Cell disruption technique - grinding with sand

 

 Aim

To demonstrate the breaking up of cell structures using abrasives like sand particles.

Principle

 In order to release microbial cell contents during downstream processing, a number of methods can be used for cell disruption to disintegrate the extremely tough cell walls. Any potential method of disruption must ensure that liable materials are not denatured by the process or hydrolyzed by enzymes present in the cell. The use of combination of different techniques release products from specific locations within yeast cell. Although many techniques are available which are satisfactory at laboratory scale, only limited number have been proved to be suitable for large-scale applications, particularly for intracellular enzyme extraction. Types of cell disruption methods are physical, mechanical and chemical.

Grinding with sand is a mechanical cell disruption method used to break microbial, plant, or animal cells to release intracellular components such as enzymes, proteins, nucleic acids, pigments etc. This technique tears up the structures of a cell using abrasive sand particles. Abrasion and shear force produced by grinding cells with fine sand particles mechanically rupture the cell wall and cell membrane. This helps to achieve the release of cytosolic contents, and allows some degree of breakup of physical structures. Physical structures are further removed by isopycnic centrifugation.

Grinding with sand is commonly used for laboratory-scale extraction from microorganisms and plant tissues. Usually, the process is carried out at low temperature to prevent denaturation of cellular components.

 

Materials Required

1.Yeast culture

2.Mortar and pestle

3.Sterile fine Sand

4. Sabouraud's dextrose Agar

5. Routine microbiological facilities

 

 

Procedure

1. Five ml of yeast suspension was taken into sterile test tube. One test tube was kept as control.

2. The yeast suspension was taken in a sterile mortar, and cloth filtered fine sand was added to it.

3. It was then ground with pestle several times and serially diluted.

4. 1 ml of suspension from 10 ^-4 and 10^-5 dilutions were plated on Sabouraud's dextrose agar and 

    CFU was counted

5.1 ml of the suspension from the control tube was also serially diluted and CFU was counted.

 

Result

Sabouraud's dextrose Agar plated with the sample ground with sand particle showed a fall in colony count, which indicate fall in cell count. 

 

Conclusion

Grinding with sand is a mechanical cell disruption technique in which the abrasive action of sand particles breaks the cell wall and membrane, releasing intracellular materials.

 

Production of Wine

 

Aim

To perform fermentation of grape juice into alcohol by the action of yeast cells.

Principle

Wine is traditionally used as an alcoholic beverage which is prepared from fermentation of grape. Other fruits such as apple, pears, plums etc. can also be used for production of wine. Usually, wine is prepared by using the microorganism Saccharomyces cerevisiae.

During wine production, yeast convert glucose and fructose present in the fruit is enzymatically converted first to aldehyde and then to ethyl alcohol. Grapes containing 20-30% sugar concentration will yield wine containing approximately 10 - 15% alcohol. Grapes also contain minerals, tannins, pigments, vitamins, enzymes and other aromatic compounds whose concentration in the final product give characteristic taste. The quality of wine depends on grape variety, fermentation conditions, aging process and microbial activity.

The science of wine production (enology) starts with collection of grapes. The protocol; for wine making include preparation of must. Fermentation is performed by adding microorganism Saccharomyces cerevisiae to the must. It is incubated at room temperature for 14-21 days. Must is collected, transferred to large bottle for settling, clarification and storage. The ageing include storage for along period (1-5 years) in a wooden tank or in other ageing tank. The chemical changes that occur during ageing is responsible for aroma. The final product, wine contain 10 - 15% alcohol.

Grape wine is of two types: red wine and white wine. Red wine comes from red and purple skin varieties of grape whereas white wine is prepared from white skin grapes or from red or purple grapes with skin removed. This experiment is a modified method in which red wine is produced from red grape juice. The fermenting wine is examined at one week interval, during incubation, for 

(1)total acidity (1% tartaric acid)

(2) volatile acidity (1% acetic acid)

(3) aroma - fruity, yeast like, sweet

(4) taste - bitter, sweet, sour

(5) pH 3-3.9

(6) alcohol (expressed in volume percentage).

 Materials Required:

Fresh grapes, Sugar, Yeast, sterile water.

Equipments: 

Burette, conical flask, pipettes, graduated cylinder, test tubes

Reagents:

1 % phenolphthalein in 0.1 N NaOH, potassium dichromate solution and sulphuric acid

 

Procedure:

1.     1 kg of grapes were washed thoroughly and crushed to obtain juice. The juice was filtered and around 750 ml was obtained, which was separated in two flasks as 700 ml and 50 ml.

2.     The flask with 50 ml juice was inoculated with yeast and placed in a mechanical shaker for 48 hrs to develop the inoculum.

3.     The inoculum was then added to the flask with 700 ml juice. 25 ml was withdrawn as the initial sample for evaluating total acidity, taste, flavour and aroma.

4.     The inoculated juice was allowed to undergo fermentation at room temperature (25- 30⁰C) for 21 days. 20 g of glucose was supplemented on the 7^th and 14^th days.

5.     During fermentation, samples were tested for acidity and alcohol content at 7 days intervals.

 Estimation of Parameters

        a)     pH of fermenting wine was tested with the help of pH paper. Aroma and taste were noted. 

        b)    Total Acidity (% of tartaric acid)

To 10 ml aliquot of fermented wine sample, 10 ml of distilled water and 5 drops of 0.1% phenolphthalein solution were added. The contents were mixed and titrated with 0.1 N NaOH, to the first persistent pink colour.

The total acidity was calculated using the formula:

% of tartaric acid = millilitre of alkali x Normality of alkali x 7.5

                                    Weight of sample in gram

1 ml = 1g

c)    Volatile Acidity

    Following titration, volatile acidity was calculated using the formula:

% of acetic acid = millilitre of alkali x Normality of alkali x 6

                                    Weight of sample in gram

1 ml = 1g

d) Estimation of Ethanol

Estimation of Ethanol was performed by adding potassium dichromate reagent and checking the OD at 600 nm

 

Potassium dichromate solution: 34 g of potassium dichromate (K₂Cr₂O₇) was dissolved in 500 ml of distilled water in 1l flask. 32.5 ml conc. H₂SO₄ was added by keeping the flask in ice bucket.

 

Preparation of standard curve for alcohol concentration

1)     1-10 % of alcohol samples were prepared.

2)     1.5 ml each of various concentration alcohol samples were taken in different test tubes.

3)     2.5 ml of potassium dichromate reagent and 1 ml of distilled water were added and test tubes incubated at 600C in water bath for 30 minutes.

4)     Optical density was measured at 600 nm

5)     A standard curve was plotted with concentration of alcohol on x-axis and optical density at 600 nm on Y -axis.

 

Determination of concentration of alcohol of fermented sample

1)     1.5 ml of wine sample was mixed with 2.5 ml of potassium dichromate reagent and 1 ml of distilled water were added and test tubes incubated at 60⁰C in water bath for 30 minutes.

2)     Optical density was measured at 600 nm

  

Dilution	Optical Density
Stock Solution
2 %
4 %
6 %
8 %
10%
Wine Sample
W1
W2

 

Result

After 21 days, % tartaric acid in the fermented wine was noted. pH and alcohol content was also noted.

 

Observation

Fermented Wine
	1st day	7th day	14th day	21st day
% tartaric acid
% acetic acid
pH
Taste
Aroma
% of alcohol