LAB 5: DETERMINATION OF ANTIMICROBIAL EFFECTS OF MICROBIAL EXTRACTS

INTRODUCTION.

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. Consumers nowadays demand food products with fewer synthetic additives but increased safety, quality and shelf-life. These demands have led to renewed interest in the use of natural antimicrobials to preserve foods. However, despite the wide range of potential antimicrobials, relatively few are suitable for use in practice in particular food products. For example the bacteriocins.

Bacteriocins are proteinaceous toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strain. They are typically considered to be narrow spectrum antibiotics. Although bacteriocins can be found in numerous Gram-positive and Gram-negative bacteria, those which are produced by lactic acid bacteria are to be given special attention due to its potential as natural bio preservatives in the food industry.

In fermented foods, 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. These bacteriocins have been reported to inhibit the growth of many pathogens. In this experiment, we are going to examine and discuss the effects of LAB strains on Escherichia coli (E.coli)  and Staphylococcus aureus (S.aureus).

E. coli is a Gram-negative rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms (endotherms).It normally lives inside your intestines, where it helps your body break down and digest the food you eat. Unfortunately, certain strains of E. coli can get from the intestines into the blood. This is a rare illness, but it can cause a very serious infection.

S.aureus is a facultative anaerobic, Gram-positive coccus, and is the most common cause of staph infections. It is frequently part of the skin flora found in the nose and on skin. S. aureus can cause a range of illnesses from minor skin infections, such as pimples, impetigo, boils (furuncles), cellulitis folliculitis, carbuncles,scalded skin syndrome, and abscesses, to life-threatening diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome (TSS), chest pain, bacteremia, and sepsis.

Part I : Determination of Bacteriocin Activity via Agar Diffusion Test.

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 is swabbed uniformly across a culture plate. A filter-paper disk, impregnated with the LAB, 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.

Part II : Determination of Bacteriocin Activity via Optical Density.

Spectrophotometers are commonly used for the measurement of transmittance or reflectance of solutions, transparent or opaque solids, such as polished glass, or gases. However they can also be designed to measure thediffusitivity on any of the listed light ranges that usually cover around 200nm - 2500nm using different controls and calibrations. Within these ranges of light, calibrations are needed on the machine using standards that vary in type depending on the wavelength of the photometric determination. This wavelength must be standardized and may need to be adjusted specifically to the material being tested. Different bacteria may not have the same maximal absorbance wavelength.

 RESULT

PART 1

E.COLI

S.AUREUS 

PART 2

E.COLI

CLICK TO ENLARGE

S.AUREUS

CLICK TO ENLARGE

DISCUSSION

PART 1

Using sterile inoculation loop, a loop full of direct Leuconostoc mesenteroides culture was touched over the swabbed plate and a small smear was made

A drop of the soft agar was dropped into the well to seal the bottom. The test organism Salmonella typhi, Escherichia coli, and Shigella flexinerrae were swabbed on the respective plates

The bacteriocin was extracted and its antagonistic activity was studied against the indicator organisms by well diffusion method

Bacteriocin was extracted by cell free supernatant method and the crude supernatant was determined for its antagonistic activity.

A well diffusion method was performed. In the activity it was observed that the bacteriocin produced by L.mesenteroides was effective against Salmonella typhi and Escherichia coli. But there was no effect against Shigella flexinerrae.

The reviews say that the bacteriocins produced by L.mesenteroides are active against Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Streptococcus faecalis, Escherichia coli, Bacillus cereus, L.monocytogenes has been considered as the major food borne pathogen and most activities were against them in food industries.

Now these studies reveal the scope for bacteriocins, not only as preservatives but also as an antibiotic for many diseases and infections.

Bacteriocin activity was determined in an agar well diffusion assay. To test the ability of the polyclonal antiserum to neutralize bacteriocin activity, serial dilutions of bacteriocin were mixed with an equal volume of undiluted antiserum in each well prior to adding the overlay.

Preimmune serum and sterile deionized water were mixed with bacteriocin in the control wells. All tests were run in duplicate.

