Characteristic of Allergen Protein of Nila baby fish (Oreochromis niloticus) and Its Processed Product

Allergen protein is a protein that could triggers allergy reaction. This study purposes to examine the characteristic of allergen protein of unripe, boiled, and fried nila baby fish. Characteristic of allergen protein were observed by electrophoresis and immunoblotting method, while the determination of protein concentration was observed by Bradford method. Identification of the used nila baby fish was accomplished in Faculty of Fishery and Marine Science of IPB. The result of identification showed that the used nila baby fish was Oreochromis niloticus. The proximate analysis of unripe, boiled, and fried nila baby fish resulted water content ranged from 19.16%-23.68%, fat content ranged from 1.03%-20.44% and carbohydrate content ranged from 0.16%-20.27%. Protein concentration of unripe, boiled, and fried nila baby fish extract respectively were 1963.45 mg/L; 699.82 mg/L; 607.79 mg/L. The band amount of allergen protein of unripe, boiled, and fried nila baby fish which was detected by electrophoresis, respectively were 16 of protein band, 26 of protein band and 16 of protein band. The immunoblotting showed that the sum of respondent who contained specific IgE that can bind with allergen protein of boiled and fried nila baby fish were more than allergen protein of unripe nila baby fish. It indicated that the processing process by boiling and frying would increase allergenicity toward nila baby fish.


INTRODUCTION
Nila Fish (Oreochromis niloticus) is one of high valuable fish species in economic. Consumption need either seed or fish from year to year tends to increase along with cultivation plant expansion (Darwisito et al., 2008). Moreover, food processing products of nila fish also start developing. One of them is food processing product comes from nila baby fish. This product has umami flavor and contains nutrient especially essential amino acid, such as leucine, histidine, glycine, alanine, and tyrosine as well as high protein and sufficiently low level of lipid (Ogunji et al., 2005, Justi et al., 2003. World Allergy Organization states that 22% of the world population suffer from allergies and continues to increase every year. Ebo et al (2010) reported that consumed nila fish could potentially causing an allergy to sensitive people toward nila fish. Allergy could trigger light symptoms such as pruritis, runny nose, and puffiness even evoked an enough heavy response like anaphylaxis that may cause death 2016). Some foods which contained allergen are fish, shell, egg, soybean, cow milk, wheat, peanut, tree nuts (as canary, walnut, hazelnut) (Gupta et al., 2013). Type of common allergen protein contained in seafood, mollusk, and fish is tropomyosin (Boye and Godefroy, 2010).
Foodstuff contained allergen protein could be detected by molecular weight and its reaction with IgE antibody, like allergen protein in goatfish (Oreochromis mossambicus) that has molecular weight about 17 kDa-114 kDa (Liu et al., 2012). Allergen protein of mackerel fish has molecular weight about 20 kDa (Misnan et al., 2005), and tuna fish's about 27-38 kDa 27-38 kDa (Hamada et al., 2001). Food processing process with heat could influence allergenicity characteristic of the foodstuff (Nowak-wegrzyn and Fiocchi, 2009). Besides the unripe nila baby fish, this study also used fried and boiled nila baby fish. The study aim is to examine the characteristic of allergen protein of unripe, fried, and boiled nila baby fish

Proximate Analysis (AOAC, 1990)
The proximate analysis carried out consisted of water content, ash content, protein concentration, fat content and carbohydrate content as describe in AOAC 1990.
Protein Extraction (Hashimoto et al., 1979) Sample of nila baby fish treated with 3 different treatments, i.e. unripe, fried, and boiled. The fried nila baby fish obtained from frying process at 130 °C-140 °C for 10 minutes, whereas the boiled nila baby fish obtained from boiling process at 100 °C for 5 minutes. 20 grams of each nila baby fish sample were weighed, added phosphate buffer saline (PBS) pH 7.5 with ion power (I) = 0.05 (15.6 mM Na 2 HPO 4 , 3.5 mM KH 2 PO 4 ) and blended for 3 x 1 minutes, next centrifuged at 4000 rpm for 25 minutes at 4 °C. The obtained supernatant separated and kept in freezer at -20 °C for the further analysis.

