Lab 5 report prepared by Teoh Leong Sin

Name : Teoh Leong Sin

Matric no: 111433

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 produced antimicrobial agents, such as bacteriocins, is on the rise, since nowadays 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 monocytogens, Staphyloccus aureus, Enterococcus faecalis and Clostridium tyrobutyricum.

 

 Objective:

To determine the antimicrobial effects of the extracelluar extracts of selected LAB strains.

Results:

Part 1: Determination of bacteriocin activity via agar diffusion test

The sample result that shows that there is inhibition zone or presence of antimicrobial effect.

The sample result that shows that there is no inhibition zone or absence of antimicrobial effect.

Strains of Lab	Strains of spoilage/pathogenicbacteria	Inhibition zone(cm)
Lactobacillus casei	S.aureus	(0.6+0.6)/2 = 0.6
	P.aeruginosa	(1.3+0.9)/2 = 1.15
	K.pneumonia	(1.0+1.3)/2 = 1.1
Lactobacillus brevis	S.aureus	0
	P.aeruginosa	0
	K.pneumonia	(0.7+0.8)/2 = 0.75
Lactobacillus planterum	S.aureus	0
	P.aeruginosa	0
	K.pneumonia	(0.9+0.9)/2 = 0.9

Part 2 : Determination of bacteriocin activity via optical density

              Serial dilution of extracellular extract

Dilutions	OD600 of spoilage/pathogenic bacteria
	Strain 1 : S.aureus	Strain 2 : P.aeruginosa	Strain 3 : K.pneunomia
2x	0.699	0.854	0.828
10x	0.698	0.990	0.943
50x	0.590	0.780	0.625
100x	0.168	0.553	0.512
Equation	y= -0.0054x + 0.7572	y= -0.0038x + 0.9468	Y= -0.004x + 0.8849
OD600 of control	0.508	0.129	1.156
50% of OD600	0.254	0.0645	0.578
AU/ml	93.19	232.18	77.85

Discussion:

1)An anti-microbial is a substance that kills or inhibits the growth of microorganisms such as bacteria, fungi, and protozoans

2)The Lactic acid bacteria(LAB) comprise a clade of Gram-positive,acid-tolerant,generally non-sporulating, non-respiring rod or cocci that are associated by their common metabolic and physological characteristics.Lactic acid bacteria are rod-shaped bacilli or cocci characterizd by an increased tolerance to a lower pH range.This aspect partially enables LAB to outcomplete other bacteria in  a natural fermentation as they can withstand the increased acidity from organic acid production. LAB are amongst the most important groups of mircroorgnisms used in the food industry.

3)Part 1 determination of bacteriocin activity is mainly due to lactic acid and other acid production.

The agar diffusion test is means of measuring effect of an antimicrobial agent against bacteria grown in culture. 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 is a measure of the compound's effectiveness: the larger the clear area around the filter disk, the more effective the compound.

4)Three types of LAB have been used in the experiments are Lactobacillus casei, Lactobacillus brevis, Lactobacillus plantirum. Three types of pathogenic microorganisms have been used are S.aureus, P.aeruginosa, K.pneumonia.

5)Spectrophotometer is a photometer that can measure the intensity features of spectrophotometers are spectral bandwidth and linear range of absorption or relectance measurement.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. When working with a particular type of cell, you would determine the optical density at a particular wavelength that correlates with the different phases of bacterial growth. Typically the OD₆₀₀ is measured.

6)Strain of L. plantarum showed the strongest inhibition effects on K. pneumonia because its OD₆₀₀ were less than the OD₆₀₀ of the positive control among the three pathogenic bacteria.

Conclusion:

Bacteriocin was extracted by centrifuge method to determine for the antagonistic activity. LAB is a useful bacteria used to produce bacteriocin that can inhibit the growth of bacteria. Bacteriocins have been defined as proteinaceous substances exhibiting bactericidal activity against closely related species. Currently they are receiving increased attention because of their inhibitory activity against food spoilage and food-borne pathogenic bacteria . Commercial nisin preparations have been evaluate in food systems. It was now widely used as bio preservatives in the food industry due to their antibacterial properties. This allowed a more strict microbial control of a variety of commercial food products.

