A-lactalbumin + sodium oleate
Read a paper but cannot find. Adding sodium oleate to water creates the buffered solution. At 12 ° C, 1 part sodium oleate readily dissolves in 10 parts water. Basic salts such as sodium oleate are generally soluble in water. The resulting solutions contain medium concentrations of hydroxide ions and pH is greater than 7.0.
A-lactalbumin + sodium oleate + PBS (Buffered Solution)
Liprotides were routinely formed by mixing calcium depleted α-lactalbumin from bovine milk (aLA, ≥85%, Sigma-Aldrich, L6010) dissolved in Milli-Q water and 125 mM sodium oleate (OA, ≥95% pure, Sigma-Aldrich, O3880) dissolved in 20% ethanol at an aLA:OA molar ratio of 1:15 in 50 mM PBS (50 mM NaHPO4, pH 7.4, 150 mM NaCl) and incubated for 1 h at 20 °C or 80 °C (leading to lip20 and lip80 respectively).
Liprotides kill cancer cells by disrupting the plasma membrane
A-lactalbumin + oleic acid + PBS (Buffered Solution)
Tris–HCL: [37,53]. Briefly, α-LA was dissolved in 20 mM Tris–HCl at pH 8 to a final concentration of 600 μM and the solution heated to 60 °C. Then, 40 mol of OA was added to the solution followed by 2 min of sonication at 30 °C (Branson 3510R-MT, 42 kHz, 130 W). The reaction mixture was stirred at 60 °C for 30 min, cooled under tap water, acidified to pH 3.5, and stirred for 24 h at 4 °C. The complex is referred to as α-LA-OA Tris–HCl 60.
A-lactalbumin + oleic acid + ethanol solution
We show that mixing bovine α-Lactalbumin (BLA) with pure OA (liquid form), followed by incubation and shaking at 37°C is the most attractive choice for preparing BLA-OA complexes (resulting complex referred to as BLAOA (pure)). Pre-dissolving OA in methanol or ethanol and then mixing with BLA also produces a complex (referred to as BLAOA (methanol) or BLAOA (ethanol)) with similar activities as BAMLET. BLA mixed with OA pre-dispersed in phosphate buffer forms little of the active complex.
A-lactalbumin + oleic acid + high PH with NAOH
NaOH: [38]. α-LA was dissolved in 5 mM NaOH and 1 mM EDTA at pH 12 to a final concentration of 600 μM. The solution was heated to 45 °C and then 40 mol of OA dissolved in 1 ml of ethanol was added followed by sonication for 2 min at 30 °C (Branson 3510R-MT, 42 kHz, 130 W). After stirring for 30 min at 45 °C the reaction was cooled under tap water to room temperature, acidified with HCl to pH 3.5, and stirred for 24 h at 4 °C. The complex is referred to as α-LA-OA NaOH 45.
A-lactalbumin + oleic acid + low PH with HCL
HCl: [39,54]. Briefly, α-LA was dissolved in 1 M HCl and by stirring for 24 h at room temperature to a final concentration of 600 μM. Next, the solution was heated to 85° and then 40 mol of OA previously dissolved in 1 ml of ethanol was added to the solution followed by 2 min of sonication at 30 °C (Branson 3510R-MT, 42 kHz, 130 W). The reaction was completed by constant stirring for 30 min at 85 °C and cooling under tap water and stirred for 24 h at 4 °C. The complex is referred to as α-LA-OA HCl 85.
Note: Can substitute Acetic Acid or Lactic Acid.
A-lactalbumin + oleic acid + lactic acid
The BAMLET complex in bovine colostrum was prepared according to the methodology described by Delgado et al. (2015). Colostrum samples from second and third milkings were adjusting to pH 5.5 using lactic acid (85%, v/v). Subsequently, the colostrum was heated until to 85 °C in a 5L digital general-purpose water bath (Fisher Scientific Co, Niles, Illinois, USA), after which oleic acid was added (0.36 g in 1 mL of ethanol, w/v). The mix was ultrasonicated for 2 min at 30 °C using an ultrasonic processor model CPX-130 (Cole-Parmer, Vernon Hills, Illinois, USA). The reaction was completed with constant stirring for 30 min at 85 °C, then the BAMLET complex reaction was stopped by cooling under running water and stirred for 24 h at 4 °C. The samples were neutralized to pH 7 with 5 M NaOH and finally ultrasonicated for 1 min at 130 W with pulsations of 10 s at 85% opening (Cole-Parmer, Vernon Hills, Illinois, USA) to finally obtain the BAMLET enriched bovine colostrum ready to make the spreadable cheese.
Spreadable cheese production
The production of spreadable cheese was carried out according to the following methodology. Colostrum from the second and third milking was heated to 38 °C, adding calcium chloride (0.25 g L−1 of colostrum) and stirred until it was completely dissolved to carry out the coagulation process. Next, 4 mL of rennet was added per liter of colostrum, homogenizing the mixture until flocculation and then left for 30 min. The curd was cut gently for 30 min at 38 °C, and was then stored at 4 °C for 12 h, after which the cheese was centrifuged using a Sorvall Legend RT centrifuge (Thermo Scientific, Mundelein, Illinois, USA) at 2980 rpm for 30 min to remove the whey. The mixture was then filtered with # 5 Whatman filter paper with the help of a vacuum pump (Air Admiral 79,202–00, Cole-Parmer, Vernon Hills, Illinois, USA) and stored under refrigeration conditions until use.
Using BAMLET complex in a functional spreadable cheese elaborated with bovine colostrum