Food Microbiology: 4th Yr Biotech (Biotechnology), Term VII

FOOD MICROBIOLOGY L T P: 3-1-0 Credit: 4 Unit I: Historical Background (8) History of Microorganisms in food, Taxonomy, role and significance of microorganisms in foods. Intrinsic and Extrinsic Parameters of Foods that affect microbial growth. Unit II: Microbes in Food (12) Microorganisms in fresh meats and poultry, processed meats, seafoods, fermented and nonfermented dairy products and miscellaneous food products. Probiotics and health benefits, microbiological examination of surfaces, air sampling, metabolically injured organisms, enumeration and detection of food-borne organisms. Bioassay and related Methods Unit III: Food Preservation (12) Food Preservation using irradiation, Characteristics of radiations of Interest in food preservation. Principles Underlying the Destruction of Microorganisms by irradiation, Physical and Chemical Methods of Food preservation, Legal Status of Food preservation and Role of FDA. Unit IV: Storage (8) Stable and clean packaging, Canning and different methods of canning, Consumer perspective and future of food biotechnology. Reference Books: Jay J.M., Modern Food Micro-Biology Aspen Publication, 2000. Doyle M.P., Beuchat L.R. and Montville T.J., Food Microbiology: Fundamentals and Frontiers, ASM Press, 2001. Lopez G.F.G., and Canovas G.V.B., Food Science and Food Biotechnology, CRC Press, 2003.

Food Microbiology
This is the study of microorganisms involved in the spoilage, contamination, and preservation of foods. This science includes those microorganisms that are essential for the production and processing of foods such as cheese, bread and wine. This study also includes pathogens associated with human diseases.

Over View of Food Microbiology (1)

Food spoilage is a main concern; it occurs during the time of food production, transport, storage, or preparation. Microbial contamination causes food spoilage; food may be contaminated from outside sources on the way from the filed to the processing plants or during slaughtering animals, storage, transport and distribution. Physical, chemical, and biological process are used in the preservation of foods.

Over View of Food Microbiology (2)

Contaminated foods often cause diseases to human (Food-borne illness or food-borne disease or food poisoning). Food-borne pathogens can be detected by cell culture techniques, immunological techniques and molecular probes. Beneficial microorganisms are involved to ferment dairy products, meats and fruits. Lactic acid bacteria are mainly involved in milk fermentation and yeasts are the main microbes involved in alcohol fermentation.

Over View of Food Microbiology (3)

Probiotics and health: Probiotics are live microorganisms (mainly bacteria) that are similar to beneficial microorganisms found in human intestine. Probiotics are administrated in adequate amount to change the microbial community of human gut.

Microbial Growth
Bacteria are single-celled organisms (Yeasts are single-celled and molds are multi-cellular fungi)
Bacteria multiply in a process called binary fission in which two cells arise from one (Yeasts are multiplied by budding, binary fission or spore formation) Growth Rate: is the change in cell number per unit time

dx/dt= X,

ln(x/x0 )= t

The interval for one cell to become two is known as the generation time or doubling time Quickest generation time is 9 mins Bacteria typically 15-30 mins, in extreme cases it can be 1yr.

Binary Fission

Growth Curves

Lag Phase
Considered as the adjustment period when the organism adapts to new surroundings

No Microbial growth
Synthesize enzymes to adapt to the environment

Recovery from stress or injury

This period may be extended in unfavorable environments In extreme cases the lag phase can last for weeks

Exponential (log phase)

Growth is stable Growth rate is constant for a given bacteria under specified conditions Catabolic processes generate energy

Anabolic processes build cell structures

Stationary Phase
Over time, essential nutrients become depleted or waste products build up to toxic levels so that logarithmic phase ceases (end) and results in stationary phase No net growth in stationary phase (cell replacing but number not increasing Cell functions such as energy metabolism may continue

Endospore-forming bacteria produce the endospore once the culture has entered the stationary phase

Death phase
Viable cell count decreases

Under certain circumstances cell death is accompanied by cell lysis

Exponential (log phase)

Growth Rate: is the change in cell number per unit time

dx/dt= X, ln(x/x0 )= t

Growth Rate: is the change in cell number per unit time

dx/dt= x, Growth rate decreases as the population density increases.
dx/dt= (m - m x/K)x

where K is the carrying capacity of the environment (the stationary phase population) and m, the maximum specific growth rate. As x increases and approaches K, the growth rate falls to zero

Factors Affecting the Growth and Survival of Micro-organisms in Foods Intrinsic Factors (Substrate Limitations)
Nutrient Content pH and Buffering Capacity Redox Potential, Antimicrobial Barriers and Constituents Water Activity

Extrinsic Factors (Environmental Limitations)

Relative Humidity Temperature Gaseous Atmosphere

Nutrient Content
Microorganisms use food as a source of nutrients and energy. They use chemical elements (from nutrients) which are essential for their growth since they can not synthesize.

They utilize a specific substrate as energy source.

Food products such as meat or casein digests, tomato juice, sugar and starch in microbiological media provides powerful testimony to their growth suitability. The concentration of key nutrients may determine the rate of microbial growth

pH and Buffering Capacity

pH is equal to the negative logarithm of the hydrogen ion activity

Activity is proportional to concentration and the proportionality constant, the activity coefficient, approaches unity as the solution becomes more dilute

where (aH) is the hydrogen ion activity and [H+] is the hydrogen ion concentration.

pH directly regulates microbial growth and metabolism because activity of enzymes is highly dependent on pH. Bacteria grow fastest in the pH range 6.08.0, yeasts 4.56.0 and filamentous fungi 3.54.0.

pH and Buffering Capacity Approximate pH ranges of some common food commodities

pH The ability of low pH to restrict microbial growth has been deliberately employed since the earliest times in the preservation of foods with acetic and lactic acids

Lower pH prevents bacterial growth and spoilage is dominated by yeasts and moulds. Addition of week lipophylic acid causes leakage across the membrane. This way H+ ion can easily penetrate the cell, and because of this pH cytoplasm decreases. In the cytoplasm, those molecules are dissociated and the anion then react with essential cell substances. With inhibition as a result.

Effect of H+ transport: Increase in denaturization of membrane proteins and transport enzymes. Affects cell permeability and decreases intracellular pH

Intrinsic factor: Redox Potential affects microbial growth in food

Oxidation Release of electrons from a compound Reduction Incorporation of electrons into a compound Always occur in pairs Oxidation - Reduction When an oxidation-reduction reaction occurs, an electric potential is created Oxidated = positive Reduced = negative Depending on the concentration of oxidizing and reducing compounds:

Eh and microbial growth

Anaerobiosis -200 mV Aerobiosis 200 mV -200 a 200 mV includes growth of microaerobic, aerobic and anaerobic organisms

Eh values of selected foods

Food Fruits, vegetables, juices Milk Eh Value(mV) 300-400 300

Meat
Ground meat Cheese

-200
200 -200

Some important redox couples and their standard redox potential

Redox potentials of some food materials

Chopping, grinding of food materials will increase the access of oxygen resulting in increase its redox potential. Since oxygen is usually the most influential redox couple in food systems. It has a high standard redox potential and is a powerful oxidizing agent; if sufficient air is present in a food, a high positive potential will result.

Redox Potential
Redox potential is expressed by Nernst Equation

where Eh and E0 are both measured at pH 7; R is the gas constant; T, the absolute temperature; n, the number of electrons transferred in the process and F is the Faraday constant.

Factors influencing of the redox potential (Eh) of Foods

Redox couples present (depends on the properties of oxidant and reductant) Ratio of oxidant to reductant pH of the medium Poising capacity :There will be some resistance (posing capacity) to change in foods redox potential when a redox condition changes. It is very much similar to buffering capacity of a medium. Availability of oxygen Microbial activity

Microbial Activity, Oxygen and Redox Potential (I)

Microbial growth depletes oxygen present in food resulting in decrease of its redox potential. Redox potential could be decreased also due to production of reducing compound such as hydrogen by micro-organism. Microbes will predominately grow in the food surface due to more air (oxygen) available in the food surface. Bacillus subtilis (Eh +100 to +135 mV) produces rope in the open texture of bread and Acetobacter species growing on the surface of alcoholic beverages, oxidize ethanol to acetic acid to produce either spoilage or vinegar.

Microbial Activity, Oxygen and Redox Potential (II)

Obligate anaerobes tend only to grow at low or negative redox potentials and often require oxygen to be absent. oxygen exerts a specific toxic effect to many anaerobes. Obligate or aero-intolerant anaerobes are unable to scavenge and destroy toxic products of molecular oxygen such as hydrogen peroxide and superoxide anion radical produced by molecular oxygen.

Redox potential is an indicator of microbial growth in foods

Decrease in redox potential indicates the microbial growth particularly in diary products. Redox dyes such as methylene blue and resazurin are used to detect the microbial growth. Methylene blue is used to determine the proportion of viable cells. Test: A cell suspension stained with methylene blue is examined under the microscope and viable cells with a reducing cytoplasm appear colorless. Non-viable cells fail to reduce the dye and appear blue. So, blue color is inversely proportional to viable micro-organism present in the food. Methylene blue [3, 7-Bis(dimethylamino) phenazo -thionium chloride, MB+] is a water soluble polyaromatic cationic redox dye and it is easily reduced by various reducing agents to the colorless hydrogenated molecule.

Procedure in Methylene Blue Testing. (1) Measure 1 ml of the methylene blue thiocyanate solution into a test tube. (2) Add 10 ml of milk. (3) Tubes may be placed in the water bath immediately at 35o C within 10 minutes. (4) When temperature reaches 36o C, slowly invert tubes a few times to assure uniform creaming. Record this time as the beginning of the incubation period. Cover to keep out from light.

(5) Check samples for decolorization after 30 minutes of incubation. Make subsequent readings at hourly intervals thereafter.
(6) After each reading, remove decolorized tubes and then slowly make one complete inversion of remaining tubes. (7) Record reduction time in whole hours between last inversion and decolorization.. Decolorization is considered complete when four-fifths of the color has disappeared.

Classification.The suggested classification (Quality of Milk) is listed. Class 1. Excellent, not decolorized in 8 hours. Class 2. Good, decolorized in less than 8 hours but not less than 6 hours.

Class 3. Fair, decolorized in less than 6 hours but not less than 2 hours.
Class 4. Poor, decolorized in less than 2 hours.

Factors Affecting the Test.Many factors affect the methylene blue reduction test. Oxygen content must be used up before the color disappears, any manipulation that increases the oxygen affects the test.

Cold milk holds more oxygen than warm milk.

The kind of organisms affect the rate of reduction. The coliforms appear to be the most rapidly reducing organisms, closely followed by Streptococcus lactis, some of the faecal Streptococci, and certain micrococci. Thermoduric (Bacillus, Clostridium) and psychrotrophic (Gammaproteobacteria, Actinobacteria ) bacteria reduce methylene blue very slowly.

The accuracy of the test is increased if the samples are periodically inverted during incubation.

Intrinsic Factors: Antimicrobial Barriers and Constituents A physical barrier such as the skin, shell, husk or coat of a food product prevents microbial invasion resulting in inhibition of its growth. These outer surfaces are usually composed of macromolecules relatively resistant to degradation and provides an inhospitable environment for microorganisms. A low water activity, a shortage of readily available nutrients and, often, antimicrobial compounds such as short chain fatty acids (on animal skin) or essential oils (on plant surfaces) in the food outer surfaces are the major constraint for microbial growth.

