Name: Yu Tiam Meng
Matric card no.: 111437
LAB 5: Determination of Antimicrobial Effects of Microbial Extracts
Introdution :
Certain groups of bacteria can produce antimicrobial substances with the capacity to inhibit the growth of pathogenic and spoilage microorganisms. Organic acids, hydrogen peroxide, diacetyl and bacteriocins are included among these antimicrobial compounds. Interest in naturally produce antimicrobial agents, such as bacteriocins, is on the rise, since nowadys consumers demand "natural" and "minimally processed" food.
Bacteriocins comprise a large and diverse group of ribosomally synthesized antimicrobial proteins or peptides. Although bacteriocins can be found in numerous Gram-positive and Gram-negative bacteria, those produced by lactic acid bacteria (LAB) have received special attention in recent years due to their potential application in the food industry as natural biopreservatives. Different classes of LAB bacteriocins have been identified on the basis of biochemical and genetic characterization. These bacteriocins have been reported to inhibit the growth of Listeria monocyotogenes, Staphylococcus aureus, Enterococcus faecalis and Clostridium tyrobutyricum.
Objective:
To determine the antimicrobial effects of extracellular extracts of selected LAB strainsResults:
Part 1:
Determination of bacteriocin activity via agar diffusion test
Strains of LAB
|
Strains of spoilage / pathogenic bacteria
|
Inhibition zone (cm)
|
L. casei
|
S. aureus
|
(0.60+0.60)/2 = 0.60
|
K. pneumonia
|
(0.60+0.60)/2 = 0.60
|
|
P. aeruginosa
|
(0.70+0.60)/2 = 0.65
|
|
L. brevis
|
S. aureus
|
No inhibition zone.
|
K. pneumonia
|
(0.70+0.80)/2 = 0.75
|
|
P. aeruginosa
|
(0.80+0.70)/2 = 0.75
|
|
L. plantarum
|
S. aureus
|
(0.60+0.60)/2 = 0.60
|
K. pneumonia
|
(0.65+0.60)/2 = 0.625
|
|
P. aeruginosa
|
(0.70+0.70)/2 = 0.70
|
Part 2 : Determination of bacteriocin activity via optical density
Serial dilution of extracellular extract
Strain of
LAB: L. plantarum
Dilutions
|
OD600 of spoilage/pathogenic
bacteria
|
||
Strain
1 :
K. pneumonia
|
Strain
2 :
S. aureus
|
Strain
3 :
P. aeruginosa
|
|
2x
|
0.994
|
0.588
|
0.609
|
10x
|
1.174
|
0.827
|
0.891
|
50x
|
0.669
|
0.563
|
0.630
|
100x
|
0.613
|
0.366
|
0.438
|
Equation
|
y = -0.0052x + 1.0719
|
y = -0.0034x + 0.725
|
y = -0.0031x + 0.7665
|
OD600 of control
|
1.156
|
0.270
|
0.150
|
50% of OD600
|
0.578
|
0.135
|
0.075
|
AU/ml
|
94.98
|
173.53
|
223.06
|
1) K.pneumonia
2) S.aureus
3) P.aeruginosa
Discussion :
1) . The lactic acid bacteria (LAB) comprise a clade of Gram-positive, low-GC, acid-tolerant, generally non-sporulating, non-respiring rod or cocci that are associated by their common metabolic and physiological characteristics. These bacteria, usually found in decomposing plants and lactic products, produce lactic acid as the major metabolic end-product of carbohydrate fermentation. This trait has, throughout history, linked LAB with food fermentations, as acidification inhibits the growth of spoilage agents. Proteinaceous bacteriocins are produced by several LAB strains and provide an additional hurdle for spoilage and pathogenic
microorganisms. Furthermore, lactic acid and other metabolic products
contribute to the organoleptic and textural profile of a food item. The
industrial importance of the LAB is further evinced by their generally recognized as safe (GRAS) status, due to their ubiquitous appearance in food and their contribution to the healthy microflora of human mucosal surfaces. The genera that comprise the LAB are at its core Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, and Streptococcus as well as the more peripheral Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Sporolactobacillus, Tetragenococcus, Vagococcus, and Weisella; these belong to the order Lactobacillales.
