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1 酛 Bifidobacterium bifidum CCRC 14146B. catenulatum CCRC Lactobacillus acidophilus CCRC ph 4.6 P B. bifidum CCRC ph cfu/ml P cfu/ml 1

2 B. bifidum CCRC 14146B 酛 Streptococcus thermophilus CCRC 12257ST1:1 43 STB P cfu/ml cfu/ml resistant starch 1:1 B. bifidum CCRC B. longum CCRC log cfu/ml 4 log cfu/ml 3 log cfu/ml 4 35 B. longum CCRC log cfu/ml : 2

3 2000Ziemer and Gibson, 1998 Chevalier et al., 1991Ziemer and Gibson, 1998 ; Steer et al., 2000 Ziemer and Gibson, 1998 ; Crittenden et al.,

4 resistant starch 4

5 1899 Tissier Bacillus bifidus 1910 Metchnikoff cfu/g cfu/g cfu/g cfu/g 2000Fooks et al., 1999Steer et al.,

6 Clostridium Staphylococcus Aureus Enteropathogenic Escherichia coli Ziemer and Gibson, Steer et al., Ziemer and Gibson,

7 Ziemer and Gibson, 1998 Steer et al., 2000 probiotics 1989 Fuller 2000Ziemer and Gibson, 7

8 1998Steer et al., 2000Bielecka et al., 2002Mattila-Sandholm et al., 2002 Krasaekoopt et al., 2003 Bifidobacterium Lactobacillus Enterococcus. 2000Brown et al., 1998 酶 Lapierre et al., 1992Sun and Griffiths, 2000 酶 phosphofructose kinase EMP Ostlie et al.,

9 2000 Krasaekoopt et al., 2003 Ostlie et al., 2003 bacteriocin 肽 Ziemer and Gibson, 1998Krasaekoopt et al., 2003 ph Ostlie et al ph 2.0 Marteau et al., 1997Wang et al. (1999) Trindade and Grosso (2002) ph ph ph 1 Tripathi et al.2002 ph 4.5 Chung et al ph

10 BK Ziemer and Gibson, 1998 Gomes and Malcata, Lapierre et al., 1992Bearson et al., 1997 Ziemer and Gibson, 1998Steer et al., 2000Sun and Griffiths, 2000 Bielecka et al., 2002Krasaekoopt et al., 2003 Hs0074 Caco-2 diffusely adhering E. coli Ouwehand and Salminen, 1998 L. plantarum 0407 B. bifidum Bb12 supernatant E. coil 9517 Salmonella enteritidis 4444 Fooks and Gibson,

11 2. Hydroxymethyglutaric-CoA reductase Homma, 1998 Xiao et al., mg/dl Lapierre et al., 1992Ziemer and Gibson, 1998 Bielecka et al., 2002Krasaekoopt et al., 2003 ; Xiao et al., lactose intolerance β- 苷酶 beta-galactosidase β- 苷酶 β- 苷酶 β- 11

12 苷酶 酶 Ziemer and Gibson, 1998 Bielecka et al., 2002Krasaekoopt et al., 2003 β- 苷酶 Ouwehand and Salminen, AIgA Ziemer and Gibson, 1998Steer et al., 2000Bielecka et al., 2002Krasaekoopt et al., 2003 IgG2a IgM ; Ouwehand and Salminen, 1998 ; Shu and Gill,

13 5. Ziemer and Gibson, 哚 Lapierre et al., 1992Ziemer and Gibson, 1998Krasaekoopt et al., BK BK Ziemer and Gibson,

14 70 Krasaekoopt et al., 2003Ostlie et al., 2003 Mattila-Sandholm et al., 2002 酛 Streptococcus thermophilus Lactobacillus bulgaricus (CNS) 10 7 cfu/ml 10 6 cfu/ml 10 8 cfu ml Krasaekoopt et al.,

15 prebiotics Brown et al., 1998Ziemer and Gibson, 1998) Ziemer and Gibson, 1998 Steer et al.,

16 Bielecka et al., 2002 synbiotics Brown et al., 1998Silvi et al., 1999Crittenden et al., 2001 ; Bielecka et al., 2002 Charalampopoulos et al., 2002 Bielecka et al., 2002Charalampopoulos et al.,

