MIKROBIOLOGI PANGAN

• DIANA NUR AFIFAH

• Outline
• Sejarah Mikrobiologi
• Pertumbuhan Mikroba
• Analisis Kuantitatif Mikrobiologi pada Bahan Pangan
• Sifat-sifat Mikroba Pangan
• Bakteri
• Kapang
• Khamir
• Sejarah Mikrobiologi
• Mikrobiologi: ilmu yang mempelajari kehidupan makhluk hidup yang bersifat
mikroskopik.
• Since the time of the Greeks,
the emergence of maggots from dead bodies
and spoiled flesh was thought to be due to
spontaneous generation.
• 1658, Athanasius Kircher reported that, using a microscope, he had seen minute living
worms in putrid meat and milk.
• 1664, Robert Hooke described the structure of molds.
• 1674, Antony van Leeuwenhoek observed bacteria in saliva, rainwater, vinegar, and
other materials; sketched the three morphological groups (spheroids or cocci, cylindrical
rods or bacilli, and spiral or spirilla); and also described some to be motile. He called
them animalcules
• 1665, Francesco Redi disproved abiogenesis theory by showing that the maggots in
spoiled meat and fish could only appear if flies were allowed to contaminate them.
• 1765, Lazzaro Spallanzani showed that boiling the meat infusion in broth in a flask and
sealing the flask immediately prevented the appearance of these microscopic organisms.
• Finally, in 1861, Louis Pasteur demonstrated that, in boiled infusion, bacteria could
grow only if the infusions were contaminated with bacteria carried by dust particles in
air. His careful and controlled studies proved that bacteria were able to reproduce
(biogenesis) and life could not originate by spontaneous generation.
• Pertumbuhan Mikroba
• Binary Fission
• Generation Time (or Doubling Time)
• G is generation time (with time unit in minutes; also expressed as doubling time, td, with
time unit usually in hours),
• 0.3 is a constant (value of log102 and indicates doubling),
• t is the duration of study (min),
• log10x is initial and log10z is final cell numbers per milliliter or colony-forming units,
(CFUs), per milliliter.
• Example:
• Under a given growth condition, the initial population of 104 cells/ml of a bacterial
species increases to 106 cells/ml in 120 min, its generation time will be:
• Growth Curve
• Fase Adaptasi
• Menyesuaikan dengan substrat dan kondisi lingkungan sekitarnya.
• Belum terjadi pembelahan sel karena beberapa enzim mungkin belum disintesis.
• Kadang tetap, ada juga yang menurun
• Lamanya fase adaptasi dipengaruhi:
• Medium dan lingkungan pertumbuhan
• Jumlah inokulum
• Fase Pertumbuhan Awal
• Setelah adaptasi, sel mulai membelah dengan kecepatan yang masih rendah.
• Fase Pertumbuhan Lambat
• Karena jumlah nutrien sangat berkurang, dan adanya hasil-hasil metabolisme yang
beracun dan menghambat pertumbuhan mikroba.
• Pertumbuhan sel tidak stabil, namun jumlah populasi masih naik karena yang tumbuh
masih lebih banyak dari yang mati.
• Fase Pertumbuhan Statis
• Jumlah populasi sel tetap karena yang tumbuh sama dengan yang mati.
• Ukuran sel lebih kecil karena nutrien sudah mulai habis.
• Sel lebih tahan terhadap keadaan ekstrim seperti panas, dingin, radiasi, dan bahan kimia
• Fase menuju Kematian dan Fase Kematian
• Sebagian mengalami kematian karena nutrien mulai habis dan energi cadangan di dalam
sel juga habis.
• Jumlah sel mati semakin tinggi.
• Kecepatan kematian dipengaruhi kondisi nutrien, lingkungan, dan jenis mikroba.
• Factors Influencing Microbial Growth
in Food
• The ability of microorganisms (except viruses) to grow or multiply in a food is
determined by the food environment as well as the environment in which the food is
stored, designated as the intrinsic and extrinsic environment of food, respectively.
• INTRINSIC FACTORS
OR FOOD ENVIRONMENT
• A. Nutrients and Growth
• 1. Carbohydrates in Foods
• 2. Proteins in Foods
• 3. Lipids in Foods
• 4. Minerals and Vitamins in Foods
• B. Growth Factors and Inhibitors in Food
• C. Water Activity and Growth
• D. pH and Growth
• E. Redox Potential, Oxygen, and Growth
• Sumber energi : untuk pertumbuhan, pergerakan, metabolisme, sintesis protein dan
proses seluler lainnya.
• Movement of Molecules
• Passive diffusion: higher to lower concentration.
• Contoh: air yang dapat keluar masuk sel secara bebas
• Facilitated diffusion: higher to lower concentration, carrier molecule (permease).
• Active transport: lower to higher, takes energy, needs enzyme.