2）. Lactic acid bacteria (LAB) display numerous antimicrobial activities. This is mainly due to the production of organic acids, but also of other compounds, such as bacteriocins and antifungal peptides. Several bacteriocins with industrial potential have been purified and characterized. The kinetics of bacteriocin production by LAB in relation to process factors have been studied in detail through mathematical modeling and positive predictive microbiology. Application of bacteriocin-producing starter cultures in sourdough (to increase competitiveness), in fermented sausage (anti-listerial effect), and in cheese (anti-listerial and anti-clostridial effects), have been studied during in vitro laboratory fermentations as well as on pilot-scale level. The highly promising results of these studies underline the important role that functional, bacteriocinogenic LAB strains may play in the food industry as starter cultures, co-cultures, or bioprotective cultures, to improve food quality and safety. In addition, antimicrobial production by probiotic LAB might play a role during in vivo interactions occurring in the human gastrointestinal tract, hence contributing to gut health.
2）. Lactic acid bacteria (LAB) display numerous antimicrobial activities. This is mainly due to the production of organic acids, but also of other compounds, such as bacteriocins and antifungal peptides. Several bacteriocins with industrial potential have been purified and characterized. The kinetics of bacteriocin production by LAB in relation to process factors have been studied in detail through mathematical modeling and positive predictive microbiology. Application of bacteriocin-producing starter cultures in sourdough (to increase competitiveness), in fermented sausage (anti-listerial effect), and in cheese (anti-listerial and anti-clostridial effects), have been studied during in vitro laboratory fermentations as well as on pilot-scale level. The highly promising results of these studies underline the important role that functional, bacteriocinogenic LAB strains may play in the food industry as starter cultures, co-cultures, or bioprotective cultures, to improve food quality and safety. In addition, antimicrobial production by probiotic LAB might play a role during in vivo interactions occurring in the human gastrointestinal tract, hence contributing to gut health.
3) . The agar diffusion test, or the Kirby-Bauer disk-diffusion method, is a means of measuring the effect of an antimicrobial agent against bacteria grown in culture.
The bacteria in question is swabbed uniformly across a culture plate. A filter-paper disk, impregnated with the compound to be tested, is then placed on the surface of the agar. The compound diffuses from the filter paper into the agar. The concentration of the compound will be highest next to the disk, and will decrease as distance from the disk increases. If the compound is effective against bacteria at a certain concentration, no colonies will grow where the concentration in the agar is greater than or equal to the effective concentration. This is the zone of inhibition. Thus, the size of the zone of inhibition is a measure of the compound's effectiveness: the larger the clear area around the filter disk, the more effective the compound.
4) . Optical density, measured in a spectrophotometer, can be used as a measure of the concentration of bacteria in a suspension. As visible light passes through a cell suspension the light is scattered. Greater scatter indicates that more bacteria or other material is present. The amount of light scatter can be measured in a spectrophotometer.
5) . For optical density method, negative-control(5ml of double strength MRS with 2ml of distilled water) is prepared for "auto-zero" via spectrophotometer. Positive-control(MRS and pathogenic bacteria grow without LAB's extracellular extract) is prepared. Any reading that lower than this positive-control reading shows antimicrobial effects.
6) . We used the distilled water to dilute the pathogenic bacteria containing broth and this greatly reduced the color density of the broth. Thus, make the reading(from spectrophotometer) not accurate. We can use other substances like peptone to carry out the dilution.
Conclusion :
The antimicrobial substances produced by lactic acid bacteria(LAB) is organic and thus not strong enough if compared with artificial antimicrobial substances. So, the antimicrobial effects produced are very small but still very useful.Refenrences :
NAME:T.SYED ISKANDAR B.T.SYED AZHAR
METRIK NO:111434
Certain groups of bacteria can produce antimicrobial substances with the capacity to inhibit the growth of pathogenic and spoilage microorganisms. Organic acids, hydrogen peroxide, diacetyl and bacteriocins are included among these antimicrobial compounds. Interest in naturally produce antimicrobial agents, such as bacteriocins, is on the rise, since nowadys consumers demand "natural" and "minimally processed" food.
Bacteriocins comprise a large and diverse group of ribosomally synthesized antimicrobial proteins or peptides. Although bacteriocins can be found in numerous Gram-positive and Gram-negative bacteria, those produced by lactic acid bacteria (LAB) have received special attention in recent years due to their potential application in the food industry as natural biopreservatives. Different classes of LAB bacteriocins have been identified on the basis of biochemical and genetic characterization. These bacteriocins have been reported to inhibit the growth of Listeria monocyotogenes,Staphylococcus aureus, Enterococcus faecalis and Clostridium tyrobutyricum.
METRIK NO:111434
LAB 5: Determination of Antimicrobial Effects of Microbial Extracts
Introdution
:
Certain groups of bacteria can produce antimicrobial substances with the capacity to inhibit the growth of pathogenic and spoilage microorganisms. Organic acids, hydrogen peroxide, diacetyl and bacteriocins are included among these antimicrobial compounds. Interest in naturally produce antimicrobial agents, such as bacteriocins, is on the rise, since nowadys consumers demand "natural" and "minimally processed" food.
