J. Fd Hyg. Safety 16(4), 264 273 (2001) Lactobacillus reuteri t w w w³ z «ûz 1 Á½ x 1 Á»Á½ Á Á Á½ Á *Áx Á y œ» w œw w w Antimicrobial Activity of Lactobacillus reuteri Against Major Food-Borne Pathogens Nam Hoon Kwon 1, So Hyun Kim 1, W. K. Bae, J. Y. Kim, J. Y. Lim, K. M. Noh, J. M. Kim, J. S. Ahn*, J. Hur, and Y. H. Park &RVBMMZDPOUSJCVUFE %FQBSUNFOUPG.JDSPCJPMPHZ$PMMFHFPG7FUFSJOBSZ.FEJDJOFBOE4DIPPMPG "HSJDVMUVSBM#JPUFDIOPMPHZ4FPVM/BUJPOBM6OJWFSTJUZ /BUJPOBM7FUFSJOBSZ3FTFBSDIBOE2VBSBOUJOF4FSWJDF ABSTRACT Antimicrobial activities of five different probiotics (Lactobacillus reuteri, L. acidophillus, L. bulgaricus, L. casei and Bifidobacterium longum) against 8 bacterial pathogens were determined on the Mueller Hinton Agar containing supernatant of probiotics obtained from 3 different growth conditions (MRS without glycerol, MRS with 0.5 M glycerol or 0.25 M glycerol solution). Though antimicrobial activity of L. reuteri in the first two conditions was not better than the others', the activity was significantly higher than that of others in 0.25 M glycerol solution. This prominent effect might be attributable to reuterin, produced by L. reuteri using glycerol. We could detect the presence of reuterin in the supernatant of 0.25 M glycerol solution with 500 MHz Nuclear Magnetic Resonance (NMR). The result of minimum bactericidal concentration (dilution fold) has revealed that reuterin showed pan-bactericidal effects against 8 major food-borne pathogens. To examine any changes of antimicrobial activities of the probiotics, the probiotics were treated with different conditions, pepsin or trypsin digestion. Antimicrobial activity of reuterin was not entirely affected by any of these treatments, while the activities of the other probiotics were significantly decreased. Key wards ý Lactobacillus reuteri, Reuterin, Probiotics, antimicrobial activity Salmonella, Staphylococcus aureus, Listeria monocytogenes š Escherichia coli O157:H7 w ³ w x wš 8,10,14,19,24,26). y y w š, œ w ƒ x q z ƒ f š. wr, ³ w w³ controlw w ƒ, û w w w z w r l xw» 8,27). 5ƒ w ü Salmonella enterica serovar Typhimurium DT104, methicillin ü y s ³ (MRSA), vancomycin ü y s ³ (VISA) 29) t š w ü ³ Author to whom correspondence should be addressed. w e ƒ ƒ w w ³ y xw ƒw. w y wì w³, w w, š probiotics w š 7,8,11,16,21,27,29). Probiotics, y s w» mw w³, jš g w 16). Probiotics Lactobacillus, Bifidobacteria x ü w y w w w w ƒ w š 16). t ƒ ù e ³ ƒ ª w 16) x ƒ» w ewš 4,5,6,13,17,28). ü, y x wš w³ w 4,5,12). w w³ w ƒ,, 264
