Food Microbiology

Elok Zubaidah

Food Microorganisms
Bacteria Yeast Mold Viruses

How Long do Bacteria Need to Grow?

Under ideal conditions, some bacteria can double in number every 15-30 minutes Example: bacteria with a 20 min doubling TIME time:
Start 20 Min

40 Min 1 Hour

Bacterial Multiplication
Time 0 20 40 80 160 420 Numbers 1 2 4 16 256 2,097,152

Bacterial Growth Curve

Stationary Phase

Number of Bacteria (log10)

Lag Phase

Death Phase

Time

Changing the Bacterial Growth Curve

Number of Bacteria (log10) Much longer Lag Phase Time

*Sub-optimal means lowered: pH, AW,Temp., etc.

Conditions for Spoilage

Water pH Physical structure Oxygen temperature

Microorganism transfer
Soil and Water Plants and animals Raw to processed food / cross contamination Person to Food Person to Person

Microorganism Growth in Foods

Intrinsic Factors
composition pH presence and availability of water oxidation-reduction potential
altered by cooking

physical structure presence of antimicrobial substances

What are the factors affecting microbial growth in foods?

FATTOM(P)-

Factors Affecting Microbial Growth in Foods

F (Food for the microbes to eat)

Nutrients in food affect microbial growth: Sources of energy (e.g., sugars, proteins) Sources of nitrogen (e.g., proteins) Vitamins Minerals In order for bacteria to grow, the food has to have the right nutrients for the bacteria and the bacteria have to be able to get to the food...

 So the biological structure of the food is important:

Plants have outer skins which protect them from microbial growth What happens when the skin is damaged (or cut into like this watermelon)?

Acid (pH)
Microorganisms grow best at pH near 7
As the pH goes lower, if microorganisms grow, they grow slower

Most pathogens do not grow or at least dont grow well at pH < 4.6
However, they may survive at least for a short time at low pH

Many yeast, molds, and spoilage bacteria can grow at pH < 4.6
Why is this important?

Clarification of Acidity (pH)

pH scale:

no matter what extracellular pH a microorganism prefers, intracellular pH is relatively near neutrality

Temperature and Time

Classifying bacteria by growth temperatures:
Thermophiles(very hot) Mesophiles (cool to very warm or hot) Psychrotrophs (cold or warm) Psychrophiles (only cold) In food microbiology, we are most concerned with mesophiles and psychrotrophs

Classification of Bacteria by Temperature Requirements

variations in temperature optima:

 Refrigeration (< 41F)

Slows or stops pathogen growth Most pathogens dont grow in refrigerated foods However, a few pathogens can grow slowly under refrigeration (they are psychrotrophs)
Listeria monocytogenes Yersinia enterocolytica Aeromonas hydrophila Clostridium botulinum type E

Many spoilage microbes are psychrotrophs

Freezing does not kill bacteria

Some may die when frozen, but this cant be counted on

 Heat
Cooking to > 165F kills most pathogens
Remember which ones arent killed/destroyed?
Spores, toxins

If food is held on a hot serving line at > 140F, pathogens can NOT grow or produce toxins

Oxygen (Oxidation -

R eduction Potential)

O-R potential generally refers to the amount of oxygen present Pathogens:

Most bacterial pathogens can grow with or without Oxygen (Facultative anaerobes) Some food pathogens can only grow when no Oxygen is present (anaerobes)
Example?

oxygen relationships of microorganisms:

 Spoilage microorganisms:
Obligate aerobes:
Pseudomonas spp. Molds How do you think food processors use this information about obligate aerobe spoilage microbes?

Microaerophiles:
Lactic Acid Bacteria

Anaerobes
Desulfotomaculum spp. (food processing concern--can spoilage) Others

Relation to Oxygen
Aerobes: use oxygen in metabolism; obligate.
Microaerophiles: require oxygen (also obligate), but in small amounts. Anaerobes: grow without oxygen; SEE NEXT

A: aerobe B: microaerophile

Capnophiles: require larger amounts of carbon dioxide than are found normally in air.

Anaerobes grow without O2

Classifications vary, but our definitions: Obligate (strict) anaerobes: killed or inhibited by oxygen. Aerotolerant anaerobes: do not use oxygen, but not killed by it. Facultative anaerobes: can

C: could be facultative or aerotolerant. D: strict anaerobe

Protections of bacteria against oxygen

Bacteria possess protective enzymes, catalase and superoxide dismutase. Catalase breaks down hydrogen peroxide into water and oxygen gas. Superoxide dismutase breaks superoxide down into peroxide and oxygen gas. Anaerobes missing one or both; slow or no growth in the presence of oxygen.
Fe3+ -SOD + O2- Fe2+ -SOD + O2

Fe2+ -SOD + O2- + 2H+ Fe 3+ -SOD + H2O2

enzymes that destroy toxic oxygen species:

toxic forms of oxygen:

enzymes that destroy toxic oxygen species:

testing microbial culture for presence of catalase:

negative positive

catalase H2O2 + H2O2

2 H2O + O2

Moisture (water)
Why is water important to bacteria? To survive? To grow? Is it the total amount of water present in the food thats important? Water in food is present in two forms:
BOUND FREE

Bound water is unavailable for bacteria to grow

Water Activity (AW) A measure of the FREE WATER- w that is ater available for microbial growth AW is simply the Relative Humidity of the food (at equilibrium with the air around the food) AW can range from:
0.00 (no free water) to 1.00 (all free water)

We dont usually measure AW values in the Air Force, but you can look them up in food microbiology books

Aw

Foods (some examples)

0.98 to 1.00 Fresh meats, fruit, vegetables, canned fruit in light syrup, canned vegetables, beverages 0.93 to 0.98 Processed cheese, bread dough, lightly salted meats (e.g., hot dogs), canned fruit in heavy syrup 0.85 to 0.93 Ham, aged cheddar cheese, most bread, dry sausage, sweetened condensed milk

*
0.60 to 0.85 Flour, cereals, jams/jellies, nuts, some cheese, heavily salted foods, dried fruit < 0.60 Chocolate, honey, noodles, crackers, potato chips, dried milk, dried vegetables

*Pathogens do not grow or produce toxins at AW < 0.85

Adapted from APHA, Compendium of Methods for the Microbial Examination of Foods, 1992.

Water activity and microbial growth

Relative growth or reaction rate

0.0

0.1

0.2

0.3 0.4 0.5 0.6 Water activity

0.7

0.8

0.9

1.0

Most bacteria cant grow below aw = 0.85 Most yeasts & molds cant grow below aw = 0.65

Low water activity: halophiles, osmophiles, and xerotolerant

Water is critical for life; remove some, and things cant grow. (food preservation: jerky, etc.) Halophiles/halotolerant: relationship to high salt. Marine bacteria; archaea and really high salt. Osmophiles: can stand hypertonic environments whether salt, sugar, or other dissolved solutes Fungi very good at this; grandmas wax over jelly. Xerotolerant: dry. Subject to desiccation. Fungi best Bread, dry rot of wood Survival of bacterial endospores.

salt tolerance:

Typical water activity of some foods

Aw examples
0.95 fruits, vegetables, meat, fish, milk 0.91 some cheeses, ham

0.87 salami, pepperoni, dry cheeses, margarine, 0.80 fruit juice concentrates, sweetened condensed milk, syrups, flour, rice, high sugar cakes 0.75 jam, marmalade
0.65 oatmeal, fudge, marshmallows, jelly, molasses, sugar, nuts 0.60 dried fruits, honey 0.50 dried pasta, dried spices

0.30 cookies, crackers

0.03 dry milk, dehydrated soups, corn flakes

Most bacterial pathogens can not grow or produce toxins at AW < 0.91 Exception: Staph. aureus can grow and produce toxin down to 0.85 How do you think AW can be decreased? 1. 2. 3.

How do these methods work?

 Growth of microbes in food follows a typical microbial growth pattern Growth rate depends on the nutritional value and temperature of the food Number of microbes depends on both inoculum size and growth rate Food spoilage occurs at high populations density (at stationary phase) - retarding microbial growth delays spoilage

Food Spoilage
Food is considered spoiled when an undesirable change in the color, flavor, odor or texture has occurred. Foreign substances in food products make foods undesirable.

Spoilage is a natural phenomenon; it occurs at varying rates depending on the storage temperature, kind of food involved, kind of microorganisms present, packaging materials used, food additives used and method of preservation.

It is a gradual process occurring because of poor sanitation, enzymatic or chemical reactions, improper temperature controls, microbial growth or physical abuse.