Bacteriocins comprise a large and diverse group of ribosomally synthesized antimicrobial proteins or peptides. Although bacteriocins can be found in numerous Gram-positive and Gram-negative bacteria, those produced by lactic acid bacteria (LAB) have received special attention in recent years due to their potential application in the food industry as natural biopreservatives. Different classes of LAB bacteriocins have been identified on the basis of biochemical and genetic characterization. These bacteriocins have been reported to inhibit the growth of Listeria monocyotogenes,Staphylococcus aureus, Enterococcus faecalis and Clostridium tyrobutyricum.
Objective:
To determine
the antimicrobial effects of extracellular extracts of selected LAB strains
Results:
Part 1:
Determination of bacteriocin activity via agar diffusion test
Strains of
LAB
|
Strains of
spoilage / pathogenic bacteria
|
Inhibition
zone (cm)
|
L.
casei
|
S. aureus
|
(0.60+0.60)/2
= 0.60
|
K. pneumonia
|
(0.60+0.60)/2
= 0.60
|
|
P. aeruginosa
|
(0.70+0.60)/2
= 0.65
|
|
L. brevis
|
S. aureus
|
No
inhibition zone.
|
K. pneumonia
|
(0.70+0.80)/2
= 0.75
|
|
P. aeruginosa
|
(0.80+0.70)/2
= 0.75
|
|
L. plantarum
|
S. aureus
|
(0.60+0.60)/2
= 0.60
|
K. pneumonia
|
(0.65+0.60)/2
= 0.625
|
|
P. aeruginosa
|
(0.70+0.70)/2
= 0.70
|
Part 2 :
Determination of bacteriocin activity via optical density
Serial
dilution of extracellular extract
Strain of
LAB: L. plantarum
Dilutions
|
OD600 of spoilage/pathogenic bacteria
|
||
Strain 1 :
K. pneumonia
|
Strain 2 :
S. aureus
|
Strain 3 :
P. aeruginosa
|
|
2x
|
0.994
|
0.588
|
0.609
|
10x
|
1.174
|
0.827
|
0.891
|
50x
|
0.669
|
0.563
|
0.630
|
100x
|
0.613
|
0.366
|
0.438
|
Equation
|
y = -0.0052x + 1.0719
|
y = -0.0034x + 0.725
|
y = -0.0031x + 0.7665
|
OD600 of control
|
1.156
|
0.270
|
0.150
|
50% of OD600
|
0.578
|
0.135
|
0.075
|
AU/ml
|
94.98
|
173.53
|
223.06
|
1) K.pneumonia
2) S.aureus
.png)
3) P.aeruginosa
.png)
.png)
.png)
DISCUSSION:
Lactic acid bacteria are a group
of related bacteria that produce lactic acid as a result of carbohydrate
fermentation. These microbes are broadly used by us in the production of
fermented food products, such as yogurt (Streptococcus spp. and Lactobacillus spp.), cheeses (Lactococcus spp.), sauerkraut (Leuconostoc spp.) and sausage.
These organisms are heterotrophic and generally have complex
nutritional requirements because they lack many biosynthetic capabilities. Most
species have multiple requirements for amino acids and vitamins. Because of
this, lactic acid bacteria are generally abundant only in communities where
these requirements can be provided. They are often associated with animal oral
cavities and intestines (eg. Enterococcus
faecalis), plant leaves (Lactobacillus, Leuconostoc) as well as
decaying plant or animal matter such as rotting vegetables, fecal matter,
compost, etc.
Lactic acid bacteria are used in the food industry for several
reasons. Their growth lowers both the carbohydrate content of the foods that
they ferment, and the pH due to lactic acid production. It is this
acidification process which is one of the most desirable side-effects of their
growth. The pH may drop to as low as 4.0, low enough to inhibit the growth of
most other microorganisms including the most common human pathogens, thus
allowing these foods prolonged shelf life. The acidity also changes the texture
of the foods due to precipitation of some proteins, and the biochemical
conversions involved in growth enhance the flavor. The fermentation (and growth
of the bacteria) is self-limiting due to the sensitivity of lactic acid
bacteria to such acidic pH.
An antimicrobial is a substance that kills or inhibits
the growth of microorganisms such as bacteria, fungi, or protozoans. Antimicrobial drugs either
kill microbes (microbiocidal) or prevent the growth of microbes
(microbiostatic). Disinfectants are antimicrobial substances used on
non-living objects or outside the body.
The
history of antimicrobials begins with the observations of Pasteur and Joubert,
who discovered that one type of bacteria could prevent the growth of another.
