International Journal of Food Microbiology 146 (2011) 111117

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International Journal of Food Microbiology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Survival of Lactobacillus plantarum in model solutions and fruit juices

Sawaminee Nualkaekul, Dimitris Charalampopoulos
Department of Food and Nutritional Sciences, The University of Reading, P.O. Box 226, Reading RG6 6AP, UK

a r t i c l e

i n f o

a b s t r a c t
The aim of the work was to study the survival of Lactobacillus plantarum NCIMB 8826 in model solutions and develop a mathematical model describing its dependence on pH, citric acid and ascorbic acid. A Central Composite Design (CCD) was developed studying each of the three factors at ve levels within the following ranges, i.e., pH (3.04.2), citric acid (640 g/L), and ascorbic acid (1001000 mg/L). In total, 17 experimental runs were carried out. The initial cell concentration in the model solutions was approximately 1 108 CFU/mL; the solutions were stored at 4 C for 6 weeks. Analysis of variance (ANOVA) of the stepwise regression demonstrated that a second order polynomial model ts well the data. The results demonstrated that high pH and citric acid concentration enhanced cell survival; one the other hand, ascorbic acid did not have an effect. Cell survival during storage was also investigated in various types of juices, including orange, grapefruit, blackcurrant, pineapple, pomegranate, cranberry and lemon juice. The model predicted well the cell survival in orange, blackcurrant and pineapple, however it failed to predict cell survival in grapefruit and pomegranate, indicating the inuence of additional factors, besides pH and citric acid, on cell survival. Very good cell survival (less than 0.4 log decrease) was observed after 6 weeks of storage in orange, blackcurrant and pineapple juice, all of which had a pH of about 3.8. Cell survival in cranberry and pomegranate decreased very quickly, whereas in the case of lemon juice, the cell concentration decreased approximately 1.1 logs after 6 weeks of storage, albeit the fact that lemon juice had the lowest pH (pH ~ 2.5) among all the juices tested. Taking into account the results from the compositional analysis of the juices and the model, it was deduced that in certain juices, other compounds seemed to protect the cells during storage; these were likely to be proteins and dietary bre In contrast, in certain juices, such as pomegranate, cell survival was much lower than expected; this could be due to the presence of antimicrobial compounds, such as phenolic compounds. 2011 Elsevier B.V. All rights reserved.

Article history: Received 15 April 2010 Received in revised form 10 January 2011 Accepted 29 January 2011 Keywords: Cell survival Storage Probiotics Fruit juices Lactobacillus plantarum Mathematical modelling

1. Introduction Probiotics are dened as Live microorganisms which, when administered in adequate amounts, confer a health benet on the host (FAO/WHO, 2002). Probiotics can exert several benecial effects, including the prevention or treatment of intestinal infections, irritable bowel syndrome and inammatory bowel disease, the stimulation of the immune system, the improvement in the digestibility of food products, the control of blood cholesterol levels, and the prevention of atopic allergies (Gionchetti et al., 2002; Kajander et al., 2005; Saggioro, 2004). However, in order to exert their health benets, the minimum concentration of live probiotic bacteria at the expiry date of the product should be around 107 CFU/mL (Ding and Shah, 2008; Homayouni et al., 2008; Krasaekoopt et al., 2003). The high numbers have been suggested because during passage through the stomach and the small intestine, a signicant number of bacterial cells die (Shah, 2000). As a result, the survival of the strains during

Corresponding author. E-mail address: d.charalampopoulos@reading.ac.uk (D. Charalampopoulos). 0168-1605/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2011.01.040

processing and subsequent storage of the food product is of paramount importance. Probiotics are mainly used in the production of fermented milks (Sanchez et al., 2009) and other dairy products, such as yoghourt (Kailasapathy, 2006), ice cream (Homayouni et al., 2008), cheese (Fritzen-Freire Muller et al., 2010), and to a lesser extent fermented meats, cereals, vegetables, and fruits (Champagne and Gardner, 2005). Other non-dairy foods that can be used as vehicles for probiotics are fruit juices, as they are suitable for consumers that are lactose intolerant (Prado et al., 2008), and contain high amounts of sugars; the latter have been shown to be advantageous for the survival of probiotics during storage (Ding and Shah, 2008). Although signicantly less amount of information is available regarding the factors inuencing probiotic survival in fruit juices compared to dairy products, the most likely parameters include the species/strain used (Kailasapathy et al., 2008), the method of preparation of the cultures (Champagne and Gardner, 2005), the composition of the product (Shah, 2000), the storage temperature and time (Saarela et al., 2006a), the presence of dietary bre in the product (Saarela et al., 2006b), the oxygen levels (Shah, 2000), and the type of container (Champagne et al., 2008). In this study, a human derived strain of Lactobacillus plantarum was used that was previously shown to be relatively acid and bile

