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For the first time, we have studied the influence of polyunsaturated fatty acids on cholesterol metabolizing activity of bifidobacteria. High levels of destruction of cholesterol in the process of cultivation of strain Bifidobacterium Longum DK 100 was noted. Based on the results, the technology of probiotic dietary supplements (DS), enriched with polyunsaturated fatty acids, has been developed. The paper reveals that the introduction of probiotic DS leads to a twofold reduction in total cholesterol in the experimental groups in comparison with the control one, which indicates high effectiveness of DS. It has been observed that against the background of the atherogenic diet, the employment of probiotic DS is accompanied by a significant reduction in triglyceride levels by 37% and 45% in the "linen" and "cedar" groups, respectively, compared to the control group of animals. It has been established that in the blood serum of animals of experimental groups, the level of high-density lipoproteins increases by 45% in "linen" and 40% in "cedar" groups, while low-density lipoproteins decreased by 19% and 23% respectively. With the introduction of probiotic DS with linseed oil, the atherogenic index decreases by 83%, and with the cedar one by 86%. The results confirm the hypocholesterinemic effect of DS during cholesterol loading.