Investigation of the quality, antimicrobial, and antioxidant effects of commercial carob molasses and determination of phenolic acids by using HPLC

Abstract

Abstract

This study examines the bioactive properties and quality of commercial carob molasses, commonly consumed by children in Turkey for tonsillitis relief, while highlighting potential risks. Total phenolic content was analyzed spectrophotometrically, and phenolic acid composition by high‐performance liquid chromatography (HPLC). For the determination of health benefits, antimicrobial and antioxidant activity methods were used, and for quality control, pH, 5‐hydroxymethylfurfural (5‐HMF), ash content, and water‐soluble solids were determined. The results showed that there were differences in the bioactive properties of the samples. Sample 2 was found to have the highest antioxidant potential (93.86 ± 8.46 ascorbic acid equivalent [AAE]) and metal chelation ability (56.99 ± 1.91%). All carob molasses samples showed antimicrobial activity against Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, and Candida krusei, and minimum inhibitory concentration (MIC) values ranged from 200 to ≥400 µg/mL, approaching bactericidal/fungistatic effects. It was concluded that 5‐HMF levels in all samples exceeded the 15 mg/kg limit and that many samples' pH values (between 4.71 and 5.18) and ash contents were outside the acceptable ranges. HPLC analysis showed that Sample 2 had the highest gallic acid content (569.423 ± 0.003 µg/100 mg), while Sample 6 had the highest cinnamic acid content (14.838 ± 0.003 µg/100 mg). As a result, it was observed that carob molasses, consumed with the thought that it was beneficial for tonsillitis, was not as effective as expected on the related microorganisms. It was concluded that better quality control in carob molasses production and storage is needed, and stricter food regulations should be implemented for public health.

Practical Application

Carob is a fruit with important nutritional value for both adults and children thanks to its high sugar, dietary fiber, mineral, and phenolic compound content. The molasses obtained from the fruit is frequently used as a food supplement or to alleviate respiratory tract disorders in children. The findings of this study suggest that carob molasses, which is widely used for its health benefits, is not very effective against pathogens associated with respiratory tract disorders and underline the critical importance of improving quality criteria related to production, quality (5‐hydroxymethylfurfural [5‐HMF], pH levels, and ash content) control, and ensuring its safety after it is placed on the market. This is particularly important for the protection of vulnerable groups, including children, who may be more sensitive to potential risks. It is, therefore, essential that the quality of food products available on the market is rigorously assessed and that health claims are supported by scientific evidence.

1. INTRODUCTION

As a medicinal and aromatic plant, Ceratonia siliqua L. is considered one of the oldest medicinal plants on earth due to its geographic origins (Ramón‐Laca & Mabberley, 2004). C. siliqua is regarded as an important plant for both economic and environmental reasons in Mediterranean coastal countries, where it is not only found growing naturally but is also widely cultivated (Goulas et al., 2016). The fruits of the plant, known as “Carob, Ballıbaba, Ballıboynuz, Hannıp, Harrup, Kaluş, Melük” in Anatolia, are consumed as food (Bakış et al., 2011).

Carob molasses, a traditional product made from the fruits of C. siliqua, has been consumed in Turkey for many years. The ripe, brown fruits are washed, shredded, and boiled in water until they form a thick consistency, from which molasses is obtained. Industrially, molasses is produced using a cold infusion method under a steam vacuum. Despite its high sugar content, carob molasses has a low glycemic index and is known for its many traditional uses (Hsouna et al., 2011).

Molasses, a traditional food product in Turkey, is made from sugar‐rich fruits such as grapes, mulberries, apricots, and carob. The juice of these fruits is concentrated to a soluble dry matter content of 70%–80%. Molasses is a highly nutritious product, containing carbohydrates, minerals, organic acids, proteins, flavonoids, and phenolic compounds. In recent years, carob products, especially carob syrup, have gained a lot of interest because of their possible health advantages (Zannini et al., 2024) Due to its high natural sugar content, carob molasses can be used as a natural sweetener and colorant in products such as ice cream and cakes (Akkaya et al., 2012; Biner et al., 2007). Carob molasses is frequently consumed by adults and children for energy‐giving, nutritious, and disease‐preventive purposes, especially in upper respiratory tract infections. It was determined that even children with cancer were given carob molasses as a complementary treatment option at a rate of 27.6% (Kemer & Dalgiç, 2023). Considering the health benefits claims, the antimicrobial activity of this carob molasses, which is commonly consumed in society to support the treatment of tonsillitis (inflammation of the tonsils) in children and is characterized by a low glycemic index, has been investigated against pathogens primarily involved in respiratory tract infections. Tonsillitis is typically characterized by inflammation of the tonsils caused by bacterial or viral agents, and during infection, elevated oxidative stress may occur due to increased production of free radicals and insufficient antioxidant defense mechanisms. Antioxidants, which neutralize free radicals and reduce oxidative stress, are of critical importance, particularly in such infections. Therefore, the antioxidant activity of the investigated food product has also been evaluated. Furthermore, considering its widespread use and concerns about its quality and safety, qualitative and quantitative analyses were performed using ultraviolet (UV) spectroscopy and high‐performance liquid chromatography (HPLC) to determine the presence of significant secondary metabolite groups, such as phenols and phenolic acids, known for their strong antioxidant effects.