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tolerant (Charalampopoulos et al., 2003; Patel et al., 2004), has been shown to survive well in vivo up to the ileum (Vesa et al., 2000), and has immune (Pochard et al., 2005) and anti-inammatory activities (Foligne et al., 2006). The overall aim of this study was to enhance the existing knowledge on the effect of the juice composition on the survival of lactic acid bacteria. For this reason, a mathematical model was developed describing the survival a potential probiotic Lactobacillus plantarum strain during refrigerated storage in model solutions, as a factor of pH, citric acid and ascorbic acid. These results were then used to explain the survival of L. plantarum in various types of fruit juices, taking also into account the compositional characteristics of the juices. 2. Materials and methods 2.1. Preparation of culture L. plantarum NCIMB 8826 (National Collection of Industrial and Marine Bacteria, UK), isolated from human saliva, was used in this study. The strain was preserved in 2 mL-cryovials containing 40% glycerol, stored at 80 C. The cells were cultivated at 200 rpm, 37 C, in 100 mL of MRS broth (Oxoid, UK) for 16 h. The cells were harvested by centrifugation at 3200 g for 15 min. The pellets were washed once with 0.1 M phosphate buffer saline (Oxoid, UK) and were resuspended in 10 mL of PBS. The cell concentration in the nal PBS/cell suspension was approximately 3 1010 CFU/mL. 2.2. Experimental design The experiments were designed using a Central Composite Design (CCD). Three factors (pH, citric acid, ascorbic acid) were studied at ve different levels. Seventeen experimental runs were performed in total; these are detailed in Table 1. The values for each of the factors were chosen so that they reect the values usually found in fruit juices (Ranken and Kill, 1993). Multiple regression analysis was performed in order to t a second order polynomial equation, described below, to the data: Y = 0 + 1 X1 + 2 X2 + 3 X3 + 11 X1 + 22 X2 + 33 X3 + 12 X1 X2 + 13 X1 X3 + 23 X2 X3 Where Y is the response [log10N0week log10N6 week], 0, 1, 2, 3,, 23 are the regression coefcients, and X1, X2, X3, are the pH, citric acid concentration, and ascorbic acid concentration, respectively.
2 2 2

The modelling was conducted using a stepwise selection method within the Minitab statistical software (Release 15, State College, PA, USA). The data were statistically treated by analysis of variance (ANOVA). Three- and higher-order interactions were neglected. In order to validate the model, six conditions were randomly selected, within the ranges of pH, citric acid and ascorbic acid that were used to construct the model; these are detailed in Table 2. The cell viability was assessed after 6 weeks of storage. The [log10N0week log10N6week] data were then compared to the ones predicted from the model, using regression analysis. 2.3. Preparation of model solutions The model solutions contained 50 g/L sucrose, 25 g/L glucose and 25 g/L fructose (Fisher Scientic, UK), and appropriate amounts of citric acid (Fisher Scientic, UK) and ascorbic acid (Acros, UK), as detailed in Table 1. Their pH was adjusted with 2 M NaOH (Sigma, UK). The solutions were philtre-sterilised (0.2 m) into 60 mL sterile bottles. The bottles were covered with aluminium foil in order to protect the solutions from light. The PBS/cell suspension was added so that the initial cell concentration was approximately 1 108 CFU/mL. The solutions were stored at 4 C for 6 weeks. Samples were collected every week and analysed for pH, viable cell counts and compositional changes. 2.4. Bacterial enumeration The plate count method was used to determine the number of viable bacterial cells. The culture was serially diluted in PBS; 100 L of the suspension was then spread onto a MRS agar (Oxoid, UK) plate, in triplicate. The plates were incubated aerobically at 37 C for about 3 days, after which they were counted and expressed as CFU/mL. 2.5. Chemical analyses Sugar concentrations (sucrose, glucose and fructose) were determined by high performance liquid chromatography (HPLC) using an Agilent HPLC 1100 system, consisting of a Rezex RCMMonosaccharide Ca + column linked to a Carbo-Ca (4 3.0 mm) guard column (Phenomenex, USA), (300 7.80 mm), and a refractive index detector (Shodex RI-71). The eluent was HPLC grade water with a ow rate of 0.5 mL/min, and the temperature was set at 84.5 C. Citric, malic, acetic, lactic and ascorbic acids were determined using an Agilent 1100 HPLC system, consisting of a Prevail organic acid column (Alltech, USA), (4.6 250 mm) and a 210 nm UV detector (Hewlett Packard Interface 35900). The eluent was 25 mM KH2PO4 (adjusted to pH 2.5 with phosphoric acid) with a ow rate of 1.0 mL/ min; the analysis was conducted at room temperature. The total dietary bre in juice samples was determined by a combination of enzymatic and gravimetric methods (AOAC, 1997), using a total dietary bre assay kit (Sigma, UK). One gramme of freeze-dried sample of each juice was dissolved into 50 mL of 0.08 M sodium phosphate buffer (pH 6.0). -Amylase (0.1 mL) was then added and the contents mixed in a water bath at 95 C for 15 min. After cooling, the pH of the solutions was adjusted to 7.5 by adding

Table 1 Central Composite Design (CCD) studying the survival of L. plantarum during refrigerated storage as a function of pH, citric acid and ascorbic acid. Solution 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 pH 1 1 1 1 1 1 1 1 0 0 1.68 + 1.68 0 0 0 0 0 Citric acid 1 1 1 1 1 1 1 1 0 0 0 0 1.68 + 1.68 0 0 0 Ascorbic acid 1 1 1 1 1 1 1 1 1.68 + 1.68 0 0 0 0 0 0 0 pH 3.24 3.96 3.24 3.96 3.24 3.96 3.24 3.96 3.6 3.6 3 4.2 3.6 3.6 3.6 3.6 3.6 Citric acid (g/L) 12.8 12.8 33.1 33.1 12.8 12.8 33.1 33.1 23 23 23 23 6 40 23 23 23 Ascorbic acid (mg/L) 282 282 282 282 818 818 818 818 100 1000 550 550 550 550 550 550 550

Table 2 Composition of the model solutions used for the validation of the mathematical model. Solution 1 2 3 4 5 6 pH 3.2 3.3 3.4 3.5 3.7 3.9 Citric acid (g/L) 15 7 25 35 17 21 Ascorbic acid (mg/L) 900 400 700 300 200 800

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10 mL of 0.275 N NaOH; then 0.1 mL of protease solution was added, and the solutions were incubated at 60 C for 30 min. Subsequently, the pH of the solutions was adjusted between pH 4.0 and 4.6 using 0.325 M HCl; 0.1 mL of amyloglucosidase was then added and the solutions were incubated at 60 C for 30 min. Four volumes of 95% ethanol were added into each solution; these were left overnight at room temperature to allow complete precipitation. The solution was ltered and the residue was washed thrice with 20 mL of 78% ethanol, twice with 10 mL of 95% ethanol, and twice with 10 mL acetone, and was then air-dried. After drying, the residue was weighed and the sample split in two. One half was used for analysing the protein content, using the bicinchoninic acid protein assay kit (Sigma, UK) and the other half the ash content. The total dietary bre was calculated as the weight of the residue minus the weights of protein and ash. The total phenol content of juices was determined using the Folin Ciocalteu assay (Lukanin et al., 2003). The juices were initially claried by centrifugation (10,000 g, 10 min, 4 C). Then, 0.5 mL of each claried juice was mixed with 0.5 mL of FolinCiocalteu reagent and 5 mL of 20% Na2CO3 solution. The solutions were diluted to 50 mL with distilled water, and mixed thoroughly. The assay mixtures were allowed to stand for about 30 min at room temperature. The absorbance was measured at 630 nm; the blank was distilled water. The total phenol concentration was calculated using a calibration curve, prepared with various concentrations of gallic acid.

3. Results 3.1. Cell survival in model solutions The evolution of the cell concentration of L. plantarum during storage in the model solutions, and the log difference between week 0 and week 6, [log10N0week log10N6week] are presented in Table 3. In all the solutions, the number of cells started to decrease slowly after the rst two weeks of storage, especially in the case of the solutions with very low pH. The cell concentrations were signicantly reduced (P b 0.05) by week 5 and week 6, although in most cases they were still higher than 107 CFU/mL. The lowest cell survival was observed for solution 11, which had that the lowest pH value (pH 3). High concentrations of citric acid seemed to support high cell survival, as suggested by the results of solutions 3, 4, 7, 8, and 14 (Table 3). 3.2. Model development ANOVA analysis of the stepwise regression demonstrated that a second order polynomial model t well the data. The R2 values of the model was R2 = 0.89. Moreover, the residual plots did not show any trend in the distribution of the residuals around the zero line, further conrming the goodness of t. Based on the regression coefcient estimates and the corresponding Prob N F values, it was deduced that the pH and citric acid concentration had a signicant effect on the model, whereas the ascorbic acid concentration did not (Table 4). The polynomial mathematic model that was used to estimate the log difference, [log10N0week log10N6 week], was: log10 N0week log10 N6week = 5 10 27:775pH + 3:678pHpH
1

2.6. Cell survival in fruit juices Seven commercial fruit juices (orange, grapefruit, blackcurrant, pineapple, pomegranate, cranberry and lemon) were purchased from a local supermarket. The PBS/cell suspension was added into 40 mL of each juice, so that the initial cell concentration was approximately 1 108 CFU/mL. The juices were stored at 4 C for 6 weeks. Samples were collected weekly and analysed for pH, viable cell counts and compositional changes. The survival experiments were conducted in triplicate for each condition, using different inoculums for each of the replicates.

0:0069pHcitric acid

Table 4 Fig. 1 shows the response surface plot of [log10N0week log10N6week] as a function of pH and citric acid concentration. Fig. 2 shows the correlation between the observed and the predicted values of the log decrease [log10N0week log10N6week], for six solutions (Table 2), as part of a model validation study. The latter result suggested that the model had a good predictive ability, as the regression coefcient, (R2), was 0.80. 3.3. Cell survival in fruit juices

2.7. Statistical analysis Comparisons between the various sets of data were carried out using the t-test. A P-value below 0.05 (presented as P b 0.05) was considered statistically signicant.

The log decrease [log10N0week log10N6week] after 6 weeks of storage in the various fruit juices is shown in Table 5, whereas the

Table 3 Evolution of the cell concentration of L. plantarum (in CFU/mL), and lactic acid concentration during refrigerated storage for six weeks. Solution 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 log10N (0 week) 8.01 0.00 8.17 0.09 8.07 0.12 8.07 0.02 8.03 0.01 8.02 0.01 8.04 0.04 8.02 0.01 8.02 0.00 8.10 0.06 8.06 0.10 8.07 0.02 8.07 0.07 8.02 0.09 8.08 0.05 8.06 0.07 8.04 0.03 log10N (1 week) 8.00 0.04 8.06 0.01 7.95 0.02 8.01 0.04 7.96 0.05 8.03 0.12 7.92 0.02 7.98 0.04 7.97 0.09 7.98 0.02 7.90 0.04 8.02 0.04 8.08 0.03 8.06 0.04 7.96 0.05 7.95 0.02 8.00 0.07 log10N (2 week) 7.99 0.09 8.03 0.04 7.96 0.04 8.05 0.04 7.86 0.04 8.04 0.03 7.93 0.11 8.03 0.02 8.07 0.08 8.03 0.05 7.53 0.05 8.01 0.07 8.01 0.07 8.05 0.05 8.07 0.03 8.08 0.03 8.08 0.05 log10N (3 week) 7.64 0.07 8.01 0.01 7.78 0.09 8.16 0.04 7.64 0.05 7.98 0.01 7.90 0.06 8.08 0.07 8.08 0.04 8.14 0.02 7.09 0.06 8.02 0.03 8.00 0.05 7.95 0.01 8.08 0.03 8.03 0.06 8.00 0.02 log10N (4 week) 7.39 0.03 7.79 0.02 7.64 0.04 8.09 0.07 7.33 0.01 7.84 0.05 7.76 0.05 7.94 0.04 8.02 0.03 8.05 0.06 6.50 0.04 7.93 0.03 7.82 0.08 8.04 0.04 8.01 0.02 8.02 0.03 8.04 0.05 log10N (5 week) 7.17 0.05 7.64 0.04 7.53 0.04 8.05 0.03 6.99 0.05 7.71 0.03 7.55 0.09 7.97 0.01 7.91 0.06 7.97 0.08 5.94 0.01 7.89 0.09 7.63 0.07 8.01 0.01 7.97 0.08 8.01 0.03 7.96 0.05 log10N (6 week) 6.93 0.08 7.37 0.09 7.20 0.12 8.02 0.04 6.58 0.07 7.53 0.07 7.30 0.15 7.93 0.01 7.67 0.05 7.89 0.05 5.20 0.14 7.82 0.03 7.32 0.04 8.08 0.02 7.94 0.04 7.96 0.03 7.93 0.02 [log10N0week log10N6 1.08 0.04 0.79 0.09 0.87 0.12 0.05 0.03 1.45 0.04 0.49 0.04 0.74 0.10 0.09 0.01 0.35 0.03 0.21 0.05 2.86 0.12 0.25 0.02 0.75 0.06 0.06 0.05 0.14 0.05 0.10 0.05 0.10 0.03
week]

Lactic acid (g/L) at week 6 0.38 0.09 0.81 0.29 0.71 0.14 1.84 0.09 0.03 0.07 0.65 0.10 0.17 0.38 1.19 0.05 1.33 0.03 0.80 0.02 0.11 0.14 1.55 0.01 0.47 0.01 1.11 0.01 0.98 0.26 0.82 0.03 0.83 0.01

Standard deviation ( S.D.) calculated with 95% condence.

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Term Constant pH pH*pH pH*citric acid

Coefcient 5 * 10 27.775 3.678 0.0069

1

Condence intervals (68.614, 37.608) ( 19.123, 36.426) (4.879, 2.478) ( 0.0029, 0.0109)

P value 0.000 0.000 0.000 0.003

Predicted value

Table 4 Regression coefcients, their 95% condence intervals and the corresponding P values for the log decrease [log10N0week log10N6 week]. The regression coefcients are based on the uncoded values of the controlling factors.

1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0 0.2 0.4

y = 1.872x + 0.0003 R = 0.7983

0.6

0.8

survival curves are shown in Fig. 3. The cell concentration in orange, blackcurrant and pineapple juices, all of which had a pH of about 3.8, decreased in all cases less than 0.4 log. The highest survival was obtained for grapefruit, albeit the fact that its pH was very low (pH ~ 3.2). The cell concentrations in cranberry and pomegranate decreased very quickly (Fig. 3). In the case of lemon juice, the cell concentration decreased approximately 1.1 log after 6 weeks of storage, albeit the fact that lemon juice had the lowest pH (pH ~ 2.5) among all the juices tested. The developed mathematical model was then used to predict the cell survival in orange, grapefruit, blackcurrant, pineapple, and pomegranate, based on the pH, citric acid and ascorbic acid concentrations of the juices. The model was able to satisfactory predict the cell survival in orange, blackcurrant and pineapple; however, it failed to predict cell survival in grapefruit and pomegranate. 3.4. Chemical changes in fruit juices during storage Fig. 4 presents the concentrations of dietary bre, protein and total phenol in the various juices. The highest concentrations of dietary bre and total proteins were observed in orange and grapefruit juices, whereas the highest concentration of total phenols was observed in pomegranate (4.3 g/L); a high total phenol concentration was also found in cranberry juice (1.5 g/L). Table 5 presents the chemical changes that took place in the fruit juices after 6 weeks of storage, including the evolution of pH, citric, malic, lactic, acetic, and ascorbic acids, as well as that of total sugars. In all cases, the pH did not change signicantly (P b 0.05). Ascorbic acid decreased between 70 and 100% during storage in all juices and their respective controls (noninoculated juices), indicating the instability of ascorbic acid. The concentration of total sugar (expressed as the sum of sucrose, glucose and fructose concentrations) increased in most cases from 5 to 50 g/L during storage. However, smaller decreases were observed for the inoculated juices compared to their respective controls.

Observed value
Fig. 2. Linear regression of the observed versus the predicted values of the log decrease [log10N0week log10N6week] from six model solutions.

4. Discussion In order for probiotics to survive the adverse conditions of the gastrointestinal tract and reach the intestine in sufcient numbers, they need to be present at a concentration of at least 107 CFU/mL in the product at the end of shelf-life; this, in the case of fruit juices, corresponds to approximately 109 CFU per portion. The aim of this work was to evaluate, through the development of a mathematical model, the effect of pH, citric acid and ascorbic acid on the survival of L. plantarum cells during refrigerated storage, and evaluate the ability of the model to predict the cell survival in various fruit juices. The reason for selecting these three factors is that they have been suggested to play an important role for the survival of lactobacilli in foods, including juices (Champagne and Gardner, 2005). It must be noted however, that in addition to citric acid, malic acid is usually present at similar amounts in most juices (Ranken and Kill, 1993); this was taken into account when applying the model to the data from the juices. Moreover, it should be noted that for a commercial application, instead of fresh cells, which were used in this study, either frozen or freeze dried cells are used. This aspect needs to be investigated further, as it is likely that the preparation of the cells will affect their survival during storage in the juices. Based on the results from the model solutions and the stepwise regression analysis that was conducted, it was deduced that pH and citric acid were the main factors inuencing cell survival during storage, whereas ascorbic acid did not. The strong inuence of pH on cell survival in various food products has been widely suggested (Champagne and Gardner, 2005). In the case of lactic acid bacteria, pH homeostasis between the intracellular and the extracellular environment is maintained by the action of a proton translocating ATPase. The enzyme hydrolyses ATP, which generates the energy for the extrusion of protons from the cytoplasm (Corcoran et al., 2005). As a result, other crucial cellular functions are deprived of ATP, and cell viability cannot be maintained. There is very little information regarding the role of citric and malic acids in the survival of lactic acid bacteria during storage. However, taking into account the fact that these organic acids are commonly used preservatives with antimicrobial properties, it was expected that citric acid would have a negative effect on cell survival in the model solutions. However, citric acid had a signicant positive effect on cell survival. A likely explanation for this could be that citric acid was metabolised by the cells, however, based on the chemical analysis data, this was not the case, as its concentration did not signicantly (P b 0.05) decrease during storage (see discussion below). Ascorbic acid has been associated with the good survival of L. acidophilus in yoghourt, which was attributed to the fact that it acts as an oxygen scavenger (Shah, 2000; Zarate et al., 2005). This is in contrast to the results of this study, which showed that ascorbic acid did not have an effect on cell survival. It is interesting also to note that

Fig. 1. Response surface plot illustrating the effect of pH, citric acid, and their interaction on the log decrease [log10N0week log10N6week].

S. Nualkaekul, D. Charalampopoulos / International Journal of Food Microbiology 146 (2011) 111117 Table 5 Compositional and microbiological changes in fruit juices after refrigerated storage for six weeks. Juice Sample pH Citric acid (g/L) 8.9 0.3 7.1 0.1 10.0 0.1 0.023* 14.1 0.1 13.5 0.3 14.4 0.5 0.054 2.3 0.1 1.3 0.4 2.5 0.2 0.035* 5.7 0.3 4.7 0.4 6.9 1.4 0.011* 6.0 0.1 5.9 0.4 6.7 0.8 0.268 3.3 0.2 3.4 0.5 3.8 0.1 0.701 9.2 0.2 10.1 0.4 13.6 1.1 0.071 Malic acid (g/L) 1.8 0.6 0.9 0.1 1.3 0.2 0.120 0.9 0.0 0.3 0.3 1.1 0.1 0.084 4.2 0.6 2.7 0.2 3.5 1.0 0.089 3.2 0.6 1.6 0.8 2.6 0.0 0.016* 1.6 0.1 1.6 0.3 2.4 1.4 0.623 2.9 0.0 2.9 0.5 3.4 0.6 0.902 1.1 0.1 0.8 0.1 1.4 0.1 0.026* Ascorbic acid (mg/L) 358 7 73 5 192 16 0.000* 217 2 20 30 133 34 0.012* 557 10 28 21 170 193 0.001* 226 3 59 7 66 13 0.003* 144 4 0 144 5 0.001* 720 1 240 20 322 39 0.001* 340 3 94 44 113 2.0 0.019* Total sugar (g/L) 88.7 1.3 96.2 1.4 97.6 1.9 0.024* 79.8 0.4 84.0 4.7 84.9 1.2 0.280 3.0 0.2 2.2 0.6 3.8 0.3 0.223 133.4 8.0 139.9 3.5 142.5 1.4 0.341 78.3 2.4 74.8 2.8 81.4 1.2 0.107 116.6 0.3 128.6 3.5 129.3 9.4 0.033* 102.5 2.1 139.0 6.3 159.1 5.9 0.010* Lactic acid (g/L) 0 2.7 0.2 0 0.001* 0 1.9 0.1 0 0.000* 0 1.4 0.2 0 0.008* 0.5 0.0 2.0 0.3 0.9 0.3 0.013* 0 0.1 0.1 0 0.102 0 0 0 0 1.3 0.3 0 0.012* Acetic acid (g/L) 0 2.8 0.9 0 0.029* 0 0.4 0.6 0 0.413 0 0.8 0.7 0 0.180 0 1.4 0.3 0 0.014* 0 0.2 0.2 0 0.172 0 0.7 1.0 0 0.339 0 1.4 0.3 0 0.014* [log10N0week log10N6 Predicteda,b 0.40
week]

115

Experimental 0.07

Orange

Grapefruit

Blackcurrant

Pineapple

Pomegranate

Cranberry

Lemon

Week 0 Week 6 Controlc P value Week 0 Week 6 Control P value Week 0 Week 6 Control P value Week 0 Week 6 Control P value Week 0 Week 6 Control P value Week 0 Week 6 Control P value Week 0 Week 6 Control P value

3.76 0.01 3.75 0.00 3.76 0.01 3.20 0.01 3.17 0.01 3.20 0.01 3.74 0.01 3.73 0.02 3.76 0.01 3.76 0.01 3.79 0.00 3.79 0.02 3.25 0.01 3.21 0.01 3.21 0.00 2.53 0.03 2.51 0.01 2.50 0.01 2.52 0.01 2.51 0.00 2.50 0.01

1.56

0.02

0.51

0.31

0.44

0.28

1.52

7.80

N/A

8.02

N/A

1.12

Standard deviation ( S.D.) and t-test between week 0 and week 6 calculated with 95% condence, *signicant (P b 0.05). N/A: Not applicable. a [log10N0week log10N6 week] as predicted by the mathematical model (Eq. (1)) and the coefcients in Table 4. b The total concentrations of citric plus malic acid were used as inputs to the model (Eq. (1)), rather than citric acid. c The control was uninoculated juice (after 6 weeks of storage).

the ascorbic acid concentrations decreased substantially over time in the model solutions (data not shown). This was expected, as a variety of factors can inuence the stability of ascorbic acid during processing and storage, including the temperature, the concentration of salt, sugars and minerals in the juice, the pH, the oxygen levels, the presence of enzymes, and light (Tiwari et al., 2009). Regarding the compositional changes taking place during storage in the model solutions, lactic acid was produced after 6 weeks of storage, suggesting that the cells were metabolising the available energy sources. The amounts produced were small though, ranging between 0 and 1.9 g/L (Table 3). The higher amounts were observed for the experimental runs that demonstrated the higher survival, suggesting that the cells most likely used the energy generated through the metabolism to maintain their viability. The pH of the

solutions however did not change (data not shown), presumably due to the high buffering capacity of the solutions. Despite the likely protection offered by citric acid, the amounts of citric acid did not change signicantly during storage (P b 0.05, data not shown), whereas no acetic acid was produced based on the HPLC data. Fermentation studies have shown that citric acid was metabolised by Lactobacillus species, such as L. plantarum and L. casei, when present in the growth media along with sugars. In all these cases, the pH of the media ranged between 4 and 5, and the main product was acetic acid, with the concomitant production of ATP (Kennes et al., 1991; Palles et al., 1998). A likely possibility for the fact that reduction in the citric acid concentration was not observed in this study, could be that the cells were maintained at 4 C and at very low pH values (in contrast to the higher temperatures and pH values of the published studies), and

9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 0 1 2 3 4 5 6

Cell concentration (log10 CFU/mL)

8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0

e e te it ng nt fru pl na ra ra ur ap pe ra kc ne O ra eg

Concentration (g/L)

Dietary fibre Protein Total phenol

rry be an

ac

Fig. 3. Evolution of the cell concentration (log10 CFU/mL) of L. plantarun in fruit juices during six weeks of storages. Symbols: orange juice, grapefruit juice, blackcurrant juice, pineapple juice, pomegranate juice, cranberry juice and lemon juice.

Fig. 4. Concentrations of dietary bre, protein and total phenol in the various fruit juices.

Po

Bl

Cr

Pi

Le

Storage time (weeks)

on

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S. Nualkaekul, D. Charalampopoulos / International Journal of Food Microbiology 146 (2011) 111117

as a result they did not metabolise high amounts of citric acid, to show statistically signicant changes in the compositional data. The application of the developed model in the juices indicated that cell survival was predicted well in some of them, including orange, blackcurrant and pineapple, although it must be noted that the total concentration of citric plus malic acid was used as an input to the model, rather than just citric acid. This indicated that in these particular juices, the pH and the level of organic acids were the main factors inuencing cell survival. On the other hand, the model failed to predict the cell survival in grapefruit and pomegranate. This suggested that besides pH and citric acid, other components present in the juices probably inuence cell survival. Likely factors include the dietary bre, protein and polyphenol contents of the juices. In order to investigate this in more detail, the juices were all analysed for the above components, aiming to understand better the experimental results. It must be also noted that the cell survival was tested in two more juices, namely lemon and cranberry. Both of these juices had pH values (pH ~ 2.5) outside the pH range used for the model development and therefore the model could not be applied to the survival data in this case. However, the survival data are presented in this study as they help to highlight the fact that certain components in the juice are likely to have a very drastic positive or negative effect. The cells survived well in orange, blackcurrant and pineapple juices; one reason for this was probably the high pH of these juices, which was around pH 3.8. Among the three juices, orange juice showed the highest cell survival; this could be due to the higher acid content (approximately 11 g/L in total of citric plus malic acid) compared to the other juices, and also due to the presence of total dietary bre. Its concentration was approximately 2.8 g/L (Fig. 4) compared to 1.6 g/L in pineapple, and 0.2 g/L in blackcurrant juice. It has been suggested that dietary bres can protect probiotic cells during processing and storage, via a mechanism that involves the physical immobilisation of the cells on to the bre (Saarela et al., 2006b). Examples of such bres include oat -glucan, the addition of which into yoghourt resulted in improved survival of B. animalis subsp. lactis during prolonged cold storage (Vasiljevic et al., 2007). Other researchers have reported that oat our with 20% -glucan was able to protect L. rhamnosus during storage in apple juice (Saarela et al., 2006b). Moreover, Sendra et al. (2008) reported the citrus bres enhanced L. acidophilus CECT 903 and L. casei CECT 475 survival during storage at 4 C for 40 days in fermented milks. It is interesting to point out that the cells survived well in blackcurrant juice, although it had a low sugar concentration (3 g/L), much lower than the rest of the juices. In the case of the lemon and grapefruit juices, the survival of L. plantarum cells was in both cases better than expected, considering that the pH of the juices were very low, i.e., 2.5 and 3.2 respectively. This could be due to the high concentrations of organic acids (approximately 11 and 15 g/L of total organic acids, respectively) and to the presence of pectin, which is common for citrus fruits (Cerda et al., 1988; Masmoudi et al., 2008); indeed, the analysis showed the presence of 1.9 and 2.8 g/L of dietary bre, for lemon and grapefruit, respectively. In addition, grapefruit juice contained the highest amount of proteins (N 6 g/L), which could have also supported cell survival. In the case of the cranberry juice, the very fast drop of cell concentration within one week of storage was partly due to the low pH (pH ~ 2.5); the low concentration of organic acids (approximately 6 g/L of malic plus citric acid) most likely contributed to it too. Another factor could be the high levels phenolic compounds (1.5 g/L), such as benzoic acid, which is present at high levels in fresh cranberry juice (Chen et al., 2001) and has been suggested as a likely factor that is likely inuencing the survival of probiotic lactobacilli during storage (Sheehan et al., 2007). Other phenolic acids, which are likely present in cranberry juice, such as coumaric acid (Chen et al., 2001), have been shown to have an inhibitory effect against L. plantarum (Landete et al., 2007; Rodriguez et al., 2009; Ruiz-Barba et al., 1990). The results