They did not know at that time that the reason one bacterium failed to grow was
that the other bacterium was producing an antibiotic. Technically, antibiotics
are only those substances that are produced by one microorganism that kill, or
prevent the growth, of another microorganism. Of course, in today's common
usage, the term antibiotic is used to refer to almost any drug that attempts to
rid your body of a bacterial
infection. Antimicrobials include not just antibiotics, but synthetically
formed compounds as well.
The
discovery of antimicrobials like penicillin and tetracycline paved the way for better health for
millions around the world. Before penicillin became a viable medical treatment
in the early 1940s, no true cure for gonorrhea, strep throat, or pneumonia existed. Patients with infected wounds
often had to have a wounded limb removed, or face death from infection. Now,
most of these infections can be cured easily with a short course of
antimicrobials.
However,
with the development of antimicrobials, microorganisms have adapted and become
resistant to previous antimicrobial agents. The old antimicrobial technology
was based either on poisons or heavy metals, which may not have killed the
microbe completely, allowing the microbe to survive, change, and become
resistant to the poisons and/or heavy metals.
Antimicrobial
nanotechnology is a recent addition to the fight against disease causing
organisms, replacing heavy metals and toxins and may some day be a viable
alternative.
Infections
that are acquired during a hospital visit are called "hospital acquired
infections" or nosocomial
infections. Similarly, when the infectious disease is picked up in the
non-hospital setting it is considered "community acquired".
CONCLUSION:
As for the results obtained the antimicrobial effect is not so effective because the production of antibodies is few.Thus the uses of the antimicrobial effect is still can be used if in large number.
NAME : AHMAD AZIZUL BIN MD SADIK
MATRIC NO : 114116
LAB 5 : DETERMINATION OF ANTIMICROBIAL EFFECTS OF
MICROBIAL EXTRACTS
Introduction
Certain
groups of bacteria can produce antimicrobial substances with the capacity to
inhibit the growth of pathogenic and spoilage microorganisms. Organic acids,
hydrogen peroxide, diacetyl and bacteriocins are included among these
antimicrobial compounds. Interest in naturally produce antimicrobial agents,
such as bacteriocins, is on the rise, since nowadys consumers demand
"natural" and "minimally processed" food.
Bacteriocins comprise a large and diverse
group of ribosomally synthesized antimicrobial proteins or peptides. Although
bacteriocins can be found in numerous Gram-positive and Gram-negative bacteria,
those produced by lactic acid bacteria (LAB) have received special attention in
recent years due to their potential application in the food industry as natural
biopreservatives. Different classes of LAB bacteriocins have been identified on
the basis of biochemical and genetic characterization. These bacteriocins have
been reported to inhibit the growth of Listeria
monocyotogenes,Staphylococcus aureus, Enterococcus faecalis and
Clostridium tyrobutyricum.
Objective :
To determine the antimicrobial effects of extracellular ectracts of
selected LAB strains.
Materials and Reagents :
- MRS broth
- Sterile filter paper disk (50mm x 50mm)
- Forceps
- Sterile universal bottles
- Cultures of LAB and spoilage/pathogenic organisms
- Bench-top refrigerated centrifuge
- Incubator 37oC
- UV/Vis spectrophotometer
- Distilled water
- Trypticase soy agar
- Brain heart infusion agar (BHI)
- Yeast extract
Observation :
Results :
Part I : Determination of bacteriocin activity via agar diffusion test
Strains
of LAB
|
Strains
of spoilage/pathogenic bacteria
|
Inhibition
zone (cm)
|
L.
casei
|
K.
pneumonia
|
(0.60
+ 0.60)/2 =0.60
|
S.
aureus
|
(0.60
+ 0.60)/2 =0.60
|
|
P.
aeruginosa
|
(0.70
+ 0.60)/2 = 0.65
|
|
L.
brevis
|
K.
pneumonia
|
(0.70
+ 0.80)/2 = 0.75
|
S.
aureus
|
No
inhibiton zone
|
|
P.
aeruginosa
|
(0.70
+ 0.80)/2 = 0.75
|
|
L.
plantarum
|
K.
pneumonia
|
(0.65
+ 0.60)/2 = 0.625
|
S.
aureus
|
(0.60
+ 0.60)/2 = 0.60
|
|
P.
aeruginosa
|
(0.70
+ 0.70)/2 = 0.70
|
Part II :
Determination of bacteriocin activity via optical density
Dilutions
|
OD600
of spoilage/pathogenic bacteria
|
||
K.
pneumonia
|
S.
aureus
|
P.
