Determination of Benzoic Acid, Sorbic Acid and HMF in Grape Molasses Produced by Traditional and Modern Methods

Sorbic acid and benzoic acid have long been widely used in the food industry to inhibit the growth of various bacteria, yeasts and fungi, especially in acidic media. The health effects have led to limitation on the concentrations that can be used in food. In most studies, HMF has been found to have carcinogenic effects such as cytotoxicity toward mucous membranes, the skin and the upper respiratory tract; mutagenicity; chromosomal aberrations; and carcinogenicity toward humans and animals. Because of these reason, the analytical determination of these compounds is important for consumer interest and protection. The aim of this study is to determine the concentration of sorbic acid, benzoic acid and HMF in grape molasses samples by using HPLC. For HPLC analysis, a Zorbax SB RP18 (150 × 4.60 mm i.d. × 5 μm) column was selected as the stationary phase at 25°C. In sixteen grape molasses samples (produced by traditional and modern method), sorbic acid, benzoic acid and HMF concentrations were determined by HPLC method. HMF was detected in all of the commercial and traditional type molasses samples. Only two commercial molasses sample, sorbic acid and benzoic acid were detected. Original Research Article Şanli and Altınbaş; JPRI, 33(38B): 330-336, 2021; Article no.JPRI.71748 331


INTRODUCTION
Preservative additives are substances used to prevent undesirable changes in foods during storage and transportation. Consumer demand tends to nutritious, artificial additive-free, readyto-eat, and long shelf-life foods. This situation is the reason for pressure on researchers to study the level of additives and harmful components to obtain safe and healthy foods [1].
In these days, benzoic and sorbic acids and their respective sodium, potassium, and calcium salts are widely used as a chemical preservatives. Especially they are generally used to for the extension of shelf life and enhancing of food quality. Sorbic acids and benzoic acids are generally active in foods of low pH value and essentially ineffective in foods at neutral pH values [2,3].
Accrued interest is given to preservatives as recent studies have reported serious side effects associated with these substances. The topical usage of potassium sorbate and sodium benzoate-containing product resulted in skin reactions such as rash, urticaria, and contact dermatitis [4,5].

5-Hydroxymethylfurfural
(5-hydroxymethyl-2furfuraldehyde, HMF, CAS No. 67-47-0) is formed during the thermal treatment of carbohydrate-containing foods, is considered as a potential carcinogen for humans. The previous literature has shown that it was concluded that sugary food heated under household cooking conditions could act as an initiator and promoter of colon cancer because of the presence of HMF [6]. The formations of HMF were inevitable in vinegar and soy sauce, which were seen as essential liquid seasonings for home cooking. Theobald et al. [6] also indicated that it was difficult to estimate the HMF content in commercial samples which was prepared in households [7].
Since the 1950s there have been reports of HMF in food. It has been identified in a wide variety of heat processed foods. Depending on production technology and storage, levels in food vary considerably. While HMF is practically not present in fresh food, high levels in the g/kg range can be found in dried fruits, coffee and caramel products. In honey and some other food, concentrations of HMF can be used as an indicator of heating and storage changes. For example, the Codex Alimentarius standard sets a maximum limit for HMF in honey of 40 mg/kg (80 mg/ kg in tropical honey) as a way of assuring that the product has not undergone heating during processing [8].
Molasses is one of our traditional foods, although it is industrial production, it is also produced in small family businesses and traditional methods. Grape molasses is Turkey's oldest sweetener used before cane sugar and honey were introduced to the Middle East and Mediterranean regions. In the past years molasses, which is one of the basic nutrients of human beings, has become less productive in the changing world conditions. In Turkey molasses is made between at the end of September and the beginning of October when the grapes are mature and this time is called molasses time.
In order to protect human health, determination of maximum permitted amounts of these additives and amount of HMF in food products is very important. Consequently, easy and reliable analysis methods for detection of these additives in foodstuffs are required for food safety.
The objective of the present study was to measure the concentration of benzoic acid, sorbic acid and HMF (Table 1) in food products such as grape molasses produced by traditional and modern methods by using of HPLC technique. HPLC detection has become the most widely applied analytical separation technique because of its superior performance and reliability, especially in the food, pharmaceutical, environmental, forensic, clinical, and flavor sciences [7].

Chemicals
All chemicals in this study were used without further purification. The standard of sorbic acid, benzoic acid and HMF were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). Sodium hydroxide and acetic acid were also obtained from Sigma-Aldrich. HPLC-grade acetonitrile (ACN), methanol (MeOH Darmstadt, Germany), acetic acid were purchased from Merck. Ultrapure water, with conductivity lower than 0.05 μS/cm, was obtained with a Milli-Q system (Millipore, Bedford, MA, USA). Stock solutions of sorbic acid were prepared by dissolving in water (50 mL) to make a 100 mg/L solution. HMF and benzoic acid standards were prepared in methanol. All solutions were protected from light, and all solutions were stored at 4° C for short time usage (daily) and at − 20°C for long-term usage (between days).