17 extrusion, droplet method emulsion, two-phase system hydrocolloids setting bath water-in-oil 25 µm-2 mm 17

18 Krasaekoopt et al., 2003 alginateκ- κ-carrageenan CAP cellulose acetate phthalate chitosan gellan gum xanthan gum resistant starch arabic gum gelatin 2000Jankowski et al., 1997Sultana et al., 2000Dembezynski and Jankowski, 2002Hansen et al., 2002 Hansen et al., 2002Sultana et al., 2000 Jankowski et al., 1997Sultana et al., 2000Dembezynski and Jankowski, 2002 Gaserod et al.,

19 Sun and Griffiths, 2000 κ- Adhikari et al., 2000 CAP CAP Favaro-Trindade and Grosso, 2002 Desmond et al., 2002 resistant starch α1-4 α1-6 pancreatic α-amylase resistant starch 1997Brown et al.1998 Yue and Waring1998 : 19

20 Yue and Waring1998 Silvi et al.1999haralampu2000 Thompson : RS1 RS1 2. RS2 RS2 20

21 RS3 RS

22 15 ( 1999Goni et. al., kcal/g RS P0.05 Cairns et al., Brown et al., 1998 ph ph Cairns et al., 1996Brown et al., 1998Silvi et al.,

23 ph Wang et al., 1999 Brown et al., 1998Wang et al., 1999Haralampu, 2000Thompson,

24 -80 Table 1. Bacteria used in experiment Bacteria Strain Bifidobacterium bifidum CCRC MRS, 37 Bifidobacterium bifidum CCRC MRS, 37 Bifidobacterium longum CCRC MRS, 37 Bifidobacterium catenulatum CCRC MRS, 37 Lactobacillus acidophilus CCRC MRS, 37 Lactobacillus brevis CCRC MRS, 37 Streptococcus thermophilus CCRC MRS, 37 Lactobacillus bulgaricus CCRC MRS, 37 24

25 AnchorNew Zealand Milk Ltd. Composition Moisture 3.3 Crude protein 33.4 Crude fat 0.8 Carbohydrate 54.1 Ash 8.4 high amylose corn starch, National Starch. and Chemical Corp.Chicago) Composition% RS3 RS2 Available starch Resistant starch Fat < 1.0 < 1.0 Protein < 1.0 < 1.0 Ash < 1.0 <

26 55% Composition % Glucose Maltose 5-10 Maltotriose 3-5 Isomaltose Panose Isomaltotriose 3-5 Isomaltotetraose Other sugar Bile salt, Casamino acid, Peptone, Yeast extract, Tween 80, Difco TM Lactobacilli MRS, M17 broth, AgarDifco Laboratories, USA 2. Meat extract (Sigma, Louis, American) 3. Potassium chloride, hydrogen chloride, glucose 4. Sodium chloride, Lithum chloridemerck, Darmstadt, Germany 5. Sodium acetate, Manganese sulfate, Di-ammonium hydrogen citrate ( 26

27 6. L-cysteine HCl, Dipotassium hydrogenphosphate, Sodium hydroxide 7. Magnesium sulfate, Sodium propionate 8. MRS brothmerck Composition Peptone 1 Meat extract 0.8 Yeast extract 0.4 Dipotassium hydrogen citrate 0.2 Tween Di-ammonium hydrogen citrate 0.2 Sodium acetate 0.5 Magnesium sulfate 0.02 Manganese sulfate Carbohydrateglucose or resistant starch or isomaltooligosaccharide 2 27

28 1 Forma Scientific, USA Deng Yng, Japan Deng Yng, Japan DBL-120 2pH Cyberscan 510, Singapore 3 Hermile Model 2323K, Germany 4 Tomin Inc., Taiwan 5 Model DV-+ Broookfield, USA 6 7 : Hitachi S

29 MRS brothmrs0.05l-cysteine HCl0.5 Casamino acid 酛 酛 ph 2.0,

30 1. Hood and Zottola1988 Wang et al.1999 Sultana et al Hansen et al µl ph (0.2 M KCl/HCl buffer) 9.9 ml ph rpm 3 1mL MRS agar Noh and Gilliland1993Wang et al.1999sultana et al. 2000Hansen et al µl 0.3 ph 7.0 (0.2 M KCl/HCl buffer) 9.9 ml ph rpm 3 1 ml MRS agar

31 3. Hood and Zottola1988Lemeland and Laroche1991 Wang et al.1999 Sultana et al.2000 Hansen et al µl ph 2.0 (0.2 M KCl/HCl buffer) 19.8 ml ph rpm rpm µl ph µl 0.3 ph ml (0.2 M KCl/HCl buffer) ph rpm 3 1 ml MRS agar

32 酛 Wang et al.1999 Crittenden et al.2001 Fooks and Gibson rpm 5 0.9% NaCl RS3RS ml MRS agar

33 酛 酛 Crittenden et al B. bifidum CCRC 14146B. catenulatum CCRC L. acidophilus CCRC MRS broth MRS0.05L-cysteine HCl0.5 Casamino acid % MRS broth (MRS0.05L-cysteine HCl0.5Casamino acid) ml MRS agar

34 Crittenden et al.2001 B. bifidum CCRC B. catenulatum CCRC L. acidophilus CCRC MRS MRS0.05L-cysteine HCl0.5Casamino acid % NaCl 2% 14% solid non fat, SNF 37 ph 4.6 Dave and Shah1997 Adhikari et al.2000 Crittenden et al.2001 Torre et al.2003 B. bifidum CCRC B. catenulatum CCRC MRS MRS 0.05% L-cysteine HCl0.5% Casamino acid % NaCl 2% 酛 14% SNF 43 ph ml MRS agar

35 2% B. bifidum CCRC % SNF 37 2% 酛 3% RS3 RS2 14% SNF 43 ph ph 0 5 酛 1. 2% S. thermophilus CCRC 12257ST L. bulgaricus CCRC 14009LB B. bifidum CCRC 14146B 14% SNF 37 STB 1% B. bifidum CCRC 14146B S. thermophilus CCRC 12257ST 7% 14% SNF 43 ph Adhikar et al.2000vinderola et al.2000tharmaraj and Shah2003 Torre et al.2003 Akalin et al.2004 酛 0h ph 4.6 AF 4 35

36 0d ph ph 4.6 AF 4 0d 4 0d 1 ml S. thermophilus CCRC ST M17agar L. bulgaricus CCRC 14009LB 1.0 N ph 4.5 MRS agar B. bifidum CCRC 14146B MRS 0.2Lithum chloride 0.3Sodium propionate MRS-LP agar Lapierre et al., 1992Vinderola and Reinheimer, 1999Adhikari et al., 2000Vinderola et al., 2000Tharmaraj and Shah,

37 Wang et al.1999 Korus and Lii2003 酛 10 ml 7000 rpm 5 0.9% NaCl 0.9% NaCl 2 ml 15% RS3 2 ml 2 ml 100 µl ph M KCl/HCl buffer9.9 ml rpm 3 1 ml MRS agar

38 Wang et al.1999 Korus and Lii2003 酛 10 ml 7000 rpm 5 0.9% NaCl 0.9% NaCl 2 ml % RS3 RS2 2 ml 2 ml rpm % NaCl Dimantov et al Wang et al.1999 a b a 25Tween 80 2 min b ab 1 ml MRS agar 48 38

39 1. Dave and Shah1997Adhikari et al.2000crittenden et al.2001 Oliveira et al.2001 Torre et al SNF 酛 S. thermophilus CCRC : L. bulgaricus CCRC =1 : 1 43 ph ml MRS agar

40 ph Cyberscan ph Ariga et al Broookfield LV 3 Rawson and Marshall g 15 ml 13 cm 3000 rpm 15 Torre et al

41 Factorial design SAS 1999 Generallinear modelglm Duncan s new multiple range test 41

42 Ziemer and Gibson, 1998Wang et al., 1999 ; Favaro-Trindade and Grosso, 2002 酛 ph 1.0 ph cfu/ml ph 7.0 P 0.05 ph P 0.05 ph cfu/ml ph P0.05 B. bifidum CCRC 14146L. acidophilus CCRC L. brevis CCRC cfu/mlb. catenulatum CCRC 14667B. bifidum CCRC B. longum CCRC cfu/mlchung et al.1999 ph

43 Wang et al. (1999) Trindade and Grosso (2002) ph ph ph 1 Crittenden et al.2001 ph 2.0 Favaro-Trindade and Grosso 2002 ph L. acidophilus B. lactis ph cfu/ml Tripathi et al.2002 ph

44 1. Fig. 1. The acid resistant ability of probiotics. Initial: initial viable count. ph 7.0, ph 1.0 or ph 2.0: incubated in ph 7.0, ph 1.0 or ph 2.0 KCl/HCl buffer for 3 hours. Probiotics: B. bifidum CCRC 11844, B. longum CCRC 11847, B. bifidum CCRC 14146, B. catenulatum CCRC 14667, L. acidophilus CCRC and L. brevis CCRC

45 0.3% cfu/ml ph 7.0 P 0.05 P P0.05 B. bifidum CCRC 14146B. catenulatum CCRC 14667L. acidophilus CCRC L. brevis CCRC cfu/ml B. bifidum CCRC B. longum CCRC cfu/mlchung et al Favaro-Trindade and Grosso

46 2. Fig. 2. The bile resistant ability of probiotics. Initial: initial viable count. 0% bile: incubated in ph 7.0 KCl/HCl buffer for 3 hours. 0.3% bile: incubated in ph 7.0 KCl/HCl buffer containing 0.3% bile salt for 3 hours. Probiotics: B. bifidum CCRC 11844, B. longum CCRC 11847, B. bifidum CCRC 14146, B. catenulatum CCRC 14667, L. acidophilus CCRC and L. brevis CCRC

47 3 ph ph cfu/ml P B. bifidum CCRC B. longum CCRC P B. bifidum CCRC 14146, B. catenulatum CCRC 14667, L. acidophilus CCRC L. brevis CCRC P B. bifidum CCRC B. longum CCRC P0.05 B. bifidum CCRC 14146B. catenulatum CCRC L. acidophilus CCRC cfu/mll. brevis CCRC cfu/ml B. bifidum CCRC B. longum CCRC L. brevis CCRC cfu/ml 10 5 cfu/ml B. bifidum CCRC 14146B. 47

48 catenulatum CCRC L. acidophilus CCRC B. bifidum CCRC B. longum CCRC

49 3. Fig. 3. The survival of probiotics in simulated gastrointestinal conditions. Initial: initial viable count. ph 7.0: incubated in ph 7.0 KCl/HCl buffer for 6 hours. GI: incubated in ph 2.0 KCl/HCl buffer for 3 hours then in ph 7.0 KCl/HCl buffer containing 0.3% bile salt for 3 hours. Probiotics: B. bifidum CCRC 11844, B. longum CCRC 11847, B. bifidum CCRC 14146, B.catenulatum CCRC 14667, L. acidophilus CCRC and L. brevis CCRC

50 2 RS3 RS cfu/ml B. bifidum CCRC B. longum CCRC 11847B. bifidum CCRC 14146B. catenulatum CCRC 14667L. acidophilus CCRC L. brevis CCRC (1994)Bielecka et al.2002 Brown et al Silvi et al.1999 Fooks and Gibson

51 2. Table 2. Effects of resistant starch or isomaltooligosaccharide on the growth of probiotics Strain Log cfu/ml Glu RS2 RS3 Oligo ±0.03 ab 9.58±0.10 ba 8.24±0.03 cab 8.77±0.03 de ±0.03 aab 8.45±0.04 bc 7.95±0.29 cc 8.85±0.01 dd ±0.05 aa 8.52±0.03 bbc 8.45±0.03 ca 9.24±0.03 da ±0.02 ac 8.60±0.03 bb 8.41±0.01 ca 9.15±0.02 ac ±0.04 ac 8.54±0.04 bbc 8.39±0.04 ca 9.18±0.01 dbc ±0.05 ab 8.25±0.09 bd 8.16±0.06 bb 9.20±0.02 ab a-d : different letters in the same row indicate significant difference (P0.05). A-E : different letters in the same column indicate significant difference (P0.05). Glu: Glucose. RS: resistant starch. Oligo: isomaltooligosaccharide. probiotics: B. bifidum CCRC 11844, B. longum CCRC 11847, B. bifidum CCRC 14146, B. catenulatum CCRC 14667, L. acidophilus CCRC and L. brevis CCRC

52 酛 酛 Crittenden et al., B. bifidum CCRC 14146B. catenulatum CCRC L. acidophilus CCRC MRS cfu/ml Critenden et al