aeruginosa
|
|
2x
|
0.994
|
0.588
|
0.609
|
10x
|
1.174
|
0.827
|
0.891
|
50x
|
0.669
|
0.563
|
0.630
|
100x
|
0.613
|
0.336
|
0.438
|
Equation
|
y
= -0.0052x + 1.0719
|
y
= -0.0034x + 0.725
|
y
= -0.0031x + 0.7665
|
OD600
of control
|
1.156
|
0.270
|
0.150
|
50%
of OD600
|
0.578
|
0.135
|
0.075
|
AU/ml
|
94.98
|
173.53
|
223.06
|
Graph of equation
:
K. Pneumonia
S. Aureus
P. Aeruginosa
Discussion :
Lactic acid bacteria refers to a large group of beneficial bacteria that
have similar properties and all produce lactic acid as an end product of the
fermentation process. They are widespread in nature and are also found in our
digestive systems. Although they are best known for their role in the
preparation of fermented dairy products, they are also used for pickling of
vegetables, baking, winemaking, curing fish, meats and sausages. Lactic acid
bacteria are therefore excellent ambassadors for an often maligned microbial
world. They are not only of major economic significance, but are also of value
in maintaining and promoting human health.
A broad number of food products, commodity chemicals,
and biotechnology products
are manufactured industrially by large-scale bacterial fermentation of various
organic substrates. Because enormous amounts of bacteria are being cultivated
each day in large fermentation vats, the risk that bacteriophage contamination
rapidly brings fermentations to a halt and cause economical setbacks is a
serious threat in these industries. The relationship between bacteriophages and
their bacterial hosts is very important in the context of the food fermentation
industry. Sources of phage contamination, measures to control their propagation
and dissemination, and biotechnological defence strategies developed to
restrain phages are of interest. The dairy fermentation industry has openly
acknowledged the problem of phage contamination,
and has been working with academia and starter culture companies to develop
defence strategies and systems to curtail the propagation and evolution of
phages for decades.
The first contact between an infecting phage and its
bacterial host is the attachment of the phage to the host cell. This attachment
is mediated by the phage's receptor binding protein (RBP),
which recognizes and binds to a receptor on the bacterial surface. RBPs are
also referred to as host-specificity protein, host determinant, and
antireceptor. For simplicity, the RBP term will be used here. A variety of
molecules have been suggested to act as host receptors for bacteriophages infecting
LAB; among those are polysaccharides and
(lipo)teichoic acids, as well as a single-membrane protein. A number of RBPs of
LAB phages have been identified by the generation of hybrid phages with altered
host ranges. These studies, however, also found additional phage proteins to be
important for successful a phage infection. Analysis of the crystal structure
of several RBPs indicated these proteins share a common tertiary folding, as
well as supporting previous indications of the saccharidenature
of the host receptor. The Gram-positive LAB
have a thick peptidoglycan layer,
which must be traversed to inject the phage genome into
the bacterial cytoplasm.
Peptidoglycan-degrading enzymes are expected to facilitate this penetration,
and such enzymes have been found as structural elements of a number of LAB
phages.
The disk diffusion method of Kirby and Bauer has been
standardized and is a viable alternative to broth dilution methods for laboratories
without the resources to utilize the newer automated methods for broth
microdilution testing.
When a 6-mm filter paper disk impregnated with a known
concentration of an antimicrobial compound is placed on an agar plate,
immediately water is absorbed into the disk from the agar. The
antimicrobial begins to diffuse into the surrounding agar. The rate of
diffusion through the agar is not as rapid as the rate of extraction of the
antimicrobial out of the disk, therefore the concentration of antimicrobial is
highest closest to the disk and a logarithmic reduction in concentration occurs
as the distance from the disk increases. The rate of diffusion of the
antimicrobial through the agar is dependent on the diffusion and solubility properties
of the drug in agar and the molecular weight of the antimicrobial
compound. Larger molecules will diffuse at a slower rate than lower
molecular weight compounds. These factors, in combination, result in each
antimicrobial having a unique breakpoint zone size indicating susceptibility to
that antimicrobial compound.
If the agar plate has been inoculated with a
suspension of the pathogen to be tested prior to the placing of disks on the
agar surface, simultaneous growth of the bacteria and diffusion of the
antimicrobial compounds occurs. Growth occurs in the presence of an
antimicrobial compound when the bacteria reach a critical mass and can
overpower the inhibitory effects of the antimicrobial compound. The
estimated time of a bacterial suspension to reach critical mass is 4 to 10
hours for most commonly recovered pathogens, but is characteristic of each
species, and influenced by the media and incubation temperature. The size
of the zone of inhibition of growth is influenced by the depth of the agar,
since the antimicrobial diffuses in three dimensions, thus a shallow layer of
agar will produce a larger zone of inhibition than a deeper layer.