Additionally, the quality control analyses of the samples were conducted based on the criteria set by the Turkish Standards Institute (TS 13717), an official regulation in Turkey, for carob molasses, including parameters such as pH, 5‐hydroxymethylfurfural (5‐HMF), total ash, and water‐soluble solids. Studies conducted on commercial carob molasses in the Turkish market are generally related to their nutritional value and adulteration (Öner et al., 2024; Peren Aykas et al., 2023). A review of the literature revealed that no prior research has been conducted on commercial carob molasses in Turkey within the scope of this study. As a result, it is thought that this study will provide valuable information in terms of forming an idea about the quality and effects of carob molasses, which is available in the Turkish market and is widely consumed especially by children and babies.

2. MATERIALS AND METHODS

2.1. Materials

Ten different commercial carob molasses samples were purchased from herbalists (aktars), pharmacies, and local markets in Ankara. Samples numbered 2, 3, 4, and 9 were obtained from pharmacies, sample number 1 was obtained from a local market, and samples numbered 5, 6, 7, 8, and 10 were purchased from herbalists (aktars). All samples were collected in 2022.

2.2. Antioxidant activity

2.2.1. Total antioxidant capacity (phosphomolybdenum method)

A 1 mL molybdate solution was added to the samples, followed by vortex mixing. The mixture was then incubated at 90°C for 90 min. After incubation, the tubes were rapidly cooled, and the absorbance was measured at 695 nm. The results were expressed as ascorbic acid equivalent (AAE) based on the method described by Prieto et al. (1999).

2.2.2. ABTS radical scavenging activity

The ABTS (2,2'‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid)) solution was prepared at a concentration of 7 mM and adjusted to a final concentration of 2.45 mM by adding potassium persulfate to generate the ABTS cation. The mixture was left in the dark at room temperature for 12–16 h to allow radical formation. It was then diluted with ethanol to achieve an absorbance of 0.70 ± 0.02. The diluted ABTS solution was combined with 10 µL of the sample solution and left to react for 6 min. The absorbance of the resulting mixture was measured at 734 nm, using gallic acid as the reference standard. Each experiment was performed in triplicate (Re et al., 1999). The percentage of inhibition (%) was determined using the formula provided below.

$$\mathrm{Inhibition}\left(\%\right)=[(A_{\mathrm{C}}-A_{\mathrm{S}})/A_{\mathrm{C}}]\times 100$$

where A _C is the absorption of blank solution and A _S is the absorption of sample solution.

2.2.3. Metal chelation capacity

A 2 mM FeCl₂ solution was added to the samples and allowed to react at room temperature for 5 min. Subsequently, 5 mM ferrozine (3,2‐(pyridyl)‐5,6‐bis(4‐phenylsulfonic acid)‐1,2,4‐thiazine) was introduced, and the absorbance of the Fe²⁺–ferrozine complex was measured at 562 nm after 10 min. Ethylenediaminetetraacetic acid (EDTA) solution, prepared at 1, 0.5, and 0.25 mg/mL concentrations, was used as a reference. After incubating at room temperature for 10 min, the color intensity was recorded in a microtiter plate reader at 562 nm and 25°C. The experiments were conducted in triplicate. The chelating capacity of the samples with Fe²⁺ was calculated using the equation provided below (Prietro et al., 1999; Figure 1).

$$\mathrm{Chelating}\mathrm{activity}\%=[1-\left(A_{\mathrm{S}}/A_C\right)]\times 100$$

where A _C is the absorption of blank solution and A _S is the absorption of sample solution.

FIGURE 1.

High‐performance liquid chromatography (HPLC) chromatograms of gallic acid (a), cinnamic acid (b), and Sample 5 (c).

2.3. Antimicrobial activity tests

The carob molasses samples were dissolved in water (H₂O) at a final concentration of 800 µg/mL, sterilized using 0.22 µm Millipore filters, and used as stock solutions. Ampicillin‐clavulanate (AMC), gentamicin (GEN), fluconazole (FLU), and ketoconazole (KET) were employed as standard agents. These standard antibiotics were prepared following Clinical and Laboratory Standards Institute (CLSI) guidelines and used at specific concentrations (µg/mL). Reference antibacterial agents were sourced from Sigma Chemical Co., dissolved in phosphate buffer (ampicillin, pH 8.0; 0.1 mol/mL), dimethyl sulfoxide (ketoconazole), or water (gentamicin, fluconazole), as outlined in CLSI (1996).