II. Microbial Growth and Food Spoilage

Meats and dairy products are ideal environments for spoilage by microorganisms because of their high nutritional value and the presence of easily utilizable carbohydrates, fats, and proteins; proteolysis (aerobic) and putrefaction (anaerobic) decompose proteins;

Fruits and vegetables have much lower protein

and fat content than meats and dairy products and undergo different kind of spoilage;
the presence of readily degradable carbohydrates in vegetables favors spoilage by bacteria; high oxidation-reduction potential favors aerobic and facultative bacteria; molds usually initiate spoilage in whole fruits

1. Frozen citrus products are minimally processed and can be spoiled by lactobacilli and yeasts 2. Grains, corn, and nuts can spoil when held under moist conditions; this can lead to production of toxic substances, including aflatoxins and fumonisins 3. Shellfish and finfish can be contaminated by algal toxins, which cause of variety of illnesses in humans

The microbial agent causing spoilage depends on the source of the food and its nutritional value:
Meats

may be contaminated by intestinal pathogens released during slaughter Dairy products - lactic acid bacteria Fruit and vegetables - soil and water microbes

Some microbes that cause spoilage may be human pathogens but the majority are not!

Growth of microbes in food follows a typical microbial growth pattern Growth rate depends on the nutritional value and temperature of the food

Number of microbes depends on both inoculum size and growth rate Food spoilage occurs at high populations density (at stationary phase) - retarding microbial growth delays spoilage

The principles discussed in Ch. 20 (microbial growth controls) are used here

Food preservation:

Temperature

Acidity

Lower: decreased growth rate - but, psychrophilic microbes Perishable food will only last for a few days at 4 oC Freezing ( - 20 oC) destroys the texture of many products and does not completely stop growth Deep freezing (- 80 oC) is costly

Most foods at neutral or acidic pH At pH < 5 microbial growth is inhibited Pickling: Decreased food pH by the addition of vinegar (acetic acid bacteria); veggies, meats, fish Fermented foods: acid is produced during food production (e.g., sauerkraut, yogurt, etc); lactic acid, acetic acid, and propionic acid bacteria; limited to pH > 4

Controlling Food Spoilage

1. Removal of microorganisms-filtration of water, wine, beer juices, soft drinks and other liquids 2. Low temperature-refrigeration and/or freezing retards microbial growth but does not prevent spoilage 3. High temperature Canning Canned food is heated in special containers called retorts to 115C for 25-100 minutes to kill spoilage microorganisms

Pasteurization-kills disease-causing organisms; substantially reduces the number of spoilage organisms Low-temperature holding (LTH)-68C for 30 min High-temperature short-time (HTST)-71C for 15 seconds Ultra-high temperature (UHT)-141C for 2 seconds

Shorter times result in improved flavor and extended shelf life

Water availability-dehydration procedures (e.g., freeze-drying) remove water and increase solute concentration Chemical-based preservation Radiation-nonionizing (ultraviolet or UV) Microbial product-based inhibition Bacteriocins-bacteriocidal proteins produced by bacteria; active against only closely related bacteria (e.g., nisin)

What are Food-Borne Diseases?

People get sick with a food-borne disease when they consume foods or beverages contaminated with disease-causing microbes, chemicals, insects or other harmful substances.

Bacteria, viruses and parasites cause most of these diseases. Toxins, poisons and chemicals can also contaminate food and cause illness.

Food-Borne Diseases

two primary types

food-borne infections food intoxications

There are a reported 76 million cases of food-borne disease occurring every year in the United States alone. Most of these cases are mild and cause symptoms for a day or two. More serious cases require 323,000 hospitalizations annually, and even cause 5,000 deaths a year. People most at risk tend to be those who are very old, very young, women who are pregnant. Even robustly healthy people are vulnerable if they are exposed to a very high dose of an unhealthy organism.

One symptom these diseases produce in common is that because they enter the body through food, the first sign of illness is usually nausea, vomiting, abdominal pain and cramps and diarrhea. the spectrum of food-borne diseases constantly changes and evolves even as the science of food safety continues to make significant advances and discoveries. Some diseases, such as cholera, tuberculosis and typhoid fever have been eradicated thanks to food safety improvements. Other diseases are just now being discovered or are adapting and evolving into new strains. E.Coli 0157:H7, for example, didn't exist 25 years ago.

Foodborne diseases and microbial sampling:

Food poisoning - Caused by preformed toxin in the food; organism may or may not be alive and growing; Clostridium botulinum and

Staphylococcus aureus

Food infection - Live cells delivered by contaminated food; organism multiply once food is ingested; Salmonella Sampling: Process food to release microbes; culturing and use of molecular probes (antibodies, gene probes, PCR) to detect specific microbes

Examples of foodborne diseases

most are infections and associated with animal products:
Organism Number of cased per year (U.S.)
1,963,000

Foods to watch
Poultry and diary products

Campylobacter jejuni

Salmonella spp.
Clostridium perfringens Giardia lamblia
Norwalk-like viruses

1,340,000
248,000

Poultry, meat, diary and eggs

Cooked and reheated meat products

200,000
9,200,000

Contaminated meat
Shellfish, other food

Staphylococcus aureus:

Common skin, respiratory, and GI tract flora Grows readily in unrefrigirated meats and creamy foods; toxins are heat resistance Produces 7 entrotoxins; the most potent is A (entA); a superantigen (T cell stimulation cytokines intestinal inflammation gastroenteritis) Severe but short response (1-6 hrs following ingestion; done by 48 hrs) Detection of toxins or the organism in food Antibiotics are useless

Clostridial diseases:

Gram positive, spore-forming, anaerobes common in soil; C. perfringens and C. botulinum C. perfringens - food poisoning: ingestion of > 108 cells (inappropriate cooking followed by unrefrigirated storage in closed containers) spore germination in the intestine leads to neurotoxin production Alteration of water permeability of intestinal lining diarrhea and intestinal cramps (no vomiting or fever); onset within 7 - 16 hrs of ingestion but gone in 24 hrs Diagnosed by isolation of microbe or detection of toxin in feces

Botulism (C. botulinum):

The most potent toxin known; few cases but high mortality (25%); destroyed by 10 min in 80 oC Flaccid paralysis of muscles Common in soil and water How? Improper canning spore germination toxin production canned food used without cooking disease Infant botulism: consumption of honey that is contaminated by spores (0 - 2 months) Treatment: antitoxin and ventilation

Salmonellosis:

Gram negative enteric bacterium; all strains are pathogenic; transmission is from sources (eggs, meats) and by food handlers Colonization of of intestinal epithelium

Two diseases:
Enterocolitis (most commonly by S. typhimurium): 105 - 108 viable cells; disease onset within 8 - 48 hrs; headaches, chills, vomiting, diarrhea and fever (2-3 days); continuous shading of organism for months/years (Typhoid Mary); treatment - none Typhoid fever (S. typhi): Septicemia leading to high fever that can last for several weeks; mortality is 15% if untreated; antibiotics Prevention: Cooked food (70 oC for 10 min); monitor for carrier state among food handlers

Pathogenic E. coli:

Some strains of E. coli; diarrhea and urinary tract infection; classification of pathogens is based on toxin and diseases Enterohemorrhagic (O157:H7) - colonization of the small intestine and verotxin production diarrhea and kidney infection; uncooked and undercooked ground meat; occasional epidemics Enterotoxigenic (Travelers diarrhea) - heat labile toxin; water and produce in developing countries; immunity Enteropathogenic - diarrhea that afflicts young children Enteroinvasive - invasive colon infection; bloody diarrhea; survival in phagosomes; in developing countries Treatment and prevention: diseases are self-contained but antibiotics help; irradiation of ground beef!

Campylobacter:

Gram negative microaerophile common in poultry and sometimes in beef C. jejuni and C. coli bacterial diarrhea; C. fetus spontaneous abortion in livestock Ingestion of 104 cells colonization of small intestine inflammation high fever (104 oC), headache, malaise, nausea, cramps, diarrhea subsides in 1 week; erythromycin to shorten infectious stage Prevention by proper cooking and hygiene (including utensils)

Listeriosis:

Listeria monocytogenes: a gram (+) bacillus; Cold and salt

tolerant; wide distribution; found in soil water and raw milk; contaminates all food products either at source or during processing; mostly in processed food Pathology (2500 per year): Uptake by phagocytes growth lysis of phagocyte infection of nearby cells Immunity due to cell-mediated TH1 cells macrophage activation In normal individuals - gastrointestinal food infection; in immuno-compromised individuals - acute bacterimia and meningitis (20% death rate) Prevention: cleanliness during food processing; avoiding outdated foods Diagnosis by culturing from blood and spinal fluid; treated with trimethoprim drugs

Other foodborne infectious diseases :

Bacterial diseases Yersinia enterocolitica enteric fever

Bacillus cereus - food

poisoning by heat stable toxin Shigella spp. - shigolosis (100,000 per year) Vibrio spp. contaminated seafood

Viral diseases - the most common cause of gastrointestinal diseases; 24-hour flu - fast and self-containing; fecal contamination

Norwalk viruses, rotaviruses, astroviruses, hepatitis A

Preventing Foodborne Disease Food infections (microbes are transferred to consumer) Food poisoning (results from the toxin consumption)

Food-Borne Intoxications

ingestion of toxins in foods in which microbes have grown include staphylococcal food poisoning, botulism, Clostridium perfringens food poisoning, and Bacillus cereus food poisoning

Toxins

ergotism

toxic condition caused by growth of a fungus in grains carcinogens produced in fungus-infected grains and nut products carcinogens produced in fungus-infected corn

aflatoxins

fumonisins