1. Why do we use factor 6.25 during protein determination by kjeldahl method?

One of the most employed methods of protein determination that has gained
universal appeal is the Kjeldahl method. Several variations and modifications are
available but all of them employ the amount of nitrogen present in the sample to
calculate indirectly the crude protein content.
In general, it is assumed that protein contains 16% nitrogen, which means that
each gram of nitrogen determined reflects a protein content of 100÷16 = 6.25g. The
factor 6.25 has been worked out based on a number of studies on amino acid profile.
In some foods, other factors give more accurate result, for example, 6.8 for flour
proteins, 5.71 for soybeans, 6.37 for milk, etc. During reporting the result, it is
therefore customary to mention the factor (usually 6.25) used in the calculation.
The nitrogen content in the sample is obtained by first oxidizing the organic
nitrogen with concentrated H2SO4 into (NH4)2SO4 as under:

Nitrogenous organic compound + H2SO4 CO2 + H2O + (NH4)2SO4 + SO2

2. Why is the hysteric gel formation property in agar-agar very important in

microbiological works?
Agar is commercially obtained from species of Gelidium and Gracilaria. It is a
mixture of at least two polysaccharides: agarose and agaropectin. Agarose is a
neutral, linear polymer of molecular weight about 120,000. The gel network of
agarose contains double helices formed from left-handed three-fold helices. These
double helices are stabilized by the presence of water molecules bound inside the
double helical cavity. Exterior hydroxyl groups allow aggregation of up to 10,000 of
these helices to form suprafibers.
Agar is insoluble in cold water but dissolves to give random coils in boiling
water to form heat-resistant gels. The gel formation properties of agar are unique. It
shows hysteresis in that gelation takes place at temperatures far below the gel-melting
temperature. The gel remains solid up to about 85°C while the melted agar sets at
around 40°C. This feature allows us to culture thermophilic organisms in agar plates.
Agar has a major use in microbiological media as it is not easy for microorganisms
to metabolize. Agar also forms clear, stable, and firm gels. In the food area, it is used
in icings, glazes, processed cheese, jelly sweets and marshmallows. It may be used
in tropical countries and by vegetarians as a substitute for gelatin (protein obtained
from animal bones and hides, which liquefies at temperatures above 25°C).
3. What is pregel condition and how can it be prevented?
Low methoxyl pectin (LMP) pregel is defined as too fast a reaction between the
LMP and the calcium ions, resulting in a non-homogeneous gel structure, which
resembles applesauce in texture. Nine times out of ten the cause of pregel is related
to the way in which the calcium is introduced to the LMP. Usually, the calcium gets
added too fast, or at the wrong point in the process, or it is simply too concentrated
when added. This indicates that the choice of LMP, calcium level and its method and
sequence of addition, and mixing temperature are all important. With LMP, the pectin
should be hydrated separately in water. To prevent such pregel condition calcium-
containing ingredients are prepared in a separate vessel and mixed only when
everything is ready.

4. Write short notes on flavor reversion.

Soybean oil and other fats and oils containing linolenic acid show the reversion
phenomenon when exposed to air. Reversion flavor is a particular type of oxidized
flavor that develops at comparatively low levels of oxidation, before the onset of
rancidity. The off-flavors may develop in oils that have a peroxide value of as little
as 1 or 2. Other oils may not become rancid until the peroxide value reaches 100.
Linolenic acid is generally recognized as the determining factor of inversion
(reversion) flavors. These off-flavors are variously described as grassy, fishy, and
painty. The origin of these flavors appears to be the volatile oxidation products
resulting from the terminal pentene radical of linolenic acid, CH3-CH2-CH=CH-CH2-
. The first perceptible reversion flavor was found to be caused by 3-cis-hexenal,
which has a pronounced green bean odor. Other flavorful aldehydes isolated were 2-
trans-hexenal (green, grassy), 2-trans-nonenal (rancid), and 2-trans-6-cisnonadienal
(cucumber flavor). These findings illustrate the complexity of the reversion flavors.
Similar problems occur with other polyunsaturated oils such as fish oil and rapeseed
oil. The conditions known to influence the onset and development of reversion
flavors are temperature, light, oxygen, and trace metals.

