18/02/2014

MICROBIOLOGY OF THERMAL
PROCESSING
Lilis Nuraida, Ph.D.
SEAFAST Center
dan
Departemen Ilmu dan Teknologi Pangan
Institut Pertanian Bogor

What causes it?

How to prevent it?

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Outline

 Microbiology of Food Processing

 Ecology of Microorganism in food
 Thermal inactivation of microorganism
 Microbioloy of low acid thermally
processed food
 Microbiology of acidified thermally
processed food
 Spoilage of themally processed food

Microbiology of Thermal Process - LN- 2014-FTSP

Microbiology of Food Processing

 The microorganisms of primary concern to

the food processor:
 Molds
 Yeast
 Bacteria.

They can grow in food and

cause spoilage and risk of
foodborne illness

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Microbiology of Food Processing

 Molds
 Multi-cell organisms forming tubular filaments.
 Reproduce by spores, which are borne in or on aerial
structures.
 Most molds have little heat resistance and cannot
survive the thermal processes for low-acid canned
foods.
 The presence of molds in canned meat and poultry products only as a
result of under-processing
under processing or as a post
post-processing
processing contamination.
contamination

The growth of mold in canned foods

indicating the food container has an opening
to the outside environment (leakage).
Microbiology of Thermal Process - LN- 2014-FTSP

Microbiology of Food Processing

 Yeasts
 Single cells, larger than bacteria
 Widely found in nature and are particularly associated with
liquid foods containing sugars and acids.
 Quite adaptive to adverse conditions such as acidity and
dehydration.
 Compared to bacterial spores, yeasts possess little resistance
to heat.
 Most yeast forms are destroyed on heating to 77ºC.

• If yeast causes spoilage of canned food, under-processing

or leakage must be suspected
• The growth of yeasts results in the production of alcohol
and large amounts of carbon dioxide gas, which swells the
container.

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Microbiology of Food Processing

 Bacteria
 Single cells, smaller than yeast
 Based on cell wall composition: Gram
positive and Gram negative
 The most important and troublesome of
all the microorganisms for the food
processor.
 Certain bacteria form endospore that
remain alive under conditions that kill
other
th cells.
ll
 Bacterial spores are heat resitant
 Formation of extracellular (capsule)
polysaccharides in certain bacteria
increase heat resistance

Microbiology of Thermal Process - LN- 2014-FTSP

Source of microorganism
 Raw materials and ingredients are the primary sources
 The ultimate source for pathogens in raw meat and
p
poultry
y is their feces.
 Soil or water can be a common source of food borne
microorganisms and spores.
 Vegetables become contaminated during production
 Those that grow close to or through the soil usually
having high numbers of bacteria and bacterial spores,
including spores of C.C botulinum.
botulinum

• The processing environment

and equipment
• Employee

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Bacteria spore (endospore)

 Spore forming bacteria: Clostridium and
Bacillus
 One cell vegetative forms a spore
 Dormant state
 Form in detrimental environment, i.e.
nutrition depletion
 Highly resistant cell to preserve the cell's
genetic material in times of extreme
stress:
 Endospores can survive environmental assaults
that would normally kill the bacterium.These
stresses include high temperature, high UV
irradiation, desiccation, chemical damage and
enzymatic destruction.
 Spore will germinate in suitable
environment, e.g. availability of nutrient
 Cocci do not form spore Microbiology of Thermal Process - LN- 2014-FTSP

Why Bacterial Endospores resistance?

 Unique cellular structure.
 The outer proteinaceous coat
surrounding the spore provides
muchh off the
h chemical
h i l andd
enzymatic resistance.
 Beneath the coat resides a very thick
layer of specialized peptidoglycan
called the cortex. Proper cortex
formation is needed for dehydration
of the spore core, which aids in
resistance to high temperature.
 A germ cell wall resides under the
cortex
 Small acid-soluble proteins (SASPs)
are only found in endospores.
 in part responsible for resistance to
UV light and DNA-damaging
chemicals https://micro.cornell.edu/research/epulopiscium/bacteria
l-endospores

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Spore germination

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Ecology of Microorganism in Food

Faktor-faktor yang mempengaruhi pertumbuhan

mikroorganisme
Kebutuhan Nutrisi
 Kandungan nutrisi
 Kebutuhan nutrisi
 Air kapang paling
 Oksigen minimal, diikuti
dengan khamir,
 Suhu kemudian bakteri
 Nilai pH gram negatif
 Senyawa  Bakteri Gram positif
antimikroba membutuhkan nutrisi paling
lengkap.
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pH dan pertumbuhan
Ketersediaan air minimum mikroorganisme
untuk pertumbuhan 1 2 3 4 5 6 7 8 9 10 11 12 13

mikroorganisme Kapang

Khamir

Bakteri Asam Laktat

Mikroorganisme
g Aw minimum Staphylococcus
p y aureus

untuk tumbuh Salmonella spp

Kapang 0.75 Escherichia colii

pH 4.6 C. botulinum

Khamir 0.88 V. parahaemolytiicus

C. perfringens

C. botulinum 0.93
• Berasam rendah (pH > 5.3) :
S. aureus 0.85 • kacang-kacangan, daging, susu, santan,
keju
keju.
Salmonella 0.93 • Berasam sedang (pH 4.5 – 5.3) :
• alpokat, nangka.
• Asam (pH 3.7 – 4.5) :
• tomat, pear, nenas.
• Asam tinggi (pH < 3.7) :
• sawi asin, yoghurt, strawberry.
Mikrobiologi Makanan Kaleng - LN- 2012

Oksigen
Mengapa Clostridium, Bacillus,
BAL dapat tumbuh pada
makanan yang dikemas
hermetis?
BAL: mikroaerofilik
Clostridium bersifat
anaerob obligat
Bacillus besifat fakultatif
anaerob
Senyawa antimikroba:
• Bahan pengawet: nitrat/nitrit,
bakteriosin, asam organik,
benzoat dll
Suhu
• Psikrofilik: toleran terhadap dingin,
tumbuh pada suhu < 4 oC
• Psikrotrofik:
– pertumbuhan optimum pada 14 - 20
oC
C, dapat tumbuh pada 4 oC
– Contoh:
• Clostridium botulinum tipe E
• strain non-proteolitik tipe B dan F
 Mesofilik:
 suhu optimum 30 -37 oC
 Contoh: C.
C botulinum
 Termofilik:
 suhu optimum 45 - 60 oC
 sporanya resisten terhadap pemanasan,
tidak memproduksi toksin
 Contoh: Bacillus stearothermophilus

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Thermal Inactivation of microorganism:

•Pasteurization
•Sterilization
Why 2 methodes of thermal processing?
 Yeast, molds and non-spore former bacteria are inactivated by
boiling/cooking
 C. botulinum forms spores and need higher temperature to
inactivate them

Cooking, vegetative cells Coolling and storage, spores may

germinate, spoil the food and produce toxin
are killed, spores survive
Microbiology of Thermal Process - LN- 2014-FTSP

Pasteurisasi
 Pemanasan untuk membunuh mikroorganisme penyebab
penyakit
 Misalnya pasteurisasi susu: 63° C; 30 min., 72° C; 16 sec.,atau
ekivalen
 Membunuh mikroorganisme yang tidak membentuk spora
 Dua kelompok mikroorganisme tahan pasteurisasi susu:
 termodurik (Streptococcus dan Lactobacillus) dan
 termofilik (Bacillus, Clostridium, Alicyclobacillus, Geobacillus,
Thermoanaerobacter)
 Produk pasteurisasi tidak “steril”
 Perlu dibantu dengan teknik pengawetan lain: pendinginan, pengasaman
 Pangan dengan pH tinggi (>4.6) yang dipasteurisasi tidak bisa
disimpan pada suhu kamar, harus didinginkan
 Umur simpan pendek tergantung suhu dan waktu pemanasan
(biasanya 2 minggu)
 Contoh: susu pasteurisasi
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Sterilisasi
“Commercial sterility means the absence of microorganisms capable of
growing in the food at normal non-refrigerated conditions at which the food
is likely to be held during manufacture, distribution and storage.”
(Ref: Codex Alimentarius Commission (WHO/ FAO) CAC/RCP 40-1993 40 1993 )
 Pemanasan di atas suhu 100 C untuk menginaktivasi spora
 Harus dijamin bahwa proses telah membunuh/menginaktivasi semua
mikroba berbahaya bagi kesehatan
 Paling berbahaya Clostridium botulinum (botulin)
 Pendekatan keamanan dan komersial : Konsep 12 D
 Dua hal kritis : suhu dan waktu
 Produk dapat disimpan pada suhu kamar dalam waktu lama (1-2
tahun)
 Contoh: Makanan kaleng (kornet, sardin dll)

Makanan yang disterilisasi jika telah dibuka kemasannya harus

disimpan pada suhu dingin (refrigerator)
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Faktor-faktor yang mempengaruhi resistensi

mikroorganisme terhadap pemanasan

 Komposisi pangan:
 Air, Lemak, Garam, Karbohidrat, Nilai pH,
Protein dan senyawa lainnya
 Jumlah Mikroorganisme pada bahan
 Suhu pertumbuhan
 Senyawa penghambat
 Suhu dan waktu pemanasan
 Perlakuan ppendahuluan sebelum
pemanasan

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• Bakteri patogen tdk tumbuh • Terdapat peluang bakteri patogen

• Spora bakteri tidak tumbuh • Spora bakteri tumbuh
• Cukup proses pasteurisasi • Sterilisasi komersial
• Target: sel vegetatif, kapang, khamir • Target: spora bakteri

Mikrobiologi Makanan Kaleng - LN- 2014-FTSP

Thermal inactivation of microorganism

 Low acid foods commercially sterile

products will require high temperatures
to inactivate the organisms
g of concern.
 Acidified meat and poultry products can
be processed at lower temperatures,
since only vegetative cells must be
inactivated;
 The pH prevents germination and
outgrowth of the spores.
 The presence of nitrite and salt reduces
the amount of heat needed to achieve
commercial sterility
 heat kills the vegetative cells of
pathogens; salt and nitrite prevent
germination and outgrowth of spores

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Jumlah mikroorganisme vs
pemanasan
Jumlah mikroorganisme
yang tinggi pada bahan
pangan memerlukan
Jumllah mikroorganisme

pemanasan yang lebih

lama

Thermal process does

not replace sanitation
and microbiological
Xt
quality of raw materials

Waktu pemanasan
Mikrobiologi Makanan Kaleng - LN- 2013

Microorganisms vs thermal process

 Thermal Death Time (TDT): waktu 100-----
yang diperlukan untuk membunuh
sejumlah mikroorganisme pada suhu
Survivors (%)

tertentu
 Nilai D: waktu yang diperlukan untuk 10-------------------
membunuh sebanyak 90%
mikroorganisme atau 1 log10 pada
suhu tertentu.
 Sterilisasi makanan tidak asam 1--
menggunakan konsep 12 D untuk C.
botulinum

The result of a heat sterilization p

process
depends on:
D value
• the initial number of microorganisms;
• the decimal reduction time (heat- Waktu (min.)
resistance) of microorganisms;
• the time and temperature of heat exposure.

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Dr value of thermophilic and mesophilic

Microorganisms Dr (minutes)
Geobacillus stearothermophillus 4.0-5.0
Thermoanaerobacterium (Clostridium) 3.0-4.0
thermosaccharolyticum
h h l i
Clostridium nigrificans 2.0-3.0
C. botulinum (tipe A dan B) 0.10-0.20
C. sporogenes (PA 3679) 0.10-1.5
B. coagulans 0.01-0.07
 In most canned products it is not the pathogen of concern that determines
the amount of heat needed but rather the spoilage microorganisms
 Spoilage is more heat resistant than the pathogens.
 As the spoilage is more resistant than pathogen, the heat applied provides
a wide margin of safety with respect to survival of pathogens in the
product.
Microbiology of Thermal Process - LN- 2014-FTSP

