bovine colostrum is an opaque white liquid which is a natural source of nutrition . five main components in bovine colostrum are water , fats , proteins ( e.g. , caseins , albumins , and globulins ) , sugars ( essentially lactose ) , and mineral salts . since bovine colostrum spoils easily , it is sometimes processed into dairy products .
the most durable form of bovine colostrum is bovine colostrum powder , which is produced by the dehydration of raw bovine colostrum . in developing countries ,
casein is the major protein in cow 's milk , and comprises about 80% of the total protein content of which the rest , 20% , are the whey or serum protein [ 1 , 2 ] .
whey proteins contain alphalactalbumin ( -la ) , betalactoglobulin ( -lg ) , immunoglobulin , lactoferrin , and bovine serum albumin .
alphalactalbumin ( -la ) and betalactoglobulin ( -lg ) are two kinds of whey protein in bovine colostrum and specifically produced in the mammary epithelial cells only during lactation . as a monomer , alphalactalbumin ( -la ) and betalactoglobulin ( -lg ) strongly bind calcium and zinc ions and may possess bactericidal or antitumor activity . high - effect capillary electrophoresis ( hpce ) ,
this technology already has applied to protein and aminofacid broadly for many years . due to the appealing advantages in separation efficiency and resolution , small amounts of solvent and sample , ce
have rapidly been applied for the assay in the protein separation field .   for a long time , the capabilities of matrix - assisted laser desorption / ionization ( maldi ) mass spectrometry for the rapid evaluation of proteins have been proved .
furthermore , it has also been tested to be a valid tool in the dairy industry .
although the technique does not determine the exact concentrations of bovine colostrum proteins , the relative proportions of several main bovine colostrum proteins can be identified . in this experiment ,
another portion was separated with cpe method and detectable differences in their maldi spectra were found .
the present work describes alphalactalbumin ( -la ) and betalactoglobulin ( -lg ) in the rehydration of bovine colostrum powder were successfully separated by cloud point extraction using a nonionic surfactant triton x-114 .
the separation was mainly based on their contrasting hydrophobicities , and our main goal was to achieve the highest concentration of alphalactalbumin ( -la ) and betalactoglobulin ( -lg ) in the surfactant - poor phase obtaining high efficiency of cpe method .
since separation of these proteins using cpe depends on several variables , such as type and concentration of surfactant , ph , net charge , size and sample volume [ 6 , 7 ] , the optimization design was concluded in .
the matrix - assisted laser desorption ionization - time of flight mass spectrometry ( maldi - tof ms ) was used to confirm the separation of alphalactalbumin ( -la ) and betalactoglobulin ( -lg ) from the rehydration of bovine colostrum powder .
  bovine colostrum powder ( sunlife bovine colostrum powder ) was purchased in a local chinese market . all the water used in
the experiment was milli - q deionized water ( millipore , billerica , ma , usa . ) .
acetonitrile ( hplc grade ) was obtained from sk chemicals ( ulsan , korea ) .
the -cyano-4-hydroxycinnamic acid ( chca ) was obtained from applied biosystems ( foster city , ca , usa ) .
trifluoroacetic acid ( tfa ) was obtained from sigma - aldrich ( steinheim , germany ) .
nacl , isooctylphenyl ether ( triton x-114 ) , and sucrose were obtained from sigma - aldrich ( steinheim , germany ) .  
the factors were triton x-114 concentration , sample volume , nacl and sucrose concentration , and ph . the surfactant solution was prepared with 10  mmol / l tris - hcl ( ph 7.4 ) , 150  mmol / l nacl , and 6% ( w / v ) sucrose .
200  l bovine colostrum sample was added to the surfactant solution , and the solution was homogenized again .
the ce was performed using a p / ace mdq capillary electrophoresis system ( beckman coulter , fullerton , ca , usa ) under a normal polarity separation mode . a capillary ( beckman coulter , fullerton , ca , usa ) with 50  cm effective length ( 60  cm total ) and
an inner diameter ( i.d . ) of 75  m was used with an applied voltage of 25  kv at 20c .
prior to use the capillary for the first time , a rinse for 1  min ( 30  psi ) with 0.1  mol / l hcl was performed .
a rinse for 10  min ( 30  psi ) with the running buffer was performed then equilibrate the capillary for 10  min ( 25  kv ) .
finally a rinse for 10  min ( 30  psi ) was performed the temperature of the separation capillary column was thermostated at 20  c. among each runs , the capillary was rinsed with 0.1  mol / l hcl for 0.5  min and then with the electrophoresis buffer for 1.5  min .
the separation buffer was 50  mm citrate buffer ( ph 3.0 ) , and the applied voltage is 25  kv . the electrophoresis buffers were filtered through 0.22  m filter before use .
the samples were dissolved in 1  ml of buffer and filtered [ 0.22  m ( millipore ) ] for ce analysis .
samples were injected into the capillary using pressure at 0.5  psi for 10  s. the absorbance was measured at a wavelength of 214  nm for the detection of protein . to prepare a saturated solution of chca matrix : 10  mg of the dry chca matrix
was added to a tube containing 1  ml solution of water / acetonitrile/0.1% tfa ( 4/5/1 , v / v / v ) .
