指導老師 : 鄭伯智老師 林宏榮老師 指導教授 : 陳啟楨 Speaker: 林季怡 報告日期 :100.05.22

1. Functional properties of Lactobacillus strains isolated from kimchiSchool of Life Sciences, Handong Global University, Pohang, Gyungbuk 791-708, Korea 指導老師:鄭伯智老師 林宏榮老師 指導教授:陳啟楨 Speaker:林季怡 報告日期:100.05.22

2. Introduction

3. Kimchi is a Korean traditional fermented vegetable product and is gaining popularity as a functional food. • It is typically consumed in a raw (uncooked) state, and serves as a side-dish with practically every meal • The magazine Health mentioned kimchi in its list of the top five "World's Healthiest Foods"

4. Kimchi, being a lactic acid-fermented vegetable product consumed raw, is therefore also considered to be a good source of potentially beneficial and useful LAB • Among these, particularly the lactobacilli are of interest as beneficial inhabitants of the intestinal tract of man and animals.

5. A wide range of such desired roles have been suggested for diverse strainsof LAB. • Including immune stimulation, pathogen exclusion, productionof bioactive substances, and general intestinal health.

6. LAB play an important role inthe taste and quality of kimchi, and many strains associated withkimchi show antimicrobial activity in addition to other usefulproperties. • The over-ripening (or over-souring) of kimchi is the most seriousdefect in kimchi, and is mainly due to the overproduction of lactic acid by some strains of LAB.

7. The best way to overcome the problem of excessive acidproduction is to control the growth of LAB without harming thequality of the end product. • The major objective of the reported work was to study thefunctional properties of LAB from kimchi

8. 實驗架構

9. strains isolated from kimchi 16S rDNA sequencing by strains isolated from kimchi Physiological properties Determination of functional properties for selection of strains 6 tests 8 tests

10. 1、Strains *16S rDNA sequencing 2、Physiological properties * Carbohydrate fermentation *Hydrolysis of Arginine *Dextran production *Presence of haem-dependent catalase activity *Determination of lactic acid enantiomers produced *Detection of meso-diaminopimelic acid (m-DAP) 3、Determination of functional properties for selection of strains *Response to simulated stomach duodenum-passage (SSDP) *Determination of cell surface hydrophobicity *Adhesion properties to human cell lines (HT29 cell cultures) *In vitro adherence assay. *Agar diffusion method for antibiotic resistance *Detection of biogenic amine production *Cholesterol-related activities *Antimicrobial activit

11. Materials & Methods V.S Results

12. Strains

13. Strains • Lactic acid bacterial strains were isolated from home-made and commercial kimchi using MRS agar for plating. • pH of MRS-broth to 4.1 - 4.2 after 24 h at 30 °C. • These strains were identified by 16S rDNA sequencing using BLAST.

14. Strains • The srains were isolated at levels between 106 and 107/ml from kimchi samples. • Fermentation ability in simulated kimchi juice was checked, and pH values between 3.4 and 3.9 were reached within 24 h. • L. sakei: 11 strain L. plantarum: 1 strain

15. Physiological properties

16. Carbohydrate fermentation • In MRS borth fermentation broth without glucose but with chlorophenol red (0.04 g/l) as pH indicator. • The individual sugars were prepared as 2.5% (m/v) solutions and filter sterilized using a 0.45 μm filter • 25 μl of sugar solution was addedto 100 μl

17. Hydrolysis of Arginine • The experiment for the production of ammonia from arginine was carried according to Harrigan and McCance (1976). • MRS broth without ammonium citrate was used as growth medium

18. Dextran production • The strains were streaked onto MRS agar supplemented with 10% sucrose (Junsei, Japan) • After 24 - 48 h incubation at 30 °C, colonies were investigated for dextran (slime) production • Non-producers showed no change, and had normal shape and color.

19. Presence of haem-dependent catalase activity • Cells were grown aerobically at 30 °C in MRS broth containing 5 g glucose/l (Sigma, USA) supplemented with 30 μg haematin (Sigma, Switzerland). • After 24 h, catalase activity was determined by the presence of effervescence when adding 0.6% H202

20. Determination of lactic acid enantiomers produced • Strains were grown at 30 °C for 18-24 h in MRS broth with and without sodium acetate. • Lactic acid enantiomers were determined using an enzymatic kit • Based on the oxidation of D(-)-lactate and L(+)-lactate to pyruvate in the presence of NAD+

21. Detection of meso-diaminopimelic acid (m-DAP) • Presence of meso-DAP in the bacterial cell wall was determined using thin-layer chromatography on cellulose plates • The test described byTamang et al. (2000).

22. Detection of meso-diaminopimelic acid (m-DAP) 有些菌種的 L-Lysine 會被 meso-Diaminopimelic acid 所置換。 比如大腸桿菌：Escherichia coli。

25. Determination of functional properties for selection of strains

26. Response to simulated stomach duodenum-passage (SSDP) • This assay was designed to represent a simplified and standardized test system • Giving predictive values for the assumed survival of LAB in the human stomach and duodenum under simulated physiological conditions • The test described by Haberer et al. (2002).