Protein Analysis (Bradford, 1976)
Bradford reagent made of the following manners: 100 mg of coomasie brilliant blue G-250 dissolved in 50 mL of ethanol 95%. Next, 100 mL phosphate acid 85% added and diluted until 1 L precisely with aquadest. Then filtered the solution using filter paper Whatman No.1, afterwards kept the solution in dark bottle at refrigerate temperature. The solution is the Bradford reagent.
An amount of protein isolate dissolved with aquadest then 100 μL was taken into dark test tube. Added 5 mL of Bradford reagent, then shaken it by vortex and incubated for 5 minutes. Protein concentration was measured by spectrophotometer at 595 nm wavelength. The used standard was BSA with various concentration of 0-1000 ppm.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) (Laemmli, 1970)
Optimum concentration of acrylamide in separating gel was 12 % while 4% for stacking gel. Added 40 µl of protein extract with sample buffer (1:1), heated for 5 minutes, then put into wells coincide with marker protein that had been heated for 15 minutes. Run SDS-PAGE for 135 minutes at 90 Volt. Gel was colored by staining solution consist of coomasie brilliant blue R-250, methanol, glacial acetate acid, and aquadest. Then washed the colored gel three times using aquadest, next rinsed by destaining solution consist of methanol, glacial acetate acid, and aquadest.
Protein molecular weight of sample could be calculated by regression equation, which obtained from curve of relation between relative mobility of marker protein (Rf or retention factor) and molecular weight logarithme of marker protein. Relative mobility of marker protein was counted in comparing protein migration rate that measured from start line of separating gel till end of protein band, and dye migration rate.

Serum Preparation (Zakaria et al., 1998)
Blood serum were taken from 8 allergy sufferers of seafood or fish and 1 normal respondent who known by respondent quisioner and agreement. Blood of respondent which taken was 10 mL. Medical personels of Science and Technology Faculty of UIN Syarif Hidayatullah Jakarta collected the respondent bloods. The blood placed in tube without EDTA. The blood of allergy sufferer carried to laboratorium for separating its serum by centrifuge. The blood incubated for 1 hour, then sentrifuge at 2500 rpm for 20 minutes. ± 5 mL of supernatant taken then kept in freezer at -20 °C for the further analysis.

Immunoblotting (Bollag and Edelstein, 1991)
Uncolored gel of electrophoresis result transferred to other container. Afterwards, gel and nitrocellulose membrane stacked in transblotting device. The arranging of transblotting device stained with transfer buffer solution using filter paper and spons as an aid. Then the arranged device included in blotting container and filled with transfer buffer solution. Blotting was held at 90 Volt for 90 minutes. The transferred membrane released from the device series and performed fixation with methanol 50% for 2 minutes, then blocked using skim milk 5% in PBST for 1 hour at room temperature. Later, membrane washed with PBST 3 x 5 minutes. 300 µL of allergy sufferer serum with dilution rasio 1: 10 in PBST added to membrane, then incubated for 2 hours at room temperature. The washery used PBST for 3 x 5 minutes, next given 2 µL of HRP antibody conjugated monoclonal mouse anti-human IgE (with dilution rasio 1:3000 in PBST) and incubated while shaking for 1 hour. Membrane rewashed with PBST for 3 x 5 minutes, and added DAB substrate. The positive result marked by forming brown band in nitrocellulose membrane.

Characteristic of Raw Material
Nila baby fish attained the age of 2 until 4 weeks with size 3-6 cm were used at this study. Identification result from Faculty of Fishery and Marine Science of IPB showed that nila baby fish implied in family of Cichlidae with Latin name Oreochromis niloticus. Figure 1 showed the appearance of nila baby fish, colored golden-black with black ribbon or line in vertical artwart at tail fin till body. Amount of spines at dorsal fin ranged from 16-18. The pectoral fin colored black. Fish's mouth aimed up with unbulged head. Nila baby fish conformed to ovale body, with golden-cream underside and bright eye colour.

