Cheese and yogurt are two kinds of nutritious dairy products that are used worldwide. The major preservatives in dairy products are sodium benzoate, potassium sorbate, and natamycin. The maximum permitted levels for these additives in cheese and yogurt are established according to Iranian national standards. In this study, we developed a method to detect these preservatives in dairy products by reversed phase chromatography with UV detection in 220 nm, simultaneously. This method was performed on C18 column with ammonium acetate buffer (
Nowadays, preservation techniques have considerable role in food industry. Generally, they are used for improving the quality with boosting durability of products and thus enhancing food shelf life. The rate of food spoilage can be controlled by many procedures such as suitable packaging for preventing available oxygen, sterilization, pasteurization, dehydration (drying), smoking, freezing, and food additive [
Dairy products as an important nutritional recommended consuming in daily and appropriate amounts. The safety of dairy products should be considered in the presence of preservatives. The acceptable daily intake (ADI) represents amount of daily consumption of substance without any risk even for a lifetime. According to ADI, the maximum permitted limit for food additives is based on mg/kg of body weight. The most commonly used preservatives in dairy products such as cheese and yogurt are benzoate, sorbate, and natamycin [
Sodium benzoate (E211) is known as the first chemical preservative approved in food products by the US Food and Drug Administration (FDA). Its solubility is more than other salt of benzoate like potassium and calcium. In an extensive review Sieber et al. (1995) investigated natural occurrence of benzoic acid in many types of dairy products [
Potassium sorbate (E202) is produced from the potassium salt of 2,4-hexadienoicacid (sorbic acid). The carboxyl group and conjugated double bonds in the sorbate structure are reactive and can have strong effect on the antimicrobial activity as well as the quality and the safety of the product [
The use of benzoate, sorbate, and natamycin in dairy products as additives is forbidden in Iran; only sorbate is permitted for additives in processed cheeses at a maximum level of 1000 mg/kg. Therefore, determination of these preservatives is needed for safety and quality control of dairy products.
There are various analytical methods for determination of these preservatives in cheese and yogurt such as thin layer chromatography, UV spectroscopy, high performance liquid chromatography (HPLC), and gas chromatography (GC). HPLC is the most common analytical technique for the detection and quantification of these preservatives. A practical method should be developed to identify all preservatives simultaneously [
The aim of the present study is to develop effective, simple, efficient, and fast RP-HPLC method to detect the three preservatives in one runtime. A simple extraction procedure was selected for measuring three preservatives from cheese and yogurt matrix. The result of the validation method parameters indicates the accuracy of the method. Finally, in order to ensure effective quality control in dairy products, the method was tested on 195 samples of commercial cheeses and yogurts in Iranian markets.
In this study, 195 samples of 15 brands and 5 types from commercial cheeses and yogurts were purchased from supermarkets located in Tehran. The cheeses samples were categorized as follows: cream, processed, Labneh (Iranian cheese), lactic, and brined cheeses, and yogurts sample were classified as follows: common yogurt and probiotic and flavored yogurt (fruit, vegetable, and strained yogurt) according to Iranian national standard (ISIRI).
Deionized water was prepared through the Thermo Scientific Barnstead Easy pure II system. Sodium benzoate and potassium sorbate used in this study were obtained from Merck (Darmstadt, Germany). High purity standard of natamycin was purchased from Sigma-Aldrich (Wisconsin, USA). The structures of these preservatives are illustrated in Figure
Chemical structures of sodium benzoate, potassium sorbate, and natamycin.
Chromatographic analysis was carried out on 1200 series HPLC from Agilent technology equipped with four solvent lines, high pressure pump, degasser, 20
Individual stock standard solutions of sodium benzoate and potassium sorbate were prepared in deionized water (1000 mg/kg) and natamycin stock standard was in methanol (1000 mg/kg). The mixed standard solutions were prepared by diluting the stock solutions appropriately with mobile phase to give a concentration between 5 mg/kg and 40 mg/kg.