53 4. 37 Fig. 4. The growth curves of probiotics incubated at MRS broth at 37. MRS broth: MRS broth + 0.5% casamino acid % L-cysteine HCl. probiotics: B. bifidum CCRC 14146, B. catenulatum CCRC and L. acidophilus CCRC

54 5. 40 Fig. 5. The growth curves of probiotics incubated at MRS broth at 40. MRS brothmrs broth + 0.5% casamino acid % L-cysteine HCl. probiotics: B. bifidum CCRC 14146, B. catenulatum CCRC and L. acidophilus CCRC

55 6. 43 Fig. 6. The growth curves of probiotics incubated at MRS broth at 43. MRS broth: MRS broth + 0.5%casamino acid % L-cysteine HCl. probiotics: B. bifidum CCRC 14146, B. catenulatum CCRC and L. acidophilus CCRC

56 7. 45 Fig. 7. The growth curves of probiotics incubated at MRS broth at 45. MRS broth: MRS broth + 0.5% casamino acid % L-cysteine HCl. probiotics: B. bifidum CCRC 14146, B. catenulatum CCRC and L. acidophilus CCRC

57 2. Ostlie et al., 2003Crittenden et al., 2001 B. bifidum CCRC 14146B. catenulatum CCRC L. acidophilus CCRC ph B. bifidum CCRC ph 6.5 ph 4.6 L. acidophilus CCRC ph 6.09 B. catenulatum CCRC ph 5.43 B. bifidum CCRC 酛 57

58 3. 37 ph Table 3. The ph value of milk fermented at 37by probiotics Time (h) ± 0.13 a 6.20 ± 0.12 b 5.33 ± 0.11 c ± 0.12 a 6.18 ± 0.12 b 5.03 ± 0.10 c ± 0.12 a 6.15 ± 0.14 b 4.71 ± 0.06 c ± 0.12 a 6.14 ± 0.14 b 4.63 ± 0.05 c ± 0.06 a 6.13 ± 0.16 b ± 0.06 a 6.11 ± 0.16 b ± 0.07 a 6.09 ± 0.16 b probiotics: B. bifidum CCRC 14146B. catenulatum CCRC and L. acidophilus CCRC a-c : different letters in the same row indicate significant difference (P<0.05). 58

59 3. 4 ph 4.6 P0.05 ph B. bifidum CCRC B. catenulatum CCRC ph cfu/ml B. catenulatum CCRC ph 4.6 B. bifidum CCRC cfu/ml Crittenden et al B. bifidum CCRC cfu/ml 9 ph ph ph

60 4. Table 4. Effects of resistant starch or isomaltooligosaccharide on the fermented characteristics of probiotics in milk at 43 Carbohydrate Time to achieve ph4.6 (h) Log cfu/ml Control ± 0.29 a ± 0.29 a 8.66 ± ± % Glu ± 0.29 b ± 0.29 b 8.51 ± ± % RS ± 0.29 b ± 0.29 b 8.54 ± ± % RS ± 0.29 b ± 0.29 b 8.50 ± ± % RS ± 0.29 b ± 0.29 b 8.50 ± ± % RS ± 0.29 b ± 0.29 b 8.56 ± ± % RS ± 0.29 b ± 0.29 b 8.61 ± ± % RS ± 0.29 b ± 0.29 b 8.62 ± ± % Oligo ± 0.29 b ± 0.29 b 8.49 ± ± % Oligo ± 0.29 b ± 0.29 b 8.55 ± ± % Oligo ± 0.29 b ± 0.29 b 8.60 ± ± 0.03 The difference between control and different carbohydrates was significant difference (P0.05). Glu: Glucose. RS: resistant starch. Oligo: isomaltooligosaccharide. Probiotics: B. bifidum CCRC 14146B. catenulatum CCRC

61 8. Fig. 8. The viable counts of B. bifidum CCRC in probiotic yoghurt containing 3% resistant starch during 35d storage at 4. 61

62 9. ph Fig. 9. The ph of B. bifidum CCRC in probiotic yoghurt containing 3% resistant starch during 35d storage at 4 62

