TOLERANCE OF LACTOBACILLUS AND BIFIDOBACTERIUM STRAINS TO LOW PH, BILE SALTS AND DIGESTIVE ENZYMES

Anna Olejnik¹, Monika Lewandowska¹, Monika Obarska¹, Włodzimierz Grajek^2
1 Department of Biotechnology and Food Microbiology, the August Cieszkowski Agricultural University of Poznań
² Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, Poznań, Poland

ABSTRACT

Probiotic strains of L. casei, L. acidophilus, L. helveticus, B. animalis, B. lactis and two strains of B. bifidum were examined for their survival at pH 3.0 and 3% bile salts. Additionally, the viability of L. casei cells was tested in presence of pepsin, trypsin, chymotrypsin, alpha-amylase and lipase, and after heating at 90ºC for 30 min. It was found that all pre-treatments caused significant decrease in bacteria survival. The most sensitive strain for stress factors was B. bifidum 1. Using Caco-2 monolayer culture as the in vitro adhesion model, the significant reduction of adherence after thermal treatment, digestion with pepsin, trypsin and chymotrypsin, and bile salts were observed. The inhibition of bacteria adhesion after thermal denaturation and proteolysis proved an hypothesis that adhesion factors are of proteinaceous origin.

Key words: survival, adhesion, lactobacilli, bifidobacteria, pH, bile, enzymes, heat treatment.

INTRODUCTION

In recent years an increasing intensification of research on microbial population of gastro-intestinal tract is noted. There are many review on this subject revealed the active and beneficial role of intestinal microflora on human health in the literature [3, 4, 7, 11, 17, 18, 20, 22, 25, 26, 29, 30]. The common used definition of probiotic bacteria, introduced by Fuller [6], described that "probiotics are live microbial feed supplements, which beneficially affect the host animal by improving its intestinal microbial balance". This term was further expanded on to food and non-food use of microbial cultures. The criteria applied to select probiotic bacteria, including microbiological and technological aspects, are presented in Table 1.

Table 1. Criteria applied in selection of probiotic bacteria for human use [3, 11, 25]

Properties	Criteria
Safety	Human origin, from healthy persons Non pathogenic - Generally Regarded as Safe status No connection with diarrhoeagenic bacteria No ability to transfer antibiotic resistance genes Genetic stability
Functional	Acid and bile stability Resistance to digestive enzymes Survival in human intestine and ability to adhere to the intestine surface. No invasion Adhesion to intestine surface Antagonistic activity against human pathogens Production of anti-microbial metabolites, especially against gram-negative pathogens Anti-carcinogenic and anti-mutagenic activity Reduction immune response in case of allergy Immuno-stimulation without inflammatory effects Improving of bioavailability of food compounds and production of vitamins and enzymes
Technological	Good sensorial properties Fermentative activity Good survival during freeze drying and spray drying Good growth and viability in food products Phage resistance High stability during long-term storage

Most definitions emphasize that the microorganisms used as probiotic should be viable. However, there are several reports revealed that non-viable probiotics are also able to adhere to intestine surface and be active in reduction of duration of diarrhoea diseases [2, 12]. The investigation on the immunological effects of probiotics showed that production of specific IgA-secreting cells is much higher when living cells were administrated. Viability of the probiotic cells is crucial for the uptake of probiotic antigens through the Peyer´s patches and to bind to M-cells in the intestine [13, 19]. Halpern et al. [10] reported that the production of gamma-interferon by T-cells was higher when living cell with yoghurt were consumed.

To benefit human health, the probiotic must survive passage trough the upper gastro-intestinal (GI) tract and arrive alive at the site, which will be colonised by this organism. Attachment of probiotic strains to the epithelial cells and intestinal mucosal is pre-requisite for the intestine colonisation. It influences the time of bacteria retention in the intestine and the functional activity of bacteria. During passage through the upper alimentary tract the microorganism are subjected to several stress factors. In stomach the probiotic bacteria are stressed by very low pH value, in the range of 1.5-3.0. The minimum pH value for lactic acid bacteria is over 3.0 what is higher than that in stomach. According to Libudzisz and Kowal [16] the minimal pH for Lactobacillus lactis is 3.6-4.4, for Lactobacillus casei 3.3-3.6. Kim et al. [14] defined for L. lactis pH 4.5 as sublethal and 2.5 as lethal, and for L. lactis subsp. cremoris pH 5.0 as sublethal and 3.0 as lethal. Extreme acidity of the stomach can be considered as a key-factor in the selection of the biotope-specific lactic acid bacteria strains. The pH value strongly influences the bacteria viability. Lee and Wong [15] showed that pH value in fermented milk decreased L. casei viable cell number by 2 log-cycle for 13 days, whereas for >30 days when pH 6.5. The same parameters for L. acidophilus CH5 were 3 days at pH 4.0, and >30 days at pH 6.6. Also B. bifidum is very sensitive for low pH and the decrease of viable cell number by 2 log-cycle was observed after 4 days at pH 4.3, and >15 days at pH 6.6. Bacteria introduced into stomach reside there at pH below 3.0 for 1-4 h, depending on the diet, what is enough long time to kill majority of microorganisms.

The second important factor influenced on the bacteria in stomach is pepsin. This proteolytic enzyme can hydrolyse the proteins of the outer-layer of bacterial cells where are localized many aggregation factors and adhesins, e.g. collagen binding proteins and mucus binding proteins. This can affect cytoplasm membrane integrity and adhesive properties of bacteria. Negative effects on bacteria survival have also other proteolytic and lipolytic enzymes secreted in small intestine. An destructive factor are also conjugated bile salts. They play a role of emulsifier, which can destroy the structure of plasmatic membrane. As results, the action of all these factors can significantly reduce the viability and the adhesive properties of the lactic acid bacteria during passage through gastro-intestinal tract.

The aim of this study was to determine the effect of low pH and exposition to bile salts on viability of examined strains and effect of proteolytic enzymes and thermal treatment on the survival of Lactobacillus casei strain and its adhesion to epithelial cell surface.

MATERIALS AND METHODS

Bacterial strains and culture condition

Seven probiotic strains of Lactobacillus (L. acidophilus, L. helveticus, L. casei) and Bifidobacterium (B. bifidum 1 and 2, B. animalis, B. lactis) isolated from regional food products were used in investigation. Pure strains were stored at -80°C in cryogenic vials in the MRS medium (Merck) and glycerol (10 %v/v). For the experiments, the strains were cultured in MRS broth at 37°C for 18 h.

Intestine cell cultures

The established cell line Caco-2 (ATTC HTB 37) was maintained in Dulbecco´s modified Eagle medium (DMEM, Sigma), supplemented with 20% heat inactivated (56°C, 30 min) fetal bovine serum (FBS, Gibco BRL), 1% non-essential aminoacids 100X (NEAA, Sigma) and 50 mg l^-1 gentamycin (Gibco BRL). For adhesion experiments, aproximately 6 x 10⁴ cells per cm² were seeded on the 6-well plates (Nunc) and cultured as monolayers for 21 days at 37°C, in a 5% CO₂/95% air atmosphere. The culture medium was changed daily.

Effect of pH, bile salts and enzymes

The pH value of the MRS medium was adjusted to 3.0 with 1M HCl and inoculated with bacteria culture of 1x10⁶ CFU/ml. The bacterial strains were incubated at this pH value at 37°C for 60, 120, and 180 min. Afterwards the pH of medium was neutralized to 6.0 with 1 M NaOH and the cells were collected by centrifugation (5000 x g), vigorously vortexed and the CFU of the sample were determined by serially diluting in saline (0.85% NaCl) and plating onto the MRS-agar medium. Percentage survival of bacteria was determined by dividing the CFU of samples treated at low pH by CFU of culture incubated at pH 6.0.

Tolerance for bile acids were determined by addition of 2 ml of bile salts (Sigma), composed of sodium cholate and sodium deoxycholate (1:1), to 8 ml of MRS medium and inoculated with bacteria for 1x10⁶ CFU/ml. For the control samples, two ml of distilled water was introduced instead of bile salt mixture. The final concentration of bile salts was 3%. The bacteria were incubated in this medium at 37°C for 60, 120, and 180 min. The samples were collected at time zero and at 60 min, 120 min and 180 min, and CFU was determined to estimate the bacteria survival.

Tolerance to digestive enzymes was tested by introduction of bacterial cells at 1x10⁶ CFU/ml to 0.01% buffered solutions of enzymes: lipase (porcine pancreas), trypsin (porcine pancreas), alpha-chymotrypsin (bovine pancreas) and alpha-amylase (bovine pancreas) in phosphate buffered saline (PBS, pH 6.9), and pepsin (porcine stomach) in citrate-HCl buffer (pH 2.5). The enzyme solutions were sterilized by membrane microfiltration (0.22 μm). The bacteria cells were treated at 37°C for 1 h, 2h, and 3h. Survival of bacteria was examined by plating on the MRS agar medium at time zero, after 1, 2 and 3 h of incubation.

In vitro adhesion assay

Bacteria cultures in MRS medium were harvested by centrifugation at 5,600 g for 10 min, washed twice with 0.85% saline solution and diluted to standardized concentration of 1x10⁶ CFU/cm³ in DMEM without any antibiotic.

The 21 day-old Caco-2 monolayers, developed in 6-well microplates, were washed twice with PBS. One mililiter of inoculum with defined bacteria concentration was added to each well of the tissue culture plate and the plates were incubated anaerobic at 37°C for 60 min. Afterwards the DMEM was removed and the monolayers were washed three times with PBS to remove non-attached bacteria cells. Following the last wash, epithelial cells were gently detached by trypsinisation, harvested by centrifugation, and incubated with 1% Triton X-100 in PBS for 5 min for lysis of epithelial cells. The adherent bacteria were counted by plating the serial 10-fold dilution of the suspensions using agar plates. Bacteria strains were cultured anaerobically on MRS agar (Merck).

Statistical analysis

The results are expressed as mean +/-SD. Student´s t-test was used to examine the significance o the difference between two mean values. When comparisons of more than two mean values were made the data were analysed with one way ANOVA (STATISTICA 6.0. software). The level of significance was set at p < 0.05.

RESULTS AND DISCUSSION

Bacteria used in this study were of commercial importance. They were isolated from fermented dairy products sold on the Polish market. Bacteria cells were pre-treated by several stress factors occurring in the gastro-intestine tract. As a research criterion was chosen the viability of bacterial cells after pre-treatment. Additionally the influence of these treatments on the adhesion of L. casei was examined. All bacterial cultures were taken from exponential growth phase and directly subjected to each of the four stress factors: low pH, bile salts, enzymes and heating.

Viability of bacteria at low pH

Low pH value significantly decreased the bacterial viability. The time of incubation at low pH value was chosen according to the residence time of nutrient in the stomach. Depending on the protein, carbohydrates and lipid composition of meal, it lasts from 1 h to 3 h. Natural concentration of HCl in stomach reaches 170 mM and the minimum pH is about 1.0. After ingestion of standard meal the pH value in stomach can increase to 7.0, which then declines to acidic values [24]. The in vivo acidity of gastric fluids decreased gradually, depending of the buffer capacity of the meals. Ekmekcioglu [5] defined the time to decline one pH value to be about 10-20 min. Full gastric emptying lasts up to 4 h, especially when meals are rich in fibre and fat.

Table 2. Percentage survival of bifidobacteria at pH 3.0 for different time of incubation

Incubation time (h)	Viability (+/-SD) [%]
	B. animalis	B. bifidum 1	B. bifidum 2	B. lactis
1	69.6 (±8.2)	59.2 (±6.2)	78.2 (±6.4)	70.3 (±16.8)
2	67.5 (±5.0)	57.6 (±7.0)	74.2 (±5.4)	69.9 (±4.0)
3	65.8 (±4.0)	52.0 (±9.3)	68.3 (±6.4)	67.8 (±2.1)
	Level of significance (p)
	0.760	0.925	0.250	0.972

Table 3. Percentage survival of lactobacilli at pH 3.0 for different time of incubation

Incubation time (h)	Viability (+/-SD) [%]
	L. acidophilus	L. helveticus	L. casei
1	68.9 (±6.1)	69.1 (±7.9)	88.3 (±5.9)
2	67.7 (±2.2)	65.2 (±4.7)	87.2 (±4.8)
3	64.0 (±5.1)	61.9 (±5.1)	85.0 (±3.6)
	Level of significance (p)
	0.470	0.392	0.719

The results of our investigation, presented in Table 2 and 3, show that viability of bifidobacteria and lactobacilli decrease significantly after incubation at pH 3.0. The highest reduction of viability appeared after the first hour of incubation, independently on the strain examined. Among bifidobacteria, the strain No 1 showed the highest sensitivity to low pH among bifidobacteria examined and its viability after 3 h of incubation decreased to 52.0% of initial population. The data presented in Table 2 showed that further prolongation of incubation from 1 h to 3 h resulted only in further small reduction of viability. Statistical analysis of results obtained in this experiment not revealed significant differences between bifidobacteria viabilities.

The experiment carried out with lactobacilli also revealed the highest reduction of bacteria viability during the first hour of incubation. The loss of L. acidophilus and L. helveticus viability was very similar and ranging in 36-38%, whereas the viability of L. casei was much higher. However, it should be stressed that statistical analysis did not proved the significance of differences observed between the survival of lactobacilli strains. The general comparison of all strains examined for significant differences in the cell viabilities after 1 and 3 hours of treatment indicated the similar pH tolerance (Table 4 and 5).

Table 4. Statistical differences between viabilities of bifidobacteria and lactobacilli incubated at pH 3.0 for 60 min

	B. animalis	B. bifidum 1	B. bifidum 2	B. lactis	L. acidophilus	L. helveticus	L. casei
B. animalis	-	0.923	0.884	1.000	1.000	1.000	1.000
B. bifidum 1	0.923	-	0.317	0.892	0.946	0.943	0.946
B. bifidum 2	0.884	0.317	-	0.917	0.850	0.854	0.850
B. lactis	1.000	0.892	0.917	-	0.999	0.999	0.999
L. acidophilus	1.000	0.946	0.850	0.999	-	1.000	1.000
L. helveticus	1.000	0.943	0.854	0.999	1.000	-	1.000
L. casei	1.000	0.946	0.850	0.999	1.000	1.000	-

Table 5. Statistical differences between viability of bifidobacteria and lactobacilli incubated at pH 3.0 for 180 min

	B. animalis	B. bifidum 1	B. bifidum 2	B. lactis	L. acidophilus	L. helveticus	L. casei
B. animalis	-	0.837	1.000	1.000	0.994	0.964	0.404
B. bifidum 1	0.837	-	0.889	0.889	0.992	0.999	0.051
B. bifidum 2	1.000	0.889	-	1.000	0.998	0.982	0.343
B. lactis	1.000	0.889	1.000	-	0.998	0.982	0.343
L. acidophilus	0.994	0.992	0.998	0.998	-	0.999	0.160
L. helveticus	0.964	0.999	0.982	0.982	0.999	-	0.101
L. casei	0.404	0.051	0.343	0.343	0.160	0.101	-

Prasad et al. [21] screened large collection of 2000 strains of lactobacilli and bifidobacteria from the New Zealand Dairy Research Institute to select strains with functional characteristics, including ability to survive at pH 3.0 for 3 h and bile concentration up to 1.0%. They select 200 strains in an initial screening, which show 80% survival, and from them 4 strains, which were compared to L. rhamnosus GG and L. acidophilus LA-1. It was demonstrated that strains selected exhibited better survival at low pH than these two known probiotic strains. However, with prolongation of incubation time from zero to 3 h survival of examined strains decreased in the manner comparable to our study. Inhibiting effect of low pH value on the L. rhamnosus and L. plantarum viability reported also Goderska et al. [8]. Haller et al. [9] also tested bacterial survival under conditions of gastrointestinal tract. The authors exposed Lactobacillus johnsonii La1, a known probiotic strains, and food fermenting strains of L. sakei, L. plantarum, L. curvatum and L. paracasei to pH 1.0-2.5 and cholic or bile salts in two-step procedure to mimic intestinal transit. They reported better tolerance of probiotic strain to gastro-intestinal fluids than food origin lactobacilli. The critical limit of L. johnsonii La1 and L. plantarum was pH 1.5. At pH 1.5 none of experimental strains survived subsequent exposure to cholic acid or human bile. The authors suggested that bacterial growth phase rather than cell densities of bacteria affect tolerance upon treatment with HCl and bile. Interesting data are described in paper of Kim et al. [14]. The authors examined survival of Lactococcus lactis cultivated over the pH range from 7.0 to 1.5. They found that during 3 h of incubation total number of bacteria was increased when pH over 4.0. Below pH 3.5 the number of bacteria in the medium decreased because of the loss of viability. At pH < 2.0 none viable bacterial cells were detected.

Viability of bacteria in presence of bile salts

The presence of bile salts in the environment of bacteria cultures is much more detrimental than effect of pH 3.0. The bile salts secreted into human small intestine consist of sodium cholate, sodium deoxycholate, and sodium chenodeoxycholate. These substances play a role of lipid emulsifier and they can destabilise membrane integrity in bacterial cells. The strains of probiotic bacteria should survive the presence of bile salts and be able to colonise the intestine surface. The concentration of bile salts used in this experiment was very high as strong selective factor. The in vivo bile concentration in the intestine is much lower.

Table 6. Percentage survival of bifidobacteria at presence of 3% bile salts for different time of incubation

Incubation time (h)	Viability(+/-SD) [%]
	B. animalis	B. bifidum 1	B. bifidum 2	B. lactis
1	65.4 (±4.2)	47.7 (±5.2)	75.1 (±7.6)	73.3 (±9.0)
2	64.5 (±5.5)	29.5 (±8.7)	66.2 (±8.6)	64.6(±6.5)
3	62.5 (±4.6)	13.2 (±2.7)	60.8 (±8.6)	60.2 (±9.0)
	Level of significance (p)
	0.767	0.002	0.096	0.198

Table 7. Percentage survival of lactobacilli at presence of 3% bile salts for different time of incubation

Incubation time (h)	Viability (+/-SD)
	L. acidophilus	L. helveticus	L. casei
1	63.1 (±4.7)	63.6 (±6.0)	88.2 (±4.6)
2	55.8 (±12.2)	63.9 (±3.7)	84.7 (±5.0)
3	53.8 (±12.0)	62.3 (±4.5)	77.4 (±5.8)
	Level of significance (p)
	0.54	0.913	0.101

Data presented in Table 6 indicate that the most sensitive strain was B. bifidum 1, which decrease its viability after 3 h of incubation with 3% bile up to 13.2%. As statistical analysis showed, this strain differs significantly from other strains tested (Table 8 and 9). The other bifidobacteria survived bile treatment with about 60-63%. Similar examination was performed with three Lactobacillus strains (Table 7). The most resistant strain for 3% bile concentration was L. casei. Statistical analysis showed significant differences between single strains (Table 8 and 9). The prolongation of incubation time from 1 to 3 h demonstrated that significant differences between all tested strains showed B. bifidum 1. Significant differences were also observed between L. casei and L. acidophilus viability (Table 8 and 9). It should be noted that all tested bacteria presented high resistance for such high concentration of bile salts what is rather characteristic for intestinal microflora.

Table 8. Statistical differences between viability of bifidobacteria and lactobacilli incubated at 3% bile salts for 60 min

	B. animalis	B. bifidum 1	B. bifidum 2	B. lactis	L. acidophilus	L. helveticus	L. casei
B. animalis	-	0.044*	0.506	0.703	0.999	0.999	0.007*
B. bifidum 1	0.044*	-	0.001*	0.003*	0.096	0.082	0.000*
B. bifidum 2	0.506	0.001*	-	0.999	0.285	0.325	0.202
B. lactis	0.703	0.003*	0.999	-	0.447	0.499	0.117
L. acidophilus	0.999	0.096	0.285	0.447	-	1.000	0.003*
L. helveticus	0.999	0.082	0.325	0.499	1.000	-	0.004*
L. casei	0.007*	0.000*	0.202	0.117	0.003*	0.004*	-

* significant differences p<0.05

Table 9. Statistical differences between viability of bifidobacteria and lactobacilli incubated at 3% bile salts for 180 min

	B. animalis	B. bifidum 1	B. bifidum 2	B. lactis	L. acidophilus	L. helveticus	L. casei
B. animalis	-	0.0002*	0.999	0.999	0.697	1.000	0.174
B. bifidum 1	0.0002	-	0.0002*	0.002*	0.0002*	0.0002*	0.0002*
B. bifidum 2	0.999	0.0002*	-	1.00	0.859	0.999	0.103
B. lactis	0.999	0.002*	1.00	-	0.901	0.999	0.085
L. acidophilus	0.697	0.0002*	0.859	0.901	-	0.720	0.011*
L. helveticus	1.000	0.0002*	0.999	0.999	0.720	-	0.163
L. casei	0.174	0.0002*	0.103	0.085	0.011*	0.163	-

* significant differences p<0.05

Many authors investigated the effect of bile on survival of lactic acid bacteria. Kim et al. [14] examined the effect of bile concentration in the range of 0-0.4% on the L. lactis survival and they reported inhibiting effect of bile at concentration over 0.04%. They detected that all bacterial cells were killed at 0.2% and higher. For L. lactis, bile salts at 0.1% was chosen as the lethal level. Comparing to this study, bacteria used in our experiments showed much more resistance to detrimental action of bile salts. Prasad et al. [21] realized a project on selection of probiotic strains. They screened 2000 lactobacilli and bifidobacteria strains for their survival at presence of 1% bile. Among this population they selected about 10% of strains, which showed 80% survival after acidic and bile treatment. Comparing both the treatments they found that the decrease of survival caused by bile salts was higher than that caused by low pH. An inhibiting effect of bile on survival of Lactobacillus brevis reported also Rönkä et al. [23]. These facts confirmed hypothesis on destructing action of bile salts on phospholipids in cell membranes what results in lost of cell integrity.

Viability of bacteria after enzymatic treatment

Human gastro-intestinal tract can be considered as enzymatic system. The main role in nutrient digestion plays pepsin secreted to the stomach fluid and pancreatic complex consists of proteolytic enzymes (trypsin, chymotrypsin A and B, elastase, carboxypeptidase A and B), lipolytic enzymes (lipase, phospholipase and esterases), and glycolytic (alpha-amylase).

Table 10. Percentage survival of L. casei cells after enzymatic treatment

Enzyme	Viability(±SD) [%]
Trypsin	51.8 (±10.5)
Pepsin	76.3 (±8.5)
Lipase	20.5 (±4.5)
Chymotrypsin	69.2 (±5.7)
Amylase	47.3 (±6.9)

Pepsin hydrolyses protein in acidic condition (optimum pH 1.0-2.5) and produces peptides and amino acids. Trypsin, an endo-enzyme, acts in alkaline condition (pH 8-9) and hydrolyses the internal peptide bounds, especially between alkaline amino acids, as lysine and arginine. The reaction products of its activity are low molecular weight peptides. The pancreatic lipase (optimum pH 7-8.5) hydrolyses triacylglyceroles to fatty acids, monoglycerols and glycerol. It acts at the interfacial water/lipids and requires a presence of bile salts emulsifying lipids and solubilising lipolytic products in micelles. Pancreatic alpha-amylase (optimum pH 6.5-7.2) hydrolyses alpha-1,4-glycosidic bounds to maltose, maltotriose and alpha-dextrins.

Taking into consideration morphological structure of bacterial cells it should be emphasized that outer layers of cells are composed of substances, which are susceptible to enzymatic hydrolysis at the presence of enzyme mentioned above. Many of these substances play important role in cell adhesion, cell integrity and cell signalling. The destroying of this subtle structure cause alteration in cell function and reduce the viability.

In our study we have chosen Lactobacillus casei for experiments with enzymatic treatment because this strain demonstrates immune stimulation and is used actually in commercial application. The results presented in Table 10 showed that enzymatic treatment caused significant reduction of L. casei viability. The highest loss of bacteria viability, reached about 80% of initial population, was achieved in incubation with pancreatic lipase. This enzyme hydrolyses phospholipides of cytoplasmatic membranes surrounding cytosol what case loss of cell integrity and cell death. The data obtained in these experiments showed also considerable effects of proteolytic enzyme action. Trypsin caused greater viability loss than pepsin what can be resulted also from specific pH condition of treatment: for pepsin very acidic and for trypsin alkaline. Unexpectedly, high viability loss was noted in the incubation with alpha-amylase where the number of viable cells amounted only 47.3% of initial population. This enzyme can hydrolyse the glycosidic bounds between N-acetylmuramine acid and N-acetylglucoseamine in cell wall.

Table 11. Significance level of L. casei viability as result of enzymatic treatment

Enzyme	Trypsin	Pepsin	Lipase	Chymotrypsin	Amylase
Trypsin	-	0.017*	0.003*	0.101	0.943
Pepsin	0.017*	-	0.0002*	0.779	0.006*
Lipase	0.003*	0.0002*	-	0.0002*	0.010*
Chymotrypsin	0.101	0.779	0.0002*	-	0.032*
Amylase	0.943	0.006*	0.010*	0.032*	-

* significant differences p<0.05

Comparing the effects of individual enzymes on cell viability it can be assumed that lipase significantly differ from action of other enzymes (Table 11). No significant differences were detected between action of pepsin and chymotrypsin. However, statistically significant differences were observed between alpha-amylase effect and pepsin, chymotrypsin and lipase.

Adhesion of L. casei to Caco-2 cells after different pre-treatment

L. casei strain showed low adherence to Caco-2 cells. It amounted only 4.79% and was comparable to the results reported by other authors [27].

Table 12. Bacteria adhesion to Caco-2 cells after different pre-treatment

Treatment	Loss of viability after treatment (%)	Adhesion of bacteria non-treated (%)	Adhesion of bacteria after pre-treatment (%)	Percentage influence of pre-treatment on bacteria adhesion (%)
pH 3.0	15.3	4.79	4.04	15.65
Bile salts 3%	23.5	4.79	0.42	91.3
Trypsin	50.3	4.79	1.42	70.36
Pepsin	24.2	4.79	0.83	82.67

As shown in Table 12, all stress factors decreased bacteria adherence, however, not in the same range. The lowest effect caused incubation at low pH, which reduced bacteria adhesion only by 15.3% in comparison to non-treated bacteria. The high adherence reduction caused exposition of bacterial cells to bile salts what exceeded 91% of initial adherence. It should be also noted that bacterial adherence was significantly reduced by proteolytic enzymes, especially pepsin. It can be explained partially by hydrolysis of adhesins on the bacterial cell surface and partially by extreme pH optima of enzymatic reactions. The involvement of bacterial surface proteins in adhesion is supported by results obtained in experiments with bacteria heated at 90°C (data not shown). The denaturation of bacterial proteinaceous adhesion factors made impossible natural adherence of bacteria to Caco-2 cells.

Bibiloni et al. [1] investigated adhesion of two strains of Bifidobacterium bifidum to Caco-2 cells after treatment with chymotrypsin and heating at 100°C for 30 min. In quantitative analysis they observed about 50% decrease of bacteria adhesion after enzymatic treatment and entire loss of adherence after thermal inactivation. The authors suggested that adhesion determinants could be glycoproteins or carbohydrate chains attached to the cell wall. This thesis is confirmed by our observations. Similar hypothesis defined Tuomola et al. [27] who examined effects of chemical, physical and enzymatic pre-treatments of probiotic lactobacilli on adhesion to the human intestinal mucus glycoproteins. They found that adhesion of L. acidophilus LC1 and L. rhamnosus GG was reduced by boiling, autoclaving and by pepsin and trypsin treatment suggesting that bacterial proteins are essential for their adhesion. According to the authors, adhesion of L. casei Shirota strain was not affected by any of the several treatments but this declaration is in contrast to experimental results reported. The adhesion of L. casei Shirota to Caco-2 cells was very low and alteration caused by treatment was very slight but significant.

Generally, this study demonstrated the negative effects of stress factors connected with the passage of lactic acid bacteria through gastro-intestinal tract. This dependence should be taken into consideration in selection of probiotic strains.

CONCLUSIONS

1. Incubation of lactobacilli and bifidobacteria at pH 3.0 resulted in large reduction of their viability. The differences in the survival between strains were insignificant.

2. Exposition of bacterial cells to 3% bile salts reduced their survival. The highest sensitivity to bile action exhibited B. bifidum 1. This strain viability was differed significantly from all others.

3. The highest reduction of bacterial survival occurred at first hour of the exposition to the stress factors.

4. All stress factors including low pH, bile salts and digestive enzymes reduced survival and adhesion of Lactobacillus casei to Caco-2 cells.

5. The decrease of bacterial adherence caused by thermal and enzymatic treatment suggests proteinaceous character of adhesion factors.

ACKNOWLEDGEMENT

This study was founded by the Ministry of Science and Informatization, Poland (project PBZ-KBN/020/P06/1999/01).

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Anna Olejnik
Department of Biotechnology and Food Microbiology,
the August Cieszkowski Agricultural University of Poznań
Wojska Polskiego 48, PL-60-627 Poznań, Poland
tel. +48 61 8466027
email: olejnik@au.poznan.pl

Monika Lewandowska
Department of Biotechnology and Food Microbiology,
the August Cieszkowski Agricultural University of Poznań
Wojska Polskiego 48, PL-60-627 Poznań, Poland

Monika Obarska
Department of Biotechnology and Food Microbiology,
the August Cieszkowski Agricultural University of Poznań
Wojska Polskiego 48, PL-60-627 Poznań, Poland

Włodzimierz Grajek
Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, Poznań, Poland
ul. Wojska Polskiego 48
60-627 Poznań
Poland

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