• Group translocation: lower to higher with chemical change
 • Facilitated Diffusion
• Active Transport
• Group Translocation
• pH
Pengelompokan mikroba:
• Acidophiles : pH 0 and 5.5
• Neutrophiles : pH 5.5 and 8
• Alkalophiles : pH 8 – 11.5
• Oxygen
• Microbe can be classified base on their oxygen requirements:
• Oxygen Tolerance
• Aerotolerant – do not use O2 but can grow when it is present
• Often ferment glucose to lactic acid
• Microaerophiles – require O2 but grow only in low concentration
• Oxygen is lethal to some organisms
• All organisms produce superoxide ( O2-)
• Superoxide is toxic to cells (steals electrons)
• Superoxide must be neutralized
Superoxide dismutase
O2- + O2- + 2 H+ --------------------------------> H2O2 + O2
• Hydrogen peroxide is also toxic to cells and it must be neutralized
catalase
2 H2O2 ----------------------> 2 H2O + O2
• Obligate Anaerobes lack of:
• Superoxide dismutase ( SOD )
• Catalase
• Mikroba anaerobik fakultatif:
• mempunyai superoksida dismutase
 • tidak mempunyai katalase
• mempunyai peroksidase
• mengkatalis reaksi antara H2O2 dengan senyawa organik menjadi senyawa
organik teroksidasi dan H2O
• Selama sel tanaman atau hewan masih hidup dan aktif, daya oksidasi-reduksi (O-R)
masih rendah atau mempunyai energi potensial (Eh) negatif karena adanya zat-zat
pereduksi didalamnya, seperti asam askorbat, dan gula-gula pereduksi, sehingga
menghambat difusi oksigen dari luar ke dalam. Mikroba yang dapat tumbuh adalah yang
bersifat anaerobik, seperti Clostridium.
• Setelah pengolahan, O-R tinggi, Eh positif, zat pereduksi berkurang, yang tumbuh
mikroba aerobik seperti kapang, khamir, dan bakteri aerobik (Salmonella, Shigela,
Pseudomonas, dll).
• Mikroba anaerobik fakultatif bisa hidup dengan O-R tinggi, sedang, maupun rendah,
misal BAL dan Bacillus sp.
• EXTRINSIC FACTORS
• Temperature,
• Relative humidity,
• Gaseous environment
• Temperature
• Pengaruh : aktivitas enzim,
• Temperature optimum
• Psychrophiles : 50C - 150C
• Mesophiles : 200C - 400C
• Thermophiles : 450C - 600C
• Analisis Kuantitatif Mikrobiologi pada Bahan Pangan
• Haemocytometry is a total count method where every cell (dead or alive) is counted. It
works by introducing a standard amount of bacteria solution in to the haemocytometer - a
glass slide with lots of grid lines. This is placed under a microscope and the number of
cells counted using a standard method.
• A far simpler technique is turbidmetry which works on the principle that a more turbid
(cloudy) solution has more cells. The light absorbed is recorded and can be compared
with reference graphs to estimate the number of cells. This is also a total cell count
method.
 • In dilution plating, 1cm3 of original bacterial solution is taken and diluted with 9cm3 of
water. The same is done with this diluted solution and so on. A sample from each dilution
is cultured; once individual colonies can be seen, it means each of those represents a
single bacterium that was in the solution. The number is multiplied by the dilution factor.
• This is a viable cells count method - meaning it only counts those bacteria which are
alive, since dead bacteria can not grow colonies. Depending on the aim of your
investigation, this could be a much more useful type of count.
• When a bacterial solution is spread out evenly over an agar surface, it will produce
a bacterial lawn. It is used in testing the effectiveness of antibiotics or disinfectants.
• B has a zone of inhibition which means it has prevented bacteria growing. A, however,
has had no affect. The size of the zone is indicative of the substance's concentration,
rather than it's effectiveness.
• Daftar Pustaka
• Ray B. 2005. Fundamental Food Microbiology. CRC Press, New York.
• Afifah DN, Sulchan M, Syah D, Yanti, Suhartono MT, Kim JH. 2014a. Purification and
Characterization of a Fibrinolytic Enzyme from Bacillus pumilus 2.g Isolated from
Gembus, an Indonesian Fermented Food. Prev. Nutr. Food Sci. 19(3):213-219.
• Afifah DN, Sulchan M, Syah D, Yanti, Suhartono MT. 2014b. Isolation and identification
of fibrinolytic protease-producing microorganisms from Red Oncom and Gembus,
Indonesian fermented soybean cakes. Malaysian Journal of Microbiology 10(4): 273-279.

MICROBIAL FOOD SPOILAGE & MICROBIAL FOODBORNE DISEASES

• Microorganisms are a problem in foods because they can cause
food spoilage or foodborne disease.
• Microorganisms may be present in the raw ingredients of the food
• or may be introduced to the food during preparation, from the food handlers or the food
production environment.
• Definition
Food Spoilage
The condition in which food becomes undesirable or possibly unsafe to eat is referred to as
spoilage.
Spoilage affects the aroma, texture
and/or appearance of food.
Examples: sour milk,
moldy cheese,
slimy, rancid meat,
or mushy,
discolored vegetables
• Definition
Microbial factors
Microorganisms which grow and reproduce,
causing unwanted changes to the odor
taste, and texture of the food.
