Departement of

Food Science and Technology

Microbiological analysis of fresh and

frozen foods, canned food, and high
carbohydrate-food
Harsi D. Kusumaningrum
Harum Fadhilatunnur

TPN300 Food Analysis

 Outline
Preparation of sample homogenate

Microbiological analysis of fresh and frozen foods

Microbiological analysis of canned food

Microbiological analysis of high content

carbohydrates-food

TPN300 Food Analysis

 Course Learning Outcomes
After completing this course, students will be able to
▪ describe the principles behind the preparation of sample homogenate as
well as the analysis of fresh and frozen foods, canned food, and high
content carbohydrates-food
▪ select appropriate microbiological analysis to assess the microbiological
quality of fresh and frozen foods, canned food, and high content
carbohydrates-food
▪ interpret data of microbiological analyses of fresh and frozen foods, canned
food, and high content carbohydrates-food

TPN300 Food Analysis

 Preparation of Sample Homogenate
Points of attention
• Cool refrigerated samples in ice at 0-4°C and transport them in a
sample chest with suitable refrigerant capable of maintaining the
sample at 0-4°C until arrival at the laboratory.
• Do not freeze refrigerated products. Unless otherwise specified,
refrigerated samples should not be analyzed more than 36 h after
collection.
• Examine refrigerated shellfish and shell stock within 6 h of collection
but in no case more than 24 h after collection.
Bacteriological Analytical Manual Online, April 2003

TPN300 Food Analysis

 Points of attention
Thawing
• Whenever possible, avoid transferring the sample to a second
container for thawing.
• Normally, a sample can be thawed at 2-5°C within 18 h. If rapid
thawing is desired, thaw the sample at less than 45°C for not more
than 15 min.
• When thawing a sample at elevated temperatures, agitate the
sample continuously in thermostatically controlled water bath.

Bacteriological Analytical Manual Online, April 2003

TPN300 Food Analysis

 Blending and diluting of samples
requiring enumeration of microorganisms https://goldenlab.co.id/

• All foods other than nut meal

• Add 450 (or 225) ml phosphate-buffered dilution water to blender jar
containing 50 (or 25) g analytical unit and blend 2 min. Make dilutions of
original homogenate promptly, using pipets that deliver required volume
accurately.
• Do not deliver less than 10% of total volume of pipet. For example, do not use
pipet with capacity greater than 10 ml to deliver 1 ml volumes; for delivering
0.1 ml volumes, do not use pipet with capacity greater than 1.0 ml.
• Not more than 15 min should elapse from the time sample is blended until all
dilutions are in appropriate media.

TPN300 Food Analysis

 Sampling of food surfaces
Swabbing
• Swabs of approximately 2 cm with the head firmly attach to an
applicator stick of 12 to 15 cm long.
• Swabs can be made of cotton, calcium alginate, Dacron, or
Rayon.
• Swabs made of calcium alginate fibres are soluble in aqueous
https://www.rapidmicrobiology.com

solutions containing 1% of sodium hexametaphosphate (or

sodium glycerophosphate, or sodium citrate, or 1% or any
mixture of these).

https://www.rapidmicrobiology.com

TPN300 Food Analysis

 • The sponge should have an area of at least 50 cm2. The
width of the sponge should be no larger that 10 cm.
• The diluent should be 0.9% unbuffered saline (sodium
chloride solution) for lab-prepared cellulose waffle www.keydiagnostics.com.au

dishcloths.
• Pre-moistened swabs are acceptable for use as long as it
can be shown any diluent residues which may remain on
the carcass after sampling are not harmful to consumers.
https://www.sodibox.com/

TPN300 Food Analysis

 Exercise 1
The analysis of TPC of meat carcass has been prepared as follows:
Carcass area of 10cmx10cm was swabbed and the swab was inserted
into 200 mL buffer dilution. 20 mL of this suspension was homogenized
in 140 mL other buffer dilution, and then 10 mL of this last suspension
was added into 50 mL buffer dilution. From this last suspension each
of 1 mL was pipetted onto Petri dishes (triplo), added with 20 mL PCA.
After incubation 154, 162, and 119 colonies were found.
What is the TPC per cm2 carcass?

TPN300 Food Analysis

 Microbiological analysis
of fresh and frozen foods
www.ghanalive.tv

TPN300 Food Analysis

 Microbiological analysis of meat & poultry
• Microorganisms on fresh meat and poultry
• Mesophillic bacteria from GI tract
• Bacteria from environments (soil/water)
• Bacteria from process equiment and human
• Bacteria: coliform, enterococci, E. coli, S.aureus, C. perfringens,
Salmonella
• Microorganisms of chilled product: changed to psychrophillic,
particularly Pseudomonas, Acinetobacter, Moraxella

TPN300 Food Analysis

 Spoilage of meat products

• Fresh meat spoilage is typically by

psychrophilic, Gram negative aerobes
(Pseudomonas) or pychrophilic, Gram
positive, facultative anaerobes (lactics)
- (vacuum-packaged fresh meat)
https://www.uma-food.com/
• Processed products spoilage is most
often by the lactic acid bacteria

TPN300 Food Analysis

 Spoilage of food products
Spoilage of food products
Temperature
relationships of 212oF 100oC
200
the major groups 90
180
of bacteria 80
160 70 Obligate thermophiles
(optimum temp.)
140 60
Clostridium thermosaccharolyticum
Facultative thermophiles 50 Bacillus stearothermophilus
Streptococcus thermophilus
Clostridium perfringens 40 Mesophiles
(optimum temp.)
30
Escherichia coli
Psychrophiles 20 Bacillus subtilis
(optimum temp.)
10
Pseudomonas geniculuta
Achromobacter guttatus 0
-10
Harsi D. Kusumaningrum, 2020

TPN300 Food Analysis

 Produk Olahan Daging, Daging Unggas dan Daging Hewan Buruan dalam Bentuk Utuh
Maupun Potongan yang Dibekukan (Diproses, Disimpan Maupun Diperdagangkan)

How do we
prepare the
sample?

TPN300 Food Analysis

TPN300 Food Analysis
 Microbiological analysis of fishery product

•Shelf life depending on initial level of

contamination and storage temperature
•Number and species affected by normal
flora and handling
https://www.netralnews.com

• Spoilage microorganisms particularly Pseudomonas, usually psychrotrophic, and

produced methyl mercaptan, dimethyl sulfida, etc
• Often found pathogens such as: Salmonella, S.aureus, C. perfringens, C. botulinum,
V. parahaemolyticus, V. cholerae, Y.enterocolitica, E. coli and proteus

TPN300 Food Analysis

 Microbiological Analysis of Vegetables
• Mold spoilage is caused by Penicillium, Phytophthora,
Alternaria, Botrytis, Fusarium, Cladosporium,
Trichoderma, and Aspergillus.
• Among the bacteria, species of Pseudomonas,
Erwinia, Xanthomonas, Enterobacter,
Flavobacterium, Chromobacter, Lactobacillus,
Bacillus, and Clostridium are the most important as
well as non fecal enterococci and lactic acid
streptococci

https://www.dreamstime.com/

TPN300 Food Analysis

 Frozen vegetables
• Major source of organisms: contaminated equipment (blanching step destroys
most contaminating microorganisms, except spores).
• Coliforms and enterococci are common contaminants and may be present in large
numbers. Their presence does not usually indicate fecal contamination. They are
part of the processing line.
• Escherichia coli is a relatively rare contaminant of blanched vegetables and its
presence may indicate fecal contamination.

TPN300 Food Analysis

PerBPOM No. 13 Tahun 2019 tentang Batas Cemaran Maksimal Mikroba dalam Pangan Olahan
 Recommended tests: Additional tests:
• Aerobic plate count. • Geotrichum count (sanitation index for fruits and
• Total coliform count. tomato products).
• E. coli. • Listeria monocytogenes (for fruits and
• Salmonella. vegetables destined for chronically ill or
immuno-compromised persons).
• Yeast and molds.
• E. coli 0157/H7.

Brackett, R. E. and Splittstoesser, D. F. 2001. Fruits and Vegetables, p. 515. In

F. P. Downes and K. Ito (eds.), Compendium of Methods for the
Microbiological Examination of Foods. American Public Health Association,
Washington, DC.
Permentan No. 53 Tahun 2018 tentang Keamanan dan Mutu Pangan Segar
Asal Tumbuhan
https://goldenlab.co.id/

TPN300 Food Analysis

 Microbiological analysis of high carbohydrate foods

• Food with high content of carbohydrate such as sugar

and flour is dry product that often contaminated by
microorganisms, due to unhygienic packaging and
storage.
• Sugar and flour often contain thermophillic bacteria
that grow at 40-60oC or higher, particularly the
sporeformer bacteria.
https://id.pinterest.com/
• Spoilage thermophillic bacteria usually belong to the
genus Bacillus and Clostridium.