The pH of samples was measured by 827 model of Metrohm pH meter according to Iranian national standard (ISIRI) [
The calibration curves of each preservative were prepared over the range of 5–40 mg/kg. The linearity and the correlation coefficient for each standard curve of benzoate, sorbate, and natamycin were calculated.
The detection limit (LOD) and quantitation limit (LOQ) were expressed as follows:
The obtained data was analyzed by MATLAB R2010b and the SPSS statistical package, version 16 (SPSS Inc., Chicago, IL, USA). Analysis of variance (ANOVA) was utilized to evaluate the differences of distribution among 5 types of cheeses and yogurts. Statistical significance was set at
195 commercial dairy products from 15 brands in 5 kinds of cheeses (cream, processed, Labneh, lactic, and brined cheeses) and yogurts (regular, probiotic, fruit, vegetable, and flavored strained yogurt) were prepared in April 2015 till August 2015.
In this study, samples were classified for checking with their relevant standards according to the name labeled on the packaging.
The separate methods for determination of benzoate, sorbate, and natamycin have been used in Iranian national standard [
To optimize the extraction, the chemical structures of these preservatives were considered (Figure
Analytical characteristics of the method validation.
Analyte | Calibration equation | Correlation coefficient | LOD |
LOQ |
Recovery (%) | RSDr ( |
||
---|---|---|---|---|---|---|---|---|
Yogurt | Cheese | Yogurt | Cheese | |||||
Sodium benzoate |
|
0.999 | 0.326 | 0.989 | 94.163 | 93.316 | ±0.549 | ±0.799 |
Potassium sorbate |
|
0.998 | 0.520 | 1.575 | 88.382 | 85.245 | ±1.332 | ±1.394 |
Natamycin |
|
0.997 | 0.511 | 1.548 | 87.853 | 87.221 | ±0.549 | ±1.646 |
There were other studies for determination of these preservatives individually by chromatographic methods [
HPLC chromatogram: (1) sodium benzoate, (2) potassium sorbate, and (3) natamycin.
Peak identification was achieved by comparing the retention times of standard compounds, and quantification was based on using the calibration curves fitted by linear regression analysis. The results of the calibration data, LOD, LOQ, and the recovery of each preservative are illustrated in Table
In this study, an HPLC method was developed and characterized with minimizing sample preparation for the determination of preservatives in dairy products. There is some similarity between our report and other works, but shorter run-time and suitable resolution of this study can reduce the cost of analysis and have some benefits in quantification as well as LOD, LOQ, and recovery.
The use of benzoate, sorbate, and natamycin as additive for cheeses and yogurts is forbidden by Iranian national standards [
The method was applied to several kinds of commercial cheeses and yogurts. Each sample was divided according to kinds and brands, as has been described in sampling and results are shown in Table
The amount of pH, sodium benzoate, potassium sorbate, and natamycin (mg/kg) in cheese and yogurt samples.
Brands | Preservatives (mg/kg) | pH | |||||||
---|---|---|---|---|---|---|---|---|---|
Sodium benzoate | Potassium sorbate | Natamycin | |||||||
|
Range |
|
Range |
|
Range |
|
Range | ||
Yogurt | Common |
|
4.573–43.101 |
|
0–34.181 |
|
0–9.640 |
|
3.76–4.72 |
Probiotic |
|
0–28.320 | ND | ND | ND | ND |
|
3.79–4.49 | |
Vegetable |
|
0–58.188 |
|
0–183.730 |
|
0–66.460 |
|
4.10–4.88 | |
Fruit |
|
8.729–40.541 |
|
117.600–240.600 |
|
0–27.190 |
|
3.70–4.59 | |
Strained |
|
2.076–22.931 |
|
0–11.880 |
|
0–6.910 |
|
3.98–4.46 | |
|
|||||||||
Cheese | Brined |
|
3.717–125.771 |
|
0–99.561 |
|
0–158.025 |
|
4.23–6.01 |
Lactic |
|
1.735–3.457 | ND | ND | ND | ND |
|
6.37–7.07 | |
Processed |
|
6.300–59.953 |
|
0–1150.320 |
|
0–62.132 |
|
4.93–6.88 | |
Cream |
|
5.988–27.275 |
|
0–247.367 |
|
0–12.132 |
|
4.50–6.63 | |
Labaneh |
|
10.241–23.617 |
|
0–232.075 |
|
0–8.622 |
|
4.93–5.35 |
45 yogurts and 19 cheeses among 195 samples contained natamycin. Concentrations of natamycin ranged from 4.355 to 66.462 mg/kg in yogurts and 5.249 to 158.025 mg/kg in cheeses. Natamycin was only detected in flavored yogurt (fruit, vegetable, and flavored strained yogurt). It seems that, for reducing the microbial contamination caused by the addition of flavorings, the manufacture added natamycin in flavored yogurt. The amount of natamycin in 21.05% of contaminated cheeses was found in higher concentration than the maximum legal limit of USA FDO (40 mg/kg) [
Illustration the natamycin content in (a) cheese and (b) yogurt samples.
