Microbiology Aspect on asepto smart 78 Commercially Aseptic filling Machine & Lamination

ASEPTO SMART -78 filling Machine
Presented By: Vishnu Kumar Gupta

Aseptic processing
Aseptic processing
Aseptic
environment
Filling & sealing
Filling of
product
Aseptically packaged
product
Sterilizations Sterilizations

Commercial sterility
The commercially sterile product must:
•Keep without deterioration, stable
and good commercial value during
storage.
•Free from micro-organisms and
toxins harmful to the health of
consumers.
•Free from any micro-organisms
liable to proliferate during storage.

A commercially sterile
product is free from micro-
organisms which grow under
the prevailing conditions.
Commercial Sterility
COMMERCIAL STERILITY
The expression “commercial sterility” is frequently used for UHT-treated
products. A commercially sterile product is defined as one which is free from
microorganisms that grow under the prevailing conditions. In low acid products –
products with pH above 4.5 – the most heat-resistant microorganisms which can
grow are spores. As their heat resistance is much higher then that of vegetative
microorganisms, sterilization processes concentrate attention to the killing effect
on spores only. The group of low-acid products comprise not only milk, but most
milk-based products and UHT liquid products..
Fig. Time-Temperature curve for
UHT treatment in direct, A, and
indirect, B system.

HTST = high temperature – short time, UHT = ultra high temperature Heat treatments – definitions
Pretreatment
Thermisation 63–65°C/15 sec Preliminary heating
Pasteurization 63°C/30 min Pasteur's method rare today
(batch pasteurization)
Heat treatment — Chilled distributed products
HTST pasteurization 72–75°C/15 sec. Milk
HTST pasteurization 85–90°C/2–5 sec. Cream
HTST pasteurization 90–120°C/2–5 sec. Fermented products
Ultra pasteurization 125–138°C/2–4 sec. Cold storage
Ambient distributed products
UHT 135–150°C/4–15 sec Ambient storage
Conventional sterilization approx. 116°C/20 min Ambient storage
Heat treatments – definitions

UHT – Ultra High Temperature
processes
Continuous processes Batch sterilisation in container
Direct
UHT
Indirect UHT
Pasteurization
Continuous processes
Temp °FTemp °CTemp °F
Temp °C
Time Minutes
Seconds
Time
300°
200°
100°
50°
100°
150°

A typical aseptic line of Filling

A CIP program for a circuit with pipes, tanks and other 'cold
components' can comprise the following stages:
1. Rinsing with warm water for three minutes
2. Circulation of a 0.5 – 1.5 % alkaline detergent at 75 °C for
about 10 minutes
3. Rinsing with warm water for about three minutes
4. Disinfection with hot water 90 – 95 °C for five minutes
5. Gradual cooling with cold tap water for about 10 minutes
(normally no cooling for tanks).
Steps for CIP cleaning of 'cold' components:
1. Rinsing with water
2. Circulation of alkaline detergent
3. Rinsing with water
4. Disinfection with hot water
5. Cooling with tap water

1.Storage tank for alkaline detergent.
2.Storage tank for acid detergent
3.Ring lines for detergents
4.Objects to be cleaned
5.Decentralized CIP units

Capacity l/h35.000 l/h
Increasing particle size
200 cP
50 cP
500 cP
100 cP
50,000 cP
ASEPTIC PROCESSING SYSTEMS
Viscosity cP

It is quite common to process both low-acid (pH>4.5) and
high-acid (pH<4.5) products in the same UHT plant.
However, only low-acid products like milk require UHT
treatment to make them commercially sterile.
Spores cannot develop in high-acid products such as juice,
and the heat treatment is therefore only intended to kill
yeast and moulds.
Consequently high temperature pasteurization at 90 - 95°C
for 15 to 30 seconds is sufficient to make most high-acid
products commercially sterile

UHT pilot plant based on tubular heat
exchangers.

Aseptic Packaging
Definition:
“Hermetically sealed to
prevent re-infection”

Product
AIR
Microorganisms
Moisture
Odors
Product
Sensory + nutritional
attributes
Barrier Properties

5
6
5
TM-00816:6Technical Training Centre 3/0503
•adhesion layer
•seals in the liquid
LDPE
mPE
Aluminium foil - oxygen, flavour and light barrier
LDPE3
4
1
2
Paper2
4•protects againstoutside moisture
•for stability and strength
•lamination layer
1 LDPE
Packaging Material
3
6

4
5
6
•Aluminium foil - oxygen, flavour and light barrier
•Polyethylene - adhesion layer
•Polyethylene - seals in the liquid
Polyethylene3
1
2
•for stability andstrength
•adhesion layer
2 Paper
3
4
5
- protects against outside moisture1 Polyethylene
6
Packaging Material

1.Polyethylene
Protection against outside moisture
2.Board
Stability
3.Polyethylene
Adhesion layer
4.Aluminium Foil
Oxygen, flavor and light barrier
5.Polyethylene
Adhesion layer
6.Polyethylene
Seals in the liquid

LS
Longitudinal Seal
TS
Transversal Seal
The Packet Sealing

Making A Seal
PULLTAB
STRIP
APPLICATION
(SA)
LONGITUDINAL
SEAL
(LS)
TRANSVERSAL
SEAL
(TS)
FLAP
SEALING
All Seals are Plastic to Plastic
HEAT - Melt the layers of plastic.
PRESSURE - Press the two surfaces
together under force until the molten plastic
layers become one.
TIME – Time that the jaws are in contact.
COOLING - Allow the molten plastic
pool (the seal) time to cool before it is
exposed to mechanical forces.