4y txthƒ gtfw4h t t qefh gfhtww ƒp pƒt4rfty Ffu ƒ9 i5 ƒypif s rpy 265», y, bacteriocins, deconjugated bile salts w š 6,13,30). L. reuteri w ü w Lactobacillus w glucoseƒ w glycerol w w w³ reuterin w w ³ 1,30). Reuterin w 1). L. reuteri w glycerol 1,3-propanediol, β-hydroxypropionic acid wì reuterin reutrerin yw β-hydroxypropionaldehyde 148 š monomer, hydrated monomer, š cyclic dimer 3ƒ xk w 30). L. reuteri w³ probiotics ³ L. bulgaricus, L. casei, L. acidophilus, B. longum w w³ w L. reuteri w³ w š w. w probiotics t ƒ ù m w w š, y x wz w w³ y wš w. x œ ³ x w ³. Lactobacillus reuteri (CHR. HANSEN) L. acidophilus (CHR. HANSEN) L. bulgaricus (CHR. HANSEN) L. casei (CHR. HANSEN) Bifidobacterium longum (CHR. HANSEN) 5 ³ w³ x w w³. Staphylococcus aureus MNEV (exfoliative toxin A+) S. aureus FRI 913 (SEA+, SEC+, SEE+ TSST-1+) Listeria monocytogenes ATCC 11285 Salmonella enteritidis ATCC 13076 S. typhimurium S. enteritica serova Typhimurium DT104 (ampicillin, chloramphenicol, streptomycin, sulfonamides š tetracycline 5ƒ w ü ³ ) Escherichia coli O157:H7 ATCC 43894 (SLTI+, SLTII+) E. coli O157:H7 ATCC 43890 (SLTI+) ³ 5 ³ d» w 0.02 M glucose (Glu) ƒw Lactobacilli MRS broth (MRS+Glu) (Difco, USA), 0.5 M glycerol (G) ƒw Lactobacilli MRS broth (MRS+G) š 0.25 M glycerol solution 3ƒ w. MRS+Glu MRS +G 37 C 24~48 o w. 0.25 M glycerol solution MRS+Glu ³ w 2,500 rpm, 30 w e pellet w š phosphate buffered saline (PBS) 2 w 0.25 M glycerol solution g 37 o C, 6 w. ³ y» e w ù B. longum x» w. ³ 30 ml w š ³ pellet d z d w 2g w ƒƒ 3ƒ ³ d 2,500 rpm, 30 mw š 0.45 µm filter (Sartorius, Germany) ³w x w ¾ -20 C o w. 8 w³ tryptic soy broth (TSB) (Difco) 37 o C, 24 w 3.0 10 CFU/ml 7 w w w w³ x w š, 1.0 10 6 CFU/ml w ³ d x w. w³ x 1. w w w³ x. 0.5, 1, 2, 3 ml (MRS +Glu, MRS+G) 1, 2, 3, 4 ml (0.25 M glycerol solution) w ƒ ³ d Mueller Hinton Agar (Difco) yww 10 mlƒ w petri dish x. 8 w³ (3.0 10 7 CFU/ ml) inoculum replicator ( : 3 10 CFU) w 4 37 C 36 o w. 2. Nuclear Magnetic Resonance (NMR) w L. reuteri d. L. reuteri w 0.25 M glycerol solution d w» w œ, w. 3. ³ d x, (minimum inhibitory concentration (MIC)), (minimum bactericidal concentration (MBC)). w w w³ x ƒ w³ L. bulgaricus, L. casei, L. reuteri ³ d x (MIC), (MBC) d w. ƒ ³ w³ { w w» w L. bulgaricus, L. casei MRS+Glu w š L. reuteri 0.25 M glycerol solution w d w. 3ƒ ³ (L. reuteri, L. bulgaricus, L. casei) d 0.5, 1, 2,
Nam Hoon Kwon et al. 266 3, 4 ml를 Mueller Hinton Broth (MHB) (Difco)와 혼합하 여 최종 10 ml가 되도록 하였다. 8종의 유해균들을 각 유산 균의 상층액이 농도별로 첨가된 시험관에 1.0 10 CFU/ml 의 농도로 접종하였다. 각 시험관들을 37 C에서 36시간 동 6 o 안 배양한 뒤 균의 성장이 관찰되지 않은 상층액의 최소농 도를 MIC로 하였고 균이 성장하지 않은 배양액을 5% 혈액 한천배지 (KOMED, Korea)로 옮겨 재 배양한 뒤 여기서도 균의 성장이 관찰되지 않은 최소 농도를 MBC로 하였다. 8가지 균이 접종 된 시험관 중 S. aureus FRI 913, S. enteritica serova Typhimurium DT104, E. coli O157:H7 ATCC 43894, L. monocytogenes ATCC 11285의 4종 균을 선택 하여 시간 경과에 따른 항균효과를 알아보기 위해 생존균수 측정을 하였다. 각 균수 측정을 2시간, 36시간 경과 시에 각 각 실시하였다. 4. 적정과 pepsin 또는 trypsin 처리에 따른 항균력 변화 시험. 항균력 시험 3과 동일한 방법으로 얻은 3가지 유산균 (L. bulgaricus, L. casei, L. reuteri)의 상층액을 각 각 4군으로 나누었다. 한 군은 1 N NaOH 용액을 첨가하여 7.3으로 조정, 의 변화에 따른 항균력의 변화를 관찰 하였고, 다른 두 군은 효소처리에 따른 항균력 변화를 보기 위해 각각 pepsin 처리군, trypsin 처리군으로 하였고 나머지 한 군은 대조군으로 하였다. Pepsin 처리군과 trypsin 처리군은 각각의 를 2.0, 7.3으로 조정한 뒤 20 unit/ml-pepsinogen (Sigma, USA)과 1 ml trypsin-edta (Sigma, USA)를 각 Fig. 1. Comparison of antimicrobial activities of 5 probiotics in agar method. A: Each number indicated a bacterial inoculation of eight food-borne pathogens. 