Investigating the chemical and microbial characteristics of Kermanshahi ghee and Azerbaijan yellow ghee during the storage period

Introduction: Ghee, also known as animal oil or milk oil, is a tasty and nutritious dairy product made by heating butter to remove water through evaporation and separating non-fat solids through sedimentation (Nekera et al. 2023). It is usually made from cow's milk, buffalo's milk, or a combination of the two (Kapadiya Dhartiben 2017). As defined by Codex, ghee is produced exclusively from the milk fat, cream, or butter of various animal species using processes that almost completely remove water and non-fat solids (Codex 2018). Ghee is a complex of triacylglycerol lipids, along with a small quantity of free fatty acids, phospholipids, sterols, hydrocarbons, carbonyl compounds, fat-soluble vitamins (A, D, E, and K), carotenoid pigments, moisture and trace elements such as copper and iron. Ghee typically consists of 99-99.5% fat with a moisture content of less than 0.5% (Kapadiya Dhartiben 2017). Due to its low moisture content, ghee has a longer shelf life compared to other dairy products (Aditya and Divya 2018). Additionally, ghee is considered nutritionally superior to other oils because it contains medium-chain fatty acids that are absorbed by the liver and used for energy. Ghee exclusively contains butyric acid, which gives it a unique taste and aids in digestion. Saturated fatty acids are the main component of fatty acids in ghee. Additionally, it also contains high levels of monounsaturated and polyunsaturated fatty acids, which offer numerous biological benefits (Kumar et al. 2015). Improper packaging and storage can lead to ghee spoiling due to exposure to bacteria, yeasts, molds, and oxygen (Antony et al. 2018). Due to oxidative rancidity, the main qualitative parameters such as color, taste, aroma, and nutritional value of the ghee change and make it unusable (Gandhi et al. 2013). Kermanshahi ghee and Azerbaijani ghee, known as yellow oil, are commonly used animal oils in Kermanshah and West Azerbaijan provinces. These types of ghees are traditionally made from cow's milk in small local workshops. Given the widespread use and nutritional significance of these ghees in consumers' diets, this study was carried out to assess changes in their chemical properties, microbial properties, and fatty acid composition over a 6-month storage period.
Material and methods: The raw cow's milk was obtained from traditional farms in Urmia and Kermanshah, and ghee samples were then produced in three replicates in local workshops. Initially, the raw milk underwent heat treatment at a temperature of 85 °C for 10 minutes in order to pasteurize it. Subsequently, the milk was cooled down to 44 °C and then inoculated with a 3% starter culture. The inoculated milk was placed in an incubator for 4 hours at the specified temperature. Once the pH dropped to 4.6, yogurt was made. The yogurt was then left at room temperature for 2 days to allow for increased lactic acid production. Afterward, the yogurt was processed in a butter-making machine, completing the butter-making process in 15-20 minutes. Finally, a two-phase liquid consisting of butter and buttermilk was produced. The upper liquid, which was butter, was separated manually from the lower liquid, known as buttermilk. In order to make ghee, the separated butter was heated at a temperature of 100-120 ˚C for a few minutes. At this stage, the moisture evaporated and brown non-fatty solids were deposited. Ultimately, the oil was filtered and the ghee samples were then placed into containers and stored at room temperature (25 °C) for 6 months. In order to assess the shelf life of ghee samples, the acidity, acid, and peroxide values were measured over 6 months at 25°C. Additionally, the microbial properties and fatty acid composition of the ghee samples were analyzed post-production. The study utilized a split plot experimental design within randomised complete blocks (RCBD). All trials were conducted in triplicate. Statistical analysis was carried out using one-way ANOVA, with significant differences between means identified through Duncan's multiple range tests at a significance level of p < 0.05.