European
Medicines Agency Evaluation of Medicines for Human Use (EMEA) in The Guideline on Allergen Products: Production and Quality Issues (EMEA/CHMP) BWP/304831/2007, explained that concentration of protein, carbohydrate, fat, and water constituted parameters used to value the raw material of protein isolate. Allergen in foodstuff commonly formed of protein or glycoprotein (Cianferoni and Spergel, 2009).  Table 1 showed that the boiled nila baby fish had protein concentration 19.16% that similar to unripe nila baby fish (19.31%), otherwise protein concentration of fried nila baby fish increased 23.68%. Justi et al. (2003) identified 4 weeks old-unripe O. niloticus possessed 17.2 % of protein concentration, whereas according to Asia et al., (2015) the fried nila baby fish had 20.27 % of protein concentration. The difference of proximate value, included protein concentration of nila baby fish in every study caused by environment condition, fish habitat, nutrition or nutrient, and different fish food. (Soekendarsi et al., 2016). Meanwhile, fluctuation of nutrient decreasing of foodstuff caused by cooking process depended on kind of foodstuff and temperature (Sundari et al., 2015).
The highest of fat content acquired at fried nila baby fish 20.44%, that caused by presence of absorbed cooking oil in the fish. Besides that, the frying process engendered the decreasing of water content of nila baby fish product become 27.45%. It happened because frying process effected the movement of mass, which signed of movement oil into product (Saguy and Pinthus, 1995). Carbohydrate content obtained by difference of calculation, so if there were one or more of content from proximate parameter changed, the carbohydrate content would also changed. In this matter, if the water content declined thus the carbohydrate content would be huge in foodstuff. Table 2 showed the protein concentration of extract from unripe, boiled, and fried nila baby fish. The protein concentration of unripe nila baby fish extract was higher than protein concentration of boiled and fried nila baby fish extract. Boiling and frying process could affect protein solubility in phosphate buffer saline solution as solvent. The decline of concentration was suspected of related to protein loss or modification because of heating. Protein heating triggered the denaturation, colour changing, derivatization of amino acid, dissolution of peptide bond, and forming of active compound in censoring (Sundari et al., 2015). Figure 2 showed the protein profile of unripe, boiled, and fried nila baby fish. The amount of detected protein bands of unripe nila baby fish were 16 protein bands with molecular weight 10 kDa, 11 kDa, 12 kDa, 15 Da, 18 kDa, 27 kDa, 34 kDa, 40 kDa, 46 kDa, 65 kDa, 80 kDa, 99 kDa, 108 kDa, 119 kDa, 250 kDa, and more than 250 kDa. The boiled nila baby fish resulted 26 protein bands with molecular weight 10 kDa, 11 kDa, 14 kDa, 16 kDa, 27 kDa, 32 kDa, 38 kDa, 44 kDa, 59 kDa, 65 kDa, 69 kDa, 72 kDa, 84 kDa, 88 kDa, 93 kDa, 99 kDa, 102 kDa, 108 kDa, 113 kDa, 119 kDa, 125 kkDa, 132 kDa, 138 kDa, 145 kDa, 250 kDa, and more than 250 kDa. The amount of detected protein bands of fried nila baby fish were 16 protein bands with molecular weight 10 kDa, 13 kDa, 15 kDa, 32 kDa, 44 kDa, 56 kDa, 65 kDa, 75 kDa 84 kDa, 99 kDa, 113 kDa, 119 kDa, 125 kDa, 132 kDa, 250 kDa and more than 250 kDa. The protein band amount of boiled nila baby fish was bigger than unripe and fried nila baby fish.

SDS-PAGE
The protein bands of nila baby fish which resistant of boiling process were 10 kDa, 11 kDa, 27 kDa, 99 kDa, 108 kDa, 250 kDa and more than 250 kDa, whereas the protein bands of nila baby fish which resistant of frying process were 10 kDa, 65 kDa, 99 kDa, 250 kDa and more than 250 kDa. The protein bands of nila baby fish which resistant of boiling and frying process were 10 kDa, 99 kDa, 250 kDa and more than 250 kDa.
Heating process caused changing of protein solubility, thus affecting the amount and type of extracted protein in process of protein extraction (Palupi, et al 2015)

