, and cows are overfed to meet the equilibrium of MP needed. This leads to AA waste and poor N efficiency [2]. In the previous literature, approximately 25% of dietary N is captured in milk, and the remaining approximately 75% is captured in urine and feces of dairy cows [3]. Therefore, feeding cows with a low protein concentration of essential and nonessential AA supplements may maintain and improve the total N efficiency [46]. In lac tating dairy cows, AAs play a significant role in mammary protein synthesis. The previous literature on AA metabolism has mainly focused on balanced diets, which are necessary to maintain and enhance milk protein synthesis in lactating cows [7]. In mammals, AAs not only work as precursors for the syn thesis of milk protein but also as signaling molecules for the regulation of protein synthesis and lactation [8]. For example, Moshel et al [9] reported that leucine supplementation in creases the βlactoglobulin synthesis in mouse and bovine mammary epithelial cells. Furthermore, isoleucine, methio nine and threonine affect milk protein (casein) synthesis when removed from the culture medium of bovine mammary epi thelial cells. So, a sufficient supply of AAs can increase the MP of milk protein and milk yield. Numerous studies have shown that AAs stimulate the synthesis of milk protein, which is mediated by mammalian target of rapamycin (mTOR) [10]. Histidine was the first AA found for the synthesis of milk protein [4]. Previous literature also demonstrated that intro duction of histidine enhanced milk protein secretion [11]. A study by Appuhamy et al [6] showed that the addition of histi dine to the cells activated the mTOR pathway and promoted milk protein synthesis, which upregulated phosphorylation of the downstream protein. Similarly, histidine was one of the transcriptional factors of the mTORC1 pathway, and it was negatively regulated by phosphorylated S6 kinase 1 and de creased the βcasein synthesis rate [12]. The consequences of essential AAs on milk protein synthesis and the mTOR sig naling pathway have been extensively studied [13].
Nanoemulsification is a promising technology that has been used in various fields, including the pharmaceutical and food industries, with several potential applications. It is defined as a process by which substances of core materials are surrounded by a wall material to obtain capsules [14]. Liposomes are nano sized vesicles consisting of a membranelike phospholipid bilayer surrounding an aqueous medium. Liposomes have been widely used in the pharmaceutical, food, and cosmetic industries, and have been successfully employed for the en capsulation of a wide range of synthetic drugs and bioactive molecules [15,16]. The application of liposomes onto bioactive compounds at the nanoscale range protects them from chemical degradation by the surrounding dispersion medium. Encapsu lation implements the controlled release of bioactive molecules at the right place and time and it also increases the shelf life of bioactive molecules. Liposomes can be prepared as Lipoid S 75 (phosphatidylcholine [PC]>75%) from soybean lecithin. Amphiphilic lipoid S 75 is composed of a hydrophilic head domain and hydrophobic tail domain. This can come from the liposome in the aqueous media having a structure of a hydrophilic surface and a hydrophobic inner layer. It would be an ideal core material to prepare carriers for a nutrient which has the biological activity itself [17]. To the best of our know ledge, there is no scientific data on the effect of nanoemulsified AAs in casein expression. Therefore, the purpose of this ex periment was to determine the effect of liposomecoated methionine and cysteine on the in vitro expression of milk protein casein in bovine mammary epithelial cells.

Production of nanoemulsion for amino acids delivery
The AAs were encapsulated in lecithin (Lipoid S 75)based nanoemulsions, produced by highpressure homogenization (HPH), as described by a previous study. Briefly, 10 wt. % of AAs were diluted with 10 wt. % of ethanol, and then the mix ture was added to 5 wt. % of lecithin, 75 wt. % of water and stirred by blender (develop preemulsion) until completely emulsified by highspeed homogenization using an Ultra Turrax T25 blender (IKA Labortechnik, Germany) for 4 min at 24,000 rpm, to achieve a primary emulsion. After that, cooled preemulsion was passed through a highpressure homoge nizer (MN400BF, Micronox, Seongnam, Korea) 3 times at 1,000 psi, resulting in nanoemulsion. The collected nanoemul sified AAs (methionine and cysteine) were used for further analysis.
Particle size and polydispersity analysis The particle size distribution and polydispersity (zeta poten tial) of the nanoemulsified methionine and cysteine were analyzed by dynamic light scattering correlation spectroscopy using a highperformance particle sizer instrument (Malvern Instruments, Malvern, UK). The droplet size distribution was www.ajas.info

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Kim et al (2019) Asian-Australas J Anim Sci 32:257-264 characterized regarding the mean droplet size (Zdiameter) and width of the distribution (polydispersity index, PDI) at a scattering angle of 190° at 25°C. Briefly, by measuring the backscattered light of 100 μL samples diluted in 1 mL of dis tilled water put into a polystyrene latex cell and measured at a scattering angle of 90°, dispersant refractive index of 1.33, and material refractive index of 1.59 at 25°.

