Screening of Lactic Acid Bacteria with Potent Adhesive Property in Human Colon using Colonic Mucin-binding Assay
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(2) ®"²æ B 36 ² B 6 ^ (2004). 960. ~ lectinç Wî*~ ÖKj Ï FÖ~ ËÚ 6OË~ & > ®º(18,19), Takahashi (19)f RCMj Ï~ polystyrene beadö z+~, FÖ~ Ö Ëj G;~º îÚ FêO»j n~, Lactobacillus acidophilus group FÖ " RCM*~ ÖËj Ò~& . Ö", L. acidophilus YN 2062& &Ë ¸f ÖËj & ~& . Þ Matsumura (18)f Lactobacillus acidophilus «" RCM*~ ÖËj Ò Ö", L. acidophilus JCM 1025, SBT 267, LA2, SBT 2062& ¸f ÖËj &rb , "~ SLPf colonic mucin~ Ú¦ ê *~ Ö~ 7ºWj ; : ® . ¾ f L. acidophilus groupöò >Ú ®Ú FÖ ²F 6OË"~ *>' jv& ÚJÚ ; . V¢B öBº *~ &ËöB Ö> 6OËj < º "¢ Fê Ï'b, 6ï[ Wî rat colonic mucin" FÖ ~ lectinç Wî*~ ÖËj G;~º colonic mucin-binding assay~ ªC j Lactobacillus acidophilus IFO 3025¢ Ï~ J;~& . $ O»j Ï ~ Lactobacillus³, Bifidobacterium³, Lactococcus³ 5 ªÒ" C 32«" ê 1" 5 3^~ Fj ªæöB ª Ò Bifidobacterium³ " º;>º 18«~ ªÒ" , C 50«~ FÖ"¢ &çb ÖËKj G;~& . $ &Ë ¸f Wj Lactobacillus species FSB-1ö & ÿ ;j >¯~& .. þÒò 5 O» ÒÏ 5 þÿb þö ÒÏB 5 ªÒ FÖ" 32«f ' V&ö B ª·A¾ ªÒ~ ãV& &ö ®b"öB & 7 ", Table 2ö BB :f ? . Þ þ öB colonic mucin-binding assay~ {ãö ÒÏB "º Lactobacillus acidophilus IFO 3025& . $ þö ÒÏB þÿbf *~ &ËÚ mucin" &Ë jÝ W(18,19)j &æ ® rJê Wister rat(ë W, 10"_, (")þæz, ãV)& . 24Û1oC 5 55% ç&Û ê .B ÒGÚöB 24-48* ÿn 'wÆ ~&b, Òò(â·Òò, Â)f bj ¶F/V . Fj~ ªæb¦8 Bifidobacterium³ ~ ªÒ ãVê >öæö "~º ê 1" 5 3^ ò~ Fj ªæj j~ ò ÒÏ~& . MRS(Difco Co, USA) ÚVæöB Fj ªæj 6V'b ³ C~, Mellissa (21)~ triple-layer diffusion O»ö V¢, ªæ ò *ç 7*[ö *~~ê r, j* 6V' Fæ> ê anaerobic systems(Becton Dickinson Lab, USA)¢ Ï ~ V·~& . r Bifidobacterium³ "& ÖF'b WË > ®ê, BL ÚVæ(Difco Co., USA)¢ FVæ ÒÏ~ 37oCöB 48* ÿn V·~& . BL ÚVæ öB WË colony 7 ;& ç colony 100B¢ 1N' b ªÒ(B1-B100)~, lithium chloride 0.5 mg/mL, sodium lauryl sulfate 20 µg/mL, sodium propionate 5 mg/mL, neomycin sulfate 10 µg/mL& FB modified VF-Bouillon agar (21)¢ Ï~ ÒN '[ r, 37oCöB 48* ÿn V ·~& . r ÒÏB £ f SigmaÒ~ B®îb, . VæöB WË C 18«~ "¢ Bifidobacterium³ º; " ~& (Table 2 ^). FÖ~ V· 5 surface lectin-like protein(SLP)~ B ËÚ 6OË Ö> FÖ"~ Fêj * ÒÏB 5 ªÒ" 50« 7, Bifidobacterium³ "º MRS ÚVæ 300 mLö * V· 1%¢ '' 7«~, 6V'b 37oCö B 48* ÿn V·~& . Lactobacillus³ æ FÖ". j ÿ¢ VæöB ÿï 7«~, 37oCöB 24* ÿn 6V V· ê þö ÒÏ~& . *öB V· ''~ V·j 4oC, 650 göB 20ª* ö ªÒ~ Ú¢ ²> ê, 10 mM phosphate buffered saline(pH 7.2, ~ PBS)b 3² ^¿~& . Ú¢ 2 M guanidine-HCl(Sigma Chemical Co., USA) Ïö *ç ê, 37oCöB 2* ÿn êû~ Úö ¦OB Wîj º Â~& . ºÂB Ïj 4oC, 1,600 göB 30ª* öªÒ~ Ú¢ B r çûj Ã~> 4oCöB 48* ÿn RC