Antagonism against Helicobacter Pylori and Proteolysis o f Lactobacillus Helveticus CU 631 and Strain Identification

The antagonistic activities of 30 strains of lactobacilli against Helicobacter pylori were determined and Lactobacillus helveticus CU631 has been selected as the strain which possesses the strongest inhibitory effect in the disc diffusion assay showing inhibition zone diameter of 10±1.5 mm, whereas those of L. plantarum and L. fermentum have been shown to be 4.0±0.6 mm. H. pylori G88016 revealed the highest vacuolating toxin producing activity among the 8 strains, the inhibitory activity of L. helveticus CU631 in vacuolating toxin producing activity of H. pylori manifested in the co-culture of two strains and in the 5:5 mixture of supernatant of the two strains. Both L. helveticus CU631 and cell free culture supernatant had a strong inhibitory activities in urease and cytotoxin producing activities of H. pylori NCTC11637 and CJH12. An accelerated proteolytic activity of water soluble peptides by L. helveticus CU631 during the refrigeration storage has been manifested in the cream cheese. DNA seqences of 16S-23S ribosomal RNA spacer region showed typical pattern among the various strains of L. helveticus, which could be used in the identification of L. helveticus CU 631. (Asian-Aust. J. Anim. Sci. 2002. Vol 15, No. 7 : 1057-1065)


INTRODUCTION
Lactobacilli have been used since decades against infectious diseases, these bacteria are supposed to compete with other microorganism on mucosal surfaces, a process named bacterial interference.In order to colonize mucosal epitherial surface, lactobacilli need to have some abilities to adhere to epitherial cells, to exclude competitive strains and to produce inhibitory substances.Antagonistic or inhibitory effect of lactic acid bacteria termed as probiotic effects, the term probiotic first used to describe organisms and substances added to animal feed to promote growth (Parker, 1974).This definition has since been broadened to include health promoting actions of microorganisms, possible mechanism of action include acid production (Wendakoon et al., 1998;Fuller, 1991) and other by-products of bacterial metabolism (Streekumar and Hosono, 2001).It has been proposed that lactic acid production by these organisms, unrelated to pH, is responsible for inhibition of Helicobacter pylori, the curved Gram negative bacterium that can cause peptic ulcer disease in man (Cover and Blaser, 1992).Considering high rate of infection of approximately 80% with this pathogenic organism in the oriental countries, it is a major public health concern in many part of world (Gill and Desai, 1993;Graham et al., 1991;Midolo et al., 1995).Treatment of H. pylori infection with antibiotics does not always eradicate the organism, and antibiotic therapy frequently produces adverse effect (NIH, 1994).H, pylori is becoming resistant to a number of antibiotics, particularly to metronidazole and clarythromycin which are currently being used to treat patients with gastric ulcers (Heatley, 1995), alternative forms of effective and simple therpeutical regimens are needed.
It is needed to screen lactobacilli probiotic strains which is antagonistic to H. pylori to characterize and identify the strains, the results of in vitro inhibitory activity of vacuolating cytotoxin and urease activity by lactobacilli could provide some clues that probiotic organisms may have a role in H. pylori treatment both through direct action against the organism and in the lessening of clinical side effects associated with antibiotics.Those informations on proteolysis of water soluble peptides could be applied in utilyzing them as a starter organism in the preparation of fermented dairy products.
Several lactobacilli strains have been identified by DNA sequencing of 16S-23S ribosomal RNA spacer region, this study was conducted to screen strains which have a strong inhibitory effects on H. pyroli and to characterize and identify probiotic effects of the strain.

Organisms and propagation
H. pylori and Lactobacillus spp.strains used in this study have been shown in table 1. Lactobacillus spp.used in this study were maintained at -80°C in skim milk/glycerol medium, an aliquot of each lactic strain from the stock at -80°C was grown at 37°C in DeMan, Rogosa, Sharpe (MRS) (Difco, Detroit, Mich.) in aerobic condition.Washed cells were prepared from the MRS broth cultures by centrifuging 10,000×g for 2 min and washing the pellet with phosphate buffered saline (PBS pH 7.2) 3 times.The stock cultures of H. pylori NCTC 11637, CH7-1, CH333-1, CH578, CJH 12, CJH57, GB8016, GB8037-1 were kept at -80°C in peptone glycerol medium.H. pylori were cultured at 37°C in Brucella media (Difco) added with 10% horse serum in the jar with CampyloPak Plus (BBL, USA).

