Immobilized cell technology (Edited by Ayan Santra)

1. IMMOBILIZED CELL TECHNOLOGY IN
BEER, WINE AND DAIRY INDUSTRY.
Submitted to : DR. RS singh
Submitted by : mumtaj begum
19011022
MSc. hons .biotechnology 2nd year
PUNJABI UNIVERSITY ,
PATIALA .

2. CONTENTS :
• WHAT IS IMMOBILIZATION.
• INTRODUCTION.
• IMMOBILIZED CELL SYSTEM
• TYPES OF SUPPORTS
• WINE PRODUCTION WITH IMMOBILIZED CELL
• BEER PRODUCTION WITH IMMOBILIZED CELL
• DAIRY INDUSTRY PRODUCTION WITH IMMOBILIZED CELL .
• ADVANTAGES
• DISADVANTAGES

3. INTRODUCTION :
• Immobilization is defined as the imprisonment of cell or
enzymes in a particular support or matrix.
• The immobilized whole cell system is an alternative to
enzyme immobilization where the target cell is immobilized.
• The support or matrix allows the exchange of medium
containing substrate or effector or inhibitor molecule.
• Allows cells to be held in place throughout the reaction,
following which they are easily separated from the products
and may be used again.

4. IMMOBILIZED CELL TECHNOLOGY
IN WINE INDUSTRY

5.  IMMOBILIZED CELL SYSTEM
• Immobilized cell systems Cells can be kept inside bioreactors in
suspension (free cells) or immobilized in various supports.
• There are four main immobilization techniques for yeast cells:
1. Attachment to a surface,
2. Entrapment within a porous matrix
3. Cell aggregation (flocculation)
4. Containment behind barriers
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6. 1.Attachment to a surface
• The attachment to a surface can be done by natural adsorption,
electrostatic forces or covalent binding, with cross-linking agents.
• The attachment of cells to an organic or inorganic support may
be obtained also by creating chemical bonds (covalent) between
cells and the support using cross-linking agents.
• However, this immobilization procedure is generally incompatible
with cell viability, since the cross-linking agents are highly toxic
for the microbial cells decreasing their activity.
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7. • As consequence, this method of immobilization is no longer
used for microbial cells but still remains suitable for the
immobilization of enzymes.
• The surface of the immobilization support is important in the
process of adsorption of cells as rough surfaces allows the cell
retention into the support’s cavities.
• This immobilization technique is often used as it is an easy and
natural process that takes place spontaneously.
• In the last years, natural adsorption is the most used technique
for yeast cell immobilization and further applied in wine making.
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8. 2.Entrapment within a porous matrix
• Entrapment within a porous matrix can be performed by two
approaches:
• a) The cells are introduced in a porous material and, after
growing, their mobility is restricted by the presence of other cells
and by the matrix
b) A solid matrix is synthesized in situ around the cells. The cells
are incorporated in the matrix of a more or less rigid polymer.
• The polymers are synthetic such as polyacrylamide, or can be
made from proteins (gelatin, collagens) and polysaccharides
(cellulose, alginate, agar, and carrageenan).
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9. 3.Cell Aggregation or Flocculation
• Cell aggregation or flocculation can occur naturally or by using
artificial flocculating agents.
• It is a complex process connected with the expression of
flocculation genes such as FLO1, FLO5, FLO8 and FLO11.
• Yeast flocculation is an attractive method because of its
simplicity and low costs.
• The flocculation depends on various parameters such as pH,
nutrients, dissolved oxygen, medium composition and
fermentation conditions (temperature and agitation) as well as
the age of the cell.
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10. • In food industry, the main applications of the flocculation
are the alcohol production, some kind of beers and
sparkling wines (secondary fermentation).
• The flocculation is very important for the brewing industry
as it is an effective, environmentally friendly, easy and
without costs method to separate the yeast cells from beer
at the end of the fermentation.
• The flocculation of the yeast is a very important
characteristic also in the traditional making of sparkling
wines.
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11. 4.Containment Behind a Barrier
• Containment behind a barrier can be achieved by two main
methods:
• Entrapment of the cells in microcapsules and by the use of
microporous membrane filters (hollow fiber) or by cell
immobilization onto an interaction surface of two immiscible
liquids.
• The method based on the entrapment of cells in microcapsule or
encapsulations, consists firstly in entrapping the cells in a
spherical gel and posterior coating with a polymer such as
polyethyleneimine.
• Then, the gel is dissolved but the cells are left in suspension,
contained behind the polymer barrier.
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12. • The microporous membranes filters are normally made of
polymers, e.g. polyvinylchloride or polypropylene.
• The containment of the cells behind a barrier allows very high
cell concentrations. For this reason, the membranes used should
be freely permeable to nutrients and products released during
the fermentation , as well as mechanically resistant.
• This method of immobilization is normally used when a cell free
product is needed.
• The main disadvantages are related to mass transfer limitations
and the possibility of membrane fouling caused by the cell
growth.
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13.  Types of supports
• For successful industrial application of this technology the
proposed supports must ideally be of food grade
quality,abundant in nature and cost effective.
• These supports are mostly natural organic polysaccharides or
inorganic material abundant in nature. They may be used
without much modification
or after minor treatment to alter their properties(porosity,
surface charges, etc.), others can be commercially synthesized.
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14. • Examples of supports and techniques proposed in wine-making
in the recent years include the following:
1) Inorganic supports for cell immobilization in wine-making
2) Organic supports for cell immobilization in wine-making
3) Membrane systems for cell immobilization in wine-making
4) Natural supports for cell immobilization in wine-making
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15.  Wine production with immobilized
cells
• Immobilization technology is used in
various fermentation processes.
• Immobilized cells were used for
bioethanol production,cider production,
vinegar and brewing as well as for wine-
making.
• In our days, the induction of alcoholic
fermentation and malolactic fermentation
is done with starter cultures of cells, i.e.
pure culture of cells isolated and
developed for conducting wine
fermentations.
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16. • Most fermenters used in the winemaking industry are of a
batch type, i.e. separate lots (batches) and are individually
fermented till conclusion of the process.
• Some industries adopted continuous methods, because of its
advantages in controlling the yeast population and activity,
keeping them in their maximum.

17. • The environmental conditions of
continuous fermentations are favorable for
the yeast growth, thus the biomass
concentration is approximately two times
larger than traditional wine-making.
• One of the most important characteristic
of the continuous process is the high
volumetric productivity but, despite of its
potential advantages, it is only profitable
when working all year-round.
• Immobilized cell systems emerged as a
technique that provides also large amounts
of cells but is more economic than the free
cells continuous wine-making.
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18. Bear

19. INTRODUCTION TO BREWING
• Brewing is one of the oldest biotechnologies with history back
more than 8000 yrs.
• Brewing is the production of beer through steeping a starch
source (commonly cereal grains) in water and then fermenting
with yeast.
• Ingredients are water, fermentable starch source, brewing yeast,
flavoring agent & secondary starch source(adjunct).
• Main steps involved are
• Malting ,Milling, Mashing
• Wort filteration and boiling ,
• Hop addition, Fermentation ,Aging etc.
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20. CONTD..
• Fermentation is the essential part of the brewing process,
responsible for the formation of most flavor compounds, while
the secondary fermentation provides beer maturation and final
beer sensory properties.
• They are most time consuming steps in the overall beer
production.
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21. IMMOBILIZED CELL TECHNOLOGY (ICT) IN BEER
INDUSTRY
• ICT has been attracting continual attention in brewing industry
over past 30 yrs.
• Reasons are:-
▶ Faster fermentation rates.
▶ Increased volumetric productivity.
▶ Continuous operation.
• It is well established now in secondary fermentation and alcohol
free & low alcohol beer production.
• Key parameters of this technology are:
 Selections of carrier material
 Method of immobilisation together with the bioreactor design.
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22. CONTD..
• ICT processes have been designed for different stages in beer
production.
• 1. Bioflavoring during maturation
• 2 . Main fermentation
• 3 . Fermentation for production of alcohal free beer and low
alcohol beer.
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23. ADVANTAGES
• Main advantages of using immobilised cells for production of
beer are:
 Enhanced fermentation productivity due to higher biomass
densities
 Improved cell stability
 Easier implementation of continuous operation
 Improved operational control and flexibility
 Facilitated cell recovery and reuse
 Simplified downstream processing.
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24. BASIC MODES OF CELL IMMOBILIZATION USED
IN BREWING
• Cell immobilisation can be classified into four categories
based on the mechanism of cell localisation and the nature of
support material:
(i) Attachment to the support surface
(ii) Entrapment within a porous matrix
(iii) Containment behind or within a barrier
(iv) Self-aggregation
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25. ATTACHMENT TO THE SUPPORT SURFACE
• Micro-organisms adsorb spontaneously on a wide variety of
organic and inorganic supports.
• Yeast cells are immobilised by ionic attraction. Adsorption
affinity of yeast cells for glass was increased by pretreating
the cells in aluminium ions.
• DEAE cellulose supports have been commonly used for the
production of alcohol-free beer and maturation of green beer.
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26. ENTRAPMENT WITHIN A POROUS MATRIX
• It can be performed by two different basic methods:
 first is gel entrapment, the porous matrix is synthesised in situ
around the cells to be immobilised.
 In the second, cells are allowed to move into the preformed
porous matrix.
• Both methods provide cell protection from the fluid shear and
higher cell densities.
• The concept is that the matrix is porous enough for substrates and
products to traverse.
• Mechanical strength of matrix is important.
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27. CONTD..
• Polysaccharides (e.g. alginate, chitosan, pectate and
carrageenan), synthetic polymers (e.g. polyvinylalcohol, PVA)
and proteins (gelatine, collagen) can be used under mild
conditions forentrapment with minimal loss of viability.
• Mainly gel entrapmet is used.
• Gels are mostly used in form of spherical beads with diameters
ranging from about 0.3 to 5 mm.
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28. CONTAINMENT BEHIND OR WITHIN A BARRIER
 It includes
 Systems with a barrier formed around cells such as microcapsules
ie. microencapsulation
• Too expensive to be used in beer production
 Systems with cells contained within a compartment separated by
a preformed membrane such as hollow fibre and flat membrane
modules.
• Polymeric microfiltration or ultrafiltration membranes, ceramic,
silicone or ion exchange membranes are used.
 Mass transfer through the membrane is dependent on the pore
size and structure as well as on the hydrophobicity/hydrophilicity
and surface charge.
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29. SELF-AGGREGATION
• Passive techniques based on sedimentation capabilities
• Based on formation of cell clumps or floccules, which can be
naturally occurring as in the case of flocculent yeast strains, or
induced by addition of flocculating agents.
• Simplest and the least expensive method.
• It has been successfully exploited over almost 40 years by
Dominion Breweries in New Zealand.
• Flocculent yeast cells are separated from the beer in a conical
settler by gravity. Recycled back into the hold-up vessel to
increase the cell density and to achieve better control of the
fermentation rates.
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30. CARRIER SELECTION AND DESIGN
A list of the various carrier materials, which have been
investigated for application in beer production, is presented.
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31. REACTOR DESIGN
• ICT bioreactors can be classified into three categories,
depending on the location of immobilised cells:
• 1. STATIONARY PARTICLE REACTOR
• 2. MOVING AND MIXED PARTICLE REACTOR
• 3. MOVING SURFACE REACTOR
For fermented food or beverage production, bioreactors of
category (i) and (ii) are usually employed
• Various modifications and combinations of stirred tank,
packed-bed, fluidised-bed, gas-lift and membrane
reactors were proposed for different phases in beer
production.
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32. CONTD..
 Selection of the appropriate reactor must be based on critical
issues such as:
 Choice of cell carrier
 Supply and Removal of gases and solutes in the liquid phase
and removal of excess biomass formed.
 Reactor sterilisation and sterile transfer of immobilised
biocatalysts.
• Reactors that can be thoroughly sterilised and directly
inoculated with cells or cell-aggregates or reactors packed
with preformed porous carriers are desireable.
• Primary beer fermentation systems are much more sensitive
to contamination than the secondary fermentation systems.
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33. CONTD..
• Enterobacteria and acetic acid bacteria have been detected in
immobilised yeast bioreactors used for primary fermentation
while lactic acid bacteria were found in reactors used for
secondary fermentations.
 Various methods are available to fight development of
contaminants in an ICT bioreactor:
• Sulphite addition (widely used in the wine industry)
• Heat treatment, are also used in suspended and immobilised
cell systems.
• Use of high dilution rates or harsher environmental conditions
(e.g. pH, temperature, salt concentrations, …)
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34. IMMOBILIZED CELL TECHNOLOGY IN
DAIRY INDUSTRY
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35. INTRODUCTION
 Lactic acid bacteria (LAB) are widely used in the production
of fermented dairy products such as cheese, yoghurts and
creams because of their technological, nutritional and
eventual health properties.
 The production of organic acids and the resulting
acidification is essential for the production, development of
typical flavour and preservation of these products.
 The transformation of lactose by lactic cultures improves the
digestibility and various metabolic and enzymatic activities
of LAB lead to the production of volatile substances, which
contribute to flavour, aroma and texture developments in
fermented dairy products.
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36.  Probiotics are defined as microbial cells which transit the
gastrointestinal tract and which, in doing so, benefit the
health of the consumer.
 Among these micro-organisms, LAB and especially
Lactobacilli and Bifidobacteria are already used in many
probiotic dairy products.
 The dairy industry involves processing of raw milk into
products such as consumer milk, butter, cheese, yogurt,
condensed milk, dried milk (milk powder), and ice cream,
using processes such as chilling, pasteurization, and
homogenization.
 Typical by-products include buttermilk, whey, and their
derivatives.
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37.  The dairy industry posses many practical difficulties:
 Increased contamination
 High down streaming cost
 Unstable products
 Coupling of biomass and metabolite productions
 In order to overcome these drawbacks, Immobilization Cell
Technology (ICT) is applied. Immobilization refers to the “
technique in which a macromolecule or cell is confined
spatially i.e. they are associated with a support material
either in soluble or insoluble form, which limits its free
movement so that it can be retained there and reused in
successive process runs”.
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38.  Immobilization modifies:
1) Physiology of cells.
2) Affects the sensitivity of LAB to salt and penicillin.
 ICT can be used:
 To produce starters for the dairy industry
 Aspects of biomass production in beads
 Continuous cell release from beads
 Continuous fermentations with filtration cell recycle are
examined.
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39.  Advantages of Cell Immobilization as compared
with Free-Cell (FC) Systems :
1) High cell density and very high volumetric productivity.
2) Reuse of biocatalysts.
3) High process stability (physical and biological) over long
fermentation periods.
4) Retention of plasmid-bearing cells.
5) Improved resistance to contamination.
6) Uncoupling of biomass and metabolite productions.
7) Stimulation of production and secretion of secondary
metabolites.
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40. 40

41. The purpose of these techniques is either to retain high
cell concentrations within the bioreactor or to protect
cells from a hostile environment.
 For industrial applications in the food industry, the
carrier material must be non-toxic, readily available
and affordable.
 It should also lead to high-cell loading and the cells
should have a prolonged viability in the support.
For food applications, the most widely used
immobilization technique is the entrapment of cells
within a food-grade porous polymeric matrix.
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42. Thermal (κ-carrageenan, gellan, agarose, gelatin) or
ionotropic (alginate, chitosan) gelation of the droplets
are used to produce spherical gel biocatalysts.
These polymers are readily available and widely
accepted for use as additives in the food and
particularly dairy.
Gel entrapment is a relatively simple method resulting
in usually spherical beads with diameters ranging
from 0.3 to 3.0 mm with high biomass concentration.
 A careful selection of polymer composition is
necessary to achieve high mechanical stability of gel
biocatalysts during long-term fermentation.
