Unit 15

Microbiological Analysis of Canned Foods

15.1. Course Objectives

This laboratory work is aimed to equip students with the principles and skills of
analysis on determining microbiological quality of canned food in general and
microbial spoilage in low acid canned foods.

15.2. Course Learning Outcomes

After completing this laboratory work, students are expected to be able to:
1. explain the principles of aseptic sampling for canned food.
2. explain the principles of microbiological quality analysis for canned food in
general.
3. explain the principles of flat sour analysis in low acid canned food.
4. explain the principles of anaerobic thermophilic spoilage analysis in low acid
canned food.
5. explain the principles of sulfide spoilage analysis in low acid canned food.
6. demonstrate the microbiological analysis of low acid canned food for aerobic and
anaerobic microorganisms.
7. demonstrate the analysis of microbial spoilage in low acid canned food.

15.3. Principle of Analysis

15.3.1. Canned Food Spoilage
Canned food is a thermal-processed food by high temperature in closed
(hermetic) container under anaerobic condition. Quality requirement for canned
foods is regulated by Indonesia’s national standard, covering standard for both
aerobic and anaerobic bacteria. One example is a standard for canned soup and
broth as shown in Table 15.1.
Spoilage in canned food is rarely happen. However, if spoilage occurred, a
laboratory test must be carried out. Quality decreasing of canned food can be
classified into physical damage, chemical deterioration, and microbiological
spoilage.

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124 Microbiological Analysis of Canned Foods

Table 15.1. Microbiological standard for canned soup

and broth (SNI 7388-2009)
Microbial Contamination Maximum Limit
Aerobic bacteria < 10 col/g
Anaerobic bacteria < 10 col/g
Clostridium sp. Negative/g

15.3.1.1. Physical damage

Physical damage happened in canned food usually is not harmful, but the
product may not be available to be consumed anymore due to the non-proper
appearance. For example: damage due to heat impact.
15.3.1.2. Chemical deterioration
Chemical deterioration can be a nutrient degradation, or the use of unsuitable
metal as can materials that leads to chemical reaction between the can and food. The
chemical spoilage are hydrogen swells, black stains, and discoloration, or rot due to
the reaction between can and corrosive substances.
(a) Hydrogen swells
Can swelling is caused by hydrogen formation from the chemical reaction
between acid from food and metal from can. This happens when acid food is
packaged in defect can, for example the tin layer of can is grazed or the metal used
for the can is not suitable for acid food.
(b) Black stains in the inner part of can
This spoilage happens frequently in corn, shrimp, crab, meat, and fish canning.
Usually it happens at sterilization process, where sulfur in protein is broken down
and then the product reacts with iron from can. Black stains actually are not harmful
to human, but it reduces product appearance.
15.3.1.3. Microbial spoilage
Microbial spoilage in canned food usually depends on the pH, i.e low acid food
with pH > 4.6 or high acid food with pH ≤ 4.6. Low acid food is easier to experience
microbial spoilage than high acid food, therefore this laboratory work focuses on the
microbial spoilage in low acid canned food.
(a) Microbial spoilage in low acid canned food
Low acid canned food is usually processed with thermal process to achieve the
commercial sterility. Spoilage in this type of food usually happens if the product is
kept at >43oC. This spoilage may be caused by heat-resistant thermophilic spore-
forming bacteria. These bacteria can grow at 55oC, and some of them still can grow
at 70oC. In addition, some of them are facultative thermophilic which may grow at
35oC or at lower temperature.
Some microbial spoilage that mostly happened in low acid canned food are: (a)
flat sour; (b) anaerobic thermophilic spoilage with swelling; and (c) sulfide spoilage.
Food Analysis Laboratory Manual 125

