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Kamali H, Teimouri F, Jambarsang S, Paydar A, Shiranian M, Naderipour Z et al . Investigating the Microbial Indicators of Extracted Chubak Juice for Halva Tahini Preparation: Insight into Production Methods. JNFS 2025; 10 (4) :533-541
URL: http://jnfs.ssu.ac.ir/article-1-1222-en.html
Investigating the Microbial Indicators of Extracted Chubak Juice for Halva Tahini Preparation: Insight into Production Methods
Hamideh Kamali , Fahimeh Teimouri * , Sara Jambarsang , Abolfazl Paydar , Mahboubeh Shiranian , Zahra Naderipour , Jalal Sadeghizadeh-Yazdi
Environmental Sciences and Technology Research Center, Department of Environmental Health Engineering, School of Public Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
Keywords: Food safety, Fungal contamination, Bacterial contamination, Hlva tahini.
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Investigating the Microbial Indicators of Extracted Chubak Juice for Halva Tahini Preparation: Insight into Production Methods
Hamideh Kamali; PhD candidate 1, Fahimeh Teimouri; PhD *2, Sara Jambarsang; PhD 3, Abolfazl Paydar; BSc 2,4, Mahboubeh Shiranian; MSc3, Zahra Naderipour; MSc2 & Jalal Sadeghizadeh-Yazdi; PhD6
1 Department of Environmental Health Engineering, School of Public Health, Shahrekord University of  Medical Sciences, Shahrekord, Iran; 2 Environmental Sciences and Technology Research Center, Department of Environmental Health Engineering, School of Public Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran; 3 Center for Healthcare Data Modeling, Departments of Biostatistics and Epidemiology, School of public health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran; 4 Student Research Committee, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.; 5 Department of Food Science and Technology, School of Public Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
ARTICLE INFO ABSTRACT
ORIGINAL ARTICLE Background: Extracted Chubak juice (Ab-chubak) is used for aeration, whitening, and increasing the consistency of Halva Tahini. This study investigated the most important microbiological quality parameters of Ab-chubak. Methods: Sampling of Ab-chubak was done from 100 Halva Tahini units (manual and boiling preparation methods) in Ardakan city. The serial dilution and pour plate method on Sabouraud Dextrose Agar were used for fungal detection, as this medium is primarily employed for the selective cultivation of yeasts and molds, and additionally for determining the microbial contamination in food. Total coliforms (TC) and Fecal coliforms (FC) were identified by most probable number (MPN) method. Results: Yeast frequencies were 31.6% and 48.8% in the manual and boiling of Ab-chubak production methods. Similarly, the results of Geotrichum and Aspergillus fungi were 31.6% and 10% in manual methods, respectively, whereas in the secondary method, Geotrichum had frequency of 11.6%, and Aspergillus was not detected.  TC were 392.52 and 508.25 MPN/100 ml in Ab-chubak prepared in manual and boiling methods, while FC coliforms were 308.31 and 434.55 MPN/100 ml in the mentioned samples, respectively. The results indicated a significant difference in mean number of fungal colonies between two Ab-chubak preparation methods (P=0.04). Also, the results showed a significant difference (P=0.002) in the morphology of the investigated fungi in two Ab-chubak preparation methods. Conclusion: The average of bacterial indicators was similar in two Ab-chubak preparation methods. However, the presence of fungal species such as Aspergillus was confirmed, which can be considered as an opportunistic pathogen.
Article history:
Received: 2 Jan 2025
Revised: 20apr  2025
Accepted: 21 May 2025
*Corresponding author:
f.teimouri@ssu.ac.ir
Department of Environmental Health Engineering, School of Public Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Postal code: 8915173160
Tel: +98 35 31492151

Keywords:
Food safety;
Fungal contamination; Bacterial contamination; Hlva tahini.

