Experimental Techniques for Bio-Chemical

Engineering
1. AGING PROCESS
(CHEESE PRODUCTION FROM MILK)
Specifically the milk component involved in cheese production is a soluble protein called casein. The
enzyme rennet can be used to catalyze the conversion of casein in milk to para-casein by removing a
glycopeptide from the soluble casein. Para-casein further clots, i.e. coagulates, in the presence of
calcium ions to form white, creamy lumps called the curd, leaving behind the supernatant called
the whey.

Rennet Ca++
casein ----- para-casein (aq.) ------ para-casein (ppt)

The precipitate is soft at this point and can be separated from the whey by the use of cheese cloth.
Filtration does not work very well; filter paper clogging is a recurrent problem.

Industrial Significance:

Industrially, the lactic acid level in the milk is increased by adding a starter culture
of Streptococci or Lactobacilli to the milk and fermenting at 32ºC for 10 to 75 minutes.

Thus, the selection of a suitable strain, the amount of starter culture, and the length of pre-ripening, is
of the utmost importance in creating the subtle differences in the final color and aroma that
distinguishes an expensive cheese from a cheap one.

Steps in cheese making:

Procuring at warm temperature Coloring Coagulation Separation

Flavoring Compression of the curd Aging Cheese

 2. HYDROLYTIC ACTION OF BACTERIAL PROTEASE

(ENZYMES IN LAUNDRY DETERGENTS)

In today’s laundry detergents, enzymes such as proteases and amylases are some of
the active ingredients. Oligosaccharides and dextrins released from the enzyme’s
hydrolytic action are soluble; thus, the stain is physically cut off from the surface of
the fabric piece by piece, with the enzyme acting as scissors. The action of proteases,
as implied by the name itself, is similar to that of amylase, except that a large protein
molecule is hydrolyzed. During the process of hydrolysis, the peptide bonds that hold
various amino acids together to form a protein molecule are broken down, releasing
smaller polypeptides and individual amino acid units. Generally, polymers made of
less than approximately one hundred amino acid monomer units are called
polypeptides and larger ones are called proteins.

This experiment is to observe the hydrolytic action of bacterial protease in removing

protein-based stains

3. DRY/WET WEIGHT METHOD

(MEASUREMENTS OF CELL BIOMASS CONCENTRATION)
Biomass concentration is one of the most critically needed measurements in
fermentation studies. It is also one of the most difficult and unreliable ones. In this
experiment, the cell density of a given sample is measured with the following four
methods:

1. Wet/ Dry weight

2. Optical density
3. Direct cell counting with a chamber
4. Successive dilutions followed by plating.

Dry/Wet weight method:

a. Dry in an oven an empty aluminum weighing pan or a sheet of cellulose

acetate filter membrane, 47mm in diameter, 0.45µm in pore size.
Weigh them and store them in a desiccator lined with Drierite
(anhydrous CaSO4).
 b. Stir the flask to suspend the culture evenly. Pour out 100 ml of the
culture into a graduated cylinder.
c. Separate the cells from the broth either by centrifugation at 10,000 g
for 5 minutes or by filtration. In the case of centrifugation, carefully
discard the clear broth and scrape the cell paste from the centrifuge
tube into a weighing pan. Rinse the centrifuge tube with a few ml of
water. Pour the rinse water into the weighing pan, as well. In the case
of filtration, the culture is poured into the holding reservoir fitted on
the filter membrane. A vacuum is applied to pull the liquid through the
membrane. Rinse the reservoir with a few ml of water and scrape any
paste adhering to the glassware. The wet weight of the culture is
measured immediately after all the water has been pulled through.
d. Dry the cell paste in an oven set at 100ºC. The cells will be charred and
the filter membrane will be burned if the temperature of the oven is set
too high. Measure the weight of the pan/filter plus the cell paste
periodically until there is no further decrease in the dry weight. It will
take 6-24 hours to dry the sample completely, depending on the oven
temperature and the thickness of the paste. Calculate the difference in
the weight, and express the dry weight in g/l.

4. COLORIMETRIC METHOD

(AMINO ACID ASSAY BY NINHYDRIN COLORIMETRIC METHOD)

The reaction between alpha-amino acid and ninhydrin involved in the development
of color are described by the following five mechanistic steps:

alpha-amino acid + ninhydrin - reduced ninhydrin + alpha-amino acid + H2O

alpha-amino acid + H2O - alpha-keto acid +NH3

alpha-keto acid + NH3 - aldehyde + CO2

Step (1) is an oxidative deamination reaction that removes two hydrogen from the
alpha-amino acid to yield an alpha-imino acid. Simultaneously, the original ninhydrin
is reduced and loses an oxygen atom with the formation of a water molecule. In Step
(2), the NH group in the alpha-imino acid is rapidly hydrolyzed to form an alpha-keto
acid with the production of an ammonia molecule. This alpha-keto acid further
undergoes decarboxylation reaction of Step (3) under a heated condition to form an
aldehyde that has one less carbon atom than the original amino acid. A carbon
dioxide molecule is produced here. These first three steps produce the reduced
ninhydrin and ammonia that are required for the production of color in the last two
Steps (4) and (5). The overall reaction for the above reactions is simply (slightly
inaccurately) expressed in Reaction (6) as follows:

alpha-amino acid + 2 ninhydrin - CO2 + aldehyde + final complex(BlUE) + 3H2O

In summary, ninhydrin, which is originally yellow, reacts with amino acid and turns
deep purple. It is this purple color that is detected in this method.

5. COLORIMETRIC METHOD

(GLUCOSE ASSAY BY DINITROSALICYLIC COLORIMETRIC

METHOD)
This method tests for the presence of free carbonyl group (C=O), the so-called
reducing sugars. This involves the oxidation of the aldehyde functional group present
in, for example, glucose and the ketone functional group in fructose. Simultaneously,
3, 5 – di nitro salicylic acid (DNS) is reduced to 3-amino, 5 – nitro salicylic acid under
alkaline conditions:
Oxidation
Aldehyde group -------- carboxyl group

Reduction
3, 5 – di nitro salicylic acid -------- 3-amino, 5 – nitro salicylic acid

Because dissolved oxygen can interfere with glucose oxidation, sulfite, which itself is
not necessary for the color reaction, is added in the reagent to absorb the dissolved
oxygen.

The above reaction scheme shows that one mole of sugar will react with one mole of
3, 5 – di nitro salicylic acid. However, it is suspected that there are many side
reactions, and the actual reaction stoichiometry is more complicated than that
previously described. The type of side reaction depends on the exact nature of the
reducing sugars. Different reducing sugars generally yield different color intensities;
thus, it is necessary to calibrate for each sugar. In addition to the oxidation of the
carbonyl groups in the sugar, other side reactions such as the decomposition of sugar
also competes for the availability of 3, 5 – di nitro salicylic acid. As a consequence,
carboxymethyl cellulose can affect the calibration curve by enhancing the intensity of
the developed color.

6. LIVE CELL IMAGING

It is the study of living cells using time lapse microscopy. It is used by scientist to obtain
understanding of biological function through the study of cellular dynamics. Live cells
was pioneered in first decade of the 20th century.
One of the first time lapse micro cinematic graphic film of cells forever was made by
Julius Ries, showing the fertilization and development of the seas urchin egg. Since then
several microscopy methods have been developed which allow researchers to study live
cells in greater detail with less effort. A newer type of imaging utilizing quantum dots
have been used as they are shown to be more stable.

7. TRANSMISSION ELECTRON MICROSCOPY DNA

Transmission electron microscopy DNA sequencing is a single molecule sequencing
technology that uses transmission, electron microscopy techniques. The method was
conceived and developed in 1960s and 1970s but lost favor when extent of damage to
the sample was recognized.
In order for the DNA to be clearly visualized under an electron microscope, it must be
labeled with heavy atoms. In addition specializing imaging techniques and abrasion
correcting, optic are beneficial for obtaining the resolution required to image the
labeled DNA molecules. In theory transmission electron microscopy DNA sequencing
could provide extremely long read lengths but issue of the electron damage may still
remain and technology haven’t been commercially developed.
 Bio-Chemical Engineering

Assignment:
Experimental Techniques for Bio-Chemical Engineering

Submitted to:
Sir Zia ul Haq

Submitted by:
Syed Mohtashim Ali (14-CHE-24)
Ahmad Hassan (14-CHE-74)
Faizan Fareed Ahmad (14-CHE-85)
Nafees Ali (14-CHE-72)

NFC IEFR Faisalabad