Immunoblotting
Immunoblotting is a standard method to detect an allergen or allergy from food using respondent serum. The chosen respondent was a respondent who has allergic to fish, seafood, or bug according to interview result. The interview of 20 respondents resulted that 8 respondents expressed allergy symptom while 12 respondents didn't express allergy symptom. Table 3 showed respondent data whose allergy symptoms, that would be taken their serum for immunoblotting test of allergen protein extract from nila baby fish. Immunoblotting test result on Figure 4 indicated that 8 respondents possessed specific IgE which could bind to allergen protein in unripe, boiled, and fried nila baby fish. It was expessed by forming brown band at nitrocellulose membrane. The brown band formed because of reaction between anti IgE human antibody labeled HRP (Horseradish Peroksidase) enzyme and DAB (3,3-Diaminobenzidine) substrate.
Respondent (1), (2), (3), (5), (6), (7) and (8) had specific IgE that could bind to allergen protein in boiled and fried nila baby fish, whereas the mentioned respondents didn't have specific IgE that could bind to allergen protein in unripe nila baby fish. Respondent (4) had specific IgE that could bind to allergen protein in unripe nila baby fish but didn't have specific IgE that could bind to allergen protein in boiled and fried nila baby fish. It is showed that 7 respondents from 8 respondents positively had allergic to boiled and fried nila baby fish but had not allergic to unripe nila baby fish, while only one respondent had allergic to unripe nila baby fish. The result indicated that the processing food by boiling and frying triggered the allergenicity increasing of nila baby fish.
According to research by Toda et al., (2014) exposed that respondents more allergic to processing food product than unripe food, e.g. extract of fried peanut linked IgE tighter than unripe peanut. The increasing of allergenicity caused by boiling and frying process which was suspected of related to tridimensional structure transformation of protein, so the epitope that formerly closed and unreactive with specific IgE antibody turned it open and reactive. The food processing product could string out the amount rise of protein epitope that reacted to specific IgE antibody in respondent serum (Boye and Godefroy, 2010). Allergenicity of allergen protein could influence on its owned epitope (Shreffler et al., 2004). Link between IgE and allergen commonly happened if allergen protein contained more than one epitope (Nowak-wegrzyn, 2003).
Molecular weight of allergen protein of boiled nila baby fish that bound to IgE antibody's respondent (1) were 95 kDa, 115 kDa, 145 kDa 250 kDa and more than 250 kDa, whereas IgE antibody's respondent (1) could bind to allergen protein of fried nila baby fish had molecular weight 93 kDa, 115 kDa, 145 kDa, 250 kDa and more than 250 kDa. IgE antibody's respondent (2) that linked with allergen protein of boiled nila baby fish had molecular weight 84 kDa, 99 kDa and more than 250 kDa, while that linked with allergen protein of fried nila baby fish had molecular weight 93 kDa, 133 kDa, 250 kDa and more than 250 kDa. Respondent (3) had IgE antibody that bound to allergen protein of boiled nila baby fish had molecular weight 127 kDa and more than 250 kDa, whereas those linked with allergen protein of fried nila baby fish had molecular weight 127 kDa, 144 kDa, 250 kDa dan lebih dari 250 kDa.
Molecular weight of allergen protein of boiled nila baby fish that bound to IgE antibody's respondent (7) were 69 kDa, 80 kDa, 94 kDa, 110 kDa, 116 kDa, 250 kDa and more than 250 kDa, whereas those linked with allergen protein of fried nila baby fish had molecular weight 43 kDa, 47 kDa, 67 kDa, 74 kDa, 83 kDa and more than 250 kDa.
IgE antibody's respondent (8) could bind to allergen protein of boiled nila baby fish had molecular weight 91 kDa, 105 kDa, 110 kDa, 116 kDa, 128 kDa, 134 kDa, 222 kDa and more than 250 kDa, while those linked with allergen protein of fried nila baby fish had molecular weight 95 kDa, 116 kDa, 121 kDa and more than 250 kDa.
The difference of molecular weight in each respondent showed that respondent 4 only sensitive to unripe nila baby fish, meanwhile respondent (1), (2), (3), (5), (6), (7) and (8) only sensitive to processed nila baby fish. The negative respondent towards nila baby fish showed to normal person that happened unbinding between IgE and allergen protein, so there wouldn't result a brown band.
Allergen come from animal (include fish and shell) commonly possessed a high thermostability and so the allergenicity of the protein would not disappear but moreover increasing (Boye & Godefroy, 2010). Heating process to nila baby fish suspected would change the protein conformation include the protein epitope, so that caused the increasing of protein allergenicity character of nila baby fish.
In other study, O. mossambicus was identified had allergen protein with molecular weight 17 kDa till 114 kDa and major protein at 32 kDa. The mentioned protein named Ore m4 implied in tropomyosin group (Liu, et al., 2012). Allergen protein in respondent (4) with molecular weight 31 kDa approached molecular weight of O.mossambicus at 32 kDa (Ore m4). The molecular weight 31 kDa suspected of allergen protein in tropomyosin group.

CONCLUSION
Type of nila baby fish used in this study was Oreochromis niloticus. Proximate characteristic of unripe nila baby fish changed through process of boiling and frying. Also, allergen protein profile of nila baby fish was influenced by boiling and frying process. The amount of protein bands of boiled nila baby fish were more than unripe and fried nila baby fish.
Nila baby fish supposed contained allergen protein which resistant of heating process with molecular weight 10 kDa, 99 kDa, 250 kDa and more than 250 kDa. The process of boiling and frying to nila baby fish would increase the allergenicity character of nila baby fish.