Introduction:
An anti-microbial 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).
Lactic acid bacteria produce various compounds such as organic acids, diacetyl, hydrogen peroxide, and bacteriocin or bactericidal proteins. The bacteriocins from the generally recognized as safe (GRAS) lactic acid bacteria (LAB) have arisen a great deal of attention as a novel approach to control pathogens. Bacteriocins are antimicrobial proteinaceous compounds that are inhibitory towards sensitive strains and are produced by both Gram-positive and Gramnegative bacteria. The antimicrobial effect of lactic acid bacteria has been appreciated by man for many years .
The bacteriocins have been reported to inhibit the growth of bacteria; in this experiment we use them to inhibit growing of Listeria monocytogens, Staphylococcus aureus, Clostridium tyrobutyrium and Salmonella.
L. monocytogenes is a gram positive, non-spore forming, motile, facultatively anaerobic, rod shaped bacterium. It is catalase positive, oxidase negative, and expresses a Beta hemolysin which causes destruction of red blood cells. The pathogenesis of L. monocytogenes centers on its ability to survive and multiply in phagocytic host cells.
S. aureus is a facultatively anaerobic, Gram-posotive coccus which can cause a range of illnesses from minor skin infections such as pimples, cellulitis, folliculitis to life-threatening diseases such as pneumonia, endocarditis and sepsis.
C.tyrobutyricum spores present in raw milk ferments lactate causing the "late-blowing" defect in high-pH. C.tyrobutyricum is a rod-shape, gram-positive bacteria that grows under anaerobic condition.
In this experiment, we use two types of technique to determine the bacteriocin activity which are agar diffusion method and the optical density method.
Part I: Agar diffusion test
In the agar diffusion test, we determine its zone of inhibition, one species of bacteria is uniformly swabbed onto a nutrient agar plate. Extracellular extract are placed on paper disks then added to the surface of the agar. During incubation, the extract diffuses from the disk containing the agent into the surrounding agar. Measurements can be made to quantify the size of the zones of inhibition around the disks. The relative effectiveness of an extract is determined by comparing the diameter of the zone of inhibition with values in a standard table.
Part II: Optical density method
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. Typically, when working with a particular type of cell, we would determine the optical density at a particular wavelength that correlates with the different phases of bacterial growth. Generally we will want to use cells that are in their mid-log phase of growth.
Objective:
1. To determine the antimicrobial effects of extracellular extracts of selected LAB strain
2. To provide the experience of using optical density spectrophotometer
3. To provide experience of preparing serial dilution
(part 1 we oredi done, u juz do part 2 result n discussion)
Results:
Part 1: Determination of bacteriocin activity via agar diffusion test
Strains of LAB | Strains of spoilage/ pathogenic bacteria | Inhibition zone (cm) |
1 | Escherichia coli | 0.7 |
Staphylococcus aureus | 0.9 | |
2 | Escherichia coli | 0.7 |
Staphylococcus aureus | 0.8 | |
Average | Escherichia coli | 0.8 |
Staphylococcus aureus | 0.7 |
Discussion:
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 andphysiological 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 humanmucosal 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. The lactic acid bacteria (LAB) are rod-shaped bacilli or coccus. LAB are characterized by an increased tolerance to a lower pH range. This aspect partially enables LAB to out compete other bacteria in a natural fermentation, as they can withstand the increased acidity from organic acid production (e.g., lactic acid). Laboratory media used for LAB typically includes a carbohydrate source as most species are incapable of respiration. LAB are catalase negative. LAB are amongst the most important groups of microorganisms used in the food industry.
Escherichia coli is a Gram-negative rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms (endotherms). Most E. coli strains are harmless, but some, such as serotypeO157:H7, can cause serious food poisoning in humans, and are occasionally responsible for product recalls. The harmless strains are part of the;normal flora of the gut, and can benefit their hosts by producing vitamin K2, and by preventing the establishment of pathogenic bacteria within the intestine.
E. coli are not always confined to the intestine, and their ability to survive for brief periods outside the body makes them an ideal indicator organism to test environmental samples for fecal contamination. The bacteria can also be grown easily and its genetics are comparatively simple and easily manipulated or duplicated through a process of metagenics, making it one of the best-studied prokaryotic model organisms, and an important species inbiotechnology and microbiology.
Staphylococcus 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. About 20% of the human population are long-term carriers of S. aureus. The carotenoid pigment staphyloxanthinis responsible for its characteristic golden colour, which may be seen in colonies of the organism. This pigment acts as a virulence factor with anantioxidant action that helps the microbe evade death by reactive oxygen species used by the host immune system. Staphylococci which lack the pigment are more easily killed by host defenses.
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. Its incidence is from skin, soft tissue, respiratory, bone, joint, endovascular to wound infections. It is still one of the five most common causes of nosocomial infections, often causing postsurgical wound infections. Abbreviated to S. aureus or Staph aureus in medical literature, S. aureus should not be confused with the similarly named and similarly dangerous (and also medically relevant) species of the genus Streptococcus.
The result of the diameter of inhibition zone obtained from Escherichia coli was 0.800cm while the diameter of inhibition zone obtained from Staphylococcus aureus was 0.700cm.
Part 2: Determination of bacteriocin activity via optical density
(Serial dilution of extracellular extract)
Strain of LAB 1: 0291
Dilutions | Strain 1: E.coli | Strain 2: S.aureus | ||||
Exp 1 | Exp 2 | Average | Exp 1 | Exp 2 | Average | |
0x | 0.398 | 0.408 | 0.403 | 0.467 | 0.689 | 0.578 |
2x | 1.013 | 1.113 | 1.063 | 1.789 | 2.217 | 2.003 |
10x | 1.095 | 1.319 | 1.207 | 2.154 | 2.394 | 2.274 |
50x | 1.118 | 1.156 | 1.137 | 2.399 | 2.497 | 2.448 |
100x | 1.591 | 1.735 | 1.663 | 2.327 | 2.403 | 2.365 |
Equation | y = 0.2915x + 0.2083 | y = 0.3259x + 0.9197 | ||||
OD 600 of control | 0.304 | 0.304 | ||||
50% of OD600 | 0.152 | 0.152 | ||||
AU/ml | -0.193 | -2.356 | ||||
Strain 1: E.coli
Strain 2: S.aureus
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| Inside of photospectrometer |
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| Photospectrometer |
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| Dilutions of spoilage (E.coli and S.aureus) |
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| Culture of LAB and Spoilage S.aureus |
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| Culture of LAB and spoilage E.coli |
Discussion:
The optical density (OD) or called as scattering intensity is The measure of transmittance of an optical medium for a given wavelength. The higher the OD the lower the transmittance, the higher protection factor by a filter (e.g. goggles, viewing windows, etc.). This factor allows us to calculate the cell density from any measured OD. A spectrophotometer can be set at a wavelength of 420 – 660 nm. In this experiment, the OD600 is measured. Typically, this wavelength must be standardized and may need to be adjusted specifically to the material being tested. Different vegetative cells and bacterial spores may not have the same maximal absorbance wavelength.
Generally, the OD value is higher for more diluted solutions. It indicates that the LAB strains have stronger antimicrobial effect on the pathogenic bacteria from more diluted solution. LAB 1 showed strongest inhibition effects on Salmonella since it has the higher value than E.coli bacteria.
Conclusion:
Some of the microorganisms can synthesis certain substances that have antimicrobial effects.
Reference:
