carmine is a natural red pigment extracted from dried scale insects ( cochineal , dactylopius coccus ) .
the main chemical component of this pigment is carminic acid ( 7--d - glucopyranosyl-9,10-dihydro-3,5,6,8-tetrahydroxy-1-methyl-9,10-dioxoanthracene carboxylic acid , figure 1 ) .
carmine is widely used for coloring food products , cosmetics , and medicines [ 2 , 3 ] . in 2012 ,
the japanese consumer affairs agency published a report on carmine detailing its potential links to dyspnea and anaphylactic reactions .
this report detailed approximately 20 articles from 1960s about anaphylaxis resulting from the use of the cosmetics or consumption of food containing carmine .
carminic acid is not the cause of these allergic reactions ; they are rather caused by protein contaminants in the carmine [ 5 , 6 ] .
some of the allergenic protein contaminants have been identified , and one of them has high homology with phospholipase a , which is a well - known allergen in vespidae venom .
hence , the use of carmine in foods is regulated both in japan and european union . for consumer safety ,
the general method for detection of protein contaminants in carmine involves suspension of the pigment in an acidic solution ( 1% aqueous solution of phosphoric acid ) and then separation of any protein contaminants from the solution by hplc .
this method is time - consuming because about ten hplc analyses are required to obtain a sufficient quantity of protein for sds - page analysis .
therefore , an alternative method that is rapid , simple , and has high reproducibility is needed . in this study , we developed a rapid and simple method for detection of protein contaminants in carmine with high reproducibility .
sample b was manufactured by kishi kasei company ( carminic acid , lot number 174302 ) , and we carried out further purification of sample b as follows .
firstly , sample b was dissolved in 5% of phosphoric acid and loaded on a spherical polymerized divinylbenzene resin .
then bound carminic acid was eluted by 50% ethanol and dried ( sample c ) . to separate the protein fraction from the carminic acid
, we used an ultrafiltration device with a molecular weight cut - off of 3,000  da .
two ultrafiltration devices were tested ; one was v - shaped , with a regenerated cellulose membrane ( amicon ultra-0.5  ml  3  k , millipore , billerica , ma ) , and the other was cylindrical , with a modified polyethersulfone membrane ( nanosep 3  k omega , pall corporation , port washington , ny ) .
the initial sample volume was 500  l , and the filtration was done by centrifugation until the samples were concentrated to about 0.40.7 of their initial volume .
after these filtration steps , the concentration of carminic acid in each sample would be diluted by at least 6  10 times mathematically .
electrophoresis was carried out using precast gels ( 15% choju gel , oriental instruments , sagamihara , japan ) and stained using silver stain ms kit ( wako chemicals , osaka , japan ) .
each carmine sample was dispersed in phosphoric acid ( 1% ( mass fraction ) aqueous solution ) at a final concentration of 50  mg / ml using ultrasonication .
a white precipitate of aggregated protein contaminants was observed when sample a ( cochineal extract , lot number m008363 , biocon ) was dispersed in phosphoric acid ( figure 2 ) .
we had confirmed low reproducibility of hplc separations , such as disappearance and shift of a peak from contaminant proteins ( data not shown ) .
the aggregation of protein contaminants in the phosphoric acid would reduce the reproducibility of protein separation . as a logical approach to prevention of aggregation of protein contaminants , we made use of protein denaturant first .
therefore , in the present method , urea ( 8  mol / l ) and guanidine hydrochloric acid ( 6 
detection of proteins is usually performed by colorimetric methods such as biuret method and ninhydrin reaction .
free carminic acid interferes with the detection of allergenic proteins in the carmine by colorimetric analysis , and it needs to be removed before analysis . in addition , to detecting the allergenic proteins , protein fractions of > 6  kda need to be collected , and determining the molecular weight of protein contaminants is also important . both sds - page and mass spectrometric analysis are generally used methods for determination of molecular weight of proteins .
bound carminic acid on protein shift in molecular weight is found in mass spectrometric analysis , and a smear band pattern is also acquired from sds - page .
consequently , in the present study , two ultrafiltration devices were compared for separating the protein fraction from the carmine solution . both of these had molecular weight cut - offs of 3,000  da and are described in section 2 .
the initial sample volume was 500  l , and the filtration was done by centrifugation until the samples were concentrated to about 0.40.7 of their initial volume .
50 , 100 , and 200  mg / ml concentrations of carmine were suspended in each solution . at 100 and 200  mg / ml of carmine ,
carminic acid suspensions were difficult to remove by both ultrafiltration devices , and a concentration of 50  mg / ml was used in subsequent experiments .
after filtration , the concentration of carminic acid in each sample was diluted by at least 6  10 times .
the amicon ultra-0.5 v - shaped ultrafiltration device could not quantitatively remove the carminic acid , and thus the sds - page results were obscured by remaining pigment ( figure 3(a ) ) .
however , the cylindrical ultrafiltration device did remove sufficient carminic acid to allow the protein contaminant band to be clearly observed at around 60  kda .
although the cylindrical ultrafiltration device provided more effective removal of carminic acid than the v - shaped device , the detection of low molecular weight proteins ( < 20  kda ) was low ( figure 3(b ) ) .
