Acidification kinetic parameters of fermentation of carrot juices by
Probiotics foods represent one of the largest sectors in functional food markets. Most of the available probiotic products are some form of dairy, despite the continuous growth of the non-dairy probiotic sector, with products like soy-based drinks, fruit-based foods, and other cereal-based products. Both non-dairy (in general) and soy-based probiotic products represent a huge growth potential for the food industry, and may be widely explored through the development of new ingredients, processes, and products. For this purpose, new studies must be carried out to: test ingredients, explore more options of media that have not yet been industrially utilized, reengineer products and processes, towards potentially meet the demands of lactose-intolerant and vegetarian consumers for new nourishing and palatable probiotic products .
Lactic acid bacteria are among the most important probiotic microorganisms typically associated with the human gastrointestinal tract. Traditionally, lactic acid bacteria have been classified on the basis of phenotypic properties, e.g. morphology, mode of glucose fermentation, growth at different temperatures, lactic acid configuration, and fermentation of various carbohydrates. However some species, like the so-called
For use in food, important criteria for probiotics must be met, in particular that they should not only be capable of surviving passage through the digestive tract, by exhibiting acid and bile tolerance, but also have the capability to proliferate in the gut.
Probiotics must be able to exert their benefits on the host through growth and/or activity in the human body. Although generally recognised as safe a probiotic strains must be characterized by a set of tests that assure its safety to consumer (1, 2, 3, 5, 6).
Inclusion of probiotic bacteria in fermented dairy products enhances their value as better therapeutic functional foods. However, insufficient viability and survival of these bacteria remain a problem in commercial food products. By selecting better functional probiotic strains and adopting improved methods to enhance survival, including the use of appropriate prebiotics and the optimal combination of probiotics and prebiotics (synbiotics), an increased delivery of viable bacteria in fermented products to the consumers can be achieved .
The fermentation of vegetable products, applied as a preservation method for the production of finished and half-finished food products, is considered as an important technology, though requiring more research, as a growing number of raw materials are being processed in this way by the food industry. The main reasons for this interest are nutritional, physiological and hygienic aspects of the process . Thus, according to Kelwicka, (2010) , the fermentation of beetroot juice requires selected starter cultures made of LAB, naturally present in this vegetable although their number is usually very small. This makes them un-appropriate to, alone, conducting a fermentation that ensures satisfying sensory properties of the fermented juice, with improved health promoting activity.
Thus, probiotic juices represent an alternative to dairy products that suits consumers who don't want to eat dairy foods or are lactose intolerant. Adding probiotics to juices is more complex than formulating in dairy products where the bacteria can be easily added to other cultures.
Despite its potential for healthy products development, there is very little research activity addressing the fermentation of vegetable juices using probiotic bacteria.
2. Materials and methods
2.1. Vegetables treatments
Fresh vegetables (carrots, cucumbers, beetroot, white cabbage, red cabbage) were purchased from a retail market and specifically processed by removing the non-edible pieces. The raw material processing was made faster, because the possibility of contamination and proliferation of microorganisms in the products is very high in comparison with their intact counterparts (Lee, 2011). Using a domestic extractor the vegetables were turned into juice. The heating treatment of the juice, applied at 800C with a view to destroy the undesirable microorganisms under the limit of detection, was followed by cooling at 400C.
2.2. Microorganisms and fermentation conditions
The lyophilized culture was aseptically inoculated into the vegetable juices and vigorously homogenized for 15 min, according to the producer’s specification. The fermentation experiments were carried out using Erlenmeyer flasks containing 50ml of juice, without pH adjustment. The flasks were incubated statically in an incubator chamber at 37±0.20C. Sampling was taken at regular interval of times for physico-chemical and microbiological analysis.
The tested supplements were: L-cysteine hydrochloride monohydrate (Merck, Darmstadt, Germany), L-lysine hydrochloride (Merck), L-valine (Merck), L-leucine (Calbiochem, San Diego, CA, USA) and yeast extract (Merck). Cysteine, lysine, valine and leucine were separately added in quantity by 0.1% (w/v) into carrot juice, while amounts by 0.2% (w/v) were tested, also individual, in the case of the yeast extract and cysteine. A control sample without supplements was carried out for each experiment.
2.3. Physico – Chemical analysis
Metabolic activity of the strain LA-5 in the conditions mentioned above was evaluated based on the dynamics of pH, respectively end products of fermentation. The pH values were measured with a HACH pH-meter. Lactic acid was determined using commercial kits (K-DLATE from Megazyme International). The calculations were made with Megazyme Mega-CalcTM and expressed as g lactic acid/l. Reducing sugars were analyzed applying the spectrophotometric method with 3.5-dinitrosalicilic acid (DNS) after the removing of other substances with reducing character using basic lead acetate and expressed as g glucose/l. Ascorbic acid was determined applying the 2,6-dichloroindophenol titrimetic method, based on the reduction of the sodium salt of the dye by ascorbic acid (AOAC method). It was expressed as mg/100ml. The amino acids content, expressed as g glycine/100ml, was determined through the Sörensen method.
2.4. Microbiological analysis
The amount of viable cells of
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 , 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.
2.5. Statistical analysis
Statistical analysis was carried out using the software SPSS (Statistical Package for the Social Science 17.0 trial version).
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).
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
Concluding, higher inoculum densities of
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
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).
|Inoculum, g/l||vmax·10-3 (units/min.)||tmax (h)||tpH 5.0 (h)||tpH 4.2 (h)|
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
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  and Kotzamanidis .
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.
3.2. Effect of temperature on the dynamics of fermentation
According to the information provided by the producer of the lactic culture, respectively to the data found in literature, two different incubation temperatures were tested: 370C and 410C respectively.
The dynamics of both pH and lactic acid (Figure 3) emphasizes the influence of the higher temperature on the rate of acidification. After 24h no significant differences between the pH values were determined, while the lactic acid content of the samples fermented at 410C was 1.24-fold higher comparatively with those fermented at 370C. This situation may be due to the higher amino acids content in the samples fermented at 410C, that act as buffer. Thus, expressed as glycin, the total amount was by 0.165g/100ml at the end of the analyzed interval, which represented an increase by 10% comparatively with the batch fermented at lower temperature.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
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
3.3. The behaviour of different raw materials during the lactic acid fermentation by Lactobacillus acidophilus LA-5
Fresh white cabbage (
pH and lactic acid dynamics during the lactic acid fermentation of vegetable juices with
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 , 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 ().
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.
 studied the fermentation of cucumber juices with a 0.5%, 1% and 2% additions of the onion juices by
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
where the constants α and β are determined by the pH of the fermentation.
A simplified presentation of the above model relates to the linear part of the equation which is presented as:
The R squared coefficient closed by the ideal value ‘’1’’ (R2 = 0.9989) in the case of the carrot juices fermented with
The metabolization of the reducing sugars after 24h of lactic acid fermentation of vegetable juices with
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
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.
3.4. Effect of growth factors on the dynamics of the lactic acid fermentation of the carrot juices by Lactobacillus acidophilus LA-5
Kinetic parameters such as the time to reach pH 5.0 and the maximum rate of acidification are important in terms of the shelf life of the fermented vegetable juices. These ones were differently modified by the presence of the amino acids or of the yeast extract at the initial moment of fermentation. From Table 2 we deduced that a highest influence on both tpH 5.0 and vmax was exerted by cysteine, added to the juice in amount by 0.2% (w/v). Compared with the other supplements, the yeast extract had a relative good effect on the analyzed parameters. At the used concentrations, the behavior of valine and lysine seems to be unobservable from this point of view, excepting the poor effect of lysine on the maximum rate of acidification. Time to complete the fermentation (tpH 4.2, h) ranged between 7.4 (YE) and 10.42 (Leu), trend that underline the statement that in the above mentioned experimental conditions
|Time-decreasing of tpH 5.02)||1.28||0.85||0||0||1.69||1.1|
|Time-increasing of vmax3)||0.82||0.84||0.98||1.05||1.1||1.05|
MRS broth used for lactobacilli enumeration often incorporates L-cysteine to improve the recovery of these ones, especially due to the fact that
Lactic acid is the major metabolite of
The values were expressed in percents by reporting the difference between sample and control to the control, at the same moment of time
Negative values shows that for the corresponding interval of time the supplements had not influence on the lactic acid production at the used levels.
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 non-polar 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 with 0.2% (w/v) cysteine seems to be undesirable.
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 conditions employed during fermentation ().
Effect of supplements on the performance of lactic acid production was evaluated based on lactic acid productivity and lactic acid yield, respectively on glucose ratio (Table 4).
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 after 24 h of fermentation in the samples with valine and lysine added, although in these ones the substrate consumption seems to be directed to the increasing of biomass, aspect emphasized by the average values of the lactic acid yield.
|Lactic acid yield2)||1.1||0.85||0.88||0.79||0.85||1.15|
|Lactic acid productivity3)||1.01||0.99||1.06||1.05||0.7||1.13|
|Glucose conversion ratio4)||1.1||0.9||1.2||1.05||0.92||1.25|
The effect of supplements (amino acids and yeast extract) on the ascorbic acid dynamics is shown in Figure 9. L-Ascorbic acid (AA), also known as vitamin C, is a representative water-soluble vitamin possessing a variety of biological, pharmaceutical, and dermatological functions; it promotes collagen biosynthesis, provides photoprotection, causes melanin reduction, scavenges free radicals, and enhances immunity ().
Due to the heat treatment applied with a view to destroy the epiphytic microbiota of the fresh vegetable juices, the losses occurred in the ascorbic acid content represented about 65%.
The presence of ascorbic acid into vegetable juices submitted to fermentation by probiotic bacteria, especially by
L-cysteine, a sulfur-containing amino acid known as a powerful reducing agent, caused the reduction of dehydroascorbic acid to ascorbic acid, which led a different behavior of the samples Cys_1 and Cys_2 by the others. The increase of this parameter was by 80% and 56.4% respectively, after 2h from the initial moment of fermentation. Subsequently, the analyzed parameter had the same diminishing tendency as in the other batches.
The losses occurred after 24h of lactic acid fermentation of carrot juices with
In order to evaluate the probiotic feature of the vegetable juices, the study of the effect of supplements on
Between the analyzed samples, those with yeast extract and 0.1% (w/v) cysteine added registered a higher increase of the number of viable cells till 14.4 - 14.5 log CFU/ml in the first 8h of the process. Concerning the yeast extract, the most possible explanation is due to an enhanced availability of minerals, which are growth promoters for
Referring to the juices with leucine, lower values were determined comparative with the control during 24h, while in the samples with 0.2% (w/v) cysteine added the trend of the survival of lactobacilli was slow down in the period 6 - 8h, the level being by 13.5 and 13.6 log CFU/ml respectively. The last observation agrees with this one of , which have shown that the increasing of cysteine concentration improved the viability of
The batches supplemented with valine and lysine had occupied an intermediate position, the growth until 14.2 log CFU/ml after 8h of fermentation making from the utilization of these amino acids a promising variant in the future, with a view to optimize the conditions of the process unfolding. In the period 8 - 24h the number of viable cells decreased, as result of the lack of tolerance at lower pH of the analyzed strain.
The correlation between the most important parameters of the lactic acid fermentation of the carrot juices with
|Analytical variables||pH||lactic acid||glucose||viable cells||glycine||ascorbic acid|
The correlations are strong between pH and lactic acid, respectively pH and glucose, while a very strong relationship pH - viable cells could be considered (Table 5). A non-existent relationship between ascorbic acid / amino acids content (expressed as glycine) and the other analyzed parameters was determined.
A firm correlation between glucose and lactic acid was expected, but on the one hand it is known that the practical yield of sugars conversion to lactic acid of the strains of the group
Factor Analysis (FA) is a multidimensional statistic method whose purpose is the analysis of the structure of mutual dependences of variables. The method is similar to the Principal Component Analysis (PCA) with the exception of the factor weights that are scaled ().
Applying FA to the experimental data, the analytical variables were reduced to two principal components, which accounted for 59.72% (PC1) and respectively 18.95% (PC2) from the total variance. According to the component matrix, respectively to the values of the component loadings expressed by the first second principal components (rotation method: Varimax with Kaiser normalization), the most notable variables were pH and lactic acid (equal loading values by 0.954). Higher values were obtained also for viable cells (loading 0.939) and glucose (loading 0.933).
The combination of PC1 and PC2 (Figure 10) underlined the lack of correlation between amino acids content / ascorbic acid and all the other parameters taking into account both control and supplemented samples. While PC1 affected the dependent and independent variables involved in the progress of the lactic acid fermentation of vegetable juices, respectively in their probiotic feature, PC2 separated the variables which contribute to the nutritional characteristics of the final products.
Applying PCA to the lactic acid fermentation of cabbage juices with various microorganisms,  established that the original 7 analytical variables were reduced also to 2 independent components that explained 88.2% from total variance of input data (PC1 66.9% and PC2 21.3%).
Cluster Analysis (CA) is a statistic method whose purpose is to join data into clusters with a view to increase their withingroup homogeneity. Usually, the FA is considered the first step of CA, with a view to reduce the data dimensionality. In order to better distinguish among experimental samples, the cluster method of the nearest neighbour was used. The distances between objects were measured as squared Euclidean distance. K-Means Cluster Analysis divided the experimental data into three groups, characterized by similar analytical properties, as follows:
cluster 1: all the carrot juices (control samples and the batches with amino acids and yeast extract added) at the initial moment of fermentation, respectively at 2th h of fermentation. Supplementary, this cluster included the control and the sample with leucine at 4th h of fermentation (C_4 and Leu_4);
cluster 2: all the carrot juices at 24th h of fermentation and the sample with lysine added at 8th h of fermentation ;
cluster 3: the carrot juices with leucine and lysine added, respectively the control, at 6th and 8th h of fermentation (Leu_6, Leu_8, Lys_6, Lys_8, C_6, C_8); the carrot juices with cysteine, valine, respectively yeast extract from 4th to 8th h of fermentation (Cys_4 - Cys_8, (Val_4 - Val_8), (YE_4 - YE_8).
The clusters in axes of two selected variables (Figure 11) denote that the samples from the first cluster were marked with a higher content of substrate, null or very lower lactate amount and pH values more than 5. The corresponding time was both 0 and 2h (C, Val, Leu, and YE) or the entire interval 0 - 4h (Leu and control).
The samples at the final moment of fermentation and those with lysine after 8h of the process were included in the second cluster, characterized through lower or average values of glucose content, higher lactic acid amount and pH values close to 4.2. This cluster marks the achievement of the optimum characteristics of the lactic acid fermented products.
The samples included in the third cluster best describes a vigorous process, being characterized through average values of the main parameters involved in the dynamics of the lactic acid fermentation of vegetable juices.
The usefulness of the methods of statistical analysis is underlined by a lot of applications of CA that could be reported: in evaluation of analytical and sensory characteristics of vegetable juices (, ), in distinguishing between wines aged a different number of months ().
4. Further research
The importance of consuming probiotic foods for the improvement of the quality of life increasingly more in the last years, being underlined by the scientific literature. The diversification of the market from this point of view could be strong correlated with the increasing of the life expectancy worldwide.
Our further researches are needed in order to optimize the level of nutrients (individually and in combination) and in the same time their influence on growth and viability of probiotics (in particular of
Different vegetable juices are suitable and alternative food matrices for the production of functional foods with
The research was funded by Executive Unit for Financing Higher Education, Research, Development and Innovation (UEFISCDI) in the frame of the Project PN-II-ID-PCE-2008-2 (ID_1359). The microorganisms were kindly provided by Chr. Hansen, Romania.