0.5  l of the sample / matrix ( 1/5 , v / v ) mixture was spotted per well on a stainless steel maldi sample plate .
all the maldi measurements were performed on a 4700 proteomics analyzer ( applied biosystems , foster city , ca , usa ) equipped with a nd : yag laser ( 355  nm ) operating at 200  hz .
ions formed by a pulsed laser beam were accelerated to 20 kilo volts in positive ion linear mode with a 740  ns delay times .
the acquisition method was set as follows : 60 subspectra were acquired on one spot and 25 shots were averaged per subspectrum , so one maldi spectrum was the accumulated spectra of a total of 1500 shots per spot . for each sample ,
five spots were equally spotted and examined , and the acquisition method ran three times per spot .
the final data were the trimmed means of the results of these 15 parallel tests as a whole .
this instrument operating parameters were optimized for ion peaks corresponding to apomyoglbin ( horse , m / z 16952 ) , thioredoxin ( e. coli , m / z 11674 ) , and insulin ( bovine , m / z 5734 ) .
to highlight the extraction of whey proteins from bovine colostrum and their separation from casein proteins by cloud point extraction , ce was used to identify -lactalbumin and -lactoglobulin extracted from the rehydration of bovine colostrum powder .
the electropherograms of standard and cloud point extraction sample from the rehydration of bovine colostrum powder were shown in figures 1(a ) and 1(b ) and figure 1(c ) , respectively .
the retention time of -lactalbumin and -lactoglobulin was about 10 and 15  min under the optimal separation condition . in figure 1(c ) , there were only two whey protein peaks ( -lactalbumin and -lactoglobulin ) .
it was proved right with maldi - tof ms in the next experiment . from figure 1(c ) , it is also proved the efficiency of the extraction procedure is successful . to highlight the extraction of whey proteins from bovine colostrum and their separation from casein proteins by cpe , maldi - tof ms to
intact samples of the two protein fractions was applied . a simple sample treatment that only involved dilution of precipitated proteins was performed .   figure
2(a ) displays the mass spectra obtained , and it summarizes different proteins identified in the surfactant poor and water phases .
the theoretical molar masses are in agreement with those previously described for whole cow bovine colostrum protein analyses by maldi - tof ms .
the mass spectra of figure 2(a ) show ions of ca.m / z11958 and 23919 corresponding to the -casein and -casein , respectively .
those ions in figure 2(b ) of higher m / z values correspond to -lactalbumin ( m / z14152 ) and -lactoglobulin ( m / z18318 ) .
finally , the ion of ca.m / z 14152 in figure 2(b ) probably corresponds to species originating from lactose addition to -lactalbumin .
the main proteins identified in the surfactant poor phase ( figure 2(b ) ) were -lactalbumin and -lactoglobulin , which are fractions of the whey proteins . besides such proteins , -casein and -casein were also detected in the water phase .
in fact , both last proteins also present amphiphilic character [ 4 , 5 ] , corroborating their presence in the surfactant poor phase .
cpe was efficiently employed to cow bovine colostrum samples being able to extract and separate whey from casein proteins using only 200  l of sample volume , sample volume without any sample pretreatment .
( 1% , w / v ) , 150  mmol / l nacl , and sucrose ( 6% , w / v ) were used at ph 7.0 for protein separations . at such conditions ,
good partition coefficient was obtained , allowing the separation of -lactalbumin and -lactoglobulin ( present in the surfactant poor phase ) from casein proteins ( present in the water phase ) in only 15  min and with minimum costs , indicating that the proposed factorial design was successfully applied in the experimental domain employed and the main objective of this work attained .
although the partition coefficient was obtained , it is important to comment that some hydrophobic proteins were achieved in the surfactant poor phase and vice versa , as demonstrated through maldi - tof ms analysis .
after cloud point extraction , the capillary electrophoresis was used to check the efficiency of the extraction procedure .
the results had been effectively confirmed by the characterization with matrix - assisted laser desorption ionization time of flight mass spectrometry ( maldi - tof ms ) . finally , the adopted strategy could also be used for separating those low - abundant proteins in bovine colostrum after properly optimizing the cpe method for such task .
 alphalactalbumin ( -la ) and betalactoglobulin ( -lg ) in the rehydration of bovine colostrum powder were successfully separated by cloud point extraction using a nonionic surfactant triton x-114 . 
 the effects of different factors , including the surfactant concentration , sample volume , electrolyte , and ph were discussed . the optimized conditions for cloud point extraction of alphalactalbumin ( -la ) and betalactoglobulin ( -lg ) 
 can be concluded that the best surfactant is 1% ( w / v ) triton x-114 , 200 l of sample volume , 150 mmol / l nacl , and 6% ( w / v ) sucrose . 
 after cloud point extraction , the capillary electrophoresis is used to check the efficiency of the extraction procedure . 
 the results had been effectively confirmed by the characterization with matrix - assisted laser desorption ionization time of flight mass spectrometry ( maldi - tof ms ) . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusion