## 2 Materials and Methods
### The Milk Thistle Bio-Ferment and Extract Preparation  
Preparation of the bio-ferment (B) of milk thistle (ground and defatted grains):  
Stage 1. Preparation of the inoculum  
A total of 750 mL of distilled water was introduced into a 1000 mL flask, the flask was heated until it boiled and kept at this temperature for 2 h (to sterilize the apparatus), and then the contents of the flask were cooled to 60 \({}^{\circ}\)C. After this time, 50.00 g of lactic acid bacteria medium (CM0359) was introduced into the flask and heated (to its boiling point) for 2 h, and then the contents of the flask were cooled to a temperature of 37.5 \({}^{\circ}\)C in order to introduce lactic acid bacteria strains (700.00 mg). The introduced bacterial strains were multiplied for 48 h at 37.5 \({}^{\circ}\)C, thus obtaining the inoculum. A mixture of lactic acid bacteria strains was used in the fermentation process because the ultimate outcome of mixed fermentation is superior to that of fermentation carried out with a single isolated strain (there is a synergistic effect from different bacterial strains). Compared to a single strain, the use of mixed bacterial cultures increases the efficiency of the process with better use of plant raw materials. Additionally, there is an increased ability to adapt the strains used to changing process conditions, with increased resistance to contamination by undesirable microorganisms. The use of mixed bacterial culture is a technique that not only enhances fermentation efficiency but also yields economic advantages. Due to the absence of the requirement to isolate a specific strain, the overall expenses of the entire procedure are reduced, and the fermentation time is shortened [29; 30].  
Stage 2. The milk thistle fermentation process and filtration of the obtained bio-ferment  
The following raw materials were introduced into a 500 mL conical flask: molasses (18.00 g); distilled water (300.00 g); mineral salts such as ammonium sulfate (2.00 g), calcium chloride (1.00 g), and potassium dihydrogen phosphate (1.00 g); Lipase AY30 enzyme (70.00 mg); ground and defatted milk thistle grains (5.00 g); and inoculum (10.00 mL). The contents of the flask were stirred for 30 min (until the introduced raw materials were dissolved), and then the fermentation process was started in a laboratory dryer at a temperature of 37.5 \({}^{\circ}\)C for an appropriate time. During the process, samples were taken every day and analyzed for polyphenol content (see Section 2.7.4: Total polyphenol content). The highest content of polyphenols was observed on the 21 st day of fermentation, after which a gradual decrease in antioxidant activity (content of active compounds) was observed. Therefore, the process was carried out for 21 days, performing three independent experiments [29; 31].  
After the process was completed, the obtained bio-ferment was subjected to 3-stage filtration: I: first, the bio-ferment was filtered in a glass funnel; II: then the bio-ferment was centrifuged in a centrifuge (5 min, 166 Hz, 10,000 \(\times\)\(g\)); III: finally, the additionally filtered and centrifuged bio-ferment was filtered using sterile syringe filters with a pore size of 0.45 \(\upmu\)m (intended for sterilizing filtration of aqueous solutions). In this way, the cosmetic raw material was free of microorganisms. The amount of bio-ferment obtained and filtered was approximately 100 mL. The bio-ferment was stored in a freezer at \(-\)15 \({}^{\circ}\)C for 3 months.  
Preparation of the extract (E) of milk thistle (ground and defatted grains):Extraction of ground and defatted grains of milk thistle was carried out using an ultrasonic method. First, 300.00 mL of distilled water was introduced into 5.00 g of the ground and defatted grains of milk thistle. Next, the extraction was carried out with the use of ultrasound bath at a frequency of 40 kHz (for 1 h at 60 \({}^{\circ}\)C), and then the extract obtained was subjected to filtration on a pressure funnel through a Whatman paper filter (codified EEA03).