27. Response to simulated stomach duodenum-passage (SSDP)

28. Determination of cell surface hydrophobicity • The test for bacterial adhesion to hydrocarbon was adopted to screen lactobacilli for cell surface hydrophobicity (Doyle and Rosenberg, 1995) • For hydrophobicity testing, bacteria were grown in MRS broth at 37 °C for 18 h.

29. Determination of cell surface hydrophobicity • Cells from 5 ml of culture were collected by centrifugation (9500×g) at 4 °C for 6 min. • The cells were washed twice with PBS. • One ml of this suspension was used to determine the OD580……(1)

30. Determination of cell surface hydrophobicity • In duplicate assessments, a further 1.5 ml of this suspension was added to an equal volume of n-hexadecane (Sigma, USA) and thoroughly mixed for 2 min using a vortex. • The phases were allowed to separate at room temperature for 30 min, after which one ml of the watery phase was removed and the OD580 was determined……(2)

31. Determination of cell surface hydrophobicity • The OD580 of duplicate assessments was meaned and used to calculate the degree of hydrophobicity. • Percentage hydrophobicity was calculated as follows: OD580 reading 1- OD580reading 2 OD580 reading 1×100=% hydrophobicity

32. Adhesion properties to human cell lines (HT29 cell cultures) • Mucus secreting HT29 cells were routinely grown in RPMI 1640 medium supplemented with 10% bovine calf serum (BCS),and 100 μg streptomycin and 100 U penicillin per mlat 37 °C in a 5% CO2 atmosphere. • For adhesion assays, HT29 monolayers were prepared in 24-well Costar® tissue plates (Corning Incorporated, USA) according to Schillinger et al. (2005).

33. Adhesion properties to human cell lines (HT29 cell cultures) • Cells were inoculated at a concentration of 5.4×105 cells per well to obtain confluence and cultured for 21 days prior to the adhesion assay.

34. In vitro adherence assay • The probiotic strain Lactobacillus rhamnosus GG was used as control

35. Determination of cell surface hydrophobicity and adhesion properties to human cell lines

36. Agar diffusion method for antibiotic resistance • The selected 12 strains were screened for their susceptibility to 8 antimicrobial agents • Lactobacillus plantarum299v as a relevant probiotic control strain

37. Agar diffusion method for antibiotic resistance • In the MRS and then these cultures were diluted with the same fresh medium to a density 105 CFU/spot for 18 hours at 37 °C. • Plates were incubated in air at 37 °C for 48 h and were examined for growth.

38. Agar diffusion method for antibiotic resistance • Minimum inhibitory concentration (MIC) was considered as the lowest concentration of the antimicrobial that inhibits bacterial growth

39. Antibiotic resistance

40. Detection of biogenic amine production • All strains were subcultured 5 to 10 times in MRS broth containing 0.1% of each precursor amino acid in order to promote the enzyme induction. • Sigma, USA *L-Tyrosine disodium salt *L-Histidinemonohydrochloride monohydrate *L-Ornithinemonohydrochloride *L-Lysine monohydrochloride

41. Detection of biogenic amine production • As a positive control, Enterococcusfaecalis ATCC 29212 was used, and a positive reaction with tyrosine was detected. • All selected strains were negative in this test

42. Cholesterol-related activities • All selected strains showed deconjugation ability of bile salts, as was indicated by the BSH test on agar plates • Cultures were screened for bile salt hydrolase (BSH) activity by spotting 10 μl of a culture grown in MRS broth onto BSH screening medium

43. Cholesterol-related activities • MRS agar plates supplemented with 0.5% (w/v) sodium salt of TDCA (taurodeoxycholic acid) and 0.37 g CaCl2/l • Plates were incubated anaerobically in an anaerobic jar (Difco, USA) at 37 °C . • After which BSH activity was semi-quantified by measuring the diameter of precipitation zones..

44. Cholesterol assimilation in vitro was determined by growing cells • In MRS broth supplemented with 0.5% w/v TDCA and 0.1 g of water soluble cholesterol/l at 37 °C . • Following incubation, bacterial cells were harvested by centrifugation (10,000 rpm, for 5 min) and the supernatant and un-inoculated control MRS broth

45. Cholesterol lowering effects

46. Antimicrobial activity • Using the paper disc method, 100 μl culture of each test strain were spread on LB agar plate by sterile swab. • Sterile 6 mm discs (Whatman,England) were placed on the agar and 40 μl of cultures of selected LAB strains were inoculated onto the disc.

47. Antimicrobial activity • After incubation for 12 h at 37 °C, the diameter of each clear zone was determined. • Hydrogen peroxide production was determined according to Eschenbach et al. (1989). • Presumptive bacteriocin formation was determined according to Choi et al. (2000).

48. Antimicrobial activity

49. Discussion

50. Carbohydrate fermentation • The 9 L(+) lactic acid producing strains of L. sakei hydrolyzed arginine, fermented a wider array of sugars, and showed diversity in their ability to ferment amygdalin, cellobiose, mannitol, trehalose, gentiobiose and salicin • The 2 DL lactic acid could only ferment 6 (galactose, glucose, fructose, mannose, N-acetyl-glucosamine, salicin) out of 49 sugars