D value of C. botulinum 62A in various foods

 Effect of pH on D value at 115 C

pH Spaghetti, tomattoes Spanish Rice

sauce and cheese
4.0 0.128 0.117
4.6 0.223 0.210
5.0 0.260 0.266
6.0 0.491 0.469
7.0 0.515 0.550

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Heat Resistance and Thermal Death of Spores and

Vegetative Cells

Microbiology of Thermal Process - LN- 2014-FTSP

Mikrorganisme vs proses thermal

100-----
 Nilai z
Log Nilai D, menit

 Suhu yang
10-------------------
diperlukan untuk
menurunkan atau
meningkatkan 1 1--
siklus logg ppada
kurva TDT (1 log
nilai D)
Nilai z
Suhu

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Fo dan konsep 12 D

 Fo adalah waktu yang diperlukan (dalam menit) pada suhu 250 oF

untuk membunuh sejumlah mikroorganisme
g
Fo = Dr (log a – log b)
Konsep 12 D:
– Pemanasan yang harus dilakukan untuk mengurangi
survival C. botulinum sampai 10 -12
Fo = Dr (log a – log b)
Fo = 0.21
0 21 (l l 10 -12
(log 1 – log 12)

Fo = 0.21 x 12 = 2.52
Pemanasan selama 2.52 menit pada suhu 250 oF
mengurangi spora C. botulinum menjadi 1 dalam 1
kaleng dari 1012 kaleng.
Microbiology of Thermal Process - LN- 2014-FTSP

The Microbiology of Low-Acid Canned Foods

 Clostridium botulinum
 Great concern to home and commercial canners
 Mesophilic,
p Gram p
positive, rods
 Produce toxin:
 Although the spores are heat resistant, the toxin is not.
 The toxin can be inactivated by boiling temperatures
(100ºC).

• Present in soil or water: easily contaminates foods

• Produce heat resistant spores
• Canning foods provides an anaerobic environment favorable to
growth of the organism if it has not been destroyed by the process.
• Strains:
• Putrefactive: producing odor during their growth.
• Non-proteolytic: more dependent on carbohydrates, such as
sugars and starch, for growth and do not produce putrefactive
odors.
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Pertumbuhan C. botulinum

Untuk bergerminasi spora C. botulinum memerlukan

kondisi:
 Lingkungan anaerobik
 Makanan asam rendah
 Suhu antara 40ºF sampai 120ºF
 Kadar air relatif tinggi
 Nutrisi kompleks
 Tidak ada bakteri lain

C. botulinum dapat ditemukan pada:

 Canned/thermally processed foods pH> 4.6
 Vacuum packed smoked fish and sausage
 Foil-wrapped baked potatoes
 Vacuum packed mushrooms

Microbiology of Thermal Process - LN- 2014-FTSP

Inactivation of C. botulinum

 Heat processes for low-acid

canned meat, poultry and other
low acid food are designed to
produce a product that is safe
with respect to C. botulinum.
  commercial strerilization

Microbial inhibitors and pH can impact the processes needed to

inactivate C. botulinum.
Inclusion of sodium nitrite and sodium chloride in meat and poultry
products (e.g., commercially-sterile, canned cured meats) can
lower the thermal process required to produce a commercially
sterile product.

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Aw vs Clostridium botulinum
 Aw (water activity = aktivitas air):
 air yang tersedia untuk pertumbuhan mikroorganisme
 Spora
S tidak
id k bergerminasi
b i i pada
d aw < 0.93
0 93
 Makanan dengan aw > 0.85:
 pemanasan seperti pada makanan asam rendah
 Makanan dengan aw < 0.85:
 pemanasan untuk membunuh sel vegetatif = makanan
asam
 Makanan dengan aw < 0.85
 pengukuran aw harus akurat dan reguler terhadap produk jadi
 kontrol harus dilakukan sejak persiapan ingredien sampai
pemanasan (pencapaian suhu yang ditetapkan)

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Garam vs Clostridium botulinum