Ten different commercial carob molasses samples, obtained from herbalists (aktars), pharmacies, and local markets in Ankara, were also dissolved in water (H₂O) at a concentration of 800 µg/mL, sterilized via 0.22 µm Millipore filters, and used as stock solutions. Antibacterial activity testing was conducted using standard strains obtained from American Type Culture Collection (ATCC) and Refik Saydam Central Hygiene Institute (RSKK). The strains included Gram‐positive bacteria (Staphylococcus aureus ATCC 6538, Enterococcus faecalis ATCC 29212), Gram‐negative bacteria (Klebsiella pneumoniae ATCC 700603, Escherichia coli RSKK 95085, Pseudomonas aeruginosa ATCC 1069), and fungi (Candida albicans ATCC 10231, Candida krusei ATCC 6258). These strains were provided by Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology.

For bacterial culture and dilution, Mueller–Hinton Broth (MHB; Difco) and Mueller–Hinton Agar (MHA; Oxoid) were used, while RPMI‐1640 medium with L‐glutamine, buffered to pH 7 with MOPS (3‐(N‐morpholino)propanesulfonic acid), was utilized for fungal suspensions as described in CLSI (1996) and Özçelik et al. (2009). Antibacterial and antifungal activities were assessed using the broth microdilution method. Test samples at 800 µg/mL were added to the first row of 96‐well microplates, followed by twofold serial dilutions (800–6.25 µg/mL for samples; 32–0.25 µg/mL for controls). The minimum inhibitory concentrations (MICs) were determined as the lowest concentration preventing visible microbial growth. Minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) were defined as the lowest concentrations yielding no regrowth in subcultures, while bacteriostatic (MBS) and fungistatic (MFS) effects were noted when regrowth occurred.

The study, performed in triplicate (Aytemir & Özçelik, 2011), evaluated the antimicrobial properties of carob molasses against commonly encountered pathogens such as S. aureus, E. faecalis, K. pneumoniae, E. coli, P. aeruginosa, C. albicans, and C. krusei. These microorganisms are known to cause contamination in food and pose health risks, as microbial contamination can lead to spoilage, degradation, and reduced efficacy of active ingredients. Regulatory agencies like the Food and Drug Administration (FDA) set microbial contamination limits for aerobic mesophilic bacteria and yeast/mold to ensure product safety. The antimicrobial activity of carob molasses highlights its potential for both preventing microbial contamination and serving as a protective agent (Food and Drug Administration, 2022a, 2022b, 2022c; Orhan et al., 2012; Scientific Committee on Consumer Safety, 2015; USP Pharmacopeia, 2009).

2.4. Total phenolic content

A 10% (w/v) Folin–Ciocalteu solution and the sample were dispensed into the wells of microtiter plates and incubated for 5 min at room temperature. Following this, a 7.5% (w/v) sodium carbonate solution was added to each well, and the plates were incubated in the dark at room temperature for 30 min. The color intensity of the reaction was then measured at 735 nm using a microtiter plate reader (VersaMax ELISA Microplate Reader). Each sample was analyzed in triplicate, and the results were expressed as milligrams of gallic acid equivalents (GAE) per gram of sample.

A calibration curve was generated using standard solutions of gallic acid (Sigma‐Aldrich) at concentrations of 1, 0.5, 0.25, 0.05, and 0.01 µg/mL. The calibration equation obtained was y = 4.0103x − 0.0413, r ² = 0.9989, as reported by Zongo et al. (2010).

2.5. Analysis of phenolic acids by HPLC

Molasses samples were diluted at a 1:4 ratio using 25% acetonitrile and filtered through 0.42 µm pore size filters before being transferred into vials. The concentrations of gallic acid (Sigma Aldrich, G7384), protocatechuic acid (Sigma Aldrich, 03930590), vanillic acid (Sigma Aldrich, 94770), syringic acid (Sigma Aldrich, S6881), cinnamic acid (Sigma Aldrich), and 2‐hydroxycinnamic acid (Sigma Aldrich, H22809) were determined at 265 nm, their wavelength of maximum absorbance. Similarly, ferulic acid (Sigma Aldrich, 1270311), chlorogenic acid (Sigma Aldrich, C3878), caffeic acid (Sigma Aldrich, C0625), p‐coumaric acid (Sigma Aldrich, C9008), and sinapic acid (Sigma Aldrich, D7927) were analyzed at 320 nm due to their maximum absorbance.

HPLC analysis was performed using the HP Agilent 1260 series LC system. The ACE 5 C18 column (5 µm, 150 mm × 4.6 mm) was maintained at 25°C, and 20 µL of samples and standards were injected. The mobile phases consisted of Solvent A (acetonitrile:water:formic acid in an 80:20:0.1 ratio) and Solvent B (water:formic acid in a 100:0.1 ratio). A gradient elution program was applied: 5%–15% Solvent A from 0 to 10 min, 15%–20% from 17 to 22 min, 20%–30% from 22 to 32 min, and 0%–100% from 32 to 35 min, holding at 100% until the 43rd minute. The flow rate was set at 0.8 mL/min, and initial conditions were re‐established within 2 min (Gök et al., 2021).