5. Discuss the fibril theory of gel formation.

A gel is a solid jelly-like soft material that can have properties ranging from
soft and weak to hard and tough. Gels are defined as a substantially dilute cross-
linked system, which exhibits no flow when in the steady-state. By weight, gels are
mostly liquid, yet they behave like solids due to a three-dimensional cross-linked
network within the liquid. It is the cross-linking within the fluid that gives a gel its
structure (hardness) and contributes to the adhesive stick. In this way gels are a
dispersion of molecules of a liquid within a solid in which liquid particles are
dispersed in the solid medium.
Jelly formation is due to the precipitation of pectin rather than its swelling. Only
when the pectin, acid, sugar and water are in definite equilibrium, the precipitation
of pectin takes place. The rate of precipitation is influenced by the following factors:
 Concentration of pectin in the solution
  Constitution of pectin
 Hydrogen ion concentration (PH) of the pectin solution
 Concentration of sugar in solution
 Temperature of the mixture
There are several theories to explain the formation of jellies.

Fibril theory:
When sugar is added to the pectin solution, it destabilizes the pectin-water
equilibrium and the pectin conglomerates forming a network of fibrils through the
jelly. This network of the fibrils holds the sugar solution in the inter-fibril spaces.
The strength of the jelly depends on the structure of the fibrils, their continuity and
rigidity.

6. Discuss the chemical method of moisture determination in foods.

Reactions between water and certain chemical reagents can be used as a basis for
determining the concentration of moisture in foods. In these methods a chemical
reagent is added to the food that reacts specifically with water to produce a
measurable change in the properties of the system, e.g., mass, volume, pressure, pH,
color, conductivity. Measurable changes in the system are correlated to the moisture
content using calibration curves. To make accurate measurements it is important that
the chemical reagent reacts with all of the water molecules present, but not with any
of the other components in the food matrix. Two methods that are commonly used in
the food industry are the Karl-Fisher titration and gas production methods. Chemical
reaction methods do not usually involve the application of heat and so they are
suitable for foods that contain thermally labile substances that would change the mass
of the food matrix on heating (e.g., food containing high sugar concentrations) or
foods that contain volatile components that might be lost by heating (e.g. spices and
herbs).

Karl-Fisher method:
The Karl Fischer titration is particularly adaptable to food products that show
erratic results when heated or submitted to a vacuum. The Karl-Fisher titration is
often used for determining the moisture content of foods that have low moisture
contents (e.g. dried fruits and vegetables, confectionary, candies, chocolate, roasted
coffee, oils and fats, or any low-moisture food high in sugar or protein.). The method
is quite rapid, is accurate, and uses no heat.
 This method is based on the fundamental reaction described by Bunsen in 1853
involving the reduction of iodine by SO2 in the presence of water:
2H2O + SO2 + I2 H2SO4 + 2HI
This reaction was originally used because HI is colorless, whereas I2 is a dark
reddish-brown color, hence there is a measurable change in color when water reacts
with the added chemical reagents. Sulfur dioxide and iodine are gaseous and would
normally be lost from solution. For this reason, the above reaction has been modified
by adding solvents which include methanol and pyridine in a four-component system
to dissolve the iodine and SO2.