10. Maintenance
4. Pre-process
7. Filler
Packaging IntegrityMICROBIOLOGICAL
Investigation1. Hygiene
12. End Product Investigation
2. CIP
11. Storage3. Raw material
INFORMATION
9. Laboratory QC/QA
5. Processing
Equipment
8. Documentation /Records
6. Aseptic
Transfer
Information collection of any trouble shoot problem

Package Integrity
Package Integrity
At the present time, tighness of packages can be
checked by three different procedures:
-Tear down
-Conductivity
-Dye-testing

1- Transversal Sealing.
2-Longitudinal Sealing Inside.
3- Layer Ruptures.
4- Pull Tab.
I. Visual Check
II. Red Ink Test
5- Package Forming.
Package Integrity Tests

Contact to
Aluminum Foil
Recommended ASEPTO SMART- 78 package integrity flow
Visual defect and
design check
Transversal Seal
Evaluation
Longitudinal
Seal Evaluation
Red Ink Leakage
Evaluation
Result
Result
Result
Acceptable PackageUnacceptable Package
Checks at machine
Checks at end of line

Preparation of sample
Transversal Seal

PE Film is stretched
along the seal
Sealing is stronger
than packaging material
Good sealing

Breakage inside the
packaging material
Watch the paper fibers
along the seal
Good sealing

Breakage inside the plastic
layer of the packaging
material
Aluminum foil becomes
visible (1)
Watch the remainings
of the stretched PE film (2)
Good sealing

Rupture in packaging
material along the seal
Combination of previous
examples
Good sealing

Cross section of transversal
and longitudinal seal is
broken inside the packaging
material
Sealing is stronger
than packaging material
Good sealing

No breakage of PE film
and/o packaging material
layers.
The seal is weaker than
the packaging material
This is a ‘blocked’ or
‘cold’ seal: the blocked
area is large
Bad sealing (1)

This is a ‘blocked’ or ‘cold’ seal:
the blocked area is small
Bad sealing (2)

Bad sealing (3)
Check for PE lumps using your finger nails before pulling
the transversal seal

Bad sealing
Another example of a bad seal.
During sealing PE lumps are pressed inside the transversal
seal

Overheating
Blistering due to over
heating
Plastic residue under the LS
Defective Longitudinal Seal

Look for leakage. Any
leakage is to be considered
a defect and acted on.
Insert the needle in the air gap
and inject the red ink.
Open the air gap with a cut. It
makes inserting the needle easier.
Ink test LS

This picture shows
wrinkles and transversal
air channels running
across the LS.
This picture shows a seal with
a leaking channel.
Leaking Longitudinal Seal

A Pull Tab opening consists of a hole
punched in the packaging material.
 The hole is closed from the outside by an
aluminium tab and the inside by a plastic
strip.
An air gap around the hole prevents
paper fibres to come in contact with the
inner patch.
Pull Tab

The inner patch seals the hole,
preventing product to come in contact
with the packaging material. This restores
the microbiological barrier and ensures
package integrity.
Look for red ink leakage on the paper
board layer in the area surrounding the PT
hole. Red ink penetration to the paper
board indicates PT application failure.
Pull Tab

The reason for insufficient PT
sealing can be various, some
examples are:
A.Poor cutting leaving paper fibres
around the hole.
B.Under or over heating.
C.Insufficient air gap
Pull Tab

Dry the package thoroughly,
wet ink left in the package could
jeopardise the result and create
a false positive reading.
Leave the ink in the
package for at least 5
minutes and remove the
excess ink with a pipette.
Apply ink well covering
the Pull Tab hole.
Test method for PULL TAP CHECK

Visually inspect the
package, if any ink has
penetrated this is
considered a defect.
When the ink has
dried, peel of the outer
layer.
Test Result of dye Test

In this picture there
is a red ink leakage,
this is a defect Pull
Tab sealing and
actions should be
taken to rectify.
This is an example
of a good Pull Tab
seal.
Pull Tab seal

Longitudinal Seal and
S.A Evaluation
What will be revealed:
•Measurement of Air Gap
•Evaluation of Heat Zone & Strip position
•Evidence of Channels
•Rupturing of material layers
Specific Tools
•10x magnifier with 0.2mm divisions (preferably illuminated) -Zonoscope.
•Red ink solution (saturated Erythrosine B in pure Iso propanol).
•Vernier Caliper or ruler
Critical Issues
•TAKE two packages and unfold to expose LS, DO NOT crease the strip
•MEASURE the strip position and heat zone
•INJECT ink into one air gap, check for leaks through longitudinal creases
•CUT up the middle of the Strip remove overlap, pull off strip at 90o
.
•EVALUATE seal quality .