Cytotoxicity of nanoemulsions
MTT was used in cytotoxicity analysis. Briefly, cells were seed ed in a 96well plate at a density of 0.5×10 4 cells/well. After a 24h interval, cells were treated with different concentrations (100 to 500 μg/mL) of nanoemulsified AAs (methionine and cysteine). After 48 h of incubation at 37°C with 5% CO 2 , cells were treated with 10 μL of MTT solution and incubated for 4 h. The optical density of each well was measured at a wavelength of 540 nm using a Spectra count enzymelinked immunosorbent assay plate reader (Packard Instrument Co., Downers Grove, IL, USA). The cytotoxicity of the blank nano encapsulated AAs was expressed as the percentage of cell viability, calculated from the ratio between the numbers of living cells treated with the nanoencapsulated AAs and that of the untreated cells. Based on the cytotoxicity data, the con centration of encapsulated AAs (18 μg/mL) was chosen for the present study.

Cell culture and treatment
Bovine mammary epithelial cells were cultured in a 12well plate at a density of 1.5×10 4 cells/well in DMEM supplemented with 10% of FBS, 100 μg/mL of penicillin and streptomycin, 5 μg/mL of insulin, 50 μg/mL of gentamicin, and 1 μg/mL hydrocortisone in a humidified atmosphere, including 5% CO 2 at 37°C. After cells reached approximately 90% to 100% confluency, they were treated with nanoemulsified AAs (me thionine and cysteine) at a concentration of 18 μg/mL for 24 h. The experiment performed in triplicate.

Milk protein gene expression quantification by quantitative reverse transcription polymerase chain reaction
At the end of the experiment, cellular mRNA was extracted from MACT cells by a RNeasy lipid tissue kit (Qiagen, Va lencia, CA, USA) according to the manufacturer's protocol.
Extracted RNA was quantified with UVS99 microvolume UV/Vis spectrometerACT gene. cDNA synthesis was per formed with 500 ng of cellular RNA using oligo (dT) primers and reverse transcriptase provided by the Superscript III first strand synthesis system for RTPCR (Invitrogen, USA). The level of caseinrelated gene mRNA expression was assessed by SYBR Greenbased realtime PCR on an ABI 7500 PCR system (Applied Biosystem, Foster City, CA, USA). The target gene expression levels were normalized against housekeeping gene glyceraldehyde 3phosphate dehydrogenase. Primers used for quantitative RTPCR are listed in Table 1.

Statistical analysis
All the experiments were conducted in triplicate. Data were statistically analyzed, and comparison was performed using a statistical package (SPSS16.0) (SPSS, Inc., Chicago, IL, USA). The results were represented as a mean±standard error of the mean. The significant difference between the mean was compared by least significant difference, and the value was considered p<0.05.

Characterization of AA nanoemulsion
The effectiveness of the core material used to prepare AA na noemulsion was determined from the total amount of AAs in nanoemulsions and AAs that were extracted from surface of nano particles. The core material of Lipoid S 75 had greater efficiency and a smaller particle size (62.81±1.37 nm) when compared to other core materials of Lipoid S 753 (171.60±4.54 nm), Lipoid S 100 (72.67±1.31 nm), Lipoid S 1003 (156.60± 3.51 nm), Lecinol S 10 (162.36±5.45 nm), and HSPC 50 (158.57 ±4.54 nm) (Figure 1). Based on these properties, we selected Lipoid S 75 for the preparation of nanoemulsified AAs. Methio nine with Lipoid S 75 was entrapped in unsaturated soybean lecithin with an encapsulation efficiency of 77.6%. Similarly, cysteine with Lipoid S 75 was entrapped in unsaturated soy bean lecithin with an encapsulation efficiency of 89.8%. The average particle size distribution of nanoemulsion of methi onine in Lipoid S 75 was in the range of 475.7 nm, whereas, the particle size distribution of the cysteine in Lipoid S 75 was in the range of 431 to 475 nm by intensity as presented in Figure 2. The results of the current study showed that high pressure homogenization 3 times at 1,000 psi was the optimal condition to prepare an AA nanoemulsion. The zeta poten tial of nanoemulsion of methionine and cysteine in Lipoid S 75 was -11.8±4.42 mV and -14.6±1.63 mV, respectively ( Fig   Figure 1. Particle size of core materials analyzed by dynamic light scattering correlation spectroscopy. The core material of Lipoid S 75 had greater efficiency and a smaller particle size when compared to other core materials of Lipoid S 75-3, S 100, S 100-3, Lecinol S 10, and HSPC 50. HSPC, hydrogenated soybean phosphatidylcholine. ure 3). The high PDI was caused by the main limitation of dynamic light scattering due to high concentration in the cuvette. This can lead to Brownian motion, such as multiple scattering with a decreased path length of particles. These phenomena can result in unfavorable particle size. Dynamic light scattering measures the hydrodynamic radius of a parti cle. A possible reason may be that Lipoid S 75 can attract water as more water is added; therefore, the dynamic diameter in creases slightly. As the sample becomes more diluted, more Lipoid S 75 may move out of the particle, causing the particle size to decrease. Fortunately, this was not the case, as this can give substantial problems when this would be used for ther apeutic applications [18]. In addition, all further studies of cytotoxicity and efficacy using methionine and cysteine in Lipoid S 75 on milk protein in MACT cells were performed based on the efficiency and particle size of nanoemulsionbased delivery systems.

