Salting Out

Aim

To precipitate out dissolved egg white protein by salting out

Principle

Salts such as ammonium sulphate and sodium sulphate ae used for the recovery and fractionation of proteins. As the salt concentration of a solution increases, more of the bulk water become associated with salt ions. The salts remove water from the surface of the protein revealing hydrophobic patches which come together causing the proteins to precipitate. Proteins which exhibit hydrophobic interactions aggregate and precipitate from the solution. This is a vital step in downstream processing of proteins. Precipitation relies on the principle that when compounds of higher affinity are added to the protein solution in a solvent, the protein get separated as a precipitate.

 Materials Required

Egg white solution, 10% ammonium sulphate solution, glass rod, routine microbiological facilities

Procedure

1. 10% ammonium sulphate solution was gently added to the solution of egg white with constant stirring, till precipitation was maximum.

2. The mixture was filtered and washed with ammonium sulphate solution in a funnel over filter paper

Result

The egg white protein was precipitated

 

Isolation of microbial flora of fermented milk

Aim

To isolate and enumerate bacteria of fermented milk.

Principle

Fermentation with certain microorganisms is necessary in the preparation of foods such as cheese, curd, yoghurt etc. Milk is fermented with lactic acid bacteria to make curd. Thus, the presence of microorganisms in food though considered harmful in some cases, it is definitely beneficial in other cases.

Materials Required

Curd, sterile tubes, nutrient agar, petri plates, Bunsen burner, pipettes, L-rod.

Procedure

1. Sterile nutrient agar plates containing 20 ml of medium were prepared.

2. Curd sample was diluted by serial dilution technique to obtain dilutions of 10^-4, 10^-5, 10^-6, 10^-7.

3. The plates were labelled corresponding to the dilutions.

4. Using sterile pipettes 0.1 ml from each dilution was placed in respective nutrient agar plates.

5. The L-shaped glass rod was sterilized with alcohol followed by flaming. The rod was cooled and gently placed on the surface of agar.

7. Petri plate was rotated in both clockwise and anticlockwise direction to uniformly spread the sample over agar surface. The plates were incubated in inverted position for 24 - 48 hours at 37⁰ C.

8. The number of colonies were counted and the total microorganisms per ml of original sample was calculated.

Observation

Each of the dilution plates were observed for colonies of bacteria. The number of colonies were counted.

Result

The number of organisms per ml was found to be  2.6 x 10⁷

 

Observation (on left side)

 Enumeration of Bacteria from Curd

Organism	Dilution	Number of Colonies	Number of Organisms per ml
BACTERIA	10-5	26	26 x 105    = 2.6 x 107 0.1
	10-6	4	4 x 106    = 4 x 107 0.1

 

The number of organisms per ml of curd can be calculated by applying the formula,

Number of organisms per ml of curd = Number of colonies ×dilution factor

                                                                           Amount plated

 

Pellicle formation

 Aim

To demonstrate pellicle formation in a broth culture 

Principle

Homogenous suspended microbial growth in a liquid medium will aid in availability of oxygen and other nutrients. This is ensured by using a rotary shaker or impeller driven bioreactor that keeps the cell, product, substrate and oxygen well mixed. When agitation stops, the culture remains static and the dissolved oxygen in the culture broth gets quickly used up, restricting growth and encouraging anaerobic physiology. At the surface of the broth, due to oxygen availability from the above gaseous phase, cells continue to multiply forming a well textured physical mat known as pellicle at the liquid surface. This further cuts off any oxygen diffusing from the top, pushing the lower part to further anaerobiosis.

Pellicle formation can result in low yields in a bioprocess industry due to unintended channelling of the substrate and efforts since unwanted by-products will be formed and will complicate the downstream processing.

 Materials Required

1. Culture: Aspergillus niger

2. Media: Doelger Prescott broth

3. Routine microbiological facilities

 Procedure

1. Aspergillus niger was inoculated in 300 ml of Doelger Prescott media.

2. It was incubated for 6 hours in a rotary shaker and then at room temperature undisturbed for about 7 days.

3. After incubation, the culture broth was observed for pellicle formation.

Observation and Result

A thick mat of microbial growth was observed on the surface of the liquid media.

Production and estimation of Citric acid

Aim

To demonstrate and estimate citric acid production by Aspergillus niger

 

Principle

Citric acid is the key intermediate of tricarboxylic acid cycle. Citric acid is used as acidulant in food and pharmaceutical industry for the production of carbonated beverages and used as plasticizer. Commercially, citric acid is produced by surface fermentation using Aspergillus niger. Beet molasses medium containing 10-20% sucrose is used in the commercial production. Doelger Prescott medium is used in this experiment to produce citric acid using Aspergillus niger. 

Estimation of citric acid is done by titration against standard NaOH using phenolphtahelin indicator. The amount of citric acid produced is expressed in gram per 100ml.

 Materials Required

1. Culture: Aspergillus niger

2. Media: Doelger Prescott broth

3. Routine microbiological facilities, Burette, pipette, conical flask, measuring cylinder

4. Reagents: 0.1 N NaOH, Phenolphthalein

 Procedure

1. 300 ml of Doelger Prescott media was prepared. pH was adjusted to 4.0 using 0.1 N HCL.

2. The medium was autoclaved and inoculated with a loopful of Aspergillus niger culture.

3. The flask was incubated at room temperature for 7 days.

4. After incubation for 7 days, the pH of the medium was noted.

5. The amount of citric acid in the culture broth was estimated by titration.

Estimation of Citric acid by titration

1. 1 ml of phenolphthalein indicator was added to 200 ml hot boiled water in a 250 ml flask.

2. 1 ml of culture filtrate was added to the above flask and titrated against 0.1  NaOH to the endpoint, in a well illuminated white background.

3. The initial and final readings were noted to calculate the volume of standard NaOH

 Result

The pH of the media was 4 before inoculation of Aspergillus niger culture and it decreased to 2 after 7 day incubation. This is due to the production of citric acid by the fungal cylture.

The amount of citric acid was estimated to be 0.30 g/100ml

  

Observation (left side)

1 N NaOH stock solution was prepared by dissolving 40 g NaOH in distilled water. From this stock solution, 0.1 N NaOH was prepared by mixing 50 ml stock solution with 450 ml distilled water.

Citric acid estimation

Conical flask: 200 ml hot boiled water + 1 ml of phenolphthalein + 1 ml of culture filtrate

Burette: 0.1 N NaOH

Endpoint: Appearance of distinct pale pink colour

	1	2	3
Initial burette reading	50	48	46
Final burette reading	49.5	47.6	45.5
Volume of 0.1 N NaOH rundown (ml)	0.5	0.4	0.5

 

Calculation (left hand side)

Citric acid (g/100ml) =

Volume of 0.1N NaOH × Normality × Equivalent molecular weight of citric acid ×100

Volume of sample taken

0.47 × 0.1 × 0.064 × 100/1

Citric acid (g/100ml) =         0.30 g/100 ml