Intrinsic Factors: Antimicrobial Barriers and Constituents

Physical damage to the physical barriers allows microbial invasion of the underlying nutrient-rich tissues resulting in microbial growth. This is the explanation why the damaged fruits and vegetables deteriorate more rapidly than entire products. Antimicrobial isothiocyanates (mustard, horseradish, watercress, cabbage) and antifungal phaseollin (green beans) present in food inhibit microbial growth. Many natural constituents of plant tissues such as pigments, alkaloids and resins have antimicrobial properties. The enzyme lysozyme of animal products catalyses the hydrolysis of glycosidic linkages in peptidoglycan responsible for the strength and rigidity of the bacterial cell wall. Destruction or weakening of this layer causes the cell to rupture (lyse) under osmotic pressure.

Water molecules may pass freely from the cytoplasm to the environment and from the environment to the cytoplasm. A living organism will only be stressed if there is a net flow out of the cytoplasm, leading to plasmolysis, or a net flow into cytoplasm leading to rupture of the membrane. A useful parameter is water activity, aw which helps us to understand the movement of water from the environment to the cytoplasm or from the cytoplasm to the environment. Water Activity (aw): The water activity of a substrate is defined as the ratio of the partial pressure of water in the atmosphere in equilibrium with the substrate.
Aw = p/p0 p = vapor pressure of food system p0 = vapor pressure of solvent (water) Important implications for the storage of foods in low aw

Intrinsic Factor: Water Activity (I)

Water Activity is a colligative property

Intrinsic Factor: Water Activity (II)

Cytoplasm is an aqueous solution and so must have a lower water activity than pure water; thus a micro-organism in an environment of pure water will experience a net flow of water molecules into the cytoplasm. If it cannot control this it will increase in size and burst. Bacteria, fungi and algae cope by having a rigid strong wall capable of withstanding the osmotic pressure of the cytoplasm which may be as high as 30 atm in a Gram-positive bacterium or as little as 5 atm in a Gram-negative species. Freshwater protozoa, on the other hand, cope with the net flow of water into the cell by actively excreting it out again with a contractile vacuole.

Minimum water activities at which growth can occur.

The limiting value of water activity for the growth of any microorganism is about 0.6 Below this value the spoilage of foods is not microbiological. It may be due to insect damage or chemical reactions such as oxidation.

Water Activities of saturated salts solution at 250C

EXTRINSIC FACTORS(ENVIRONMENTAL LIMITATIONS) AFFECT MICROBIAL GROWTH OF FOODS a)Relative Humidity b)Temperature c) Gaseous Atmosphere

Relative Humidity
Relative humidity and water activity are interrelated. As relative humidity increases, water activity increases. Extrinsic factors regulates intrinsic factors Relative humidity is essentially a measure of the water activity of the gas phase. Water will transfer from the gas phase to the food when food commodities having a low water activity are stored in an atmosphere of high relative humidity and the water activity of food will increase resulting in increase of microbial growth. If micro-organisms starts to grow and become physiologically active they will produce water as an end product of respiration. Thus they increase the water activity of their own immediate environment which will eventually increase the growth of micro-organisms and spoil a food.

Relative Humidity
The storage of fresh fruit and vegetables requires low relative humidity. If it is too low then many vegetables will lose water and become flaccid.

If it is too high then condensation may occur and microbial spoilage may be initiated.

Temperature
Microbial growth; -80C up to 1000C Water should be present Bacteria growth are normally at 35-370C, moulds rather less, about 300C

Important temperatures for microbial growth

Mesophilic and psychrotrophic organisms are generally of greatest importance in food microbiology. Mesophiles are frequently of human or animal origin (common foodborne pathogens such as Salmonella, Staphylococcus aureus and Clostridium perfringens). Mesophiles grow more quickly at their optima than psychrotrophs.

Temperature regulates microbial growth

Temperature regulates enzyme activity Membrane structure is changed in low Temperature High temperature increases protein denaturation and ruptures cell membrane

Gaseous Atmosphere
Oxygen increases Redox Potential which supports microbial growth CO2 inhibits microbial growth The mechanism of CO2 inhibition is yet to be determined. CO2 decreases pH of the food.