Original Article Evaluation of Microbial Contamination of Dried Black Curd (Gharahghorut) Produced in Iran Mojtaba Chizari ABSTRACT (MSc) Department of Microbiology Background and objectives: Dried Black Curd (DBC), also and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, known as Gharahghorut (Persian), is a dairy product produced Shahid Beheshti University, Tehran, from curd of yogurt or doogh. The aim of this study was to Iran evaluate microbial contamination of industrially and traditionally Seyed Masoud Hosseini produced DBC in Iran. (PhD) Department of Microbiology and Microbial Biotechnology, Faculty Methods: Four DBC brands holding certification of the Institute of Life Sciences and Biotechnology, of Standards and Industrial Research of Iran (ISIRI), Iran Shahid Beheshti University, Tehran, Ministry of Health and Medical Education (IMHME) and ISO Iran were purchased from a market in Tehran, Iran. Microbial Ali Mohammadi monitoring for presence of coliform, Escherichia coli, coagulase- (PhD) Department of Microbiology, Faculty of Biological Sciences, positive Staphylococcus aureus, mold and yeast was performed Alzahra University, Tehran, Iran using different basic, enriched, selective and differential media Javad Fakhari including peptone water agar, MacCankey agar, blood agar and (MSc) Department of Microbiology brain heart infusion agar, etc. according to the ISIRI standards and Microbial Biotechnology, Faculty (No.13299). of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Results: None of the tested samples had microbial Iran contamination. However, in one traditional DBC sample, the Mojtaba Mashhadi Mohammadzadeh- Vazifeh population of mold and yeast was higher than the acceptable (MSc) Department of Microbiology level defined by the ISIRI standard. and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, Conclusion: We found no contamination with coagulase- Shahid Beheshti University, Tehran, positive S. aureus, E. coli and coliforms in four brands of Iran traditionally and industrially produced DBC in Iran. This Corresponding author: Seyed Masoud Hosseini indicates that the hygienic practices designated by the IMHME Email: [email protected] are well-implemented in the industrial sector. However, the Tel: +9829905913 mold and yeast contamination in traditionally produced DBC Address: Department of Microbiology should be prevented by applying hygienic practices during the and Microbial Biotechnology, Faculty process of manufacturing and distribution. of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Keyword: Dried black curd (DBC), Gharahghorut, Microbial Iran Contamination Received: 2020/04/24 Revised: 2020/06/14 Accepted: 2020/07/26 This work is licensed under a Creative Commons Attribution 4.0 License. DOI: 0.29252/mlj.14.6.48 Medical Laboratory Journal, Nov-Dec, 2020; Vol 14: No 6 49/ Chizari and colleagues INTRODUCTION However, ignoring food hygiene regulations Dried black curd (DBC), also known as can result in increased risk of DBC gharehghorut, gharaghorut, black kashk or contamination with foodborne pathogens, such qara, is a traditional milk product with black, as Escherichia coli, Staphylococcus aureus brown, white or yellow color and a very sour and molds. The present study aimed to assess taste. This stiff, sour and chewable dairy microbial contamination of traditionally and product is basically produced by boiling the industrially produced DBC in Iran. yogurt whey derived from mixture of kashk, MATERIALS AND METHODS buttermilk and whey. It is used alone or along All reagents and microbial culture media with other raw materials for producing fruit used in the experiments were based on the leather. It is rich in lactic acid and contains recommendations of ISIRIs (No. 2461-1, some minerals such as calcium and vitamins, 2946, 6806-3 and 10899-2) (4–7). Sampling especially vitamin B2 (1). Based on the was performed based on the standard sampling preparation method provided in the Moeen protocol for agricultural products that are Encyclopedia, doogh is boiled and dried in a consumed orally (ISIRI No. 2836) (8). thick bag. Then, tuff or the remaining matter is Characteristics of DBC samples from shaped as bullets and further dried under manufacturers in Tehran (Iran) as well as the sunlight. Finally, the sour water extracted from type of certifications issued by different the bag is exposed to sunlight or boiled dry in authorities including the Iran Ministry of order to obtain gharaghorut. Health and Medical Education (IMHME), The hardness, softness, taste and color of DBC ISIRI, HACCP, ISO 9001 and ISO 22000 are depends on the amount and method of heating, summarized in table 2. Different DBC brands the level of fat, evaporated water, acidity and were purchased from a market in Tehran and sour. The composition of DBC is summarized were kept at 18-25 ˚C (Figure 1). in table 1. A healthy and standard DBC should The pH of DBC samples was determined and be free from mold and warehouse pests, microbial features were assessed using unpleasant and unnatural taste and