ê, ÿÖ~ SLP¢ B~& . Biotinylated SLP~ B SLP 1 mgj 0.15 M NaCl¢ F 0.1 M Na2HPO4 Ï(pH 9.0)ö Ï~, Vö biotinz £, D-biotinyl-ε-aminocaproic acid N-hydroxysuccinimide ester(Roche Diagnostics Co., USA) 1 mgj 40 µL~ DMSO(Sigma Chemical Co., USA)ö Î & ê, 4oCöB 16* ÿn >wV . ê >wÏj 10 mM PBS(pH 7.2) 4oCöB 24* ÿn RCj >¯~ biotin-conjugated SLP¢ B~& . Biotinylated SLP Ï 7 ~ Wî ïf bovine serum albumin(Sigma Chemical Co., USA)j &bî ~ Micro BCA protein assay kit(Bio-rad Lab., Canada)j Ï~ G;~& . Rat colonic mucin(RCM)~ B RCMf Perret (22)~ O»ö V¢ Wister rat~ &Ë Úã b¦V B>î . 10"_~ ëW, Wister ratj ~ÒB ¦¢ .B~ &Ëj ' ê, &Ë Úãj slide glass ' ÚÚÚ 6ï[j ²>~& . ²>B 6ï[j Folch’s Ï (CHCl3 : CH3OH = 2 : 1, V/V, £ 1/)ö *ç~ îæÎ ê, 4oC, 1,600 göB 20ª* öªÒ¢ >¯~& . *bj Folch’s Ïb 2² ^¿~, diethyl ether(£ 1/) 3² ÒN ^¿~ îæV . îæB ²> 6ï[ö 6 M guanidineHCl Ïj &~ 37oCöB 2* ÿn êû~B >wÎ. r mucinj ºÂ~, çV öB öªÒ~ çûj ªÒ~& . ªÒB çûj Ã~> 4oCöB 24* ÿn RC ê, ÿÖ~ RCMj B~& . Colonic mucin-binding assay Microtiter plate(Maxisorp, Nunc, Denmark)~ wellö RCM Ï(50 µg/mL, 6 M guanidine-HCl) 75 µL¢ '' ª"~, 4oCöB ~!J ÿn >wB mucinj well ö ¦OV. . rÆ plate¢ PBST(PBS-0.05% Tween 20, £ 1/) 3 ² ^¿ ê, blocking buffer(0.05% Tween 20" 1% BSA¢ F PBS, pH 7.2) 100 µL¢ Ò ª" r, çNöB 2 * ÿn O~~ RCM ¦O>æ pf well j blocking ~& . ê ' wellf PBST¢ Ï~ 4² ^¿~, biotinylated SLP Ï 50 µL¢ Ò ª"~ 1* ÿn çNöB.
(3) ËÚ 6OË Ö> FÖ"~ Fê. 961. >wV . >w~æ pf biotinylated SLP¢ B~V * >wçûj B ê, PBST¢ Ï~ ' wellj 3² ^ ¿~& . -² ¾ÒB plateö '.® CB horseradish peroxidase(HRP)-conjugated streptoavidin(Parmingen, Lab., USA) 50 µL¢ ª"~, çNöB 1* ÿn >wV . Ö~æ pf HRP-conjugated streptoavidinj B~V *, çûj ªÒ PBST 7² ^¿ ê, 3,3',5,5'-tetramethylbenzydine (TMB) liquid substrate system(Sigma Chemical Co., USA) 60 µL¢ Î&~ BïÎ r, 2 N H2SO4(£ 1/) 60 µL ¢ ª"~ >wj ;æV . Bï ;êº 450 nmöB Microtiter plate reader(Molecular Devices, USA)¢ ÒÏ~ 7ê¢ G;~& .. ¦Æ~& . Þ ;{ ÿ;j * L. species FSB-1~ 16S rDNA~ "VBªCj >¯~& . 16S rDNAº 2«~ ~ universal primer(5'-GAGTTTGATCCTGGCTCAG-3', 5'AGAAA GGAGGTGATCCAGCC-3')¢ ÒÏ~ PCRö ~ Ã~(27), QIA Quick PCR Product Purification kit(Quiagen, Germany)ö ~ ;B r, ABI PRISM Big Dye Terminator 5 Genetic Analyzer 377(Applied Biosystems, CA, USA)j Ï~ Bj ªC~& . FêB " L. species FSB-1~ 16S rDNA Bf CLUSTRAL W software(28)¢ Ï~, Lactobacillus³ " 5 EMBL/Gene Bank database¦V áf F"~ B" ;RÊ, çÿWj Ò~& .. Colonic mucin-binding assay~ ªC J; Colonic mucin-binding assay~ Ö"ö 'Ëj ~º ''~ Ö;¶¢ 'z~V *~, &' FÖ" L. acidophilus IFO 3025~ SLP¢ Ï~ j¾~ þj >¯~ « ª C j J;~& .. Ö 5 V. Biotinylated SLP~ ¾Ò³ê Ö;: ' biotinylated SLP~ ¾Ò³ê¢ Ö;~V *~, ³ê¢ '' 0, 0.625, 1.25, 2.5, 5.0, 10.0 µg/mL ; r, TMB Bï* 10ªöB ªC j >¯~, Ö"¢ jv~& .. ËÚ 6OW Ö> FÖ Fêj * colonic mucinbinding assay~ ªC R'z RCM~ ¦Oö ~º pH~ Ë: ËÚ 6OË Ö> FÖ"¢ Fê~V * >b colonic mucin-binding assay ¢ Ï~V *, ªCO»~ Ö"ö 'Ëj > ®º '« j 'z ~¶ ~& . Colonic mucin-binding assayöB microtiter plate~ wellö & RCM~ ¦Of ª C»ö ®Ú Ö"f çÖ>º 7º >w 7 ~¾, RCM~ ¦Oö ~º pH~ 'Ëj Ò~& (Table 1). RCM~ ¦O ;ê¢ wellö ¦OB Wîïb jv Ö", ¦OB RCMf pH 4.8, 7.0 5 10.0~ öB '' 7ê 0.36, 0.35 5 0.34¢ ¾æî . Ö"º RCM~ microtiter plate ö & ¦O ;ê& pH~ æzö V¢ N& ìrj ¾ æÚº ©b, ê~ þöBº RCM B ÒÏB Ï. 6 M guanidine-HCl~ pH 4.8öB ç7 ¦OB ªCö Ï~& . f FÒ þÖ", Shi (29)f 4&æ~ W ª¶bî(polyurethane, polystyrene, silicone, methyl methacrylate)~ ö ²~ ~F mucinj ¦O~º þöB, mucin~ ¦O &Ë F& mucin 7 Wî 7ÒÒ(naked protein backbone)~ ²>' Wî ; Wª¶ ö Ö ~V r^¢ ~&b, mucinf Wî~ 7ÒÒ ö ê>zb çÒÒ(carbohydrate cluster) Ö>Ú ®º >Ú® ~& . $ Ohara (30)ê mucin~ microtiter plateö & ¦O;êº pH 2-9~ º*öB N & ì :ê ® (31). Ö"º þ"ê ¢~~º Ö" .. TMB Bï* Ö;: Colonic mucin-binding assayöB TMB ~ ¾Ò*ö V 'Ëj ¦Æ~V *~, TMB ¾Ò 0, 5, 10, 20, 40ª êö Bïj ;æÊ 450 nmöB 7ê¢ j v~& .. Colonic mucin-binding assayö* HRP-streptoavidin~ R' ¾Ò³ê Ö;: Colonic mucin-binding assay~ ' ªC j J;~V *, pH 4.8~ öB RCMj microtiter plate ö ¦OÎ ê, L. acidophilus IFO 3025¦V áf bio-. ËÚ 6OË Ö> FÖ~ ÿ; þöB ËÚ 6OË Ö> " FêB Lactobacillus species FSB-1~ ÿ;j *~, S4700 SEM(Hitachi, Japan) "Ò; *¶* ãö ~ ;' &V(23)j >¯ ~& . Î"ï, Ö² ºW, ÚÿW, BÎ' ßW 5. · Vîö & β&>ª W ~ z' ßWj Cowan" Steel(24) 5 Macfaddin~ O»(25)ö V¢ ¦Ò~, Bergey’s Mannual of Systemic Bacteriology(26)~ ÚÏ" jv. Table 1. Effects of pH on mucin-coating onto wells of microtiter plates in colonic mucin-binding assay. ' pHö 8 RCM~ ¦O : Microtiter plateö & RCM ¦O ;êö ~º pH~ 'Ëj Ò~V *~, 6 M guanidine-HCl Ïö 0.1 N NaOH(£ 1/) $º 0.1 N HCl (£ 1/)j &bB, pH¢ 4.8, 7.0, 10.0b ;~& . ' pH~ 6 M guanidine-HCl Ïö RCMj Î&~ 50 µg/ mL~ ³ê& >ê B~& . j microtiter plateö ' ' 75 µLO ª" r 4oCöB 16* ÿn ;~~ RCM j ¦OV . Microtiter plateö ¦OB colonic mucin~ ï j mucin7 Wî ³ê jv~¶, Micro BCA Protein assay kitj Ï~ G;~& . HRP-conjugated streptoavidin~ ¾Ò³ê Ö;: Colonic mucin-binding assayö ®ÚB HRP-conjugated streptoavidin~ ¾Ò³ê& ~º 'Ëj G;~V *~, biotinylated SLP~ ³ê¢ 5.0 5 10.0 µg/mL ;~, 6 HRP-conjugated streptoavidin~ ³êê Cj Ï~ ªCj >¯ r, Ö"¢ jv~& . r TMB Bï*f 10ªb ~& .. Mucin-coating pH. 4.8. 7.0. 10.0. Bound protein (O.D., meanÛSD). 0.36Û0.03. 0.35Û0.01. 0.34Û0.01. 1). Protein contents were measured by Micro BCA protein assay kit and expressed as meanÛSD of quadruplicate tests (Absorbance at 600 nm)..