Cytotoxicity of nanosomes
Colorimetric MTT assays were performed 24 h after treatment with or without nanoemulsified of AAs to highlight a possible cytotoxic effect of these emulsions ( Table 2). Results of the present study showed that cell viability was slightly agitated at AA concentrations up to 500 μg/mL compared with con trol cells (p<0.05). Findings of the current study support those of Basirico et al [19], who previously reported that no fatty acid treatment on mammary epithelial cells reduced cell viability. However, at the same time, nanoemulsified AAs significantly increased the cell viability at concentrations up to 500 μg/mL when compared with nonemulsified AAs (p<0.05). Although the AAs had no significant effect regardless of the dose used, the doseresponse curve showed that the administration of 100 to 500 μg/mL of nanoemulsified AAs increased the cell viability from 10.1% to 11.9%, respectively when compared with free AAs.

Effects of methionine and cysteine nanoemulsion on the expression of caseins
Several studies have evaluated the contribution of AAs to mam mary gland metabolism and protein secretion and synthesis [20]. In this experiment, the potential effect of nanoemulsified methionine and cysteine on milk protein casein expression in bovine epithelial mammary cells was studied. The current study showed that, compared with the nonemulsified me thionine group, the supplementation of methionine to Lipoid S 75 generated significantly increased effects on the casein expression. Figure 4 shows that nanoemulsified methionine had stimulatory effects of 2fold on the α s2 casein, 5fold on the βcasein, and 2fold on the κcasein expression when com pared with the nonemulsified methionine group, which sustained the notion that methionine is a key limiting factor for milk protein synthesis. Our study results confirmed that nanoemulsified methionine promoted milk protein αcasein, βcasein, and κcasein expression in cultured mammary epi thelial cells as compared with nonemulsified methionine. Our current study findings agreed with those reported pre viously that histidinecontaining dipeptide promoted milk protein production compared with supplementation of free histidine [21]. However, the mechanism of nanoemulsified AA utilization is complicated and unclear. It has been previ ously demonstrated that peptides containing methionine can easily utilize free methionine supplementation for mammary tissue and milk protein synthesis in cultured mammary epi thelial cells [22]. A rat model experiment clearly showed that dipeptides were hydrolyzed extracellularly when compared with free AAs [23]. However, it remains unclear why supple mentation with dipeptides containing methionine to cultured mammary epithelial cells significantly increased efficiency of α s2 casein gene expression, but α s1 casein gene expression was not increased when supplementation of free methionine was higher than 60 μg/mL in cultured bovine mammary epithelial cells [24]. The free methionine transporter may be saturated at 60 μg/mL, but dipeptides containing methionine may be absorbed through different transporters located on bovine mammary epithelial cell membranes [25]. This transporter may be from a different species, so bovine mammary epithelial cell membranes containing the transporter have a high affinity and absorb dipeptide methionine directly for milk protein casein synthesis. Figure 5 shows the effect of nanoemulsified cysteine supplementation on casein expression. Compared with nonemulsified cysteine, the expression of α s2 casein, βcasein, and κcasein was decreased by emulsified cysteine. The cysteine content is lower in cow's milk than in human milk because methionine content is higher in cow's milk. Therefore, the methionine/cysteine ratio is 2 to 3fold greater in cow's milk than that of other mammals, but 7fold greater  in human milk. Two considerable characteristics of AA com position of human milk are the ratio between the sulfur containing AAs methionine and cysteine, and the low level of aromatic AAs phenylalanine and tyrosine because of the low levels of the specific enzymes required to metabolize them. Wu et al [26] demonstrated that AAs, including arginine, cysteine, glutamine, and leucine act as both cell signaling candidates and regulators for gene expression and protein phosphorylation pathways. The expression of the α s2 casein, βcasein, and κcasein genes was decreased when nanoemul sified cysteine was used to supplement the bovine mammary epithelial cells. Our study results agree with those of Raggio et al [27], who reported that the efficiency of the absorbed AAs into milk protein decrease markedly as protein delivery in creases. However, further studies are needed to clarify the nature of the response by mammary cells to increase AA supply.

CONCLUSION
In conclusion, the medium (soybean lecithin) used for mixing AAs (methionine and or cysteine) for emulsification enhanced encapsulation capability, size, and PDI. Addition of core ma terial Lipoid S 75based nanoemulsion of methionine to bovine mammary epithelial cells showed the potential to influence milk protein α s2 casein, βcasein, and κcasein when com pared with free methionine and cysteine. Therefore, further study is needed to determine how a nanoemulsionbased model may help to clarify the regulation mechanism of me thionine and cysteine on casein expression through the mTOR pathway in bovine mammary epithelial cells.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manu script.