References:

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

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

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1622751/

http://www.pjbs.org/pjnonline/fin202.pdf

http://dictionary.reference.com/browse/arbitrary

Lab 5 by Tang Su Xian

Name : Tang Su Xian
Matric's card no: 111431

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 produced antimicrobial agents, such as bacteriocins, is on the rise, since nowadays 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 monocytogens, Staphyloccus aureus, Enterococcus faecalis and Clostridium tyrobutyricum.

  Lactobacillus casei, Lactobacillus brevis and Lactobacillus plantarum are the examples of lactic acid bacteria. L casei, is a lactic acid producing bacteria that occurs naturally in the human gut and mouth. It is a transient bacterium that usually lives for about ten days at a time in the intestines but it is resistant to gastric acid, so unlike some beneficial bacteria, it can survive quite happily in the intestinal tract. Lactobacillus casei is considered a probiotic safe for consumption. Lactobacillus casei is typically the dominant species of nonstarter lactic acid bacteria (NSLAB) present in ripening Cheddar cheese, and, recently, the complete genome sequence of L. casei ATCC 334 has become available. L. casei is also the dominant species in naturally fermented Sicilian green olives. Whereas, Lactobacillus brevis can be found in many different environments and in fermented foods such as sauerkraut and pickles. It is also one of the most common causes of beer spoilage. Ingestion has been shown to improve human immune function, and it has been patented several times. Lactobacillus plantarum is a widespread member of the genus Lactobacillus, commonly found in many fermented food products as well as anaerobic plant matter. It is also present in saliva (from which it was first isolated). It has the ability to liquefy gelatin. L.plantarum has one of the largest genomes known among the lactic acid bacteria and is a very flexible and versatile species. The ability of L. plantarum to produce antimicrobial substances helps them survive in the gastro-intestinal tract of humans. The antimicrobial substances produced have shown significant effect on Gram-positive and Gram-negative bacteria.

  There are two methods have been introduced to determine the bacteriocin activity, namely diffusion test and optical density method. 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. Whereas, 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:

To determine the antimicrobial effects of the extracelluar extracts of selected LAB strains.

Results:

Part 1: Determination of bacteriocin activity via agar diffusion test

The sample result that shows that there is inhibition zone. 

The sample result that shows that there is no inhibition zone.

Strains of LAB	Strains of spoilage / pathogenic bacteria	Inhibition zone (cm)
L. casei	S. aureus	(0.60 + 0.60)/2 = 0.60
	K. pneumonia	(1.00 + 1.30)/2 = 1.15
	P. aeruginosa	(1.30 + 0.90)/2 = 1.10
L. brevis	S. aureus	No inhibition zone.
	K. pneumonia	(0.70 + 0.80)/2 = 0.75
	P. aeruginosa	No inhibition zone.
L. plantarum	S. aureus	No inhibition zone.
	K. pneumonia	(0.90 + 0.90)/2 = 0.90
	P. aeruginosa	No inhibition zone.

Discussions:

Part 1: Determination of the bacteriocin activity via agar diffusion method.

1.)    From the results of this experiment, we found that, among the three strains of LAB used, the bacteriocin from L. casei was the most effective against towards the three pathogenic bacteria, namely S. aureus, K.pneumonia and P.aeruginosa. Whereas, the bacteriocin from L.brevis and L. plantarum were effective against K. pneumonia only. This is because the extracts of the lactic acid bacteria gave zones of inhibition onto the indicator pathogen strains tested. In the agar diffusion assay, a linear relationship existed between area of the zone of inhibition.

2.)   Generally bacteriocin production is strongly dependent on pH, nutrient sources and incubation temperature, and activity levels do not always correlate with cell mass or growth rate of the producer strain. Higher bacteriocin levels are often obtained at temperatures, nutrient sources and pH values lower than required for optimal growth. Maximal bacteriocin production may be obtained by supplementing the medium with growth limiting factors, such as sugars, nitrogen sources, vitamins and potassium, phosphate, or by regulating the medium pH. The culture medium pH and temperature might be the reasons of L. pneumonia and L. brevis failed to display their antimicrobial activity in this experiment.

