FACTORS THAT INFLUENCE MICROBIAL GROWTH IN FOOD

FACTORS THAT INFLUENCE MICROBIAL GROWTH IN FOOD

Characteristics of the food itself is called intrinsic factors (substrate limitation) Factors that are external to the food is called extrinsic factors (environmental factors) Implicit factors Processing factors

INTRINSIC FACTORS

Nutrient content pH and buffering capacity Redox potential Water activity Antimicrobial constituents Antimicrobial structures

Nutrient Content

Foods contains nutrients: as a source of water, source of energy, source of nitrogen, source of vitamins and related growth factors and source of minerals. Nutrients are chemical in nature (organic and inorganic) Microorganisms need the nutrients in food for their biomass, essential nutrients for growth that the organisms cannot synthesize, and substrate for energy source.

Nutrient content

An organisms that is unable to utilize a major component of a food material will limit its growth in that food than those that can. Simple substances can be utilized by the microorganisms. Microbes have enzymes to breakdown complex chemicals (nutrients) in foods.

Nutrient Content Carbohydrates: Monosaccharides: (Hexoses and Pentoses) Disaccharides: Lactose, Sucrose, Maltose Oligosaccharide:
Raffinose: glucose + fructose + galactose) Stachyose: glucose + fructose + galactose + galactose

Polysaccharides: starch, glycogen, cellulose, hemicellulose, dextrins, pectins, gums and mucilage, inulin

Nutrient Content

Carbohydrates as source of energy through several metabolic pathways Metabolic end products to produce amino acids, CO2, organic acids. Synthesize complex carbohydrates e.g. dextrins (desirable), slime (undesirable) Sugars are used in biochemical identifications of microorganisms isolated from foods.

Nutrient Content

Proteins:
Simple proteins: polymers of amino acids (albumins,
globulins, lutelins, prolamins, and albuminoids.

Conjugated proteins: protein + -----Metal (hemoglobin and myoglobin) Carbohydrates (glycoprotein such as mucin) Phosphate (phosphoproteins such as casein) Lipids (lipoproteins)

Nutrient Content

Peptides: amino acids (few) Nonprotein nitrogenous (NPN) compounds: (amino acids, urea, ammonia, creatinine, trimethylamine) Degree of solubility determines the ability of microorganisms to utilize the protein. E.g. albumin soluble in water; collagens insoluble in water. Specific microbes.

Nutrient Content Lipids in foods: Free fatty acids, glycerides, phospholipids, waxes and sterols. Plant sources vs animal sources. Lipids are in general less preferred as source of energy and cellular materials.

Nutrient Content

Many microbes produce extracellular lipases that hydrolyzed fats. Some produce extracellular lipid oxidase to produce different aldehydes and ketones from unsaturated FAs. Some produce endocellular lipases and oxidases. Food spoilage (rancidity), flavour development, metabolize cholesterol (probiotic).

Nutrient Content

Minerals and vitamins: Minerals in small amount and present in foods. Most microorganisms can synthesize B vitamins. Some fastidious Lactobacillus species require some essential amino acids, B vitamins that may be added to foods. It is not possible or practical to control microbial growth in food by restricting nutrients.

Nutrient content

Amylolytic enzymes to breakdown starches. Proteolytic enzymes to breakdown proteins. Lipolytic enzymes to breakdown lipids and fats. Pectinase?, Gelatinase? , Cellulases? Specific microorganisms.

How nutrients affect growth?

Addition of certain nutrients to certain will favor certain microbes. E.g. adding fruits containing sucrose in yoghurt increase the range of carbohydrates available and allows the development of more diverse microflora e.g. yeasts. The amount or concentration of nutrient available, to some extent the determine the rate of microbial growth; more easily utilizable nutrients, the faster the growth rate.

How nutrients affect growth?

Presence of antimicrobial factors, or processing methods that may cause deficient in essential nutrients, may limit microbial growth.

Nutrient metabolism

Starch hydrolysis, sugar fermentation, production of hydrogen sulphide and indole, or nitrate reduction can be used to identify microorganisms. Also measurements of microbial metabolism or end production of end products can be used to estimate the bacterial population. E.g. use of dyes (methylene blue, resazurin, tetrazolium). The rate at which the indicator change colour, is related to the metabolic rate of the organisms in the sample. The greater the number of bacteria, the faster is the colour change. Useful in dairy industry.

pH and Buffering Capacity

pH = -log (aH) = log 1 / (aH)~ log 1 / [H+] Where aH is hydrogen ion activity [H+] is hydrogen ion concentration

pH values below 7 is acidic, above 7 is alkaline environment. Difference in pH of 1, 2 and 3 units correspond to 10- , 100- , and 1000- fold differences in hydrogen ion concentration.

Importance of pH of an environment

Effect the activity and stability of enzymes. Plotting microbial growth rate against pH produces a approximately symmetrical bell-shaped curve. In general: bacteria grow fastest in the pH range 6.0 8.0 Yeasts 4.5 to 6.0 Filamentous fungi 3.5 to 4.0 Exceptions: lactobacilli and acetic acid bacteria, can grow at low pH, optimum usually pH 5.0 to 6.0. Food with alkaline pH: egg white, gamat extract.

pH and microbial ecology

plant products e.g. vegetables have moderately acid pH, spoilage organisms are soft-rot producing bacteria such as Erwinia carotovora and pseudomonads Fruits lower pH prevents bacterial growth and spoilage dominated by yeast and moulds. Fresh milk? Meat? pH growth range of microorganisms on food (Jay pg 36)

Keeping quality of meat

Meat from rested keeps longer than meat fatigued animals Direct result of final pH of meat attained upon completion of rigor mortis. Upon death of well-rested animals, 1% of glycogen in meat is converted to lactic acid, pH reduces from about 7.4 to 5.6 (depending on type of animals) Fish: pH attained upon complete rigor mortis is about 6.2 to 6.5. Meat have better keeping qualities than fish, spoilage mainly by bacteria.

Inherent vs added acidity

Inherent acidity by activity of microorganisms e.g. fermented foods, means of preservation, presence of weak organic acids. Partial dissociation of weak acids plays an important role in their ability to inhibit microbial growth. E.g. of common food acids: acetic, propionic, lactic, sorbic, citric, benzoic, phosphoric, carbonic, nitrous and sulfurous.

Microbial inhibition by weak organic acid

weak organic acids do not dissociate completely into protons and conjugate base in solution but establish an equilibrium. HA H+ + Aequilibrium constant Ka = [H+] [A-] / [HA] Rearrange: 1/ [H+] = 1/Ka . [A-] / [HA] Log base 10: pH = p Ka + log [A-] / [HA] Henderson-Hasselbach equations

1.

2.

Organic acids enter the cell only in the protonated form (undissociated) form. Once inside the cell, they dissociate, releasing H+ (proton) into the cell cytoplasm. The cytoplasm becomes acidic, accumulation of anions that cannot passed back across the cell membrane. Change in pH results in a change in transmembrane proton gradient (difference in pH between inside and outside of cell), can serve as sensor to stop or start energy-dependent reactions.

1.

The changes in acidity inside the cell may result in protonation or deprotonation of amino acids in proteins. May alter secondary and tertiary structures of proteins, changing functions and signaling change of pH to the cell.

pH

When the pH is equal to acids pKa, then half of the acid present will be undissociated. If pH is increased then dissociation of the acid will be increased. Weak acid is used to inhibit growth of microorganisms. High extracellular H+ and concentration of undissociated acid.

Three mechanisms bacteria maintain the internal pH (pHi)

E.g. Salmnella enterica serovar Typhimurium. 3 mechanisms:

1. homestatic response 2. acid tolerance response (ATR) 3. synthesis of acid shock proteins

Homeostatic response
At external pH is >6.0, salmonella cell adjust the internal pH; the homeostatic response maintains the pH by increasing the activity of proton pumps to expel proton from cytoplasm. The mechanism is always on and functions in the presence of protein synthesis inhibitors.

ATR

ATR is triggered by a pH outside of 5.5 to 6.0. This mechanism is sensitive to protein synthesis inhibitors. ATR appears to involve the membrane-bound ATPase proton pump and maintains internal pH at >5.0, even external pH values as low as 4.0. Loss of ATPase activity abolish by gene mutation or or metabolic inhibitors abolishes the ATR but not pH homeostatic mechanism.

Synthesis of acid shock proteins

The synthesis of these proteins is triggered by outside pH of 3.0 to 5.0. A set regulatory proteins.

Buffering capacity

those that tend to resist changes in pH are said to be buffered. In general meats are more highly buffered than vegetables. Proteins in meat contribute to the buffering capacity. Vegetables are gen. low in protein, lack buffering capacity to resist changes in pH during growth of microorganisms.

Approximate pH values of some foods FOOD pH

Milk Cheddar cheese Ground beef Chicken Fish Shrimps Cabbage Spinach Tomatoes Apples Bananas Honeydew Limes 6.3 to 6.5 4.9; 5.9 5.1 to 6.2 6.2 to 6.4 6.6 to 6.8 6.8 to 7.0 5.4 to 6.0 5.5 to 6.0 4.2 to 4.3 2.9 to 3.3 4.5 to 4.7 6.3 to 6.7 1.8 to 2.0

Minimum pH values for the growth of some foodborne bacteria Microorganisms pH

Clostridium botulinum Group I C. botulinum Group II C. Perfringens Escherichia coli O157:H7 Lactobacillus brevis Listeria monocytogenes Pseudomonas fragi Salmonella spp Staphylococcus aureus Vibrio parahaemolyticus Zygosaccharomyces bailii
4.6 5.0 5.0 4.5 3.16 4.1 ca 5.0 4.05 4.0 4.8 1.8

refer to Jay Table 3-1, 3-2 and 3-3 pp 38 and 39.