1. Listeria monocytogenes ATCC 11285; 2. Salmonella enteritidis ATCC 13076; 3. Escherichia coli O157:H7 ATCC 43890; 4. E. coli O157:H7 ATCC 43894; 5. S. enteritica serova Typhimurium DT104; 6. S. typhimurium; 7. Staphylococcus aureus MNEV; 8. S. aureus FRI 913 B: 1 ml supernatant of Lactobacillus reuteri; C: 4 ml supernatant of L. acidophillus; D: 4 ml supernatant of L. bulgaricus; E: 4 ml supernatant of L. casei; F: 4 ml supernatant of Bifidobacterium longum All probiotics were incubated in 0.25 M glycerol solution before supernatants were collected. As figure showed, growth of every pathogen was inhibited by 1 ml supernatant of L. reuteri. But, supernatants of the other probiotics were not able to suppress the growth of 8 pathogens even when 4 ml supernatants were applied. Journal of Food Hygiene and Safety, Vol. 16, No. 4
4y txthƒ gtfw4h t t qefh gfhtww ƒp pƒt4rfty Ffu ƒ9 i5 ƒypif s rpy 267 ƒ ƒw. 2 37 C gš o z 5.0 w z yy w. 5.0 w z w w w³ y ƒ š w. z d filter ³w š z w w w³ x. ƒ w ³ 3.0 10 CFU 4 37 C o 36 w. 5. m. SAS V8 gj Q (Cochran Q test) v w (Fredman twoway ANOVA by ranks), m w. w w w³ x: ³ ƒ MRS+Glu broth w w³ x, L. bulgaricus w 4 ³ d 0.5 ml ƒ w³ w w. L. bulgaricus L. monocytogenes w. L. reuteri, L. acidophilus, B. longum d ƒƒ 1ml ƒw w³ w w. ù L. bulgaricus, L. casei d 1ml 8 w³ z w. 2 ml 3ml d ƒ ³ w³ w w³., MRS+Glu broth w 5 ³ w³ x L. bulgricus L. casei w³ ³ w. MRS+G w d w³ Fig. 2. Proton NMR spectroscopy of Lactobacillus reuteri supernatant. x MRS+Glu broth w w ùk û. ù B. longum d 1ml ƒ S. aureus w w³ š, L. reuteri d 1ml ƒ MRS+Glu broth x w³ w. w³ x L. bulgaricus L. caseiƒ ƒ w w³. 0.25 M glycerol solution w w³ x 9) L reuteriƒ k w w³ š w š (Fig. 1) m w (p< 0.05). L. reteuri d ƒ (1, 2, 3, 4 ml) w³ z w. ù w ³ x w³ ùkü. Table 1. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Lactobacillus reuteri, L. bulgaricus and L. casei. Pathogens MIC and MBC (dilution fold) * L. reuteri a L. bulgaricus b L. casei b Salmonella c 10 2.5 5 E. coli O157:H7 d 10 2.5 5 S. aureus e 10 2.5 5 L. monocytogenes 5 2.5 5 Each probiotic was incubated at concentration of 2 g/30 ml and then its supernatant was collected. Concentration of each supernatant was adjusted to 1.0 10 6 CFU/ml. *: Antimicrobial activity of each probiotic was intended to be compared with on the basis of same bacterial concentration, so OD value of each supernatant was not calculated. a: Its supernatant was obtained from 0.25 M glycerol solution. b: Their supernatant were obtained from MRS broth containing 0.02 M glucose without glycerol. c: Salmonella used in this study were S. typhimurium, S. enteritica serova Typhimurium DT104 and S. enteritidis ATCC 13076. d: Escherichia coli O157:H7 used in this study were Escherichia coli O157:H7 ATCC 43894 and E. coli O157:H7 ATCC 43890. e: Staphylococcus aureus used in this study were S. aureus MNEV and S. aureus FRI 913.