2.6. Analysis of some chemical properties

The pH, 5‐HMF, total ash, and water‐soluble solids (Brix) of molasses samples were analyzed following the Turkish Standards (TS 13717). The pH values were measured using a pH meter. For determining the 5‐HMF content, test solutions were prepared by diluting the samples with water. A portion of the test solution was treated with p‐toluidine and barbituric acid solutions, and the red color developed in the presence of 5‐HMF was measured spectrophotometrically at 550 nm. The total ash content was determined using a muffle furnace. The Brix value, representing the water‐soluble solids, was measured with a refractometer, and the results were reported as °BX (Turkish Standards Institution, 2016).

2.7. Statistical analysis

The Microsoft Excel program was used to evaluate the experimental results. All values are given as mean ± SD (standard deviation). Statistical analysis was performed using analysis of variance (ANOVA; Dunnett's test) with GraphPad Prism software. Results with p‐values less than 0.05 were considered statistically significant.

3. RESULTS AND DISCUSSION

This study provides valuable insights into the bioactive properties of carob molasses, focusing on its antioxidant, antibacterial, and antifungal activities. The findings contribute to the growing body of research on carob, a natural product widely consumed for its potential health benefits. The novelty of this study lies in its comprehensive evaluation of various carob molasses samples, including their chemical composition, phenolic content, and bioactivity, which have not been extensively studied in the context of carob molasses. In particular, this research highlights the diverse antioxidant capacities of the samples, with some exhibiting significant metal chelation and antioxidant activities.

The antioxidant activity of the carob molasses samples in this study, assessed through total antioxidant capacity, ABTS radical scavenging, and metal chelation tests, provides important insights into their bioactive properties. The total antioxidant capacity, measured by the phosphomolybdenum method, showed that Sample 2 had the highest antioxidant potential (93.86 ± 8.46 AAE), surpassing other samples, though the overall values were lower than those reported in previous studies, such as Tounsi et al. (2020), which found average values of 107.41 ± 7.63 mg AAE/g in homemade carob molasses and 111.88 ± 5.50 mg AAE/g in commercial samples (Table 1). This difference highlights possible variations due to factors such as production methods and sample sources. In contrast, the ABTS radical scavenging activity was minimal across all samples, aligning with findings from Zannini et al. (2024), who reported a broad range of ABTS scavenging potential in carob syrups. Zannini et al. reported that the ABTS radical scavenging potential of the samples ranged from 4500 to 7500 Trolox equivalents/100 g (Zannini et al., 2024). Dhaouadi et al. reported that adding sugar to carob syrup reduced the antiradical potential of ABTS by 28% (p < 0.05; Dhaouadi et al., 2014). Fidan et al. found the ABTS radical scavenging potential of carob syrup as 441.11 ± 4.57 µM TE/g (Trolox Equivalent) dry weight (Fidan et al., 2019; Table 2).

TABLE 1.

Total antioxidant capacity results of carob molasses samples.

Sample code	Total antioxidant capacity (AAE mg/g) ± SD
1	57.99 ± 7.45
2	93.86 ± 8.46
3	75.30 ± 7.40
4	41.22 ± 5.30
5	68.34 ± 6.25
6	39.26 ± 4.79
7	60.31 ± 9.17
8	85.30 ± 7.94
9	69.24 ± 6.06
10	74.95 ± 8.34

Abbreviations: AAE, ascorbic acid equivalent; SD, standard deviation.

TABLE 2.

ABTS radical scavenging activity and metal chelation capacity results of carob molasses samples.

