CHEESE STRUCTURE

1. Cheese
Structure
SAAD HALEEM

2. Cheese is a versatile nutrient-
dense dairy product
Its end-product characteristics
includes:
• flavor
• physicochemical prop.
• functional prop. (texture and
melting properties)
• Quality
Cheese structure is highly
affected by its end-product
characteristics
Introduction

3. 01
Cheese microstructure is of great importance to
its manufacturer & consumer
02
Understanding the location & interaction of the
various cheese components
03
Cheese microstructure comprises of the casein
matrix in which the fat globules are entrapped
Importance of Cheese Microstructure

4. Light Microscope
1. Bright Field LM
Gives information on chemical composition or structural and spatial relationships
Sample Preparation
• The sample is sectioned to sections of 5 lm in thickness before examination
1. Embedding (using paraffin or a low viscosity resin)
• In this method, dehydration is achieved by using alcoholic formalin and acetone
• Sample is then embedded and sectioned using a glass ultra microtome.
2. Freezing (used in the microstructure analyses of cheese)
• Small rectangular pieces of samples are frozen in liquid nitrogen and mounted on an object disk with a cryo-protective
embedding agent, usually a water-based glycol such as a medium known as optimal cutting temperature compound
(OCT).

5. Specific stains
Specific stains are used for
identification of proteins,
fats and carbohydrate
components
Frequent satins for
carbohydrate
To identify the
carbohydrates, such as
starch, within the cheese
matrix iodine is used
Frequent stains for
protein
• Fast green
• Acridine orange
• Methylene blue
Frequent stains for fats
• Oil red O
• Sudan dyes

6. 2. Polarized LM
Polarised microscopy is used to examine food components within the cheese
matrix that exhibit birefringence, i.e. an ordered crystalline structure.
• When a polarised light is passed through a birefringent material, this light vibrates in different
planes and travels at different speeds . The material has different refractive indices according
to its thickness and this difference is termed birefringence
• Many food components are birefringent.
• Processed cheese containing starch was observed under the polarized LM in order to detect
whether the starch has been gelatinized. Micrographs of the cheese did not show
birefringence in the case of non-gelatinised starch, whereas birefringence was observed in
microwave-heated cheese products indicating the presence of gelatinized starch

7. Light microscope images, at a
magnification of 400, of microwave-
heated cheese under normal (left)
and polarized (right) light showing
birefringence. On the left image,
blue and black represent the
protein matrix and starch,
respectively
(Arimi et al., 2008).
The white patches (in the left
image) represent the pores formed
due to the evaporation of moisture,
as a result of microwave-heating.

8. 3. Fluorescence microscopy
• This employs use of fluorescent light as a unique method of illumination
• Definition:
• It is defined as the emission of photons by atoms or molecules whose electrons are
transiently stimulated to a higher excitation state by radiant energy from an outside source
(Nichols, 2006)
• Micrographs obtained by fluorochromes emit light from exited molecules of the
components of interest based on the dyes used
• Fluorescence provides a sensitivity that is not available in other forms of LM
• allowing the detection of fluorescing compounds present in amounts as little as 10^18 mol
(Lichtman and Conchello, 2005)

9. Conti..
• Fluorescence induces a wide range of compounds such as fluorescent dyes that
fluoresce only in specific chemical environments
Nowadays, it is not commonly used in the microstructural analysis of cheeses
• due to the fact that such staining techniques are already applied in the
analyses by the confocal microscopy, in particular the confocal scanning laser
microscopy (CSLM)

10. 4. Confocal laser scanning microscopy (CLSM)
• CLSM is one of the most useful microscopy technique for studying the microstructure of a wide
variety of foods,in particular dairy products such as cheese (Auty et al., 2001 ; Romeih et al., 2012).
• It is considered as a powerful tool since CLSM penetrates the cheese surface to visualize internal
structure without disturbing it.
• CLSM can identify several cheese components, such as fat and protein, and the location of
microorganisms simultaneously using specific fluorescent labels or strains
(McMahon et al., 2009 ; Abhyankar et al., 2011 ; Wadhwani et al., 2011 ; Panouille et al., 2011).
• The most frequently used fluorescent dyes used in the cheese microstructure are fast green, rhodamine
and fluorescein isothiocyanate for the protein component and oil red O and Nile red for the fat.
• The drawback of using CLSM is related to the high costs of the equipments used and their
maintenance required
(Nichols, 2006).

12. EM is use for
measuring
Higher
resolution
include
• Protein
structure
• Topology
• Morphology
• Composition
of cheese.
Principles of
electron
microscope
In (EM) an electron gun
generates electron beams
that interact with the
specimen and as a result a
number of derived
radiations are emitted;
called secondary electrons
(McMullan, 2006).
Electron Microscopy EM
The first electron microscope,
built in 1931 by Ruska and
Knoll use beam of
accelerated electrons as a
source of illumination.
EM has a much higher
resolution of 0.1–5 nm
compared to LM techniques

13. Scanning Electron Microscope(SEM)
• A scanning electron microscope(SEM) is a type of electron microscope that
produce images of a sample by scanning it with a focused beam of electrons.
• The electron interact with particles in the sample of cheese, producing various
signals that contain information about the sample’s surface topography and
composition.
• SEM divided into three types
1. Conventional SEM
2. Cryo Scanning Electron Microscope
3. Environmental SEM (ESEM)

14. 4-These emitted
radiations called
secondary electrons
3-As a result a number
of derived radiations are
emitted
2-Interact with
Specimen
(McMullan, 2006).
1-Electron gun
generates
Electron beams
Working step of scanning electron microscopy
1. Conventional SEM

15. SEM technique operates with these electrons, these technique require
microscopically examination is under vacuum then specimen is completely dry
to generate an image providing sample information including a topographical
view.
After sample preparation, the microstructure examination of the cheese using
conventional SEM of the prepared specimens is undertaken immediately, at an
acceleration voltage of about 5–15 kV and a magnification of 500–10,000X.
cryo-SEM is more suitable for full fat cheese and in processed cheese, very low
temperature used during the sample preparation and microscopically examination
can lead to the shrinkage of the cheese resulting in showing the protein matrix as
smooth surfaced in the micrographs obtained. (Kalab, 1980).
SEM/Cryo Scanning Electron Microscope

16. 3:-Environmental SEM (ESEM)
• ESEM differs from conventional SEM in that ESEM uses a gaseous environment in
the chamber in which the sample is being microscopically examined.
• In ESEM the specimen to be examined at a relatively higher pressure and not
required to samples completely dry .
• In ESEM sample chamber water vapor gas is used to hydrate sample can form
imaged and not require very low temperature.
• The ESEM micrographs showed a porous protein matrix rather than a smoothed
surfaced matrix in the cryo-SEM micrographs.
• ESEM was not useful for distinguishing starch and fat globules. (Donald, 2003).

17. Conventional (left) and cryo (right) scanning
electron microscopy images
(Romeih et al., 2012; Noronha et al., 2008a,
respectively).
Cheddar and processed cheese manufactured
using casein and vegetable oil, respectively. In
the left figure, black arrows indicate the
bacterial cells and white arrows indicate voids
of removed fat globules. The sample
preparation, which includes defatting using
ethanol solutions, allows for the localization of
bacteria without interruptions from fat
globules. In the right figure, P: protein, F: fat
globule and S: starch.

18. Transmission electron microscopy (TEM)
• Image formation based on scattered electrons
• Specimen should be very thin (0.1–0.2 µm)
• TEM techniques used for cheese are:
a) Thin sectioning
b) Replica method
a) Thin sectioning technique:
• Small samples are chemically fixed, embedded and sectioned
• Magnifications similar to those in SEM techniques
• Higher acceleration voltage (50–70 kV)

19. • Sequential fixation of protein & fat by
aqueous solutions of:
- glutaraldehyde
- osmium tetra oxide
• Dehydration done, using ethanol
mixed with acetone
• Low-viscosity epoxy resin i.e, Epon,
Araldite or Spurr’s used for embedding
• Acetone increases miscibility
• Thin sections of 0.1–0.2 µm are cut
using ultra-microtome with glass knife
• Shedding of sections done in a copper
• Stained with 2% aqueous uranyl
acetate and lead citrate
• Microscopically examined

20. Replica technique:
Used as an alternative to thin sectioning
Used for microstructural analysis of high fat cheeses
Cheese samples soaked in glycerol overnight before being plunged into liquid nitrogen
Freeze-fracturing, sublimation and coating with platinum and carbon done to form replica
Carbon-platinum replica of cheese surface prepared by evaporating platinum at 20º angle to the
surface
Cleaning of replica by placing in acid solution i.e, chromic acid, for 2–3 days
Washing replica in water, then in acetone, and mounted on copper/rhodium grids to be examined
immediately