C5H5N·I2 + C5H5N·SO2 + C5H5N + H2O 2C5H5N·HI + C5H5N·SO3

C5H5N·SO3 + CH3OH C5H5N (H) SO4·CH3

These reactions show that for each mole of water, 1 mol of iodine, 1 mol of
SO2, 3 mol of pyridine, and 1 mol of methanol are used. For general work, a
methanolic solution is used that contains these components in the ratio of 1 iodine: 3
SO2: 10 pyridines, and at a concentration so that 3.5 mg of water =1ml of reagent.
The food to be analyzed is placed in a beaker containing solvent and is then titrated
with Karl Fisher reagent (a solution that contains iodine). While any water remains
in the sample the iodine reacts with it and the solution remains colorless (HI), but
once all the water has been used up any additional iodine is observed as a dark red
brown color (I2). The volume of iodine solution required to titrate the water is
measured and can be related to the moisture content using a pre-prepared calibration
curve. The precision of the technique can be improved by using electrical methods to
follow the end-point of the reaction, rather than observing a color change. Relatively
inexpensive commercial instruments have been developed which are based on the
Karl-Fisher titration, and some of these are fully automated to make them less labor
intensive.
In a volumetric titration procedure, iodine and SO2 in the appropriate form are
added to the sample in a closed chamber protected from atmospheric moisture. The
excess of I2 that can-not react with the water can be determined visually. The end
point color is dark red-brown. Some instrumental systems are improved by the
inclusion of a potentiometer (i.e., conductometric method) to electronically
determine the end point, which increases the sensitivity and accuracy. The volumetric
titration can be done manually or with an automated unit. The automated volumetric
titration units (used for 100 ppm water to very high concentrations) use a pump for
mechanical addition of titrant and use the conductometric method for endpoint
determination (i.e., detection of excess iodine is by applying a current and measuring
the potential). The volumetric titration procedure described above is appropriate for
samples with a moisture content greater than∼0.03%. A second type of titration,
referred to as coulometric titration, is ideal for products with very low levels of
moisture, from 0.03% down to parts per million (ppm) levels. In this method, iodine
is electrolytically generated to titrate the moisture as:
2I- I2 + 2e-
The amount of iodine required to titrate the moisture is determined by the
current needed to generate the iodine. Just like for volumetric titration, automated
coulometric titration units are available commercially. In a Karl Fischer volumetric
titration, the Karl Fischer reagent (KFR) is added directly as the titrant if the moisture
in the sample is accessible. However, if moisture in a solid sample is inaccessible to
the reagent, the moisture is extracted from the food with an appropriate solvent (e.g.,
methanol). Then the methanol extract is titrated with KFR.
Before the amount of water found in a food sample can be determined, a KFR
moisture equivalence (KFReq) must be determined. The KFReq value represents the
equivalent amount of moisture that reacts with 1 ml of KFR. Standardization must be
checked before each use because the KFReq will change with time. The KFReq can
be established with pure water, a water-in-methanol standard, or sodium tartrate
dihydrate. Pure water is a difficult standard to use because of inaccuracy in measuring
the small amounts required. The water-in-methanol standard is pre-mixed by the
manufacturer and generally contains 1mg of water/ml of solution. This standard can
change over prolonged storage periods by absorbing atmospheric moisture. Sodium
tartrate dihydrate (Na2C4H4O6·2H2O) is a primary standard for determining KFReq.
This compound is very stable, contains 15.66% water under all conditions expected
in the laboratory, and is the material of choice to use. The KFReq is calculated as
follows using sodium tartrate dihydrate:
𝟑𝟔 𝐠 𝐇𝟐 𝐎/𝐦𝐨𝐥 𝐬𝐨𝐝𝐢𝐮𝐦 𝐭𝐚𝐫𝐭𝐚𝐫𝐚𝐭𝐞 × 𝐒× 𝟏𝟎𝟎𝟎
KFReq (mg H2O/ml) =
𝟐𝟑𝟎.𝟎𝟖 𝐠/𝐦𝐨𝐥 × 𝐀
Where, KFReq = Karl Fischer reagent moisture equivalence.
S = weight of sodium tartrate dihydrate in gram.
A = ml of KFR required for titration of sodium tartrate dihydrate.
Once the KFReq is known, the moisture content of the sample is determined as
follows:
𝐾𝐹𝑅𝑒𝑞 × 𝐾𝑆
% H2O = × 100
𝑆
Where, KFReq = Karl Fischer reagent moisture equivalence.
Ks = ml of KFR used to titrate sample.
S = weight of sample in mg.
The major difficulties and sources of error in the Karl Fischer titration methods
are as follows:
  Incomplete moisture extraction: For this reason, fineness of grind (i.e., particle
size) is important in preparation of cereal grains and some foods.
 Atmospheric moisture: External air must not be allowed to infiltrate the reaction
chamber.
 Moisture adhering to walls of unit: All glass-ware and utensils must be carefully
dried.
 Interferences from certain food constituents: Ascorbic acid is oxidized by KFR
to dehydroascorbic acid to overestimate moisture content; carbonyl compounds
react with methanol to form acetals and release water to overestimate moisture
content (this reaction also may result in fading end points); unsaturated fatty
acids will react with iodine, so moisture content will be overestimated.

7. What is Retinol Activity Equivalents (RAE)?

Vitamin A can be obtained from food as preformed vitamin A in animal
products or as provitamin A carotenoids in fruits and vegetables. Yet, while
preformed vitamin A is effectively absorbed, stored, and hydrolyzed to form retinol,
provitamin A carotenoids like β-carotene are less easily digested and absorbed, and
must be converted to retinol and other retinoids by the body after uptake into the
small intestine. The efficiency of conversion of provitamin A carotenes into retinol
is highly variable, depending on factors such as food matrix, food preparation, and
one’s digestive and absorptive capacities.
The most recent international standard of measure for vitamin A is retinol
activity equivalents (RAE), which represent vitamin A activity as retinol. It has been
determined that 2 micrograms (μg) of β-carotene in oil provided as a supplement
could be converted by the body to 1 μg of retinol giving it an RAE ratio of 2:1.
However, 12μg of β-carotene from food are required to provide the body with 1μg of
retinol, giving dietary β-carotene an RAE ratio of 12:1. Other provitamin A
carotenoids in food are less easily absorbed than β-carotene, resulting in RAE ratios
of 24:1.
RAE is the measure of vitamin A activity based on the capacity of the body to
convert provitamin carotenoids containing at least one unsubstituted ionone ring to
retinaldehyde. 1 microgram RAE = 1 mg retinol = 12 mg β-carotene = 24 mg other
vitamin A precursor carotenoids.
A retinol activity equivalent (RAE) is a measure of the amount of vitamin A that
can be actively absorbed by the body. Because of the variation in the conversion rate
of carotenoids to retinol, daily vitamin A requirements are expressed in micrograms
(μg) of Retinol Activity Equivalents (RAE), a unit that takes into consideration the
ease of absorption depending on the source of vitamin A.
8. Discuss the salting out of proteins.
Salting out also known as salt-induced precipitation, salt fractionation, anti-
solvent crystallization, precipitation crystallization, or drowning out is an effect
based on the electrolyte-non electrolyte interaction, in which the non-electrolyte
could be less soluble at high salt concentrations. It is used as a method of purification
for proteins as well as preventing protein denaturation due to excessively diluted
samples during experiments. The salt concentration needed for the protein to
precipitate out of the solution differs from protein to protein. This process is also used
to concentrate dilute solutions of proteins. Dialysis can be used to remove the salt if
needed.
Salt compounds dissociate in aqueous solutions. This property is exploited in
the process of salting out. When the salt concentration is increased, some of the water
molecules are attracted by the salt ions, which decreases the number of water
molecules available to interact with the charged part of the protein.
There are hydrophobic amino acids and hydrophilic amino acids in protein
molecules. After protein folding in aqueous solution, hydrophobic amino acids
usually form protected hydrophobic areas while hydrophilic amino acids interact with
the molecules of solvents and allow proteins to form hydrogen bonds with the
surrounding water molecules. If enough of the protein surface is hydrophilic, the
protein can be dissolved in water. As a result of the increased demand for solvent
molecules, the protein–protein interactions are stronger than the solvent-solute
interactions; the protein molecules associate by forming hydrophobic interactions
with each other. After dissociation in a given solvent, the negatively charged atoms
from a chosen salt begin to compete for interactions with positively charged
molecules present in the solution. Similarly, the positively charged cations compete
for interactions with the negatively charged molecules of the solvent. This process is
known as salting out.
Salt precipitation can be a very powerful tool to purify proteins by precipitation.
Ammonium sulfate is usually the salt of choice since it is cheap, very soluble in water
and is able to become much more hydrated (interacts with more water molecules)
than almost any other ionic solvent. In practice, ammonium sulfate is either added
directly as a solid or added as a saturated solution to precipitate the desired proteins.
As different proteins have different compositions of amino acids, different
protein molecules precipitate at different concentrations of salt solution. Unwanted
proteins can be removed from a protein solution mixture by salting out as long as the
solubility of the protein in various concentrations of salt solution is known. After
removing the precipitate by filtration or centrifugation, the desired protein can be
precipitated by altering the salt concentration to the level at which the desired protein
becomes insoluble.
One demerit of salting out in purification of proteins is that, in addition to
precipitating a specific protein of interest, contaminants are also precipitated as well.
Thus, to obtain a purer protein of interest, additional purification methods such as ion
exchange chromatography may be required.