The results show the presence of benzoate in most of yogurts ranging from 2.076 to 58.188 mg/kg, while benzoate was not detected in two probiotic samples and the lower amount of benzoate in other probiotic yogurts was observed. Therefore, it requires more studies on the probiotic yogurt.
All of the cheese samples contained benzoate ranging from 1.735 to 125.771 mg/kg, while they had not mentioned adding benzoate on the product labels. In other similar studies it was reported that the concentration of benzoate in Iranian dairy products was lower than 30 mg/kg [
Illustration of benzoate content and the pH in each cheese sample. The samples that located in the violet ellipse have the standard concentration and pH level.
Illustration of the benzoate content and pH in each yogurt sample. The violet rectangle shows the permitted range for concentration of benzoate and the suitable pH for the activation of it.
Sieber et al. analyzed benzoate in many kinds of dairy products and their report showed that benzoic acid occurred naturally by microbial degradation or addition of an herbal essence which contains benzaldehyde [
In Iranian national standards, benzoate is not considered as a food additive for dairy products. It seems that a maximum permissible quantity of this preservative should be defined for cheese and yogurt.
The concentration of sorbate in yogurts and cheeses ranged between 3.809–240.600 and 4.190–1150.320 mg/kg, respectively. 27 yogurt samples and 41 cheese samples were containing sorbate (Figures
Illustration of the concentration of Sorbate and pH for each cheese sample. The samples that are located outside the violet rectangle have more than allowed concentration of sorbate and pH.
Illustration of concentration content of sorbate in the yogurt samples.
It seems that adding preservatives in flavored yogurt was common as it was seen about natamycin and preservative was added for microbial growth control. Sorbate which was detected in processed cheese could be explained with maximum legal limit in Iran (1000 mg/kg).
All achieved results in this study are shown in the abstract space of data that is obtained by applying the principle component analysis. In Figures
Biplot of first three principal components for the cheese data.
Biplot of first three principal components for the yogurt data.
As is clear in Figure
The same analysis was done for the yogurt data. As is shown in Figure
In this research we aim to extract and evaluate the amount of preservatives including sodium benzoate, potassium sorbate, and natamycin among different cheeses and yogurts. Proposed method was demonstrating RP-HPLC method due to its simplicity, reliability, sensitivity, rapidness, and selectivity for detection at very low concentrations. It can recognize all preservatives at one wavelength in less than 12 min and involves minimal sample preparation. The results of this investigation showing the presence of sodium benzoate in dairy products such as cheeses and yogurt became evident naturally, while we observed natamycin and sorbate in some samples that had conflict to authorized level of these preservatives in Iranian national standards. Therefore, it is recommended that a maximum permissible limit of preservatives should be defined for cheese and yogurt.
None of the authors had any personal or financial conflicts of interest.
The authors would like to thank the participants for their enthusiastic support. This work was a student thesis supported by a grant from the Pharmaceutical Quality Assurance Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, no. 92-02-144-23027. Drug and Food Control Laboratory, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran, is the laboratory in which work was carried out.