TPN300 Food Analysis

TPN300 Food Analysis
 • Thermophillic spores often found in sugar and flour, caused specific spoilage
such as:
(a) spores causing ’flat sour’ → for example B. stearothermophilus that grows at
low acid food.
(b) spores of anaerobic bacteria non-producing H2S → for examples: spores of C.
thermosaccharolyticum which is thermophilic; C. botulinum and C.
sporogenes which are mesophilic.
(c) spores of anaerobic bacteria causing sulfide spoilage (producing H2S) → for
example C. nigrificans. There are also spores of facultative anaerobic
bacteria that can cause sulfide spoilage, for example B. betanigrificans.

TPN300 Food Analysis

 Principles of analysis
• To enumerate the number of thermophilic spores in sugar and flour, the sample
must be heated to kill the vegetative cells.
• However, since flour will be gelatinized when it is heated and cause problems
during pipetting, flour suspension is mixed with media prior to heat treatment.
• 3 analyses:
1. analysis of flat-sour spores → DTBPA
2. analysis of anaerobic thermophilic spores → Thyoglycolate layered with NA
3. analysis of anaerobic spores causing sulfide spoilage in sugar and flour → sulfite agar

TPN300 Food Analysis

 Exercise 2
Excercise 3:
For microbiological analysis of cane sugar, 20 g was brought in a
sterile 250-mL flask and added by sterile distilled water to a final
volume of 100 mL. After boiled for 5 min, the sample was cooled
immediately in cold waterbath and the volume was brought
back to 100 mL with sterile water. Two mL of the sample was
inoculated into each of 5 petri plates, added by Dextrose
Tryptone Agar, and incubated at 55oC for 48-72 h. If after
incubation of the plates at 55oC for 48-72 hr, 10 colonies were
found in 5 plates, what is the spores count of the product
(spores/10g)?

Harsi D. Kusumaningrum, 2020

TPN300 Food Analysis

 Microbiological analysis of canned food
• Canned food is a thermal-processed food by high temperature in closed
(hermetic) container under anaerobic condition.
• In Indonesia, canned foods are regulated under BPOM regulation No. 24/2016
about the requirement of commercially sterile foods.
• Commercially sterile foods are low acid products (pH >4.6 and Aw >0.85)
hermetically packaged (sealed packaging to avoid the entry of microbes during
and after heat processing) and commercially sterilized for storage at room
temperature.
• The regulation states that commercial sterilization must be
carried out to an F0 value for at least 3.0 minutes in order
to destroy Clostridium botulinum.

TPN300 Food Analysis

 Microbiological analysis of canned food
• Incidence of spoilage in canned foods is low, but when it occurs it must be
investigated properly.
• When can contains a spoiled product and no viable microorganisms, spoilage
may have occurred before processing or microorganisms causing the spoilage
may have died during storage.

TPN300 Food Analysis

 Classification of canned food products according
Classification of Canned food products according to acidity
to acidity
Low acid Acid
pH greater than 4.6 pH 4.6 and below
Meats Tomatoes
Seafoods Pears
Milk Pineapple
Meat and Vegetable Mixtures Other fruit
Spaghetti Sauerkraut
Soups Pickles
Vegetables Berries
Asparagus Citrus
Beets
Pumpkin
Green beans
Corn
BAM Online, January 2001
Harsi D. Kusumaningrum, 2020

TPN300 Food Analysis

 Spoilage of canned food
• Swollen cans often indicate a spoiled product, cans may progress from normal
to flipper, springer, soft swell, and hard swell.
• Flat – a can with both ends concave; it remains in this condition even when the
can is brought down sharply on its end on a solid, flat surface.
• Abnormal cans are not always caused by spoilage but also overfilling, buckling,
denting, or closing while cool.
• Flipper, a can that normally appears flat; when brought down sharply on its end
on a flat surface, one end flips out. When pressure is applied to this end, it flips
in again and the can appears flat
• Springer, a can with one end permanently bulged. When sufficient pressure is
applied to this end, it will flip in, but the other end will flip out