(4) 962. ®"²æ B 36 ² B 6 ^ (2004). L. acidophilus IFO 3025 Vö~ SLP¢ B r, biotinylated SLP~ ³êö V colonic mucin"~ ÖËö & ' Ëj jv 5 G;~& (Fig. 2). Biotinylated SLP~ ³ê& à &ö V¢ colonic mucinö & Ö;êê Ã&~º ãË j & . ß® colonic mucinö & biotinlyated SLP~ Ö ;ê& çFçb Ã&~, « 7ê& 1.0-2.0j ¾æÚ º ³ê biotinylated SLP 5.0 µg/mL& ªCO»j * ' ¢ 6>î . $ biotinylated SLP~ ³êö V ¢ colonic mucin"~ Ö F~W ®º N¢ "º . þÖ"º, · b~ ËÚ 6OË G;j * G ;O» FÎ~² ÒÏF &ËW ®rj {B "î .. Colonic mucin-binding assayö* biotinylated SLP~ ¾Ò ³ê Ö;: Colonic mucinö & '. Ö;ê¢ ¾æÚº biotinylated SLP~ ³ê¢ Ö;~V *~, &' FÖ. Colonic mucin-binding assayö* TMB Bï* Ö;: ç~ þöB áÚê 'öB colonic mucin-binding assay ~ ªC Ö"ö ~º TMB Bï*~ 'Ëj Ò Ö", Fig. 3ö ¾æ :f ? . TMB £~ ¾Ò* ^Úî> colonic mucinö & L. acidophilus IFO 3025 Vö~ biotinylated SLP ÖËê& Ã&~º Ö"¢ ¾æÚî . V¢ B þê çV Ö"öBf ÿ¢~², TMB ¾Ò*ö V colonic mucinö & Ö;ê& çFç~ ãËj , « 7ê& 1.0-2.0~ Ò¢ ¾æÚº 10ª ¾Ò& ªC~ ' ¢ 6>î . V¢B æ.ræ~ Ö"¢ « " r, FÖ~ ËÚ ç ÒWj ï&~V * FÖ ~ lectinç Wî" W î colonic mucin*~ ÖËj G;~º colonic mucin-binding assay¢ ÒÏ ãÖ, mucin~ ¦Of pH 4.8, HRP~ ³êº 6B®~ 24,000V C, biotinylated SLP~ ¾Ò³ êº 5.0 µg/mL, TMB~ Bï*f 10ª '~ ªC ªj { > ®î . Takahashi (19)f RCMj &æ polystyrene bead¢ z+ ~ FÖ~ ÖËj G;~º îÚ FêO»j : ®b¾, polystyrene beadf "~ SLP*~ jß' Ö ãËj , ËÚ çÒWj ï&~º >bº ² ¦'. ©b rJ^ ® . Þ Matsumura (18)f RCM " Lactobacillus acidophilus group*~ ÖËj jv 5 G;. Fig. 2. Effects of biontinylated surface lectin-like protein on adhesive property in colonic mucin-binding assay. 1) Adhesion to colonic mucin was measured by colonic mucinbinding assay and expressed as meanÛSD of quadruplicate tests.. Fig. 3. Effects of TMB scintillation time on adhesive property in colonic mucin-binding assay. 1) Adhesion to colonic mucin was measured by colonic mucinbinding assay and expressed as meanÛSD of quadruplicate tests.. Fig. 1. Effects of horseradish peroxidase conjugated streptoavidin on adhesive property in colonic mucin-binding assay. 1) Adhesion to colonic mucin was measured by colonic mucinbinding assay and expressed as meanÛSD of quadruplicate tests.. tinylated SLP~ ³ê¢ 5.0 5 10.0 µg/mL '' ¾Ò r, ªCÖ"ö ~º HRP-conjugated streptoavidin~ 'Ëj Ò ~& . Fig. 1ö ¾æ :f ?, HRP-conjugated streptoavidin ³ê& 6²> colonic mucinö & Ö Ô² G ;>º Ö"¢ & . ß® 6 HRP-conjugated streptoavidin Ï 3,000-36,000V C~ öB biotinylated SLP 5.0 5 10.0 µg/mLf colonic mucin*~ Öf ~ çFçb 6² ~º Ö"¢ & . V¢B þöBº colonic mucinö & biotinylated SLP~ Ö çFç~ ãËj ¾æÚ, 7 ê& 1.0-2.0j ¾æÚº HRP-conjugated streptoavidin 24,000V Cj ' ³ê Ö;~, ê~ þö ÒÏ~& ..
