industrial lecture 10

8/4/2019 Industrial Lecture 10

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BIO 425

Industrial Microbiology

Lecture 10

Major secondary metabolites ofthe microorganisms


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Content Outline

Definition

Action mechanism of the bacteriocins

Different examples Colicin

Nisin

Pediocin

The others


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Definitionof Metabolites

Concept developed by John Bulock (1961),

Arnold L.Demain (1965-1975)

Primary metabolites Secondary metabolites


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Primary metabolites

Metabolic products of the cells:

Fo

rmed after the pathways by catabo

lic action

form the precursors of the secondary

metabolites as low molecular weight


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Primary metabolites

Primary metabolites are microbial products

made during the exponential phase of growth

whose synthesis: an integral part of the normal growth process.


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Primary metabolites

They include intermediates and end-products

of anabolic metabolism,

which are used by the cell as building blocksfor essential macromolecules

(e.g. amino acids, nucleotides) or are

converted to coenzymes (e.g. vitamins)..


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General definition for secondary

metabolites

Natural organic compounds that have a

restricted distribution

unique to a small no.oforganisms and that have noobvious function in cell

growth

an

d that are pro

duced by the cells that slo

wdownor stop dividing.


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Bulock and Demains concept

Tropophase: Log phase definition-primarymetabolite formation, simply.

Idiophase: The phase for secondary metabolite

formation Idiolite: a synonym for secondary metabolite

Idiotroph: a mutant that cannot synthesizesecondary metabolite, but grow in minimal medium,

unless the necessary precursor molecule is provided.


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Some properties of secondary

metabolites

More than 8000 secondary metabolites is known

Not all of them are produced commercially

General usage

Plant protection and plant growth hormones(to

initiate, speed up seed germination)

Animal nutrition(growth improvement, veterinary

medicine) Human medicine, The most important market


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Some important precursors of secondary metabolites-Low

molecular weight intermediates

aminoacids lead to: peptide AB, penicillins,

cephalosporins,

to

xin

s, precurso

rs fo

rA

lkalo

ids Sugars: aminoglycoside ABs

Acetyl Co-A: Tetracyclines

Terpenes: Lead derivationofGibberellins,alkaloids.


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Distinctive characteristics of secondary

metabolites

Primary metabolites are essential to cells, like

aminoacids, sugars, nucleotides,

carbohydrates.

Secondary metabolites are always non

essential for the cells


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metabolites

As non-essential,

theyve a restricted distribution

and theyre not universal, produced only by certain species of a given

genera,

or by even certain strains of species.


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metabolites-2

Chemically diverse, very complex

Contain so many different chemicals instructure,

contain amino sugars, nucleosides, epoxides,

indoles, pyrrolles, terpenoids, naphtalene,lactone,

macrolides, phenazines, cyclic peptides(somany different chemicals)


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metabolites-3

Exist as the members of closely-related chemical

families

E.g. 3 different neomycinAB, 4 diff. Tyrocidine, 10

bacitracin, >50 actinomycins(for cancer therapy)

Difference: the constituent aas are different, related

with cultural manipulation that scientists do in

culture media(add appropriate precursor for

adequate synthesis-directed fermentation)


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metabolites-4

All bioactive, have biological activity,

have tremendous economical importance,

imp. fo

r health an

dn

utrition


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metabolites-5

produced only in production phase,

restricted to this phase-mostly related with

nutritionally rich media

If poor media is used-secondary metabolite

synthesis overlaps with primary metabolite

synthesis.


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metabolites-6

A metabolite is secondary only

if it has no function in vegetative growth of

the cell an

d the metabo

lism


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metabolites-7

All secondary metabolites are externalized by

the cells;

thats given

to

extracellular fluid an

d themedia,

transported out immediately after their

function, ext. % is 90-99%.

Evolutionally cells donot like those

metabolites inside.


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metabolites-7

Ribosomally synthesized;

Most are synthesized by

enzymes(Enzymatically produced.);

Synthesase enzymes,

general enzymes of primary metabolites;

or specific enzymes are responsible in their

synthesis.


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General overproduction methodology of primary and secondary metabolites:

Recombinant DNA technology


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Different examples:

Toxins

Antibiotics

Bacteriocins


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A toxin producer:

Bacillus thurngiensis a Gram-positive,

soil-dwelling bacterium of the genus Bacillus.

