Preservatives, MOAs and Bacterial Resistance

2. Preservatives, MOAs and Bacterial Resistance
Raheem Bahadar
PhD Scholar
Department of Pharmacy, COMSATS Institute of Information Technology,
Abbottabad.

3. Introduction
 Preservatives
 A preservative is a substance or a chemical that is added to products such as food,
beverages, pharmaceutical drugs, paints, biological samples, cosmetics, wood,
and many other products to prevent decomposition by microbial growth or by
undesirable chemical changes.
 In general, preservation is implemented in two modes, chemical and physical.
o Chemical preservation entails adding chemical compounds to the product.
o Physical preservation entails processes such as refrigeration or drying.

4.  Food preservatives have been around since centuries ago, as in pickled onions,
salted meat and fish, sweetened fruit and spiced foods.
 As people move away from the countryside and demand for food increased many
people rely on processed items as part of their daily sustenance.
 Food preservatives help maintain the freshness and shelf life of such food
products because without them, they would spoil quickly due to exposure to air,
moisture, bacteria or mould. Either natural or synthetic substances may be added
to avoid or delay these problems.

5. History Of Preservatives
 Preservatives have been used since prehistoric times. Smoked meat for example
has phenols and other chemicals that retard spoilage.
 The preservation of foods has evolved greatly over the centuries, and has been
instrumental in increasing food security.
 The use of preservatives other than traditional oils, salts, etc. in food began in the
late 19th century, but was not widespread until the 20th century.

6. Importance Of Preservatives
 Food preservatives are essential for many reasons.
 They are used to:
 Maintain consistency and texture of foods
 Improve or retain nutritional properties
 Delay spoilage
 Enhance flavors, textures, and color

7. Major Applications
 Bakery products
 Dairy and frozen foods
 Snack foods
 Meat, poultry and seafood
 Confectionery and condiments
 Beverages

8. Types of Preservatives
Natural Food Preservatives
Salt
Sugar
Alcohol
Vinegar
Honey
Onion
Sorbic Acid

9. Chemical preservative agents
 Weak Organic Acids
 Mode of Action
 The most common classical preservative agents are the weak organic acids.
 E.g:
 Acetic acid
 Lactic acid
 Benzoic acid
 Sorbic acid.

10.  These molecules inhibit the outgrowth of both bacterial and fungal cells.
 Sorbic acid is also reported to inhibit the germination and outgrowth of bacterial
spores.
 In solution, weak acid preservatives exist in a pH-dependent equilibrium between
the undissociated and dissociated state.
 Preservatives have optimal inhibitory activity at low pH because this favours the
uncharged and undissociated state of the molecule, which
is freely permeable across the plasma membrane and is thus able to enter the cell.

11.  Therefore, the inhibitory action is classically believed to be due to the compound
crossing the plasma membrane in the undissociated state.
 Subsequently, upon encountering the higher pH inside the cell, the molecule will
dissociate resulting in the release of charged anions and protons which cannot
cross the plasma membrane.
 So, the preservative molecule diffuses into the cell until equilibrium is reached in
accordance with the pH gradient across the membrane resulting in the
accumulation of anions and protons inside the cell.

12.  Therefore, inhibition of growth by weak acid preservatives has been proposed to
be due to a number of actions including,
 Membrane disruption,
 Inhibition of essential metabolic reactions,
 Stress on intracellular pH homeostasis and
 The accumulation of toxic anions.

13.  Resistance Mechanisms
 Microbial resistance to weak organic acids can involve various mechanisms.
 For bacteria, significant knowledge exists on their intrinsic, non-inducible
resistance mechanisms against these compounds.
 Gram-positive bacteria do not possess an outer membrane like Bacillus
megaterium, hence preservatives can easily enter these cells and their intrinsic
resistance is relatively low.

14.  While, in gram negative bacteria, resistance mechanisms are more complicated
since these organisms possess an inner and an outer membrane.
 The outer membrane has a clear role in modulating the accessibility of a cell to
preservatives and other small molecules; the lipopolysaccharides is of crucial
importance in this respect.
 In some cases microorganisms are able to degrade the added preservatives by
making use of specific enzymes.
E.g:
 Degradation of methyl para(4)-hydroxybenzoate by Pseudomonas aeruginosa.

