## 2 Materials and Methods
### Batch Fermentation  
3.1 Batch Fermentation with Strains in Suspension Using _Enterococcus faecalis_ and _Kluyveromyces lactis_  
Batch fermentation was carried out using inactivated strains (due to the fact that using activated strains in the fermentation process did not result in a higher LA production in both samples) and untreated (proteinized) and treated (deproteinized) cheese. In order to deproteinize the cheese whey (to eliminate not only the proteins but also the fats), it was put into an autoclave at 121 \({}^{\circ}\)C and 1.03 bar pressure for 15 min. After it was passed through a 60 sieve (0.050 nm) to eliminate the majority of the proteins, it was centrifuged at 4500 RPM for 10 min to finally go through vacuum filtration.  
The fermentation culture medium was composed of 0.5% p/v yeast extract, 0.9676% p/v ammonium phosphate, a neutralizer (5 g/L calcium carbonate), and 200 mL of deproteinized and proteinized cheese whey, which was mixed and placed in a water bath at 65 \({}^{\circ}\)C for 10 min. It was cooled and inoculated with a separately native strain (3 grades). The fermentation parameters were as follows: 32 \({}^{\circ}\)C and 120 RPM for 7 days.  
The proteinized cheese whey used in the fermentation process had the following physicochemical characteristics: 0.45% acidity, 0.50% ash, 3.41% lactose, 0.81% proteins, 0.85% fats, and a pH of 5. Meanwhile, the deproteinized cheese whey had the following characteristics: 0.38% acidity, 0.52% ashes, 3.57% lactose, 0.04% proteins, 0.38% fats, and a pH of 4.84.  
3.2 Batch Fermentation with Strains Immobilized Using _Enterococcus faecalis_ and _Kluyveromyces lactis_  
Batch fermentation using strains immobilized in alginate beads was carried out in deproteinized and proteinized cheese whey. The culture medium was composed only of the deproteinized and proteinized cheese whey without adding other additives. This was due to the fact that in preliminary tests, it was observed that the alginate beads lost their stability, so it was decided to use the cheese whey without any additives.  
To produce the alginate beads, each strain was cultivated individually in MRS broth at 32 \({}^{\circ}\)C and 120 RPM for 24 h. Then, to concentrate the bacteria in a pellet, a volume of this broth was taken and centrifuged at 4500 RPM for 20 min. Then, this pellet was washed three times with a saline solution that was discarded, and 1 mL of saline solution was added to the biomass pellet to dissolve it. After the biomass solution was mixed in a 2.5% sodium alginate solution (1 mL of biomass in 25 mL of sodium alginate) and then drip-added to a 4% CaCl\({}_{2}\) solution for 1 h for its stabilization, and these conditions were for _Kluyveromyces lactis_. This was carried out with 5% CaCl\({}_{2}\) and 3% sodium alginate solution for 1 h of stabilization for _Enterococcus faecalis_.  
The calcium chloride solution was prepared and placed in a beaker on a magnetic stirrer at 120 RPM. Then, the biomass solution was added to a burette and dropped into the beaker with the calcium chloride solution, and when all the mixture passed, it was left in contact for one hour h for its stabilization. Finally, the beads were washed with sterile distilled water to remove excess calcium ions before use, for which the calcium chloride was recovered. Table 1 shows the fermentation systems using batch fermentation with suspended cells and immobilized cells.