# A Model for Probiotic Fermented Food Production
## 2 Material and Method
### Real-Time PCR  
We used a commercial probioticic strain, _Lactobacillus rhamns_ GG (ATCC 53103), as a reference strain for the real-time PCR standard curve preparation. In this study, 12.5 mg of lyophilized _L. rhamns_ GG was diluted in 1 L of pasteurized milk (an inoculum level of 6 Log CFU/mL), and five subsequent serial dilutions were performed in 20 mL of a solution of pasteurized milk (10-8-6-4-2 mL) and trisodium citrate at 2% (Sigma Aldrich) (10-12-14-16-18 mL, respectively). The lyophilised product suspension was centrifuged (3500\(\times\)\(g\), 10 min), and DNA was extracted from the pellet by using the QIAamp DNA Stool Mini Kit (Qiagen) following the manufacturer's protocol. The five dilutions from _L. rhamns_ GG genomic DNA preparations were amplified using real-time PCR.  
After the PCR reaction optimization, the standard curve was established using the serial dilutions of _L. rhamns_ GG samples from the initial concentration of 10\({}^{6}\) CFU/mL of viable bacterial cells to simulate the cheese product inoculation. Aliquots of the standard samples prepared for DNA extractions were plate counted. Based on standard curves, the reaction efficiencies were determined, and the possible inhibition of PCR reactions was examined.  
The correlation between Ct values and CFU/mL was determined by using Design and Analysis Software v2.6, QuantStudio (Thermo Fisher, Monza, Italy).  
The standard curves' parameters obtained by the fivefold dilution samples series showed an R\({}^{2}\) = 0.9925, an amplification efficiency of 109.64%, and a slope of \(-\)3.1206.  
The absolute quantification of the _L. rhamns_ strains (HN001 and LR06) was performed on milk samples before inoculation as negative controls, on samples collected right after inoculation with 10\({}^{6}\) CFU of probioticic, on the discarded whey samples, in cheese after 3 days of inoculation and in cheese after one month of ripening.  
PCR amplifications were performed in a 25 \(\mu\)L reaction volume containing SYBR(tm) Green PCR Master Mix (Applied Biosystems, Monza, Italia), 0.2 \(\mu\)M of each primer, and 1 \(\mu\)L of genomic DNA extract. The primers used were _L. rhamns_ specific, PoxProm FW 'TGAAAGGGYTTGCATTGTTAT' and PoxProm RV 'AATGCGCCYACTTCTCATG' [68]. The PCR amplification was performed using a QuantStudio(tm) 3 Real-Time PCR (ThermoFisher). Bacterial detection was achieved with a 3-step qPCR with the initial denaturing step of 50 \({}^{\circ}\)C for 2 min and 95 \({}^{\circ}\)C for 2 min, 40 cycles of 95 \({}^{\circ}\)C for 30 s, 60 \({}^{\circ}\)C for 15 s, 72 \({}^{\circ}\)C for 20 s, and then 95 \({}^{\circ}\)C for 1 min and 55 \({}^{\circ}\)C for 30 s. All samples were also subjected to melting curve analysis in the temperature from 60 to 95 \({}^{\circ}\)C with an increase of 0.5 \({}^{\circ}\)C for 1 s to establish the specificity of the amplification. DNA from the _L. rhamns_ GG bacteria was included in the PCR standard curve assay. For the DNA quantification, triplicate samples were used, and the mean of CFU (colony-forming unit) quantity per gram wet weight was calculated, also considering the correspondence with the plate count for the possible presence of multiple putative _box_ genes [69].  
\[\text{Copies/g}=\frac{\text{DNA concentration }(\text{ng/\mu L})\times 6.02 \times 10^{23}\text{ molecules/moles}}{660\text{g/mole}\times(\text{target DNA length }(\text{bp}))\times 1\times 10^{9}\text{ ng/g}}\]