• Definition
Natural food enzymes
The discoloration of
fruits and vegetables caused by enzymes.
Enzyme
A complex protein molecule that stimulates or speeds up a specific chemical
reaction without being used up itself.
• Microbial food spoilage
• Effect of temperature and time on the growth of bacteria.
• Safe and dangerous temperatures for foodstuffs.
• Some End Products from Microbial Metabolism
of Food Nutrients
• CFUs: Colony Forming Units
APC: Aerobic Plate Count
• Some factors to be considered in selecting a microbial or chemical indicator for a product
(or several similar types of products) are:
• In a good fresh product, it can be present in low numbers (microbial) or absent
(chemical).
• Under normal conditions of storage (temperature, time, packaging), it should increase
(microbial or chemical) in quantity to reach a very high level.
• When spoilage occurs under normal storage conditions, it should be the predominant
causative agent (microbial or chemical).
• It can be detected rapidly (microbial or chemical).
• It can be used reliably to predict shelf life and spoilage status (microbial or chemical).
• It should have a good relationship with the sensory criteria of spoilage of the particular
product (microbial or chemical).
• Food-borne Illness
• The sickness resulting from eating food contaminated with either bacterial toxins or by
certain bacteria in the food, often resulting in vomiting, diarrhea and prostration.
• Food-borne diseases are most often caused by several species of bacteria, although
viruses, parasites, amoebas and other biological as well as chemical agents may be
responsible.
• Food-borne Illness
• Food poisoning would include illness caused by naturally poisonous foods, like certain
wild mushrooms, or from chemical contaminants in the food.
• Common Bacterial Food-borne
Pathogens
• Bacillus cereus
• Campylobacter jejuni
• Clostridium botulinum
• Clostridium perfringens
• Listeria monocytogenes
• Salmonella serovars
• Staphylococcus aureus
• Vibrio cholera
• Vibrio vulnificus
• Vibrio parahaemolyticus
• Yersinia enterocolitica
• Bacillus cereus and other Bacillus species
• Genus Bacillus are Gram-positive, aerobic, spore forming rods, Those causing foodborne
illness fall into the Bacillus subtilis group
• facultatively anaerobic with large vegetative cells, typically 1.0 pm by 3.0-5.0 pm in
chains.
• It grows over a temperature range from 8 to 55 0C, optimally around 28-35 0 C, and
• does not have any marked tolerance for low pH (min. 5.0-6.0, depending on the
acidulant) or water activity (min. 0.95).
 • The ability to produce spores resistant to factors such as drying and heat means that the
food-poisoning bacilli are widely distributed in foods.
• In most circumstances however they are only a small part of the total flora and are not
present in numbers sufficient to cause illness
• Staphylococcus aureus
• Staphylococcus aureus
• S. aureus is a Gram-positive, small round bacterium (coccus), that can produce a heat-
stable toxin.
• Less than 1µg of toxin can cause illness.
• S. aureus is found all over, but the most common source of contamination of food is via
humans, i.e. food handlers.
• Staphylococci are found in the nasal passages, throat, on the skin and in the hair of more
than half of healthy people.
• Staphylococcus aureus
• Intoxication is caused by contaminated food being kept either not hot enough (i.e. at
<60°C) or not cold enough (i.e. at >8°C), which allows the organism to grow and produce
its toxin.
• The symptoms (nausea, vomiting, cramping) of staphylococcal food poisoning come on
very rapidly (within a few hours) and are usually acute.
• Salmonella spp.
Food Sources
Meat, poultry, and egg products
Other foods via human carriers
Symptoms
Abdominal pain, diarrhea, fever, chills, vomiting
Dehydration, headache
• Salmonella spp.
Incubation Time
5 to 72 hours; usually 12 to 48 hours
Controls
Cook thoroughly
 Chill rapidly
Enforce good personal hygiene rules
Prevent cross-contamination
• Shigella spp.
Food Sources
Moist mixed foods, liquids, contaminated produce
Symptoms
Abdominal pain, diarrhea, fever, chills, blood in feces, nausea, dehydration
• Shigella spp.
Incubation Time
Usually less than 4 days
Controls
Chill and heat foods rapidly
Enforce good personal hygiene rules
Control flies
Prepare foods safely
• Vibrio parahaemolyticus
Food Sources
Raw seafood, sushi, saltwater fish, shellfish,
Fish products, salty foods, cucumbers
Symptoms
Abdominal cramps, diarrhea, nausea, vomiting
Mild fever, chills, headache
• Vibrio parahaemolyticus
Incubation Time
Usually 10 to 20 hours
Controls
Use proper cooking and chilling procedures
 Separate raw from cooked foods
Do not use sea water to rinse food
• Vibrio parahaemolytices
• Escherichia coli
Food Sources
Any food exposed to sewage-contaminated water
Symptoms
Similar to Shigellosis—abdominal pain, diarrhea, fever, chills, blood in feces, nausea,
dehydration
• Escherichia coli
Incubation Time
About 11 hours
Controls
Chill and heat foods rapidly
Enforce good personal hygiene rules
Control flies
Prepare foods safely
• Escherichia coli
• Escherichia is the type genus of the Enterobacteriaceae family and E.coli is the type
species of the genus.
