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Probiotic and technological characteristics of microorganisms isolated from a natural ecosystem, the Kefir grain. - CIDCA UNL P - Ctedra de Microbiologa General, Facultad de Ciencias Exactas, UNLP Graciela L. De Antoni - CIC-PBA -

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SLIDE 1
  • CIDCA
  • Cátedra de

Microbiología General, Facultad de Ciencias Exactas, UNLP

  • CIC-PBA
  • CONICET

Graciela L. De Antoni Probiotic and technological characteristics of microorganisms isolated from a natural ecosystem, the Kefir grain.

UNLP

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SLIDE 2

Researchers: Analía Abraham Graciela Garrote Pablo Pérez María Serradell Patricio de Urraza PHD students: Martin Humen Micaela Medrano Judith Piermaría Fernanda Hamet PHD students: Emiliano Kakisu Marina Golowczyc Pablo Mobili Fernando Trejo Ayelén Hugo Technicians: Lucia Brandi Mario Barrera

UNLP

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SLIDE 3

Kefir Fermented milk from the Caucasian mountains Described as a food in:

  • Código Alimentario Argentino Art

576 - (Res. MSyAS N° 295 del 14.04.99)

  • International Dairy Federation

Bulletin

UNLP

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SLIDE 4

Kefir was use in

* Treatment of gastrointestinal disorders * Treatment of respiratory disorders Kefir properties:

  • Antibacterial, antifungic and

antitumoral (in vitro and in animals)

  • Immunomodolatory (in animals)

Probiotic fermented milk

UNLP

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SLIDE 5

Homemade: with grains Industrial production in east Europe and Asia

“Kefir: a millenary fermented

milk with probiotic properties”

UNLP

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SLIDE 6

UNLP

Lactobacillus Lactococcus Leuconostoc Saccharomyces Candida Kluyveromyces Torula

Kefir grains Matrix polysaccharide-protein Yeast Lactic Acid Bacteria Acetic Acid Bacteria

108 – 109 microorganisms/gram

Source of strains safe and potentially probiotic

Acetobacter

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SLIDE 7

UNLP

Identification of isolates

  • Sugar fermentation
  • Whole-cell proteins by SDS-PAGE
  • Sequence of spacer 16S-23S rRNA
  • ARDRA
  • RAPD-PCR
  • FT-IR
  • Dra. Liliana Semorile UNQ
  • Dr. Osvaldo Yantorno CINDEFI
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SLIDE 8

UNLP UNLP

  • Lb. kefir
  • Lb. parakefir

22 Heterofermentative Lactobacilli

  • Lc. lactis subsp. lactis
  • Lc. lactis subsp. diacetylactis

22 lactococci

Kefir grains

20 Homofermentative Lactobacilli

Lb plantarum

40 yeasts

Kluyveromyces Saccharomyces

Selection of 5 strains resistant to gastrointestinal conditions : Lb. plantarum CIDCA 83114, Lb. kefir CIDCA 8348, Lc. lactis CIDCA 8221, Sc. cerevisiae CIDCA 8112, K. marxianus CIDCA 8154

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SLIDE 9

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1 2 3 4 5 6 7 8 9 10

Kluyveromyces marxianus CIDCA 8154

1- Glucose 2- Lactose 3- Milk (diluted 1:10) 4- Milk fermented with Lb. plantarum 83114 5- FM with yeast 8154 6- 83114 + 8154 (1: 1) 7- 83114 + 8154 (1: 3) 8- 83114 + 8154 (1: 5) 9- 83114 + 8154 (1: 8) 10- Commercial milk without lactose (diluted 1:10) Thin layer chromatography (TLC)

Milk fermented with Sugar content (g/100ml) ± DS*

  • L. plantarum (48 hs)

3,91 ± 0,13

  • K. marxianus (48 hs)

0,07 ± 0,02

  • L. plantarum + K. marxianus (48 hs)

0,07 ± 0,01 Non fermented milk Comercial lactose-free milk 4,03 ± 0,46 0,9

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SLIDE 10

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Log cfu/ml

5

  • 1

1 2 3 4 5 6 7 8

Bactericidal effect of SCS of

  • Lb. plantarum against EHEC 71935

time(h)

8338 8336 8318

83114

Strain CIDCA 83114

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SLIDE 11

SEM of Hep-2 monolayer incubated during 3 h with:

Control Lb. plantarum 114 106 Control buffer EHEC 106

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EHEC 106 EHEC 108 114 + EHEC 106 114 + EHEC 108

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LDH liberated by Vero cells by EHEC SCS preincubated or not with Lb. plantarum CIDCA 83114 and their isolated walls

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Porcentaje de LDH liberado

4 8 12 16 20 24 28 32

* *

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SLIDE 14
  • Lb. kefir

S-layer: Hydrophobic protein 66-71 kDa S-layer protein in SCS: 0.1 mg/ml

S-layer S-layer

SDS-PAGE

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SLIDE 15

Invation inhibition of Caco-2 cells by Salmonella preincubated with Lb. kefir SCS

Salmonella en PBS MRS # SN 8321 SN 83113 SN 8344 SN 8348 SN 8345

10 10

1

10

2

10

3

10

4

10

5

Invasión de Salmonella sp (UFC/ml)

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Caco-2 Caco-2 + Salmonella Caco-2 + Salmonella + S-layer

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SLIDE 16

Invasion inhibition of Caco-2 by Salmonella preincubated with S-layer protein

A A M R S . 1 m g / m l . 5 m g / m l . 1 m g / m l

10

3

10

4

*

Salmonella (CFU/ml)

*

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S-layer protein was detected on Salmonella surface by Ag-Ac reaction

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SLIDE 17

Adhesion of trophozoites of Giardia intestinalis WB on Caco-2 cells

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SLIDE 18

6.10 6.103

3

1.10 1.104

4

Trofozoites/ml Intestine content in infected animals

8/20 8/20 (40 %) (40 %) 2/20 2/20 (10 %) (10 %)

Infection rate (7 days post-inoculation 2,6 .107

control

Trofozoites per animal

treatment

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In In-

  • vivo assays

vivo assays

Mouse C57BL/6 (S. lipolytica CIDCA 812, L. plantarum CIDCA 83114,

  • L. kefir CIDCA 8348)
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SLIDE 19

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80 días a 6ºC el número de microorganismos viables :

  • L. plantarum CIDCA 83114: 1011 UFC/g
  • L. kefir CIDCA 8348: 108 UFC/g
  • S. cerevisiae CIDCA 82112: 106 UFC/g

Spray drying: microorganismos del tricepa

  • Leche en polvo reconstituída

al 11%

  • Temperaturas de salida: 70ºC
  • Temperatura de entrada: 180ºC
  • Presión: 5 Bar
  • 6
  • 5
  • 4
  • 3
  • 2
  • 1

ASD 15 30 45 60 80 time (d) log (N/No)

  • Dra. Paula Teixeira, Universidad Católica de Oporto
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SLIDE 20

Muchas Gracias

UNLP