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Nutritional enhancement of probiotics and prebiotics: technology aspects on microbial viability, stability, functionality and on prebiotic function

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During the consumption probiotics are exposed to various conditions in the upper GI-tract which may affect detrimentally their viability. The single most detrimental factor to probiotic viability in the GI-tract is the low pH of the stomach. In addition, e.g. bile secreted to the upper part of the small intestine has antimicrobial properties. VTT: Influence of various test parameters on the acid and bile tolerance test result was evaluated to optimise the testing protocol: the parameters studied (strain and test) were cell density (L. rhamnosus E800, acid and bile tolerance), different bile reagents (B. animalis subsp. Lactis Bb-12), sterilisation technique for the bile solution (B. animalis subsp. lactis Bb-12 L. rhamnosus E800 and GG), molarity of the buffer solution (B. animalis subsp. lactis Bb-12; acid and bile tolerance), growth conditions (fermenter vs. test tube grown B. animalis subsp. lactis Bb-12, bile tolerance).Influence of processing conditions on acid and bile tolerance of B. animalis subsp. lactis Bb-12 was studied by using fermenter grown (15 and 22 h) cells prior and after freeze-drying with different cryoprotectants (sucrose and RSM as carriers for non-neutralised and neutralised cells). From the same freeze-dried preparations influence of food matrix storage on acid and bile tolerance of B. animalis was studied after harvesting the cells from milk, fruit juice or PBS (control) at baseline or after two weeks storage in milk and PBS. Kinetic changes in the membrane functions during acid treatment of B. animalis were further investigated by using a membrane potential-sensitive fluorochrome, DiBAC4(3), in conjunction with a fluorometer. VTT: B. animalis subsp. lactis Bb-12 fresh cells tolerated well pH3.0 and poorly pH 2.5; pepsin supplementation had clearly protective effect against the pH of 2.5. The same phenomenon was observed with sucrose- and RSM-protected freeze-dried cells. Different freeze-drying conditions or fermentation times did not affect the acid tolerance of the cells. Milk showed clearly protective effect of the cells against low pH and some protection against bile acids. No significant differences in the bile tolerance of differently produced fresh or freeze-dried B. animalis cells were detected. However, the type of bile used had a large impact, bile extract being much more detrimental to the cells than bile acids. The DiBAC4(3) fluorescence measurements showed a rapid decrease of fluorescence during the acid exposure of the cells (more profound in pH 2.5 treated cells). This change in the membrane integrity suggests hyperpolarisation of the cell membrane during acid stress which can be associated to the activity of proton pumps.
It has been demonstrated for many bacteria that responses induced by one type of stress may then confer protection to the bacteria against other different stress challenges. This phenomenon is known as cross tolerance. The protection responses induced by bile salts, heat or NaCl improved the capacity of the probiotic bacteria in withstanding stressful processing techniques such as spray drying were investigated. Log-phase cells of probiotic lactobacilli strains were re-suspended in 20% RSM and were either spray dried immediately or exposed for one hour to sub-lethal levels of heat (48°C), NaCl (5%), bile salts (1mM GDCA) or heat (4ºC) + NaCl (5%) prior to spray drying. Cells were spray dried at two different outlet temperatures, 90°C and 95°C. Higher levels of survival of the spray drying process relative to the un-induced cells were obtained. Heat afforded the lowest level of survival of the induced cells, with the greatest level of survival being observed for cells induced with heat + NaCl. Moreover, in the frame of the work it was observed that even though induction of a heat tolerance response prior to spray drying increases the ability of the cells to survive the drying process, it did not significantly effect survival of the dried cells during storage. The effect of mild pressure pre-treatments on technological behaviour of L. rhamnosus GG was evaluated. With respect to heat tolerance pressure pre-treated cells (at 100MPa and 37°C) showed higher survivability than untreated ones when both were exposed to lethal heat treatment. Flow cytometric analysis showed that the acquisition of pressure-induced heat tolerance was related to membrane stabilization and protein biosynthesis. Pressure induced thermotolerance occurred as a consequence of stabilization of cellular membranes - presumably by incorporation of heat shock proteins into cytoplasmic membrane - which in turn led to an enhanced transient protection against degradative effects of heat on cell membrane. The absence of induced thermal tolerance upon addition of Chloramphenicol suggested that the proteins expressed during pressure adaptation was involved in the prevention of thermal degradation on cell membrane. Pressure pre-treated LGG showed also an improved tolerance against chemical compounds which are able to degrade cytoplasmic membranes, such as bile acid and nisin. This result confirmed the positive role of protein synthesis during pressure adaptation in protecting this vital cellular component. The potential of utilising pressure shock response in increasing the survivability of LGG during spray drying, which poses multiple environmental stresses (including heat, osmotic and oxidative stress) was also demonstrated.
Bifidobacteria are among the first species to colonise the human gastrointestinal tract and as such are believed to play an important role in gut homeostasis and normal development. Nowadays, bifidobacteria have become economically important since they are added in high numbers as live bacteria in numerous food preparations with various health-related claims. These bacteria are not naturally found in food but are purposely added to it as live bacteria because of their claimed health benefits. Examples of such products include infant formulas, cheese, dietary supplements and fermented dairy products. However, survival of bifidobacteria in food products remains sometimes problematic. In consequence, active research is needed to gain an insight into the different factors that may impact on bacterial survival and resistance to stress. In the past few years the complete genome sequence of a number of bacterial strains isolated from the human gastrointestinal tract has been established, including that of a specific B. longum strain isolated from the faeces of a healthy infant. Knowledge of this genome sequence allowed the design of DNA based arrays, containing a set of B. longum genes. These DNA arrays were used for global expression profiling in response to environmental changes. By compiling expression profiles from a range of different environmental stresses it was possible to identify genes that are commonly induced in response to these unfavourable conditions. The proteins encoded by these genes are thought to provide non-specific protection to the cell in the event of adverse conditions, whereas additional genes can be transcribed in response to a specific type of stress. To learn more about the general stress responses of B. longum NCC2705, expression profiles obtained after three different stresses namely, oxidative stress, starvation and heat shock were compiled. In all cases, cells were grown under identical controlled conditions and subjected to either a heat shock, exposure to H2O2 or transition into stationary phase. The gene expression profiles observed in response to these stresses were compared to those obtained from exponentially growing cells. Twenty-one genes were identified as being induced by a factor greater than 2.5-fold including genes such as dnaK, grpE, dnaJ, hspR, groEL, groES, clpB, hrcA. Also of interest are several upregulated genes encoding proteins with no predicted function that could possibly play a general role in protecting B. longum cells from unfavourable conditions. Noteworthy, we identified a gene rapidly and highly induced under different conditions that may be developed as a stress marker. Stress markers can be used to monitor the physiological status of the cells or help to identify experimental parameters that will lead to high induction of the general stress response.
This work deals with the application of flow cytometric analysis to evaluate the mechanisms of leading to viability loss of Lactobacillus rhamnosus GG (LGG) following exposure to physical treatments. A multiple staining strategy, which is composed of physiological dyes carboxyfluoresceindiacetate (cFDA) and propidium iodide (PI) was applied to examine specific cellular metabolic activities and their relative changes following various treatments, which may lead to loss of viability of probiotic organisms. It was expected that additional insights on process-induced changes in cellular integrity or metabolic activities, which were not explicitly assessable by culture techniques, could be achieved using this measurement technique. From the results of flow cytometric measurement following exposure to heat and pressure one could elucidate the mode of action of these physical stressors on cellular activities or integrity. Ultimately, the use of this technique might open a possibility to an improved design of bacterial inactivation processes, i.e. one could then choose a certain type of cellular damage preferred and then select the type of treatments required to achieve this goal. Particularly in the field of probiotic research, the importance of the ingestion of viable bacteria in eliciting health effect is sometimes questioned, since non-viable bacteria were reported to be effective as well. Non-viable probiotic cells are of interest due to easy-handling and longer shelf life. However, systematic studies on the efficacy of inactivation methods to produce non-viable cells are still lacking. In this context, high pressure killed cells might be one of the promising candidate to be investigated, since the fluorescence pattern of pressure inactivated cells - which is indicative for a lower extent of damage on metabolic activity and on membrane - is quite similar to the one of viable cells. Furthermore, flow cytometric analysis in combination with liposomes loaded with fluorescence marker was used to evaluate the protective effect of different types of sugars on model membranes. With this technique the sugars present in the spray drying media was evaluated on their stabilizing effect on liposomes during dehydration. Principally, the size distribution and the retention of incorporated flurochrome in lipid vesicles in the presence of lactose, polydextrose, Raftilose®P95 or in absence of sugar before and after dehydration can be monitored. In the case of drying without sugar, an increase in liposome size was found, as evidenced by a broadening of the histogram in the direction of higher liposome sizes. This behaviour was thought to be highly related to fusion and/or aggregation of liposomes. On the other hand the size distributions of liposomes dried in the presence of all evaluated sugars were nearly identical to the one before dehydration. This indicates that the addition of sugars could effectively prevent fusion and/or aggregation. On the other hand, not all sugars tested had the good capacity in preventing leakage of cF out of liposomes. In the absence of sugar there was only a small fraction of liposomes which still had fluorescence intensity values equivalent to that before dehydration. It is obvious that drying increased the permeability of the phospholipid bilayer, thus allowing cF to leak from the liposomes.
The objective of this work was to evaluate the applicability of spray drying in the production of skim milk-based preparations containing probiotic bacteria. Furthermore, oligofructose-based or polydextrose-based prebiotic substances were also included in the carrier matrix to assess their protection capacity and to examine the possibility of generating synbiotic powder products. Initially, the thermal tolerance, an indicator of probiotic survival during spray-drying, of three strains of probiotic lactobacilli were compared. L. rhamnosus E800 was the most heat resistant strain, followed by L. salivarius UCC 500, with L. rhamnosus GG being least heat resistant. Although L. rhamnosus GG exhibited the poorest thermal tolerance of the three Lactobacillus strains studied, it was the best survivor during spray-drying, indicating that thermal tolerance alone is not an accurate predictor of performance during spray-drying and that other phenomena, such as dehydration affect cell viability during drying. When reconstituted skim milk was used as spray drying carrier, a microbial survival rate of 60% to 80% was achieved at an outlet temperature of 80°C. This results in probiotic powders harbouring high numbers of viable microorganisms (~1E9CFU/g) generated with stationary phase cultures. The incorporation of commercial prebiotic substances such as Raftilose®P95 or polydextrose in the skim milk powder did not exert any adverse effect on bacterial survival upon spray drying. Probiotic cultures retained good viability during storage in powders containing skim milk/prebiotics at 4 and 15°C. However, stability of bacteria during long term storage at 37°C was impaired by partial substitution of skim milk with either of the prebiotic substances evaluated. This might be caused by the inability of the oligosaccharides in these prebiotic substances in adequately replacing water molecules on dehydration. The spray drying media were also calorimetrically characterised in terms of their glass transition temperatures so as to evaluate the contribution of glassy state on the maintenance of bacterial survival during storage. Although all evaluated carriers were in the glassy state, differences were still observed in their capacity to confer protection on the probiotic bacteria. The presence of the dried medium in the glassy state appeared to have had only little effect on bacterial stability in the spray dried powder and thus is not sufficient for preservation. Moreover, storage survival was affected by the phase of growth of the spray-dried culture with stationary phase best, followed by lag and log phase. Flow cytometric assessment in combination with functional dyes (carboxyflurescein diacetate and propidium iodide) was applied as a diagnostic tool to evaluate the type of cellular injuries which occurred upon spray-drying. Cell death was caused mainly by damage to cell membranes and the degree of membrane disintegration increased progressively as the outlet temperatures increased.

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