**3.3. The behaviour of different raw materials during the lactic acid fermentation by Lactobacillus acidophilus LA-5**

Fresh white cabbage (*Brassica oleracea* L.), red cabbage (*Brassica oleracea* var. *capitata* f. *rubra*), red beet (*Beta vulgaris* var. *vulgaris*), cucumbers (*Cucumis sativus*) and red onion (*Allium cepa*  var. *ascalonicum*) were chosen in order to perform different experimental batches, as follows: Cb - cabbage juice, RCb - red cabbage juice, Rb - red beet juice, Cc - cucumber juice, CcO cucumber juice with 0.1% (v/v) onion juice added after the heating and cooling of the batches.

pH and lactic acid dynamics during the lactic acid fermentation of vegetable juices with *Lb. acidophilus* are shown in Figure 5 and Figure 6 respectively. The pH values ranged from 6.29 to 3.74, no significant differences between the analyzed batches being observed, excepting the red beet juice. Thus, after one day a higher value by 4.28 was determined, the prolongation of the time of fermentation with other 24h hadn't a positive influence on this parameter.

After 24h, the highest decrease of pH was determined in the case of the cucumber juice (2.51 units), correlated with the increase of the lactic acid amount until 9.36g/l. Although the pH values of the samples Cc and Cb were close during the process development, the maximum rate of acidification vmax registered a better value of 9.33·10-3 units/min. in the case of the cucumber juice. This could explain the fermentation slowdown in the batch Cb the interval 6 - 8 hours. Correlated with the results of the microbiological analysis, it seems that this time the process was directed towards the growth of the useful microbiota. A minimum value of the maximum rate of acidification, by 6.66·10-3 units/min., was determined in the case of CcO, while the time to reach pH 5.0 (tpH 5.0, hours) ranged between 1.9 (Cb) to 3.5 (CcO).

A relative distinct behaviour was observed in the case of red cabbage juice, red beet juice and cucumber juice with onion juice added, in the sense of the slowdown of the metabolism objectified in the dynamics of the parameters that describe the process unfolding. The differences could be explained through the presence of some chemical constituents which can act as inhibitors on useful bacteria, like anthocyanins in the red cabbage, betacyanins in red beet, respectively constituent sulfides in the onion juice. According [14], sulfides, especially those with three or more sulfur atoms, apparently possess potent antimicrobial activity. However, concerning the batch with onion juice added the initial trend was attenuated after 6 hours of fermentation, the oils and their sulfides constituent showing weak antimicrobial activity ([15]).

**by Lactobacillus acidophilus LA-5** 

batches.

parameter.

between 1.9 (Cb) to 3.5 (CcO).

weak antimicrobial activity ([15]).

incubation temperature applied in the further studies was by 37±0.10C.

The faster consumption of the carbon source, correlated with the growth of the useful microbiota at higher temperature, respectively with the increase of the lactic acid content until the value by 9.1g/l, was followed at 24h by the decline of the viability of *Lactobacillus acidophilus*. Taking into account the dynamics of all the above mentioned parameters, the

**3.3. The behaviour of different raw materials during the lactic acid fermentation** 

Fresh white cabbage (*Brassica oleracea* L.), red cabbage (*Brassica oleracea* var. *capitata* f. *rubra*), red beet (*Beta vulgaris* var. *vulgaris*), cucumbers (*Cucumis sativus*) and red onion (*Allium cepa*  var. *ascalonicum*) were chosen in order to perform different experimental batches, as follows: Cb - cabbage juice, RCb - red cabbage juice, Rb - red beet juice, Cc - cucumber juice, CcO cucumber juice with 0.1% (v/v) onion juice added after the heating and cooling of the

pH and lactic acid dynamics during the lactic acid fermentation of vegetable juices with *Lb. acidophilus* are shown in Figure 5 and Figure 6 respectively. The pH values ranged from 6.29 to 3.74, no significant differences between the analyzed batches being observed, excepting the red beet juice. Thus, after one day a higher value by 4.28 was determined, the prolongation of the time of fermentation with other 24h hadn't a positive influence on this