268 GfxA ydˆ yp fw 0.25 M glycerol solution ùkù k w L. reuteri w³, L. reueriƒ glycerol w w reuterin w³ d w» w œ w. 500 MHz NMR, proton w» (-HC=O) ƒ reuterin y w (Fig. 2). ³ d x, (MIC), (MBC): w w ƒ w³ x ƒ z L. reuteri L. bulgaricus, L. casei w MIC MBC d w» w x w w. xw ƒ ƒ ³ ƒ w³ ùkü w d w, ³ w w ww L. reuteriƒ ƒ w w³ ƒ y w (Table 1). 4ƒ w³ w ³ d x ww L. reuteri w³z ƒ ùkû (Fig. 3)., pepsin trypsin w³ y x: 3ƒ ³ w³ y Table 2» w., L. bulgaricus L. casei w³. ù L. reuteri d, reuterin w³ y w w m w (p<0.05). Pepsin ù trypsin ƒ ww w³ y r, reuterin w³ yƒ (Table 3, 4). L. bularicus L. casei w³ ùkü. L. bulgaricus, Salmonella typhimurium, E. coli O157:H7 (ATCC 43894, ATCC 43890), S. aureus (FRI 913, MNEV) š L. monocytogenes Fig. 3. Viable count graph of four major food-borne pathogens incubated with supernatant of 3 probiotics. A: Viable count graph of Salmonella enteritica serovar Typhimurium DT104 (with 1 ml supernatant added); B: Viable count graph of Escherichia coli O157:H7 ATCC 43894 (with 1 ml supernatant added); C: Viable count graph of Staphylococcus aureus (with 1 ml supernatant added); D: Viable count graph of Listeria monocytogenes ATCC 11285 (with 2 ml supernatant added). Supernatant of L. reuteri was collected from 0.25 M glycerol solution. Three pathogens, S. enteritica serova Typhimurium DT104, S. aureus and E. coli O157:H7 didn't survived 1 ml supernatant of L. reuteri. L. monocytogenes could grow in the broth containing 1 ml supernatant of L. reuteri though 2 ml supernatant of L. reuteri showed bactericidal effect on it.
4y txthƒ gtfw4h t t qefh gfhtww ƒp pƒt4rfty Ffu ƒ9 i5 ƒypif s rpy 269 Table 2. Influence of adjustments on antimicrobial activities of 3 probiotics. Probiotics Supernatant volume a Salmonella d E. coli O157:H7 e S. aureus FRI913 S. aureus MNEV Listeria monocytogenes L. reuteri b L. bulgaricus c L.casei c 5.0 7.3 5.0 7.3 5.0 1 + + + + + 2 - - - - ± 3 - - - - - 1 + + + + + 2 - - - - ± 3 - - - - - 1 + + + + + 2 + + + + - 3 - - - - - all volume all positive 1ml + + + + + 2ml + + + + + 3ml - - + - - 4ml - - - - - all volume all positive 7.3 +: Growth ±: Weak growth -: No growth a: The volume of supernatants added in MHB (total 10 ml). b: Its supernatant was obtained from 0.25 M glycerol solution. c: Their supernatants were obtained from MRS broth containing 0.02 M glucose without glycerol. d: Salmonella used in this study were S. typhimurium, S. enteritica serova Typhimurium DT104 and S. enteritidis ATCC 13076. e: E. coli O157:H7 used in this study were two strains; E. coli O157:H7 ATCC 43894 and E. coli O157:H7 ATCC 43890. Table 3. Change of antimicrobial activity of Lactobacillus reuteri after pepsin or trypsin treatment Enzyme Supernatant volume a Salmonella b E. coli O157:H7 c S. aureus FRI913 S. aureus MNEV Listeria monocytogenes No treatment Pepsin Trypsin 1ml + + + + + 2ml - - - - + 3ml - - - - - 1ml + + + + + 2ml - - - - + 3ml - - - - - 1ml + + + + + 2ml - - - - + 3ml - - - - - Supernatant of L. reuteri was obtained from 0.25 M glycerol solution. +: Growth -: No growth a: The volume of supernatants added in MHB (total 10 ml). b: Salmonella used in this study were S. typhimurium, S. enteritica serova Typhimurium DT104 and S. enteritidis ATCC 13076. c: E. coli O157:H7 used in this study were two strains; E. coli O157:H7 ATCC 43894 and E. coli O157:H7 ATCC 43890.