Sample code	Concentration (mg/mL)	ABTS radical scavenging activity % ± SD	Metal chelation capacity % ± SD
1	0.5	8.22 ± 5.50*	–
	1	9.10 ± 0.21*	–
	2	9.86 ± 2.81*	5.20 ± 13.89 ns
2	0.5	15.63 ± 1.10**	35.16 ± 2.98***
	1	8.64 ± 1.03*	35.48 ± 2.16***
	2	14.03 ± 2.44*	56.99 ± 1.91***
3	0.5	14.75 ± 0.86*	25.16 ± 16.27*
	1	14.29 ± 0.31*	36.94 ± 6.57**
	2	19.38 ± 3.32**	50.59 ± 0.73***
4	0.5	4.97 ± 2.81*	–
	1	4.68 ± 3.42 ns	–
	2	11.29 ± 1.08*	–
5	0.5	1.38 ± 2.26 ns	4.05 ± 3.24 ns
	1	0.14 ± 0.58 ns	21.81 ± 4.95**
	2	7.76 ± 2.09*	35.14 ± 3.99***
6	0.5	40.88 ± 3.43***	–
	1	4.85 ± 4.49 ns	–
	2	–	–
7	0.5	12.68 ± 1.76*	8.80 ± 3.09*
	1	9.99 ± 0.72*	31.10 ± 0.30***
	2	12.9 ± 1.75*	48.63 ± 9.00***
8	0.5	12.81 ± 2.65*	10.83 ± 2.14*
	1	16.90 ± 1.70**	25.49 ± 3.63**
	2	21.32 ± 0.99**	51.03 ± 1.69***
9	0.5	9.82 ± 2.84*	14.11 ± 8.89*
	1	1.70 ± 2.60 ns	21.74 ± 5.45**
	2	5.74 ± 3.83 ns	35.07 ± 5.73***
10	0.5	2.62 ± 0.49 ns	12.23 ± 3.07**
	1	3.03 ± 2.08 ns	25.84 ± 1.39**
	2	11.8 ± 1.41*	49.59 ± 6.30***
Gallic acida/ethylenediaminetetraacetic acid (EDTA)b	0.5	99.86 ± 0.19***a	86.13 ± 5.09***b
	1	98.62 ± 0.68***a	103.43 ± 1.2***b
	2	98.85 ± 0.17***a	92.79 ± 2.58***b
F‐value		17.980	7.541
Critical value (CV)		1.599	1.567

^a ABTS radical scavenging activity reference.

^b Metal chelation capacity reference.

Abbreviations: –, no activity; ^ns, not significant; SD, standard deviation.

^*** p < 0.001; ^** p < 0.01; ^* p < 0.05 significance compared to control group.

Furthermore, the metal chelation capacity, a novel aspect of this research, revealed that Sample 2 exhibited the highest chelation capacity (56.99 ± 1.91%), with Samples 3, 7, and 8 (50.59 ± 0.73, 48.63 ± 9.00, and 51.03 ± 1.69, respectively) showing similar values, a result that contributes new knowledge to the field, as no previous studies have evaluated the metal chelation potential of carob molasses (Table 2). These findings collectively underscore the complex antioxidant profile of carob molasses, suggesting potential health benefits, though further studies are necessary to explore the mechanisms and broader applications of these bioactivities.

In the current study, it was aimed to investigate the effectiveness of ten carob molasses samples against some microorganisms (S. aureus, E. faecalis, K. pneumoniae, E. coli, P. aeruginosa, C. albicans, and C. krusei) that are both pathogenic in humans and cause contamination and quality problems in foods. The results showed that MICs values range from ≥200 to ≥400 µg/mL, which are the concentration values close to bactericidal/statical (MBC/‐MBS) and fungicidal/statical (MFC/‐MFS) effects (Table 3). Previously published study, Carob‐mediated calcium hydroxide nanoparticles were found against the Gram‐negative aerobic strains (P. aeruginosa, E. coli) more susceptible than the Gram‐positive stains (S. aureus; Alayed et al., 2022). In another study, ethanol extract of carob pulp was obtained using microwave‐assisted extraction, and MIC values against S. aureus and E. coli were found to be 227.27 µL/mL and MBC was found to be 454.54 µL/mL using the microdilution method (Zahorec et al., 2023). Fidan et al. reported that carob extract obtained from the carob plant using the well diffusion method had significant antibacterial activity against S. aureus and E. coli, taking into account the inhibition zone value (13–12 mm; Fidan et al., 2019). Another study found that yogurt containing carob molasses showed strong antimicrobial activity against S. aureus, E. coli, and E. faecalis by the well diffusion method (Shalabi, 2022). Attia et al. tested the antimicrobial activities of aqueous, methanolic, and ethanolic extracts of carob fruits by disk diffusion method (Kirby–Bauer method) and reported that they had antimicrobial activity against S. aureus, K. pneumoniae, E. coli, and C. albicans (Attia et al., 2014). Ikram et al. assumed that polyphenols in carob may be secondary metabolite group compounds responsible for the activity (Ikram et al., 2023). In our study, carob molasses samples showed MIC values of ≥200–≥400 µg/mL against S. aureus, E. faecalis, K. pneumoniae, E. coli, P. aeruginosa, C. albicans, and C. krusei, and these values are concentration values close to bacteriostatic and fungistatic effects. Especially carob molasses displayed MIC values of 200 µg/mL against S. aureus (Sample 8), E. faecalis (Samples 7 and 8), E. coli (Sample 4), and P. aeruginosa (Samples 1, 4, 5, 7, and 10), which are concentration values generally close to bactericidal effect (MBC; 200 µg/mL; Table 3). In our study, antifungal activity was observed against C. albicans and C. krusei with MIC values of 200 µg/mL for Sample 7; additionally, significant fungicidal effects were noted for Samples 1–10 with MFC values of 200 mg/mL. As a result, the antimicrobial activity of carob molasses samples in the range of ≥200–≥400 µg/mL confirms its potential as a natural remedy for folkloric use; this is also consistent with the findings of antimicrobial studies conducted using different methods and in various countries.

