## 4 Discussion
### Probiotic Bacterial Encapsulation as a Method of Protection from External Factors
They demonstrate an antimicrobial effect, and when added to the cosmetic, they inhibit the development of bacteria, mould and fungi, thus extending the shelf life. The list of substances with antimicrobial activity and their permissible concentrations when used in cosmetic products are listed in Annex V to Regulation (EC) No. 1223/2009 of 30 November 2009 [38]. The most commonly used antibacterial preservatives can be divided according to their chemical composition, namely, organic acids and their derivatives, alcohols and phenols, aldehydes and formaldehyde-releasing agents, isothiazoliones, biguanides, quaternary ammonium compounds (QACs), nitrogen compounds, derivatives of heavy metals and inorganic compounds [56; 57]. In general, the ideal preservative ingredient should be stable, compatible, effective at low levels, non-toxic, compliant with cosmetic regulations and inexpensive [58; 59; 60]. However, in the case of probiotics-loaded cosmetics, it is difficult to meet the above-mentioned microbiological requirements while maintaining viable cells. The aim of our study was to develop an effective method of introducing live bacteria (strain of _L. casei_) into formulations. It was important to select the adequate preservative system that would guarantee both of these requirements: to ensure the survival of probiotic bacteria in the microspheres and the protection of the products from pathogenic microorganisms, i.e., to ensure the microbiological purity and stability of the cosmetic formulations. In the case of our research study, some preservatives from the group of organic acids and their derivatives were used, specifically dehydroacetic acid, salt of benzoic acid (sodium benzoate) and sodium levulinate--sodium salt of levulinic acid--combined with sodium salt of anisic acid, which are examples of \"natural preservatives\" [61]. Additionally, for comparison purposes, the most popular cosmetic preservatives were checked, namely, parabens (also belonging to the group of organic acid derivatives). Among alcohols, phenoxyethanol in combination with ethylhexylglyeverin was used. As an example of ketones, hydroxyacetophenone was selected. The antimicrobial effect of organic acid derivatives (sodium benzoate combined with sodium salts of levulinic and anisic acid, or dehydroacetic acid and sodium benzoate) is related to the reduction in pH [62]. Acids and derivatives of organic acids, including benzoic acid and sorbic acid, inhibit the development of microorganisms through several mechanisms: firstly, they lower the pH of the external environment to values unfavourable for the development of microorganisms [63]; secondly, they cause acidification of the cytoplasm due to the penetration of uncharged organic acids into the cells (resulting in the internal pH causing their dissociation into anions, which results in the lowering of the internal pH), which affects the isoelectric pH of enzymes involved in glycolysis and active transport [62]. Alcohols and phenols (a system based on phenoxyethanol and ethylhexylglicerin) are agents with effective antimicrobial properties against both bacteria and fungi. Their action is bactericidal, especially in the case of acid-resistant mycobacteria. The mechanism of action of alcohol is related to protein denaturation or the inhibition of protein synthesis through several mechanisms [64]. [PERSON] et al. [65] showed an effect of oxidative stress in _Pseudomonas putida_ KT2440 as a result of exposure to phenol. At low concentrations, benzyl alcohol and phenoxyethanol lyse the bacterial cell membrane. In this way, they can denature the structure of proteins by binding to amino acid residues [66; 67]. However, aldehydes also have a broad spectrum of activity against both bacteria and fungi. They can react with chemical groups (amine, amide, imine, thiol, carboxyl and hydroxyl substituents) on biomolecules, including proteins and DNA. This leads to protein aggregation, which results in their irreversible chemical modification, leading to the inhibition of metabolism and cell division [68; 69].