smell, general, enriched, selective and differential external materials such as pebble, hair and media. In addition, presence of E. coli, burnt particles, color, additives and coagulase-positive S. aureus, coliforms, mold preservatives (1). and yeast was evaluated based on the protocols Gharaghorut is mainly produced traditionally provided by the ISIRI (No. 13299) (9). The at homes and less frequently in small factories. chemical composition of two DBC samples Accordingly, it is essential for producers to was analyzed. All measurements were carried receive an official hygiene code. Food out three times. spoilage can easily occur in the production Coliforms were isolated by using the most process, while physical, chemical and probable number (MPN) and colony counting biological contamination can have both health methods using enterobacteria enrichment and economic consequences. Spoilage also (EE) broth (Cat No. 651702, Quelab Inc., decreases the shelf life of the product and may Canada), violet red bile glucose agar (No. lead to food poisoning (2). Therefore, 651702, Quelab Inc., Canada) and nutrient microbial control of food products is a critical agar (Cat No. 105450, Merck, Germany) and o step to increase shelf life while maintaining incubation for 24 hours at 37 C. Glucose quality (3). The pH of DBC is low due to the oxidase fermentation test was conducted using high concentration of lactic acid, which limits glucose agar (Cat No. 651135, Quelab Inc., the growth of microorganisms. Canada) for further confirmation (ISIRI No. 11165 & 1,2-2461) (10–12). Table 1. Sensory and physical characteristics of DBC based on the Institute of Standards and Industrial Research of Iran (ISIRI, No. 13299) Indicator Description Color The product should be uniform with a cream to dark blackish-brown color. Taste and smell DBC should possess sour taste without any unpleasant and unnatural smell. Consistency and The product should have a uniform texture in form of concentrated liquid to solid phase. texture Medical Laboratory Journal, Nov-Dec, 2020; Vol 14: No 6 50/ Evaluation of Microbial Contamination … Table 2. Characteristics of the purchased DBC brands Sample code A B C D Number of sample Three Three Three Three Production method Industrial Industrial Traditional Traditional Color White Black White Black Hygiene code (IMHME) x x ISO 9001:2008 x x x x ISO 22000:2005 x x x x HACCP x x x x Standard logo (ISIRI) x x Production date x x Expiry date x x Note: the characteristics of samples A and B were written on their packages. Figure 1. Color and appearance of the purchased DBC brands Sample A Sample B Sample C Sample D The MPN technique and lauryl sulfate broth In addition, rabbit plasma (Cat No. 113306, (No. 652406, Quelab Inc., Canada), EC broth Merck, Germany) was used for confirmatory (Cat No. 110285, Merck, Germany) and test (ISIRI No. 3-6806) (13). The spread plate peptone water indole free (Cat No. 652106, technique was used to isolate and count molds Quelab Inc., Canada) were applied to isolate E. and yeasts. In this regard, samples were coli, which was confirmed by indole (ISIRI incubated in dichloran glycerol (DG18) agar (Cat No. 620238, Liofilchem, Italy) for 5-7 No. 2946) (6). Coagulase-positive S. aureus o were isolated using the MPN technique (5) and days at 25 C (ISIRI No. 2-10899) (7). After evaluating microbial contamination, the Giolitti-Cantoni broth (Cat No. 836930, obtained values were compared to standard Quelab Inc., Canada), Baird Parker agar (Cat levels (14). No. 105406, Merck, Germany) and brain heart The protein content of samples was infusion (BHI) broth (Cat No. 180239, Quelab determined via the Kjeldahl method based on Inc., Canada). Medical Laboratory Journal, Nov-Dec, 2020; Vol 14: No 6 51/ Chizari and colleagues the ISIRI standard (No. 1-9188) with standard to measure phosphorus level based on the acceptance level (SAL) of 8-12 (g/percent) ISIRI standard (No. 1808 (18). The method is (15). The fat content of samples was measured based on digestion of DBC samples in using the Soxhlet method based on the ISIRI sulphuric acid and hydrogen peroxide, then standard (No. 760) with SAL of ≤1 (g/percent) adding molybdate solution and ascorbic acid, (16). The ash content of samples was and finally measuring phosphate in the determined through ash analysis in a furnance solution at 820 nm (19). (550 oC) based on the ISIRI standard (No. RESULTS 1755) with SAL of ≤9 (g/percent) (17). Table 3 presents the results of culturing Moisture level was determined based on the DBC samples in culture media with different ISIRI standard (No. 1753) with SAL of 15-30 conditions. No microbial growth was observed (g/percent) (15). in culture of peptone water, plate count agar, The calcium content of DBC was determined MacConkey agar, blood agar, MRS agar, M17 using a Young Lin AAS 8020 atomic agar, nutrient agar, glucose agar, YM agar, absorption spectrometer (South Korea) with two phases, BHI agar, EMB and peptone water 99.7% burnt acetylene based on the ISIRI agar under different pH and aerobic/anaerobic standard (No.