63 酛 酛 S. thermophilus, ST L. bulgaricus, LB H 2 O 2 Dave and Shah, 1997Crittenden et al., 2001Oliveira et al., 2001 酛 S. thermophilus CCRC ST B. bifidum CCRC 14146B 1:1 43 STB S. thermophilus CCRC 12257ST L. bulgaricus CCRC 14009LB 1:1 (STLB) 1. STB STLB ph S. thermophilus CCRC 酛 0h 10 6 cfu/ml ph 4.6 AF 4 0d 10 7 cfu/ml L. bulgaricus CCRC B. bifidum CCRC

64 酛 0h 10 6 cfu/ml ph 4.6 AF 4 0d 10 8 cfu/ml 10 8 cfu/mldave and Shah cfu/ml 1995 Crittenden et al.2001 Lactobacillus acidophilus Bifidobacterium 10 8 cfu/ml 64

65 5. Table 5. The viable counts of probiotic yoghurt and traditional yoghurt during fermentation period Time ST ( 10 5 cfu/ml) LB ( 10 5 cfu/ml) B ( 10 5 cfu/ml) STLB STB STLB STB 0h 19.00± ± ± ±7.09 AF ± ± ± ± d ± ± ± ± h: after the addition of starter culture into the milk. AF: after completion of yoghurt fermentation (ph 4.6). 0d: after overnight storage of yoghurt samples. ST: S. thermophilus CCRC LB: L. bulgaricus CCRC B: B. bifidum CCRC STLB: S. thermophilus CCRC mixed with L. bulgaricus CCRC STB: S. thermophilus CCRC mixed with B. bifidum CCRC Yoghurt milk: 14% SNF reconstituted milk with 7% sucrose. 65

66 2. 6 STB ph 酛 0h ph 6.5 ph 4.6 AF 4 0d ph 4.4 Dave and Shah1997 L. acidophilus 4 0d ph STB STLB 酛 0h 0.75 cp ph 4.6 AF cp 4 0d cp ph 4.6 AF % 4 0d % ph 4.6 AF 4 0d ph P

67 6. Table 6. The physicochemical characteristics of probiotic yoghurt Treatment Time ph Viscosity (cp) Syneresis (%) 0h 6.53 ± ± 0.06 STLB AF 4.63 ± 0.05 a ± ax ± 2.00 bx 0d 4.45 ± 0.15 ax ± 4.40 ay ± 1.88 by 0h 6.52 ± ± 0.03 STB AF 4.64 ± 0.04 a ± bx ± 1.76 ax 0d 4.42 ± 0.05 bx ± 4.62 by ± 2.79 ay a-b: different letters in the same STLB or STB indicate significant difference (P<0.05). x-y: different letters in the STLB and STB indicate significant difference (P<0.05). STLB: S. thermophilus CCRC mixed with L. bulgaricus CCRC STB: S. thermophilus CCRC mixed with B. bifidum CCRC Yoghurt milk: 14% SNF reconstituted milk with 7% sucrose. 67

68 3. 7 STB STLB P0.05 B. bifidum CCRC 酛 S. thermophilus CCRC B. bifidum CCRC Torre et al.2003 (S. thermophilus, ST) 68

69 7. Table 7. Sensory evaluation of probiotic yoghurt Treatment Appearance Odour Texture Thick Acidity Flavor Overall STLB 4.60 ± ± ± ± ± ± ± 0.42 STB 4.30 ± ± ± ± ± ± ± 0.42 The appearance, odour, texture, thick, acidity, flavor and overall acceptability of samples were scored on hedonic scale of 1-7; 7 was considered excellent, 4 acceptable and 1 extremely poor. The difference between STLB and STB was no significant difference (P0.05). STLB: S. thermophilus CCRC mixed with L. bulgaricus CCRC STB: S. thermophilus CCRC mixed with B. bifidum CCRC Yoghurt milk: 14% SNF reconstituted milk with 7% sucrose. 69

70 10 B. bifidum CCRC B. longum CCRC cfu/ml ph cfu/ml P 0.05 Wang et al

71 10. Fig. 10. The effects of resistant starch on the acid resistant ability of probiotics. Initial: initial viable count. ph 2.0: B. bifidum CCRC and B. longum CCRC in ph 2.0 KCl/HCl buffer for 3 hours. RS: mixed with resistant starch 3. 71