Apparatus
The HPLC analysis was carried out on an Agilent 1260 series HPLC system with ternary solvent pump, online degasser, automatic injection system, column heater, and multi wavelength detector [7]. UV detection was used (235 nm for sorbic acid and benzoic acid; 284 nm for HMF). Analyses were run at a flow rate of 1.0 mL·min − 1 .
Because of the peak shape and analysis time, an Zorbax SB RP18 (150 × 4.60 mm i.d. × 5 μm) column was selected as stationary phase at 25°C. 35% (v/v) ACN-water containing acetate buffer at pH 4.7 was used as a mobile phase for sorbic acid and benzoic acid. For HMF determination, %10 (v/v) MeOH-water containing 0.1 (v/v) acetic acid was selected as a mobile phase. Isocratic elution was used for all analysis.

Preparation of Molasses Samples for HPLC Analysis
Sixteen grape molasses samples were collected from Turkey, Usak (Eight commercial type and eight traditional type). Five grams of grape molasses samples were dissolved in 25 ml of water, transferred quantitatively into a 50 ml volumetric flask, added by 0.5 ml of Carrez solution I and 0.5 ml of Carrez II and make up to 50 ml with water. The resulting filtrate was used for 20 μL per injection for chromatographic analysis. The peak area was obtained for each solution on the ordinate against the compounds concentration, in milligrams per liter, on the abscissa.

RESULTS AND DISCUSSION
The use of sorbic acid and benzoic acid in processed foods is extremely important. Not using this antimicrobial agent may cause microbial activities that lead to food poisoning [18]. However, there are some limitations [7]. Fermented products are the foremost food groups that have limitations for food additives because of their importance in healthy nutrition, prevention, and curing effects [7]. Turkish Food Codex that is prepared considering scientific truths and conclusions of Codex Alimentarius and in accordance with European Union directives is effective in such applications in Turkey [19]. [20,21] and is also formed from the degradation of sugars at high temperatures [22]. Furfural and Methylfurfural are also furanic compounds formed in non-enzymatic browning reaction during thermic treatments.

Hydroxymethylfurfural (HMF) is an intermediate product in the Maillard reaction
In this paper sorbic acid, benzoic acid and HMF contents of sixteen grape molasses sample were determined by HPLC method.
A set of sorbic acid, benzoic acid and HMF standards were tested to determine the validation parameters (linearity, range, detection limit, and quantitation limit) [23]. The linearities were calculated by plotting the peak area versus concentration of compounds. Five standard solutions were prepared for claibration of compounds. Each solution was injected in duplicate. The calibration curves were obtained by linear leastsquares regression. The validation data are reported in Table 2 , respectively, where s is the standard deviation of the response and m is the slope of the corresponding calibration curve [24,25].
In Table 3, sorbic and benzoic acids contents of eight commercial type grape molasses samples were given. For six grape molasses samples, sorbic acid and benzoic acid could not be determined. In only two commercial molasses samples, they could be detected and calculated as a highest value 1970 mg.kg -1 for sorbic acid, 988 mg.kg -1 for benzoic acid.   Table 4. HMF content in commercial type grape molasses samples

Sample
Grape molasses 1 (commercial type) Grape molasses 2 (commercial type) Grape molasses 3 (commercial type) Grape molasses 4 (commercial type) Grape molasses 5 (commercial type) Grape molasses 6 (commercial type) Grape molasses 7 (commercial type) Grape molasses 8 (commercial type) For eight traditional molasses sample, sorbic acid and benzoic acid were not detected. In Fig. 1, grape molasses sample for sorbic and benzoic acid chromatogram were given.
In all of the commercial and traditional type molasses samples were detected HMF. In commercial type of grape molesses sample, it was found between 6.8-102.4 mg.kg The chromatogram of molasses analysis were given in Fig. 2.

CONCLUSIONS
Sorbic acid and benzoic acids are widely used in food sample in Turkey. Therefore, the use of these compounds should be supervised by the Turkish public health authorities. In this study, the determination of sorbic acid and benzoic acid in 16 commercial and traditional molasses samples were done by HPLC method. Also evaluation of HMF content of 16 different traditional and commercial molasses samples could be achieved in this paper.

DISCLAIMER
Also there is no conflict of interest between the authors and producers of the products. The research was funded by personal efforts of the authors.

CONSENT
It's not applicable.

ETHICAL APPROVAL
It's not applicable.