Comparative between Ammonia Ion Selective Electrode and Dye Binding Method to study effect of Processing Methods on Protein Content of Plain Yogurt


Heba Kashour*, Lina Soubh

Department of Analytical and Food Chemistry, Faculty of Pharmacy, Damascus University, Damascus, Syria.

*Corresponding Author E-mail:



In this study, two analytical methods were used to determinate the protein, the ammonia ion selective electrode method and dye binding method using orange G and the spectrophotometer at λmax 478 nm by determining the linearity, accuracy, precision, limit of detection and limit of quantitation of each. In comparison, the dye binding method was chosen for its accuracy, repeatability, sensitivity (LOD, LOQ) and speed of performance. After that, it was applied to samples of prepared plain yogurt to study effect of different properties (source, heat treatment and type) of used milk on protein content of plain yogurt.


KEYWORDS: Protein, Dye binding, Orange G, Ammonia ion selective electrode, Plain yogurt.




The protein content of milk is of outstanding importance. Milk proteins are of the highest quality, both in digestibility and in content of essential amino acids. The protein content of milk is a major factor in determining the nutritive value and palatability of nearly all other manufactured dairy products. It is also recognized that 80% of the nitrogen of milk is attributable to the caseins (αs1, αs2, β, κ) and 20% to whey proteins (α-lactalbumine, β-lactoglobulin, immune-globulins)1,2.


The original production of fermented milk products derived from the need to prolong the shelf life of milk instead of being disposed. Yogurt is defined as the product being manufactured from milk with a gel structure that results from the coagulation of the milk proteins, due to the lactic acid secreted by defined species of bacteria cultures3. Until a few years ago no simple and practical method was available for determining protein with sufficient accuracy to use it.



The Kjeldahl method, the official method of determining protein, is impractical for routine use, because it is so costly, complicated, and time-consuming. In the last 30 years, many approximate methods (based on diverse principles) have been proposed2.


The dye binding method is an official AOAC procedure for determining protein in dairy products, including fluid milk, half-and-half, nonfat dry milk, ice cream mix, chocolate drink and buttermilk4,5.


Udy first applied the dye binding procedure to the determination of flour proteins. Later, he applied the same procedure to the determination of milk proteins6.


Orange G dye is a disulfonic acid which binds the basic groups of proteins near pH 2.0. Fraenkel-conrat and Cooper showed that in buffers at pH 2. 2 the acid dye, orange G, combined stoichiometric with these basic groups7,8.


Figure 1: Structure of Orange G dye


During the past few years there has been an active interest in potentiometry, and especially in ion-selective electrodes, to provide quantitative analytical techniques. An ammonia probe has been used to replace the distillation-titration procedure for estimating the ammonium content of Kjeldahl digest solutions. A procedure is described for estimating the total nitrogen content of barley, malt, wort and beer9.



Comparison between ammonia ion selective electrode method and dye binding method by using Orange G dye, and choosing the best one to be applied on the yogurt samples to study effect of different properties (source, heat treatment and type) of the milk used in the preparation on protein content of plain yogurt.



Reagents and solutions:

Dye binding method:

Orange G dye was purchased from Titan Biotech Ltd, Citric acid, Thymol. Orange G dye reagent is prepared by dissolving 1 g of the dye in a solution containing 21 g of citric acid and 2.5 ml of a 10% thymol solution in alcohol (used as a preservative). The final volume is made up to 1000 ml6.


Ammonia ion electrode method:

Ammonium sulfate (NH4)2SO4 99.5% (Panreac, Spain). Sulfuric acid H2SO4 (95-98%) (Panreac Quimica, Spain). Copper catalyst solution CuSO4.5H2O we prepared solution (0.05 g/mL H2O) (Rhodia, France). Sodium hydroxide solution NaOH 10 N (Panreac, Spain). Potassium sulfate K2SO4.


Samples: 3 samples of each cow, sheep, goat and soy milk.

3 samples of each pasteurized milk (Heating at 85 °C for 20–30 min/at 90–95 °C for 5 min)3.

