Department of microbiology microbial food technology group a diploma in quality assurance in microbiology diploma



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4.Explain about the factors influencing the growth of micro-organisms.

FACTORS INFLUENCING THE GROWTH OF MICRO-ORGANISMS


  • Interactions between microorganisms and our foods are sometimes beneficial. Food is the substrate, for the growth of microorganisms.

  • The type of microorganisms present and the environmental conditions are also important. The food or substrate dictates what can grow and cannot grow.

  • The characteristics of the food or substrate one can make predictions about the microbial flora that may develop.

  • The factor that favor or inhibit the growth of microorganisms is essential for the principles of food spoilage and preservation.

  • The chief compositional factors of a food that influence microbial activity includes:




  1. INTRINSIC FACTORS  1. pH

2.Moisture content

3. Oxidation –reduction potential (Eh)

4. Nutrient content

5.Antimicrobial constituents

6.Biological structures


  1. EXTRINSIC FACTORS  1. Temperature

2. RH of the environment

3. Presence or concentration of gases in the environment

INTRINSIC FACTORS:

The parameters of plant and animal tissues that are inherent part of the tissues are referred to as intrinsic factors.

1. Hydrogen ion concentration pH:

PH is one of the main factors affecting the growth of survival of microorganisms in culture media

and in foods.

Example:- When pure water ionizes equal number of OH – and H+ are produced. Only a small amount of water ionizes so that the concentration of these ions is very small, -1 X 10^-7 mol/L. This can be summarized as follows:

H2O > OH- + H+

[H+] =[OH-] = 1 X 10^-7 mol/L

S solution containing equal number of H+ and OH – ions is neutral in reaction. A solution containing more H+ ions than OH- ions is acid.

A solution containing more OH- than H+ ions is alkaline.

Example:-


  • A solution containing 10^-7 mole H+/L has a pH of 7 and is neutral.

  • A solution containing 10^-5 mole H+/L has a pH of 5 and is acid.

  • A solution containing 10^-8 mole H+/litre has a pH of 8 and is alkaline.

pH RANGES FOR MICRO-ORGANISMS:-

All microorganisms have a pH range in which they can grow and an optimum pH at which they grow best. Saccharomyces cerevisiae, for example has a pH range of 2.35 – 8.6 with an optimum at pH 4.5.



pH not only influence the growth rate of an organism within its pH range but is also has an overall influence on the growth curve. This is illustrated in Fig. 6.10, which shows the effect of pH on the growth curve. Notice that is pHs below the optimum:

EFFECT ON pH ON THE GROWTH RATE OF BACTERIA, YEAST, AND MOULDS



  • growth rate decreases;

  • the maximum number of cells produces drops;

  • the length of the lag phase increases;

  • the length of the stationary phase shortens;

  • the death rate increases.

The temperature of the environment (incubation determines the pH minimum for an organism

temperature in the laboratory), the nutrients that are available, the water activity and the presence of inhibitors.


HOW DOES pH AFFECT MICROBIAL CELLS?

The internal pH of cells is maintained near to pH 7.0(this may be lower in some organisms,

e.g., yeasts in which the cells pH has been measured at pH 5.8) and is the pH at which cells metabolism works best.

Cells membranes are impermeable to H+ and OH- ions and, in addition, cells may have a mechanism to pump out H+ ions.


When organisms are subjected to pHs outside their optimum but within the growth range, K+ and OH- ions affect the outer layers of the cells but not the internal pH. pHs above and below the optimum for growth may affect the following:

  • The enzymes (permeases) need for the uptake of nutrients, including essential ions.

  • The production of extra cellular enzymes and their subsequent activity when released.

  • The mechanism of ATP production in the bacteria, which involves the cell membrane.

When the microbial cell is subjected to extreme pHs cell membranes become damages. H+ and OH- ions

can then leak into the cell where enzymes are denatured and nucleic acid molecules are denatured, leading to cell death.

The effect of weak acids on microbial cells is temperature dependent. At concentrations that inhibit growth and cause cell death, they have less effect as the temperature is lowered.

The order of activity of acids in terms of their antimicrobial effect is

Propionic > acetic > lactic > citric > phosphoric > hydrochloric.

pH and the growth of micro organisms in foods.

Foods are quire variable in terms of their pHs. Most are acidic ranging from the very acidic to almost neural in reaction.

pH changes in foods due to the activity of micro-organisms. Milk sours as a result of latic acid production by streptococi and lactrobacilli.



pHs of foods


Food

pH

Food

pH

Lemon

2.2-2.4

Meat

5.4-6.9

Strawberry

3.1-3.9

Halibut

5.6

Tomato

3.9-4.6

Lettuce

6.0

Pear

3.7-4.7

Cod

6.2-6.6

Banana

4.5-4.7

Milk

6.3-6.6

Carrot

5.0-6.0

Egg white

8.6-9.6

Potato

5.3-5.6







Strong inorganic acid is not often included in processed foods but hydrochloric and phosphoric acids are used in the manufacture of carbonated and non-carbonated drinks. Coals, for example, contain phosphoric acid.
pH ranges for food poisoning bacteria.

Organism Minimum Optimum Maximum

Staph, aureus 4.0 6.0-7.0 9.8

Clostridium perfringens 5.5 7.0 8.0

Listeria monocytogenes 4.1 6.0 – 8.0 9.6

Samlonella spp 4.05 7.0 9.0

Vibro parahaemolyticus 4.8 7.0 11.0

Bacillus cereus 4.9 7.0 9.3

Campylobacter 4.9 7.0 9.0

Yersinia 4.6 7.0-8.0 9.0

Clostridium botulinum 4.2 7.0 9.0
2. Water activity

Water in the liquid state is essential for the existence of all living organisms. The cells of living organisms have very high water content, i.e., more than 75%. The amount of water is required to maintain the cell in an active state, and without liquid water living organisms, including micro-organisms, will not grow or reproduced.

The ways in which water can become unavailable for growth are:


  • The water contains dissolved solute such as sugar or salts.

  • The water is crystallized as ice.

  • The water is present as water of crystallization or hydration.

  • The water is absorbed on to surface (matrix effects).

The amount of water available for microbial growth in terms of the water activity is the amount of water

available in a food(or other materials) for microbial growth. More precisely:


Vapour pressure of a substance or solution

Water activity = ------------------------------------------------------------

Vapour pressure of water at the same temperature


The amount of water available to microorganisms in foods is normally indicated in terms of water

Activity the water content is referred to as Equilibrium relative humidity (ERH) atmosphere above a food at equilibrium with the food and is equal to the aw X 100%. Raoult’s law be used to calculate the water activities.



THE WATER ACTIVITY OF FOODS:-

The water content of a food may be bare little relationship to its water activity. Fresh meat, for example, has a water content of 75% but a water activity of 0.98. Muscle protein and fat are the bulk of the solids present. These are not soluble in water, have little surface effect and therefore do not contribute in any major way to the water activity. Water soluble materials (glucose, amino acids, mineral salts and vitamins) are present in such small quantities that the water activity of fresh meat is very high.

Foods may have low salt content but low water activity.

THE EFFECT OF WATER ACTIVITY ON MICRO-ORGANISMS:-

According to Raoult’s law:

n

aw = ------------



N + n
Where n is the number of moles of solute and N the number of moles of solvent (water)

Another, more useful way of writing the equation is :





  • Xerophiles (Organism loving dry conditions). This term applied specifically to a group of moulds (Xerophilic moulds) that can grow under very dry conditions, i.e., environments with water activities as low as 0.61. They will not grow at water activities higher than about 0.96 and their optimum water activity is in the region of 0.9 – 0.85. These organisms can cause spoilage of dries and salted fish, for example, the mould Xeromyces bisporus.

  • Halophiles (Salt-loving organisms).

  1. Moderate halophiles are organisms that require sodium chloride but will grow only at moderate concentrations, i.e. between 1 and 10% Sodium ions are believed to be involved with the transport mechanisms associated with the cell membrane and the uptake of materials from the environment. For example, Vibrio parahaemolyticus, 1-8% sodium chloride.




Effect of water activity on microorganisms:

  1. Extreme halpohiles are organisms that will only grow at high sodium chloride concentrations. Unlike most other bacteria, their cell walls are made of protein. Na + ions appear to form ionic bonds that maintain the stability of these proteins and therefore the structure of the wall. At high salt concentrations the cell wall is rigid and the cells take on a cylindrical shape. As the concentration of Na+ in the environment decreases the cell shape becomes more and more rounded until the cell wall disintegrates and the cells lyse. This happens when the sodium chloride concentration in the environment reaches about 12% Halobacterium Salinarum is associated with the spoilage of salted fish.

  • Halotolerant (haloduric) organisms: These organisms are able to grow at high sodium chloride concentration but do not have a specific requirement for sodium chloride like the halophiles.

Example:- Staphylococcus aureus can grow at sodium chloride concentrations as high as 20%(aw 0.83). Pediococcus halophilus can grow at 20% sodium chloride (aw 0.83)

Osmophilic yeasts:- ( yeasts loving high osmotic pressures) certain yeast that will grow where the water activity is low. Example:- Saccharomyces rouxii (Zygosaccharomyces rouxii) will grow at sugar concentration of 70% and above (aw 0.62).Saccharomyces rouxii can be responsible for the spoilage of foods with high sugar concentrations, eg., soft-centered chocolates.



  • Osmotolerant organisms:-This terms is applied to organisms (mainly yeasts) that grow best at high water activities but are also tolerant of high sugar concentration can grow at sugar concentrations of 60% and above.


The effect of water activity on the growth curve is

Produces a slower growth rate;

Increases the length of the lag phase;

Causes the production of fewer cells when the stationary phase starts;

Causes cells to die more rapidly during the death phase.