TPN300 Food Analysis

 • Soft Swell, a can bulged at both
ends, but not so tightly that the
ends cannot be pushed in
somewhat with thumb pressure.
• Hard Swell, a can bulged at both
ends, and so tightly that no
indentation can be made with
thumb pressure. A hard swell
will generally "buckle" before
the can bursts. Bursting usually
occurs at the double seam over
the side seam lap, or in the
middle of the side seam

https://eriecountypa.gov/

TPN300 Food Analysis

 Spoilage of canned foods
• Usually caused by growth of microorganisms following leakage or
underprocessing
Leakage
• Occurs from can defects, punctures, or rough
handling.
• Contaminated cooling water sometimes leaks to
the interior through pinholes or poor seams and
introduces bacterial spoilage.
• A viable mixed microflora of bacterial rods and
cocci indicates of leakage, which may usually be
confirmed by can examination.

TPN300 Food Analysis

 Spoilage of canned foods
Underprocessing, may be caused by:
• undercooking;
• retort operations that are faulty because of inaccurate or improperly functioning
thermometers, gauges, or controls;
• excessive contamination of the product for which normally adequate processes
are insufficient;
• changes in formulation or handling of product resulting in a more viscous
product or tighter packing in the container, with consequent lengthening of the
heat penetration time;

TPN300 Food Analysis

 Hazards of canned foods
• Underprocessed and leaking cans are of major concern and both pose potential
health hazards.
• Naturally, if Clostridium botulinum (spores, toxin, or both) is found, the hazard is
obvious.
• Intact cans that contain only mesophilic, Gram-positive, sporeforming rods should
be considered underprocessed, unless proved otherwise.

TPN300 Food Analysis

TPN300 Food Analysis
 Spoilage microorganisms that cause high and low acidity
in various vegetables and fruits
Spoilage type pH groups Examples
Thermophilic
Flat-sour >5.3 Corn, peas
Thermophilic(a) >4.8 Spinach, corn
Sulfide spoilage(a) >5.3 Corn, peas
Mesophilic
Putrefactive anaerobes(a) >4.8 Corn, asparagus
Butyric anaerobes >4.0 Tomatoes, peas
Aciduric flat-sour(a) >4.2 Tomato juice
Others
Lactobacilli 4.5-3.7 Fruits
Yeasts <3.7 Fruits
Molds <3.7 Fruits
TPN300 Food Analysis
 Spoilage by Thermophilic Bacteria
• Under processing of canned foods results in spoilage by thermophilic
bacteria and the three types of spoilage by thermophiles are:
• (i) flat sour,
• (ii) thermophilic acid (TA) spoilage, and
• (iii) sulphide spoilage.

TPN300 Food Analysis

 Spoilage by thermophilic bacteria
(i) flat sour (asam tanpa gas)
• In flat sour, the ends of the can remain flat but the pH decreases.
• Spoilage cannot be detected unless the can is opened.
• Usually caused by facultative anaerobic sporeforming bacteria
• The spores germinated at thermophilic temperature, the vegetative cells grow at
both mesophilic and thermophilic temperature
• The spoilage occurs in low acid foods and is caused by thermophilic species of
Bacillus; such as B. coagulans and B. stearothermophilus. These organisms
produce acid without gas.

TPN300 Food Analysis

 Spoilage by thermophilic bacteria
ii) thermophilic acid (TA) spoilage
• the can swells due to production of CO2 and H2 by
Clostridium thermosaccharolyticum.
• Spoilage mostly occurs in low and medium acid foods.
• C. thermosaccharolyticum is an obligate thermophile
and therefore needs hot storage for its development.

https://www.sasionline.org/

TPN300 Food Analysis

 Spoilage by thermophilic bacteria
(iii) sulphide spoilage
• Sulphide spoilage (sulphur stinker) is caused by Clostridium nigrificans in low acid
foods.
• Spores of this bacterium are not very heat resistant and their appearance is an
indication of under processing. Spoilage is indicated by the presence of H2 and
blackening of the material.
• Sources of all these organisms are generally, the plant equipment, sugar, starch,
and other material or soil.

