LACTOBACILLUS PROTEASES GENERATION ANALYSIS

Various Lactobacilli strains were tested for their capability in generating proteases with potential ability in interacting and cleaving bPG. Lactobacilli were grown in three different media and consequent proteolytic activity examined through skim milk agar plates.

First, various strains of Lactobacilli, some of which we knew from the literature to be highly hydrolytic such as L. helveticus, or commonly used in the production of cheese such as L. acidophilus (Salimen Seppo et al. 1998), were grown in 10 ml MRS (5.5% MRS, 0.05% cysteine solution autoclaved at 121°C for 15 minutes (De Man J. C.

34 et al. 1960)) overnight at 37°C in a CO2 chamber. Subsequently, 100 µl of each bacteria

were transferred into falcon tubes containing 10 ml of either 20% NFDM solution, UHT milk or MRS containing 1% skim milk (Figure 7), and allowed to grow overnight, always at 37°C in a CO2 chamber. Meanwhile, skim milk agar plates were prepared (10% v/v

skim milk, 1.5% w/v agar solution autoclaved at 121°C for 15 minutes and poured 20 ml into Petri dish plates (Maunsell Blaithin et al. 2006) ). After bacteria were allowed to grow and skim milk agar solidified, sterile filter paper disks (5 mm diameter) were arranged on the surface of each agar plate. Fifteen µl of each bacteria-containing media were carefully added to one of the filter paper disk. These procedures were conducted in triplicates. Plates were incubated over night at room temperature. The proteolytic activity was quantified by measuring the visible zone of clearance surrounding the filter paper disk. If proteases were produced by the microorganisms and capable of hydrolyzing the surrounding casein, a clear halo would be observed around the filter disk, on the contrary if no proteolytic activity occurs, the area of interest would remain white.

Figure 7: Experimental design to determine Lactobacillus proteolytic enzymes generation in different growth media.

2.3.4.1.1 PROTEASES INTERACTION WITH BOVINE PLASMINOGEN

It is known that LAB produce two types of proteases: extracellular or cell wall associated. These enzymes are needed by the microorganisms to hydrolyze bigger proteins that the cell will then uptake and use as a source of peptides and amino acids for metabolic purposes (Salimen Seppo et al. 1998). After analyzing which media and which strains resulted in higher generation of proteases, Lactobacilli were grown again under ideal conditions and the solution to be analyzed for enzymes activity was incubated with

L.casei 10 L.helveticus MR220 L.reuteri 11284 L.reuteri 23272 L.acidophilus NCFM L.paracasei 25302 L.casei 661 L.johnsonii 11088 L.reuteri T1 MRS +1% SKIM MILK 20% NFDM UHT bPG (0.5 mg/ml) hydrolysis

Skim milk agar plate hydrolysis

35 bPG. A series of experiments were conducted in order to determine whether the proteolytic enzymes released outside the cell or associated with the cell wall of the microorganism, showed ability in bPG hydrolysis. Analysis of the products obtained from this reaction was conducted through SDS-PAGE as described in section 2.3.2.3.1.

Since best hydrolytic activity observed in skim milk agar plates resulted for microorganisms grown in UHT milk and MRS-1% skim milk, these media were used to test for bPG hydrolytic enzymes.

After inoculating UHT milk with each microorganism, and allowed Lactobacilli

to grow overnight, 1 ml of UHT milk after inoculation was transferred into a 1.5 ml Eppendorf tube. Samples were centrifuged at 10 rpm between 5 and 10 minutes until a clear supernatant was observed. The supernatant obtained after centrifugation, was added to bPG (1mg/ml, 11.36 μM) at a 1:1 volume ratio and allowed to interact for 3 hours at 37°C. After 3 hours the reaction was stopped by addition of Laemmli buffer solution (1:1 volume ratio) and samples were boiled for 5 minutes and analyzed through SDS-PAGE.

Due to UHT high protein content, it was not possible to use this media to test for the cell wall associated enzymes ability in interacting with bPG since high dilutions were needed prior or after interaction with the substrate, causing poor results through electrophoresis analysis. In order to be able to have a better understanding on the ability of either extracellular or cell wall bound proteins in hydrolyzing bPG, MRS with 1% skim milk was used as growth medium instead.

In a similar way, LAB were grown in MRS overnight and then transferred into fresh MRS plus 1% skim milk growth medium. Lactobacilli were allowed to grow and subsequently 1 ml of LAB in MRS-1% skim milk was transferred into a 1.5 ml Eppendorf tube. Tubes were centrifuged at 10000 x g for 5 minutes to allow all the pellets to reach the bottom of the tube and obtain a clear supernatant. The supernatant was transferred into a new Eppendorf tube, and the pH was adjusted to 6-7 with 0.1-1 N NaOH. Subsequently, bPG (1mg/ml, 11.36μM) was added at 1:1 (v/v), and the solution was incubated at 37°C for 3 hours to test for extracellular enzymes presence and ability in bPG proteolysis.

On the other hand, cell wall bound enzymes were purified following McGavin et al. (1993) procedures with modifications. The pellets previously obtained were first

36 resuspended in 1 ml of phosphate buffered saline (PBS x1) using a rotor. Once the pellets were completely dissolved in PBS, samples were centrifuged at 10000 x g for 5 minutes. PBS was discarded and replaced by 1 ml 5M LiCl. Pellets were resuspended again using a rotor for approximately 1h. When no visible pellets were observed in the tube, the solution was centrifuged again for 30 seconds at 5000 rpm. At this point the supernatant obtained should contain the proteins that were previously bound to the surface of the microbes‟ cell wall. Dialysis was conducted by covering and sealing the top of the Eppendorf tube with a membrane (MWCO 3500 Da) and placing the tubes upside down in a water bath for a period of 3 hours. During this step, diffusion of LiCl outside the sample occurs while water molecules will travel in the opposite direction until a balance is reached. After the dialysis, samples‟ pH was adjusted to 6-7 with 0.1 HCl. Samples were concentrated using an Eppendorf vacufuge at 30°C until obtaining 100 µl solution. At this point 20 µl of the concentrated solution were added to 20 µl of bPG (1mg/ml, 11.36 μM) and incubated for 3 hours at 37°C to test for ability of cell wall bound enzymes to hydrolyze bPG which was analyzed through SDS-PAGE and western blot.