(1) Flat sour

In this type of spoilage, can is flat, but pH of the product decreased. Flat sour is
caused by Bacillus stearothermophilus, facultative anaerobic bacteria. Its spores can
germinate only at thermophilic temperature, but the growth of its vegetative cells
can happen at both thermophilic and mesophilic temperature.
(2) Anaerobic thermophilic spoilage with swelling
The symptom of this spoilage is swelling and if strong swelling occurred it
sometimes can cause explosion. It is commonly caused by obligate anaerobic-
thermophilic spore-forming bacteria, such as Clostridium thermosaccharolyticum,
which produces a lot of H2 and CO2 gas, resulting in can swelling. Canned food
contaminated by these bacteria usually has cheesy odor. The vegetative cells may
still grow at temperature lower than thermophilic temperature.
(3) Sulfide spoilage
This spoilage is indicated by flat can, but the product inside turns into black and
has the smell of rotten egg. It is commonly caused by obligate anaerobic-thermo-
philic spore-forming bacteria, i.e. Desulfotomaculum nigrificans which produce H2S
gas. The can will not swell because H2S is highly soluble in food, but it will react
with iron in food producing iron sulfide with black color.
(b) Microbial spoilage in high acid canned food
Microbial spoilage in high acid canned food is generally caused by micro-
organisms that can grow at pH ≤ 4.6 or heat resistant microorganisms. This may
happen if there is a high level of contamination at raw materials and/or inadequate
thermal process due to maintaining product’s quality, such as the hardness, vitamin
content, etc. There are some groups of microorganisms causing spoilage in high acid
canned food:
(1) Anaerobic butyric acid-producing bacteria
Example of this microbe is Clostridium pasteurianum. This anaerobic bacterium is
a mesophilic and spore-forming bacteria. It does not only produce butyric acid, but
also carbon dioxide and hydrogen.
(2) Aciduric acid-forming bacteria
Example of this type of bacteria is Bacillus coagulans. This bacterium usually
grows in tomato and its products. It is a facultative anaerobic bacterium and can
grow at 30-35oC or 55oC.
(3) Heat resistant mold
This mold usually contaminates fruits juice and concentrate. Some examples of
heat resistant mold are Byssochlamys fulva or some identical species producing
ascospores which is very heat resistant. The spoilage is indicated by moldy taste and
odor, faded color, presence of mold’s mycelium, and sometimes mild swelling on
the lid.
126 Microbiological Analysis of Canned Foods

(4) Yeast and/or non-spore forming bacteria

These microorganisms grow in canned food with extreme inadequate heating or
can leakage.
15.3.2. Principles of Microbiological Analysis for Canned Food
Analysis of canned food is usually carried out for aerobic and anaerobic
bacteria, both mesophilic and thermophilic bacteria. However, sometimes analysis
for spore-forming bacteria Clostridium sp. is also needed. For aerobic and anaerobic
bacteria analysis, Plate Count Agar (PCA), Nutrient Agar (NA) atau Tryptone Soy
Agar (TSA) are used. The incubation is conducted at 30°C for mesophilic bacteria or
at 55°C for thermophilic bacteria. For anaerobic bacteria analysis, the incubation is
carried out in anaerobic condition.
15.3.3. Principles of Microbial Spoilage Analysis of Low Acid Canned Foods
Microbial spoilage occurred in low acid canned food is commonly caused by
acid-forming non-gas-forming bacteria (flat sour), thermophilic anaerobic gas-
forming bacteria, and/or sulfide-forming bacteria.
Acid-forming non-gas-forming bacteria analysis is done by inoculating sample
in Petri dish containing Dextrose Tryptone Bromcresol Purple Agar (DTBPA). In this
media, the acid-forming bacteria will grow by forming colony surrounded by yellow
area due to the acid production.
Analysis of anaerobic thermophilic gas-forming bacteria is done by inoculating
sample in Thioglycolate media. During incubation, Thioglycolate media should be
layered by Nutrient Agar (NA) or parafin. The positive growth of anaerobic bacteria
is indicated by turbidity with or without gas. If the NA or paraffin layer comes up to
the top, it indicates that there is gas formation.
Sulfide-forming bacteria analysis is done by inoculating sample in test tubes
containing sulfite agar. Sulfide formation is indicated by black spot in the media. The
incubation is carried out at two temperatures, i.e. 30-35oC for mesophilic bacteria
and 55oC for thermophilic bacteria.
15.3.4. Anaerobic Condition for Microbiological Analysis of Canned Food
There are some methods to create anaerobic condition during incubation. Some
of them are:
15.3.4.1. Anaerobic jar with catalyst
This jar must be in vacuum condition but then filled with hydrogen gas (H2) or
gas mixtures containing H2. The presence of hydrogen indicates that the catalyst is
active. The most common catalyst used for this method is palladium, which becomes
hot within 1 minutes when it is active. The catalyst can be re-activate by heating it at
160oC for an hour.
15.3.4.2. Jar with H2 and CO2 generator
The most common jar with H2 and CO2 generator is Gas Pak (BBL Microbiology
Systems) (Figure 15.1). This jar does not need vacuum condition. After generator
unit is activated by water addition, the jar is closed quickly, then water condensation
Food Analysis Laboratory Manual 127

