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The disposal of whey is a major problem for the dairy industry, which demands simple and economical solutions. The main objectives of the paper were to study the effects of nutrient supplementation of whey on the growth of Lactobacillus johnsonii (NRRL B-2178). Because of the short lifespan of probiotics, study has been carried out aiming at the evaluation of the contribution of nutrients addition in the improvement of the viability of used microorganism. Maximal cell growth of 8.7 log (CFU mL-1) and shelf life of 5 days were achieved during the production of beverage in the presence of yeast extract (3%) and inulin (1%) including the synergistic effect of temperature 39 °C. The ejection of inulin out of fermentation process reduces the biomass production for 0.2 log (CFU mL-1) but the addition of 1% inulin after the fermentation extends the shelf life of the product for 10 days.
Key words: Whey, Probiotics, Lactobacillus, Supplementation, Inulin, Response Surface Methodology
Whey is a by-product of the cheese industry which was often disposed as a waste in the past, causing high environmental contamination (González-Martínez, Becerra, Cháfer, Albors, Carot & Chiralt, 2002) and significant loss of protein and energy source. The biochemical oxygen demand (BOD) of whey varies from 30,000 to 50,000 mg g-1 depending upon the wastage of milk in the whey (Mukhopadhyay, Chatterjee & Guha, 2003). Because of these high values, the pressure of antipollution regulations forces the dairy industry to develop the new technologies that can change whey from waste to a valuable product (Castillo, 1990). In accordance with these requirements, considerable efforts have been made over the past years to find new outlets for the whey utilization and the reduction of environmental pollution (González-Martínez et al., 2002).
Since the treatment of whey as an effluent is very expensive the conversion of the liquid whey into beverage (i.e. fermented or not) is one of the most attractive options for the utilization of the whey due to the simplicity of the process, use of the same equipments of milk treatment, and the excellent functional properties of the whey and its proteins (Gandhi & Patel, 1994).
There is lot of published articles about fermented whey beverages with different kinds of microorganism based on UF concentrated whey (KrÅ¡ev & Tratnik, 1984), and articles about beverages with probiotics based on reconstituted whey in powder (Hernandez-Mendoza, Robles, Angulo, De La Cruz & Garcia, 2007). However, the production of products with characteristics of probiotic food is still challenge for food industry. It is important to give new healthy-safe food to consumers because they pay attention to safe food more and more each day. Hence, lactic beverages could be considered as an important vehicle for the delivery of probiotic micro-organisms to the human gastro intestine (Oliveira, Sodini, Remeuf & Corrieu, 2001). In this way, several products are being developed using mixtures of whey, milk and fruit juice with probiotic bacteria (DjuriÄ‡, CariÄ‡, MilanoviÄ‡, TekiÄ‡ & PaniÄ‡, 2004).
To produce therapeutic benefits, a sufficient number of viable microorganisms must be present throughout the entire shelf life of the product. According to the literature (Schuller-Malyoth, Ruppert & Muller, 1968) a good probiotic culture should contain between 106 and 108 viable cells per milliliter. However, these organisms often show poor viability in market preparations. Several factors have been involved in affecting the viability of probiotic cultures in fermented milks such as pH, acidity, the presence of other microorganisms, temperature, nutrient supplementation and others (Shah, 2000). A possible method of ensuring adequate numbers of probiotic bacteria in cultured dairy products is supplementation with substances stimulatory toward the growth of probiotic bacteria. In recent years, there has been more focus on synbiotics, a combination of prebiotics and probiotics in a single product. There is a considerable interest in the use of prebiotics to enhance the survival and colonization of probiotic bacteria that are added in food products. Inulin, non-digestible carbohydrate which naturally contains fructo-oligosaccharides, possesses characteristics of dietary fiber, and as such is of particular interest for its metabolic properties.
Since LAB have complex requirements for growth (Hebert, Raya & De Giori, 2000) they require high level of nutrient supplementation for increasing their growth and viability including nitrogen source, prebiotics, vitamins and microelements and adequate environmental parameters that affect the lactic acid production process and growth of LAB. In many fermentation studies, yeast extract is considered to be an essential nutrient for lactobacilli for efficient lactic acid fermentation and cell growth (Amrane, 2005).
However, in the literature a little attention has been paid to the possibilities of probiotic beverage production by Lb. johnsonii NRRL B-2178 and there are no data concerning the influence of nutrient supplements in synergy with temperature on growth and viability of Lb. johnsonii in the probiotic whey-based beverage. Also there are no data for influence of inulin as a promoter of bacterial surviving in the produced beverage when it was added after the fermentation process.
In this study Lb. johnsonii was selected for whey fermentation as top beverage producer due to its good characteristics as a probiotic, excellent beverage sensory characteristics profile and satisfactory fermentation dynamic (BulatoviÄ‡, Rakin, MojoviÄ‡, NikoliÄ‡, VukaÅ¡inoviÄ‡ SekuliÄ‡ & ÄukiÄ‡ VukoviÄ‡, 2012). In accordance with the literature this strain also showed good viability in the presence of inulin (Salem, Abd El-Gawad, Hassan & Effat, 2007). The aim of this study was the determination of the significant nutrient supplements and their levels that affect the growth of Lb. johnsonii. The synergistic effect of the key suplements and the temperature in order to achieve a maximal viable cell count of Lb. johnsonii was also investigated. This study also reported the results of the influence of inulin addition during and after the fermentation on viable cell count and their stability during the storage.
2. Materials and methods
2.1. Culture and media
The strain Lb. johnsonii NRRL B-2178 used in this work was obtained from the ARS Culture Collection (NRRL), National Center for Agricultural Utilization Research, United States Department of Agriculture (USDA). Stock culture was stored at -20 oC in 3 mL vials containing De Man, Rogosa, Sharpe (MRS) broth (Sigma-Aldrich) and 50% (v/v) glycerol as a cryoprotective agent. For the preparation of laboratory culture, a drop of stock culture was transferred in 3 mL of the MRS broth and incubated for 18 h under anaerobic conditions at the optimal growth temperature (37 °C). The working culture was pre-cultured twice in MRS broth prior to experimental use. Initial cell count for all experiments was about 7.0 log (CFU mL-1).
Sweet whey powder (Lenic Laboratories, Belgrade, Serbia), with following composition: proteins 12.11% (w/w), lipids 1.0% (w/w), and carbohydrates 69.62% (w/w), was reconstituted to contain 8% (w/v) of dry matter. The reconstituted whey with pH = 6.2 was pasteurized at 60 Â°C for 60 min, stored at 4 Â°C (no longer than one day) until its usage as fermentation medium.
Chemicals and nutrients were obtained from Sigma-Aldrich and were used in solutions form with following concentrations: yeast extract (c=0.5 g mL-1), inulin (c=0.33 g mL-1), sucrose (c=0.33 g mL-1) and pyridoxal (c=0.33 mg mL-1). The solutions were pasteurized for 20 min at 90oC, except the vitamin solution which was filter-sterilized and added to pasteurized whey prior the inoculation. When the effects of nutrient supplements were studied reconstituted whey was supplemented by addition of the required amounts of nutrients (Table 1). When the synergistic effects of significant nutrients and temperature were studied, only yeast extract and inulin were added (Table 2).
The experiments were conducting in 100-mL Erlenmeyer flasks containing 50 mL of reconstituted whey. The samples were inoculated by adding 8% (v/v) of bacteria and incubated at 37 oC during the investigation of nutrient supplements effects, or at three different temperatures (35, 39 and 43 oC) during the investigation of synergistic effects of significant nutrients and temperature. During the incubation time samples were withdrawn every 2h for determination of pH value. The fermentations were carried out until pH = 4.6 was attained. When pH = 4.6 was reached fermentations were stopped by quick cooling. The analysis of the key nutrient supplements and optimal fermentation conditions were carried out by determination of viable cell count of Lb. johnsonii (log (CFU mL-1)).
2.3. Shelf life evaluation
The optimized values of key supplements and temperature were then selected to carry out shelf life analysis of the fermented product. The fermentation was carried out in 300-mL Erlenmeyer flasks containing 200mL media at selected optimal conditions. After the fermentation, the beverage was refrigerated at 4 â-¦C. The fermented beverage was withdrawn for sampling at regular intervals for 25 days and analyzed for viable cell count, pH value and titratable acidity.
2.4. Analytical methods
2.4.1. Viable cell enumeration
The enumeration of viable cells of Lb. johnsonii (log (CFU mL-1)) was performed by the determination of colony-forming unit number on MRS agar plates after the incubation at 37 â-¦C for 48 h by pour plate technique on MRS agar (VrbaÅ¡ki & Markov, 1993).
2.4.2. Titratable acidity and pH
Titratable acidity was determined by Soxhlet-Henkel method (Varga, 2006), while the pH value was measured by pH meter (Inolab, WTW 82362, Wellheim).
2.5. Experimental design
This step of the study involved the determination of the significant nutrient supplements and their levels that considerably affected the growth of Lb. johnsonii in the process of whey-based beverage production. Statistically based experimental designs provide an efficient approach to optimization. A combination of factors generating a certain optimal response was identified through factorial designs and the use of response surface methodology (RSM).
2.5.1. Screening of Significant Nutrient Supplements
A 2(4-1) fractional factorial design (FFD) was employed to determine the key nutrient supplements that affected the growth of Lb. johnsonii significantly. The individual and interactive effects of the yeast extract, inulin, sucrose and pyridoxal on viable cell count of Lb. johnsonii (log (CFU mL-1)) as response variable were studied. The 2(4-1) design is a Resolution IV design. This design provides very good information about the main effects and also provides some information about two-factor interactions.
The coded and uncoded variables and their levels used in the 2(4-1) FFD design are listed in Table 1. The variables were evaluated by conducting of 20 runs which included two replicates of factorial experiments and four center points, as shown in Table 1.
2.5.2. Optimization of Fermentation Condition
A Box-Behnken design was employed to further investigate the synergistic effect of the key supplements and the temperature in order to optimize the fermentation process in terms of achieving a maximal viable cell count of Lb. johnsonii as the response variable. The codified levels of the variables and their real values are shown in Table 2. The Box-Behnken design with three factors at three levels (Table 2) including three replicates at the centre point was composed of 15 assays (Table 2) and specifically selected since it requires fewer runs than a central composite design in cases of three or four variables. Experiments in the centre of the design were performed to make the estimation of possible pure error. Due to systematic errors, all the experiments were carried out at random to minimize the effect of unexplained variability on the responses observed. The dependent variable (response) was the viable cell count of Lb. johnsonii (log (CFU mL-1)).
2.6. Statistical analysis
The model evaluated the effect of each independent variable to a response. The analysis of the experimental design was carried out using Design Expert Software (version 8.0, trial, Statease Inc., Silicon Valley, CA, USA) to estimate the response of the independent variables. Subsequently, three additional confirmations of experiments were conducted to verify the validity of the statistical experimental strategies.
The values obtained from the shelf life analysis experiments were means of triplicate readings. OriginPro 8 (1991-2007) computer package was employed to analyze the experimental data generated. Microbial viability data were analyzed using Two Way ANOVA. The Tukey post hoc test was performed for means comparison. A Pvalue < 0.05 was considered statistically significant for all analyses.
3. Results and discussion
3.1. Effect of nutrient supplements
The reconstituted whey contained limited amounts of protein (about 1.0%, w/v) and carbohydrate (about 5.0%, w/v). Amounts of vitamins in whey are also generally limited and confined to only about 0.001% (w/v) of pyridoxal (Frévier & Bourdin, 1977). Several studies showed that productivity of most Lactobacillus strains is significantly improved by the addition of some commercially available growth supplements such as yeast extract, inulin or vitamins which can improve the nutritional quality of the medium, because they contain growth promoting compounds (Demirci, Pouetto, Lee & Hinz, 1998). In this study, the effects of nutrient supplements including nitrogen, carbon and vitamin sources on viable cell count of Lb. johnsonii were studied using 2(4-1) FFD.
The fermentation time of 4 h achieved during the production of probiotic whey-based beverage which contained Lb. johnsonii as probiotic and nutrient supplements was different from the results reported by others (Dave & Shah, 1998) for fermented milk with probiotics, whose variation was of 7.5-9 h. However, these results were similar to those obtained by Thamer and Penna (2006) and Almeida, Bonassi and Roça (2001), whose fermentation times were 4-4.5 h, for fermented lactic beverages, containing about 50% of whey in substitution to milk and between 3 and 4.25 h, for lactic beverages containing probiotic and prebiotics (1-3%), respectively. The approximately the same results were found by Fuchs, Tanamati, Antonioli, Gasparello and Doneda (2006) in yoghurts, containing Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus casei and prebiotics (1% of inulin and 5% of oligofructose), in which the average fermentation time was 6 h.
As shown in Table 1, about 7.78 log (CFU mL-1) was obtained in the samples without nutrient supplements (Table 1, runs 13 and 14). Among the carbon sources, inulin in combination with the vitamin had a quite small negative effect on viable cell count (Table 1, runs 15 and 17) while sucrose in combination with the vitamin expressed a very small positive effect on viable cell count (Table 1, runs 3 and 18). In the combination of carbon sources inulin also expressed its negative effect, so the viable cell count in this samples was about 7.67 log (CFU mL-1) (Table 1, runs 4 and 20) what is lower than in samples fermented without suplements. Based on the literature Lb. johnsonii might not be able to metabolize the other carbohidrates with high efficiency in the presence of inulin (Amaretti et al., 2007).
The highest viable cell count of about 8.47 log (CFU mL-1) was obtained in samples that contain combination of yeast extract and vitamin (Table 1, runs 5 and 10). Slightly lower, but still high, cell count was obtained in samples that contained yeast extract and sucrose (Table 1, runs 2 and 6). In comparison with the samples fermented without supplements viable cell count in samples supplemented with yeast extract was increased for 0.7 log (CFU mL-1). That is in consistence with the literature because the several studies already showed that addition of yeast extract can positively affect the growth of Lactobacillus species (Gomes, Malcata & Klaver, 1998).
A Pareto chart (Figure 1), consisting of bars, was employed to analyze the influence of nutrient supplements on the growth of Lb. johnsonii.
As indicated in Figure 1, yeast extract, inulin and their combination influenced the viable cell count. Among these factors, the yeast extract had significant (p<0.05) positive effect on cell growth of Lb. johnsonii. Since the yeast extract had the most positive effect on cell growth the concentration of the yeast extract should be increased in further investigation. Given the significant (p<0.05) negative effect that inulin manifested, its concentration should be reduced in further investigation. Also, since the inulin is already known as prebiotic, which affects the stability of cells during the storage there is a possibility that his addition after the fermentation could affect the maintenance of viable cell count during the storage without affecting the cell count during the fermentation.
No significant (p>0.05) influence on viable cell count was obtained for other nutrients which means that the whey probably contains enough vitamins required for cell growth. Also, this result might partially be attributed to vitamins that are present in the yeast extract and are required for cell growth. In term of no utilization of sucrose, this is probably due to the inability of effective sucrose utilization in the presence of inulin (Amaretti et al., 2007).
The yeast extract and the inulin were selected as key nutrient supplements in the following study as they had the most significant influence on viable cell count of Lb. johnsonii.
3.2. Synergistic effect of the key nutrient supplements and temperature
In further investigation it was necessary to explore the synergistic effect of the yeast extract, inulin and temperature in order to maximize the viable cell count of Lb. johnsonii. Also it was necessary to see whether the decreasing of inulin concentration and increasing of yeast extract concentration have the higher influence on cell growth when the higher temperatures were used.
This part of the study was conducted as a result of the aforementioned findings, in an attempt to optimize the levels of temperature, yeast extract and inulin a Box-Behnken design was used. The inulin level was reduced to 1.0% (w/v) whereas the amount of yeast extract was increased to 3.0% (w/v). The experimental design and results are shown in Table 2.
In Table 2 it can be seen that the lowest viable cell count of 7.14 log (CFU mL-1) was observed at 43 oC when the concentrations of yeast extract and inulin were 1.5 % and 0.0 %, respectively (Table 2, run 4). At the same temperature, in samples supplemented with 3.0 % of yeast and 0.5 % of inulin, the cell count was 7.70 log (CFU mL-1) (Table 2, run 8). This cell count was lower than count reached at 35 oC (7.88 log (CFU mL-1), Table 2, run 10) at the same concentrations of supplements and count reached at 39 oC (8.0 log (CFU mL-1), Table 2, run 1) without addition of supplements. That means the very high and very low temperatures have negative influence on cell growth despite the presence of the suplements in comparison with samples fermented without supplements at temperature 39 oC.
The highest cell count 8.7 log (CFU mL-1) (Table 2, run 11) was observed at temperature 39 oC in the presence of 3% yeast extract and 1.0% inulin. This count was higher for 0.7 log (CFU mL-1) than count achieved at the same temperature without persence of supplements. Also it was higher for 1.0 log (CFU mL-1) than count reached at 35 oC (Table 2, run 10) and 43 oC (Table 2, run 4) with supplement addition. Observed results mean that the sinergy of middle temperature (39 oC) and supplement addition has the most significant influence of cell growth (Table 2, run 11). Under these conditions, an increase in cell count compared to the initial count is about 1.7 log (CFU mL-1), which can be considered as a moderate to approximately high value of growth (Pescuma, Hébert, Bru, Font de Valdez & Mozzi, 2012) compared to the strains which possess a high proteolytic activity.
It is also interesting to note that the cell growth was directly related to yeast extract concentration when the inulin concentration (1%) and temperature (39 oC) were kept at a constant level. The difference in the cell count between samples with 0.0 % and 3.0 % of yeast extract was about 0.7 log (CFU mL-1) (Table 2, runs 11 and 13). The stimulatory role of some substances, e.g. yeast extract, peptones, corn steep liquor, on bacterial growth has been related to their nucleotide content (Mikhlin & Radina, 1981) and disulphide-bonds in their peptides or proteins. The bacterial growth increased directly with the nucleotide content of the respective yeast extracts so its amount required for the optimal bacterial growth was highly dependent on diferent suppliers. Unfortunately, development of bad flavours and colour changes are usually observed in the fermented dairy products supplemented with high amount of yeast extract. On the other hand, based on the our previusly investigations (Rakin, Vukasinovic, Siler-Marinkovic & Maksimovic, 2007) this problem can be solved by fortification of fermented beverage with the various fruit bases additions that can neutralize the negative impact of yeast extract on sensory characteristics, and thus enable its use without consequences on the sensory profile of the beverage. It should be noted that the increasing of viable cell count of 1.7 log (CFU mL-1) for 4 h fermentation can be considered as an improvement over the results reported in the literature (Elli, Zink, Reniero & Morelli, 1999) for fermentation of milk, whereas the cell count was increased for 2.0 log (CFU mL-1) after the 24 h of fermentation.
As indicated in Table 1 the difference in cell count between the samples suplemented with 3 % of yeast extract fermented at 39 oC in the persence and absence of inulin was minimal (0.2 log (CFU mL-1)). Comparing the samples fermented with the presence of 1.5 % of inulin at 37 oC (Table 1, run 15) and 1.0 % of inulin at 35 oC (Table 2, run 14) it could be said that the decreased concentration of inulin has positive effect on viable cell count. In the presence of 1.5 % of inulin viable cell count was lower for 0.25 log (CFU mL-1) regardless the higher fermentation temperature.
The regression analysis was performed to fit the response function with the experimental data. The obtained data were fitted to a quadratic polynomial model. A general second order model is shown in Eq. (1) and the actual obtained model in Eq. (2).
The sinergistic effect of temperature, yeast extract and inulin on viable cell count of Lb. johnsonii can be described by the following second order polynomial equation:
where the following can be found: Y (viable cell count, log (CFU mL-1)) is the measured response associated with each factor level combination; A (temperature, °C), B (yeast extract, %), and C (inulin, %) are the independent variables; Î²0 is an intercept term; Î²1, Î²2, and Î²3 are the linear effects; Î²11, Î²22, and Î²23 the square effects; and Î²12, Î²23, and Î²13 are the interaction terms. This equation represents an empirical model, in which the response functions allow the estimation of response due to changes in the dependent variables. In order to determine the significance of the quadratic model, ANOVA analysis was conducted (Table 3).
The P-values were used as a tool to check the significance of each co-efficient, which also indicated the interaction strength of each parameter. The smaller the P-values are, the bigger the significance of the corresponding co-efficient is. Corresponding P-values suggest that, among the test variables used in this study, AC (temperature Ã- inulin) and BC (yeast extract Ã- inulin), are non-significant model terms with P-values greater than 0.05. Therefore, it can be said that the inulin concentration can not act as limiting nutrient and a small variation in its concentration will not alter the growth of Lb. johnsonii to a considerable extent. The goodness of fit of the model was examined by F-test and the determination coefficient R2. The greater the F-value is from unity, the more certain it is that the factors explain adequately the variation in the data around its mean, and the estimated factor effects are real. The analysis of variance (Table 3) showed that this regression model was highly significant (P < 0.01) as is evident from the Fisher, F-test (F-value, the ratio of mean square regression to mean square residual is 37.3) and has a very low probability value (Pmodel = 0.00049). The value of 0.202 for lack of fit implies that it is not significant comparing to the pure error and that the model equation was adequate for predicting the Lb. johnsonii growth. The fitness of the model was further confirmed by a satisfactory value of determination coefficient, which was calculated to be 0.985, indicating that 98.5% of the variability in the response could be predicted by the model. The low coefficient of variation (CV= 1.08%) suggested that the model was precise and reliable.
The three-dimensional response surfaces were generated to study the interaction among the three factors tested and to visualize the combined effects of factors on the growth of Lb. johnsonii in whey-based media (Fig. 2).
As shown in Figure 2 an increase in viable cell count was achieved with an increase in yeast extract concentration and temperature. The highest viable cell count (8.5-8.75 log (CFU mL-1)) was obtained with the inulin concentration of 1.0% (w/v) and the yeast extract concentration of 2.5-3.0% (w/v) at temperatures 38-39 oC.
It was predicted that a maximal viable cell count of 8.69 log (CFU mL-1) could be achieved when inulin and yeast extract concentrations were 1.0 and 3%, respectively in samples fermented at 39 oC. For validation of this prediction, whey samples was suplemented with 1.0% of inulin and 3% of yeast extract and fermented at temperature 39 oC. An obtained viable cell count was 8.60 log (CFU mL-1), which is 98.4% of the predicted value.
At temperature of 39 oC in the samples without suplements viable cell count was 8.0 log (CFU mL-1). Adding 3% of yeast extract and 1.0% of inulin, increase cell count to 8.62 log (CFU mL-1), which means that the increase in the cell count was generated. This increase in cell count is very important because the probiotic beverage should contain as many as possible viable probiotic bacteria, but not less than 6.0 log (CFU mL-1) to stimulate beneficial effects on health what is a precondition for regular consumption.
3.3. Effect of inulin addition moment on shelf life of fermented probiotic beverage
Based on the literature, presence of whey proetins by itself increasing the viability of Lb. johnsonii cells at low pH values (EL Shafei, Tawfik, Nadia, Dabiza, Sharaf & Effat, 2010). Taking into consideration that keeping is the most sensitive period for the survival of microorganisms due to low pH of produced beverage, combined effect of inulin and whey protein could increase the shelf life of probiotic beverage.
Based on previous results, the decrease of inulin concentration from 1.5 to 1.0% and the increase of temperature from 37 oC (Table 1, run 17) to 39 oC (Table 2, run 13) during the fermentation lead to the increase in viable cell count for 0.43 log (CFU mL-1). On the other hand, absence of inulin in samples fermented at 39 with 3% of yeast extract lead to the decreasing of cell count for only 0.2 log (CFU mL-1). This result was starting point in the analysis of inulin addition during and after the fermentation on viable cell count and their stability during the storage.
For shelf life analysis the influence of inulin (1%) on the viable cell count during the storage when it was added during and after the fermentation was investigated. As a control, the sample without inulin addition was used. All samples were supplemented with 3% of yeast extract and fermented 4h at 39 °C.
The samples were withdrawn every 5 days during the storage period for viable cell count determination. Figure 3 shows the comparasion of viable cell count in whey samples without inulin (control), with inulin added during fermentation (sample 1) and inulin added after the fermentation (sample 2), during the storage at 4 oC.
As shown in Figure 3 in all samples significant (P < 0.05) reduction of about 8.0 log (CFU mL-1) was seen at the end of the 25 days storage period. The viable cell count at the end of the 4h fermentation period was found to be 8.7 log (CFU mL-1) in sample 1 (inulin added during the fermentation) and 8.5 log (CFU mL-1) in sample 2 (inulin added after the fermentation). This was expected, because of the higher amount of carbohydrates present in the samples supplemented with inulin added during the fermentation. However, a difference in the viable cell count at the end of fermentation (0 day) in samples fermented with and without inulin addition was not statistically significant (P=0.14585 > 0.05).
In comparison with the control in both samples the difference in the viable cell count during the whole storage period was statistically significant (P<0.05) what confirms the fact that the inulin had a strong influence on the cells viability.
As indicated in Figure 3 the rate of population reduction was lower in the samples supplemented with inulin added after the fermentation. This could be due to stopped diminishing nutrient supply by addition of inulin after the fermentation. According to the literature (Voragen, 1998), in acidic environments or prolonged storage, inulin added to food may be hydrolyzed and that can lead to receiving a new amount of carbohydrates which could be used up for maintenance of microbial metabolism. Also, some strains were able to degrade inulin during the storage period and in this way the population reduction can be slowed. In a few research papers (Cardarelli, Buriti, Castro & Saad, 2008; García Fontán, Martínez, Franco & Carballo, 2006) a reduction by 2.7% and 2.4% in inulin content was observed in probiotic petit-suisse cheese and yoghurt, respectively, both stored for 28 days.
The cells viability in sample 1 decreased faster during the storage in comparison to the sample 2. After 5 days of storage the sample 1 had 6.2 log (CFU mL-1) of viable cell count. That is near to the necessary range of cell count for probiotic beverage, which means that the shelf life of 5 days was estimated for sample 1. The higher viability for Lb. johnsonii cells was observed in sample 2 with a count greater than 6.0 log (CFU mL-1) after 15 days of storage, so this period was estimated as shelf life of sample 2.
Based on the results of Two-way ANOVA statistically significant difference in viable cell count was found between the samples with inulin added during and after the fermentation during the whole storage period. After the 15 days of storage, estimated as shelf life of sample 2, the difference in viable cell count between sample 1 and sample 2 was statistically significant (P<0.05).A correlation between storage time and moment of inulin addition were found to be statistically significant at 5% level.
That suggests that for the whey fermented with Lb. johnsonii is better to be supplemented with inulin after the fermentation. This kind of supplementation leads to the storage without reduction in the viable cell count below 6.0 log (CFU mL-1) during the 15 days of storage.
The pH values were not considerable lower, ranging from 4.1 to 4.3 in all samples (data not shown) compared with pH values obtained after the fermentations. It is also interesting to note that the titratable acidity in sample 1 was lower (22.6 oSH) than in the sample 2 (23.6 oSH) at the end of fermentation. That is probably due to fact that in the presence of inulin during the fermentation cells were not able to use the yeast extract in sample 1, which is obviously promoter of growth and production of lactic acid in sample 2. The addition of inulin after the fermentation in sample 2 leads to the post acidification which is not considerable, so the titratable acidity in sample 2 was 27.6 oSH in contrast to the 26.0 oSH achieved in sample 1 after 25 days of storage.
The sample 2 which was supplemented with 3% of yeast extract and 1% of inulin added after the fermentation was triple stable (15 days) than sample 1 supplemented with both nutrients during the fermentation (5 days).
The effect of different nutrient supplements (yeast extract, inulin, sucrose, pyridoxal) and temperature on lactic acid fermentation of whey by Lb. johnsonii NRRL B-2178 was studied in this paper. The temperature of 39 °C in synergy with the yeast extract (3%) and inulin (1%) as key supplements were selected as optimal conditions for the whey fermentation in probiotic beverage production process. These conditions resulted in an increase in viable cell count and the maximal cell count of 8.7 log (CFU mL-1) was achieved in beverage which exhibited shelf life of 5 days. On the other hand, the ejection of inulin out of the fermentation process reduces the biomass production for 0.2 log (CFU mL-1) but its addition after the fermentation extends the shelf life of the product for 10 days.
The results suggest that the best way for the production of probiotic beverage by Lb. johnsonii NRRL B-2178 is the fermentation of whey supplemented with 3% of yeast extract at 39 °C followed by the addition of 1% inulin after the fermentation. These conditions provide production of probiotic beverage with the shelf life of 15 days.
This work was funded by the Serbian Ministry of Education and Science (TR 31017).