**2.4. Microbiological analysis**

The amount of viable cells of *Lactobacillus* sp. was determined by serial tenfold dilution with sterile peptone water. Aliquots of 1ml were plated, in duplicate, in plates with Man-Rogosa-Sharpe agar, enriched with L-cysteine HCl. The Petri plates were incubated for 48-72h at 37°C and the results were expressed as log colony forming units (CFU)/ml juice.

The optical density of biomass was measured with the UV-Visible spectrophotometer at 610nm. In the preparation of the calibration curve for optical density vs. dry cell weight several dilutions of the juices were made. According Altiok [8], for each dilution 2 ml of sample was used to obtain optical densities at 610 nm wavelength and 15 ml of sample was filtered with a pre-weighed cellulose acetate membrane filter having a pore size of 0.45 µm using a vacuum pump. The biomass collected on the filters was washed with 15 ml of water and the filters were dried at 1000C for approximately 24 h until constant weight was observed. The results were expressed as g.

Fermentation of Vegetable Juices by *Lactobacillus Acidophilus* LA-5 177

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

**biomass (g/l)**

The initial concentration of reducing sugars of the carrot juices, by 25.2g/l, was favourable for the growth of *Lactobacillus acidophilus* LA-5. Testing two strains of Lactobacillus (one genetically selected Mont4+ and the other genetically altered, Mont4+pxyAB-mod). Kiouss [9] established that the Mont4+ had the highest yield of lactic acid fermenting with six percent concentration of glucose, whereas the L strain utilized the sugar best at the four percent concentration. In the same time temperature and pH seemed to play the largest role

Concluding, higher inoculum densities of *Lactobacillus acidophilus* LA-5 were not significantly influenced the survival yield of the useful microbiota in the lactic acid fermented juices after 24h. In the same time, no parallel relationships between lactic acid concentration and the inoculum size were determined. The result agrees to those obtained by Agarwal, Dutt, Meghwanshi and Saxena [10] using *Enterococcus flavescens* for production of lactic acid. In their opinion, beyond a certain concentration lactic acid yield dropped due to high cell density resulting in fast depletion of essential nutrients, limiting further growth and reducing the yield. Referring to bifidobacteria, Dave and Shah [11] reported also that a higher inoculum did not always improve their viability to a satisfactory level. No data

**Figure 2.** pH and biomass evolution during lactic acid fermentation of carrot juice with different inoculum of *Lactobacillus acidophilus* LA-5: 0.2g/l ( and ▲); 0.3g/l ( and ■); 0.4 g/l (

Although the pH dynamics was quite different in the first 6h of the process, the initial amount of the pure culture did not affect the subsequent evolution or the final value of this

0 2 4 6 8 24 **Time (h)**

The sharp decrease in biomass from 6 to 8h has been correlated with the viable cells tendency, as result of reaching pH values by 4.34 to 4.47. Being known that *Lb. acidophilus* is more sensitive in acidic environment, this result underlines the necessity to manage the size of inoculum in order to obtain a balance between the lactic acid accumulation and the

in the organisms ability to grow and thus affecting its production of lactic acid.

referring to *Lactobacillus acidophilus* were found in the literature.

and ●); columns - pH values, lines - biomass

**pH**

survival of the probiotic microorganisms.

parameter (Figure 2).

### **2.5. Statistical analysis**

Statistical analysis was carried out using the software SPSS (Statistical Package for the Social Science 17.0 trial version).