Tools:
1-Zonoscope
Heat distribution (hot air application)
1. 2--Manual tearing
To verify tightness
3-Red Ink and syringe
To confirm tightness
Critical areas:
• Top and Bottom creases
• Sample preparation
Avoid mechanical stress
Definition:
•The sealing is good if, when torn, it breaks in
any layer EXCEPT where the sealing occurs
•No channels allowed
Longitudinal Sealing

The seal is defective if the strip comes
off leaving the inner coatings of the
package unaffected
The seal is acceptable since the strip
delaminates (separates) when it is
pulled off
The seal is acceptable since both inner
coatings come off with the strip, leaving
the Alu-foil bare
The seal is acceptable since one of the
two inner coatings comes off with the
strip, leaving a ruptured edge along
the seal
LS Teardown
The seal is acceptable since all the
inner coatings including the Alu-foil
come off with the strip, although leaving
paper board fibers

Transversal Seal
Evaluation
Tear Down Test
•Rupturing of the material layers
•Consistency of the Seal quality
Specific Tools
•Stretch/Seal Pliers
•10x magnifier with 0.2mm divisions
(preferably illuminated)
Method
•TAKE two sample packages
•EMPTY packages of product
•CUT off the top and bottom Transversal
Seals
•CUT no more than 1mm off the ends of
the seals
•USE the Stretch Pliers, and (if necessary)
the magnifier, to evaluate the seal quality

Plastic lumps/Ridge Cold Channel
TS Tear Down Test

Package Digestion & Red Ink
What will be revealed:
Heat distribution along the TS
TS tightness using red ink.
Specific Tools
Acid or Alkaline Dissolving Agent
10x or more magnifier with at least 0.2mm divisions (preferably illuminated)
Red Ink.
Method
PREPARE the packages for digestion
DISSOLVE the packages using an acid or alkaline solution
DRY the digested packs completely
PUT SOME RED INK with a plastic pipette and allow to dry
EVALUATE seal quality according to the
Transversal Seal Evaluation

Critical Measurement Parameters

Sealing is smooth and even
►Appearance of bubbles is
absent/minimal
►No red dye leak
Good TS Seal

Plastic Lumps on 2 areas of the TS
Defective Seal

Defective Seal
Large, numerous bubbles and ridge
breaking indicative of a hot seal

Product residue (e.g.
fruit fibers) trapped into
the TS causing
leakage.
 Particles greater than
600µm (30 Mesh
Sieve) present an
increased risk of
package integrity
failures
Defective Seal

TS Heat Pattern by Dissolving
Take the cut package and
unfold their top and bottom
flaps
Flatten package.
Note! Cut with a distinctive cut
type part of the sample to
easily identify the top TS side
of the sample.
Cut out the pieces A and B to
leave an I shaped sample, as
shown in the drawing.

At the side not including the LS
cut .away the pieces to leave the
final shape.
 Carefully wash and dry the
samples,inside and outside.
Mark each sample, on the inner
PE, with progressive numbers to
identify from which jaw pairs the
package comes.

Carefully remove the
outer layer of
polyethylene, splitting
the packaging material.
The outer
polyethylene layer and
some paper board will
come off to obtain the
sample to utilize for the
test.

It uses 30-35%NaOH solution in water as the
aluminum foil dissolving agent: the speed of
reaction is lower (at least 10 hours).
The microbiological quality of the TS is
checked by chemically exposing the inner
coating (dissolving method) and checking its
tightness by means of the leak detection
fluid (red ink).
Immerse the sample in30% NaOH solution
Bath with the aid of the tongs and leave it there
for approximately 24 hours for aluminum foil to
dissolve.
Remove the sample with the tongs and
immerse it in water to neutralize them.

The sample now needs to
be neutralized with water
and then rinsed carefully
with tap water.
Dry the samples with
paper towel and leave them
on filter paper.

Before rinsing the samples
make sure that the aluminum foil
has disappeared. If not leave the
samples in the solution a little
longer or prepare a fresh
solution.
Carefully rinse and split the
samples under tap water .
 The inner coating pouch of
the sample becomes very easy
to peel off and separate from the
rest of the packaging material .
Leave the samples until dry on
filter paper before checking their
integrity by means of the leak
detection fluid.

TS integrity by red ink
Note! Do not dry the sample by reversing the
bags. A sample can be spoiled easily. Dry the
inside of the bags by carefully using paper
towel.
Take one sample at a time.Dry it
carefully by inserting paper towel in
the bag and moving it carefully
along the TS and in the bag
corners.
Be sure that the sample is
completely dry. Water prevents a
good check.
Take note of the appearance of
the sealing. Critical spots like
bending off at the corners, plastic
lumps, irregularity of the sealing are
very easy to spot.