and disappearance of color on methylene blue strip indicator must be observed

within 30 minutes at transparent jar. This condition indicates that there is no more
oxygen in the jar. Catalyst can be re-activated by heating it at 160oC for an hour.
Water addition into Gas Pak generator will result in hydrogen release. Hydro-
gen reacts with oxygen on catalyst surface producing H2O, so the condition will be
anaerobic. The generator will also release CO2 in sufficient amount because some
anaerobic bacteria cannot grow without the presence of CO2.

Figure 15.1. Gas Pak anaerobic system

15.3.4.3. Anoxomat system

There are some methods to create anaerobic condition, such as anoxomat system
(Figure 15.2). Anoxomat is connected to gas tube containing 10% O2, 10% CO2, and
other gases (N2 and H2 gas), so that the total gas mixture is 100%. The anaerobic gas
mixture in anoxomat system is regulated as 0.2% O2, 8% CO2, and 90% N2.

Figure 15.2. Anoxomat System

128 Microbiological Analysis of Canned Foods

The principles work of anoxomat system is as follows: first anoxomat will

regulate gas from gas cylinders with pressures above 1.6 bar. If the pressure has
been reached, anoxomat will check for leakage and followed by vacuuming process.
After that, anoxomat will include anaerobic gas mixture into the jar.