TABLE 3.

Antibacterial activity against bacteria and yeast‐like fungi of the samples as minimum inhibition concentration (MICs; in µg/mL) as well as minimum bactericidal/‐statical (MBC/‐MBS) effect, fungicidal/‐statical (MFC/‐MFS) that obtained by using both microdilution method.

	S. aureus ATCC 6538		E. faecalis ATCC 29212		K. pneumoniae ATCC 700603		E. coli RSKK 95085		P. aeruginosa ATCC 1069		C. albicans ATCC 10231		C. krusei ATCC 6258
Sample code	MIC	MBC/MBS	MIC	MBC/MBS	MIC	MBC/MBS	MIC	MBC/MBS	MIC	MBC/MBS	MIC	MFC/MFS	MIC	MFC/MFS
1	400	‐/>400	200	‐/>200	400	‐/400	400	‐/>400	200	‐/200	400	400/‐	>400	‐/>400
2	>400	‐/>400	>400	‐/>400	>400	‐/>400	>400	‐/>400	>400	‐/>400	>400	‐/>400	>400	‐/>400
3	>400	‐/>400	>400	‐/>400	>400	‐/>400	>400	‐/>400	>400	‐/>400	>400	‐/>400	>400	‐/>400
4	400	‐/400	400	‐/400	400	‐/400	200	‐/200	200	‐/200	400	‐/400	>400	‐/>400
5	400	‐/400	>400	‐/>400	400	‐/400	400	‐/400	200	‐/200	>400	‐/>400	>400	‐/>400
6	>400	‐/>400	>400	‐/>400	>400	‐/400	>400	‐/>400	>400	‐/>400	>400	‐/>400	>400	‐/>400
7	400	‐/>400	200	‐/>200	400	‐/400	400	‐/400	200	‐/200	200	‐/200	200	‐/200
8	200	200/‐	200	‐/>200	>400	‐/400	400	‐/400	400	‐/400	400	‐/400	>400	‐/>400
9	>400	‐/>400	>400	‐/400	>400	‐/400	400	‐/400	400	400/‐	>400	‐/>400	>400	‐/>400
10	400	‐/400	400	‐/400	400	‐/400	200	‐/200	200	‐/200	400	‐/400	400	‐/400
AMP	0.5		4		64		16		64		–		–
GEN	2		64		32		4		1		–		–
KET	–		–		–		–		–		2		0.5
FLU	–		–		–		–		–		1		32

Abbreviations: AMC, ampicillin‐clavulanate; ATCC, American Type Culture Collection; C. albicans, Candida albicans; C. krusei, Candida krusei; E. coli, Escherichia coli; E. faecalis, Enterococcus faecalis; FLU, fluconazole; GEN, gentamicin; KET, ketoconazole; K. pneumonia, Klebsiella pneumonia; P. aeruginosa, Pseudomonas aeruginosa; RSKK, Culture collection of Refik Saydam Central Hygiene Institute; S. aureus, Staphylococcus aureus.

The total phenolic content of the carob molasses samples in this study exhibited significant variation, with Samples 8 and 2 showing the highest phenolic concentrations (41.81 ± 3.09 and 38.24 ± 0.51 mg GAE/g, respectively), while Samples 6 and 1 had the lowest levels (13.80 ± 0.31 and 16.04 ± 1.02 mg GAE/g, respectively; Table 4). These findings are in contrast to previous studies such as Küçük and Velioğlu (2022), who reported a range of 5.80–18.00 mg GAE/g for carob molasses, and Tetik et al. (2010), who found much lower values (1.62 ± 0.29 mg GAE/g). However, in alignment with Tounsi et al. (2020), who identified a polyphenol content of 1600 mg GAE/100 g in carob molasses, our results suggest that the phenolic content of our samples is notably higher than those reported in earlier studies. This trend is further supported by findings from Ioannou et al. (2023), where carob pulps, powders, and syrups ranged from 7.20 to 23.18 mg GAE/g. Collectively, these results emphasize the relatively high phenolic content of the carob molasses samples in our study, highlighting their potential as a valuable source of bioactive compounds, and indicating a positive shift in the phenolic content of commercially available carob products.

TABLE 4.

Total phenolic content of carob molasses samples.

Sample code	Total phenol content ± SD (mg GAE equivalent/g sample)
1	16.04 ± 1.02
2	38.24 ± 0.51
3	32.67 ± 1.06
4	15.21 ± 0.41
5	28.76 ± 0.51
6	13.80 ± 0.31
7	32.34 ± 1.12
8	41.81 ± 3.09
9	22.28 ± 0.12
10	28.51 ± 0.82

Abbreviations: GAE, gallic acid equivalents; SD, standard deviation.