21. Other microscopy techniques
01
02
03
04
.
- EF (Energy Filtering)TEM:
having ability to select the class
of electron interactions to image
and record as a spectrum.
- cryo-TEM: TEM combined with a cryo-chamber
LM used for detecting calcium
crystals using calcium specific dyes
Fourier transform infrared
imaging (FT-IR):
Used in cheese for examination
of microstructure
Images of processed cheese, with
4 different types of starch,
obtained
These images showed valuable
information on distribution of
protein, fat and starch

22. Software used in Microstructural Image Analyses of Cheese
Image software used in
microstructural analyses is dependent
on the desired endpoint, i.e. to
capture, archive and/or process
images and perform sophisticated
image analyses.
There are various commercially
available software, that are able to
perform these functions.
Examples of these software
programmes are
• Adobe Photoshop
• Image Pro
• MIP (Microstructure Image
Processing)
• Imaris.
Adobe Photoshop is the most
sophisticated, difficult to use,
and accordingly the most
expensive, software for
image analysis

23. Conti…
General steps of applying image analysis software
Image acquisition
Calibration
Image enhancement
Segmentation or thresholding
Counting or measuring
Data analyses

24. Steps
Image acquisition
• This refers to functions resulting in storage
of a digital file representing an image in the
hard disk of a computer.
Calibration
• Calibration refers to the process of defining a
size or colour scale that can be correlated
back to the image.
Image enhancement
• Image enhancement involves modifying the image
with the aim of defining and increasing the visibility
of the regions of interest.
• This enhancement typically includes a number of
adjustments, such as
 contrast
 sharpness
 background
 correction and
Edge enhancement

25. Segmentation or thresholding
• This is mainly to separate the objects of intrerests from other regions of the image, which can be
considered as background.
• The segmentation results in its ultimate form as black and white, which is called binary image
Original Binary Skeletonised
Original (left) and the corresponding binarised (middle) and skeletonised (right) micrographs, obtained using ImageJ
software with Line8 plug-in, from TEM performed at 5800 magnification. Images show the protein network in cheese
grains (Geng et al., 2011).

26. Conti..
Counting or measuring
• This the process of acquiring quantitative data based on the desired measurements.
• In most imaging programmes, there is a choice of up to 20–30 measurement parameters.
• Examples of common measurements include parameters such as diameter, shape and size
distribution related to the fat globules or pores.
Data analyses
• The data obtained are exported, using image analyses software, directly to other programmes
such as Excel or in a format that is recognisable by other programmes via dynamic data
exchange.
• These data are further processed in order to summarise or visualize the relevant aspects.

27. • The localization and distribution of bacterial
cells in cheese micrographs has mainly been
studied using CSLM techniques.
• In Cheddar cheese, used CSLM together with
SEM to localize the bacterial colonies of the
starter culture L. lactis and Lb. casei within the
protein matrices of the cheese.
• Clusters of starter culture cells were of uneven
size and had different shapes (blue color spots)
and distributed randomly in the cheese matrix.
Romeih et al. (2012) and Martinovic et al. (2013).
Localization &
distribution of bacteria
in cheese
microstructure

28.  SEM micrograph of Cheddar cheese,
where black arrows indicate the
bacterial cells.
 CSLM micrograph , where the protein,
fat and bacterial cells are labelled
using fluorescent dyes of Fast Green
FCF (grey), Nile Red (green) and
Hoechst 33342 (blue), respectively

29. In another study CSLM
micrographs of UF cheese
showing the protein and fat
matrix in brown/yellow and
black colour respectively.
The viable bacterial cells
appear in green colour
(left) and the compromised
nonviable cells (right) in red
colour.
(Hannon et al., 2006).

30.  In another study , In Cantal cheese, a
French Cheddar-like cheese, and
Emental cheese, lactic acid bacterial
localization was determined by
CSLM.
 CSLM micrographs showing the
Localization of the bacteria in Cantal
cheese (left), where the bacteria
appear as bright areas, fat appears in
grey levels and proteins in white, and
Emental cheese (right), where fat and
proteins appear in red and grey
respectively
(De Freitas et al., 2007;
Lopez et al. 2007)