Pour some red ink fluid , by means
of a pipette in the pouch of the inner
coating.
Spread the red ink in the pouch with
the finger without applying pressure.
Let it stand for some time and then
check the sample.

The sealing is defective if: –
any channel of red ink is
present through the sealing.
In case channels are present,
the fluid will pass through
and stain the outside of the
inner coating pouch. Check
again when the ink inside the
sample is completely dry

LS test by red ink injection
Note! Be careful not overstress
samples during their preparation.
Cut open the packages as illustrated. Cut
the corner at one end of the LS seal.

Rinse and dry the packages with a paper
towel, without folding the top and bottom
creases in the reverse direction.

Rinse and
dry the
packages with
a paper towel,
without folding
the top and
bottom creases
in the reverse
direction

Prepare some
red ink fluid in a
syringe.
Place the
needle of the
syringe into the air
channel of the LS.

Cover the injection point
with a piece of paper towel to
absorb any ink spilled and
inject the red ink along the
entire LS length.
The LS is defective if red
inkchannels occur (the ink
corners to the inside layers).

•Detects contact with Aluminum layer
Specific Tools
•Conductivity meter
•Salt bath(10g NaCl/ltr water)
•Glass or plastic beaker
Critical Issues
•CUT packages in half but do not cut LS
•FOLD package at the LS
•PLACE package in Salt Bath
•POUR Salt solution into both halves of the package using beaker (Do not
wet the cut edges)
•PLACE one probe in the bath and one in one half of the package
•CHECK for continuity, repeat for other half of the package.
IF ANY SAMPLE SHOWS POSITIVE TO CONDUCTIVITY CONTINUE WITH
RED INK TEST
Conductivity Test

Package Integrity by Conductivity
If occasional package
leakages are suspected, larger-scale investigation can be
carried out by measuring the electrical conductivity.
The following equipment is required:
1– plastic container approximately
30 cm diameter, 10 cm depth.
2– ammeter with battery and
electrodes.
3– 1% table salt solution (e.g. 10g
table salt in 1 Litre water). The
water is rendered electricallyconducting
by the addition of table salt.
If the is defective or if the internal
polyethylene coating is damaged,
electric current will flow and the ammeter
will display a reading.

Cut the package in half on
the front side panel (the
one without the LS), using
the scissors.
Empty out the packages
content. Wash carefully the
two halves of the
package with water.
Dry thoroughly the cut edges of
the package with paper towel.

Pour the salt solution
into the container.
Insert the electrodes of
the ammeterinto the salt
solution and check if the
ammeter displays a
reading.
Pour some of the salt
solution into both halves
of the package and dry
out the cut edges again
with a paper towel.
Place the halves of the
package into the salt
solution

 Immerse one electrode of the ammeter in
the salt solution andthe other electrode in the
salt solution inside the package .
Check the ammeter display.
 If the ammeter does not display a reading,
the inner layer of PE is integer
 If the ammeter displays a reading,either the
package is defective or the inner layer of PE is
damaged: red inktest is needed.
Test both packages halves in the same way.
Note! The salt solution can be usedseveral times.
Change the solution when it is not clear or once a week.
•If there is no deflection of the ammeter, continue testing
the package from item TS Heat Pattern by Dissolving.
•If there is a deflection of the ammeter, test the package
with red ink, from the next item.

PE 2
PE 1
Aluminum Foil
Lamination PE
Paper Board
Outer PE
How Conductivity Test Works

Copper Test
► Done when package shows positive to
conductivity
•Shows location of the contact to aluminum foil
Specific Tools
•Copper Sulfate solution
•Copper tester
Critical Issues
•DRY packages of water (before introducing solution)
•COVER all critical spots
Note!
If the red ink test is going to be performed on the packages, do it before
the copper test. The copper test can cause an enlargement of the cracks in
the packaging material, compromising the results of the ink penetration.

► Performed when package shows positive to
conductivity
•Ruptures of the Microbiological barrier
•Pull-Tab integrity
Specific Tools
•Red ink solution (saturated Erythrosine B in pure
Isopropanol)
•Pipette
Critical Issues
•DRY packages of water (before introducing ink)
•COVER all critical spots
•EXPOSURE to the ink (5 minutes)
•ASPIRATE excess ink with pipette and dry with paper towel
Red Ink Test

Check Leakages by Red Ink
Dry carefully the inside
of the halves of
packages with a paper
towel.
Cut the package to
separate the two halves.

Pour some red ink
inside the half package
that displayed a
deflection. Swirl the red
ink around.
Leave the red ink inside
the package for at least
5 minutes.
Remove any excess of
red ink from the inside of
the package with a
plastic pipette.

 Rinse out the red ink residue and
dry theinside of the half package
with a paper towel and with
compressed air from the filling
machine.
Let it dry before carefully
removing the outer layer of PE,
splitting the packaging material.
The outer PE and some paper
board will come off.
Note! If ink is still in the package
when this operation is performed,
artifact defects may be created.