For HPLC analyses, gallic acid, protocatechuic acid, vanillic acid, syringic acid, cinnamic acid, and 2‐hydroxycinnamic acid were analyzed at 265 nm, while ferulic acid, chlorogenic acid, caffeic acid, p‐coumaric acid, and sinapic acid were analyzed at 320 nm. Quantitative analysis was performed only for cinnamic acid and gallic acid, as the amounts of the other compounds were too small for accurate evaluation. The HPLC analysis of phenolic compounds in carob molasses provided valuable insights into the presence of specific phenolic acids, including gallic acid and cinnamic acid, which are known for their bioactive properties. The highest concentrations of gallic acid were observed in Sample 2 (569.423 ± 0.003 µg/100 mg extract), followed by significant amounts in Samples 3, 4, and 6, while cinnamic acid levels were highest in Samples 6 and 5 (Table 5). These findings align with the results from Zannini et al. (2024) and Ioannou et al. (2023), who identified varying levels of gallic acid and cinnamic acid in carob syrups, but the concentrations in our study were generally lower. This discrepancy may be attributed to differences in collection locations, times, and processing methods. The study also found that the molasses samples in our research exhibited lower gallic acid and cinnamic acid contents compared to those reported in earlier works such as Şanlı et al. (2020) and Papagiannopoulos et al. (2004). Nevertheless, Sample 2 emerged as a particularly rich source of gallic acid and total phenolic compounds, further enhancing its potential antioxidant and metal‐chelating properties. This comprehensive HPLC analysis highlights the importance of phenolic content in determining the functional qualities of carob molasses and offers a deeper understanding of their potential health benefits.

TABLE 5.

Gallic acid and cinnamic acid amounts (µg/100 mg sample) in the carob molasses samples.

Sample codes	Gallic acid (µg/100 mg sample)	Cinnamic acid (µg/100 mg sample)
1	107.658 ± 1.216	1.473 ± 0.111
2	569.423 ± 0.003	7.707 ± 0.003
3	488.340 ± 0.587	7.021 ± 0.004
4	452.241 ± 0.737	7.867 ± 0.004
5	168.901 ± 0.042	11.208 ± 0.005
6	466.100 ± 0.243	14.838 ± 0.003
7	96.288 ± 0.084	1.575 ± 0.004
8	49.471 ± 0.803	0.878 ± 0.009
9	312.386 ± 1.159	4.605 ± 0.006
10	32.128 ± 0.010	0.417 ± 0.001

In recent studies, the pH values of carob molasses have been examined across a range of samples, showing variation based on geographical origin, production methods, and storage conditions. Küçük and Velioğlu (2022) analyzed 12 different carob molasses samples and reported that the pH values ranged from 4.90 to 5.28, which aligns closely with the results found by Tounsi et al. (2020) who studied both homemade and commercially available samples. Their analysis revealed that homemade molasses had an average pH of 4.33 ± 0.07, while commercially sold molasses had a slightly higher average pH of 4.56 ± 0.00. These values indicate a consistent trend in pH levels, albeit slightly lower than those found by Şimşek and Artık (2002), who observed a range of 5.31–5.40 across 25 commercial molasses samples. However, Öner et al. (2024) reported higher pH values (5.70–5.91) for spreadable carob molasses. In this context, our results indicate that only two of the analyzed samples (Samples 4 and 6) met the pH range specified by the TS 13717 standard (5–6; Table 6), with the rest showing potential issues linked to storage conditions, particularly related to temperature and duration, which are known to reduce pH levels (Toker et al., 2013).

TABLE 6.

Some chemical properties of molasses samples.

Sample codes	pH ± SD	5‐Hydroxymethylfurfural (5‐HMF; mg/kg) ± SD	Total ash (%) ± SD	Water‐soluble solids (Brix; °BX) ± SD
1	4.92 ± 0.07	77.32 ± 1.63	0.50 ± 0.03	76.00 ± 0.75
2	4.95 ± 0.01	65.54 ± 1.63	3.20 ± 0.08	83.00 ± 1.50
3	4.88 ± 0.03	106.91 ± 1.23	3.34 ± 0.07	79.00 ± 0.75
4	5.18 ± 0.06	52.24 ± 1.05	2.83 ± 0.01	70.00 ± 0.75
5	4.74 ± 0.03	300.82 ± 1.17	2.44 ± 0.02	72.50 ± 0.75
6	5.06 ± 0.00	80.03 ± 0.42	3.06 ± 0.04	79.00 ± 1.50
7	4.83 ± 0.08	112.33 ± 1.47	0.83 ± 0.01	74.00 ± 1.50
8	4.71 ± 0.01	108.68 ± 2.11	3.00 ± 0.05	72.00 ± 1.50
9	4.81 ± 0.02	215.02 ± 2.34	2.86 ± 0.08	72.00 ± 0.75
10	4.83 ± 0.05	99.87 ± 1.89	0.50 ± 0.01	73.50 ± 0.75

Abbreviation: SD, standard deviation. n = 3.