Determination of inhibitory activity of Lactobacillus spp. by agar diffusion
Agar diffusion method (Cooper, 1964) were used to determine the antagonistic activity of lactobacilli aginst H. pylori, washed cells of lactobacilli were diluted with PBS and plated on MRS plates and individual colonies of lactobacilli were appeared on MRS agar plate by incubation at 37°C for 48 h.
The plates with the colonies were allowed to dry, 2 ml of Brucella media added with 10% horse serum containing 0.75% soft agar with Helicobacter pylori NCTC11637 was spreaded over the plate and incubated 10% CO 2 incubator at 37°C for 24 h and the diameter of inhibition zone was measured in mm.

Inhibition of urease activity of H. pylori
H. pylori was broth cultured for 48 h at 37°C and pelletized at 16,000×g for 5 min.3 ml of Christensen urea broth containing 0.012 g/L of phenol red was added with 30 µl H. pylori in destilled water as a urease control, another cuvette contains 30 µl H. pylori and 30 µl of L. helveticus CU 631 cell suspension in 3ml of Christensen urea broth containing 0.012 g/L of phenol red, optical density of the media determined at 550 nm wavelenth every 10 min for 3 h by spectrophotometer based on the method described by Coconnier and Lievin (1998).L. helveticus CU 631 was cultured in modified MRS broth and pelletized at 16,000×g for 5 min.

Cell culture and Inhibition of vacuolating cytotoxin of H. pylori by L. helveticus CU 631
RK-13 cells were cultured in RPMI1640 (GIBCO BRL,USA) supplemented with 10% fetal bovine serum in 10% CO2/ 90% air atmosphere at 37°C.H. pylori were inoculated in Brucella broth added with 10% horse serum for 7 days at 37°C in microaerobic atmosphere.Vacuolating cytotoxin (VT) activity was determined by Kamiya et al. (1994), 100 µl of RK-13 cell suspension of 3.42×104/ml was put in the each wells of 96 well plate cultured 24 h at under 10% CO 2 atmosphere.On the next day 100 µl of H. pylori culture supernatant diluted to 2X, 4X, 8X, 16X, 32X, 64X, 128X were added to each well and incubated 24 h at 37°C, VT titer was determined by the observation of vacuole in the RK cell by invert microscope, and VT titer stands the largest dilution factor which shows vacuole.Inhibitory activity by L. helveticus CU 631 was determined by the addition of mixture of H. pylori culture supernatant and L. helveticus CU 631 supernatant, the ratio of the mixture was 5:5, 6:4, 7:3, 8:2, 9:1.

Proteolytic activity of L. helveticus CU 631 by HPLC
Ten grams of cheese slurry were mixed in 20 ml of deionized destilled water in a mixer for 30s.resulting mixtures were tempered in a bath 40°C for 20 min., the extracts were centrifuged at 10,200×g for 35 min (SR20.22,Jouan INC.France) at 9°C.The solidified fat layer was removed and aqueous layer was filtered through glass wool into a clean container.The remaining cheese pellet was reextracted with 20 ml of deionized destilled water and the aquous portin was combined with the previous extract.Peptide profiles of slurries were obtaineed using the following procedure.Ten milligram of freeze dried water soluble extract from each slurry was reconstututed in 1 ml of solvent A and filtered through a 0.45 µm filter.A 50 µl loop was used to introduce the sample onto the HPLC (Model 305;Gilson Co., France).Gradient elution was used in a C 18 analytical column (Lichrospher 100 RP-18, 5 µm (Merck, USA) for which solvent A was 0.1% trifluoroacetic acid in 99.9% HPLC grade water and solvent B was 0.1% trifluoroacetic acid 90% acetonitrile, 9.9% HPLC water.
Peptides were detected using a UV detedctor (UV 119; Gilson, France) at 220 nm, the peptide profile data was processed and stored on a computer.