UHT milk (Heating at 145 °C for 1–2 sec)3.

Sterilized milk (Heating at 110 °C for 30 min/at 130 °C for 40 sec)3.

3 samples of each full fat and low fat milk

were obtained from many local markets and farms in Damascus.



Kjeldahl Digestion Apparatus (FOSS), NH3 ISE Ammonia Sensing Electrode (ionode), Spectrophotometer UV-VIS model HTACHI U-1800, sensitive balance was produced by Sartorius, pipettes titration, balloons titration, filter paper 0.33 mm.


Experimental Procedures:

Preparation of Plain Yogurt:10

The milk samples was filtered of impurities using clean gauze. Then, the temperature of milk was gradually increased to 45 ºC for the purpose of adding 1.5 - 3% (w/v) of starter culture and stirring for 4 min, where they were kept at 42 ºC for 4 h until the completion of coagulation. Manufactured yogurt was stored in a refrigerator at 5 ºC.


Analytical methods:

1. Dye Binding method:

Polar groups in proteins can bind oppositely charged dyes to form insoluble protein-dye complexes. A known excess of dye is required, and the protein content is estimated from the amount of unbound dye. The dye concentration can be measured spectrophotometrically7.


Determination the wavelength:

The absorbance spectrum of the Orange G solution (10 ppm) was scanned between (380-700) nm against the corresponding blank. The maximum absorbance was at 478 nm. The spectrum was shown in figure (2).


Figure 2: Determination of λ Max of Orange G dye.


Analysis procedure:7

A standard curve relating absorbency at 478 nm to concentration of orange G was established on serially diluted dye solutions. The operating range of this curve was between 2 - 10 ppm of dye concentration.


The yogurt sample (1 g) was mixed with 25 ml of the dye reagent. The mixture was stoppered and shaken for about 15 sec and allowed to stand for 30 min before filtering through a 0.33 mm filter paper.


The concentration of unbound dye was read from the standard curve. This value was multiplied by the total sample volume to give the total amount of unbound dye. The bound dye was found by subtracting this amount from the total amount of dye added (25 mg).


The dye binding capacity was then calculated as the ratio of dye bound per unit of protein . From this ratio , its reciprocal was used as a factor for multiplying the amount of dye bound by unknown samples to find the amount of protein present.


The protein content of the yogurt was determined by the following formula:

Percent protein =  ×100

Where: Q1 = original dye amount (25 mg)

Q2 = amount of unbound dye in filtrate (mg)

DBC = dye binding capacity (mg dye/ g protein)


2. Ammonia Ion Selective Electrode method:

A silver-silver chloride electrode is used as an internal reference electrode and the sensing electrode is a flat-ended pH electrode separated from the sample solution by a gas permeable hydrophobic membrane. The passage of ammonia through the membrane alters the pH of the thin film of ammonium chloride solution trapped between the membrane and the flat end of the pH electrode. The ammonia content of the sample will therefore determine the E.M.F produced by the cell. The response of the probe is logarithmic and follows the Nernst equation9.


Analysis procedure:

The yogurt sample (1 g) was digested in a mixture containing sulphuric acid (20 ml) and potassium sulphate (12 g) with Copper (1 ml) as catalyst in order to convert the amino-nitrogen of the organic materials into ammonium hydrogen sulphate. The sample was then diluted with distilled water to 100 ml and made alkaline using sodium hydroxide 10 N (2 ml) and the ammonia released measured with the ammonia ion-selective electrode11,12.



Validation of the two analytical methods:


Five-level protein series was established for known protein concentration diluted yogurt. The absorbance was measured. The absorbance is plotted against the protein amount.


Eight-level standard series was established for ammonium ion. The practical concentration was measured. The practical concentration is plotted against the theoretical concentration.


Both methods had good linearity as shown in (Fig. 3,4).


Figure 3: Calibration Curve of dye binding method



Figure 4: Calibration Curve of ammonia ion selective electrode



Accuracy should be reported as relative error by measuring three known concentrations of protein (0.022, 0.033, 0.044 g) and ammonium ion (0.4, 0.6, 0.8 ppm).