Check for any red marks over the
paper board layer over the package
sample.
The presence of red marks
indicates that the package leaks or
that the inner PEpouch is
damaged.

How the Ink Test Works
PRINCIPLE
•The red ink is a visible, penetrating liquid.
Due to the rupture through all the internal
layers, the red ink will reach the paper board
and, free from any barrier, will be visible from
the outside.
This test indicates if there is any rupture through the inside layers of PE,
Aluminum Foil and the PE laminate.
PE 2
PE 1
Aluminum Foil
Lamination PE
Paper Board
Outer PE
Outer PE
Red Ink

What is a “Tight Package”?

Conductivity and Ink test comparison
Red ink
Conductivity Test +
+
++-
- - -
PE 2
PE 1
Aluminum Foil
Lamination PE
Paper Board
Outer PE

Classification of bacteria by temperature preference
45 °C
20 °C
7 °C
Thermophilic
Mesophilic
Psychrophilic
Psychrotrophic

6
1.Viable bacterium
2.Nuclear content
gathered
(at bad growing
conditions) A thick wall is
formed around the
nucleus (the spore)
3.The cell decomposes
and the spore is liberated
4.The spore is freeAt
favorable conditions the
membrane of the spore
bursts and a new cell will
be formed
1
2
3
4
5
Bacterial formation of spores

Bacteria can be divided into two groups:
1.Those existing as vegetative cells only (easy to
kill by heat or other means).
2.Those existing in a vegetative state and as
spores as well, i.e. spore-forming bacteria.
While these bacteria are easily killed as long as
they are in the vegetative state, their spores are
difficult to eliminate.

High spore counts may be found in
products with low total counts, and vice
versa, so total count determination
cannot serve as a reliable basis for
enumeration of spores in food products.

The shelf life of a product is generally defined as the time
for which the product can be stored without the quality
falling below a certain minimum acceptable level. This is
not a very sharp and exact definition and it depends to a
large extent on the perception of “minimum acceptable
quality”. Having defined this it will be raw material
quality, processing and packaging conditions and
conditions during distribution and storage, which will
determine the shelf life of the product.
SHELF LIFE

Product Shelf life Storage
Pasteurized milk 5 to 10 days Refrigerated Temperature
Flexi- Pouch packaging 45 to 90 days Ambient Temperature
UHT milk 3 to 6 months Ambient Temperature .
Milk is a good example of how wide a span the concept of shelf life covers:

Long-life products
Low-acid pH >4.5
High-acid pH <4.5
Milk& Milk products, tea, coffee, liquid
food products containing vegetables
juices, fermented milk
products, fruit products

Shelf-life of milk
“The time the product can be stored
before the quality falls below an
acceptable and minimum level”
Subjective criteria:
•Taste – Sedimentation
•Colour – Fat separation
•Smell – Viscosity
•Gelation

In order to sell ASEPTIC PACKAGING products, the FDA
requires that the manufacturer prove the sterility and viability
of the product on the expiration date prior to selling the
product.
That means if you market a product "use before June 17,
2019", you must prove that the product is viable on that date
before you begin selling it today.
To accomplish this, manufacturers employ a derivation of the
Arrhenius equation which states (in essence) that molecular
activity doubles in organic molecules for every 10° C. rise
in temperature above ambient.
Accelerated Aging Calculator- Packed product self life estimation

Since ambient temperature is (conservatively) considered to be 25°
C., storing a product for six months at 35° C. will result in a one-
year accelerated aging.
Similarly, storing a product at 45° C. for three months will result in
an accelerated aging period of one year, and so on. Obviously there
is a limit to the upper temperature at which this process is valid
and, as a practical matter, most accelerated aging occurs at
temperatures at or below 55° C.
To validate this information, most practitioners use a process of
real-time aging wherein samples subjected to accelerated aging are
compared to those from an equivalent “real-time”.

Accelerated Aging Time (AAT) Calculator
Reference ASTM F1980-07

Time period it takes for the unsterilization to
development ?

Time period it takes for the un-sterility to develop:
The time between production and
development of spoilage. Valuable
information in identifying possible
areas from which the problem may
have arisen.
The time it takes for the un-sterility to
develop can be as short as 24 hours,
or as long as 3-4 weeks.
Rapid development of spoilage, 24-
72 hours, may be an indication of an
air- waterborne re-infection.

Slow development, 3-4 weeks, may be an
indication of process survivors/a cleaning
problem or a package integrity problem.
On an average, and depending to a certain
extent upon the method of evaluation,
about 50% of the total number of
defectives can be detected after 3 days of
incubation. This percentage increases to
about 75 and 85% after five or seven days
of incubation respectively at 30-35oC .