• It is a catalase-positive, oxidasenegative, fermentative, short, Gram-negative, non-sporing
rod.
• typical mesophile growing from 7-10 0C up to 50 0 C with an optimum around 37 0 C
• Pathogenesis and Clinical Features
• four major categories of diarrhoeagenic E. coli based on distinct, plasmid-encoded
virulence properties.
• Enterotoxigenic E. coli (ETEC)
– occurs between 12 and 36 h after ingestion of the organism.
– Symptoms can range from a mild afebrile diarrhoea to a severe cholera like
syndrome of watery stools without blood or mucus, stomach pains and vomiting.
 • Enteroinvasive E. coli (EIEC)
• invades and multiplies within the epithelial cells of the colon causing ulceration and
inflammation
• Clinical features
– are fever, severe abdominal pains, malaise and often a watery diarrhoea which
precedes the passage of stools containing blood, mucus, and faecal leukocytes.
• Enteropathogenic E. coli (EPEC)
• Symptoms of EPEC infection, malaise, vomiting and diarrhoea with stools containing
mucus but rarely blood, appear 12-36 h after ingestion of the organism
• Pathogenesis appears to be related to the ability of EPEC strains to adhere closely to the
enterocyte membrane.
• Enterohaemorrhagic E. coli (EHEC) or
Verotoxin-producing E. coli (VTEC)
• E.coli 0157:H7 is the most common EHEC serotype reported, although others do occur.
Nonmotile (H negative) 0 1 11 and 01 57 are more common.
• EHEC has attracted attention not only because foodborne transmission is more common
than with other diarrhoeagenic E. coli,
• but because the illness it causes can range from a non-bloody diarrhoea, through
haemorrhagic colitis, to the life threatening conditions haemolytic uraemic syndrome
(HUS) and thrombotic thrombocytopaenic purpura (TTP).
• Association with Foods
• Faecal contamination of water supplies and contaminated food handlers
• EHEC serotype 0 157:H7 have mostly involved undercooked ground meat products and
occasionally raw milk
• EHEC have been reported with other foods.
– Lettuce has been associated on several occasions and unpasteurised apple juice
was the vehicle in a large outbreak in the US
• appear to have a more marked ability to survive at low pH values than
some other bacteria
• Listeria monocytogenes
Food Sources
Vegetables fertilized with contaminated manure
Milk contaminated after pasteurization
 Contaminated cheeses and meat
Symptoms
Headache, vomiting, other flu-like symptoms
In pregnant women and people with compromised immune systems: Possible death,
meningitis, abortion and/or prenatal septicemia
• Listeria monocytogenes
Incubation Time
4 days to 3 weeks
Controls
Pasteurize or heat-process foods
Avoid re-contaminating foods
Refrigerate or freeze all dairy products
Use proper equipment cleaning and food safety procedures.
• Yersinia enterocolitia
Food Sources
Contaminated raw pork or beef
Drinking water, milk products, tofu
Symptoms
Children and adolescents: digestive upset, severe abdominal pain resembling acute
appendicitis
Adults: acute abdominal disorders, diarrhea, fever, arthritis
Both groups: skin and eye infections
• Yersinia enterocolitia
Incubation Time
3 to 7 days
Controls
Pasteurize or heat-process foods
Enforce good personal hygiene rules
Sanitize equipment and utensils
 Always purchase foods from approved sources
• Campylobacter jejuni
Food Sources
Raw or inadequately cooked or processed foods of animal origin
Unchlorinated water
Symptoms
Diarrhea, abdominal pain, fever, a vague unhealthy feeling
Less frequent: nausea, headache, urinary tract infection, reactive arthritis
• Campylobacter jejuni
Incubation Time
1 to 7 days or longer
Controls
Cook food thoroughly
Handle food properly
Dry or freeze foods
Add acids
• Clostridium botulinum
• Gram-positive, motile with peritrichous flagella, obligately anaerobic, straight or slightly
curved rods 2-10 pm long, and form central or subterminal oval spores.
• (Latin: botulus = sausage)
• Botulism is an example of bacterial food poisoning in its strictest sense: it results from
the ingestion of an exotoxin produced by Clostridiurn botulinum growing in the food.
• The botulinum toxins are neurotoxins; unlike enterotoxins, which act locally in the gut,
they affect primarily the cholinergic nerves of the peripheral nervous system.
• Eight serologically distinct toxins are recognized (A, B, C1, C2, D, E, F, and G, though
C2 is not a neurotoxin),
• Most cases of botulism in humans are due to toxin types A, B or E and the incidence of
other types in human illness is extremely rare
• The minimum pH at which C. botulinum will grow depends very much on factors such as
temperature, water activity and the acid used to adjust the pH.
