biological and technological application of kefiran, …...biological and technological application...
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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
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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
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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.
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Kefir grains
Incubation at 20 °C
Milk or whey
Filtration plastic sieve
Fermented product
grains
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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
)
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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
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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
)
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Polysaccharides and oligosaccharides
Functional Aspects
•Prebiotic
•Non-digerible fibers
•Immune system stimulation
•Antiulcer
•Antitumoral
Polysaccharide and biological function
Dietary fibersFOSsGOSsinulinelactulose
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Kefiran
Effect of kefiran on cytopathicevents triggered by Bacillus
cereus on Caco 2 cells (In vitro study)
Polysaccharide and biological function
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B. cereus: virulence factors
VEGETATIVES CELLS EXTRACELLULAR FACTORS
High haemolyticand citotoxic
activity
Association to epithelial cells
Damages in enterocytes
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Bacillus cereus on Caco 2:
Caco 2 preincubated with kefiran
Bacillus cereus on Caco 2 cells
Scanning electron microscopy
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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
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B. cereus: virulence factors
VEGETATIVES CELLS EXTRACELLULAR FACTORS
High haemolytic and citotoxic
activity
Association to epithelial cells
Damages in enterocytes
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Withoutkefiran
Scanning electron microscopy
Propidium iodide labelling
with kefiran
Effect of Supernatant of Bacillus cereus on Caco 2 cells
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*
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
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8g%
Fermented
250mg/LGrain
Kefiran
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Kefiran as food
additive
Does Kefiran improve texture
of fermented food?
Technological application of kefiran
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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
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Kefiran as food
additiveKefiran improves
texture of fermented food
Technological application of kefiran : functional properties
Gel forming ability
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Kefiran cryogels :
Water holding capacity (WHC)
90.43 + 1.51%Microscopic aspect of cryogels(magnification 200X)
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Kefiran as food
additiveKefiran improves
texture of fermented food
Technological application of kefiran : functional properties
Gel forming ability
Edible films formation
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kefiran
Edible films formation
5g/l
7,5g/l
10g/l
57 cm2
Dry in oven at 50ºC
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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
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Fermented product
8g%
250mg/LGrains
Kefiran
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Frozen Kefir
Fermented milk
Microorganisms with probioticproperties
High inhibitory capacity of pathogens and contaminant microorganisms.
Low lactose concentration (3.2%)
Functional components Kefiran
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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
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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)
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Fermentedproducts
Kefir grains
Kefiran
Development of novel functional foods
Biological functionality Biological functionality
Technological functionalityTechnological functionality
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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 [email protected]
ANPCyTCONICETUNLP
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