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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. METHODS and MATERIALS
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

It has been suggested that probiotics should be viable in order to elicit beneficial health effects. Inactivation of probiotics has been suggested to interfere with the binding to the mucosa and thereby with the immune modulating activity of probiotics. The effect of different inactivation methods on the mucus adhesion of nine probiotic strains was studied. Inactivation by heat or γ-irradiation generally decreased the adhesive abilities. However, heat treatment increased the adhesion of Propionibacterium freudenreichii and γ-irradiation enhanced the adhesion of Lactobacillus casei Shirota. Inactivation by u.v. was not observed to modulate the adhesion of the tested strains and it was concluded to be the most appropriate method for studying non-viable probiotics and preparing control products.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. METHODS and MATERIALS
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Probiotics are generally defined as viable micro-organisms that, when applied to humans or animals, beneficially affect the health of the host by improving the indigenous microbial balance (Fuller 1989; Havenaar et al. 1992). Adhesion of probiotic micro-organisms to the intestinal mucosa is considered important for many of the observed probiotic health effects. Adhesion is regarded a prerequisite for colonization (Alander et al. 1999), antagonistic activity against enteropathogens (Coconnier et al. 1993a, 1993b), modulation of the immune system (Schiffrin et al. 1997) and for increased healing of damaged gastric mucosa (Elliot et al. 1998). Therefore, adhesion is one of the main selection criteria for probiotic micro-organisms (Ouwehand et al. 1999a). Recently the literature was reviewed on the possible activity of non-viable probiotics and it was concluded that certain probiotic effects can also be obtained with inactivated probiotics. However, the method of inactivation appears to influence the efficacy of inactivated probiotics (Ouwehand and Salminen 1998). Non-viable probiotics would have economic advantages in terms of longer shelf-life and reduced requirements for refrigerated transport and storage. This would also expand the potential use of probiotics to areas where strict handling conditions cannot be met, e.g. in developing countries.

Physical treatments aimed at inactivating probiotic micro-organisms may alter the adhesive abilities of the probiotics. This could affect the efficacy of the probiotics; especially the immune-modulating properties (Kato et al. 1994). In order to test this hypothesis, selected probiotic strains were inactivated by heat (80 °C, 100 °C), u.v. and γ-irradiation, and subsequently their adhesive abilities to immobilized intestinal mucus were assessed. The other aim of the study was to determine which method of inactivation would be most appropriate for investigating the potential health properties of non-viable probiotics. This would also suggest which method would be appropriate for preparing control products in the study of probiotics in general. An inactivation method that causes little secondary changes indicates the effects from the probiotic better than effects from the inactivation.

METHODS and MATERIALS

  1. Top of page
  2. Abstract
  3. Introduction
  4. METHODS and MATERIALS
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Bacteria and growth conditions

The probiotic strains used and their culture conditions are listed in Table 1. The strains were grown from stocks stored at −75 °C in 40% glycerol (1% inoculum). To the culture media 10 µl ml−1 of [methyl-1,2–3H]thymidine was added to metabolically label the strains. The strains were washed twice with phosphate-buffered saline (PBS; pH 7·2 10 mmol l−1 phosphate) and resuspended in PBS. The absorbance at 600 nm was used to adjust the viable count to approximately 107−108 cfu ml−1. Lactobacillus (Lact.) johnsonii La1 which was subsequently diluted 10 times with PBS (2·7 × 105 cfu ml−1) in order to avoid saturation of the substratum (Tuomola et al. 1999).

Table 1.  Strains and culture conditions
Micro-organismProductGrowth conditions
MediumTimeTemperature (°C)
  • a

    A generous gift from Dr M. Saxelin, Valio Ltd, Helsinki, Finland.

  • b

    A generous gift from Dr B. Grenov, Chr. Hansen A/S, Hørsholm, Denmark.

  • c

    de Man, Rogosa and Sharpe Broth.

  • d

    Yeast extract, glucose broth.

  • e

    Whey-based broth.

Lactobacillus johnsonii La1aLC1®MRScO/N37
Lactobacillus rhamnosus LC-705aBio Profit®MRSO/N37
Lactobacillus rhamnosus GGaGefilus®MRSO/N37
Bifidobacterium lactis Bb12aNutrish®MRSO/N37
Lactobacillus acidophilus La5bNutrish®MRSO/N37
Lactobacillus casei ShirotaaYakult®MRSO/N37
Lactobacillus bulgaricus a YoghurtMRSO/N37
Lactococcus lactis subsp. cremorisa‘Viili’MRS2 d30
Saccharomyces boulardii (nom. inval.)Precosa®YGdO/N37
Propionibacterium freudenreichii subsp. shermanii JSaBio Profit®WBe2 d30

In vitro adhesion assay

Mucus was prepared from faecal samples, obtained from healthy adult subjects, essentially as described earlier (Ouwehand et al. 1999b, 2000). Adhesion of the radioactively labelled strains to the immobilized mucus was performed as described previously (Ouwehand et al. 1999b, 2000). The adhesion was expressed as the percentage of radioactivity recovered after adhesion, relative to the radioactivity in the microbial suspension added to the immobilized mucus.

Treatments of the microbial suspensions

The microbial suspensions were divided into 1-ml aliquots and incubated for 10 min in a water-bath at 80 and 100 °C. For u.v. treatment, 5 ml of the microbial suspension was transferred to a glass dish and irradiated for 5 min at a distance of 5 cm from a 30-W u.v.c. lamp (Philips, The Netherlands). Aliquots of 1 ml were irradiated with 2 kGy using a Cobalt-60 source (Radiotherapy unit, Alcion II, General Electric, France) (source–specimen distance 20 cm with a dose rate of 15 Gy min−1). Because the γ-irradiation took a relatively long time, separate controls were used for this treatment. The treatment conditions were specifically chosen to leave less than 1 cfu ml−1 (detection limit) while avoiding secondary treatment effects to the probiotic micro-organisms.

Statistical analysis

The results from the adhesion experiments are expressed as the average of three to six independent experiments. Each experiment was performed with four parallels. A two-tailed t-test was used to evaluate the statistical significance (P < 0·05) of the differences in adhesion of a strain after treatment compared with the control.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. METHODS and MATERIALS
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Of the tested strains, five were found to adhere well when viable; Lactobacillus rhamnosus GG (32%), Lactococcus (L.) lactis subsp. cremoris (26%), Lact. delbrueckii subsp. bulgaricus (26%), Bif. lactis Bb12 (13%) and Propionibacterium freudenreichii subsp. shermanii JS (14%), Fig. 1. This corresponds to 106−107 cfu well−1. Although Lact. johnsonii La1 exhibited a high percentage of adhesion (30%), the number of bacteria per well was 0·8 × 104 cfu due to the low numbers of bacteria in the initial suspension. Saccharomyces boulardii and Lact. casei strain Shirota also exhibited a low level of adhesion; less than 5% of the applied cells adhered (< 3·5 × 105 cfu well−1).

image

Figure 1. The adhesion of probiotic strains to immobilized intestinal mucus, non-treated, u.v.-inactivated and heat-treated (at 80 and 100 °C). Adhesion is expressed as the percentage of bacteria adhered to mucus compared with the amount of bacteria added to the mucus. *Significantly different (P < 0·05) from the untreated control. LGG –Lactobacillus rhamnosus GG, La1 –Lact. johnsonii La1, LcS –Lact. casei strain Shirota, L. lactisLactococcus lactis subsp. cremoris, Lact. bulgaricusLact. delbrueckii subsp. bulgaricus, S. boulardiiSaccharomyces boulardii, Bb12 –Bifidobacterium lactis Bb12, PJS –Propionibacterium freudenreichii subsp. shermanii JS, and La5 –Lact. acidophilus La5

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The adhesion of the tested strains was not affected by inactivation with u.v. (Fig. 1). Inactivation by heat treatment of Lact. rhamnosus GG and Lact. johnsonii La1 tended to decrease the adhesion. This did, however, not reach significance. The adhesion of L. lactis subsp. cremoris and Lact. casei strain Shirota was significantly reduced by heat treatment, both at 80 °C and 100 °C (P < 0·05). Interestingly, the adhesion of Bif. lactis Bb12 and especially P. freudenreichii subsp. shermanii JS was significantly increased upon inactivation at 80 °C; from 13·3% to 17·2% (P < 0·05) and from 14·0% to 23·9% (P < 0·0001), respectively. The inactivation of P. freudenreichii subsp. shermanii JS at 100 °C also significantly increased the adhesion; from 14·0 to 23·3% (P < 0·0001), Fig. 1.

Inactivation of the tested micro-organisms with γ-irradiation caused a decrease in adhesion in all strains, with the exception of Lact. casei strain Shirota. The adhesion of Lact. johnsonii La1, L. lactis subsp. cremoris, Bif. lactis Bb12, P. freudenreichii subsp. shermanii JS and Lact. acidophilus La5 was significantly reduced after inactivation by γ-irradiation (P < 0·05). By contrast, the adhesion of Lact. casei strain Shirota was increased from 3·2 to 11·4% (P = 0·01), Fig. 2.

image

Figure 2. The adhesion of probiotic strains to immobilized intestinal mucus, non-treated (control) and γ-irradiated (gamma; 2000 Gy). Adhesion is expressed as the percentage of bacteria adhered to mucus compared with the amount of bacteria added to the mucus. *Significantly different (P < 0·05) from the untreated control. LGG –Lactobacillus rhamnosus GG, La1 –Lact. johnsonii La1, LcS –Lact. casei strain Shirota, L. lactisLactococcus lactis subsp. cremoris, Lact. bulgaricusLact. delbrueckii subsp. bulgaricus, S. boulardiiSaccharomyces boulardii, Bb12 –Bifidobacterium lactis Bb12, PJS –Propionibacterium freudenreichii subsp. shermanii JS, and La5 –Lact. acidophilus La5

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. METHODS and MATERIALS
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

It is generally considered that probiotics should be viable in order to exert beneficial health effects. This assumption has been evaluated in a review of available literature and it was concluded that, although viable probiotics have more documented health effects than non-viable probiotics, the latter are not without effect (Ouwehand and Salminen 1998). However, orally administered non-viable probiotics are less effective in modulating the immune system than viable probiotics (Conge et al. 1980; de Simone et al. 1987; Kaila et al. 1995; Perdigón et al. 1995). Non-viable probiotics may be less able to bind to the intestinal mucosa, translocate less and thereby modulate the immune system less (Kato et al. 1994). To test this hypothesis in vitro, the effect of different methods of inactivation was tested on the adhesion of nine selected probiotic strains isolated from commercial products. Different inactivation methods were evaluated in order to be able to select an appropriate method for testing the health effects of non-viable probiotics in vivo and to prepare control products with little secondary change due to the inactivation method for studying the efficacy of viable probiotics.

When viable, the observed adhesion of the tested strains was similar to earlier reported observations, with the exception of S. boulardii. This strain had earlier been found to adhere well (approximately 17%; Ouwehand et al. 1999c).

Inactivation of the strains with heat was observed to affect the adhesion in many of the tested strains. Similar observations have been made by other workers. Fontaine et al. (1994) showed that heating Bif. bifidum at 100 °C for as short as 30 s reduced adhesion by 40% compared with viable bacteria. Similarly, Tuomola (1999) showed that boiling and autoclaving significantly reduced the adhesion of Lact. rhamnosus GG and Lact. johnsonii La1 to intestinal mucus. In the current study, both strains exhibited only a slight tendency to reduced adhesion following heat treatment. These differences can be explained by the fact that in the former study the bacteria were boiled for 2 h or autoclaved for 45 min. This suggests that such treatments will not only inactivate the micro-organisms, which was the purpose of the current study, but will also change their physicochemical properties (El-Nezami et al. 1998), indicating that minimal treatment is important for preparing appropriate inactivated control products.

An increased adhesion of Bif. lactis Bb12 and P. freudenreichii subsp. shermanii JS was observed after heat inactivation. Increased adhesion of probiotics upon heat inactivation has not been reported earlier and may suggest an increased interaction with the intestinal mucosa after heat inactivation for certain strains. Heat-treated lactobacilli were able to bind more aflatoxin (El-Nezami et al. 1998) and thus it can be speculated that a similar mechanism is causing an increased adhesion to immobilized intestinal mucus.

To our knowledge, no studies have been reported the adhesive properties of probiotics inactivated by γ-irradiation. In general, inactivation by γ-irradiation decreased the adhesive abilities of the tested strains, with the exception of Lact. casei strain Shirota. This strain exhibited an almost fourfold increase in adhesion upon inactivation by γ-irradiation. The mechanism behind this increase is not understood and would require more investigation. It can be speculated that the radicals generated by the γ-irradiation expose more adhesins or alter the structure of cell envelope components enhancing the adhesive properties. This also indicates that Lact. casei strain Shirota may exhibit different adhesion and colonization properties following other treatments influencing the cell envelope structure.

Inactivation of probiotics by heat decreases the adhesion of many, but not all, of the tested strains, partially supporting the hypothesis that inactivation of probiotics by heat reduces their adhesive abilities. This may explain the often-observed reduced immune modulation of inactivated strains (Kato et al. 1994). This may be due to changes in the cell envelope of the inactivated strains rather than due to the fact that they are dead. Inactivation by γ-irradiation causes similar effects on adhesion as heat treatment and does therefore not seem to be a better alternative. To date, no studies have been published on the adhesive abilities of probiotic micro-organisms inactivated by u.v. irradiation. This method of inactivation did not significantly affect the adhesion of the tested strains compared with the corresponding viable organisms (Fig. 2). Suggesting that inactivation with u.v. is the most appropriate method to study the health effects of non-viable probiotic strains without the interference of secondary effects caused by the inactivation. u.v.-inactivated probiotics may also be more appropriate controls in the testing of viable probiotics when compared with the more commonly used heat-inactivated probiotics.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. METHODS and MATERIALS
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Financial support was obtained from the Academy of Finland.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. METHODS and MATERIALS
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References
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