Identification of the L. helveticus CU 631 strain by sequencing of 16S-23S spacer ribosomal RNA and alignment comparison
Overnight culture of L. helveticus CU 631 was pelleted and washed twice with 50 mM EDTA, chromosomal DNA was isolated by using the Wizard genomic DNA purification kit (Promega USA), described by Alatossava and Timiskjarvi (1997).
First used to PCR was performed in a DNA thermal cycler 480 (Perkin Elmer, Norwalk) with a AccuPower PCR premix (Bioneer, Korea).Oligonucleotide primer used to amplifying the 16S-23S ribosomal RNA gene spacer region were Hel I 5'-GAAGTGATGGAGAGTAGAGTAG AGATA-3' Hel II 5'-CTCTTCTCGGTCGCCTTG-3' a specific primer for L. helveticus used (Alatossava and Timiskjarvi, 1997).A reaction mixture (50 µl) for PCR of the 16S-23S ribosomal RNA gene spacer region consisted of reaction buffer (end concentration 1.5 mM MgCl 2 ), 200 µM each dNTP, 1 µM of Hel I and Hel II primer, 50 ng of bacterial DNA and 0.6 U of Bioneer DNA polymerase.The amplification profile was at 92°C for 30s, 62°C for 30s, 72°C for 30s, This is repeated for 30 cycles.The program also included a preincubation at 92°C for 2 min before the first cycle and an incubation at 72°C followed by a cooling step down to 4°C after last cycle.Amplification products were analyzed with an agarose gel electrophoresis.Amplified DNA products were then purified free from primers and nucleotide with a AccuPrep PCR purification kit (Bioneer, Korea).
PCR products from the spacer regions were sequenced directly by cycle sequencing method using Automatic seqencer (New England Biolab.Beverly) and radioactive labeling.
Sequences were read and analyzed with Generunner software and compared with those of Genebank.

Statistical analysis
Within the same treatment groupe, for the comparison of the inhibitory halo diameter by lactobacilli strains and comparison of peak number and peak area between the treatments values were compared using SAS Duncan's multiple-range test.

Antagonistic activity of Lactobacilli against H. pylori
A total of 30 dairy cultures obtained from either cultured dairy products or various culture collections were screened against 8 strains of H. pylori.In the screenig the organism, which showed a clear halo around the lactobacillus colony (figure 1) were taken as positive cultures.The extent of the inhibitory effect was found to be strain dependent, table 1 shows the average radius of inhibition zone of H. pylori by L. helveticus CU631, a strain L. acidophilus CU 620 did not make any inhibiotion zone of H. pylori, all the other lactobacilli revealed positive results; made a halo ranged from 3.0 mm to 10.0 mm.L. helveticus CU631, L. helveticus CU632, L. paracasei CU 480 were found to have higher inhibitory action on H. pylori NCTC1637, the largest halo diameter was appeared by L. helveticus CU631 averaging 10.04±1.51.However one strain of L. acidophilus CU 620 did not revealed inhibitory action on H. pylori NCTC 1637, L. delbrueckiiCU 693, L. fermentum ATCC 14931 and L. plantarum CU722 showed low degree of inhibitory activity of under 4.0 mm.Of the ten strains of L. acidophilus tested, nine were found to be inhibitory to H. pylori NCTC 1637.Fermented milks are claimed to contain a number of biologically active compounds which may contrubute to the inhibition of bacterial growth.These include diacetyl, organic acids, peptides, bacteriocins and some low molecular weight compounds (Bernet-Camard et al., 1997).The effect of probiotic organism such as L. acidophilus on H. pylori in the gastric mucosa remains to be elucidated.The acid resistance of Lactobacillus helveticus CU 631 demonstrated by the survival in skim milk broth below pH 3.5.and production of organic acid could be the the base of antagonism.