The relative standard deviation RSD% (coefficient of variation) were reported by measuring six solutions at the same concentration (0.022 g ptotein) and (0.6 ppm ammonium ion).


Table 1 shows all two validation results.


Dye binding method was chosen to apply it to samples because of its good linearity, accuracy and precision. It is also simple to operate. Other than being rapid (30 min), the dye binding method has the advantage of directly estimating the protein content in the sample rather than the ammonium content as measured by ammonia ion electrode procedure.


Table 1: Results of analytical methods validation


Dye binding method

Ammonia ion selective electrode


R2 =0.9971

y = -4.3636 x + 0.448

R2 =0.9957

y = 0.9909 x - 0.0345

Accuracy (Relative error)



Precision (RSD%)



LOD ppm

3.54 mg protein

0.28 ppm protein

LOQ ppm

10.74 mg protein

0.85 ppm protein


30 min

2.5 h

Effect of source of used milk:

The average protein content of sheep, goat, cow and soy yogurt were 6.71, 3.04, 3.5 and 3.28%, respectively [Table (2)].


The differences in protein contents of yogurt may be due to the gross composition of milk shows large inter-species differences, Because the nutritional requirements of the neonate depend on its maturity at birth, its growth rate and its energy requirements, which depend mainly on environmental temperature1.


Milk composition traits of sheep and goat milk from 10 studies show that sheep milk is richer in protein than goat milk13.


Sheep yogurt has higher protein content and good textural characteristics (firmness and viscosity) than cow, goat and soy yogurt, while Goat yogurt has lower protein content and present poor textural characteristics (weak gel), which may be due to has lower amounts of αs1-casein, resulting in softer gel products, a higher water holding capacity and a lower viscosity14.


Although soy yogurt has low protein content, it has good texture (hard gel). It may be due to soy proteins have many of the chemical and physical properties required for use in the dairy industry, which contributes to increased viscosity and gel strength15.


Table 2: The protein content of samples

Source of milk

Sample 1

Sample 2

Sample 3


















Effect of heat treatment of used milk:

The average protein content of pasteurized, UHT and sterilized yogurt were 3.496, 3.49 and 3.473%, respectively [Table (3)].


The protein content of the different yogurt samples is generally close to each other, and this is due to The caseins (present 80% from milk protein) are very heat-stable. Milk may be heated at 100°C for 24 h without coagulation and withstands heating at 140°C for up to 20- 25 min. The heat stability of the whey proteins (present 20% from milk protein) is typical of globular proteins and they are denatured completely on heating at 90°C for 10 min. The remarkably high heat stability of the caseins, which is probably due to their lack of typical stable secondary and tertiary structure1,16.


The reason may be to the protein content of sterilized yogurt samples are less because sterilization causes considerable changes in the proteins including casein and whey proteins, while pasteurization and UHT cause only denaturation in whey proteins17.


Table 3: The protein content of samples

Heat treatment of milk

Sample 1

Sample 2

Sample 3














Effect of type of used milk:

The average protein content of full fat and low fat yogurt were 3.49 and 3.65, respectively [Table (4)].


Low fat yogurt has higher protein content and has good texture (hard gel) than full fat yogurt, which is probably due to the cutting of the protein network, which form the gel, by the contained fat globules16.


These results agree with the study of Guinee and O’callaghan which studied effect of fat level on properties of processed cheese product18.


Table 4: The protein content of samples

Type of milk

Sample 1

Sample 2

Sample 3

Full fat




Low fat






As shown in this study, both of ammonia ion selective electrode and dye binding method has good linearity, accuracy, precision and Sensitivity but the dye binding method is rapidly (30 min) determine protein and does not need additional steps like digestion which takes more than 2 h.


The protein content in yogurt is influenced by source and type of used milk, while it is not significantly affected by the type of heat treatment.



The authors declare that there is not any conflict of interest related to this work.



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Received on 03.01.2021            Modified on 10.04.2021

Accepted on 06.07.2021           © RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(12):6257-6261.

DOI: 10.52711/0974-360X.2021.01082