these results indicate that the nanosep 3  k omega ultrafiltration device is better than the amicon ultra-0.5 for the removal of carminic acid , but protein recovery may be an issue .
the loss of the protein could be caused by damage to the ultrafiltration device from the high specific gravity solution used for dispersion of the carmine ( e.g. , 8  mol / l urea and 6  mol / l guanidine hydrochloric acid ) .
therefore , sds - page running buffer , 25  mmol / l tris(hydroxyamino)methane , 200  mmol / l glycine , and 1% ( mass fraction ) sds , was trialed for dispersion of the carmine samples .
this buffer has the following benefits to our analysis : it contains sds , which is a surfactant having strong protein - denaturing ability ; the specific gravity is much lower than that of 8  mol / l urea and 6  mol / l guanidine hydrochloric acid ; and it does not interfere with protein migration in acrylamide gel .
the three carmine samples were each dispersed at a final concentration of 50  mg / ml in sds - page running buffer by ultrasonication ( figure 4(a ) ) .
the nanosep 3  k omega was used to remove carminic acid , and this was followed by sds - page and silver staining ( figure 4(b ) ) . for sample
a , not all of the carminic acid was removed by ultrafiltration , but the remaining carminic acid had little effect on the electrophoresis .
several protein bands were observed at 5070  kda for all of the carmine samples .
the intensities of these bands were highest in sample a , followed by sample b , and lowest in sample c. protein bands were also observed in a lower molecular weight region ( < 20  kda ) for the partially purified carmine .
we confirmed the results of the rank order of intensities of protein bands ' exhibited high reproducibility .
the amicon ultra-0.5 was also tested for removing carminic acid from a suspension of carmine in sds - page running buffer .
compared with the nanosep 3  k omega device , much of time was required ( several days ) and more of the carminic acid remained in the solution after amicon ultra-0.5 filtration .
remaining carminic acid was able to be confirmed by view of acrylamide gel before staining ( figure 5(a ) ) and the electrophoretic pattern of protein contaminants showed more smearing by binding carminic acid on proteins ( figure 5(b ) ) . based on the sds - page results , the dependence on purity of carmine was obvious with the nanosep 3  k omega protocol .
although the detection limit of the low molecular weight proteins ( < 20  kda ) seemed to be lower with the nanosep 3  k omega than with the amicon ultra-0.5 , all of the bands were clearly visible still .
also the proteins detected at low molecular weight ( < 20  kda ) by silver staining with the amicon ultra-0.5 were also possible artifact due to the effect of the remaining carminic acid . to examine the effect of the shape of the ultrafiltration device on the isolation and detection of the protein contaminants , we also tested a microsep advance centrifugal device 3  k ( pall corporation ) . like the nanosep 3  k omega
however , this device is v - shaped rather than cylindrical , and the membrane is 10 times larger than that in the nanosep ; filtration with this device took a long time ( several days ) .
the loss of low molecular weight proteins was similar to that with the nanosep 3  k omega ( data not shown ) .
these results suggest that the cylindrical filtration device is more suitable for removal of carminic acid than the v - shaped ultrafiltration device .
carminic acid can form strong bonds with proteins through both electrostatic and hydrophobic interactions . therefore
the method commonly used for detection of protein contaminants in carmine involves suspension of the carmine in an acidic solution .
the acidic solution results in protein denaturation and protein aggregation , which cause analytical and separation difficulties . in this study
, we found that sds - page running buffer worked as an effective solvent for dispersion of carmine for removal of protein contaminants . the ultrafiltration method used in the present study had sufficient sensitivity and precision .
this method is simpler and less expensive than hplc separation and requires only a centrifuge .
when selecting an ultrafiltration device for the separation of proteins from carminic acid , a cylindrical device containing a modified polyethersulfone membrane gives good results , and the protein contaminants can also be detected by silver staining sds - page .
this method could potentially be applied to other natural pigments and to the removal of unreacted dyes used for protein labeling .
a rapid and simple method was developed for the analysis of protein contaminants from carmine .
method 's development focused on preventing protein aggregation under acidic protein denaturation conditions and the use of an ultrafiltration device . to prevent protein aggregation ,
sds - page running buffer was used for dispersion of the carmine . a cylindrical ultrafiltration device containing a modified polyethersulfone membrane provided the best separation of protein contaminants , which could then be observed as bands by sds - page .
 protein contaminants in carmine can cause dyspnea and anaphylactic reactions in users and consumers of products containing this pigment . 
 the method generally used for detection of proteins in carmine has low reproducibility and is time - consuming . in this study , 
 a rapid , simple , and highly reproducible method was developed for the detection of protein contaminants in carmine . 
 this method incorporates acidic protein denaturation conditions and ultrafiltration . to prevent protein aggregation , sodium dodecyl sulfate containing gel electrophoresis running buffer 
 was used for dispersing the carmine before filtration . 
 an ultrafiltration device was used to separate the protein contaminants from carminic acid in the carmine solution . 
 two ultrafiltration devices were compared , and a cylindrical device containing a modified polyethersulfone membrane gave the best results . 
 the method had high reproducibility . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusion