INDUSTRIAL FOOD

MICROBIOLOGY
DECEMBER 7, 2022
DOWNSTREAM PROCESSING

q DETECTION AND ASSAY OF

FERMENTATION PRODUCTS
q SEPARATION AND PURIFICATION
PROCESSES
-THE VARIOUS PROCEDURES INVOLVED IN
RECOVERING USEFUL PRODUCTS AFTER
FERMENTATION OR ANY OTHER PROCESS
TOGETHER CONSTITUTE DOWNSTREAM
PROCESSING (DSP)

- A crucial stage in producing different products in the

pharmaceutical and food industries, etc.
DOWNSTREAM PROCESSING
- refers to the procedures required to
recover and purify fermentation-produced
products.
- the product is either present in the cell, in
the medium or in broth.
CRITERIA OF A GOOD RECOVERY
PROCESS
1. The intracellular or extracellular location of the product.
2. The concentration of the product in the fermentation broth.
3. The physical and chemical properties of the desired product (as
an aid to select separation procedures).
4. The intended use of the product.
5. The minimally acceptable standard of purity.
6. The magnitude of biohazard of the product or broth.
7. The impurities in the fermenter broth.
8. The marketable price for the product.
FOUR STAGES IN DSP AFTER
FERMENTATION
1. SOLID-LIQUID SEPARATION
2. RELEASE OF INTRACELLULAR PRODUCTS
3. PURIFICATION BY CHROMATOGRAPHY
4. FORMULATION
STAGE 1. SOLID-LIQUID
SEPARATION

1.FLOTATION
2.FLOCCULATION
3.FILTRATION
4.CENTRIFUGATION
FLOATATION
- The recovery of surface
active products
- Important variables: pH, air-
flow rates, surfactants and
surface-active collector ratios

Surfactants are termed,

collectors.
•B. subtilis - surfactin
•E. coli
•Chlorella
•Chlamydomonas
• Saccharomyces cerevisiae
FLOCCULATION
- the cells (or detritus) form huge clumps so they
can be easily removed.
- this process depends on the cell type and the
medium's ionic composition.
- requires the addition of flocculating agents
(inorganic salt, organic polyelectrolyte, mineral
hydrocolloid).
FLOCCULATION
§ They must react rapidly with the cells
§ They must be non-toxic.
§ They should not alter the chemical constituents of the cell.
§ They should have a minimum cohesive power to allow for
effective
§ Subsequent water removal by filtration.
§ Neither high acidity nor high alkalinity should result from their
addition.
§ They should be effective in small amounts and below in cost.
§ They should preferably be washable for reuse.
FILTRATION
- the most used method for separating
biomass and culture of bacteria and
filamentous fungi
- variables that affect filtration: the organism’s
size, the presence of other organisms, the
viscosity of the medium, and the
temperature.
FILTRATION
DIFFERENT TECHNIQUES OF
FILTRATION
1.ABSOLUTE FILTERS
2.DEPTH FILTRATION
3.MEMBRANE FILTERS (SURFACE
FILTRATION)
4.ROTATING DRUM VACUUM FILTRATION
ABSOLUTE FILTERS
- The particular pore diameters of these
filters are smaller than the particles to be
removed.
- Absolute filters can remove bacteria
from the culture media.
DEPTH FILTERS
- consists of a matrix of
filaments, such as glass wool,
asbestos, or filter paper.
- the matrix captures the
particles while the fluid
escapes.
- most efficient for filamentous
fungi
ROTARY DRUM VACUUM FILTER

• Simple and
consumes little
energy
• e.g., yeast cells and
filamentous fungus
• Filter aid -
Kieselguhr
MEMBRANE FILTERS

Dead-end filtration/static/frontal Cross-flow/tangential filtration

- the direction of suspension -the direction of suspension flow is
flow is normal to the filter parallel to the filter surface
surface.
CENTRIFUGATION
-It may be used for bacteria, usually
protein precipitates.
- It is predominantly employed to separate
solid particles from the liquid phase
(fluid/particle separation).
CENTRIFUGATION
Advantages:
1. Filtration is slow and difficult.
2. The cells or other suspended matter must
be obtained free of filter aids.
3. Continuous separation to a high standard
of hygiene is required.
Decanter centrifuge

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STAGE 2. PRIMARY PRODUCT
ISOLATION

Release of intracellular products

- several biotechnological products
(vitamins, enzymes) contained within
cells.
- Strategies: physical, chemical, and
enzymatic
II. PRIMARY PRODUCT ISOLATION

Release of intracellular products (cell

disruption)
- Strategies: physical, chemical, and enzymatic
- The selection of a particular approach depends
on the nature of the cells
- e.g., Gram-Negative bacteria and filamentous
fungi are easier to destroy than Gram-Positive
bacteria and yeasts.
Release of intracellular products
Physicochemical methods:
1. Liquid shear
2. Solid shear
3. Agitation with abrasives
4. Freeze-thawing
5. Ultrasonication
6. Hydrodynamic cavitation
Release of intracellular products
Chemical and biological methods:
1. Detergents
2. Osmotic shock
3. Alkali treatment
4. Enzyme treatment
5. Solvents
PHYSICOCHEMICAL: LIQUID SHEAR
(HIGH-PRESSURE HOMOGENIZERS

§ Often used in the processing of milk and other

products in the food industry
§ It involves forcing a high-pressure cell suspension
through a very narrow orifice to atmospheric
pressure.
SOLID-LIQUID SHEAR

§ Applicable for temperature labile enzymes

§ Frozen samples of microorganisms are passed at
very low temperatures (-25oc) through a small
orifice at very high pressure.
§ The breakage occurs due to the liquid shear and
the presence of ice crystals.
AGITATION WITH ABRASIVES

§ Cells are disrupted with the help of

mechanically resistant beads (glass, alumina
or titanium compounds).
§ The process is exothermic – an efficient
cooling system must be associated to
prevent thermal denaturation of the enzyme.
ULTRASONICATION
§ It is widely utilized but not appropriate for
industrial application on a broad scale due to
its high cost.
§ It is achieved with a high-frequency sound
produced electronically and transported
through a metallic tip to an appropriately
concentrated cellular suspension.
FREEZE-THAWING
§ A very mild process.

§ Due to repeated freezing and thawing of the

microbial cells, ice crystals generate, create pores
in the cell wall and facilitate the release of
enzymes.

§ The process is applicable in combination with other

methods.
HYDRODYNAMIC CAVITATION

§ It can also be generated by fluid flow.

§ When fluid flows through an orifice, an increase in

velocity is accompanied by a decrease in the
pressure of the fluid.
CHEMICAL METHODS: OSMOTIC
SHOCK
§ It is applied for the mild release of enzymes
from the cells.
§ It is very efficient and unique for releasing
luciferase enzymes from Photobacterium
fischeri.
CHEMICAL METHODS: OSMOTIC
SHOCK

§ A sudden change in the salt concentration changes

the osmotic balance within the cells, and the cell is
disrupted.

§ Not very efficient for microbial cells, which have tough

cell walls.

- Thus, the cell wall is first made weak by some other

method.
ALKALI TREATMENT
§ used for hydrolysis of microbial cell wall material
provided that the desired product will tolerate a ph
of 10.5–12.5 for up to 30 min.
§ Chemical costs can be high in terms of alkali
required and neutralization of the resulting lysate.
§ Darbyshire (1981) has reported the use of this
technique in the extraction of l-asparaginase.
DETERGENTS
§ Several detergents will damage the lipoproteins of the
microbial cell membrane and lead to the release of
intracellular components
§ e.g., quaternary ammonium compounds, sodium lauryl
sulfate, sodium dodecyl sulfate (SDS) and triton X-100.
§ anionic detergents such as sds disorganize the cell
membrane while cationic detergents are believed to act on
lipopolysaccharides and phospholipids of the membrane
§ nonionic detergents such as triton X-100 cause partial
solubilization of membrane proteins
DETERGENTS

§ Disadvantage: it may interfere with

purifying processes (salt precipitation).

