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An ecologically acceptable “green” method of extraction cold-pressing is applied in order to reduce the use of organic solvents and preserve bioactive substances from raw material. Therefore, the work aimed to characterize cold-pressed oils obtained from non-intensively grown walnuts. Walnut samples were collected on the territory of Western and Central Serbia. 12 cold-pressed oils were used for the analysis. The content of water and volatile substances, index of refraction and specific absorbances, content of total phenols and antioxidant activity of the hydrophilic and lipophilic fraction of the oil were examined. Microbiological analyses included: the total number of bacteria, the total number of molds, and the presence of sulphite-reducing clostridia and Escherichia coli. In 11 oil samples, the content of water and volatile substances was in accordance with the national Rulebook. The refractive index varies from 1.4725 to 1.4745 for walnut oils. Cold-pressed walnut oils differ in the ranges of specific absorbances (1.08–1.75 at 232 nm and 0.04–0.15 at 270 nm). The content of total phenols in walnut oil was from 3.25 to 5.41 mg GAE/100 mL. The lipophilic fraction of oils showed a significantly higher antioxidant potential compared to the hydrophilic fraction. The results of DPPH• radical scavenging activities the lipophilic fraction was from 17.53% to 39.21%. In all tested walnut oil samples, the total number of bacteria and molds was low. The presence of sulfite-reducing clostridia and Escherichia coli was not detected. In addition to the general quality and biological value of the oils, it is important that they are microbiologically safe.

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