This is an area around a paper disk or colony of bacteria(LAB) or mold where no other organisms are growing.  If you are testing antibiotic sensitivity for  example, you can impregnate paper disks with antibiotic and then put them on an  agar plate of growing bacteria.  The antibiotic then diffuses into the agar  away from the disk.  If the bacteria are sensitive to the antibiotic, they  will not grow near the disk.  The size of the zone is proportional to how  sensitive the organism is.  If the organism is resistant to the antibiotic,  it will grow right up to the disk.

PART2

Many lactic acid bacteria (LAB) produce a high diversity of bacteriocins, with a fairly broad inhibitory spectrum (19).

In recent years, many bacteriocins from LAB belonging to different groups have been described, characterized, and purified.

Nisin, produced by several strains of Lactococcus lactis, is the most studied bacteriocin, and it is used as a commercial food preservative.

The role of LAB and their bacteriocins as food biopreservatives is expected to grow in the future as a result of consumer awareness of the potential risks derived from food-borne pathogens as well as from the artificial chemical preservatives currently used to control them.

Why is the result more than 1?

This was possibly because of the supplements in MRS medium was suitable for bacteriocin production.

Why is the graph inceasing?

This is probably because of the lower growth at 5.0% and 10.0% glucose because of end-product  inhibition of lactic acid. This could be seen when total acidity was higher than 1.0%, the cell growth was stopped, even the substrate still available.

SPECTROMETER.

A spectrometer (spectrophotometer, spectrograph or spectroscope) is an instrument used to measure properties of lightover a specific portion of the electromagnetic spectrum, typically used in spectroscopic analysis to identify materials. The variable measured is most often the light's intensity but could also, for instance, be the polarization state. The independent variable is usually the wavelength of the light or a unit directly proportional to the photon energy, such as wavenumber or electron volts, which has a reciprocal relationship to wavelength. A spectrometer is used in spectroscopy for producing spectral lines and measuring their wavelengths and intensities. Spectrometer is a term that is applied to instruments that operate over a very wide range of wavelengths, from gamma rays and X-rays into the far infrared. If the instrument is designed to measure the spectrum in absolute units rather than relative units, then it is typically called a spectrophotometer. The majority of spectrophotomers are used in spectral regions near the visible spectrum.

In general, any particular instrument will operate over a small portion of this total range because of the different techniques used to measure different portions of the spectrum. Below optical frequencies (that is, at microwave and radio frequencies), the spectrum analyzer is a closely related electronic device

CONCLUSION

The possible use of bacteriocins as food biopreservatives could lead to the replacement of synthethic chemical preservatives, which have their antimicrobial action reduced due the continued appearance of multiresistant microbial lineages. The increasing occurrence of classic and or emerging food borne disease and it is possibly related to the indiscriminate use of chemicals preservatives favoring the selection of microbial lineages more and more resistant and therefore of difficult control. Bacteriocins are agents that could act on the microbial cell through different ways when compared to conventional chemical food preservatives, provoking the formation of an inhospitable environment to microbial survival. In addition, these molecules present characteristics of resistance to heat, acidity, low water activity and oscillations of temperature. However, there is the necessity to develop studies involving the establishment of the some bacteriocins characteristics such as antimicrobial spectrum, isolation, toxicity and stability use as control agents to the growth and microbial survival in food. Lactic acid bacteria and their products are more effective and flexible in several applications. Most inhibitory substances produced by lactic acid bacteria are safe and effective natural inhibitors of pathogenic and food spoilage bacteria in various food.

 REFERENCES
http://mywebspace.quinnipiac.edu/erdonnald/BI298/Bacteriocins.pdf

http://en.wikipedia.org/wiki/Antimicrobial

http://medical-dictionary.thefreedictionary.com/Agar+diffusion+test

http://en.wikipedia.org/wiki/Escherichia_coli

http://en.wikipedia.org/wiki/Staphylococcus_aureus

http://www.ruf.rice.edu/~bioslabs/methods/protein/spectrophotometer.html

http://www.scielo.br/pdf/babt/v48n4/25718.pdf