 • The consensus has long been that a pH around 4.7 represents an absolute minimum and
this fact has had important practical implications for the canning industry.
• Experiments in animals have shown that toxin ingested with food and surviving
inactivation is absorbed in the upper part of the small intestine and reaches the
bloodstream via the lymphatics.
• It binds to the nerve ending at the nerve-muscle junction, blocking release of the
acetylcholine responsible for transmission of stimuli, thus producing a flaccid paralysis.
• Initial symptoms of botulism occur anything from 8 h to 8 days, most commonly 12-48 h,
after consumption of the toxin-containing food.
• Symptoms include vomiting, constipation, urine retention, double vision, difficulty in
swallowing (dysphagia), dry mouth and difficulty in speaking (dysphonia).
• The patient remains conscious until, in fatal cases, shortly before the end when the
progressive weakness results in respiratory or heart failure.
• This usually occurs 1-7 days after the onset of symptoms. Surviving patients may take as
long as 8 months to recover fully.
• Four common features are discernible
in outbreaks of botulism
(1) The food has been contaminated at source or during processing with spores or vegetative
cells of C. botulinum.
(2) The food receives some treatment that restricts the competitive microflora and, in normal
circumstances, should also control C. botulinum.
(3) Conditions in the food (temperature, pH, Aw) are suitable for the growth of C. botulinum.
(4) The food is consumed cold or after a mild heat treatment insufficient to inactivate toxin.
• Clostridium perfringens
• Gram-positive, rod-shaped anaerobe which forms oval subterminal spores, encapsulated
and nonmotile, a catalase-negative anaerobe
• Growth occurs over the temperature range 12 to 50 "C although it is very slow below
about 20 "C. At its temperature optimum, 43-47 "C
• a minimum aw for growth of 0.95-0.97, depending on the humectant, and will not grow
in the presence of 6% salt.
• Generally a self-limiting, non-febrile illness
– Characterized by nausea, abdominal pain, diarrhoea and, less commonly vomiting
• Onset is usually 8 to 24 h after consumption of food containing large numbers of the
vegetative organism.
 • In otherwise healthy individuals, medical treatment is not usually required and recovery
is complete within 1-2 days,
– Although occasional fatalities occur in the very old or debilitated.
• Typical scenario C. perfringens food poisoning outbreak
(i) a meat dish containing spores of C. perfringens is cooked;
(ii) the spores survive the cooking to find themselves in a genial environment from which much
of the competitive flora has been removed;
(iii) after cooking, the product is subjected to temperature time abuse, such as slow cooling or
prolonged storage at room temperature. This allows the spores to geminate and multiply rapidly
to produce a large vegetative population;
• (iv) the product is either served cold or reheated insufficiently to kill the vegetative cells.
Some of the ingested cells survive through into the small intestine where they sporulate
and produce enterotoxin.
• Aspergillus flavus
• Aspergillus flavus is the most common species producing aflatoxins, occurring in most
kinds of foods in tropical countries.
• This species has a special affinity with three crops, maize, peanuts and cottonseed, and
usually produces only B aflatoxins.
• Only about 40% of known isolates produce aflatoxin.
• Aspergillus flavus
• Aflatoxin
• Aflatoxins are potent carcinogens (Class 1; JECFA, 1997) affecting man and all tested
animal species including birds and fish. Four compounds are commonly produced in
foods: aflatoxins B1, B2, G1 and G2, named for the colour of their fluorescence under
ultra violet light, and their relative position on TLC plates.
• Preventing Cross-Contamination
• Separate raw animal foods during storing, preparing, holding, and display from raw
ready-to-eat food and cooked ready-to-eat food.
• Separate types of raw animal foods from each other.
• Clean and sanitizing equipment and utensils.
• Store food in packages, covered containers, or wrappers.
• Preventing Cross-Contamination
• Clean hermetically sealed containers of food of visible soil before opening.
 • Protect food containers that are received packaged together in a case or overwrap from
cuts when the case or overwrap is opened.
• Store damaged, spoiled, or recalled food separately.
• Separate fruits and vegetables before they are washed.
• Handwashing
Before:
Handling food
Handling clean utensils
Handling clean equipment
After:
Eating
Drinking
Smoking
Touching the face or hair
Using the toilet
Handling raw meat, poultry, or seafood
Handling soiled utensils or equipment
• Factors Affecting Bacterial Reproduction
• Moisture
• Oxygen
• pH
• Time and temperature
• Three groups of foods:
based upon rate of spoilage
• highly perishable
– meat
– fruit
– milk
– vegetables
 – eggs
• semi perishable
– potatoes
– nuts
• stable
– rice
– flour
– dry beans
• How to reduce water?
• drying
– sun
– heat
– freeze - dried (expensive!)
• How to reduce water?
• addition of salt or sugar
– water needed to keep salt and sugar in solution
• Preservation of food by preventing microbial growth
• pH
– very few bacteria grow below pH 5.0
– How to make food acidic?