270 GfxA ydˆ yp fw Table 4. Change of antimicrobial activities of Lactobacillus bulgaricus and L. casei after pepsin or trypsin treatment. Probiotics L. bulgaricus L.casei Enzyme No treatment Pepsin Trypsin No treatment Pepsin Trypsin Supernatant S. aureus Salmonella b E. coli O157:H7 c volume a FRI913 S.aureus MNEV Listeria monocytogenes 2 + + + + - 3 - - - - - 2 + + + + + 3 + 1 + + + - 2 + + + + + 3 - + 2 + + - 2 + + + + + 3 - - + - - 2 + + + + + 3 - + 2 + + - 2 + + + + + 3 - - + + - Supernatants of L. bulgaricus and L. casei were obtained from MRS broth containing 0.02 M glucose without glycerol. +: Growth -: No growth 1: S. typhimurium grew only. 2: E. coli O157:H7 ATCC 43894 grew only. a: The volume of supernatants added in MHB (total 10 ml). b: Salmonella used in this study were S. typhimurium, S. enteritica serova Typhimurium DT104 and S. enteritidis ATCC 13076. c: E. coli O157:H7 used in this study were two strains; E. coli O157:H7 ATCC 43894 and E. coli O157:H7 ATCC 43890. ATCC 11285 w w³ z (P<0.05) ƒ L. casei E. coli O157:H7 ATCC 43894, S. aureus MNEV w w³ ùkü. š t w w L. reuteri w³ 4 ³ L. bulgaricus, L. casei, L. acidophilus, B. longum w w. L. reuteri t w w w³ y w, ƒƒ z probiotics y š ³ w ³, w» w. MRS broth 0.02 M Glucose ƒ g ³, d w w³ xw, L. bulgaricus L. caseiƒ L. reuteri w w³. MRS+0.5 M glycerol w w. L. reuteri reuterin bacteriocin w L. reuteriƒ glycerol w w š 9). x MRS broth glycerol ƒw x ww ù L. reuteri w³ w. MRS+0.5 M glycerol broth w³ z w j w reuterin» w» ƒ. reuterin MRS +0.5 M glycerol 4.0 w ùkü š MRS broth ƒ». MRS+Glu solution w³ MRS+0.5 M glycerol d reuterin w. Reuterin j» w» w MRS+Glu broth w z 0.25 M glycerol solution w d w w, L. reuteri w³ w. 0.25 M glycerol solution reuterin ƒ. x, L. reuteri
4y txthƒ gtfw4h t t qefh gfhtww ƒp pƒt4rfty Ffu ƒ9 i5 ƒypif s rpy 271 sww ³ 0.25 M glycerol solution 6 w 4.0 w ùkü. ù ³ w³ x w w³ w q w, w 0.25 M glycerol solution L. reuteri k w w³ reuterin» w. wù y w 0.25 M glycerol solution w L. reuteri d NMR w, reuterin» p w peak y w. ³ d x MIC, MBC L. reuteriƒ Ÿ w³ y w w. L. monocytogenes w reuterin MIC, MBC ( t w ) w³ w 2 û ùkû, L. monocytogenes w³ w reuterin w w ƒ. wr, w w w³ x ƒ z ùkü L. bulgaricus MIC MBC d L. casei û w³. L. bulgaricusƒ L. casei w wù w. x mw, L. reuteriƒ k w w³z š ³ w probiotics wš y w. ù ³ m w ü ù wz w z w³z {w. w z z w³ y x w š reuterin w³ w w y ùkü L. bulgaricus L. casei w³ ùkü š, p y w. Reuterin w z ù w š w³ ù bacteriocin w ³ w ü y w³ w ƒ. L. bulgaricus L. casei w z r 3ml ƒ w³ z ƒ ƒ yw û. 4 ml ƒ w ùkû w ƒ. 4ml ƒ w³ yy w w w w³ ùkù ƒ. 23) L. bulgaricus bacteriocin w» w L. acidophillus lactacin B, acidocin B Ÿ bacteriocin w 2,20). L. casei lactocidin 705 bacetriocin w 32). L. reuteri reutericidin 31) 6 bacteriocin w bacteriocin w w 25) ù L. reuteri strain ƒ. x w ù L. reuteri MRS+G d w wz w w³ ƒ. x w strain bacteriocin w ƒ w š w. L. reuteri bacteriocin reuterin reutericyclin w³ w 22). ³ w Ÿ w³ ƒ ³ w w³. ù ³ Salmonella, E. coli w ³ w w³ ùkü. ww ³ L. reuteri w³ ƒ w Ÿ wš w L. reuteriƒ w w³, reuterin, reutericyclin, reutericidin 6 reuterin ƒ w ù ƒ z ƒ š q w. L. reuteri glucoseƒ w glycerol w reuterin w. Glycerol 1,3-propanediol, β-hydroxypropionic acid wì reuterin reutrerin yw β-hydroxypropionaldehyde 148 š monomer, hydrated monomer, š cyclic dimer 3ƒ xk w 30). w x L. reuteri ³ w y ü w», ü y reuterin w 9). w s Caco-2 s w x w L. reuteri ³ w s w ƒ š x 18). Reuterin DNA w w ribonucleotide reductase w z {w ³, protozoa, q, s¾ w š 9). L. reuteri ü ³ ³ w³ ùkü ƒ ƒ probiotics wš w 18). Reuterin glycerol w ü w w œ v. L. reuteri ³»» L. reuteri ü ƒ
272 GfxA ydˆ yp fw w ƒ. L. reuteri probiotics, œ w z» L. reuteri p reuterin ƒ š ƒ. œ yw»x, š x x w y Ì mw w. NMR w w» w 5 ³ (Lactobacillus reuteri, L. acidophillus, L. bulgaricus, L. casei and Bifidobacterium longum) ³ w w³ w. ƒ ³ 3ƒ (MRS+glucose, MRS+0.5 M glycerol, 0.25 M glycerol solution) w z d sw Meuller Hinton Agar 8 w³ w. MRS+glucose, MRS+0.5 M glycerol d w x L. reuteri w³ ³ w ù 0.25 M glycerol solution k w ùkü (p<0.05). 0.25 M glycerol solution ùkù w³ L. reuteriƒ glycerol w w reuterin ƒ, 0.25 M glycerol solution d 500 MHz Nuclear Magnetic Resonance (NMR) mw w reuterin y w. 3ƒ w w³ x ƒ w³ ùkü 3ƒ ³ (Lactobacillus reuteri, L. bulgaricus, L. casei), ƒƒ š w³ ùkü w (minimum bactericidal concentration) d w, L. reuteriƒ w reuterin Ÿ w³ y w. w, pepsin y trypsin w z 3ƒ ³ w³ y w L. bulgaricus L. casei w³ w w reuterin w³ w (p<0.05). ³ L. reuteri w³ ƒ w Ÿ w L. reuteriƒ w w³ reuterin» w. w reuterin w³ k ³ ü ù wz w w ù ƒ z ƒ š q w. š x 1. Axelsson, L., and S. E. Lindgren: Characterization and DNA homology of Lactobaillus strain isolated from pig intestine. J. Appl. Bacteriol., 62, 433-440 (1987). 2. Barefoot S. F., Klaenkammer T. R.: Detection and activity of lactacin B, a bacteriocin produced by Lactobacillus acidophilus. Appl. Environ Microbiol., 45(6), 1808-1815 (1983). 3. Barefoot S. F., Klaenkammer T. R.: Purification and characterization of the Lactobacillus acidophilus bacteriocin lactacin B. Antimicrob. Agents. Chemother., 26(3), 328-334 (1984). 4. Bernet, M. F., D. Brassart, J. R. Neeser, and A. L. Servin: Adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells and inhibition of enteropathogen-cell interactions. Appl. Environ.Microbiol., 59, 4121-4128 (1993). 5. Bernet, M. F., D. Brassart, J. R. Neeser, and A. L. Servin: Lactobacillus acidophilus LA1 binds to cultured human intestinal cell lines and inhibits cell attachment and cell invasion by enterovirulent bacteria. Gut, 35, 483-489 (1994). 6. Bernet-Camard, M. F., V. Lievin, D. Brassart, J. R. Neeser, A. L. Servin, and S. Hudault: The human Lactobacillus acidophilus strain LA1 secretes a nonbacteriocin antibacterial substance(s) active in vitro and in vivo. Appl. Environ. Microbiol., 63, 2747-2753 (1997). 7. Brassart, D., and E. J. Schiffrin: The use of probiotics to reinforce mucosal defence mechanisms. Trends Food Sci. & Technol., 8, 321-326 (1997). 8. Carlson, S. A., R. M. Willson, A. J. Crane, and K. E. Ferris: Evaluation of invasion-conferring genotypes and antibioticinduced hyperinvasive phenotypes in multiple antibiotic resistant Salmonella typhimurium DT104. Microb. Pathog., 28, 373-378 (2000). 9. Dobrogosz W. J., Raleigh N. C., and Sven E. Lindgren: Method for inhibiting microorganism growth, US patent 5, 849, 289. 1998 Dec 15.
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