After 24h, the highest decrease of pH was determined in the case of the cucumber juice (2.51 units), correlated with the increase of the lactic acid amount until 9.36g/l. Although the pH values of the samples Cc and Cb were close during the process development, the maximum rate of acidification vmax registered a better value of 9.33·10-3 units/min. in the case of the cucumber juice. This could explain the fermentation slowdown in the batch Cb the interval 6 - 8 hours. Correlated with the results of the microbiological analysis, it seems that this time the process was directed towards the growth of the useful microbiota. A minimum value of the maximum rate of acidification, by 6.66·10-3 units/min., was determined in the case of CcO, while the time to reach pH 5.0 (tpH 5.0, hours) ranged

A relative distinct behaviour was observed in the case of red cabbage juice, red beet juice and cucumber juice with onion juice added, in the sense of the slowdown of the metabolism objectified in the dynamics of the parameters that describe the process unfolding. The differences could be explained through the presence of some chemical constituents which can act as inhibitors on useful bacteria, like anthocyanins in the red cabbage, betacyanins in red beet, respectively constituent sulfides in the onion juice. According [14], sulfides, especially those with three or more sulfur atoms, apparently possess potent antimicrobial activity. However, concerning the batch with onion juice added the initial trend was attenuated after 6 hours of fermentation, the oils and their sulfides constituent showing

**Figure 5.** pH dynamics in vegetable juices obtained from different raw materials, during fermentation with *Lactobacillus acidophilus* LA-5

Referring to the red cabbage juice, although after 24 hours of fermentation the pH values were similar, the lactic acid content was lesser with about 1.5g/l compared with the white cabbage juice. This can be due to the amphoteric nature of the anthocyanins.

**Figure 6.** Lactic acid accumulation in vegetable juices obtained from different raw materials, during fermentation with *Lactobacillus acidophilus* LA-5

[16] studied the fermentation of cucumber juices with a 0.5%, 1% and 2% additions of the onion juices by *Lb. plantarum* CCM 7039. It was found that in the initial stages of fermentation, the presence of onion in the juices positively influenced lactic and acetic acid production. However, in further course of fermentation, slight inhibition effects of onion in the fermented juices were observed, especially at elevated onion/cucumber ratio.

The correlation between the biomass amount and the production of lactic acid (Figure 7) in the case of lactic acid fermentation of red beet juices with *Lactobacillus acidophilus* in the first 24 hours, was described using the Luedeking & Piret model [17]. According to this model, the instantaneous rate of lactic acid formation (dP/dt) can be related to the instantaneous rate of bacterial growth (dN/dt), and to the bacterial density (N), throughout fermentation at a given pH, by the expression:

$$dP/dt = \alpha \, dN/dt + \beta \, N$$

Fermentation of Vegetable Juices by *Lactobacillus Acidophilus* LA-5 183

the fact that the cucumber juice underwent a tumultuous fermentation although its content was only with 15.09% higher, it seems that other chemical constituents of the raw materials

The initial content of sugars in cucumber juice was situated at the maximum limit determined by [19], while in the case of the white cabbage juice was close to that one

**Figure 8.** Correlation between the substrate consumption, lactate production and viable cells Cb (a),

● - glucose, ■ - lactate, ▲ - viable cells (points - experimental data, smooth lines - predicted values)

The metabolization of the reducing sugars after 24h of lactic acid fermentation of vegetable juices with *Lb. acidophilus* LA-5 ranged between 26.66% (Rb) to 54.09% (Cc). Relative close values were obtained by other authors in lactic acid fermentation of vegetable juices. Thus, the utilization of sugar during fermentation in a mixture of beetroot juice and carrot juice and different content of brewer's yeast autolysate with *Lb. plantarum* A112 and with *Lb.* 

The tested pure culture, routinely used for dairy products, was found to be capable of growing on pure vegetable juices without nutrients added. In the batches obtained from cabbage, respectively cucumber, the maximum volumetric productivity was determined after 8 hours as follows: 19.25x1014 CFU/(l·h) for Cb, 11.9x1014 CFU/(l·h) for RCb, 18.6x1014

are responsible for the above mentioned differences.

determined by [20].

