**4.1 Antioxidant activity**

 Peptides released during the enzymatic or thermal hydrolysis of buttermilk proteins have known antioxidant properties. An investigation studied samples of biofilms, with antioxidant potential, produced from buttermilk. The results were expressed as trolox equivalent antioxidant capacity (TEAC), that is, the concentration of buttermilk in g/L that produces the same inhibition of a Trolox solution (analogous solution of vitamin E) at 1 mmol/L. When analyzing the antioxidant character of the biofilms, it was observed that the effect was only noticed in the samples that went through heating, which was justified by the release of the peptides during the heat treatment of the biofilms [13].

#### **4.2 Hypocholesterolemic activity**

The pathophysiology of coronary diseases is well established in the literature and is related, among other factors, to high cholesterol and low-density lipoprotein (LDL).

 Considering the importance of the control of serum levels for greater clinical benefit, different studies have been looking for nonpharmacological measures in patients of low risk, among them the consumption of buttermilk [22, 23]. The hypocholesterolemic action of buttermilk was studied using different methodologies that demonstrated a reduction in total cholesterol and other variables with consumption of the dairy derivative in the short term [22–24]. The effect is explained by phospholipids present in buttermilk that may be responsible for the hypocholesterolemic effect. The effect occurs through an intervention in the pathway of cholesterol synthesis, as well as in the intestinal absorption of cholesterol, lowering their blood levels. A study conducted a double-blind, randomized, placebo-controlled study. Volunteers between 18 and 65 years and body mass index (BMI) of 35 kg/m2 were submitted to two consecutive treatments of 4 weeks each, in random order. Participants should maintain their diet, medication, weight, alcohol consumption, and smoking habits normally, with the exception of the days before blood sampling for the exams. Vitamins and food supplementation products were banned from interfering with the research. The formulations tested and ingested by participants contained 22.5 g buttermilk or placebo, which should be mixed with 250 mL water and sucralose in a shaker provided to the participants. The placebo contained varied dairy ingredients; however, such ingredients do not have in their MFGM composition present in the milk fluid. The usual food intake was evaluated through a specific questionnaire for dairy foods in three moments: at the beginning, after the first treatment, and after the second treatment. After the blood draw procedure volunteers, tests of total cholesterol, triglycerides and serum concentrations of LDL and HDL, C-reactive protein, latosterol, β-sitosterol, campesterol and plasma levels of PCSK9, also called convertase 1, an enzyme that interacts directly with LDL receptors, decreasing its metabolism [25]. In conclusion, consumption of short-term buttermilk significantly reduced total serum cholesterol and triglyceride concentrations.

A manuscript evaluated 100 participants aged from 18 to 65 years regarding the lipid profile of individuals who ingested buttermilk. The study was randomized and placebo-controlled for a period of 12 weeks. The buttermilk drink was traditionally prepared with increments such as vanilla sugar in order to increase the acceptability of the dairy product. Participants were instructed not to change their usual diet, level of physical activity, and alcohol use. Total cholesterol, HDL, LDL, triglycerides, liver, and kidney function parameters, as well as cholesterol precursors, were quantified using HPLC. The author also noted a reduction in cholesterol and LDL levels [23].