15. Hydrogen peroxide (H2O2)
 Mode of Action
 Hydrogen peroxide can be added to the foods that are to be preserved, under
suitable experimental conditions, the reaction generates a short lived singlet
oxygen species which is extremely biocidal.
 The system requires hydrogen peroxide and thiocyanate for optimal activity and
is thus primarily active against microorganisms.
 Furthermore, during incomplete reduction of molecular oxygen, the superoxide
radical is generated.
 Hydrogen peroxide on its own is also known to be bactericidal.

16.  Resistance Mechanisms
 Bacteria and fungi protect themselves against hydrogen peroxide in various ways.
 Some of the most universal systems include glutathione with associated enzymes
and catalase.
 Indeed when yeast cells are treated with low concentrations of hydrogen peroxide
a number of adaptive systems are activated which will protect against a
subsequent stress with higher H2O2 concentrations.

17. Naturally occurring preservatives
Small organic biomolecules
 Mode of Action
 Some of these compounds are naturally present in spices.
E.g:
 Oregano,
 Thyme,
 Dill,
 Clove and
 Their essential oils.

18.  These compounds are generally hydrophobic and can have membrane perturbing
or even membrane rupturing characteristics.
 Hence, in practice most of the above mentioned compounds are used at
concentrations that may have only growth inhibitory effects on microorganisms.
 The essential oils from plants such as basil, cumin, caraway and coriander have
inhibitory effects on organisms such as, Aeromonas hydrophila, Pseudomonas
fluorescence and Staphylococcus aureus.

19.  The antimicrobial properties of isothiocyanates derived from onion and garlic has
been hypothesised that their antimicrobial activity is related to inactivation of
extracellular enzymes through oxidative cleavage of disulphide bonds.

20.  Resistance mechanisms
 The resistance mechanisms of microorganisms against these naturally occurring
antimicrobial compounds are not much known .
 It has described that efflux pumps in Pseudomonas putida that are involved in
toluene tolerance.
 In gram-negative bacteria, the known stock of multidrug resistance proteins
includes pumps for neutral compounds (EmrAB) as well as pumps for
amphipathic cations and anions (AcrAB).

21. Membrane perturbing proteins and peptides
 Mode of action
 The inhibitory action of most naturally occurring antimicrobial proteins and
peptides is thought to be due to membrane disruption.
 The exact mode of action of antimicrobial peptides from plants is not known.
 The specificity of many of these peptides for microorganisms as in the case of
prokaryotes, proposed to be due to the high content of anionic phospholipids such
as phosphatidyl-glycerol, phosphatidyl-serine and posiphosphatidic acid present
in bacterial membranes.

22.  Resistance mechanisms
 The secretion of proteolytic enzymes by the target microorganism.
 Recent data have shown that the extracytoplasmic protease (OmpT) is key to the
resistance development of E. coli against the antimicrobial peptide protamine.
 The cells may alter their membrane composition as a response to the stress
imposed upon exposure to a membrane active antimicrobial peptide.

23. Cell wall perturbation
 Mode of action
 Microbial cell walls are both characteristic and crucial cell survival. Most of
these structures do not occur in man and hence form ideal targets for microbial
inactivation.
 Perturbing the cell wall structure of bacteria is the principal inhibitory action of
many commercial antibiotics commonly produced by the pharmaceutical industry
like penicillin against staphylococci.
 Enzymes that degrade bacterial walls from the outside such as lysozyme have
been applied in the preservation of foods.

24.  Resistance mechanisms
 Bacteria possess two basic mechanisms of membrane mediated signal
transduction
 Firstly, the bacterial cells deal with outside ‘‘attack’’ on their cell wall by either
enzymes or environmental factors such as temperature, pH and aw.
 Bacterial cells activate a sequence of enzymatic reactions that transmit the
detection of cell wall problems to the nucleus and enhanced incorporation of cell
wall protein (Cwp1p) and other process.

25.  In an alternative process the signalling molecule enters the cell via passive
membrane diffusion or active uptake via a transporter.
 Changes at the level of the cell wall or membrane induced by either lytic
enzymes, membrane actives or changes in the physico–chemical parameters.

26. Combination preservation
 In preservation processes, which form about a third of the unit operations used in
food processing, the general aim is to employ combination processes
 Where, like a mild heat stress and a low concentration of preservatives are
combined in order to fulfill all of the previous listed objectives.
 Additionally, there is currently significant interest in using alternative physical
treatments such as
 Ultra high pressure (UHP) or
 Pulsed electric fields (PEF).

27.  Combinations of preservation treatments allow the required level of protection to
be achieved while at the same time retaining the organoleptic qualities of the
product such as, colour, flavour, texture and nutritional value