obtained in this study were in agreement with the results of the study by Sheehan et al. (2007), which showed that probiotic Lactobacillus and Bidobacterium strains survived better in orange and pineapple juice than in cranberry juice. In the latter, the viability of the cells decreased drastically, as also observed in this study. In the case of the pomegranate juice the cell concentration decreased sharply, immediately after inoculation, and reached zero after four weeks of storage. The main factors inuencing this were most likely the low pH of the juice (pH ~ 3.25) and the low organic acid concentration. Moreover, the pomegranate juice was rich in polyphenols (4.3 g/L); these were most likely mainly tannins, which according to the study of Bialonska et al. (2009) inhibited the growth of certain intestinal bidobacteria, as well as pathogenic clostridia and Staphyloccocus aureus. Regarding the chemical changes taking place in the juices during storage, the sugar concentration in most cases increased signicantly, presumably due to the hydrolysis of polysaccharides through the action of enzymes (Hoebler et al., 1989; Mohammad et al., 2010). This however, does not exclude that some of the available fermentable sugars, such as glucose, fructose and sucrose were metabolised during storage, as was previously suggested (Ding and Shah, 2008). This was likely, taking in to account that the control week 6 sample had higher sugar concentration that the week 6 sample containing the L. plantarum cells. Lactic and acetic acids were the main acids produced during storage. Interestingly, the highest amounts were produced in the juices that exhibited the best cell survival, further indicating that metabolism of the available carbon sources was taking place. Lactic acid could have been produced from the metabolism of sugars (Ding and Shah, 2008), or the metabolism of malic acid through the action of the malo-lactic enzyme, which has been identied in L. plantarum (Garcera et al., 2006), Acetic acid was probably produced from the metabolism of citric acid and sugar, as previously shown for L. plantarum (Kennes et al., 1991; Palles et al., 1998). In contrast to the results obtained from the model solutions, a statistically signicant decrease in the citric acid concentration was observed for certain juices; however in all cases the decrease was small (b 1 g/L). Despite the increase in the organic acid concentrations during storage, the pH of the fruit juices remained unchanged over the six week storage, indicating that the buffering capacity of the juices was high. A similar result was reported by Champagne and Gardner (2008) for a number of probiotic Lactobacillus strains, stored for 28 days in fruit juices under refrigeration. 5. Conclusions According to the developed mathematical model, high levels of pH and citric acid supported the survival of Lactobacillus plantarum in model solutions during refrigerated storage. Among the various fruit juices tested the cells survived well in orange, grapefruit, blackcurrant, pineapple and lemon juices, whereas they decreased rapidly in cranberry and pomegranate juices. The mathematical model was able to predict in some cases the cell survival in the fruit juices, although in some other cases it did not, most likely because other components of the juices were playing a role, such as proteins, dietary bres, or various antimicrobial compounds. References
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