Sour coagulation Sweet coagulation Flat sour
Pigmentation
Blown packs
Types of spoilage (Visible)
EnzymesAcidGas

Product changes can result in following
types of spoilage in long life milk:
Blown packs.
Development of microbial growth may result in gas
formation. Gas formation results in blown packages
and can easily be detected in the storage area. As the
first action always verify package integrity.
Sour coagulation
Development of microbial growth may result in acid
formation. A pH drop down to about 5.5 results in
aggregation of milk fat globules. It is quite common
with a combination of sour coagulation and blown
packs.

Flat sour
Development of microbial growth may result in
an acid formation, which is not enough to give
coagulation. Very often the pH-drop is in the
range 0.2 to 0.5 units. The product is visually not
changed. Careful measurement is needed to find
such a small pH-drop.

Sweet coagulation
Development of microbial growth may result in production of
enzymes into the milk. Lipolytic and proteolytic enzymes cause
aggregation of the milk fat globules. Sweet coagulation can also
occur as a result of solely enzymatic activity. If Pseudomonas got
the possibility to grow to high numbers in the raw milk, which
Pseudomonas can do at refrigeration temperatures as it is
psycrotrophic, then proteolytic enzymes are produced in the raw
milk. Pseudomonas is very heat sensitive and is killed already by
pasteurisation, but the enzymes are very heat stable and survive
UHT- treatment. This type of sweet coagulation is a very slow
reaction, which takes months.

Sample of UHT milk suffering from strong enzymatic activity.

1. Pigmentation
Bacteria may change the color off the milk. It is very rare
but when it happens it is often easy to identify the
unsterility.
Product changes can result in following types of
spoilage in long life milk:
Note! Product changes are only possible to
develop after multiplication of bacteria to at least
10 to 6 per ml. Most spoilage bacteria then need
an incubation time of at least 5 days, at 30 – 35
°C. At lower temperature it takes longer time.

Spoilage types with coagulation are easiest
visible in full fat milk. If the fat content is low, the
coagulation will be slower and weaker.
In flavored products with added sugar, product
changes are more rapid and clear than in white
plain milk. Unsterilities with flat sour reaction in
white plain milk might in flavored milk develop
spoilage type sour coagulation and sometimes
also with blown packs.
Product changes can result in following types of
spoilage in long life milk:

Usual reason to miss unsterilities is too short incubation time!
•Streak plate method
•Sensoric test
Invisible spoilage

No visible changes
Sensoric changes
A lot of enzymatic changes of the milk are possible,
which result in off- flavour. Sometimes the off-flavour is
easy to detect like the fruity and fishy smell from
Pseudomonas. Growth of almost all micro-organisms
result in off-flavor, which can be detected with a
sensoric evaluation, where the product is tasted by an
educated staff. Sensoric evaluation is considered to be
very reliable method if performed by an experienced
staff

Micro-organism/s causing the spoilage ?

ENTEROBACTERIACEAENeg ative
PseudomonasPositive
Pos; irregular coccobacilli
Neg; cells in chains
Gram Negative
No spores
Pos; filamentous
Gram Positive
SPORE FORMER
RODS
Nigrosine
Negative
Negative
OxidaseCatalaseCOCCI
PositivePositive
ROUGH IDENTIFICATION OF BACTERIA
Low acid aseptic production
Oxidase
Catalase
Gram reaction
Lactobacillus
Corynebacterium
Actinomycetes
Bacillus
Micrococcus
Staphylococcus
Streptococcus

Start the identification with a
nigrosine slide to find out if the cells
are spherical or rod-shaped. If the
cells are rod shaped continue with a
test to find out the gram reaction
etc.
Rough Identification of Bacteria (RIB)

Changes in low acid product
- Staphylococcus/Micrococcus
TM-00032:16
Product changes
• No visible spoilage
• Coagulation (sweet and
sour)
Production source
• Splices - handling
• CIP/Filler presterilisation
Natural habitats
• Skin/Udder - Staphylococcus
• Airborne - Micrococcus
Resistance
• Withstand dry conditions
• Some fairly heat resistant (thermoduric)

Mastitis bacteria
Product changes
• Bitterness,
• Gelation.
• Sedimentation of UHT milk or product.
• Coagulation (sweet and sour)
• Producing heat resistant and
proteolytic enzyme Plasmin.
Production source
 Raw material
Natural habitats:
cow or animal under infection.
Skin/Udder – Staphylococcus aureus
Resistance
• Mastitis will cause an increase of somatic cells, white blood cells, in the milk.
• heat resistant (thermoduric)

Product changes
• Sour coagulation
• pH < 5
• Gas formation
Production source
• Reinfection - Streptococcus
• CIP - Lactobacillus
Natural habitat Raw milk
Resistance
Withstand dry conditions; some fairly heat resistant
(thermoduric)
- Streptococcus/Lactobacillus

Product changes
• Flat sour
- small drop in pH
• Sour coagulation
• Gas formation
Production source
• Process survivors
• Pre-sterilization
• Packaging material
sterilization
• CIP
• Pre-processing
Natural habitats
Airborne/Raw materials/powders
Resistance
Very resistant against heat, chemicals and dry conditions
- Bacillus

