Biological and technological application of kefiran, an EPS from

kefir grains

Analía G Abraham

Centro de Investigación y Desarrolloen Criotecnología de Alimentos

UNLP- CONICET. La Plata. Argentina

CIDCA

Kefiran

O

H

HO

H

HO

H

OH

OHHH

OH

O

OH

H

H

HO

H

OH

OHHH

OH

glucose

galactose

Kooiman 1968, Micheli et al 1999

Non-digerible polysaccharide composed of glucose and galactose

Produced by lactic acid bacteria (GRAS) included into kefir grains

Kefir grains

Lactic acid bacteria (lactococcus, homo y hetero fermentatives lactobacillus)

Yeast

Acetic acid bacteria

Kefir grains are natural immobilized starter consisting of a complex association of microorganisms

Microorganisms are embedded in a proteins and polysaccharide matrix.

Kefir grains

Incubation at 20 °C

Milk or whey

Filtration plastic sieve

Fermented product

grains

Kefir grains

Grain weight increases a function of subcultures

0

2

4

6

8

10

12

0 1 2 3 4 5 6 7 8 9 10Subcultivos

Peso

(g)

lechesuero

Subcultures

MilkWhey

Weigh

t (g

)

Protein

Polysaccharide

water

LAB

Yeast

AAB

Kefir grain composition

Milk

5-6%

8-9%

86-87%

Whey

6-8%

8-9%

83-84%1.8 108 cfu/g

2.1 107 cfu/g

2.5 105 cfu/g

6.0 ± 0.05 dL/g

Kefiran: Physicochemical properties

7 8 9 10

Tiempo de retención

MW> 4 106

Molecular mass by gel permeation chromatographyIntrinsic Viscosity

-100

1900

3900

5900

0 100 200 300 400 500

Velocidad de deformación (1/s)

Esfu

erzo

de

corte

(mPa

) 0,3g/l1,0g/l2,0g/l3,0g/l6,0g/l

Shear rate (s-1)

Shea

r st

ress

(Pa

)

Polysaccharides and oligosaccharides

Functional Aspects

•Prebiotic

•Non-digerible fibers

•Immune system stimulation

•Antiulcer

•Antitumoral

Polysaccharide and biological function

Dietary fibersFOSsGOSsinulinelactulose

Kefiran

Effect of kefiran on cytopathicevents triggered by Bacillus

cereus on Caco 2 cells (In vitro study)

Polysaccharide and biological function

B. cereus: virulence factors

VEGETATIVES CELLS EXTRACELLULAR FACTORS

High haemolyticand citotoxic

activity

Association to epithelial cells

Damages in enterocytes

Bacillus cereus on Caco 2:

Caco 2 preincubated with kefiran

Bacillus cereus on Caco 2 cells

Scanning electron microscopy

Control

preincubation

B. cereus

Kefiran antagonize cell death events triggered by B. cereus

Labelling of necrotic cells with Labelling of necrotic cells with PropidiumPropidium iodide iodide

B. cereus + kefiran

B. cereus: virulence factors

VEGETATIVES CELLS EXTRACELLULAR FACTORS

High haemolytic and citotoxic

activity

Association to epithelial cells

Damages in enterocytes

Withoutkefiran

Scanning electron microscopy

Propidium iodide labelling

with kefiran

Effect of Supernatant of Bacillus cereus on Caco 2 cells

*

In vivo studies: Balb C miceIncrement in mucus producing cells in small gut

Increment in number of IgAproducing cells in lamina propria

oral administration

of kefiran 0

1020304050

1

*

*

60

11Num

bero

fglo

betc

ells

/10

vello

sida

des

Control2 días7 días

Control 7 days EPS Control 2 days 7 days

8g%

Fermented

250mg/LGrain

Kefiran

Kefiran as food

additive

Does Kefiran improve texture

of fermented food?

Technological application of kefiran

0 100 200 300 400 5000

10

20

30

40

50

60

Visc

osid

ad a

pare

nte

a 30

0 se

g-1

Kefiran (mg/L)

Rheological properties of

glucono- δ-lactone induced skim milk gels

Kefiran (mg/L)

App

aren

t vis

cosi

ty a

t 300

seg

-1(m

Pa/s

)Kefiran improves

texture of acid milk gels

Kefiran as food

additiveKefiran improves

texture of fermented food

Technological application of kefiran : functional properties

Gel forming ability

Kefiran cryogels :

Water holding capacity (WHC)

90.43 + 1.51%Microscopic aspect of cryogels(magnification 200X)

Kefiran as food

additiveKefiran improves

texture of fermented food

Technological application of kefiran : functional properties

Gel forming ability

Edible films formation

kefiran

Edible films formation

5g/l

7,5g/l

10g/l

57 cm2

Dry in oven at 50ºC

Films: Water vapour barrier properties

FilmsFilmsWater vapour Water vapour permeability permeability

(WVP)(WVP)

ChitosanChitosan (10g/L)(10g/L) 9.03 9.03 ×× 1010--1111

Kefiran (10 g/L)Kefiran (10 g/L) 5.43 5.43 ×× 1010--1111

CellophaneCellophane 8.4 8.4 ×× 1010--1111

LDPELDPE 9.14 9.14 ×× 1010--1313

LDPE: low density polyethylene.

Perfiles de tracción

0

1

2

3

4

5

6

7

8

9

0 50 100 150 200 250 300 350 400 450

0g/l1,25g/l2,5g/l3,75g/l

Elongation (%)

Traction profiles Films: mechanical propertiesTensile tests of films plasticized with glycerol

TexturometerTa xT2i (TA.XT2i–Stable Micro Systems).

kefiran 1.25g/l Glycerol

2.5 g/l Glycerol

3.75 g/l Glycerol

Fermented product

8g%

250mg/LGrains

Kefiran

Frozen Kefir

Fermented milk

Microorganisms with probioticproperties

High inhibitory capacity of pathogens and contaminant microorganisms.

Low lactose concentration (3.2%)

Functional components Kefiran

Stability Crystal size

Frozen kefir 30.3±1.8 99±41

Frozen kefir with cream

36.8±0.6 98±40

Frozen yogurt 18.8±1.1 87±28Commercial ice

cream 27.7±1.6 81±32

Frozen kefir without cream has higher stability than frozen yogurt and similar to commercial formulation

Kefiran

Frozen Kefir

105

106

107

25120

CFU

/ ml

Almacenamiento a-20 °C (dias)) Lactic Acid Bacteria Yeasts

Kefir helado

Non-trained sensorial panel

High acceptability

Kefir microflora maintains viable at least 12 days at –20°C

Frozen kefir

0 12 25Storage at -20°C( days)

Fermentedproducts

Kefir grains

Kefiran

Development of novel functional foods

Biological functionality Biological functionality

Technological functionalityTechnological functionality

Judith Piermaria

Micaela Medrano

Fernanda Hamet

Pablo Rimada

Jessica Minnaard

Silvia Racedo

Ivanna Rolny

Pablo Pérez

Graciela Garrote

Analia G Abraham

Alejandra Londero

Marina Golowczyc

Pablo Mobili

Emiliano Kakisu

Martin Humen

Fernando Trejo

David Romanin

Maria Serradell

Graciela De Antoniaga@biol.unlp.edu.ar

ANPCyTCONICETUNLP