 Tumbuh pada kadar garam 7% tetapi tidak
memproduksi toksin
 Germinasi spora terhambat pada kadar garam 10%
(= aw 0.93)
 Aplikasi pada produk kuring
 menggunakan garam nitrat/nitrit dan NaCl

water

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pH vs Clostridium botulinum

 Spora tidak bergerminasi pada pH < 4.8

 pH 4
4.6
6 merupakan batas antara
makanan asam dan asam rendah
 Pada makanan dengan pH > 4.6:
 pemanasan untuk membunuh spora C.
botulinum
 Pada makanan dengan pH < 4.6:
4 6:
 pemanasan untuk membunuh sel
vegetatif

Microbiology of Thermal Process - LN- 2014-FTSP

Efect of pH on heating requirement

Mild heat required, since spores are

inhibited by acid High heat required to destroy spores

Mild Heat Acid Mild Heat

X
Vegetative
cells (bacteria) spore
spore

Bacteria with
X X
Vegetative
cells (bacteria) spore
spore

Bacteria with

pH 4.6
High acid food Low acid food

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Other Microorganisms Important in

Low-Acid Canned Foods
 Clostridium sporogenes
 Its spores have higher heat resistance
than those of C. botulinum,
 Processes targeted to destroy spores of C.
sporogenes will also destroy C. botulinum
spores.
 Bacillus stearothermophilus and Clostridium
thermosaccharolyticum,
 Thermophilic bacteria
 Can survive processes that achieve commercial sterility
sterility.
 Not harmful to humans,
 Cannot grow under normal conditions of storage.
 If product is properly cooled and stored, generally the
spores will not grow.

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Bacillus
 Gram-positive aerobic atau facultative endospore-forming
bacteria".
 Tumbuh pada kondisi ada O2.
 Membentuk kapsul
 Dextran dan levan merupakan polisakarida yang umum diproduksi
Bacillus selain polisakarida kompleks
 Re-klasifikasi
 Bacillus stearothermophilus menjadi Geobacillus stearothermophilus
 Suhu optimum 400 to 550C
 Tumbuh pada 65o C dan toleran terhadap asam.
asam
 Jarang tumbuh pada 38 C 0

 Terdapat pada tanah, gurun, air di kutub, mata air panas, sedimen
laut, pangan dan kompos.
 Penyebab flat sour
 D250 values >4.00 menit Microbiology of Thermal Process - LN- 2014-FTSP

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Microbiology of acidified canned food

 Heat processes for acidified foods target

organisms other than C. botulinum, since the
pH prevents its growth.
growth
 The safety of acidified foods is achieved by
controlling the pH to 4.6 or less and applying
a mild heat treatment to inactivate the
vegetative cells of pathogens

The type of organism to be destroyed by the process will depend on

the p
pH of the pproduct.
If the pH is below 4.2, only vegetative cells need be considered;
If the pH is 4.2-4.6, processes may need to take into account
sporeforming organisms such as Bacillus coagulans and the
butyric acid anaerobes (Clostridium pasteurianum and
Clostridium butyricum).

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Bacterial spoilage of thermally process food

 Pembentukan gas (kaleng kembung)

 Perubahan penampakan makanan
 Perubahan bau dan rasa makanan
 Produk makanan hancur
 Larutan yang seharusnya bening
menjadi keruh

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Bacterial decomposition of thermally

processed product .....1

 Incipient spoilage – growth of bacteria before

processing;
 Typically incipient spoilage manifests itself as
low or no vacuum in the container and a slight
change in pH of the product.
 Generally no viable microorganisms are
recovered in subculture media.
 Although the product presents no risk to public
health, if there is sufficient growth, the product
may be considered to be adulterated

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Bacterial decomposition of thermally

processed product .......2

 Contamination after processing – leakage of bacteria

iinto
t the
th container
t i
 Inadequate heat processing: the growth of mesophilic
 Growth of thermophilic bacteria in the processed food:
temperature abuse, improper cooling process
 Spoilage by acid-tolerant spore-forming bacteria
((specific
p to p
products with reduced p
pH))
 Spoilage due to improper curing (specific to cured
products).