(5) ËÚ 6OË Ö> FÖ"~ Fê. 963. Table 2. Screening of lactic acid bacteria with highly adhesive property to colonic mucosa Strain name of lactic acid bacteria Lactobacillus acidophilus IFO 3025 Lactobacillus acidophilus Lactobacillus amylophilus KFRI 238 Lactobacillus casei KCTC 3109 Lactobacillus confusus KFRI 227 Lactobacillus delbrueckii KCTC 1047 Lactobacillus fermentum ATCC 14931 Lactobacillus helveticus ATCC 8018 Lactobacillus helveticus Lactobacillus hilgardii KFRI 229 Lactobacillus pentosus KCTC 3120 Lactobacillus pentosus KCCM 35472 Lactobacillus plantarum KCTC 3099 Lactobacillus plantarum KCTC 3104 Lactobacillus plantarum KCTC 3105 Lactobacillus plantarum KCTC 3108 Lactobacillus plantarum ATCC 14917 Lactobacillus species FSB-1 Lactobacillus plantarum FSB-2 ML4 Streptococcus thermophilus Streptococcus thermophilus ATCC 14485 Lactococcus garvieae KCTC 3772 Lactococcus lactis ssp. lactis ATCC 7962 Enterococcus casseliflavus KCTC 3079 Enterococcus faecalis KCTC 2011 Pediococcus pentosaceus KFRI 833 Bifidobacterium catenulatum KCTC 3358 Bifidobacterium infantis KCTC 3127 Bifidobacterium breve KCTC 3220 Bifidobacterium longum KCTC 3421 Bifidobacterium bifidum KCTC 3357 B1 B8 B18 B21 B35 B40 B49 B51 B52 B53 B54 B55 B58 B62 B64 B66 B67 B68 1). Protein (µg/mL). Binding affinity1) (MeansÛSD, O.D. at 450 nm). Relative binding affinity2) (MeansÛSD). 44.08 243.43 55.49 101.75 20.73 38.42 42.00 234.11 156.61 19.65 57.80 104.59 106.00 51.09 282.55 16.80 27.60 143.57 69.36 154.52 131.08 22.54 181.44 40.26 66.35 46.07 32.58 69.17 39.67 98.58 82.32 68.30 122.83 208.30 65.20 50.40 167.27 109.54 169.47 128.32 123.19 95.15 113.01 140.26 75.00 190.86 135.47 60.40 74.80 43.50. 1.35Û0.11 0.62Û0.18 1.31Û0.07 1.30Û0.18 1.40Û0.04 0.88Û0.12 1.38Û0.06 1.05Û0.22 0.94Û0.06 2.31Û0.11 1.95Û0.07 1.13Û0.04 0.83Û0.03 0.87Û0.18 0.53Û0.03 0.68Û0.06 0.82Û0.13 2.54Û0.08 0.63Û0.10 1.45Û0.05 1.05Û0.04 0.99Û0.10 1.55Û0.11 0.93Û0.03 1.29Û0.07 1.33Û0.39 1.22Û0.12 0.74Û0.06 0.93Û0.07 1.69Û0.05 0.78Û0.10 1.19Û0.30 0.68Û0.04 1.20Û0.04 1.17Û0.06 1.14Û0.01 1.24Û0.13 1.53Û0.09 1.41Û0.05 0.91Û0.04 1.09Û0.07 1.38Û0.05 0.96Û0.03 0.78Û0.05 1.92Û0.07 1.29Û0.04 0.80Û0.04 0.87Û0.03 0.87Û0.05 0.99Û0.04. 0.60Û0.05 1.52Û0.45 0.73Û0.04 1.32Û0.18 0.29Û0.01 0.34Û0.05 0.58Û0.03 2.46Û0.51 1.47Û0.09 0.46Û0.02 1.13Û0.04 1.18Û0.04 0.88Û0.04 0.44Û0.09 1.50Û0.09 0.11Û0.01 0.23Û0.04 3.64Û0.12 0.43Û0.07 2.25Û0.07 1.38Û0.06 0.22Û0.02 2.82Û0.19 0.37Û0.01 0.86Û0.05 0.61Û0.18 0.40Û0.04 0.51Û0.04 0.37Û0.03 1.66Û0.05 0.64Û0.08 0.81Û0.20 0.84Û0.05 2.49Û0.09 0.77Û0.04 0.58Û0.01 2.07Û0.21 1.68Û0.10 2.39Û0.08 1.17Û0.05 1.34Û0.09 1.32Û0.04 1.09Û0.03 1.10Û0.08 1.44Û0.05 2.46Û0.08 1.08Û0.05 0.53Û0.02 0.65Û0.04 0.43Û0.02. Binding affinity to mucin was measured by colonic mucin-binding assay and expressed as meanÛSD of quadruplicate tests. Relative binding affinity = Protein (µg/mL)ÜBinding affinity/100.. 2).