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43. LAB Immobilization By Entrapment
technique
SUPPORT SPECIES MAXIMUM CELL
CONCENTRATION
1) Ca-alginate Lactococcus lactis ssp. 2 x 1011 cfu ml-1
2) Ca-alginate Lactococcus lactis ssp. 3.8 x 1011 cfu ml-1
3) k-carrageenan-LBGb Lactobacillus casei 5.1 x 1011 cfu ml-1
4) k-carrageenan-LBGb Lactococcus lactis ssp. 1.3x1011 cfu g-1
5) Gellan gum Bifidobacterium longum 6.8 x 1010 cfu g-1
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44. Various dairy products
 Yogurt production:
• Yogurt posses organoleptic, nutritional and eventually
therapeutic qualities. Continuous yogurt prefermentation of
milk in a stirred tank reactor by entrapped cells in Calcium
alginate was proposed to increase performance.
 Cheese manufacturing:
 ICT demonstrates
 High productivity
 Better control of mixed culture with impact in the quality
 Economic process
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45. Quarg Cheese
 A soft, unripened, cow's milk cheese with the texture
and flavor of sour cream. Quarg comes in two versions-
low-fat and nonfat .
 The method of Quarg Cheese through ICT have been
proposed:
 Direct Vat inoculation with alginate immobilized freeze
dried starter
 Inoculation of milk
 Proliferation of milk to pH 5.5 in a bioreactor
containing bead with subsequent fermentation to ph
4.6 carried out by cell released by beads.
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46. Cream fermentation:
Advantages Of ICT:
• Better control of pH of final product
• Can use of a continuous system upto month
Frozen desserts
Yogurts cultures, Bifidobacterium , Lb.
acidophilus are used for this preparation.
ICT is responsible for improving quality of such
products by increasing properties of living cells
in the product.
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47. • The preparation of lactose-hydrolysed milk and whey,
using β -galactosidase.
• This is of great significance in a country like India where
lactose intolerance is quite prevalent.
• Lactose hydrolysis also enhances the sweetness and
solubility of the sugars, and can find future potentials in
preparation of a variety of dairy products.
• Lactose-hydrolysed whey may be used as a component
of whey-based beverages, leavening agents, feed stuffs,
or may be fermented to produce ethanol thus converting
an inexpensive byproduct into a highly nutritious, good
quality food ingredient.
• The first company to commercially hydrolyse lactose in
milk by immobilized lactase was Centrale del Latte of
Milan, Italy, utilizing the Snamprogetti technology. The
process makes use of a neutral lactase from yeast
entrapped in synthetic fibres.
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48. • Specialist Dairy Ingredients, a joint venture between the Milk
Marketing Board of England and Wales and Corning, had set up
an immobilized β -galctosidase plant in North Wales for the
production of lactose-hydrolysed whey.
• The β -galactosidase of fungal origin has been used for this
purpose.
• An immobilized preparation obtained by cross-linking β -
galactosidase in hen egg white (lyophilized dry powder) has
been used for the hydrolysis of lactose.
• A major problem in the large-scale continuous processing of
milk using immobilized enzyme is the microbial contamination
which has necessitated the introduction of intermittent
sanitation steps.
• A co-immobilizate obtained by binding of glucose oxidase on
the microbial cell wall using Con A has been used to minimize
the bacterial contamination.
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49. Control of contaminants
Yeast contamination
 Yeast is present due to introduction of unwanted microorganism.
Yeast introduction in bioreactor containing lactobaccilli
immobilized alginate beads, is not able to grow in system because
of rapid substrate turnover.
Bacteriophages contamination:
 It is a major concern for dairy industries in lactic fermentations.
Most bacteriophages aren’t destroyed by temperatures used for
milk pasteurization and raw milk will introduce phages in the
plant.
 When LAB are immobilized in alginate beads, the presence of
bacteriophages doesn’t significantly affect the acidification rate.
For ICT processes designed for the continuation of milk ,free cells
are not protected from phages. Thus phage control strategies have
to be developed or phage resistant strain are used.
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50. Use of Recycled immobilized cell
• Use of immobilized LAB as affinity matrices for removal of
unwanted compounds in liquid.
• Removal of heavy or radioactive metals from waste streams.
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51. REFERENCES
 C P Champagne, C Lacroix, I Sodini-GallotCritical Reviews in
Biotechnology1994; 14(2):109-34
 http://www.nisco.ch/download/27.pdf
 Cell Immobilization For The dairy Industry by Yann Doleyres
and Christophe Lacroix
 www.sciencedirect.com
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52. Thank you
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