Introduction
Recent data from the CDC shows that around 48 million Americans experience foodborne illnesses annually, leading to 128,000 hospitalizations and 3,000 deaths (Mahasti Shotorbani and Momeninejad, 2019). This is a major issue in public health that can be mostly avoided. There is a strong possibility of food being contaminated with different types of fungi, resulting in the development of mycotoxins during food production, preparation and storing processes (Akbarian and Babaei, 2019). While developing food, fungi decrease both the quantity and nutritional value of food by removing contaminated portions and affecting its nutrients. These microorganisms produce mycotoxins, secondary metabolites with significant impacts like causing cancer, birth defects, stunting growth, weakening the immune system, and inducing genetic mutations in organisms (Henao et al., 2010).
Coliform bacteria and Escherichia coli are the other important indicators of food safety and hygiene. Their presence in food and beverages often signifies poor sanitation practices (Sholichah et al., 2022). Non-Escherichia coli may also pose potential health hazards, as they can colonize the human intestine and produce enterotoxins (Twedt and Boutin, 1979).
Monitoring coliform levels can help validate compliance with food safety regulations and assess the potential presence of related pathogens. However, in some processed food items, more resistant organisms may serve as supplementary markers. Overall, controlling coliform contamination is essential for maintaining food quality and preventing foodborne illnesses (Mossel and Struijk, 1995).
Halva Tahini is a well-known product in Iran, Turkey, Lebanon, Greece, and Saudi Arabia. In Iran,  soapwort extract (from Saponaria officinalis) is used to enhance its texture and color, while in other countries, they tend to rely on ingredients like glucose or egg whites instead (Shaker Ardakani et al., 2012). Due to its nutritional content, ease of consumption, and affordable price, it is especially popular in hot and dry regions as a high-energy food source. In the production steps, proportions of sugar-glucose mixture, Ab-chubak, and Tahini  are combined in such a way that a solid content is formed from the emulsion of sesame oil (Tahini) in the sugar syrup mixture (Alpaslan and Hayta, 2002, Arslan et al., 2005).
Chubak is the popular term for certain plants belonging to Caryophyllaceae family. The Chubak plant is characterized by its warm and dry nature, and it is a compact shrub. Acanthophyllum genus plants, characterized by being shrubby with thick roots, are predominantly located in Iran (Hossini tabatabaei et al., 2017). The Saponaria genus includes other plants that are herbaceous and can be annual or perennial, most commonly found in Europe and are sometimes seen in Iran. Saponins and saponin extracts from plants are utilized for multiple purposes in food industry. In Iran and Turkey, Chubak extract and other similar plants are used as emulsifiers in the preparation of halva (Sezgin and Artik, 2010). Several research studies were conducted with the aim of structure identification and physicochemical and biological properties determination (Azadbakht et al., 2005, Gaidi et al., 2004). Saponins and saponin extracts of plant origin are used for various purposes, especially in food industry (Güçlü-Üstündağ and Mazza, 2007). For example, to achieve a whiter color, prevent oil separation from the mixture, improve textural properties, and increase the volume of Halva Tahini, an extracted liquid of saponin (Ab-chubak) is added to the contents.
Ab-chuback is produced in two ways: Manual and boiling methods. In the manual process, Chubak plant is mixed with water and preserved in plastic containers. After several months, the produced extract (Ab-chubak) is ready for use. in contrasted the boiling method, involves the extract is produced via heating Chubak plant. Since this extract is stored in a refrigerator, it has a longer shelf life and better preservation capabilities.
As standard NO. 2695 guidance, Escherichia coli should be negative in 1 g of Tahini sample, consequently the coliform count should not exceed 50 CFU per 1 g, and the mold and yeast should not exceed 100 CFU per g of Tahini sample.
So far, studies have been conducted on the microbial parameters of Halva Tahini, but no survey has been investigated on Ab-chubak used in Halva Tahini preparation. The results of this study can determine the necessity of developing standards for this food product. Therefore, the main objective of this research was to determine the most important microbial parameters, with an emphasis on identifying characteristics of fungi present in the extracted choubak juice (Abchoubak).