The 5‐HMF content is another critical indicator of the quality of carob molasses, particularly because elevated levels can indicate excessive heating and potential mutagenic risks. The results of Küçük and Velioğlu (2022) found that the 5‐HMF content in market‐sold samples ranged from 0.79 to 50.25 mg/kg, while Tounsi et al. (2020) reported higher average values for homemade (33.46 ± 1.45 mg/100 g) and commercially purchased molasses (31.98 ± 1.67 mg/100 g). In contrast, studies by Şimşek and Artık (2002) and Tetik et al. (2010) reported much lower 5‐HMF contents, ranging from 4.0 to 7.0 mg/kg and 1.53 ± 0.41 mg/kg, respectively. Our study found that the 5‐HMF levels in all of the analyzed samples exceeded the TS 13717 standard of a maximum of 15 mg/kg (Table 6). This suggests that long‐term storage under improper conditions likely led to the formation of 5‐HMF, posing potential health risks associated with consuming these molasses products, confirming concerns about the safety of certain molasses samples in the market (Toker et al., 2013).

Ash content in molasses is a critical quality parameter, and in the studies of Şimşek and Artık (2002) and Küçük and Velioğlu (2022), the ash content ranged from 1.33% to 3.40%, with the addition of glucose and fructose syrups lowering ash values. According to the TS 13717 standard, the total ash content should be between 2% and 3%. Our findings showed that only four samples (Samples 4, 5, 8, and 9) met the standard, while some of the others contained ash values above the threshold (2, 3, and 6) suggesting an elevated inorganic matter content, and the remains (1, 7, and 10) were below the threshold (Table 6). This could be due to improper raw material handling or excessive use of glucose or fructose syrups during production (Güçlü et al., 2023).

The water‐soluble solids content, represented as °BX, is another important parameter for determining molasses quality. Previous studies (Küçük & Velioğlu, 2022; Öner et al., 2024; Tounsi et al., 2020) reported variations in °BX values from 69.0% to 79.4%. TS 13717 sets the acceptable range between 70% and 74% for carob molasses. Our findings showed that most of the molasses samples (Samples 4, 5, 7, 8, 9, and 10) were within this range, while others (Samples 1, 2, 3, and 6) had higher °BX values, indicating excessive sugar content (Table 6). The higher °BX values are consistent with findings from previous research indicating that additives such as glucose and fructose syrups can increase the °BX values (Güçlü et al., 2023).

The results from our study highlight several issues with carob molasses quality in the market. The discrepancies in pH, 5‐HMF, ash content, and °BX levels suggest that factors such as the source and quality of raw carob, as well as production and storage conditions, are contributing to molasses quality degradation. These findings are consistent with previous research, but our study provides a more comprehensive evaluation of various physicochemical parameters, identifying specific samples that do not meet national standards.

Carob molasses, which is widely consumed in many countries due to its high nutritional value, is also used as a prophylactic in some diseases (such as tonsillitis). Therefore, considering the health benefits and quality problems of carob molasses with high 5‐HMF and ash content, it was concluded that studies should be carried out under the responsibility of the food industry in order to develop methods to preserve the stability and quality of carob molasses during production and storage processes in terms of public health.

4. CONCLUSION

This study revealed significant differences in the bioactive properties (use in tonsillitis) and quality parameters of commercial carob molasses consumed in Turkey. Although the samples showed promising antioxidant activities, the molasses samples were not as potent as expected against microorganisms associated with tonsillitis. Furthermore, high levels of 5‐HMF and pH and ash contents did not meet acceptable standards, indicating significant quality problems. These findings emphasize the need to reassess the health benefits attributed to carob molasses and its role in terms of the potential risks associated with its use. In conclusion, (1) its claimed beneficial effects on tonsillitis have not been scientifically substantiated. (2) More rigorous quality assurance practices should be adopted by manufacturers of this widely used product, especially in children, to improve product safety by ensuring that acceptable limits for 5‐HMF, pH, and ash content are respected. (3) Food authorities should implement stricter regulations and periodic inspections of carob molasses production and storage to protect public health.

In light of all these findings, it is of great importance that the quality of food products available in the market is carefully evaluated and health claims, if any, are supported by scientific evidence, especially for vulnerable groups such as children.

AUTHOR CONTRIBUTIONS

Didem Deliorman Orhan: Conceptualization; writing—review and editing; resources; supervision. Ergun Murat Sulak: Investigation. Sultan Pekacar: Investigation; writing—original draft. Hasya Nazlı Gok: Investigation; writing—original draft. Burçin Ozüpek: Investigation. Berrin Özçelik: Supervision.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

Orhan, D. D. , Sulak, E. M. , Pekacar, S. , Gok, H. N. , Ozüpek, B. , & Özçelik, B. (2025). Investigation of the quality, antimicrobial, and antioxidant effects of commercial carob molasses and determination of phenolic acids by using HPLC. Journal of Food Science, 90, e70000. 10.1111/1750-3841.70000