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Advanced Food Microbiology

Introduction
Harsi D. Kusumaningrum

Laboratory of Food Microbiology


Department of Food Science and Technology
FacultyFMof2014
Harsi D. Kusumaningrum, Agricultural Engineering, Bogor Agricultural University

Deskripsi Singkat

Membahas pertumbuhan, ekologi dan


genetika mikroba pangan, peranannya
dalam keamanan pangan, kerusakan
dan fermentasi pangan, serta inaktivasi
mikroba dengan berbagai proses
konvensional maupun alternatif dalam
pengawetan pangan, termasuk
mekanisme pembentukan spora dan
ketahanan spora serta kerusakan sub-
letal mikroba.
Kontrak perkuliahan

Harsi D. Kusumaningrum, FM 2014

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Standar Kompetensi:
Setelah menyelesaikan mata kuliah ini, mahasiswa diharapkan
dapat menjelaskan peran mikroba dalam pengolahan, kerusak-
an dan keamanan pangan, serta mekanisme inaktivasi mikroba
dengan berbagai proses konvensional maupun alternatif dalam
pengawetan pangan. Mata kuliah ini juga berkontribusi pada
pengembangan kecakapan hidup mahasiswa, terutama dalam
mengembangkan dan memutakhirkan iptek bidang mikrobiologi
pangan.

Peraturan akademik IPB

Harsi D. Kusumaningrum, FM 2014

Pengajar

Dr.Ir. Harsi D. Kusuma- Prof.Dr.Ir. Betty S.L. Prof.Dr.Ir. Winiati P.


ningrum (HDK) Jenie MS (BSL) Rahayu MS (WPR)

Prof.Dr.Ir. Lilis Nuraida, Prof.Dr.Ir. Ratih Dewanti- Dr. Siti Nurjanah STP, MSi
MSc (LNU) Hariyadi MSc (RDH)
Harsi D. Kusumaningrum, FM 2014

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Daftar Pustaka:
1. Jay, J.M. 2005. Modern Food Microbiology, 7th Ed.
AVI, Van Nostrand Reinhold, NY
2. FDA. 2000 Last Updated: 03/16/2013. Kinetics of
Microbial Inactivation for Alternative Food Processing
Technologies. Available online at http://www.fda.gov
/Food/ScienceResearch/ ResearchAreas/
SafePracticesforFoodProcesses/ucm100158.htm.
Last Updated: 03/16/2013
3. Jurnal-jurnal ilmiah terkait

Harsi D. Kusumaningrum, FM 2014

Jadwal Perkuliahan

Topik Tanggal Pokok Bahasan Pengajar Pengajar


(Kelas A) (Kelas B)
1 04/09 Pendahuluan HDK HDK
Pertumbuhan dan ekologi mikroba
pangan
2 11/09 Mikroba dan keamanan pangan HDK LNU
3 18/09 Mikroba dan kerusakan pangan WPR LNU
4 25/09 Mikroba dalam fermentasi pangan WPR LNU
dan produksi ingredien pangan
5 02/10 Genetika mikroba dan aplikasinya HDK SNU
dalam mikrobiologi pangan
16/10 UJIAN 1 (Materi 1-5)

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Jadwal Perkuliahan

Topik Tanggal Pokok Bahasan Pengajar Pengajar


(Kelas A) (Kelas B)
6 09/10 Mekanisme inaktivasi mikroba oleh HDK SNU
pH, asam dan suhu rendah
7 23/10 Mekanisme inaktivasi mikroba HDK SNU
dengan perlakuan panas secara
konvensional dan alternatif
8 30/10 Pengaruh pengawet kimia terhadap HDK RDH
mikroba, resistensi mikroba, serta
penentuan efektivitas penghambatan
oleh antimikroba
9 06/11 Mekanisme inaktivasi mikroba WPR LNU
dengan atmosfir termodifikasi,
pengeringan dan penurunan aw
13/11 UJIAN 2 (Materi 6-9)
Harsi D. Kusumaningrum, FM 2014

Jadwal Perkuliahan

Topik Tanggal Pokok Bahasan Pengajar Pengajar


(Kelas A) (Kelas B)
Mekanisme inaktivasi mikroba WPR LNU
10 20/11 dengan iradiasi dan tekanan
hidrostatik
Mekanisme inaktivasi mikroba oleh BSL RDH
11 27/11
pengawet alami
12 Kerusakan subletal bakteri dalam BSL RDH
04/12
keamanan pangan
13 Endospora dalam pengolahan BSL RDH
11/12
pangan
14 18/12 Presentasi paper HDK LNU
08/01 UJIAN 3 (Materi 10-13)

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Tugas Paper dan Presentasi

 Bentuk paper: Mini review


 Minimum me-review 3 paper jurnal internasional
 Spasi 1,5; font 11 arial
 Minimum 4000 kata, maksimum 6000 kata

Grup: 4-5 orang


Presentasi: 10 menit presentasi , 5 menit diskusi

Harsi D. Kusumaningrum, FM 2014

PENILAIAN Paper
KRITERIA 90-100 80 70 60
Diskripsi /isi Sangat Jelas Jelas dan Kurang Jelas Tidak jelas dan
makalah (70%) dan didukung didukung walaupun ada tanpa didukung
pustaka yang pustaka yang pustaka pustaka
cukup cukup
Kesimpulan Dapat Dapat Kesimpulan Tidak
(10%) menyimpulkan menyimpulkan cukup memadai menyimpulkan
dengan sangat dengan baik
baik
Format (10%) Penulisan Penulisan Penulisan Penulisan
dengan format dengan format dengan format dengan format
yang benar dan yang benar yang benar yang salah dan
bebas salah tetapi ada salah tetapi banyak banyak salah
cetak cetak cetak cetak
Pustaka (10%) Pustaka cukup Pustaka cukup Pustaka kurang Tidak ada
dan baru pustaka
Harsi D. Kusumaningrum, FM 2014

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Penilaian

Kriteria Penilaian Rentang Nilai Bobot (%)


Ujian 1 0-100 25
Ujian 2 0-100 30
Ujian 3 (UAS) 0-100 30
Tugas dan presentasi 60-100 15

Nilai Dalam Huruf Point Rentang Skor


A 4,0 ≥ 80
AB 3,5 75-79
B 3,0 66-74
BC 2,5 60-65
C 2,0 50-59
D 1,0 40-49
E 0,0 <40

Harsi D. Kusumaningrum, FM 2014

Capaian 2013/2014

Nilai Huruf Rentang Skor Jumlah

A ≥ 80 23
AB 75-79 17
B 66-74 17
BC 60-65 5
C 50-59 0
D 40-49 0
E <40 0
Total 62

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Research Progress in
Food Microbiology

Harsi D. Kusumaningrum, FM 2014

Why Food Microbiology?

Solid knowledge of Food Microbiology


is necessary to control food safety and quality
from primary production to consumption
(From Farm to Fork Concept)

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Preservation and
Fermentation Spoilage
Antimicrobials

Novel Foods New Methods


Research in
Food Microbiology
Stress Response Predictive
Modelling

Antibiotic
(Emerging) Risk Analysis
and Desinfectant
Pathogens and Food Safety
Resistance
Harsi D. Kusumaningrum, FM 2014

FOOD FERMENTATION
Fermentations of
food products :
desired changes
in food products
induced by living
or dead micro-
organisms and
their enzymes.

Recent and future research:


• improved shelf-life of a product
• improved nutritional value and digestibility
• removal of anti nutritional factors from some plant
derived base materials.
Harsi D. Kusumaningrum, FM 2014

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History and development
of probiotic research

End of 19th century:


LAB preserved meat against spoilage, which are harmless to the GI
tract of man, might benefit by reducing putrefaction in the GI tract
1920s -1930s L. acidophilus was 1930s Isolation of L. casei strain Shirota;
used in the form of acidophilus milk Enterprise founded under the name Shirota
to treat constipation and diarrhea in Institute for Research on Protective
the USA Bacteria

Recent and future research


1) isolation and characterisation of potential probiotic Lactobacillus and
Bifidobacterium strains and development of functional synbiotic pairs;
2) technological properties of probiotics;
3) stability of probiotics in various conditions and enhancement of stability;
4) beneficial impacts on health in animal and human trials, including
improved protection against intestinal pathogens and modulation of the
immune system
(Innovations in Food Technology, 2004)
Harsi D. Kusumaningrum, FM 2014

Food safety control in


food chains

Emphasis:
Providing and producing
safe - wholesome food

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Recent and future research
Influence of the food system The role and significance of
on the growth and survival microbial inactivation,
of microorganisms adaptation and environmental
factors (i.e., aW, pH,
temperature) on growth and
response of microorganisms in
various environments.
Control of microorganisms Be able to identify the conditions,
including sanitation practices,
under which the important
pathogens and spoilage
microorganisms are commonly
inactivated, killed or made
harmless in foods.
Food safety and microbiology (IFT 2001 revised)
Harsi D. Kusumaningrum, FM 2014