The most common spectrophotometers are used in the UV and visible regions
of the spectrum, and some of these instruments also operate into the near-infrared region
as well. Visible region 400–700 nm spectrophotometry is used extensively
in colorimetry science.
Ink manufacturers, printing companies, textiles vendors, and many more, need
the data provided through colorimetry. They take readings in the region of every 5–20
nanometers along the visible region, and produce a spectral
reflectance curve
or a data stream for alternative presentations. These curves can be used to
test a new batch of colorant to check if it makes a match to specifications.
Conclusions :
References :
- http://www.medicalnewstoday.com/releases/14023.php
- http://en.wikipedia.org/wiki/Lactic_acid_bacteria
- http://www.microbelibrary.org/component/resource/laboratory-test/3189-kirby-bauer-disk-diffusion-susceptibility-test-protocol
- http://en.wikipedia.org/wiki/Spectrophotometry
NAME:
MUHAMMAD AIZAT B MAT SAAD
MATRIC
NO: 111385
LAB
5: Determination of Antimicrobial Effects of Microbial Extracts
Introdution
:
Certain groups of
bacteria can produce antimicrobial substances with the capacity to inhibit the
growth of pathogenic and spoilage microorganisms. Organic acids, hydrogen
peroxide, diacetyl and bacteriocins are included among these antimicrobial
compounds. Interest in naturally produce antimicrobial agents, such as
bacteriocins, is on the rise, since nowadys consumers demand
"natural" and "minimally processed" food.
Bacteriocins comprise a
large and diverse group of ribosomally synthesized antimicrobial proteins or
peptides. Although bacteriocins can be found in numerous Gram-positive and
Gram-negative bacteria, those produced by lactic acid bacteria (LAB) have
received special attention in recent years due to their potential application
in the food industry as natural biopreservatives. Different classes of LAB
bacteriocins have been identified on the basis of biochemical and genetic
characterization. These bacteriocins have been reported to inhibit the growth
of Listeria monocyotogenes,Staphylococcus aureus, Enterococcus faecalis and Clostridium tyrobutyricum.
Objective:
To determine the antimicrobial effects of extracellular
extracts of selected LAB strains
Results:
Part 1: Determination of bacteriocin
activity via agar diffusion test
|
Strains
of LAB
|
Strains
of spoilage / pathogenic bacteria
|
Inhibition
zone (cm)
|
|
L.
casei
|
S. aureus
|
(0.60+0.60)/2
= 0.60
|
|
K. pneumonia
|
(0.60+0.60)/2
= 0.60
|
|
|
P. aeruginosa
|
(0.70+0.60)/2
= 0.65
|
|
|
L. brevis
|
S. aureus
|
No
inhibition zone.
|
|
K. pneumonia
|
(0.70+0.80)/2
= 0.75
|
|
|
P. aeruginosa
|
(0.80+0.70)/2
= 0.75
|
|
|
L. plantarum
|
S. aureus
|
(0.60+0.60)/2
= 0.60
|
|
K. pneumonia
|
(0.65+0.60)/2
= 0.625
|
|
|
P. aeruginosa
|
(0.70+0.70)/2
= 0.70
|
Part 2 : Determination of bacteriocin
activity via optical density
Serial dilution of extracellular extract
Strain of LAB: L. plantarum
|
Dilutions
|
OD600 of
spoilage/pathogenic bacteria
|
||
|
Strain 1 :
K. pneumonia
|
Strain 2 :
S. aureus
|
Strain 3 :
P. aeruginosa
|
|
|
2x
|
0.994
|
0.588
|
0.609
|
|
10x
|
1.174
|
0.827
|
0.891
|
|
50x
|
0.669
|
0.563
|
0.630
|
|
100x
|
0.613
|
0.366
|
0.438
|
|
Equation
|
y
= -0.0052x + 1.0719
|
y
= -0.0034x + 0.725
|
y
= -0.0031x + 0.7665
|
|
OD600 of
control
|
1.156
|
0.270
|
0.150
|
|
50% of
OD600
|
0.578
|
0.135
|
0.075
|
|
AU/ml
|
94.98
|
173.53
|
223.06
|
Graph of equation :
1) K. Pneumonia
Discussion:
An antimicrobial is a
substance that kills or inhibits the growth of microorganisms such as bacteria,
fungi, or protozoans. Antimicrobial drugs either kill microbes (microbiocidal)
or prevent the growth of microbes (microbiostatic). Disinfectants are
antimicrobial substances used on non-living objects or outside the body. The
history of antimicrobials begins with the observations of Pasteur and Joubert,
who discovered that one type of bacteria could prevent the growth of another.
