Biological and technological application of kefiran, an EPS from - PowerPoint PPT Presentation

Biological and technological application of kefiran, an EPS from kefir grains Analía G Abraham Centro de Investigación y Desarrollo en Criotecnología de Alimentos CIDCA UNLP- CONICET. La Plata. Argentina

Kefiran Non-digerible polysaccharide composed of glucose and galactose OH H H glucose O HO HO H H OH H OH OH OH H O galactose H HO H H OH H OH Produced by lactic acid Kooiman 1968, Micheli et al 1999 bacteria (GRAS) included into kefir grains

Kefir grains Kefir grains are natural immobilized starter consisting of a complex association of microorganisms Microorganisms are embedded in � Lactic acid bacteria a proteins and (lactococcus, homo y hetero polysaccharide fermentatives lactobacillus) matrix. � Yeast � Acetic acid bacteria

Kefir grains Milk or whey Incubation at 20 °C Filtration plastic sieve grains Fermented product

Kefir grains Grain weight increases a function of subcultures 12 Milk leche 10 Whey suero Weight (g) 8 Peso (g) 6 4 2 0 0 1 2 3 4 5 6 7 8 9 10 Subcultures Subcultivos

Kefir grain composition Milk Whey � Protein 5-6% 6-8% � Polysaccharide 8-9% 8-9% � water 86-87% 83-84% 1.8 10 8 cfu/g � LAB 2.1 10 7 cfu/g � Yeast 2.5 10 5 cfu/g � AAB

Kefiran: Physicochemical properties Molecular mass by gel Intrinsic Viscosity permeation chromatography 6.0 ± 0.05 dL/g MW> 4 10 6 7 8 9 10 Tiempo de retención Esfuerzo de corte (mPa) 0,3g/l Shear stress (Pa) 1,0g/l 5900 2,0g/l 3,0g/l 6,0g/l 3900 1900 -100 0 100 200 300 400 500 Shear rate (s -1 ) Velocidad de deformación (1/s)

Polysaccharide and biological function Polysaccharides and oligosaccharides Functional Aspects • Prebiotic Dietary fibers • Non-digerible fibers FOSs GOSs • Immune system stimulation inuline • Antiulcer lactulose • Antitumoral

Polysaccharide and biological function Kefiran Effect of kefiran on cytopathic events triggered by Bacillus cereus on Caco 2 cells (In vitro study)

B. cereus : virulence factors EXTRACELLULAR VEGETATIVES CELLS FACTORS Association to High haemolytic epithelial cells and citotoxic activity Damages in enterocytes

Bacillus cereus on Caco 2 cells Bacillus cereus on Caco 2: Caco 2 preincubated with kefiran Scanning electron microscopy

Labelling of necrotic cells with Propidium Propidium iodide iodide Labelling of necrotic cells with Control B. cereus B. cereus + kefiran preincubation Kefiran antagonize cell death events triggered by B. cereus

B. cereus : virulence factors EXTRACELLULAR VEGETATIVES CELLS FACTORS Association to High haemolytic epithelial cells and citotoxic activity Damages in enterocytes

Effect of Supernatant of Bacillus cereus on Caco 2 cells Without kefiran with kefiran Scanning electron Propidium iodide microscopy labelling

In vivo studies: Balb C mice Increment in mucus producing cells in small gut Number of globet cells oral * 60 /10 vellosidades 50 administration * 40 30 of kefiran 20 10 0 Control 2 días7 días 1 1 1 Increment in number of IgA producing cells in lamina propria Control 2 days 7 days Control 7 days EPS *

Kefiran Fermented Grain 250mg/L 8g%

Technological application of kefiran Kefiran as Does Kefiran improve food texture additive of fermented food?

Rheological properties of glucono- δ -lactone induced skim milk gels Apparent viscosity at 300 seg-1(mPa/s) 60 Viscosidad aparente a 300 seg-1 50 40 30 Kefiran improves 20 texture of acid milk gels 10 0 0 100 200 300 400 500 Kefiran (mg/L) Kefiran (mg/L)

Technological application of kefiran : functional properties Kefiran as food Kefiran improves texture additive of fermented food Gel forming ability

Kefiran cryogels : Water holding capacity (WHC) 90.43 + 1.51% Microscopic aspect of cryogels (magnification 200X)

Technological application of kefiran : functional properties Kefiran as food Kefiran improves additive texture of fermented food Gel forming ability Edible films formation

Edible films formation 5g/l kefiran 7,5g/l 10g/l 57 cm2 Dry in oven at 50ºC

Films : Water vapour barrier properties Water vapour Water vapour Films permeability Films permeability LDPE: low density polyethylene. (WVP) (WVP) Kefiran (10 g/L) 5.43 × × 10 10 - Kefiran (10 g/L) 5.43 -11 11 Chitosan (10g/L) (10g/L) 9.03 × × 10 10 - Chitosan 9.03 -11 11 Cellophane Cellophane 8.4 × 8.4 × 10 10 - -11 11 LDPE LDPE 9.14 × 9.14 × 10 10 - -13 13 Films: mechanical properties Traction profiles Perfiles de tracción Tensile tests of films plasticized 9 with glycerol 8 kefiran 1.25g/l Glycerol 7 6 0g/l 2.5 g/l Glycerol 1,25g/l 5 2,5g/l 4 3.75 g/l Glycerol Texturometer 3,75g/l 3 Ta xT2i (TA.XT2i– 2 Stable Micro 1 Systems). 0 0 50 100 150 200 250 300 350 400 450 Elongation (%)

Kefiran Fermented product Grains 250mg/L 8g%

Fermented milk � Microorganisms with probiotic properties � High inhibitory capacity of pathogens and contaminant microorganisms. � Low lactose concentration (3.2%) � Functional components Kefiran Frozen Kefir

Stability Crystal size 30.3 ± 1.8 99 ± 41 Frozen kefir Frozen kefir with 36.8 ± 0.6 98 ± 40 cream 18.8 ± 1.1 87 ± 28 Frozen yogurt Commercial ice 27.7 ± 1.6 81 ± 32 cream Frozen kefir without cream has higher stability than frozen Kefiran yogurt and similar to commercial formulation

Frozen Kefir Kefir microflora maintains Kefir helado Frozen kefir viable at least 12 days at 7 10 –20°C CFU/ ml 6 10 5 10 0 12 25 25 0 12 Almacenamiento a-20 °C (dias) ) Storage at -20°C( days) Lactic Acid Bacteria Yeasts Non-trained sensorial panel High acceptability

Kefir grains Biological functionality Biological functionality Kefiran Technological functionality Technological functionality Fermented products Development of novel functional foods

Judith Piermaria Micaela Medrano Fernanda Hamet Pablo Rimada Jessica Minnaard Silvia Racedo Ivanna Rolny Alejandra Londero Pablo Pérez Marina Golowczyc Graciela Garrote Pablo Mobili Analia G Abraham Emiliano Kakisu Martin Humen Fernando Trejo ANPCyT David Romanin CONICET Maria Serradell UNLP Graciela De Antoni aga@biol.unlp.edu.ar