Additionally, B. thuringiensis alsooccursnaturally,

in the gut of caterpillars of various types of

mo

ths an

d butterflies, as well as on the dark surface of plants.


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Another toxin producer:

Bacillus cereus

has been recognized as an agent of food

poisoning since 1955.

T

here areon

ly a fewo

utbreaks a yearreported,

Between 1972 and 1986:

52

o

utbreakso

f foo

dbo

rn

e disease asso

ciatedwith B. cereus were reported in US (in 2003,

there were only two).


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A toxin producer:

Bacillus thurngiensis a Gram-positive,

soil-dwelling bacterium of the genus Bacillus.

Additionally, B. thuringiensis alsooccursnaturally,

in the gut of caterpillars of various types of

mo

ths an

d butterflies, as well as on the dark surface of plants.


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Zearalenone

Gibberella zeae: producer strain;

Synonyms FES, Compound F-2, Toxin F2.

Description: a naturally occurring oestrogen, a mycotoxin from resorcylic acid lactone

group.

Mo

lecular weight: 318.4 g


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Characteristics:

Molecular formula: C18H22O5

Solubility

Solubility of Zearalenone in water: about0.002 g / 100ml.

slightly soluble in hexane and progressively

mo

re in

benz

en

e, aceton

itrile, dichloromethane, methanol, acetone.


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The molecular formula of

Zearalenone


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Zearalenone

Zearalenone (ZEA), also known as RAL and F-2

mycotoxin,

a po

ten

t oestro

gen

ic metabo

lite pro

duced bysome Fusarium species.


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Zearalenone

Several Fusarium species produce toxic

substances of:

considerable concern to livestock and

poultry producers:

namely, deoxynivalenol,

T-2 toxin, HT-2 toxin,

diacetoxyscirpenol (DAS)

and zearalenone.


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Zearalenone

the primary toxin causing infertility,

abortion,

or other breeding problems, especially in swine.


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Zearalenone

heat-stable;

and found worldwide in a number of cereal

cro

ps, such as maize,

barley, oats, wheat, rice,

sorghum; and also in bread.


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Chemical and physical properties

o

fz

earalenon

e a white crystalline solid.

exhibits blue-green fluorescence when excited

by long wavelength UV light (360 nm).

and a more intense green fluorescence when

excited with short wavelength UV light (260

nm).


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Chemical and physical

properties of zearalenone

In methanol, UV absorption maxima occur at 236 nm

274 nm

and 316 nm.


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of zearalenone

Maximum fluorescence in ethanol:

occurs with irradiation at 314 nm;

and with emission at 450 nm. Solubility in water: about 0.002 g /100 ml.


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of zearalenone

slightly soluble in hexane;

and progressively more so in:

benzene, acetonitrile,

methylene chloride,

methanol, ethanol and acetone. also soluble in aqueous alkali.


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Aflatoxins

It is arguably the law of unintended

consequences that:

led to the discovery of aflatoxins. Peanut meal began to be incorporated into

animal feed;

as a protein sources in the 1940's. Veterinarians in the Southern US:

began seeing cases of a noninfectious liver

disorder in pigs, cattle and dogs.


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Aflatoxins

including a particularly devastating outbreak

in a hunting dog kennel.

all ass

o

ciated with so

me fo

rmo

f feedcontaminated with fungi,

but only the dog outbreak:

directly associated with peanut meal.


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Aflatoxins

The first documented outbreak of:

what we now know of aflatoxicosis;

occurred in a guinea pig colony in England in1957


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Aflatoxins

The first appearance of aflatoxins:

often dated to a shipment of contaminated

gro

un

dn

ut (pean

ut) meal; delivered to Britain from Brazil in 1959.

The meal was used in poultry feed that:

killed turkeys, ducklings and game birds(pheasant and partridge).


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Aflatoxins

The syndrome was called:

"Turkey X disease"

and characterized by extensive liver damage including fatty change;

and subcutaneous haemorrhage.

Further imports of contaminated meal: killed calves and pigs in Britain.


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Aflatoxins:

Contaminated cottonseed meal:

causing liver cancer in farm-raised trout,

found to be due to the same agent.


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Aflatoxins:

Reexaminationof these earlier cases and;

a number of mass kills of livestock,

especially of farmed fish in the US; in earlier years suggested that the same agent

had played a role there too.