Redox Potential Eh

Oxidation-reduction (redox) reaction occurs as a result of transfer of electrons between atoms or molecules. [Oxidant] + H+ + ne [Reductant]

Where n is number of electrons e transferred e.g. electron transport chain, and generation of release of energy by oxidative phosphorylation

Redox

Is the tendency of a medium to accept or donate electrons i.e to oxidize or to reduce. When electrons are transferred from one compound to another, a potential difference is created between the two compounds. This difference can be measured by instrument and expressed as millivolts (mV). Positive potential indicates oxidizing environment; negative potential indicates reduced environment. Zero potential when concentration of oxidant and reductant is equal.

Factors influencing the measured Eh of foods

Redox couple present Ration of oxidant to reductant pH Poising capacity i.e. the resistance to change in potential of the food Availability of oxygen (physical state, packaging) Microbial activity

Redox potential of some food materials Food Raw meat (post-rigor) Raw minced meat Cooked sausages and canned meats Wheat (whole grain) Spinach Pear Grape Lemon

E (mV)
-200 +200 -20 to -150 -320 to -360 +74 +436 +409 +383

pH 5.7 5.9 ca 6.5 6.0 6.2 4.2 3.9 2.2

Hydrogen ion concentration affect the Eh For every unit decrease in pH the Eh increases by 58 mV. E.g. fruit juice high positive Eh reflection of their low pH. Couples in foods: Gluthione and cystein in meats, ascorbic acid and reducing sugars tend to establish reducing conditions.

Oxygen is a powerful oxidizing agent; if sufficient air is present in a food, a high positive potential will result, the redox couples present are in oxidized state. E.g. chopping, grinding, or mincing increase access of air to food, increase Eh Modified vacuum packaging or canning will reduce Eh . Microbial growth in food reduces Eh. (oxygen depletion, production of hydrogen by microorganisms). E.g. testing quality of dairy products use methylene blue or resazurin (redox dyes). Yeast appear colourless (reuced, viable), or blue (non-viable) when stained with methylene blue.

Effect of redox on microrganisms

Physiological groups: Obligate or strict aerobes: oxidative phosphorylation, oxygen as final electron acceptor. Pseudomonas fluorescens and other gram negative Eh bacteria rods, slime and off-odours on meat surfaces Eh of +100 to +500 mV. Bacillus subtilis produces rope in bread Acetobacter grows on the surface of alcoholic beverages, oxidize ethanol to acetic acid.

Obligates anaerobes: oxygen must be absent, grow at low or negative redox. Clostridium botulinum causing botulinum grow in anaerobic conditions such as deep in meat tissues and stews, in vacuum packs and canned foods. Anerobically vs aerobically grown Saccharomyces cerevisiae showed differences in lipid and sterol contents.

Aerotolerant anaerobes are incapable of aerobic respiration but can grow in the presence of air. E.g. lactic acid bacteria, lack catalase and superoxide dismutase, but able to in the presence of oxygen because they have the mechanism to destroy superoxide. Superoxide dismutase: convert free radicals + hydrogen to water and oxygen catalase break hydrogen peroxide to water and oxygen

Antimicrobial Barriers and Constituent

Physical barriers: skin, shell, husk, rinds of product. (dry, antimicrobial compounds on surfaces). Physical damage to these barriers allows microbial invasion of nutrient rich tissues. Certain plant antimicrobials produced as a result of physical damage eg isothiocyanate, allicin

Natural antimicrobials
Mustard oils Alicin Euganol Thymol Cinnamic aldehyde Benzoic acid Sorbic acid Oleuropein Mustard, cabbage, brassicas Garlic, onions Allspice, cloves, cinnamon Thyme, oregano Cinnamon

Cranberries Moutain ash berries Green olives

Antimicrobials in hens egg a

Albumin
Nutrient Status:
High pH Low levels of available nitrogen Moderate pH High levels of protein, carbohydrate and fat Lactoferrin Lysozyme Lactoperoxidase Immunoglobulin

Milk

Antimicrobials:
Ovotransferrin (conalbumin) Lysozyme Avidin Ovoflavoprotein Ovomucoid &ovoinhibitors

Lysozymes most effective against Gram positive bacteria, also Gram negative bacteria if their outer membrane is damaged. Ovotransferrin and lactoferrin scavange iron. Avidin and ovoflavoprotein sequester biotin and riboflavin Milk generate antimicrobial in the presence of hydrogen peroxide. Lactoperoxidase in milk catalyse the oxidation of thiocyanate by hydrogen peroxide to produce hypothiocyanate, kill Gram-negative bacteria and inhibit Gram-positives. The antibacterial effect increase with acidity, target the cytoplasmic membrane. Lactoperoxidase system can be used to preserve raw milk where refrigeration is uncommon.