Inhibitin of urease and vacuolating cytotoxin production activity of L. helveticus CU 631
The effects of L. helveticus CU631 culture and cell free culture supernatant on the urease activity of H. pylori NCTC11637 were measured as shown in figure 2A.
Urease activity is proportional to the red color intensity (550 nm), urea of the Christensen urea broth is turned into ammonia by the urease on culture.
A prominent urease activity was shown in H. pylori NCTC11637 and CJH12, the activity was strain dependent, hence the two strains were used to the urease inhibition test by L. helveticus CU631.The urease activity of H. pylori NCTC11637 was inhibited prominently by both L. helveticus CU631 culture and culture supernatant of L. helveticus CU631 (figure 2B).The inhibitory effect of helveticus CU631 on H. pylori NCTC11637 urease activity was heat stable, Coconier and Lievin (1998) claimed that lactic acid does not participate in the action of L. acidophilus against H. pylori urease.The urease of H. pylori is a surface protein component of H. pylori producing ammonia which allow survival by neutralizing the acidic environment of stomach.(Arthurton et al., 1995).
Vacuolating cytotoxin production is encoded by 259 bp VacA gene and the presence of the gene in all the test strains was evidenced by the PCR amplification results (Atherton et al., 1995).VT titer was determined by the observation of vacuole in the RK cell by invert microscope, figure 3A   cell formed due to the presence of approxomately 500,000 dalton protein (Leunk et al., 1988).RK cell line is very sensitive to vacuole formation which forms vacuole under the induction of both cytotoxin m1 and m2, as the cytotoxin known as vacuolating toxin is produced in vivo by the presence of antibody to vaculoating toxin in the sera of patients infected with H. pylori, this toxin has been proposed as a new virulence factor of H. pylori.(Pagliaccia et al., 1998).

Proteolytic activities of L. helveticus CU 631
Water soluble peptide pattern of cream cheese and L. helveticus CU631 culture added probiotic cream cheese has been determined by HPLC and the results were shown in figure 4 and in table 3, we evaluated total peaks and total peak areas of the chromatograms to determine whether L. helveticus CU 631 had an impact on these parameter.The HPLC analysis of water-soluble extracts showed some differences between with and without L. helveticus CU 631 products.In all of the chromatograms, there are noticeable differences in the heights of peaks and differences in the number of small peaks collected between 20 to 50 min.Comparions of the water soluble peptide pattern between cream cheese with L. helveticus CU 631 resulted the number of peptide peaks and peak area from the cream Table 3.Total number of peaks and total peak areas of peptides determined by HPLC for probiotic cream cheeses from 1 to 4 weeks of ripening at 4°C.

Number
Ripening time (week) Total peaks 2 Total peak areas 1, 2 1 80 a 2.0 a C-1 4 86 a 2.9 a 1 85 a 2.9 a C-3 4 100 b 4.5 b 1 Arbitrary units (×10).cheese without L. helveticus CU 631 were 80 and 2.0 arbitrary unit whereas those with L. helveticus CU 631 were 85 and 2.9 respectively, which indicates peptidases originated from L. helveticus CU 631 proceeded intensive proteolysis.During the ripening of 4 weeks at 4°C the viable counts increased to 3.3×107 cfu/g of L. helveticus CU 631, the number of peptide peaks and peak area from the probiotic cream cheese were 100 and 4.5.arbitrary unit respectively (p<0.05) whereas those without L. helveticus CU 631 were 86 and 2.9 respectively.Statistically significant differences were observed in total peak areas and total number of peaks in terms of ripening.Table 3 shows that as the sllurries ripened, and as the L. helveticus CU 631 added to the slurries, the total peak area increased, and the total number of peaks increased.indicating a greater quantity of peptides or compounds being absorbed at 220 nm.The height differnces indicate variation in the quantity of specific peptides and represent the proteolytic ability to release greater amounts of specific peptides from protein (Muehlenkamp-Ulate; 1999).Each peak may represent more than one peptide because of coelution, and other methods could be needed to separate these compounds, the identities of the peaks were not determined in this study.The results of this studies are in agreement with Muehlenkamp-ulate (1999) andFenelon (2000).

C G G T T C A A C G A C C A T A C T A T A A T G G T T G C C A A C C C G T N T G
161 2 3 4 5 6 7 8 9 0 171 2 3 4 5 6 7 8 9 0 181 2 3 4 5 6 7 8 9 0 191 2 3 4 5 6 7 8 9 0

Table 1 .
Inhibition of H. pylori NCTC11637 by Lactobacillus spp.and source of strains (diameter:mm)

Table 2 .
Effect of L.helveticus CU631 and culture supernatatnt on the Vacuolating Toxin (VT)titer of H.