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Aflatoxins:

Using the toxicity to ducklings;

to monitor purification,

the toxin was obtained from Aspergillus flavusfound in groundnuts;

from Uganda and was dubbed an aflatoxin.


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Aflatoxins:

There are fewer aflatoxins (about 10);

than trichothecenes,

all based on the same core chemical structure; and further divided into three groups:


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Major types of aflatoxins:

Aflatoxin B:

fluorescing blue in ultraviolet light.

AflatoxinG: fluorescing green in ultraviolet light.

Aflatoxin M:

secreted in the milk of animals exposed toother aflatoxins.


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Other mycotoxins

Although trichothecenes and aflatoxins:

the best knownof the mycotoxins,

and those with the greatest weaponspotential,

they are not the only ones that are of concern,

especially in

agriculture.


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Other mycotoxins

Anumber ofother fungal toxins;

sicken and kill livestock every year and;

make meat or grains unfit for consumption. These other groups include:


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Other mycotoxins

Ergot alkaloids,

Fumonisins,

Ochratoxins, Sterigmatocystins,

Zearalenones


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Ergot alkaloids:

any of a large number of alkaloids;

obtained from the ergot fungus Claviceps

purpurea

or semisynthetically derived;

secondary metabolites.


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Ergot alkaloids:

examples include ergotamine,

ergonovine,

dihydroergotamine, lysergic acid diethylamide (LSD),

methysergide.


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Ergot alkaloids:

While death has been reported from

ergotamine toxicity in clinical practice;

the major problem:

ischaemia.

This canoccur in any vascular bed, but is most

commonly reported in the limbs.


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Ergot alkaloids:

Ischaemia canoccur idiosyncratically within

the normal therapeutic dose range;

as well as inoverdose.

The incidence of adverse effects increases

with increasing dose.


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Ergot alkaloids:

Ergotamine:

causes vasoconstruction by peripheral alpha

agonism in drugs.

In addition it has been shown that:

a synergistic effect with other

vasoconstructive neuropeptides (angiotensin,

catecholamines, nicotine)


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Ergot alkaloids:

Histology of the small vessels:

shows endothelial damage with associated

thrombosis.

this may be a primary effect of ergotamine;

rather than an effect secondary to prolonged

vasoconstruction.


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Antibiotics(ab)

Most important secondary metabolites.

Some groups of m/o produce:

as many as 5000-5500 antibiotics. Expected to be increase every year due to

extensive research.


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Some important producers

Pseudomonas spp.

Bacillus spp. Actinomyces spp.(best represented by genus

Streptomyces)

Filamentous fungi


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Actinomyces

Fungi-like

Produce hypha

Still form colonies, somehow cottony

Produce exospores in contrast to endospore

formation by Bacillus species.


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Producers (%)

Pseudomonas 2,5 % of the AB known

Bacillus 8,5 % of the AB known

Actinomyces 65 % of the AB known

Filamentous fungi 24 % of the AB known


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Current AB research

Search for new AB

Screening programs


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Hot topics for current research

1- New AB search is designed / made for

ab to have a more potent inhibition.

mean

s mo

re effective than

the presen

t;due to increasing resistance.


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2-New AB search is designed / made for

eliminating side effects.

Less to

xic, havin

gno

side effecto

r less sideeffect.


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increasing the spectrum of efficiency.

No

t to

be effectiveon

a limited gen

era.


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elimin

atin

g po

ssible &n

ew diseases.


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Formationofnew semi-synthetic ABs

Naturally produced ABs are chemicallymodified (side group substitution).

Mutasynthesis(Modified Abs are obtained).


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Formationofnew semi-synthetic ABs:

Select for mutants that cannot produce the

precursor of that AB.

Provide precursor into the medium and select

a specific enzyme for efficient production for

mutasynthesis.


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Formationofnew semi-synthetic ABs:

Creationofnew and hybrid ABs:

Instead of time consuming & expensive

production.

Genetic recombination (Hybrid gene makingby genetic engineering).

Protoplast fusion(Introduce new precursors

and new enzymes) Site directed mutagenesis.


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Overproductionof commercially

produced ABs

A) Optimizationof culture:

by breaking down the controls (elaborate

controls to minimize the cell growth);

Understood by studying physiological

condition-regulation;

Optimizationof both medium and cultivation

conditions.