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Tanda-tanda kebusukan makanan kaleng

karena kurang pemanasan dan kebocoran
Penampakan Kurang Bocor
pemanasan
Kaleng Kaleng datar atau Datar
gembung; seam
umumnya normal
Penampakan produk Berlendir atau Kental dan terfermentasi
terfermentasi
Odor Normal, asam, bau Asam, bau busuk,
busuk, seragam semua umumnya bervariasi
kaleng antara kaleng
Mikroskopik dan kultur Pembentuk spora Campuran

Kebusukan terjadi pada Kebusukan acak

beberapa batch atau
bagian kemasan

Mikrobiologi Makanan Kaleng - LN- 2012

Spoilage by Acid-Tolerant Sporeformers

 Acidified foods (those products with a pH 4.6 or below) do not

require a severe thermal process to assure product safety.
Therefore a variety of acid-tolerant
acid tolerant sporeformers may survive the
process.
 A thermal process scheduled for acidified foods is designed to
inactivate a certain level of aicd-tolerant sporeformers.
 Their survival :
 A result of excessive pre-processing contamination.
 Under-processing
 The organisms of spoilage significance:
 butyric-acid producing anaerobes
 aciduric flat sour sporeformers.

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Spoilage by Acid-Tolerant Sporeformers

 The butyric-acid producing anaerobes:

 Spores Clostridium butyricum and Clostridium pasteurianum are
capable of germination and growth at pH values as low as 4 4.2-
2
4.4 and consequently are of spoilage significance in acidified
foods, particularly if the pH is above 4.2.
 Growth of these organisms in foods is characterized by a butyric
odor and the production of large quantities of gas.
 Aciduric “flat sours”
 Facultative anaerobic sporeformers
 Seldom
S ld produce
d gas iin spoiled
il d products.
d t
 Spoiled cans remain flat  “flat sour.”
 Causing spoilage in acid foods such as tomato products (by
Bacillus coagulans)

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Bacillus pembusuk makanan asam

 Bacillus coagulans (B. thermoacidurans)

 Termofilik
 D250 values 0.1 menit atau kurang
 Paling penting pada produk tomat
 Toleran terhadap asamn, tumbuh pada pH 4.0 atau lebih rendah.
 Bacillus macerans dan Bacillus polymyxa (Paenibacillus polymyxa).
 Penting sebagai pembusuk produk buah
 Termofilik

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Bacteria causing spoilage of thermally processed foods

Low acid Foods pH >4.6
Thermofilik flat sour Bacillus (Geobacillus) stearothermophilus
B. coagulans
Pembusuk Sulfida Desulfotomaculum (Clostridium nigrificans), C, bifermentans
Pembentuk gas Thermoanaerobacterium (Clostridium) thermosaccharolyticum
Mesophilic Putrefaktif anaerob: P.A. 3679, C. botulinum
Acid Foods 4 – 4.6
Thermophilic B. Coagulans
Mesophilic B. polymixa, B. Betanigrificans,
C. Pasteurianum, C. Butyricum (produksi asam butirat),
C. Sporogenes, C. Putrefaciens (busuk sulfida dan penggembungan
kaleng) Thermoanaerobacterium (Clostridium) thermosaccharolyticum
kaleng),
(pembentuk gas)
laktobasili, kamir,
Kapang (jika cukup oksigen): Byssochlamys fulva, Talaromyces flavus
dan Neosartorya fischeri
High Acid Food: 3.7-4
Mesophilic Molds, Yeast and lactic acid bacteria

Microbiology of Thermal Process - LN- 2014-FTSP

Kapang pembusuk makanan kaleng

• Byssochlamys fulva, Talaromyces flavus, Neosartorya

fischeri
•spora tahan panas sedang
•pencegahan dengan sanitasi yang baik
• Claviceps purpurea
•membentuk sklerotium (kumpulan miselium)
• Pembusuk sari buah kaleng dan produk mengandung buah
• Kebusukan karena kurang pemanasan atau kaleng bocor
(kapang bersifat aerob)

Microbiology of Thermal Process - LN- 2014-FTSP

23
 18/02/2014

Terima Kasih
lilis@nuraida.com; lilis@seafast.org

Mikrobiologi Makanan Kaleng - LN- 2014-FTSP

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