(6) 964. ®"²æ B 36 ² B 6 ^ (2004). ~V *~, þ" FÒ O»j : ®º, . þ~ Ö"f ç ® FÒ ãËj "î . &Ë mucinö & '« FÖ~ ÖË jL Lactobacillus³, Bifidobacterium³, Lactococcus³ 32«~ "f ê 1" 5 3^~ Fj ªæöB ªÒ Bifidobacterium³b º;>º 18«~ " , C 50«~ FÖ "¢ &çb, colonic mucin-binding assay¢ Ï~ &Ë mucinö & ÖËj G;b, ËÚ 6OËj jv 5 ªC~& . Ö" Table 2ö ¾æ :f ?, SLP V&³ ê 5.0 µg/mLöB mucinö &~ ç&'b ¸f ÖË (binding affinity)j ¾æÞ "º Lactobacillus hilgaridii KFRI 229, Lactobacillus species FSB-1, Bifidobacterium breve KCTC 3220& '' 2.31, 2.54, 1.69 ¸f Wj "î . Fj ªæ ªÒ" 7öº B40, B49 5 B58"& '' 1.53, 1.41, 1.92 jv' ¸f Wj ¾æî . ¾ colonic mucin-binding assayö ~ ÖË G;f SLP ³ê 5.0 µg/mLöB mucinö & ÖË~ jvæ, "ö V¢ ö Ò~º lectinç Wî~ B ï ôj ãÖ, ÖË ² Ôz¢ê *Ú' ÖËf ç&' b Ö> > ®º ^B6j < ®î . V¢B SLP ß;. Fig. 4. A quantitative analysis of adhesive property to mucin by lactic acid bacteria selected in first screening procedure. 1) (A) Type culture strains, (B) Isolated strains from infant feces. 2) Adhesion to colonic mucin was measured by colonic mucinbinding assay and expressed as meanÛSD of quadruplicate tests.. ³êöB G; mucin ÖË~ Bjv º, ' "~ Wî~ ïj J î' ÖËj ï&~º © z '. ©b Òò>î . ' "¦V SLP ò 1 mgö FB B Wîïj G;~& . Table 2ö ¾æ :f ?, "öBº Lactobacillus acidophilus, Lactobacillus helveticus ATCC 8018, Lactobacillus plantarum KCTC 3105, Lactococcus garvieae KCTC 3772& '' 243.43, 234.11, 282.55, 181.44 µgb ¸f Wî >Nj &b, Fj ªæ ªÒ"öBº B8, B35, B49, B62"& ç&'b ¸f Wî Öïj & . V¢B ' 5 ªÒ"~ î' ÖË(total binding affinity)~ jvº ' FÖ~ SLP V&³ê 5.0 µg/mLöB mucinö & ÖËj G; r, ' FÖ~ lectinç Wî >Nj J~ j v 5 ªC~& . Ö" "~ ãÖº L. helveticus ATCC 8018, L. species FSB-1, L. garvieae KCTC 3772& ' ' 2.46, 3.64, 2.82 Ö> ËÚ 6OËj &ê ©b ï& >î . $ Fj ªæ ªÒ"~ ãÖº B8, B49, B62" & '' 2.49, 2.39, 2.46b ç&'b ¸f ËÚ 6OËj "î (Table 2). Ö"º FÖ ö Ò~º SLPf mucin*~ ÖË öò jî¢ SLP 7 B Wî > Nê ËÚ 6OË ï&ö ®Ú 7º º²ªj ¾æÚº Ö "& . *~ &ËÚ b~ Cf ÚÂ æ ¢"¢Úö ;W B >Ú zb(1,9), &Ë b 7 probiotics~ ËÚ 6ïö & 6O(adhesion)f C ;Wj * F¯ b ;>Ú z (1,9-11,13). ËÚ 6ïö & probiotics ~ Öf 6ï 7 ß;Wª $º ¦*f "ö Ò~ º lectinç Wî &>Ú ® > ® . ß®, Ë Ú 6ïj W~º ê Wª 7º j r J^ ® (32). Kenji (33)f Lactobacillus casei IFO 3425~ ËÚ ÖË &Ë 6ï 7 · ê ö V¢ N& ®rj ~, mucin~ ê & ËÚ 6OËö 'Ëj & º Ò j ~& . $ Ouwehand (32)ê &¦ª~ ËÚ ^f mucin 7 ê ¦*ö V¢ ËÚ 6OË ¢ ê ~& . ËÚ ^~ colonic mucinö & ÖËj ï&~º O»f " FÖ ~ lectinç Wî j OÒF bî æ~ ~º © &¦ªî. (17,31,32,34). He (31)f ; Fjf r.V¢ < ®. Fig. 5. Scanning electronic microphotograph (SEM) of the isolate, Lactobacillus brevis FSB-1..