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Overproductionof commercially

produced ABs

Genetic engineering techniques:

(Increase copy no. of genes, using promoter

strength);

Applicationof mutational programmes.


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Industrial productionofAB

Two methods:

Batch cultures,

Submerged cultures, Continuous cultures: not used as to keep the

cells in high specific growth rate(Not

effective).


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Two different media:

Propagation media

Production media


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Propagation medium

Aim

Propagate cells at high density;

an

d tran

sfer them to

a defin

ite medium to

usein production.


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Submerged culture

According to the media ingredients,

Substrate concentration is low; Liquid content is high:

Opposite is: Solid state cultivation


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Batch culture

The most common culture system is the batch

culture, due to its simplicity and low cost.

a closed system in which there is no input or

output of materials.


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Batch culture

The cell properties:

such as size,

in

tern

aln

utrien

t co

mpo

sition

, and metabolic function vary considerably

during the growth phases.

Di d f i


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Disadvantages of continuous

cultivation:

Expensive to do it in continuous cultures,

Industrial strains used as overproducers;

show reduced growth rate compared to its

parental strain.


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Biosynthesis ofAB

In most cases,

prone toCarboncatabolite repression:

Lacto

se, Sucro

se, Dextrin

s:C

-so

urces fo

rA

Bproduction.

Other cheap sources: Nitrogen, Soybean meal,

Corn steep liquor.


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General salts used inAB fermentations

MgSO4

CaCO3

N

aC

l KH2PO4

Trace metals are so important in

fermentations like Mn, Fe, Cu, Zn.


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Biosynthesis ofAB

Important factors other than:

pH control

Steriliz

ation

:N

eatly un

dertaken

; Aeration.


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Example for biosynthesis

A current process for penicillin production:

at the beginning the producer is: Spores

A

dd them to

flasks, fo

r 4 days, 25 C

incubation period

Here: Penicillum chrysogenum.


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After 4 days transfer to 8 L flasks,

25 C, 250 rpm.

Tran

sfer to

pro

pagation

medium,500 L to

get ahigh cell density.

After than transfer to 200,000 L for 6 days

Here control the pH & aeration.


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AFTER BIG FERMENTATIONTANK

TRANSFER

FILTRATION for separating Fungi from Fermentation Liquor

Fermentation solids

Extraction & Drying

(for animal feed)Solvent Extraction Evaporation

Drying Market(0.12 g

per C-source used)


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Yield ofAB

When penicillin is firstly produced its yield is

1-10 g/mL E.g. yield is enhanced to 85000 g/mL by

perfecting the organism by mutagenesis &

genetic recombination

Other examples for commercially


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Other examples for commercially

produced ABs

Bacitracin (Topical AB)

Chloramphenicol

According to chemical structure


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classificationofAB

CH containing AB

Macrocyclic lactones

Qui

no

lon

es & relatedA

B Aminoacid and peptide AB

Heterocyclic AB containing N

Heterocyclic AB containing O



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classificationofAB

A

licyclic derivatives Aromatic AB


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Definition

Bacteriocins :

proteinaceous substances produced by

bacteria;

to inhibit the growth of similar or closely

related bacterial strain(s).

typically considered to be narrow spectrum

antibiotics,

though this has been debated.


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Structure of the bacteriocins

They are phenomenologically analogous to

yeast and paramecium killing factors,

and are structurally, functionally, and

ecologically diverse.

first discovered by A. Gratia in 1925.


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Discovery of bacteriocins

Gratia:

involved in the process of searching for ways

to kill bacteria,

also resulted in the development of antibiotics

and the discovery of bacteriophage,

all within a spanof a few years.


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Discovery of bacteriocins

He called his first discovery:

colicine

because it killed E. coli.

Classification of bacteriocins:


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Classification of bacteriocins:

categorized in several ways,

including producing strain,

common resistance mechanisms,

and mechanism of killing.

There are several large categories of

bacteriocin;

which are only phenomenologically related.


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Large categories of bacteriocins

These include the bacteriocins:

from gram-positive bacteria,

the colicins ,

the microcins,

and the bacteriocins from Archaea.


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Colicin

The bacteriocins from E. coli:

called colicins (formerly called 'colicines,'

meaning 'coli killers').

They are the longest studied bacteriocins.


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Colicin

They are a diverse group of bacteriocins;

and donot include all the bacteriocins

produced by E. coli.