Product changes
• Flat sour
Production source
• Reinfection
Natural habitats
• Air/Milk/Man
Resistance:
Withstand dry conditions and
fairly heat resistant (thermoduric)
- Corynebacterium

Product changes
• Flat sour
Production Source
• Pre-processing
Natural habitat
• Powders(chocolate, coffe)
Resistance:
Withstand dry conditions;
fairly heat resistant (thermoduric),
Thermoactinomycetes has quite heat resistant endospores
- Actinomycetes/Thermoactinomycetes

Product changes
• No changes within a few weeks
• Sweet coagulation (slowly)
• Fruity or fishy off- flavour
• Somewhat increased pH
• Fluorescing in UV-green
Production source
• Reinfection
• Package integrity
• Other ways of leakage
Natural habitat
• Water
Resistance
Very heat sensitive; can not survive dry conditions.
- Pseudomonas

Product changes
• Gas formation
• Low pH
• Coagulation
Production source
• Reinfection
• Package integrity
• Other ways of leakage
• Non hygienic design
Natural habitats
• Water/mammalians including man
Resistance
Very heat sensitive; can not survive dry conditions
- ENTEROBACTERIACEAE

Gram-negative psychrotrophic bacteria
Product changes
• Fat sepration
• Off- Flavours
• Coagulation
• Gelation formation.
Production source
• Re infection dirty water,
• Non hygienic design.
• Raw milk or product.
• high refrigeration time.
Natural habitats: dirty water
• Gram-negative psychrotrophic bacteria may attach to the surface of milking equipment and form
very thin layers, called biofilms.
• Most of them do not ferment lactose to lactic acid, and thus do not lower the pH of milk.
Resistance
They do not survive the pasteurization process but they can, however, produce heat-stable
enzymes that are not inactivated in UHT processes, thus causing off-flavours, fat separation and
early gelation formation.
To minimize the problem with psychrotrophic bacteria it is important to have a good cleaning
routine for milking equipment and storage tanks/containers, and to use clean water.
In order to maintain good raw milk quality, keep the milk properly refrigerated but limit the time at
refrigerated temperature before processing at the dairy.

Gram-negative psychrotrophic bacteria
UHT milk based on raw material with different number of psychrotropic
bacteria after 2.5 month storage at ambient temperature 22-23 °C.

Yeast and mould
Yeast and moulds can be present in raw milk.
They grow slowly at low temperature and relevant species are inactivated by
pasteurization at 72°C/15 s.
Presence of mould toxins in milk is a feed-related problem.
Aflatoxin B1 is a liver toxic and carcinogenic mould toxin that can be present in
high amounts in feed. The cow metabolizes the aflatoxin B1 into aflatoxin M1
and secretes it into the milk.
Aflatoxin M1 is also liver toxic and carcinogenic .
Aflatoxin M1 is not inactivated by UHT processing conditions.

No. TEST NAME BENEFIT
1 pH value Identify microbiological spoilage as well as chemical
contamination.
2 Milk acidity by titration
Identify the result of an intense microbiological
metabolism in the sample and obtain a rough estimate
of the milk quality.
3 Alcohol You can assess the stability of milk proteins
4 Freezing point Helps you detect milk that has been diluted by water,
as well as judge milk stability.
5 Density Allows you to estimate the solid content.
Physical and chemical tests

No. TEST NAME BENEFIT
1 Methylene blue reduction
(Resazurin)
Rough identification of the total amount of microorganisms
present in raw milk.
2
Total Aerobic Plate Count for
mesophilic aerobes
Determining the risk of spoilage and lower quality products from organisms
growing at temperatures between 30-40°C. Relevant to milk heat
sensitivity.
3
Total Spore count and Heat-
resistant spore count
To enumerate total spores and heat-resistant spores associated with
spoilage. Important for choosing right conditions for high temperature
treatment.
4
Total counting of psychotropic
aerobes
Detecting organisms growing at temperatures between 0-30°C.
Relevant to enzymatic spoilage during storage.
Microbiological tests

In doing so the number of viable Cells/ml is determined
before and after passage of the sterilization and packing
device of ASEPTO SMART -78 and from the difference in
microorganism counts the killing rate is determined by taking
mean logarithmic of initial and final survival cell/ml count.
Logarithmic reduction test by Cell Count
Procedure :
In the logarithmic reduction test by cell count, the
packaging Material is infected with the test microorganism
artificially and passed through the aseptic packaging Machine
ASEPTO SMART -78.

At least 25 packaging units which for the test have been
inoculated with artificially infected bacteria by spray or
swab method under the same conditions. packaging unit
has an initial microorganism count of at least 106 cells/ml
concentration for the test.
Test method:
Determination of the initial count( IC ) cell/ml by
hamacytometer cell count method under microscope of
artificially infected packaging Material . To get out the
microorganisms concentration from the inner surface of
the packaging material . On sheet packaging material
the microorganisms are removed by swabs in
accordance with DIN 10113-2.