§ Purification by ultrafiltration or ion-

exchange chromatography can circumvent
this constraint.
ENZYME TREATMENT
§ Lysozyme - the most widely utilized and commercially
available enzyme (produced from hen egg white).
§ Others from leycocytes, Streptomyces spp., Staphylococcus
spp., Micromonospora spp. Penicillium spp., Trichoderma spp.,
and snails.
§ Gram-positive bacteria (those with a high concentration of cell
wall mucopeptides) are more vulnerable to lysozyme activity.
§ Lysozyme plus edta can kill gram-negative bacteria by
destroying their cells. As lysozyme digests the cell wall, the
osmotic effects rupture the periplasmic membrane, releasing
the intracellular contents.
ENZYME TREATMENT
§ Lysozyme with proteases, glucanase and mannanase - used
to lyse yeast cell walls.
§ Disadvantage: the presence of the enzyme(s) may complicate
further downstream purification processes.
§ Enzyme lysis in large-scale operations is limited by the
availability and cost of appropriate enzymes.
§ The use of immobilized lysozyme may provide the solution to
such problems.
SOLVENTS
• It extracts the lipid components of the cell membrane.

• Applied across a wide range of microorganisms.

• e.g., alcohols, dimethyl sulfoxide, methyl ethyl ketone, and

toluene. – these are toxic, flammable, and can cause protein
denaturation, which requires careful consideration.

• Enzymes may also be used as a pretreatment to hydrolyze cell

walls before cell disruption by mechanical methods partially.
COMBINATION OF METHODS

A combination of physical, chemical, and enzymatic

processes are utilized to increase the efficiency of
cell disintegration in a cost-effective manner.
STAGE 3. PURIFICATION
§ The methods used are finer and further
eliminate the impurities thus leaving the
desired product purer.
STAGE 3. PURIFICATION BY
CHROMATOGRAPHY
§ Used to isolate and purify relatively low
concentrations of metabolic products from a
mixture of similar molecules (e.g., antibiotics-
homologous) and macromolecules (enzymes).

§ Liquid chromatography: the passage and separation

of different solutes as liquid (the mobile phase) is
passed through a column
CHROMATOGRAPHY TECHNIQUES:
1. Adsorption chromatography
2. Ion-exchange chromatography
3. Gel permeation chromatography
4. Affinity chromatography
5. Reverse phase chromatography
6. High performance liquid chromatography.
STAGE 4. FORMULATION

§ Refers to the preservation of biotechnology

products’ activity and stability during storage and
delivery
§ The final isolation of the product is done in one of
the two following ways:
(i) Processing of crystalline products.
(ii) Drying of products direct from solution.
CRYSTALLIZATION

§ The final purification method for those materials

which can stand the heat.
§ Principle: the initial water removal is made by
heating at reduced pressure or lowering the
temperature to form crystals that can be
centrifuged, leaving a concentrated liquor.
CRYSTALLIZATION
§ To obtain crystals:
i. Supersaturated solution is produced
ii. The solution is heated to evaporate most of the solvent
iii. The hot solution is allowed to cool – the solid appears as
pure crystals
iv. The cold solution is poured off to obtain the crystals

§ Crystalline particles from a previous preparation may be

deliberately introduced to produce the nuclei.
DRYING
§ Liquid removal (either organic solvent or water) from wet or
from solids or cells isolated from the very earliest operation.
§ It is often the last stage of the manufacturing process.
§ Approaches: liquid-phase moisture removal and solid-phase
moisture removal.
§ Purpose: i. to reduce the cost of transport, ii. for ease
handling and packaging of the material, and iii. the package
can be stored more conveniently in the dry state.
LIQUID-PHASE MOISTURE
REMOVAL

§ This involves drying by heat.

§ The processes may be broken down to the supply

of heat to the material and the removal of the
resulting water vapour.

§ Methods: Tray driers, drum dryers and spray

drying.
DRUM DRYERS
§ The product is
contacted with a
heated surface.

§ Used for more

temperature-stable
bioproducts

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0no%203.pdf
SPRAY DRYING
§ It is used extensively for
drugs, plasma and milk.

§ Little damage may occur

during the drying.

§ Convenient because of its

continuous nature.
SPRAY DRYING
§ It involves passing small droplets
of liquid containing the product
through a nozzle that directs them
over a hot gas stream.

§ The water evaporates, leaving

behind solid particles.
SOLID-PHASE MOISTURE REMOVAL

Vacuum drier vs freeze-drying:

§ The material is first frozen
§ In this frozen state, the water evaporates straight from the
material.
§ It is useful for heat-labile materials such as enzymes,
bacteria, and antibiotics.
Downstream Processing