RCb (b), Cc (c) and CcO (d)

*acidophilus* NCDO 1748 varied from 19.4 to 24.1% ([21]).

CFU/(l·h) for Cc and 10.25x1014 CFU/(l·h) for CcO respectively.

where the constants α and β are determined by the pH of the fermentation.

**Figure 7.** The correlation between the lactic acid production and viable cells count of *Lactobacillus acidophilus* LA-5 growing on red beet juices

A simplified presentation of the above model relates to the linear part of the equation which is presented as:

$$\left(p - p\_0\right) = \alpha \left(\mathbf{x} - \mathbf{x}\_0\right)$$

where *p0* and *p* are the concentrations of lactic acid (g/l) initially and at time *t*, respectively, and *x0* and *x* are the increases of the biomass (log CFU/mL) initially and at time *t*, respectively.

The R squared coefficient closed by the ideal value ''1'' (R2 = 0.9989) in the case of the carrot juices fermented with *Lactobacillus acidophilus* LA-5 (data not shown) highlights a better linear correlation, respectively a strong connection between the lactic acid production and the lactic acid bacteria growth. Not the same situation has registered in the lactic acid fermentation of the red beet juices with the same strain. The highest value of the coefficient (1 – R2) it is caused by the increase of the lactic acid amount in the first 4 hours, followed by a steady interval of evolution of this parameter. From the other hand, according [18], the deviations from the linear dependence are mostly caused by nutritive limitations of the substrates, and are related to the specific bacterial species. Not at least, the initial content of reducing sugars of the red beet, by 21.2g/l, could be limiting. However, taking into account the fact that the cucumber juice underwent a tumultuous fermentation although its content was only with 15.09% higher, it seems that other chemical constituents of the raw materials are responsible for the above mentioned differences.

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

a given pH, by the expression:

*acidophilus* LA-5 growing on red beet juices

is presented as:

respectively.

The correlation between the biomass amount and the production of lactic acid (Figure 7) in the case of lactic acid fermentation of red beet juices with *Lactobacillus acidophilus* in the first 24 hours, was described using the Luedeking & Piret model [17]. According to this model, the instantaneous rate of lactic acid formation (dP/dt) can be related to the instantaneous rate of bacterial growth (dN/dt), and to the bacterial density (N), throughout fermentation at

> *dP dt dN dt N* / /

**Figure 7.** The correlation between the lactic acid production and viable cells count of *Lactobacillus* 

A simplified presentation of the above model relates to the linear part of the equation which

 *pp xx* – – 0 0 

where *p0* and *p* are the concentrations of lactic acid (g/l) initially and at time *t*, respectively, and *x0* and *x* are the increases of the biomass (log CFU/mL) initially and at time *t*,

The R squared coefficient closed by the ideal value ''1'' (R2 = 0.9989) in the case of the carrot juices fermented with *Lactobacillus acidophilus* LA-5 (data not shown) highlights a better linear correlation, respectively a strong connection between the lactic acid production and the lactic acid bacteria growth. Not the same situation has registered in the lactic acid fermentation of the red beet juices with the same strain. The highest value of the coefficient (1 – R2) it is caused by the increase of the lactic acid amount in the first 4 hours, followed by a steady interval of evolution of this parameter. From the other hand, according [18], the deviations from the linear dependence are mostly caused by nutritive limitations of the substrates, and are related to the specific bacterial species. Not at least, the initial content of reducing sugars of the red beet, by 21.2g/l, could be limiting. However, taking into account

where the constants α and β are determined by the pH of the fermentation.

0 0,5 1 1,5 2 2,5 3

**p - p0 , g/l** **R2 = 0,7533**

1,38 1,49 1,73 1,99 **x-x0, lg CFU/ml**

 The initial content of sugars in cucumber juice was situated at the maximum limit determined by [19], while in the case of the white cabbage juice was close to that one determined by [20].