72 Wang et al

73 Fig. 11. The appearance of RS-microencapsulated cultures. probiotic 12. Fig. 12. SEM (scanning electron microscopy) images of uncooked RS3-microencapsulated cultures. 73

74 probiotic 13. Fig. 13. SEM (scanning electron microscopy) images of uncooked RS2-microencapsulated cultures. 74

75 probiotic 14. Fig. 14. SEM (scanning electron microscopy) images of precooked RS3-microencapsulated cultures. 75

76 probiotic 15. Fig. 15. SEM (scanning electron microscopy) images of precooked RS2-microencapsulated cultures. 76

77 16 25Tween 80 P cfu/ml Wang et al Tween 80 P

78 Log cfu/ml 16. Fig. 16. The viable counts of microencapsulated of B. bifidum CCRC and B. longum CCRC Tween 80: mixed with Tween

79 1. 17 B. bifidum CCRC cfu/ml ph cfu/ml 10 7 cfu/ml log B. longum CCRC cfu/ml ph cfu/ml 10 7 log B. longum CCRC B. bifidum CCRC B. bifidum CCRC B. longum CCRC Brown et al. (1998) Wang et al. (1999) 79

80 Log cfu/ml 17. Fig. 17. The acid resistant ability of precooked RS-microencapsulated cultures. Initial: initial viable count. ph 2.0 : B. bifidum CCRC and B. longum CCRC in ph 2.0 KCl/HCl buffer for 3 hours. RS: microencapsulation with precooked RS. 80

81 2. 18 B. bifidum CCRC cfu/ml cfu/ml cfu/ml 4 log 5 log B. longum CCRC cfu/ml cfu/ml cfu/ml 2 log 5 log B. longum CCRC B. bifidum CCRC ; Brown et al. (1998) 0.05 Wang et al

82 18. Fig. 18. The bile resistant ability of precooked RS-microencapsulated cultures. Initial: initial viable count. 0.3% bile: B. bifidum CCRC and B. longum CCRC in ph 7.0 KCl/HCl buffer containing 0.3% bile salt for 3 hours. RS: microencapsulation with precooked RS. 82

83 3. 19 B. bifidum CCRC cfu/ml 3 ph cfu/ml 10 4 cfu/ml 3 log B. longum CCRC cfu/ml 10 2 cfu/ml cfu/ml 3 2 log B. bifidum CCRC B. longum CCRC Wang et al CAP 83

84 Log cfu/ml 19. Fig. 19. The survival rate of precooked RS-microencapsulated cultures in simulated gastrointestinal conditions. Initial: initial viable count. GI: incubated in ph 2.0 KCl/HCl buffer for 3 hours then in ph 7.0 KCl/HCl buffer containing 0.3% bile salt for 3 hours. Probiotics: B. bifidum CCRC and B. longum CCRC RS: microencapsulation with precooked RS. 84

85 L. bulgaricus, LB H 2 O 2 Bifidobacterium bifidum CCRC S. thermophilus CCRC L. bulgaricus, LB 20 B. bifidum CCRC B. bifidum CCRC B. longum CCRC log 2 log B. longum CCRC Sultana et al

86 Adhikari et al.2000 κ- Hansen et al

87 20. B. bifidum CCRC Fig. 20. The viable counts of B. bifidum CCRC of microencapsulation with precooked RS in yoghurt stored at 4 during 35 days. Free: without microencapsulation. RS: microencapsulation with precooked RS. 87

88 21. B. longum CCRC Fig. 21. The viable counts of B. longum CCRC of microencapsulation with precooked RS in yoghurt stored at 4 during 35 days. Free: without microencapsulation. RS: microencapsulation with precooked RS. 88