Preservation and
Antimicrobials

Potency and production from


indigeneus resources

Antibiotic
and Desinfectant
Resistance

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Beberapa Judul Tesis Magister Sains IPN 2009/2013
 Deteksi senyawa antibakteri daun kesum (Polygenum minus Huds)
secara KLT-Bioautografi dan pengaruhnya terhadap membran E. coli
dan S. aureus
 Modifikasi tepung pisang Tanduk (Musa paradisiace Formatypica)
melalui Proses Fermentasi Spontan dan Pemanasan Otoklaf untuk
Meningkatkan Kadar Pati Resisten
 Evaluasi Aktivitas Antidiare Isolat Lactobacillus dari Air Susu Ibu

 Salmonella Species Occurrence Related to Colifrom Counts and The


Hygiene and Sanitation Conditions in Ready-To-Eat-Food Outlets
 Effects Of Thymus Pallescens and Origanum Floribundum Essential
Oils on The Growth Of Staphylococcus aureus and Salmonella
Typhimurium and Their Application In Minced Meat
 Enterobacter sakazakii Isolat Asal Susu Formula dan Makanan Bayi:
Karakterisasi Gen 16S rRNA dan Perilaku Bakteri Rekonstitusi
 Pemodelan pertumbuhan Staphylococcus aureus pada sosis dengan
Response Surface Model
Harsi D. Kusumaningrum, FM 2014

Food Microbiology

• Pertumbuhan Mikroba
• Ekologi Mikroba Pangan

Laboratory of Food Microbiology


Department of Food Science and Technology
Faculty of Agricultural Engineering
Bogor Agricultural University

Harsi D. Kusumaningrum, FM 2014

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MICROBIAL GROWTH

POPULATION GROWTH

 Growth
 increase in the number of microbial cells
 increase in microbial mass
 Growth rate
 change in cell number or mass per unit time
 Generation
 interval for formation of two cells from one
 Generation time (doubling time)
 time required for formation of two cells
 vary widely among organisms

Harsi D. Kusumaningrum, FM 2014

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Growth curves for the individual pathogens Salmonella serovar Enteritidis (A), E. coli O157:H7 (B), and L.
monocytogenes (C) in SEL inoculated at two different concentrations (10 and 1,000 CFU/ml). The growth of
each pathogen in SEL was compared with that in the respective individual selective enrichment broth: RV
broth for Salmonella, mEC+n for E. coli, and FB for Listeria. The broths were inoculated at the indicated
concentrations, and the cultures were incubated at 37°C in a shaker incubator. The top panels show the
actual growth
Harsi curves, and the plots
D. Kusumaningrum, in the bottom panels are corresponding Gompertz fitted curves
FM 2014

Fase Adaptasi

 Penyesuaian dengan substrat dan


kondisi lingkungan
 Belum terjadi pembelahan sel
  sel tetap, kadang  selnya
menurun
 Cepat / lambatnya tergantung
kecepatan penyesuaian dengan
lingkungan sekitarnya

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Fase Adaptasi
 Lamanya fase adaptasi dipengaruhi antara lain oleh :
 Medium dan lingkungan pertumbuhan
 Jumlah inokulum
 Kemungkinan berjalan lambat antara lain karena :
 Kultur dipindahkan dari medium kaya nutrien 
nutrien terbatas
 Mutan yang baru terbentuk : sesuaikan
lingkungan
 Kultur dipindahkan dari fase statis ke medium
baru

Harsi D. Kusumaningrum, FM 2014

Fase Pertumbuhan Awal


Sel membelah diri dengan kecepatan rendah (baru
selesai tahap penyesuaian diri)

Fase Pertumbuhan Logaritmik


 Sel membelah dengan cepat dan konstan
 Kecepatan pertumbuhan sangat dipengaruhi oleh :
 medium pertumbuhan : pH, nutrien
 lingkungan : suhu, RH
 Perlu energi lebih banyak
 Sel paling sensitif terhadap
lingkungan

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Fase Pertumbuhan Lambat
 Nutrisi dalam medium mulai 
  sel tumbuh masih   sel mati

Fase Pertumbuhan Statis / Tetap


 Populasi sel tetap ( sel tumbuh =  sel mati)
 Ukuran sel lebih kecil karena zat nutrisi mulai habis
 Sel lebih tahan terhadap keadaan ekstrim (panas, dingin,
radiasi, bahan kimia)
 No growth occurs, but many cell functions may continue,
including energy metabolism and biosynthesis
 Certain microorganisms produce secondary metabolites
such as toxins, antibiotics
Harsi D. Kusumaningrum, FM 2014

Fase Menuju Kematian / Fase Kematian


Populasi mikroba mulai menurun karena :
 Nutrien di dalam medium sudah habis
 Energi cadangan di dalam sel sudah habis
  sel mati makin lama semakin banyak
 Kecepatan kematian dipengaruhi oleh :
 kondisi nutrien
 lingkungan
 jenis mikroba

Lag phase, exponential phase, stationary


phase and death phase do not apply to
individual cell but only to population of cells
Harsi D. Kusumaningrum, FM 2014

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MEASUREMENT OF MICROBIAL GROWTH
Measure:
 changes in number of cells or
 weight of cell mass

 Total count: Direct Microscopic Count (DMC)


 quick way of estimating microbial cell number
 limitations:
 Dead cells are not distinguished from living cells
 Small cells are difficult to see
 Precision are difficult to achieve
 A phase contrast microscope is required when the sample
is not stained
 not suitable for cell suspensions of low density (for bacteria
should be > 106 cells/ml)
Harsi D. Kusumaningrum, FM 2014

Viable Count
determine the number of cells in the sample capable of
forming colonies on a suitable agar medium also called
plate count or colony count
 Spread Plate  Pour Plate
 Sample pipetted onto surface of  Sample pipetted into steril plate
agar plate (0.1 ml or less) (1 ml)
 Sterile medium added and
 Sample is spreaded evenly over
mixed well with sample
surface of agar using sterile glass
 Allow to solidify
spreader
 All plates incubated upside
 All plates incubated upside down
down  Typical pour plate results:
 Typical spread plate results: surface colonies and sub-surface
Surface colonies colonies

Harsi D. Kusumaningrum, FM 2014

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Two methods of performing a viable count

Spread plate

Pour plate

Harsi D. Kusumaningrum, FM 2014

MEASUREMENT OF GROWTH
 Cell Mass
 Net weight measurement
 centrifuging the cells and weighing the pellet
 dry weight obtained by drying the centrifuge mass,
usually by placing it overnight at 100 - 105oC
 dry weight usually 10-15% of wet weight
 filtration of culture media on a membrane filter, the filter
is oven dried.
 Turbidity measurement
 expressed as unit absorbance
 used widely to follow the rate of growth of the culture

Harsi D. Kusumaningrum, FM 2014

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Ecology of microorganisms in food

Factors that affect the survival and growth of


microorganisms in food:
 Intrinsic factors : parameters that are
inherent to the food
 Extrinsic factors : factors that people can
readily control involving food

Harsi D. Kusumaningrum, FM 2014

 Intrinsic factors
 nutrient content
 water activity
 pH
 Oxidation-reduction potential
 biological structures
 antimicrobial constituents

 Extrinsic factors
 temperature
 relative humidity
 gases in the environment

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Water activity
• All microorganisms require
water; the amount necessary
for growth varies between
species
• Amount of water that available
in food is expressed as water
activity (aw); aw of pure water
is 1.0.
• Each microorganism has a maximum, optimum, and
minimum aw for growth and survival.
• Generally bacteria dominate in foods with high aw
(minimum approx. 0.90 aw)
• Yeasts and moulds, which require less moisture, dominate
in low aw foods (minimum 0.70 aw).
Harsi D. Kusumaningrum, FM 2014

Minimum
Organism Habitat water activity
for growth
Caulobacter Dilute fresh and sea water 1.00
Pseudomonas Ubiquitous low salt 0.91
environments
Salmonella/E. coli Animals 0.91
Lactobacillus Animals and plants 0.90
Bacillus Soil 0.90
Staphylococcus Animals 0.85
Halobacterium High salt lakes such as Great 0.75
Salt Lake or the Dead Sea

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• Halophiles : microbes that require high concentrations of salt in
their environment
• Mild halophiles require 1-6% salt,
• moderate halophiles require 6-15% salt,
• extreme halophiles require 15-30% NaCl for growth.
• Halotolerant : bacteria can endure moderate salt
concentrations, but grow best in its absence.
• Halophiles are osmophiles and halotolerant organisms are
osmotolerant; however, the term osmophiles is usually
reserved for organisms that are able to live in environments
high in sugar. Most of these microbes tend to be yeast strains.
• Xerophiles : organisms that prosper in dry environments

Harsi D. Kusumaningrum, FM 2014

Hydrogen ion concentration (pH) and microbial growth

pH: hydrogen ion


concentration, relative acidity
or alkalinity
pH range of a microorganism
is defined by a minimum
value (at the acidic end of the
scale) and a maximum value
(at the basic end of the
scale).

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Different microbes have different pH optima:

Acidophiles : pH 1 to 5.5
Neutrophiles : pH 5.5 to 8
Alkalophiles : pH 8.5 to 11.5
Extreme alkalophiles : pH 10 or greater

 Most microorganisms have


approx.neutral pH optimum (pH 6-7.5).
 Yeasts are able to grow in a more acid
environment compared to bacteria.
 Moulds can grow over a wide pH range
but prefer only slightly acid conditions.

Harsi D. Kusumaningrum, FM 2014

Available Oxygen

Microorganisms can be classified according to their oxygen


requirements necessary for growth and survival:
• Obligate aerobes: oxygen required
• Facultative: grow in the presence or absence of oxygen
• Microaerophilic: grow best at very low levels of oxygen
• Aerotolerant anaerobes: oxygen not required for growth
but not harmful if present
• Obligate anaerobes: grow only in complete absence of
oxygen; if present it can be lethal

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• Strict aerobes and facultative anaerobes are able to use
oxygen in metabolic processes and generate more energy per
mole of energy source consumed.
• Aerotolerant anaerobes and strict anaerobes do not use
oxygen in their metabolism and typically have a lower energy
yield and slower growth rates.

• All cells contain enzymes that can be damaged by reacting


with oxygen and any organism that wants to live in its
presence must have methods to protect itself

Harsi D. Kusumaningrum, FM 2014

• Oxygen will react in the cell to form two major toxic products,
hydrogen peroxide and superoxide. These molecules can
cause uncontrolled reductions of cellular components.

• Superoxide is detoxified by superoxide dismutase, and is


present in all organisms capable of surviving in air.
• Hydrogen peroxide is dealt with by the enzyme catalase,
which takes two hydrogen peroxides and forms one molecule
of water and one molecule of oxygen.
• Aerotolerant anaerobes will have peroxidase instead of
catalase, reducing hydrogen peroxide with NADH + H+ and
producing water.

Harsi D. Kusumaningrum, FM 2014

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Detoxification of
oxygen.
Reaction pathways
for detoxifying
superoxide and
hydrogen peroxide

Strict anaerobes are unable to survive in the presence of


oxygen because they lack enzymes capable of
detoxifying superoxide and hydrogen peroxide. These
chemicals accumulate rapidly and kill the cell.

Harsi D. Kusumaningrum, FM 2014

Temperature

• Microorganisms have a
minimum and maximum range
with an optimum temperature
for maximal growth.
• The rate of growth at extremes
of temperature determines the
classification of an organism
(e.g., psychrotroph,
thermotroph).
• The optimum growth
temperature determines its
classification as a thermophile,
mesophile, or psychrophile.

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Psychrophiles : optimum typically
15oC or lower.
Psychrotrophs: optimum growth
temperatures 20 to 30° capable
of growth at temperatures less
than 7° C

Mesophiles : optimum from 30-


40oC, minimum around 15-20oC.

Thermophiles : optimum 55oC or


higher.
Some are hyperthermophiles;
optimum temp is 80oC or higher
(mostly Archaea ). Found in hot
springs, deep-sea hydrothermal
vents, other locations.

Harsi D. Kusumaningrum, FM 2014

Thank you

Harsi D. Kusumaningrum, FM 2014

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No Topik
1 Mikroba dalam produksi ingredien pangan
2 Mikroba terkait keamanan pangan: Cronobacter sakazakii
3 Mikroba dalam fermentasi pangan: probiotik
4 Ketahanan spora bakteri terhadap perlakuan pemanasan
5 Resistensi mikroba terhadap bahan pengawet dan antibiotik
6 Kerusakan subletal dan pengaruhnya terhadap survival bakteri
7 Pengaruh antimikroba alami terhadap mikroorganisme perusak pada pangan
8 Pengaruh bakteriosin terhadap mikroorganisme
9 Pengaruh bahan pengawet kimia terhadap mikroorganisme
10 Pengaruh perlakuan pengeringan terhadap pertumbuhan mikroorganisme
11 Pengaruh perlakuan penurunan aw terhadap pertumbuhan mikroorganisme
12 Pangaruh perlakuan atmosfir termodifikasi terhadap mikroorganisme
13 Pengaruh pH dan asam terhadap pertumbuhan mikroorganisme
14 Pengaruh penyimpanan suhu rendah terhadap pertumbuhan mikroorganisme
15 Pengaruh perlakuan iradiasi terhadap pertumbuhan mikroorganisme
16 Pengaruh perlakuan tekanan hidrostatik terhadap mikroorganisme
17 Pengaruh perlakuan microwave terhadap mikroorganisme
18 Pengaruh perlakuan ohmic heating terhadap mikroorganisme
19 Pengaruh perlakuan high pulse-technique terhadap mikroorganisme
20 Pengaruh perlakuan manotermosonikasi terhadap mikroorganisme
harsi kusumaningrum-PS IPN

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