Introduction of at least 20-40 infected
packaging units into the filling machine.
Carrying out the test run. If possible the
packaging units should be filled during the
test run to 25 % of the nominal filling
volume with sterile skimmed milk cooled to
room temperature or a sterile, filterable
liquid. The packaging units are to be cooled
immediately after filling. The test data are
then documented.

If during the test run the packs are
not filled with a test medium the
sterilized packaging units are to be
passed on as quickly as possible
after the test run for
microbiological analysis in order to
avoid falsification of the test
results.

Determination of the survivor count (SC)
for each of the artificially infected
packaging units and determination of the
initial count (IC) of retain sample. ix)
Calculation of the microorganism count
reduction .
(Mean logarithmic count reduction)
= log[ΣIC)] – log[ΣSC]
ΣIC: mean initial count
ΣSC: mean survivor count

Definition of D-value
D-value (decimal reduction time) is the time at a specific
temperature necessary to reduce the number of micro-organisms to
1/10 or 1 log reduction of the original value.
NumberofMicroorganisms
105
104
103
102
101
100
10-1
10-2
tim
e, t
Micro-organisms
D121 °C
B.Cereus 2.3 sec.
Cl.botulinum. 12.25 sec.
B.Stearothermophilus 408 sec.
time, t
D

Definition of z-value
z-value is the increase in temperature, necessary
to obtain the same lethal action or the same effect
in 1/10 of time or achive1 log reduction.
Temperature dependance
z-value [°C]
B. stearothermophilus 10.5
Color changes 29.0
Losses of vitamin B1 31.2
Losses of lysine 30.9
103
102
101
100
10-1
time, t [s]
z
temperature

Definition of F-value
Fo Value at particular temperature other then 1210C is the
time in minutes requires to provide the lethality equivalent to
that provide at 1210C for a stated time.
where:
t = sterilization time most often expressed in minutes, e.g.
the holding time in seconds divided by 60
T = sterilization temperature in °C
Fo =1 when heated one minute at 121.1°C

Q10 for flavor changes is in the order of 2 to 3, which means that a
temperature increase of 10°C doubles or triples the speed of the
chemical reactions.
Q10 value.
The sterilizing effect of heat sterilization increases rapidly with the
increase in temperature, This also applies to chemical reactions,
which take place as a consequence of an increase in temperature.
The Q10 value has been introduced as an expression of this
increase in speed of reactions and specifies how many times the
speed of a reaction increases when the temperature is raised by
10°C.
A Q10 value calculated for killing bacterial spores would
range from 8 to 30 depending on the sensitivity of a
particular strain to the heat treatment.

B* AND C* VALUES
The effective working range of UHT treatments is
also defined in some countries by reference to
two other parameters:
Bacteriological effect: B* (known as B star)
Chemical effect: C* (known as C star)
These values are based on experiments
performed by Horak (1980) with natural milk
incubated at 55 °C to enumerate thermophilic
microorganisms.

The results were presented in the
form of straight lines relating log of
time with temperature for a constant
sterilizing effect.
These data were extrapolated to give
the line that would correspondent to
9 decimal reductions of this natural
thermophilic spore population -
B*value 1.

B* is based on the assumption that
commercial sterility is achieved at 135 °C
for 10.1 sec. with a corresponding z -
value of 10.5 °C. This reference process is
given a B* value of 1.0.
Similarly, the C* value of 1 is based on the
conditions for 3 % destruction of
thiamine. This is equivalent to 135 °C for
30.5 seconds with a z - value of 31.4 °C.

B* is based on the assumption that commercial sterility is achieved
at 135°C for 10.1 seconds with a corresponding Z-value of 10.5°C;
this reference process is giving a B* value of 1.0, representing a
reduction of thermophilic spore count of 109 per unit (log 9
reduction). The B* value for a process is calculated similarly to the
F0 value:
B* = 10 ( T - 135 ) / 10.5 · t / 10.1, where
T = processing temperature (°C)
t = processing time (seconds)
The C* value is based on the conditions for a 3 percent
destruction of thiamine (vitamin B1); this is equivalent to 135°C
for 30.5 seconds with a Z-value of 31.4°C. Consequently the C*
value can be calculated as follows:
C* = 10 ( T - 135 )
/31.4 · t / 30.5
T = processing temperature (°C)
t = processing time (seconds)

A UHT process operates satisfactorily
with regard to the keeping quality of the
product when the following conditions
are fulfilled:
B* > 1
C* < 1

Limiting lines for destruction of spores and effects on milk. The values within brackets (30 °C
and 55 °C) express the optimal growth temperatures of the vital types of corresponding spore
forming microorganisms.Source: Kessler

Heat effects
Bacteriological destruction
Enzyme inactivation
Chemical destruction
Temp, °C
110 120 130
10 6
10 5
104
10 3
Decimal reduction time,
sec

THANK YOU
Prepared By
Vishnu Kumar Gupta
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