Water activity

Water requirements of microorganisms is best described in terms of water activity aw in the environment. Defined as ratio of the partial pressure of water in the atmosphere in equilibrium with the substrate, P, compared with partial pressure of the atmosphere in equilibrium with pure water at the same temperature, P0 This is numerically to the equilibrium relative humidity (ERH) expressed as: Aw = P /P0 = 1/100 ERH

Pure water has aw of 1.00, 22% NaCl (w/v) has aw of 0.86 Saturated NaCl has aw of 0.75 Depends on the number of molecules or ions present in solution rather than their size. Salt better than sucrose at reducing water activity on mole-to-mole basis.

Minimum water activities at which active growth can occur

Groups of micro-organisms
Most Gram-negative bacteria Most Gram positive bacteria Most yeasts Most filamentous fungi Halophilic bacteria Xerophilic fungi Osmophilic yeast

Minimum aw
0.97 0.90 0.88 0.80 0.75 0.61 0.61

Approximate minimum aw for growth of specific organisms

Names aw 0.97 0.96 0.95 0.94 0.94 0.86 0.81 0.81 0.78 0.70 0.62 0.61

Clostridium botulinum type E Escherichia coli Bacillus subtilis Clostridium botulinum types A and B Vibrio parahaemolyticus Staphylococcus aureus Penicillium patulum Aspergillus flavus Aspergillus ochraceus Aspergillus glaucus Zygosaccharomyces roixii Xeromyces bisporus

Water activity

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

Effect of low water activity

General effect: Lowering aw below the optimum is to increase the length of lag phase of growth. Influenced of lowered water on all metabolic activities (also influenced by other environmental factors: pH, temperature of growth, and Eh. As water activity is lowered, osmotic pressure in the environment is increased, cytoplasm increase osmotic pressure by increasing concentration of compatatible solutes that do not interfere with cytoplasmic function.

E.g. polyols glycerol, arabitol, and mannitol in fungi; amino acids and amino derivatives accumulate in bacteria. Salt tolerant Staphyloccus aureus accumulate proline as a response to low water activity. Listeria monocytogenes accumulates carnitine, glycine betaine (osmotically and chilled stress)

Germination of Bacillus and Clostridium spores were strongly inhibited when water activity was controlled by NaCL or CaCL but less inhibition when glucose or sorbitol was used, very little inhibition when glycerol, ethylene glycol, acetamide or urea was used. Osmophilic yeasts accumulate glycerol under stress. Reduce water activity results in cessation of enterotoxin B production by S. aureus even though high numbers of cells are produced at the same time.

Lowered water activity affect nutrient availability. Affect the function of cell membrane which must be kept at in a fluid state. Drying of internal part of cell. Ability to concentrate compatible solutes.

Methods to reduce water activity

Adding solute Adding ions Adding hydrophilic colloids Freezing and drying

Relationship between water content and water activity

The relationship is affected by temperature, and May seem to depend whether the water is added or remove from the substrate. Adding water to the dry substrate will result in higher water activity (adsorption). Removing water from the substrate will result in lower water activity (desorption) Refer to

Water activity of some foods

Cereals, crackers, sugar, salt, dry milk Noodles, honey, chocolate, dry eggs Jam, jelly, dried fruits, parmesan cheese, nuts 0.1 - 0.20 <0.60 0.60 0.85

Fermented sausage, dry cured meat, sweetened condensed milk, maple syrup
Evaporated milk, tomato paste, bread, fruit juices, salted fish, sausage, processed cheese Fresh meat, fish, fruits, vegetables, milk, eggs

0.85 0.93

0.93 0.98

0.98 - 0.99

Understanding water activity

Staphylococcus aureus min water activity is 0.85, halophilic bacteria is 0.75. Higher water activity is needed for bacteria to sporulate and germinate and produce toxins than for the minimum water activity for growth. Under ideal condition the minimum water activity for growth is lower than in nonideal condition e.g. ideal condition for bacteria pH 6.8 min aw is 0.91; at pH 5.5 it can be 0.95 or more.

If aw is reduced below the minimum level for growth of a microorganism, the cells remain viable for a while; If aw is reduced drastically, microbial cells lose viability generally rapidly at first, then more slowly. Very important in food processing, controlling spoilage and pathogen as well as isolating microbes e.g. adding salt in cured meat, salt in media to isolate S. auereus