**Figure 8.** Correlation between the substrate consumption, lactate production and viable cells Cb (a), RCb (b), Cc (c) and CcO (d)

● - glucose, ■ - lactate, ▲ - viable cells (points - experimental data, smooth lines - predicted values)

The metabolization of the reducing sugars after 24h of lactic acid fermentation of vegetable juices with *Lb. acidophilus* LA-5 ranged between 26.66% (Rb) to 54.09% (Cc). Relative close values were obtained by other authors in lactic acid fermentation of vegetable juices. Thus, the utilization of sugar during fermentation in a mixture of beetroot juice and carrot juice and different content of brewer's yeast autolysate with *Lb. plantarum* A112 and with *Lb. acidophilus* NCDO 1748 varied from 19.4 to 24.1% ([21]).

The tested pure culture, routinely used for dairy products, was found to be capable of growing on pure vegetable juices without nutrients added. In the batches obtained from cabbage, respectively cucumber, the maximum volumetric productivity was determined after 8 hours as follows: 19.25x1014 CFU/(l·h) for Cb, 11.9x1014 CFU/(l·h) for RCb, 18.6x1014 CFU/(l·h) for Cc and 10.25x1014 CFU/(l·h) for CcO respectively.

The relationship between the growth of *Lactobacillus acidophilus*, the substrate metabolization and the lactic acid accumulation is shown in Figure 8. The prediction functions of the values of the analyzed parameters in all the samples were defined as polynomial, the R squared being very close to unit.

Fermentation of Vegetable Juices by *Lactobacillus Acidophilus* LA-5 185

Lactic acid is the major metabolite of *Lactobacillus acidophilus*, influencing both the preservation of the fermented products and the sensorial characteristics of these ones. The effect of the amino acids and of the yeast extract on the dynamics of the lactic acid, assessed against the control, is underlined through the data from Table 3. The buffering capacity of the amino acids prevented a direct proportionality between the pH values and the lactic

Time, h Cys\_1 Leu Val Lys Cys\_2 YE 2 8.737864 -12.6214 21.52778 -29.8611 107.6923 15.38462 4 17.66784 -23.6749 -5.55556 -2.77778 28.125 12.5 6 16.98113 -1.50943 11.71717 3.636364 1.818182 5.454545 8 20.63492 -1.5873 8.571429 1.428571 -11.1111 15.87302 24 0.925926 -0.92593 5.076142 4.568528 -14.433 11.34021

**Table 3.** Time-increasing of lactic acid during 24h of lactic acid fermentation of carrot juices by

The values were expressed in percents by reporting the difference between sample and

Negative values shows that for the corresponding interval of time the supplements had not

Analyzing the whole process, only the samples with a minimum amount of cysteine added and those with yeast extract have been a great effect on the time-increasing of lactic acid. At the other opposite were found the samples with leucine added, this amino acid with nonpolar hydrophobic chains clumsying the fermentation. From the viewpoint of increase the lactic acid content in the final stages of the process, the supplementation of the carrot juices

The beneficial effect of cysteine on the lactic acid accumulation in vegetable juices can occur due to its buffering capacity, which may diminish the toxic effects of organic acids on lactobacilli. Referring to the yeast extract, which contains more cell growth factors, being used generally as a source of assimilable nitrogen, vitamins and minerals, its influence at the level of 0.2%(w/v) on the time-increasing of lactic acid could be characterized as moderate. If some authors reported different maximum lactic acid concentration in media supplemented with yeast extract, several possible explanations include the strain of microorganism, the chemical composition of the substrate, the fermentation system, and generally the

Effect of supplements on the performance of lactic acid production was evaluated based on

The previous conclusion referring to the positive influence of the yeast extract and cysteine (in minimum amount) on the development of the lactic acid fermentation of vegetable juices is confirmed by the data from Table 4. Good values of lactic acid productivity were obtained

lactic acid productivity and lactic acid yield, respectively on glucose ratio (Table 4).

acid content.

*Lactobacillus acidophilus* LA-5

control to the control, at the same moment of time

with 0.2% (w/v) cysteine seems to be undesirable.

conditions employed during fermentation ([12]).

influence on the lactic acid production at the used levels.

Correlating the number of viable cells with the dynamics of the lactic acid, the values were lower until 6 hours in the red cabbage juice and cucumber juice with onion juice added respectively. The differences were lessened in the next period of the process. However, the final yield of the lactic acid production was better in the sample CcO, by 0.78, comparatively with 0.7 in the sample Cc.