TPN300 Food Analysis

 Spoilage by mesophilic bacteria
• Spoilage of canned foods by mesophilic organisms is indicative of under
processing and is caused by species of Bacillus, Clostridium, yeast and fungi*.
* If product misses retort completely

TPN300 Food Analysis

 Spoilage by mesophilic bacteria
• Clostridium butyricum and C. pasteurianum
– produce butyric acid with swelling of the container by production of CO2
and H2
– putrefactive anaerobes, generally grow in low acid foods such as peas,
corn, meat, poultry etc, but sometimes may also spoil medium acid
foods.
• B. subtilis and B. mesentroides
– have been found to grow in poorly evacuated cans of sea food, meat and
milk.
• Gas forming B. polymyxa and B. macerans
– reported to cause spoilage of canned peas, spinach, peaches and tomato

TPN300 Food Analysis

 Spoilage by mesophilic bacteria
• Thermoduric bacteria including enterococci, Streptococcus
thermophilus etc.
• nonspore forming bacteria, the presence in canned food is indicative of a leak
or under processing.
• Heterofermentatives, produce CO2 and swell the can.
• Some other non-gas forming, non-spore forming bacteria causing
spoilage in cans are species of Pseudomonas, Micrococcus, and
Proteus.

TPN300 Food Analysis

 Spoilage by mesophilic bacteria

• Molds and their spores and yeasts are destroyed at the

pasteurization temperature. Their presence in canned
food is the result of either gross under processing or
leakage.
• Spoilage of canned fruits and fruit products or milk by
yeasts may result in CO2 production and swelling of the
cans.
• Film yeasts and fungi grow on the surface and cause
degradation of the products summarizes the types of,
spoilage commonly encountered in canned foods.

TPN300 Food Analysis

 Canner’s test
• Flat Sour Spores
• Mesophilic Bacteria
• Sulfide Spoilage Spores
• Thermophilic Anaerobic Spores
• Total Thermophilic Spores
• Yeast & Mold

TPN300 Food Analysis

 Examination of canned foods

U.S. Food & Drug Administration

Center for Food Safety & Applied Nutrition
Bacteriological Analytical Manual Online
January 2001
Chapter 21A
Examination of Canned Foods

https://www.rpcau.ac.in/ https://www.elgalabwater.com/

TPN300 Food Analysis

 Opening the can
Hard swells, soft swells, and springers.
• Chill hard swells in refrigerator before opening.
• Scrub entire uncoded end and adjacent sides of can using abrasive cleanser, cold water,
and a brush, steel wool, or abrasive pad.
• Rinse and dry with clean sterile towel.
• Sanitize can end to be opened with 4% iodine in 70% ethanol for 30 min and wipe off
with sterile towel.
• DO NOT FLAME.
• Badly swollen cans may spray out a portion of the contents, which may be toxic.
• Take some precaution to guard against this hazard, e.g., cover can with sterile towel or
invert sterile funnel over can.
• Sterilize can opener by flaming until it is almost red, or use separate presterilized can
openers, one for each can.

TPN300 Food Analysis

 Opening the can
Hard swells, soft swells, and springers (cont’d)
•At the time a swollen can is punctured, test for headspace gas, using a qualitative
test or the gas-liquid chromatography method. For a qualitative test, hold mouth
of sterile test tube at puncture site to capture some escaping gas, or use can-
puncturing press to capture some escaping gas in a syringe.
•Flip mouth of tube to flame of Bunsen burner. A slight explosion indicates
presence of hydrogen.
•Immediately turn tube upright and pour in a small amount of lime water. A white
precipitate indicates presence of CO2.
•Make opening in sterilized end of can large
• enough to permit removal of sample.

TPN300 Food Analysis

 Opening the can
Flipper and flat cans.
•Scrub entire uncoded end and adjacent
sides of can using abrasive cleanser, warm
water, and a brush, steel wool, or abrasive
pad.
•Rinse and dry with clean sterile towel.
•Gently shake cans to mix contents before • Sterilize can opener by flaming until it is
sanitizing. almost red, or use separate pre-
•Flood end of can with iodine-ethanol sterilized can openers for each can.
solution and let stand at least 15 min. • Make opening in sterilized end of can
•Wipe off iodine mixture with clean sterile large enough to permit removal of
towel. sample.

TPN300 Food Analysis

 Exercise 3
Excercise 2:
During microbiological analysis of canned crabmeat,
50g sample is homogenized with 50ml dilution. Then,
1mL and 0.1mL are pipetted to each of 2 Petri dishes
(duplo) and added by 15 ml PCA. After incubation at
30oC, 30 and 20 colonies are found on plates with 1 mL
sample; and 1 and 0 colony are found on plates with
0.1 mL sample. The can is still intact without swell but
spoilage is found by rod bacteria.
What is the TPC (cfu/g) of the product?

Harsi D. Kusumaningrum, 2020

TPN300 Food Analysis