They did not know at that time that the reason one bacterium failed to grow was
that the other bacterium was producing an antibiotic. Technically, antibiotics
are only those substances that are produced by one microorganism that kill, or
prevent the growth, of another microorganism. Of course, in today's common
usage, the term antibiotic is used to refer to almost any drug that attempts to
rid your body of a bacterial infection. Antimicrobials include not just
antibiotics, but synthetically formed compounds as well. The discovery of
antimicrobials like penicillin and tetracycline paved the way for better health
for millions around the world. Before penicillin became a viable medical
treatment in the early 1940s, no true cure for gonorrhea, strep throat, or
pneumonia existed. Patients with infected wounds often had to have a wounded
limb removed, or face death from infection. Now, most of these infections can
be cured easily with a short course of antimicrobials. However, with the
development of antimicrobials, microorganisms have adapted and become resistant
to previous antimicrobial agents. The old antimicrobial technology was based
either on poisons or heavy metals, which may not have killed the microbe
completely, allowing the microbe to survive, change, and become resistant to the
poisons and/or heavy metals. Antimicrobial nanotechnology is a recent addition
to the fight against disease causing organisms, replacing heavy metals and
toxins and may some day be a viable alternative.
There
are several classes of antimicrobial drugs:
1)
Antibiotics
- Antibiotic are generally used to treat bacterial infections. Antibiotics are
among the most commonly used drugs. For example, 30% or more hospitalized
patients are treated with one or more courses of antibiotic therapy. However,
prolonged use of certain antibiotics can decrease the number of gut flora,
which can have a negative impact on health. Some recommend that, during or
after prolonged antibiotic use, one should consume probiotics and eat
reasonably to replace destroyed gut flora.
2)
Antivirals
- Antiviral drugs are a class of medication used specifically for treating
viral infections. Like antibiotics, specific antivirals are used for specific
viruses. They are relatively harmless to the host, and therefore can be used to
treat infections. They should be distinguished from viricides, which actively
deactivate virus particles outside the body. Many of the antiviral drugs
available are designed to treat infections by retroviruses, mostly HIV.
Important antiretroviral drugs include the class of protease inhibitors.
Antiviral
drugs work by inhibiting the virus before it enters the cell, stopping it from
reproducing, or, in some cases, preventing it from exiting the cell.
3)
Antifungals
- An antifungal drug is medication used to treat fungal infections such as
athlete's foot, ringworm, candidiasis (thrush), serious systemic infections
such as cryptococcal meningitis, and others. Antifungals work by exploiting
differences between mammalian and fungal cells to kill off the fungal organism
without dangerous effects on the host. Unlike bacteria, both fungi and humans
are eukaryotes. Thus, fungal and human cells are similar at the molecular
level, making it more difficult to find a target for an antifungal drug to
attack that does not also exist in the infected organism. Consequently, there
are often side effects to some of these drugs. Some of these side effects can
be life-threatening if the drug is not used properly.
4)
Non-pharmaceutical
antimicrobials - A wide range of chemical and natural compounds are used as
antimicrobials. Organic acids are used widely as antimicrobials in food
products, such as lactic acid, citric acid, acetic acid, and their salts,
either as ingredients, or as disinfectants. For example, beef carcasses often
are sprayed with acids, and then rinsed or steamed, to reduce the prevalence of
E. coli.
Lactic
Acid Bacteria (LAB)
Lactic
acid bacteria (LAB) occur naturally in several raw materials like milk, meat
and flour used to produce foods . LAB are used as natural or selected starters
in food fermentations in which they perform acidification due to production of
lactic and acetic acids flavour. Protection of food from spoilage and
pathogenic microorganisms by LAB is through producing organic acids, hydrogen
peroxide, diacethyl, antifungial compounds such as fatty acids or phenullactic acid
and/or bacteriocins. LAB play an important role in food fermentation as the
products obtains withtheir aid are characterized by hygienic safety, storage stability
and attractive sensory properties. Many bacteria of different taxonomic
branches and residing in various habitats produce antimicrobial substances that
are active against other bacteria. Both Gram negative and Gram positive
bacteria produce bacteriocins. Bacteriocins are proteinaceous antibacterial
compounds, which constitute a heterologous subgroup of ribosomally synthesized antimicrobial
peptides.
