**3. Results and discussions**

### **3.1. Effect of inoculum size on the lactic acid accumulation and biomass growth**

A comparative study of the dynamics of lactic acid fermentation of carrot juice using three different concentrations of lyophilized pure culture was realized (Figure 1).

**Figure 1.** Correlation between lactic acid production by *Lactobacillus acidophilus* LA-5 and number of viable cells during fermentation of carrot juice with different inoculum size ▲ 0.2g/l; ■ 0.3g/l; ● 0.4 g/l (smooth lines - lactate, dashed lines - viable cells count)

Relative higher differences concerning the lactate increasing were observed between the variant with 0.2g/l pure culture initial added and the other two within 24 hours of fermentation. Thus, at the end of this interval, the excess was by 7.06% in the juice with 0.3g/l inoculum and 12.06% in the juice with 0.4g/l inoculum respectively. However, in all the batches the lactic acid accumulation, higher than 9g/l, could be considered satisfactory for the shelf life of the final products. From the other part, the number of viable cells is decisive for the probiotic feature of these ones. A direct proportionality between the amount of the lyophilised culture initial added and the viable cells was observed only in the first 4h of the fermentation. As a general characteristic, in the interval 6 - 24h pH values less than 4.5 have become inhibitory for the useful microbiota in all the experimental samples.

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

176 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

observed. The results were expressed as g.

**2.5. Statistical analysis** 

Science 17.0 trial version).

**3. Results and discussions** 

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

Statistical analysis was carried out using the software SPSS (Statistical Package for the Social

**3.1. Effect of inoculum size on the lactic acid accumulation and biomass growth** 

A comparative study of the dynamics of lactic acid fermentation of carrot juice using three

**Figure 1.** Correlation between lactic acid production by *Lactobacillus acidophilus* LA-5 and number of viable cells during fermentation of carrot juice with different inoculum size ▲ 0.2g/l; ■ 0.3g/l; ● 0.4 g/l

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

0

2

4

6

**viable cells (log CFU/ml)**

8

10

12

14

Relative higher differences concerning the lactate increasing were observed between the variant with 0.2g/l pure culture initial added and the other two within 24 hours of fermentation. Thus, at the end of this interval, the excess was by 7.06% in the juice with 0.3g/l inoculum and 12.06% in the juice with 0.4g/l inoculum respectively. However, in all the batches the lactic acid accumulation, higher than 9g/l, could be considered satisfactory for the shelf life of the final products. From the other part, the number of viable cells is decisive for the probiotic feature of these ones. A direct proportionality between the amount of the lyophilised culture initial added and the viable cells was observed only in the first 4h of the fermentation. As a general characteristic, in the interval 6 - 24h pH values less than 4.5

have become inhibitory for the useful microbiota in all the experimental samples.

(smooth lines - lactate, dashed lines - viable cells count)

0

2

4

6

**lactic acid (g/l)**

8

10

12

different concentrations of lyophilized pure culture was realized (Figure 1).

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

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

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 parameter (Figure 2).

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 survival of the probiotic microorganisms.

The maximum rate of acidification vmax was calculated as the time variation of pH (dpH/dt) and expressed as pH units/min (Table 1). Other kinetic parameters were also calculated: time to reach vmax (tmax, hours), time to reach pH 5.0 (tpH 5.0, hours), time to complete the fermentation (tpH 4.2, hours).

Fermentation of Vegetable Juices by *Lactobacillus Acidophilus* LA-5 179

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5

**OD610**

**lactic acid (g/l)**

**Figure 3.** pH and lactic acid dynamics during the lactic acid fermentation of carrot juice at different temperatures: 370C ( and ▲) and 410C ( and ●); columns - pH values, lines - lactic acid

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

The rate of acidification has been correlated with the glucose consumption: 38.9% in the case of the juice fermented at 370C, respectively 53.89% in the case of the juice fermented at 410C. The different tendency of this parameter became obviously after 4h of fermentation (Figure 4), being the consequence of the different rate of growth of *Lactobacillus acidophilus*,

**Figure 4.** Glucose consumption and microbial evolution during the lactic acid fermentation of the carrot juices at different temperatures: 370C ( and ▲) and 410C ( and ●); columns - glucose, lines -

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

Although close, the yields of glucose conversion to lactic acid have inclined the balance in favour of the juices fermented at 370C, the corresponded value being by 0.5, unlike 0.45 in

content

expressed as optical density at 610nm.

**glucose (g/100ml)**

0

1

2

3

4

**pH**

5

6

7

optical density at wavelength by 610nm

0 0.5 1 1.5 2 2.5 3 3.5 4

the case of the juices incubated at 410C.


**Table 1.** 1. Acidification kinetic parameters of fermentation of carrot juices by *Lactobacillus acidophilus* LA-5

A double amount of inoculum had an insignificant influence on the time to reach pH 5.0, important parameter from the shelf life of the fermented juices. Thus, tpH 5.0 (h) was 1.1-fold higher in the case of the batch with 0.2g/l lyophilized pure culture initial added to juice than that one with 0.4g/l. A different situation was registered concerning the maximum rate of acidification (vmax) and the time to reach this rate (tmax). Thus, a polynomial equation of the form *y = -108.5x2 + 81.75x – 4.93* correlated the size of inoculum with the corresponding values of vmax at R squared = 1. Although at the initial moment of fermentation seems to be advantageous to use a higher amount of pure culture, this aspect lessen in time, from the economic point of view being important to obtain a balance between the quantity of inoculum and the targeted parameters which ensure the preservation of the final product.

The values of the biomass content became close after about 6h of fermentation. No parallel relationship between lactic acid concentration and biomass was observed, result that agrees to those obtained by Amrane [12] and Kotzamanidis [13].

However, taking into account the lactic acid accumulation and the dynamics of the number of viable cells, it was obvious that the utilization of higher amount of inoculum is not justified.