15.4. Procedures
15.4.1. Experiment 1. Can Observation and Sample Preparation for Microbiolo-
gical Analysis of Canned Food
Samples
 Canned corn
 Canned sardine
 Canned mushroom
 Canned fried rice
Equipment
 Sterile Petri dish
 Sterile beaker glass 50 mL
 Sterile pipette 1 mL
 Sterile spatula/spoon
 Bunsen burner
 Can opener
Media
 Sterile distilled water 10 mL
 Distilled water
Working Procedure
15.4.1.1. Can observation
1. Record all important information from the label or can before can is opened.
2. Take the label off and then marked (give a code) the can with marker.
3. Observe the physical damage outside of cans, such as the presence of rust, dents
from impact, swelling, closure is not good/imperfect sealing or leakage, loose
connections edge cans, or other damages.
15.4.1.2. Sample preparation for canned food
1. Wash the second layer of the outer part of can with soap and water and rinse it
with clean water (do not wash leakage can).
2. For normal can, before open the can, hold the bottom of the can. The lid will be
opened by flame the lid with rotation, so that the heat will be distributed evenly.
3. Open the lid by using sterile can opener, lift the lid, and change it with sterile
Petri dish lid.
4. Do not flame swelled can because it may explode, but clean it with cloth or
cotton containing alcohol 70%. After that, open the lid using sterile can opener,
lift the lid, and change it with sterile Petri dish lid.
5. Liquid canned food, such as soup, meat in broth, milk, tomato paste, canned
fruits, etc., can be analyzed directly. Solid canned food (meat, fish) without
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liquid must be diluted with sterile distilled water (1:1) by adding 10 mL of water
into 10 g of sample, and then mixed.
15.4.2. Experiment 2. Microbial Spoilage Analysis of Low Acid Canned Food
Samples
 Canned corn
 Canned sardine
 Canned mushroom
 Canned fried rice
Equipment
 Sterile Petri dish
 Sterile beaker glass 50 mL
 Sterile pipette 1 mL
 Sterile spatula/spoon
 Bunsen burner
 Incubator at 30oC and 55oC
 Can opener
Media
 Sterile liquid Tryptone Soy Agar (TSA)
 Sterile liquid Dextrose Tryptone Bromcresol Purple Agar (DTBPA)
 Thioglycolate medium 10 mL
 Sterile liquid sulfit agar 10 mL
 Sterile distilled water 100 mL
 Sterile liquid Nutrient Agar or liquid parafin
 Dilution water 9 mL
Analytical procedure
15.4.2.1. Mesophilic and thermophilic aerobic bacteria analysis
1. Prepare a dilution of 10-1 from the sample. Take 1 mL of each sample from the
dilution of 100 and 10-1 and put it into sterile Petri dish.
2. Pour the TSA into the Petri dish, distribute it well by rotating the plate and let it
solidify.
3. After the agar becomes solidified, incubate 1 set at 30°C (mesophilic) and at
55°C (thermophilic) for 2 – 3 days.
4. Count the colonies that grown on the plate using standard method.
15.4.2.2. Mesophilic and thermophilic anaerobic bacteria analysis
1. Prepare a dilution of 10-1 from the sample. Take 1 mL of each sample from the
dilution of 100 and 10-1 and put it into sterile Petri dish.
2. Pour the TSA into the Petri dish, distribute it well by rotating the plate and let it
solidify.
3. After the agar becomes solidified, put the Petri dishes in anoxomat jar, and
create anaerobic condition following the anoxomat system.
4. Incubate 1 set at 30°C (mesophilic) and at 55°C (thermophilic) for 2 – 3 days.
5. Count the colonies that grown on the plate using standard method.
130 Microbiological Analysis of Canned Foods

15.4.2.3. Mesophilic and thermophilic acid-forming non-gas-forming bacteria ana-

lysis
1. Prepare a dilution of 10-1 from the sample. Take 1 mL of each sample from the
dilution of 100 and 10-1 and put it into sterile Petri dish.
2. Pour DTBPA into the Petri dish, distribute it well by rotating the plate and let it
solidify.
3. After the agar becomes solidified, incubate 1 set at 30°C (mesophilic) and at 55°C
(thermophilic) for 2 – 3 days.
4. Count the positive colonies surrounded by yellow area, indicating the acid
formation in DTBPA.
15.4.2.4. Mesophilic and thermophilic anaerobic gas-forming bacteria analysis
1. Prepare a dilution of 10-1 from the sample. Take 1 mL of each sample from the
dilution of 100 and 10-1 and put it into test tube containing thioglycolate medium.
2. Pour liquid NA or paraffin on thioglycolate medium.
3. Incubate 1 set at 30°C (mesophilic) and at 55°C (thermophilic) for 2 – 3 days.
4. Report the positive tube(s) indicated by turbidity with or without gas formation.
15.4.2.5. Bacteria causing sulfide spoilage analysis
1. Prepare a dilution of 10-1 from the sample. Take 1 mL of each sample from the
dilution of 100 and 10-1 and put it into test tube containing liquid sulfite agar.
2. Mix it well by spinning or shaking the tube mildly.
3. After the agar becomes solidified, incubate 1 set at 30°C (mesophilic) and at 55°C
(thermophilic) for 2 – 3 days.
4. Report positive tube(s) with black color in sulfite agar media.

15.5. References
U.S. Food & Drug Administration, Center for Food Safety & Applied Nutrition. 2001,
Bacteriological Analytical Manual Online, Chapter 21A, Examination of Canned Foods
BSN. 2009. SNI 7388-2009 Batas maksimum cemaran mikroba dalam pangan.