Effect of Prebiotic Compounds on the Growth and Survival of Bifidobacteria in a Laboratory Medium

This study focuses on the effects of 5 prebiotics (low and high molecular weight dextrans, fructooligosaccharides, inulin and lactulose) on 6 bifidobacteria strains (Bifidobacterium animalis subsp. lactis, B. animalis subsp. animalis, B. bifidum, B. breve, B. longum subsp. infantis and B. longum) in a laboratory medium. Two different assays were conducted (a growth experiment-for all the strains-and a survival assay-for B. animalis subsp. lactis and B. breve). Glucose was used as positive control. Growth assay pinpointed that lactulose could be successfully used to promote bifidobacteria and attain a growth index for B. animalis subsp. animalis and B. longum subsp. infantis higher than with glucose. Concerning the bifidogenic effect, it is not clear if it could be observed also during the death phase, as we recovered a beneficial effect for B. breve but not for B. animalis subsp. lactis.


INTRODUCTION
According to the definition of Gibson and Roberfroid (1995) a prebiotic is a non-digestible food ingredient which beneficially affects the host by selectively stimulating the growth and/or activating the metabolism of one or limited number of health promoting bacteria in the intestinal tract, thus improving the host's intestinal balance.
Prebiotics are generally carbohydrates, ranging in the size from small sugar alcohols and disaccharides, to oligosaccharides and large polysaccharides (Guo, 2009), sharing some common criteria: • A prebiotic should be neither hydrolyzed nor adsorbed in the intestine • It should be a selective substrate for some probiotic microorganisms • It alters qualitative composition of intestinal microflora, by promoting beneficial bacteria • It induces luminal or systemic effects that are advantageous to the host.Guo (2009) reported some compounds in the list of prebiotic: fructans, inulin, fiber gums, isomaltooligosaccharides, lactitol, lactosucrose, lactulose, oligofructose, pyrodextrins, soy-oligosaccharides, transgalacto-oligosacharides, xylo-oligosaccharides.
The interest toward prebiotic compounds increased in the last decade, as they were proposed and used as functional compounds in the "symbiotic" products or as ingredients in different foods (dairy products, frozen desserts, table spreads, baked goods and breads, breakfast cereals, fruit preparations, meat products and chocolate) (Bottazzi, 2004;Debon et al., 2010;Gonzalez et al., 2011;Guo, 2009;Lee et al., 1999;Rodrigues et al., 2011).
A symbiotic is defined as a mixture of prebiotics and probiotics that beneficially affects the host by improving the survival and implantation of live microbial dietary supplements in the gastrointestinal tract by selectively stimulating the growth and/or promoting the metabolism of one or of a limited number of health-promoting bacteria and thus improving the health of the host (Gibson and Roberfroid, 1995).

Prebiotic compounds and laboratory medium:
For each assay the media containing glucose (5 g/L) or without carbohydrates were used as positive and negative controls, respectively.
Growth assay: Aliquots of 20 mL of the laboratory medium, containing prebiotic compounds, glucose (positive control) or without carbon source (negative control), were inoculated separately with ca.6 log cfu/mL of each strain.The samples were incubated at 37°C for 4 days.Microbial growth was evaluated through the measurement of the absorbance at 600 nm by a spectrophotometer UV-visible Beckman DU 640 (Beckman DU 600, Fullerton, CA).
Data were modeled as Growth Index, as proposed by Blaszyk and Holley (1998), modified by Bevilacqua et al. (2009): where, for each time of analysis Abs s is the absorbance of the samples with different prebiotics and Abs c the absorbance of the positive control.The experiments were performed at least in duplicate, over two different batches.
Survival assay: This assay was conducted using as test strains B. animalis subsp.lactis (DSM 10140) and B. breve (DSM 20213).Aliquots of the laboratory medium of Liew et al. (2005), modified by Bevilacqua et al. (2008), containing prebiotics, glucose (positive control) or no carbon source (negative control) were inoculated with ca.5-6 log cfu/mL of each strain separately.The samples were incubated at 37°C for at least 15 days.The survival of the microbial targets was evaluated periodically through the pour plate method, using MRS agar added with 0.05% cysteine, incubated under anaerobic conditions at 37°C for 48-72 h.In addition, pH of the sample was evaluated periodically by a pHmeter Crison (Crison instruments, Barcelona, Spain).
The experiments were performed in duplicate over two different batches.

Statistical analyses:
Data were analyzed through oneway-analysis of variance and Tukey's test (p<0.05)through the software Statistica for Windows (Statsoft, Tulsa, OK).

RESULTS AND DISCUSSION
Figure 1 shows the results of the growth assay after 24 h of incubation; data were modelled as Growth Index (GI), referred to the sample containing glucose (positive control).As expected GI value of negative control was ca.2-9%; the growth was inhibited also in presence of dextrans, FOS and inulin as carbon sources.A partial growth was observed only for the strain 20088 (i.e., B. longum subsp.longum) with high molecular weight dextran, which resulted in GI of 17.32±13.84%.An interesting result was recovered when lactulose was added to the laboratory medium; in fact, it was able to promote the growth of bifidobacteria and the strains 10140, 20456 and 20213 (B.animalis subsp.lactis, B. bifidum and B. breve, respectively) showed GI values of 84.97-92.94%.These values were not statistically different from the growth of the positive control (GI of 100%); concerning the interpretation of GI values, Bevilacqua et al. (2009) proposed a simple criterion, as follows: GI<25% stands for a complete inhibition of the microorganism; GI included in the range 25-75% underlines a partial inhibition.Finally GI>75% stands for a growth kinetic similar to that reported for the optimal conditions.Therefore, we could assume that lactulose was able to support the growth of the three strains of bifidobacteria as well as glucose.
Concerning the strains 20104 and 20088 (B.animalis subsp.animalis and B. longum subsp.infantis), they showed GI>100% in presence of lactulose; the statistical analysis (data not shown) revealed that these values were different from GI in the positive control, thus underlining a stimulating effect of the prebiotic.
In a second step, the research focused on the effects of prebiotic compounds on the death kinetic of B. animalis subsp.lactis (DSM 10140) and B. breve (DSM 20213), chosen as they were the most vigorous microorganisms amongst the strains studied in the first step.

Positive control
The results are reported in Table 1.Concerning the strain 10140, cell number was below the detection limit after 6 days of storage for the samples containing glucose and lactulose; otherwise, the microorganism showed a prolonged viability both in the negative control and in the sample containing the other prebiotics.
B. breve (DSMZ 20213) showed a similar trend, with only one difference; lactulose resulted in a slower death kinetic than glucose, as after 6 days in the sample containing the prebiotic a viable count of 5 log cfu/mL was recovered.Oliveira et al. (2011) reported a beneficial effect of lactulose both in growth and death phases; in the death phase, in fact, skim milk added with the prebiotic showed also higher counts of B. lactis than the controls.However, we recovered a bifidogenic effect only for B. breve DSMZ 20213 after 6 days, but not for B. animalis subsp.lactis DSMZ 10140.Saarela et al. (2003) observed that lactulose improved the cold-storage stability of L. salivarius at 4°C for 22 days, probably due to splitting of cellchains.An enhancement by lactulose of β-glucosidase and β-galactosidase activities on intestinal microbiota was also reported (Juśkiewicz and Zduńczyk, 2002;Pham and Shah, 2008).These effects could be responsible of both the beneficial effect of lactulose in the growth and death phases.
Concerning the results obtained for the other prebiotics (FOS, inulin and dextrans), the trend was similar to that observed in the negative control, with a prolonged viability throughout the storage.We can suggest that this result was probably due to the fact that bifidobacteria were not able to use these compounds, as evidenced by the slight decrease of the pH (ca.0.5-0.7),whereas in the samples containing glucose and lactulose, the pH attained a minimum value of 4.2-4.5 after 2 days (data not shown).This low acidification could be responsible of the prolonged viability of the target strains in presence of FOS, dextrans and inulin; otherwise, the decrease of pH determined the loss of viability in the media containing glucose and lactulose.

CONCLUSION
This study suggests that lactulose could be used successfully to promote the growth of bifidobacteria and attain, for some strains (e.g., B. animalis subsp.animalis and B. longum subsp.infantis), a growth index higher than that recovered with glucose.Concerning the bifidogenic effect, it is not clear if it could be observed also in the death phase, as we recovered a beneficial effect for B. breve but not for B. animalis subsp.lactis.These results can be considered as the first step to developing a symbiotic product, containing bifidobacteria and lactulose.

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This study focused on six different strains of