89 B. bifidum CCRC 酛 S. thermophilus CCRC

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91 Ariga, H., T. Urashima, E. Michihata, M. Ito, N. Morizon, T. Kimura, and S. Takahashi Extracellular polysaccharide from encapsulated Streptococcus salivaricus subsp. thermophilus OR 901 isolated from commercial yogurt. J. Food Sci. 57: Bearson, S., B. Bearson, and J. W. Foster Acid stress responses in enterobacteria. FEMS Microbiol. Letters 147: Bielecka, M., E. Biedrzycka, and A. Majkowska Selection of probiotics and prebiotics for synbiotics and confirmation of their in vivo effectiveness. Food Res. Int. 35: Brown, I. L., X. Wang, D. L. Topping, M. J. Playne, and P. L. Conway High amylose maize starch as a versatile prebiotic for use with probiotic bacteria. Food Australia. 50(12): Cairns, P., V. J. Morris, R. L. Botham, and S. G. Ring Physicochemical studies on resistant starch in vitro and in vivo. J. Cereal Sci. 23: Charalampopoulos, D., R. Wang, S. S. Pandiella, and C. Webb Application of cereals and cereal components in functional foods: a review. Int. J. Food Microbiol. 79: Chevalier, P., D. Roy, and L. Savoie X-α-Gal-based medium for simultaneous enumeration of bifidobacteria and lactic acid bacteria in milk. J. Microbiol. Methods. 13: Chung, H. S., Y. B. Kim, S. L. Chun, and G. E. Ji Screening and selection of acid and bile resistant bifidobacteria. Int. J. Food Microbiol. 47: Crittenden, R. G., L. F. Morris, M. L. Harvey, L. T. Tran, H. L. Mitchell, and M. J. Playne Selection of a Bifidobacterium strain to complement resistant starch in a synbiotic yoghurt. J. Appl. Microbiol. 90:

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97 The Acid and Bile Resistant Ability of Probiotics and Its Application in Yoghurt Shu-Hui Yu The healthy benefits of probiotics have been demonstrated in recent years, but most probiotics are low acid- and bile-resistant. The survival of probiotics passing through the digestive tract is very low, and these probiotics do not grow well in milk. Therefore the cultures for probiotic yoghurt need to be selected or microencapsulated appropriately to produce healthy probiotic products. The purpose of this study was to investigate the acid and bile resistant ability of probiotics and its application in yoghurt. The acid and bile resistant probiotics were selected under ph 1.0, 2.0 and 0.3% bile potassium chloride buffer. The ability to hydrolyze resistant starchrs by these probiotics which grew on broth containing RS as the sole carbon source was measured. In addition, the growth ability of the acid and bile resistant probiotic culture in milk at 37 and 43was tested. Finally, the yoghurt was manufactured by using the strains that showed the best survival under gastric fluid tests and growth rate in milk in combination with yoghurt starter. The physicochemical characteristics and sensory evaluation were investigated. The results demonstrated that these probiotics utilized RS with different ability and three probiotics Bifidobacterium bifidum CCRC 14146, B. catenulatum CCRC 14667, and Lactobacillus acidophilus CCRC 10695had the highest survival under gastric fluid tests. Further study showed that the B. bifidum CCRC grew well in milk at 37 and 43and reached the ph 4.6 after 21 and 12 hours, respectively, with a viable count of 10 8 cfu/ml. The viable counts of B. bifidum CCRC could t enhance in probiotic yoghurt 97

98 containing resistant starch during 35d storage at 4. There were no significant differences of the sensory evaluation scores between probiotic yoghurt and traditional yoghurtp0.05. The viable counts of yoghurt starter and probiotics were 10 7 cfu/ml and 10 8 cfu/ml, respectively. According to this study, B. bifidum CCRC might be a promising new yoghurt culture for probiotic yoghurt. The low acid- and bile-resistant probiotics were microencapsulated by mixing with equal volume of the uncooked or precooked RS. Then, the viable counts of free and microencapsulated probiotics in the simulated digestive tract and yoghurt during storage were measured respectively. This study aimed to test how microencapsulation with RS affected the acid- and bile-resistant properties of probiotic culture. The results demonstrated that low acid- and bile-resistant probiotics were B. bifidum CCRC and B. longum CCRC Microencapsulation with precooked RS improved the acid- and bile-resistant by 1 and 4 log cfu/ml respectively compared with those of the free cultures. Furthermore, the counts of precooked RS-microencapsulated cultures were 3 log cfu/ml higher than free cultures in the simulated digestive tract test. The counts of precooked RS-microencapsulated B. longum CCRC were enhanced by 1 log cfu/ml in yoghurt stored at 4 for 35 days. Therefore the low acid- and bile-resistant probiotics could be microencapsulated by mixing with precooked resistant starch and application in probotic yoghurt. Key words: Probiotics, Acid and bile resistant, Resistant starch, Microencapsulation, Yoghurt. 98

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