Materials and Methods
Sample size and characterization
In this study, the sample size was initially estimated using the following formula (Machin et al., 2011).
  n1=n2=(p1(1-p1)+p2(1-p2))(Z1-α2+Z1-β)2(p1-p2)2         Equation 1
n: Sample size
p: Proportion of fungi in preparation method
Z1-α2  : Significance level α
α : Critical value (for 95% confidence level)
 Z1-β:  Test power 1-β
β : Probability of Type II error (for 80% power)
Using Equation 1, the sample size of 50 was calculated. So, the number of samples was 50 for each manual and boiling preparation methods, separately.

Ab-chubak is a brown liquid with relatively high viscosity that is added to Halva Tahini to prevent oil separation from the texture and to improve its characteristics. Figure 1 shows the used Chuback plant and Ab-chuback in the present study.

Microbial test performance
As microbial national standards, Total coliforms (TC), fecal coliforms (FC) and fungal parameters were selected to examine in the present study.
For fungal test examination, samples were transferred to the laboratory. The molds and yeasts enumerations were performed by the colony counting method. The primary suspension (initial dilution) and other dilutions were prepared according to the Iranian National Standard No. 10899-3, using the method of serial dilutions and pour plate count in Sabouraud Dextrose Agar (SDA) medium. Then, Plates were incubated at 25 °C for 3 to 5 days (Frances et al., 2021, Iranian National Standardization Organization, 2013).
Colonies were also investigated as yeast or mold cells by an optical microscope. For the morphological survey, a piece of grown fungus was prepared with lactophenol reagent and investigated under high magnification microscope (Ajobiewe, 2021, Elgarhi et al., 2020, Iranian National Standardization Organization, 2013). The morphology of fungal genera, such as Geotrichum and Penicillium, was examined with respect to their distinct characteristics: Geotrichum colonies typically appear creamy, yeast-like, and arthrosporic, often with a powdery surface, while Penicillium exhibits characteristic brush-like conidiophores and powdery green or blue-green colonies. Mentioned characteristics were confirmed microscopically, aligning with standard mycological identification protocols (Samson, 2011)
For bacterial test examination, TC and FC examinations were conducted as No. 2946 and 11166 of national standards (Institute of Standards and Industrial Research of Iran, 2008, 2021). The multiple-tube fermentation method was used for coliform detection. Initially, 0.1 ml of the sample was added to tubes containing 10 ml of Lauryl Sulfate Broth (LSB broth) at standard concentration. The inoculated media were incubated at 37 °C for up to 48 hours, and the gas production was checked at 24 and 48 hours. In the presence of turbidity and gas in the tube, the contents were transferred to tubes containing selective liquid media, EC, and BGB Broth medium. The EC Broth tubes were incubated at 44 °C as well and the BGB tubes were incubated at 37 °C with gas production assessed after 24 and 48 hours and compared with the Most Probable Number (MPN) table (Urooj et al., 2018).
Ethical considerations
This study was approved at Ethics Committee of Shahid Sadoughi University of Medical Sciences. (ID: IR.SSU.SPH.REC.1402.015)
Data analysis
SPSS software version 26 was used for data analysis. The parameters of total coliforms, fecal coliforms and fungal contamination levels were assessed. To investigate differences in categorical distributions, Chi-square test (Fungal species) and to compare differences in mean colony counts between two preparation methods, Mann-Whitney test was used.
Results
Fungal quantity and quality identification
Table 1 shows results of fungi colony enumeration in Ab-chubak prepared by boiling and manual methods.
Table 1. Fungal colony enumeration as CFU/ml.
Production method Mean±SD Min Max Median
Manual 46.63±53.90 0 240 42
Boiling 30.98±45.66 0 200 15

As shown in Table 1, mean numbers of fungal colonies in manual and boiling methods were 46.63±53.90 CFU/ml and 30.98±45.66 CFU/ml, respectively. Results of Mann-Whitney test indicated a significant difference in mean number of fungal colonies between two Ab-chubak preparation methods (P=0.04). In other words, the abundance of fungal numbers was not the same in two preparation methods.
The morphology of four fungal species identified in this study is shown in Figure 2 and the Frequencies of fungal types are shown in Figure 3.