(7) ËÚ 6OË Ö> FÖ"~ Fê. º Fj¦V '' bifidobacteria¢ ªÒ~ ~ ËÚ 6 OËj OÒF ;ê jv Ö", ; Fj¦V ªÒ Bifidobacterim bifidum H-2-4f Bifidobacterium breve H-1 ¸ f 6OËj ¾æÚî ~& . Ouwehand (32)f _ ç þ(j, ê 2Bú, ê 6Bú, 25-52^, 74-93^) ~ ªæb¦V Ë 6ïbî(mucin)j ªÒ~, Bifidobacterium " 4«~ 6OËj jv Ö", FjöB ªÒ mucinö & þö ÒÏB " ¸f 6OËj & : ® . Þ Ouwehand (34)f Bifidobacterium lactis Bb 12& Lactobacillus GG $º Lactobacillus delbrueckii subsp. bulgaricusf þ Ò~&j r, mucin"~ ÖË çß® ~B, "*~ ç^·Ïö ~. 965. ËÚ 6OË çß~¾ ~£ > ® ~& . f ?f þÖ"¢ « " r, ËÚ 6ï[~ mucin ~ ê f FÖ~ ~ lectinç Wî~ Öj Ï colonic mucin-binding assayº ËÚ 6OË ï&¢ * Ò*W ® ÖW ¸f þO»¢ > ® . SLP~ ³êö 8 Fê~ ËÚ 6OßW Fê";öB ËÚ 6OË ç&'b Ö> ©b ï &B L. helveticus ATCC 8018, L. species FSB-1, L. garvieae KCTC 3772f Fjªæ ªÒ" B8, B49, B62"ö &~ biotinylated SLP³êö V ÖËj Ò Ö"º Fig. 4~ A) 5 B)ö ¾æ :f ? . ³êö V ËÚ 6OËf L.. Table 3. Comparison of morphological, physiological, and biochemical characteristics between strain FSB-1 and Lactobacillus brevis Taxonomical factors Cell morphology Shape Cell size (µm) Motility Spore Colony Found in Gram staining Culural characteristics Oxygen requirement Growth at 5oC Growth at 15oC Growth at 45oC Growth in 4.0 g/L NaCl Growth in 6.5 g/L NaCl Growth in 8.0 g/L NaCl Physiological characteristics CO2 from glucose Catalase Arginase Sugar fementation Cellobiose Trehalose Mannitol Sorbitol Arabinose Lactose Melibiose Raffinose Xylose Galactose Mannose Ribose Salicine Saccharose Rhamnose Maltose Fructose Glucose Esculine. Lactobacillus species FSB-1. Lactobacillus brevis. rods 0.5-0.6Ü3.0-5.0 irregular, white, rough, raised center kimchi +. rods 0.7-1.0Ü2.0-4.0 irregular, white, rough human feces +. facultative anaerobes + + + + -. facultative anaerobes + + + + -. + -. + +. + + N.D. + + + -. + d + d d d + d + + + d. +: positive reaction, -: negative reaction, N.D.: not determined, d: 11-89% strains positive..
(8) ®"²æ B 36 ² B 6 ^ (2004). 966. Table 4. Levels of 16S rDNA sequence similarity for strain FSB-1 and type strains of some Lactobacillus species Strain. Accession No.. Similarity (%). Nucleotide differences/compaired. Lactobacillus brevis ATCC 14869T Lactobacillus collinoides JCM 1123T Lactobacillus pentosus JCM 1558T Lactobacillus paraplantarum DSM 10667T Lactobacillus plantarum JCM 1149T Lactobacillus vermiforme ATCC 13133 Lactobacillus hilgardii ATCC 8290T Lactobacillus buchneri ATCC 4005T Lactobacillus fructivorans DSM 20203T Lactobacillus mali ATCC 27053T Lactobacillus arizonensis NRRL B-14768T Lactobacillus sanfranciscensis ATCC 27651T. M58810 AB005893 D79211 AJ306297 D79210 M59295 M58821 M58811 X76330 M58824 AF093757 X76327. 98.50 94.13 92.52 92.52 92.38 91.97 91.74 91.63 90.89 89.78 89.67 89.64. 12/799 47/800 60/802 60/802 61/801 63/785 65/787 65/777 73/801 80/783 82/794 83/801. garvieae KCTC 3772f L. species FSB-1 &b ÒÏ &' probiotics FÖ" L. acidophilus IFO 3025 R Ö> Wj "î . L. helveticus ATCC 8018~ ã Öº . ² Ôf &" FÒ ;ê~ ËÚ 6OË j ¾æÚîb, Î "öB SLP~ ³êö ~'b W ¸jæº ãËj "î . Þ Fj ªæ ªÒ"ö Bº B8, B49, B62" Îv &"f FÒ ;ê~ ËÚ 6OËj "î . ¾ þ~ «Ï& ËÚ çÒW Ö> probiotics ~ BB¢º Gj J r, L. garvieae KCTC 3772º B Vöç ¦'. ©b 6>îb, Fj ªæ ªÒ " ~ ãÖ, Ë ² Ìf6 Ò' ßW" Bifidobacterium jò b ÿ;B "& BÒ>º ^B6 ® î . V¢B n;'b ¸f ËÚ 6OËj , probiotics ~ Ï&ËW ¸f L. species FSB-1j «'b Fê ~, ê~ þj >¯~& . ËÚ 6OË Ö> FÖ Lactobacillus species FSB-1~ ÿ; ËÚ 6OË Ö> " « Fê>î~ L. species FSB-1f ãV& &ö ®b"öB f~¦V ªÒ ~ & 7 "B, ;' &V, Ò 5 z' ß W" 16S rDNA "VBªCj Û ÿ;j >¯~& . " Ò; *¶* ã(SEM)j Ï~ L. species FSB-1j &V Ö"(Fig. 5), çã £ 0.5-0.6 µm, ^ 3.0-5.0 µm~ *î b colonyº MRS VæçöB 7¦*& £* [V ï ~ ®(irregular) ;& . Ò 5 z' ßWj ¦ Æ Ö" Table 3ö ¾æ :f ?, Gram ·W, catalase r W 5 jÚÿW~ ßWj < ®îb, heteroBÎ;~ arginine rWj ¾æÚî . Ö" 6 " r, L. species FSB-1f Æ;'b Lactobacillus³ö ³~º ^b 6> î . L. species FSB-1~ BÎßW &¦ª z' ßWf Bergey’s Mannual of Systematic Bacteriology(26)öB Kandler f Weiss& >Ò Lactobacillus brevis~ ßW"ê ç ¦ª ¢ ~~& . Þ ;{ ÿ;j *~, 802 bp ^~ 16S rDNA ¦ª "VB ªC(dataº B~æ p~r)j >¯~, &N data base¢ Æ& FÒWj jv~& . L. species FSB-1~ 16S rDNA~ "VBf Table 4ö ¾æ :f ?, Lactobacillusö ³~º 99B~ « 5 j« 7 ß® Lactobacillus. brevis ATCC 14869T &"f 98.5%~ ¸f çÿWj &. . Ûç 16S rDNA gene~ "VB 97%¢ V&(16S rDNA full sequence: £ 1,500 bp)b «j 6~º " º^(36,37) " r, "º Lactobacillus brevis $º F" Òò>î . ç~ ª~' ßWj J r, ËÚ6OW Ö> " « Fê>î~ L. species FSB-1f Lactobacillus brevis « ÿ;>î .. º. £. *~ &ËÚ 6ïö &~ Ö> 6OßWj <º probiotic FÖ"¢ Fê Ï'b, colonic mucin-binding assay ¢ n~ '~ ªC j ¦Æ Ö", microtiter plate ~ wellö & colonic mucin~ ¦Of pH 4.8, biotinylated SLP~ ³êº 5.0 µg/mL, 6 HRP-conjugated streptoavidinf 24,000V CÏ, TMB~ Bï*f 10ª~ öB G; '~ Ö"¢ ¾æî . ÿ öB assay systemj Ï ãÖ, ËÚ 6OË G; 5 Ö> FÖ"~ Fêö ®Ú ³~ Ò*W ®º Ö"¢ B > ®b, *~ &Ë ö & FÖ~ 6OßWj ;ï'b ªC > ®rj "î . " 32« 5 Fj ªæ F¾~ ªÒ" 18« j C 50«~ FÖ"ö &~, colonic mucin-binding assay¢ Ï~ &Ë mucinö & ÖËj jv Ö", L. species FSB-1 &Ë ¸f ÖËj "î . V¢B L. species FSB-1j &çb ;' ßW, Ò 5 z' ß W" 16S rDNAö & ¦ª "VB ªCj ÿ;. þj >¯ Ö", ËÚ 6OË Ö>" FêB L. species FSB-1f Lactobacillus brevis « ÿ;>î .. ^. ò. 1. Fernandes CF, Shahani KM. Anticarcinogenic and immunological properties of dietary Lactobacilli. J. Food Prot. 53: 704-710 (1990) 2. Fang H, Elina T, Heikki A, Seppo S. Modulation of humoral immune response through probiotic intake. FEMS Immunol. Med. Microbiol. 29: 47-52 (2000) 3. Goldin BR. Health benefits of probiotics. Br. J. Nutr. 80: s203s207 (1998) 4. Salminen S, Ouwehand A, Benno Y, Lee YK. Probiotics: how should they be defined? Trends Food Sci. Technol. 10: 107-110 (1999) 5. Sherwood L, Gorbach MD. Probiotics and gastrointestinal health..
(9) ËÚ 6OË Ö> FÖ"~ Fê Am. J. Gastroenterol. 95: s2-s4 (2000) 6. Ouwehand AC, Kirjavainen PV, Shortt C, Salminen S. Probiotics: mechanisms and established effects. Int. Dairy J. 9: 43-52 (1999) 7. Gill HS. Probiotics to enhance anti-infective defences in the gastrointestinal tract. Best Prac. Res. Clin. Gastroenterol. 17: 755773 (2003) 8. Meydani SN, Ha WK. Immunologic effects of yogurt. Am. J. Clin. Nutr. 71: 821-872 (2000) 9. Coconnier MH, Klaenhammer TR, Kerneis S, Bernet MF, Servin AL. Protein-mediated adhesion of Lactobacillus acidophilus BG2FO4 on human enterocyte and mucus-secreting cell lines in culture. Appl. Environ. Microbiol. 58: 2034-2039 (1992) 10. Ouwehand AC, Tuomola EM, Tolkko S, Salminen S. Assessment of adhesion properties of novel probiotic strains to human intestinal mucus. Int. J. Food Microbiol. 64: 119-126 (2001) 11. Ouwehand AC, Niemi P, Salminen S. The normal faecal microflora does not affect the adhesion of probiotic bacteria in vitro. FEMS Microbiol. 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