– Add acid e.g. acetic acid
– Allow bacteria to make acid from natural food components
• lactic acid bacteria
• Low-Temperature Food Preservation
• Chilled storage: 50˚F (10˚C) to 59˚F (15˚C)
• Refrigerated storage: 32˚F (0˚C) to 45˚F (7˚C)
• Freezer storage: 0˚F (–18˚C) or below
• Pasteurization
High-temperature food preservation
 Food product heated to 145˚F (63˚C) for 30 minutes or to 161˚F (72˚C) for 15
seconds then immediately cooled to 50˚F (10˚C) or less.
• Sterilization
High-temperature food preservation
Virtually kills all microorganisms and their spores.
Heating usually takes place in a large container which is pressurized according to the
food product, its ability to withstand heat, and packaging.
• The basic principles
• Eat food as soon after harvesting as possible.
• Physically protect food from pests by storing in sealed containers.
• Preserve by drying, salting or adding spices.
• References
• Ray B. 2005. Fundamental Food Microbiology. CRC Press, New York.
• Adams MR & Moss MO. 2005. Food microbiology 2nd ed. Cambridge. The Royal
Society of Chemistry.

ANALYTICAL TECHNIQUES FOR NUTRIGENOMICS

• Diana Nur Afifah
• Definitions (Rimbach et al. 2007)
• Nutrigenomics – short for nutritional genomics – refers to the study of the impact of
specific nutrients or diets on gene expression.
• Nutrigenetics investigates how genetic variability influences the body’s response to a
nutrient or diet.
• Hence, nutrigenomics and nutrigenetics are closely related disciplines but approach
the interplay of diet and genes from opposing starting points.
• The term ‘genomics’ refers to the study of all nucleotide sequences in the
chromosomes – the ‘genome’ – of an organism.
• structural genomics
• DNA sequence analysis and mapping of the genome of an organism
• functional genomics
 • system-wide experimental approaches to study gene function.
• Transcriptomics is used to describe methods that measure the relative amounts of
messenger RNA (mRNA) in order to determine patterns and levels of gene
expression, and their regulation.
• Proteomics is the study of the complete set of proteins expressed in a cell, tissue, or
organism
• Metabolomics investigates the profile and functions of all metabolites generated in a
simple (e.g. cell) or complex (e.g. entire-organ or organism) system.
• Traditionally accepted central dogma of molecular biology (a).
A schematic of the “ome” analysis and the method used are provided in the upper
field (b).
• Mano et al. 2009
• Protein Synthesis
http://greatneck.k12.ny.us/gnps/shs/dept/science/krauz/handouts/genetics_diagrams
_2.htm
• http://www.wiley.com/legacy/college/boyer/0470003790/animations/translation/trans
lation.htm
• DNA Extraction
• rice.plantbiology.msu.edu/training/DNA_extraction_overview.ppt
• http://www.ijppsjournal.com/Vol6Issue6/9478.pdf
• Polymerase Chain Reaction (PCR)
• PCR
• Developed by Kary Mullis in 1983: Winner of Noble Prize in
Chemistry in 1993
• A technique for amplifying DNA sequences in vitro by separating the DNA into two
strands and incubating it with oligonucleotide primers and DNA polymerase.
• PCR can amplify a specific sequence of DNA by as many as one billion times
• PCR is a technique that takes a specific sequence of DNA of small amounts and
amplifies it to be used for further testing
• The targets in PCR are the sequences of DNA on each end of the region of interest,
which can be a complete gene or small sequence
• Application of PCR
PCR is commonly used in
 • Biotechnology
• Forensics
• Medicine
• Genetic research
• Biomedical Application of PCR
• Diagnostics
• Vaccine Development
• Drug Development
• Pathogenesis studies
• Epidemiological studies
• Genome sequencing
• Use of PCR for Diagnostic Purposes
• Infectious Diseases:
• Detection
• Evaluation of Therapy
• Example:
• Quantitative nucleic acid analysis (HIV viral load etc.)
• Inherited Genetic Diseases
• Cancer
• Forensics
• Step in PCR Machine
• DNA denaturation (~95oC)
• Annealing (50oC – 60oC, depend on Tm of primer)
• Extension (72oC)
• Reverse Transcriptase PCR
 RT-PCR is a variant of PCR, a laboratory technique commonly used in molecular
biology to generate many copies of DNA sequence from a particular RNA
(intracellular or extracellular/viral DNA)
 RNA strand is first reverse transcribed into its DNA complement (complementary
DNA, or cDNA) using the enzyme reverse transcriptase, and the resulting cDNA is
amplified using traditional or real-time PCR
 The RT step can be performed either in the same tube with PCR (one-step PCR) or
in a separate one (two-step PCR) using a temperature between 40°C and 50°C,
depending on the properties of the reverse transcriptase used.
• Real-Time PCR
• Conventional vs Real-Time PCR
• Conventional vs Real-time PCR
• Real-Time PCR
• Detection of amplification product during amplification process
• Increased level of sensitivity
• Measurement of nucleic acid quantity/copy number:
• Viral load
• Expression level
• In real-time PCR, fluorescent molecules are used to monitor the PCR reaction while
amplification is taking place.