In
general these substances are cationic peptides that display hydrophobic or
amphiphilic properties and the bacterial membrane is in most cases the target
for their activity. Depending on the producer organism and classification
criteria, bacteriocins can be classified into several in which classes I and II
are the most thoroughtly studied. Class I, termed lantibiotics, constitue a
group of small peptides that are characterized by their content of several
unusual amino acids. The class II bacteriocins are small, nonmodified, heat
stable peptides. Many bacteriocins are active against food borne pathogens. A
large number of bacteriocins have been isolated and characterized from lactic
acid bacteria and some have acquired a status as potential antimicrobial agents
because of their potential as food preservatives and antagonistic affect against
important pathogens. The important ones are nisin, diplococcin, acidophilin, bulgarican,
helveticins, lactacins and plantaricins. The lantibiotic nisin which is produced
by different Lactococcus lactis spp. is the most thoroughtly studied
bacteriocin to date and the only bacteriocin that is applied as an additive in
food. One of the reason for increased consumption of fermented milk products is
that fermented dairy products containing probiotics which have many proposed
health benefits are available on the market. In this paper the diversity of
bacteriocins their appliction and lactic acid bacteria used are probiotics are
reviewed.
|
Genusa
|
Shape
|
Catalase
|
Nitrite reduction
|
Fermentation
|
Current
genera
|
|
Betabacterium
Thermobacterium
Streptobacterium
Streptococcus
Betacoccus
Microbacterium
Tetracoccus
|
Rod
Rod
Rod
Coccus
Coccus
Rod
Coccus
|
-
-
-
-
-
+
+
|
-
-
-
-
-
+
+
|
Hetero
Homo
Homo
Homo
Hetero
Homo
Homo
|
Lactobacillus
Weissella
Lactobacillus
Lactobacillus
Carnobacterim
Streptococcus
Enterococcus
Lactococcus
Vagococcus Leuconostoc Oenococcus Weissella
Brochothrix
Pediococcus
Tetragenococus
|
Classification of bacteriosins
The bacteriocins produced by Gram-positive bacteria like
LAB are small peptides. On a sound scientific basis three defined classes of
bacteriocins have been established: Class I, the lantibiotics; class II, the
small heat stable non lantibiotics; and class III, large heat labile bacteriocins.
A fourth class of bacteriocins is composed of an undefined mixture proteins,
lipids and carbohydrates. The existence of the fourth class was supported
mainly by the observation that some bacteriocin activities obtained in cell
free supernatant, exemplified by the activity of Lb plantarum LPCO 10 were
abolished not only by protease treatements, but also by glycolytic and
lipolytic enzymes. Most of the Gram positive bacteriocins are membrane active
compounds that increase the permeability of the cytoplasmic membrane. They
often show a much broader spectrum of bactericidal activity than the colicins
(Gram negative bacteriocins which are produced by Esherichia coli). They fall
with in two broad classes, the lantibiotics and the non
lantibiotic bacteriocins. Nisin prevents clostridal spoilage spoilage of processed
and natural cheeses, inhibits the growth of some psychrotropic bacteria in cottage cheese,
entends the shelf life of milk in warm countries, prevents the growth of
spoilage lactobacilli in beer and wine fermentations and provides additional
protection against Bacillus and clostridial spores in canned foods. Nisin is a permitted food additive in more than 50 countries
including the US and Europe under the trade name Nisaplin. Nisin is active
against many gram positive bacteria icluding Listeria spp.
|
Group I: Modified bacteriocins (the
lantibiotics)
|
Group II: Unmodified bacteriocins
|
||
|
Type A Type
B
|
One peptide bacteriocins
|
Two peptide bacteriocins
|
|
|
Nisin NK a
Lactocin
Lacticin
481
Carnocin
UI 49
|
Pediocin-like
bacteriocins b,
Pediocin
PA1, Leucocin A,
Sakacin
P, Curvacin A,
Mesentericin
Y105,
|
Lactococcin
G
Lactacin
F
Plantaricin
J/K
Plantaricin
E/F
|
|
|
Cytolysin
|
|
Carnobacteriocin
BM1,
Carnobacteriocin
B2,
Enterocin
A, Piscicolin 126,
Bavaricin
MN, Piscicocin V1a
|
Lactobin A
Plantaricin Sc
Pediocin L50d
Thermophilin 13
|
|
|
|
Nonpediocin-
like bacteriocins:
Lactococcin A
and B, Crispacin A,
Divergicin
750, Lactococcin 972,
AS-48e,
Enterocin B,
Carnobacteriocin A
|
|
Conclusion:
As a conclusion, the results show that lactic acid
bacteria(LAB) may act as a bio preservatives. From the experiment, the LAB
succesfully shows its bio preservatives properties on both gram-positive and
gram-negative bacteria which are Escherichia coli and Staphylococcus aureus.
Antimicrobial compounds produced by LAB have provided these organisms with a
competitive advantage over other microorganisms.
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