Results and discussion: The data analysis results indicated a significant increase (p<0.05) in acidity and acid value of both ghee samples during 6 months of storage at room temperature. These values were also significantly higher (p<0.05) compared to the initial values on the first day. The acidity levels of Azerbaijani yellow ghee rose from 0.73% to 1.88%, while Kermanshahi ghee experienced an increase from 0.19% to 0.73%. The acid value of Azerbaijani yellow ghee showed a significant increase (p<0.05) from 1.46 mg KOH/g on the first day to 3.54 mg KOH/g after six months, while Kermanshahi ghee rose from 0.37 mg KOH/g to 1.45 mg KOH/g over the same period (p<0.05). Over a 6-month storage period, the Kermanshahi ghee exhibited significantly lower acidity and acid value compared to the Azerbaijan yellow ghee (p<0.05). The Azerbaijani yellow ghee remained within the acceptable acidity limit for the first three months, but exceeded the limit in the sixth month. On the other hand, the Kermanshahi ghee maintained an acceptable acidity level throughout the six-month storage period. The variation in acidity and acid value between Azerbaijani and Kermanshahi ghee samples may be due to unique characteristics in the milk of the animals studied and the specific composition of their diets. The peroxide value of both ghee samples showed a significant increase after 6 months of storage at 25 ˚C (p<0.05). No significant difference was found in the peroxide value of the two ghee samples after 6 months. Researchers have attributed the increase in peroxide value of ghee during storage to the oxidation of fatty acids, which can be caused by factors such as packaging in light-permeable containers, loose lids on containers, and improper storage and transportation methods (Sulieman et al. 2017). The oxidation rate of fatty acids is likely to be influenced by their structural variations, such as differences in chain length, degree of unsaturation, and the positioning of double bonds. Additionally, the presence of a high concentration of free fatty acids in the oil increases its susceptibility to oxidation (FAO/WHO, 1993). In present study, the rise in peroxide values in the ghee samples is likely a result of the passage of time. The fatty acid profile analysis revealed that both ghee samples were predominantly composed of saturated fatty acids, with palmitic acid, myristic acid, and stearic acid being the major fatty acids present. The saturated fatty acid content in Azerbaijani yellow ghee was 67.93%, while in Kermanshahi ghee it was 64.98%. On the other hand, the total content of monounsaturated and polyunsaturated fatty acids in Azerbaijani yellow ghee was 32.07%, whereas in Kermanshahi ghee it was 35.02%. This indicates that the Kermanshahi ghee had a higher proportion of unsaturated fatty acids. Oleic acid has been identified as the primary unsaturated fatty acid in both ghee samples, with no significant difference in its concentration observed between the two samples. The content of lauric acid (C12:0) was found to be significantly (p<0.05) higher in Kermanshahi ghee compared to Azerbaijani yellow ghee, whereas the content of pentadecanoic acid (C15:0) was significantly higher in Azerbaijani yellow ghee (p<0.05). The linoleic acid levels in Azerbaijani and Kermanshahi ghee were 1.95% and 2.77%, respectively. Significantly higher linoleic acid content was observed in Kermanshahi ghee compared to Azerbaijani ghee (p<0.05). Variations in fatty acid profiles of ghee samples may be due to differences in the animals' diet or microbial ecosystem (Khiaosa-ard et al. 2015). There was no significant difference found in the conjugated linoleic acid content between Azerbaijani yellow ghee and Kermanshahi ghee. Based on the findings of the microbial analysis, it was observed that the total count of microorganisms in Azerbaijan yellow ghee (0.76 log10 CFU/g) was significantly lower compared to Kermanshahi ghee (1.32 log10 CFU/g) (p<0.05). Furthermore, the investigation and enumeration of coliforms, coagulase-positive staphylococcus aureus, mold, and yeasts in the ghee samples revealed no contamination with these microorganisms. This could be attributed to the low moisture and high fat content of the ghee, which inhibit the growth of such microorganisms. The microbial characteristics of the samples analysed in this study met the standards set by the Iranian national standard for butter.
Conclusion: Based on the findings of this study, there was a significant difference between the two ghee samples in certain chemical properties such as fatty acids content, acid value, and peroxide value. The oxidative stability of both ghee samples declined over time during storage at room temperature. Overall, the ghee samples had higher levels of saturated fatty acids and lower levels of unsaturated fatty acids. The microbial content in the ghee samples was within acceptable limits, with no contamination of coliforms, coagulase-positive staphylococcus aureus, mold, or yeasts detected. Further research is recommended to investigate the impact of natural and synthetic antioxidants on the shelf life and oxidative stability of animal oils.