3.)    L. caseisecretes a substance called “peptidoglycan”, which supports the natural defences of the body and stimulates immune responses in the intestinal tract. L. casei has demonstrated effectiveness in increasing circulating IgA (immunoglobulin A) in infants infected with rotavirus and has shortened the duration of associated diarrheal episodes. In a 2003 issue of the Journal of Nutrition Health and Aging, fermented milk containing the probiotic L. casei had a positive effect on lessening winter infections in the elderly. It is a highly prolific organism and has strong resistance to digestive enzymes.

4.)    L. plantarum has been studied for the treatment of recurrent Clostridium difficile-associated diarrhea and for Candida yeast infections. A particular strain called “299v”, derived from sour dough and used to ferment sauerkraut and salami, has demonstrated that it can improve the recovery of patients with enteric bacterial infections. The adherence of this bacterium reinforces the barrier function of the intestinal mucosa, thus preventing the attachment of the pathogenic bacteria. Eradicating such pathogens as Staphylococcus aureus from fermented food is one of the functions of L. plantarum too. Hence, the experiment conducted shown that strains of L. plantarum against the S. aureus might be due to the pH factor.

5.)    L. brevis is a lactic acid-producing organism important in the synthesis of vitamins D and K. Research studies have shown that L. brevis decreases intestinal permeability (leaky gut syndrome), improves intestinal micro flora, and has a positive effect on the intestinal immune system. A recent study showed that this friendly bacterium also has a positive effect in eliminating the ulcer-causing bacteria Heliobacter pylori.

Results:

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: S. aureus	Strain 2: P. aeruginosa	Strain 3: K. pneumonia
0x	-	-	-
2x	0.699	0.854	0.828
10 x	0.698	0.990	0.943
50 x	0.590	0.780	0.625
100 x	0.168	0.553	0.512
Equation	Y = -0.0054x+0.7572	Y = -0.0038x+0.9468	Y = -0.004x+0.8894
OD600 of control	0.508	0.129	1.156
50% of OD600	0.254	0.0645	0.578
AU / ml	93.19	232.18	77.85

1) S.aureus




2) P.aeruginosa



 3) K.pneumonia




 Discussions:

1.)    The optical density (OD) or called as scattering intensity is proportional to the cell density. Thus, we can determine a proportionality factor and get an equation. 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, the 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. For instance, the wavelength in this experiment was set as 600nm.

2.)    The positive control which showed the growth of bacteria without extracellular extract of lactic acid bacteria has been set up for each pathogenic bacteria. The OD₆₀₀ of the positive control was then measured in order for us to investigate whether there is inhibition of pathogenic bacteria activity by comparing the OD₆₀₀ of the samples. If the OD₆₀₀  of the sample is less than OD₆₀₀ of the positive control, there will be inhibition of spoilage bacteria by the strains of lactic acid bacteria, like L. plantarum used in this experiment.

3.)    Strain of L. plantarum showed the strongest inhibition effects on K. pneumonia because its OD₆₀₀ were less than the OD₆₀₀ of the positive control among the three pathogenic bacteria.

Conclusion:

In conclusion, the results from the agar diffusion method corresponded with the results from the optical density method. Both of the results showed that the strain of L. plantarum against the K. pneumonia, a pathogenic bacteria effectively. In addition, the results clearly suggest the potential usefulness of the bacteriocins produced by three lactic acid bacteria (LAB) as bio preservatives against K. pneumonia effectively. Generally, the L. casei might be the best bio preservative since it showed against the three strains of pathogenic bacteria in part one of the experiment. 

References:

1.)http://www.udea.edu.co/portal/page/portal/bibliotecaSedesDependencias/unidadesAcademicas/CorporacionAcademicaAmbiental/BilbiotecaDiseno/Archivos/Potential.pdf

2.)    http://en.citizendium.org/wiki/Lactobacillus_casei

3.)    http://en.wikipedia.org/wiki/Lactobacillus_casei

4.)    http://en.wikipedia.org/wiki/Lactobacillus_brevis

5.)     http://en.wikipedia.org/wiki/Pathogenic_bacteria

6.)     http://www.innvista.com/health/nutrition/biotics/proborg.htm

7.)     http://en.wikipedia.org/wiki/Agar_diffusion_test

8.)    http://microbiologyprojects.blogspot.com/2008/10/determination-of-bacteriocin-activity.html