7/8/2019

Biomass production of probiotics

Dr. Gabriel Vinderola
Independent Researcher from CONICET
Dairy Products Institute (INLAIN, UNL-CONICET)
Associate Professor – Food Engineering Department
Faculty of Chemical Engineering
Universidad Nacional del Litoral
Santa Fe
gvinde@fiq.unl.edu.ar

Lactic Acid Bacteria, Microbiological and Functional Aspects (5th edition). April 2019.
Editors: Vinderola G, Salminen, S., Ouwehand A. and Von Wright A.
Taylor and Francis, CRC Press.

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Large scale biomass production of LAB/probiotics

Biomass Biomass Biomass

production harvesting storage

Transparent medium Freezing

Pelleted cultures
Continuos
Milky media Block cultures
centrifugation
Milk
Cheese whey Dehydration
Membrane
Ricotta whey Freeze-drying
filtration
Whey permeate Spray-drying

Typical growth curve in broth

8-12 h of culture 108-109 CFU/ml

Lag
period

Stationary
phase

3-6 h Exponential or log Death

phase phase

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Culture medium

MRS g/L
Polypeptone 10 70% of the cost
Meat extract 10
Yeast extract 5
Milky media
Glucose 20
(confidential
Tween 80 1,08 mL industrial formulation)
K2HPO4 2
Na acetate 5 Yeast extract
NH4 citrate 2 Glucose
Milk
Mg sulfate 0,2
C Vitamin
Mn sulfate
Mn sulfate 0,05
Mg sulfate
Fe sulfate
Na citrate
Na phosphate

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1 euro/L

UF

Milk Cheese whey (Lact, WP) Whey permeate (lactose)

No cell washing

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Commercial types of LAB/probiotic

cultures for use in foods
Frozen cultures: concentrated blocks or
drops/pellets (can be aliquoted) that must be
kept at -20°C / -70°C (1010-1011 CFU/g or ml).

Freeze-dried cultures: concentrated and dehydrated cells

that must be kept at 5°C / -20°C (1011-1012 CFU/g)

Live cells

Dead cells

LIVE/DEAD® Cell Viability Kit - nvitrogen

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The culture is not the same along the growth curve

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Technological factors that affect functionality: amount of

carbon source.

T. Sashihara et al. / Int. J. Food Microbiol. 120 (2007) 274–281

Technological factors that affect functionality: pH-

controlled growth and moment of harvesting.

T. Sashihara et al. / Int. J. Food Microbiol. 120 (2007) 274–281

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Large scale biomass production of probiotic bacteria

Freeze-drying: technologically and microbiologically feasible, very expensive

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Large scale biomass production of probiotic bacteria

Spray-drying: technologically feasible, strain-dependant, not expensive

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Freeze-drying: is the process of choice to preserve cultures without

metabolic activity.

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Freeze-drying: water removal below the triple point

(sublimation)

0,01 °C
6 mBar

Freeze-drying at the laboratory: freezing (-70°C) +

vacuum (20-24 hs 0,045 mbar)

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Freeze-drying in the industry: freezing (-196°C in N2) +

vacuum (24-72 h)

Bifidobacterial culture GV2 on nuclepore track edge membrane – Dr. Gabriel Vinderola
FEI Company XL30 SEM (10 KV, high vacuum mode) MBT – MRI, Nov 20098

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Freeze-dried bifidobacterial culture P12 (pH 5.0, 22 h) – Dr. Gabriel Vinderola

FEI Company XL30 SEM (10 KV, high vacuum mode) MBT – MRI, Dec 2009

Freeze-dried bifidobacterial culture P12 (pH 5.0, 22 h) – Dr. Gabriel Vinderola

FEI Company XL30 SEM (10 KV, high vacuum mode) MBT – MRI, Dec 2009

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Influence of growth phase

Influence of the composition of the culture medium

2% Glu, 0,5% NaCl

1% Glu, 1% Sac, 0,5% NaCl

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Application of a mild stress during growth

0,3 M NaCl

45 °C
50 °C 0,7 M NaCl

55 °C

Application of a mild stress during growth

Stress térmico
Fase estacionaria

Stress térmico
Fase log

Sin stress

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Protectants
Skim milk + ….

Compounds that penetrate the cell wall and the membrane: DMSO, glycerol.
They reduce inner ice formation

Compounds that penetrate cell wall: oligosacch., aa, polymers of low MW

They form a buffer layer between the membrane and the cell Wall, avoiding ice formation

Compounds that remain outside the cell: proteins and polysaccharides

They adsorb on the cell to form a viscous layer, they maintain an amorphous ice
structure close to the cell

Rate of freezing

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General conditions to achieve high rates of

survival to freeze-drying
Harvesting of cells in late stationary.
Apply some mild stress (pH, salts, heat).
Additon of non assimilable solutes (NaCl, extra
sugars).
Use of an “universal” protectant (skim mlik,
trehalose).
Fast freezing.
Highr relation freezing-surface/depth.

Factors that affect survival suring storage

Storage temperature
Moisture and aw
Environmental humidity
Oxygen
Light

Cryoprotectant composition
Tg (glass transition T) viability

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Moisture vs. Water activity and Glass Transition Temp.

100%
Moisture: % of water (w/w)
Water activity (aw)
Partial vapor pressure of water in a
substance in relation to pure water at the .
same temperature.

Glass transition temperature (Tg) Sólido

Temperature above which a glassy subtance
becomes rubbery (transition from a solid state to a
rubbery state, allowing water movement). Below Tg Fluido
water mobility is restricted.
Trehalose has one of the highest Tg, then its big protectant capacity. Proteins are quite stable above their
Tg, that is why mix of proteins and carbohidrates are better protectants than carbohidrates alone.

Influence of storage temperature

aw 0.22
12 months

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Influence of storage temperature

BB12

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Influence of aw at 21°C

Influence of cryoprotectant after 12 months of storage

aw 0,22

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Influence of atmosphere at 21°C

BB12

Combined effect of the container and the storage

temperature
25°C 4°C

glass

Vacuum, low permeability

N2 – high permeability
N2 – low permeability
Vacuum, high permeability

Vacuum, high permeability

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Incorporation into foods with different aw

Avena
Maltodextrina
Arena

25°C

Storage of L. acidophilus LA-2 in soy bars

4° C

25° C

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Conditions to maximize survival during storage

Low storage temperature

Low water activity

Vaccum sealing

Low permeability packaging

Protectants with high Tg

Spray drying is a continuous method

of producing a dry powder from a
liquid or slurry by rapidly drying with
a hot gas (air).

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Spray-drying setting

JBurgain et al. / J. Food Engin. 104 (2011) 467–483

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Survival to spray-drying

Spray-drying protectants and costs

Protectants (10-20%)
skim milk, whey proteins, maltodextrin, starch, trehalose,
betaine, adonitol, sucrose, glucose, lactose, dextran, polyethylene
glycol, gum acacia...

Costs

Domínguez, J.M. 2011 – Drying, Comprehensive Biotechnology, 2nd Edition

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Spray-drying of lactobacilli
in 10% skim milk

Inlet T: 170°C
Outlet T: 84°C
Air flux: 600 l/h
Feeding rate: 7ml/min
Aspiration rate: 38m3/h

SEM

TEM

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Humedad versus actividad de agua

aw: 0,11 aw: 0,11

Freeze drying
aw: 0,33
Spray drying
aw: 0,33
aw: 0,52 aw: 0,52

SD
FD

Next challenge…scaling-up production

Laboratory
Pilot plant

Industry

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Material Transfer Agreement (MTA)

12 monts, from march 2018.

Optimization of the biomass production. Survival to freeze-

drying and resistance to compression (capsules) and storage.

…some take home messages

Biomass production
Slight acidic medium (5-5.8), pH controlled, batch/fed batch fermentation.
Pay attention to the growth curve: when to harvest cells.
Freeze-drying
Reference method to preserve strains.
Technologically more demanding and costly.
Few variables condition survival along storage (wáter activity and storage
termperature).
Spray-drying
Not adequate for all strains.
Low cost, continuous method, highly available in the food industry.
Lots of variable must be managed at the same time during the process (inlet
and outlet T, feeding rate, air flux).

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Biomass production of probiotics

Dr. Gabriel Vinderola
Independent Researcher from CONICET
Dairy Products Institute (INLAIN, UNL-CONICET)
Associate Professor – Food Engineering Department
Faculty of Chemical Engineering
Universidad Nacional del Litoral
Santa Fe
gvinde@fiq.unl.edu.ar

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