Principle groups of foods and their water activity




S. No

Aw valve

Food involved

S. No

Aw valve

Food involved

1

0.98 and above

Fresh meat and fish

Fish fruits and vegetables

Milk and most beverages

Canned vegetables in brine

Canned fruits in light syrup


4

0.60-0.85

Dried fruits

Flour


Cereals

Jams and jellies

Nuts

Some aged cheese



Intermediate moisture foods

2

0.93-0.98

Evaporated milk

Tomato paste

Processed cheese

Bread


Canned cured meats

Fermented sausage

Canned fruits in heavy syrup

Gouda cheese



5

Below 0.60

Chocolate

Honey


Biscuits

Crackers


Potato chips

Dried eggs

Milk and vegetables


3

0.85-0.93

Dry or fermented sausage

Dried beef

Raw ham

Aged cheddar cheese



Sweetened condensed milk





Factors affecting the water activity of foods:

  1. Kinds of solute:

  1. Nutritive value of food:

  • The better the medium for growth the lowest the limiting aw.

  1. Temperature:

  • Temperature increases  aW decreases

  • Temperature decreases  aW increases

  1. Oxygen supply:

  • Oxygen increases  aW increases

  • Oxygen decreases  aW decreases

  1. pH:

  • pH decreases and aW increases  survive

  • pH decreases and aW decreases  organism donot survive.

  1. Inhibitors:

  • Salt / sugar concentration inhibits aW.

  • Water tie up with ions so aW decreases.

  • Organism donot survive due to osmosis.


3.Oxidation – reduction potential:

  • Oxidation reduction potential or redox potential (OR or Eh) is a measure of whether microbial/material has a tendency to gain electrons 9become reduced) or lose electrons (become oxidized).

  • Microorganisms vary in their requirement for oxygen and their response to the presence of oxygen in the environment.

    1. Aerobic -Requires oxygen in order to generate cellular energy in the form of ATP.

    2. Anaerobic:(negative Eh values) - Generate cellular energy without oxygen.

    3. Obligate aerobe: (positive Eh value)

Requires oxygen for growth.

Energy production is by glycolysis, Kreb’s cycle.

Organic substrate oxidize to give CO2 and H2O (38 ATP).

Eg: Pseudomanas fluorescens, Penicillium sp., Pichia sp., Hansenula sp..

4. Microaerophiles :

Requires oxygen in minimum quantity eg. Campylobacter sp.1 – 10%, Optimum – 6%

Oxygen concentration above 10% is toxic & kills the organism.

5. Facultative anaerobes:

Grows in the absence of oxygen. Energy production is by glycolysis, Kreb’s cycle Eg. Saccharomyces cerevisiae produces 38 ATP. Eg. for food poisoning bacteria – S. aureus, E.coli.

6. Obligate anaerobes:

Donot require oxygen eg. Clostridium botulinum, Cl. perfringens.
Redox of foods & Microbial growth:

The actual redox of food will depend on a number of factors :



  1. the oxygen concentration in the environment of the food & its access to the food.

  2. Density of the food structure, which affects the ability of oxygen in the environment to penetrate.

  3. Concentration & types of reducing substances in the food that resist changes in redox towards the positive. Resistance to change in redox in a food is known as poising capacity.

  4. The way in which the food is processed.

  5. The pH of food. For every unit decrease in pH the Eh increases +58mV.

    • The surface of solid foods in contact with the air will have a positive redox whereas the interior may be negative.Eg. Carcass meat  exterior - +ve200 mV [aerobes, facultative anaerobes]

Interior - -ve150mV

  • Processing & mixing may alter the redox Eg. Milk during milking & processing [microaerophiles]

  • Heating drives off oxygen & may increase quantity of reducing substances in a food.

  • Eg. Canned foods  negative redox (obligate anaerobes, facultative anaerobes, oxygen independent organism).

  • Spoilage of canned foods  Eg. Rhizopus sp. Byssochlamys fulva .

4. Nutrient content:

These are based on :



  • Foods for energy – Carbohydrates, Fats, Proteins, Esters, Alcohols, Peptides, Aminoacids, organic acids.

  • Foods for growth _ Nitrogen containing foods.

  • Accessory food substances or vitamins.

5. Inhibitory substances & Biological structure:

  • Generally foods have some inhibitors:

  • Eg. Freshly drawn milk – Lactinins, Anticoliform factors.

  • Egg white – Lyzosyme.

  • Canberries – Benzoic acid

  • Propionibacterium – Propionic acid in Swiss cheese inhibits molds.

  • Streptococcus lactis – Nisin which inhibits lactate fermenting organism. Lactobacillus inactivates nisin..

  • Yeast – Resistant to SO2

  • Heating lipids leads to autooxidation & concentrated sugar syrups during browning results in production of furfural & hydroxy methyl furfural which are inhibitory to fermenting organisms.

  • Food has certain shell / outer covering which prevents the entry of organisms called as biological structures. Eg. Egg shell (vitelline) , Fish[scales], Fruits & vegetables[outer skin].

EXTRINSIC FACTORS:

The extrinsic parameters of those properties of the storage environment that affect both the foods and their microorganisms.



  1. Temperature:-
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