• Fluorescence Detection
• Intercalating Dyes
• Intercalating Dyes
• Quantitative PCR
• End Point Measurements
• Threshold Cycle, CT
• Threshold Cycle, CT
• Threshold Cycle, Ct
• Southern Blotting and Northern Blotting
• Northern Blot Procedure
• Northern blots allow investigators to determine the molecular weight of an mRNA
and to measure relative amounts of the mRNA present in different samples.
 • RNA (either total RNA or just mRNA) is separated by gel electrophoresis,
usually an agarose gel. Because there are so many different RNA molecules
on the gel, it usually appears as a smear rather than discrete bands.
• The RNA is transfered to a sheet of special blotting paper called
nitrocellulose, though other types of paper, or membranes, can be used. The
RNA molecules retain the same pattern of separation they had on the gel.
• 3) The blot is incubated with a probe which is single-stranded DNA. This probe will
form base pairs with its complementary RNA sequence and bind to form a double-
stranded RNA-DNA molecule. The probe cannot be seen but it is either radioactive
or has an enzyme bound to it (e.g. alkaline phosphatase or horseradish peroxidase).
• 4) The location of the probe is revealed by incubating it with a colorless substrate
that the attached enzyme converts to a colored product that can be seen or gives off
light which will expose X-ray film. If the probe was labeled with radioactivity, it can
expose X-ray film directly.
• Microarrays: A Powerful Tool for Studying the Functions of Food and Its Nutrients
(Mano et al. 2009)
• A microarray is a high-throughput genomic tool (Elliott et al. 2007; Mutch et al.
2005).
• It can be used for profiling and monitoring the expression levels of tens and
thousands of genes (entire genomes).
• It can also be used to determine the influence of food nutrients and/or bioactive
compounds (food factors) on metabolic pathways and to understand how food
nutrients and factors maintain homeostatic control of gene expression levels.
• Microarray technology is a “nutrigenomics” tool and can be used to investigate the
levels of transcripts in particular.
• A typical DNA microarray experiment follows a characteristic series of steps:
1) RNA extraction from a sample;
2) reverse transcription of the RNA in order to obtain complementary DNA (cDNA)
a) labelling of the cDNA with specific dye(s) (usually fluorophores, such as
Cyanine 3 and 5),
b) Or reverse transcription of the cDNA in order to obtain cRNA and labelling
of the cRNA;
3) hybridization of the labelled cDNA or cRNA onto the microarray under defined
conditions (e.g. time, temperature, etc.);
4) Washing of the slides to remove non-hybridized labelled oligonucleotides;
5) signal detection (e.g. using an appropriate scanning device);
6) data analysis.
• Microarray
• Microarray Chips
• GeneChip (Affymetrix)
• Agilent OligoMicroarray Kit (Agilent Technologies)
• Illumina Microarray Core (Oregon Health & Science University)
• CodeLink (GE).
• The expression data of tens and thousands of genes are visualized and statistically
analyzed using specific software programs such as:
• Expression Consol (Affymetrix)
• GeneSpring (Agilent Technologies)
• Spotfire (Spotfire Inc.).
• The obtained expression data are interpreted using software programs that analyze
biological information, such as:
• KeyMolnet (originally developed by Institute of Medical Molecular Design
Inc.)
• Pathway Studio (Ariadne Genomics)
• Thereafter, these data are used for the analysis of metabolic pathways and in
bioinformatics.
• EXPERIMENTAL DESIGN
• In general, whole animals (in vivo) and cultured cells (in vitro) are used as samples
for analyzing the functions of food factors and nutrients (Mano et al. 1994;
Nakatani et al. 2007).
• However, animal experiments should be ethically performed, and animal use in the
experiments should be in keeping with the guidelines of the “Institutional Animal
Care and Use Committee.”
• Animal experiments are suitable for analyzing all metabolic pathways involving
food factors and nutrients.
• Whole animals (in vivo) are used as analytical samples: Rodents such as mice and
rats, including gene-modified (transgenic and knockout) mice, are frequently used
in microarray analysis.
• This is because ready-made microarray chips for these animals can be obtained
from a number of platforms, and considerable information regarding the influence
of food factors and nutrients on metabolic pathways and their homeostatic control
of gene expression is also available.
• Other species such as human, chicken, Caenorhabditis elegans, and Drosophila may
also be used in microarrays.
• Research (Mano et al. 2009)
• Adult male mice (4-week-old C57BL/6J or model mice) or adult male rats (4-week-
old [Sprague-Dawley] SD or model rats).
• The animals were housed individually in an environment-controlled room under
constant temperature (25±3oC) and a 12-h light/dark cycle.
• Divided into two groups: the first group was treated with food factors or nutrients
and the other group was the control group.
• Each group comprised five to seven animals.
• In the long-term dose experiment, food factors or nutrients were continually
administered for several weeks (1–4 weeks) to clarify the late and/or secondary
biological effects of the administered food factors or nutrients.