 
As shown in Figure 2; the prevalence of Geotrichum, Penicilium, and Aspergillus was 31.6, 26.3 and 10.5%, in the manual method, while Geotrichum and Penicilium were found at a prevalence of 11.6 and 9.3% in boiling methods. However, Aspergillus was not detected in Ab-chubak prepared by the last method. Overall, manual method showed a higher prevalence of Geotrichum, Penicillium, and Aspergillus compared to boiling method, whereas yeasts were more prevalent in boiling method. The results of chi-square test also showed a significant difference (P=0.002). In other words, the morphology of investigated fungi differed between two Ab-chubak preparation methods.
Total and fecal coliforms enumeration
Table 3 shows the average total and fecal coliforms in Ab-chubak produced using boiling and manual methods.
Table 3. Total and Fecal coliforms in Ab-chubak samples (MPN/100 ml).
Bacterial indicator Mean±SD Min Max
Total coliform
  Manual		 392.52±1065.68 0 3600
  Boiling 508.25±1053.62 0 3700
Fecal coliform
  Manual		 308.31±878.24 0 3600
  Boiling 434.55±899.48 0 3200

According to Table 3, the mean of total coliform in manual and boiling preparation methods were 392.52±1065.68 and 508.25±1053.62 MPN/100 ml, respectively. Similarly, fecal coliform counts were 308.31±878.24 and 434.55±899.48 MPN/100 ml, respectively. However, results of statistical analysis using Mann-Whitney test indicated that mean values of these two parameters (P=0.94 for TC and 0.66 for FC) did not differ significantly between two Ab-chubak preparation methods.
Discussion
Analysis of microorganisms gives vital data on cleanliness of food, its expiration date, and possible health hazards (Lima et al., 2023). Detecting pathogens, spoilage microbes, and other contaminants in complicated food matrices is crucial (Ferone et al., 2020). This study examined microbial parameters in Tahini production units and identified three different types of yeast fungi including yeast, Aspergillus, Geotrichum, and Penicillium. This is consistent with a study conducted by Mahdian Yazdi et al. on Halva Tahini, which reported that among  60 samples (halva ardeh), three were contaminated with molds, one with yeast, four with Escherichia coli, and one with Enterobacteriaceae (Yazdi et al., 2018).The results of fungal counts indicated that 31.6% of manually prepared samples were contaminated with yeast, whereas 48.8% of samples prepared by boiling showed yeast contamination. Similar results were obtained from conducted studies by Sengun et al. and Kotzekidou; 1.5×102 and 4.9×103 CFU/g, respectively (Kotzekidou, 1998, Sengun et al., 2005). Additionally, a study by Kilci et al. on various types of Halva Tahini  revealed that microbiological analyses for yeast and mold contamination showed an average of 10 CFU/g, 90 CFU/g in hazelnut Halva Tahini, 45 CFU/g in carob Halva Tahini, and 25 CFU/g in vanilla Halva Tahini  (Kilci and Çetin, 2022).
This difference may be attributed to characteristics of yeasts, such as their mesophilic and thermophilic properties, which make them more resistant compared to other fungi like molds. Additionally, aerobic conditions may inhibit mold growth, which could explain the higher prevalence of yeasts in boiling method compared to manual method (Sengun et al., 2005). Another reason for the identification of yeast in boiling samples could be related to the storage of Ab-chubak in refrigerator after boiling method. This may allow for the transfer of yeast contamination through the air inside the refrigerator.
Regarding Geotrichum and Aspergillus fungi, results indicated that their prevalence in manual samples was 31.6% and 10.5%, respectively, whereas, in boiling method, Geotrichum was present at 11.6% and Aspergillus was not detected. Manual method showed a higher prevalence of Geotrichum, Penicillium, and Aspergillus compared to boiling method, while yeasts were more prevalent.
In the study by Kahraman on Halva Tahini samples, it was found that 36.66% of samples had poor quality based on microbiological and chemical standards recommended by Turkish Food Codex. The microbiological quality criteria included aerobic bacteria, Staphylococcus aureus, coliforms, Escherichia coli, molds, yeasts, Salmonella species, and staphylococcal enterotoxins. Chemical quality criteria included sesame oil, protein, moisture, ash content, acidity, total sugar content, and peroxide levels. The predominant level of contamination was 10.38% for molds, which was lower than the contamination level for yeasts at 38.20%. This could be attributed to factors such as inadequate hygiene of raw materials, production area, and sample storage conditions (Kahraman et al., 2010).