For example the bacteriocins produced by

Staphylococcus warneri:

called as warnerin

or warnericin.


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Examples to colicins

In fact, one of the oldest known so-called

colicins :

was called colicin V

now known as microcin V.


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Lantibiotics

The bacteriocins of lactic acid-fermenting

bacteria:

called lantibiotics.

This naming system: problematic for a number

of reasons.


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Lantibiotics

First, naming bacteriocins by what they

putatively kill,

would be more accurate;

if their killing spectrum were contiguous with

genus or species designations.


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Lantibiotics

The bacteriocins frequently possess spectra;

that exceed the bounds of their named taxa,

and almost never kill the majority of the taxa,

for which they are named.


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Lantibiotics

Further, the original naming is generally

derived not from the sensitive strain,

the bacteriocin kills,

but instead the organism that produces the

bacteriocin.

This makes the use of this naming system a

problematic basis for theory;

thus the alternative classification systems.

Alternative methods of classification


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Alternative methods of classification include:

method of killing (pore forming, DNAse,

nuclease, murein production inhibition, etc),

genetics (production by large plasmids, small

plasmids, chromosomal),



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molecular weight and chemistry (large

protein, polypeptide, with/without sugar

moiety);

containing atypical amino acids like

lanthionine;

and method of production (ribosomal, post

ribosomal modifications, non-ribosomal).


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Classification

One method of classification fits the

bacteriocins into

Class I

Class II a/b/c,

and Class III.

l


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Class I

Class I bacteriocins:

small peptide inhibitors

e.g. include nisin.

h l b


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The class II bacteriocins

are small heat-stable proteins.

The actionofClass IIa bacteriocins seems to

involve:

disruptionof mannose transport into target

cells.

Class II b bacteriocins form pores in the

membranes of target cells;

and disrupt the proton gradient of target cells.

Cl


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Class II

Other bacteriocins can be grouped together:

as Class IIc.

These have a wide range of effects on

membrane permeability,

cell wall formation,

and pheromone actions of target cells.

Class III bacteriocins


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Class III bacteriocins

Large,

Heat-labile.

Inhibition of microorganisms by


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Inhibitionof microorganisms by

bacteriocins

Bacteriocins are only one category of

substances

produced by bacteria that are inhibitory to

other bacteria.

These potent inhibitors offer potential

alternatives to antibiotics,

and may also replace chemical preservativesin food.

M b b i i


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More about bacteriocins

Bacteriocins differ greatly with respect to

sensitivity to pH.

Many of them are considerably more tolerantof acid than,

the alkaline pH values.

M b t b t i i


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More about bacteriocins

Mostly active in growth process of bacteria,

at pH values between 3-7.

Maximum inhibitory activity was

demonstrated at;

pH=4;

and pH=5.

A typical example:Nisin


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yp p

An antibacterial polypeptide;

produced by some strains ofLactococcus lactis

subsp. lactis.

Not only Gram-negative organisms;

and molds insensitive tonisin,

but its effectiveness against even sensitive

Gram-positive organisms,

depends on the bacterial load.

Nisin


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Nisin

As the number oforganisms increases ,

so the inhibitory effectiveness ofnisin

decreases.

Ni i


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Nisin

At neutral and alkaline pH values,

nisin is practically insoluble,

and irreversible inactivationoccurs even at

room temperature

Large molecules from milk or broth:

protect nisin from heat inactivation.

C li i


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Colicin

E.colibacteriocins are called colicins.

They are the longest studied bacteriocins.

They are a diverse group of bacteriocins;

and donot include all the bacteriocins

produced by E.coli.

B t i i d h ki


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Bacteriocins and cheese making

Some are only active as partners with a

second peptide (e.g. Class IIb).

Lactic acid;

(-hydroxypropionic acid) is a chemical

compound :

that plays a role in several biochemical

processes.

e.g. Cheese making.

Ch


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Cheese

Cheese is a solid food made from the curdled

milk of various animals;

e.g. most commonly cows;

but sometimes goats, sheep, reindeer, and

water buffalo.

LITERATURE CITED


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LITERATURECITED

Forsythe, S.J. The Microbiology of Safe Food,

Blackwell Science, Cambridge, 2000.

Wood, B. J. B., Microbiology of Fermented

Foods, Vol.1 and 2, Blackie Academic andProfessional, London, Second edition, 1998.

industrial lecture 10

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