• In the short-term dose experiment, the animals were administered a food factor or
nutrient for 2–24 h.
• The other food factors or nutrients were administered subsequently to clarify the
early biological effect of the administered food factor or nutrient.
• Dosage was decided according to human consumption
• (Ch´avez et al. 2003).
• The mice were killed by cervical dislocation either with or without overnight food
deprivation.
• They were carefully dissected to obtain tissues for isolating total RNA.
• Sample Preparation
• Total RNA was isolated using Trizol reagent (Invitrogen) or an RNeasy kit
(QIAGEN) according to the manufacturer’s instructions (Mano et al. 2000).
• The RNA was dissolved in diethyl pyrocarbonatetreated
• distilled water.
• The concentration of the total RNA was estimated from the absorbance at 260 nm,
and the quality of the sample RNA was determined by agarose electrophoresis and
reverse transcriptase polymerase chain reaction (RT–
• PCR).
• If RNA degradation was detected by agarose electrophoresis and/or no
amplification of genes such as glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) or actinwas detected byRT-PCR, theRNA samples were not used for
microarray analysis.
• Highquality RNA isolated from the cells and tissues of the experimental animals
were used for analysis.
• MICROARRAY HYBRIDIZATION AND SCANNING
• Microarrays enable the analysis of each individual RNA obtained from an animal
from a group or that of pooled RNA obtained from all animals in a group.
• For the analysis of pooled RNA, equal amounts of the total RNA from several
animals in each group were pooled.
• All experiments and analyses were performed according to the protocol of the
microarray platform (Mano et al. 2006).
• The GeneChip Expression Analysis Technical Manual (Affymetrix).
• The isolated mRNAs were reverse transcribed with the T7-(dT)24 primer and
copied to yield double-stranded cDNAs (SuperScript Choice System, Invitrogen).
• Further, biotin-labeled cRNAs were synthesized (High Yield RNA Transcript
Labeling Kit; Affymetrix) and fragmented by heating at 94◦C for 35 min.
• The fragmented cRNAs were hybridized using GeneChip
• (Affymetrix).
• After hybridization, washing and staining were performed using the Affymetrix
Fluidics Station 400.
• Thereafter, these arrays were scanned using an Affymetrix array scanner, and the
fluorescence intensity was measured with Microarray Suite 5.0 (Affymetrix).
• “One-color” or “two-color” microarrays from Agilent Technologies may also be
used.
• A one-color microarray is almost the same as theGeneChipmicroarray from
Affymetrix.
• A two-color microarray uses a single microarray slide for the comparison of two
groups.
• In a two-color microarray, the cRNA of one group is labeled with Cy5 (green
fluorescence) and that of the other group is labeled with Cy3 (red fluorescence).
• Common slide scanners such as GenePix (Axon Instruments) and ScanArray
(Perkin Elmer), which can detect fluorescence, can scan slides prepared using
several platforms.
• The relative intensities of each type of fluorescencemay then be used for ratio-based
analysis to identify the upregulated and downregulated genes.
• DATA MANAGEMENT AND BIOINFORMATICS
• The scanned hybridized microarray image data need to be converted to numerical
data for biological analysis.
• The considerable, noisy, and complex numerical data are visualized and statistically
analyzed using suitable software programs (Werner 2008; Zhang et al. 2008).
• The data management cycle comprises quality control (filtering), biological
hypothesis (normalization, determination of parameters, and interpretation),
statistical analysis (t -test, analysis of variance [ANOVA], and clustering), and
biological interpretation (construction of pathway maps or similar lists).
• In the quality control step, the unreliable data are eliminated using different filters
such as “filter on expression level,” “filter on error,” and “filter on flags.”
• In the biological hypothesis step, reliable numerical data are normalized for
comparative analysis by using the guided steps recommended by each platform.
• APPLICATIONS
• EFFECTS OF THE DIETARY INTAKE OF “NIGANA” (Crepidiastrum
lanceolatum, AN EDIBLE PLANT FROM OKINAWA)
• EFFECTS OF THE DIETARY INTAKE OF GLYCOSPHINGOLIPIDS FROM
RICE (Oryza sativa)
• Studying the functions of foods and nutrients is rather difficult.
• Typically, food is a complex and variable mixture of nutrients and other
components.
• Most food factors are weak dietary signals and must be considered in the context of
chronic exposure (Lee and Go 2005).
• Microarray analysis clearly indicates the effects exerted by food factors and
nutrients on metabolic pathways via transcriptome modifications.
• Moreover, the results of microarray analysis suggest that food factors and nutrients
influence the metabolome because alterations in the transcriptome cause changes in
the metabolome.
• Microarray analysis is one of the most convenient tools for inferring the proteome
and metabolome (Endo et al. 2002; Kato and Kimura 2003).
• Microarray technology is a typical tool used in nutrigenomics. This technology will
enhance the understanding of the manner in which food and nutrition influence
metabolic pathways and howthese factors maintain homeostasis under normal
conditions or diet-related or non-diet-related disease conditions.