In this study, the mean of number of TC was 392.52±1065.68 and 508.25±1053.62 MPN/100 ml for manual and boiling methods, respectively. Additionally, the number of FC in mentioned samples was 308.31±878.24 and 434.55±899.48 MPN/100 ml, respectively. In other words, TC and FC counts were approximately similar in two methods, with no significant differences. Given the relatively long storage duration, one reason for the higher coliform counts could be attributed to the storage of these samples in the refrigerator after boiling. This storage method may allow for the transfer of coliform contamination through secondary contamination. Another reason could be related to secondary contamination via human preparation or the preserved dishes that transferred bacterial contamination.
Most similar studies have mentioned that Halva may become contaminated with various microorganisms due to direct or indirect contact with external sources of contamination during production (Abu-Jdayil et al., 2002).  the results of the present study, indicate  that such contamination may result from the microbial flora of raw materials, such as Ab-chubak, insufficient thermal treatment, and inadequate personal hygiene during production (Aydin and Başaran, 2018).
In a study conducted by Karam et al. multiple outbreaks of S. bovismorbificans, S. Montevideo, and S. Mbandaka infection were related to Tahini consumption that were reported in the United States. Moreover, Escherichia coli was found in 17% of Tahini  samples in Lebanon (Karam, 2010).
In a study conducted by Aydın et al. on Tahini  halva samples, the results showed that coliforms and Escherichia coli were found at acceptable levels (28 MPN/g) (Aydin and Başaran, 2018). Sengun et al. reported coliforms levels of 41 CFU/g, and Kotzekidou reported 30 CFU/g, which are consistent with the findings of the present study regarding the identification of coliforms and Escherichia coli (Kotzekidou, 1998, Sengun et al., 2005).
In a study conducted by Eshaghi on Halva Tahini in Yazd and Ardakan, it was found that 8.3% of Halva Tahini samples from Ardakan and 12.5% of samples from Yazd were contaminated with Escherichia coli (Eshaghi, 2010). Since the presence of Escherichia coli in food indicates significant fecal contamination, inadequate personal hygiene may be a significant factor contributing to Escherichia coli contamination in Halva (Namiki, 1995). However, the results of studies conducted on Halva Tahini are promising, in the sense that the process of preparing Halva Tahini can reduce the bacterial indicators identified in Abchoubak, but it is still recommended to monitor this product regarding fungal surveillance.
Despite valuable findings of this study, a few limitations should be acknowledged. Molecular identification and mycotoxin analysis were notable limitations of this study, and their investigation is recommended for future research.
Conclusion
The present study underscores the need for microbiological quality control in Ab-chubak, a key additive in Halva Tahini production. The findings highlighted the presence of opportunistic fungal species and fecal coliforms, indicating potential health risks. Given the absence of regulatory standards for Ab-chubak, establishing effective contamination control measures is crucial. Implementing strict hygiene protocols, heat treatments, and improved monitoring practices across production and distribution can significantly reduce microbial contamination. These measurements will enhance food safety, minimize health risks, and ensure the hygienic quality of Ab-chubak in food applications.
Acknowledgment
The authors thank Shahid Sadoughi University of Medical Sciences for supporting the current research (Code number: 14627).
Authors' contributions
Design study was conducted by Teimouri F, Sadeghizadeh-Yazdi J.  Experimentations were performed by Paydar A, Shiranian M, Naderipour Z. Data analysis was conducted by Jambarsang S. The draft manuscript was written by Kamali H and Teimouri F. All authors read and approved the final manuscript.
Conflict of interest
No potential conflict of interest is reported by the author(s).
Funding
No specific funding was used to do this.
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Type of article: orginal article | Subject: public specific
Received: 2025/01/2 | Published: 2025/11/19 | ePublished: 2025/11/19
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