9. Discuss the principle of moisture determination in foods.

Food moisture analysis involves the whole coverage of the food items in the
world because foods comprise a considerable amount of water rather than other
ingredients. Moisture content of the food material is important to consider the food
is suitable before the consumption, because moisture content affects the physical,
chemical aspects of food which relates with the freshness and stability for the storage
of the food for a long period of time and the moisture content determine the actual
quality of the food before consumption and to the subsequent processing in the food
sector by the food producers.
Food moisture analysis involves the amount of moisture content and the
concentration of moisture by measuring qualitatively and quantitatively. The food
processing companies and the "Research and Development" (R&D) people of the
food producing company, food related peoples, graduates, undergraduates and the
high school students must be well aware of the moisture analysis of the food which
do a lot in the food safety and storage conditions and the shelf life of foods and
products.
Legal and labeling requirements
Legal limitations regarding the amount of water present in the food is necessary
for producing some of the specific products. For example, there should be less than
40% of moisture should be controlled during the production of cheddar cheese.
Economically important requirements
Moisture present in the food material is related with some type of food is
dealing with economical values and the big business among industries therefore; food
moisture analysis plays a significant role in the modern world.
Shelf life of the food or food products
Moisture rich foods are easily susceptible to the microbial attack and got rotted
and damaged. Thus, the shelf life of the food material is determined by the moisture
content in the food. Low moisture foods usually slow down growth of
microorganisms.
Food quality measurements
 Quality of the food is determined in terms of the food texture, taste, and
appearance but moisture content of the food is a determination factor of the quality
and the stability of the processed food products.
Food processing operations
Food processing operations are involved with the amount of moisture content
present in the food item which is going to be processed for a specific purpose.
It is therefore important for food scientists to be able to reliably measure
moisture contents. A number of analytical techniques have been developed for these
purposes which vary in their accuracy, cost speed, sensitivity, specificity, ease of
operation etc. The choice of an analytical procedure for a particular application
depends on the nature of the food being analyzed and the reason: the information is
needed. The total water content of food involves the concepts of ‘free’ and ‘bound’
water, equilibrium moisture content, moisture adsorption, moisture desorption etc.
The most important term is ‘bound water’ on which the ultimate accuracy of a method
for moisture content determination is related. Bound water can be physically
adsorbed or chemically bound with proteins, fats or polysaccharides. The method
applied can be classified into two groups: direct or indirect methods. The existing
reference methods for food analysis are all direct methods and are Oven-drying,
Vacuum oven-drying, Azeotropic distillation and Karl-Fischer distillation.

10. Write short notes on: Salad Oil.

Liquid oils are used both as cooking oil and salad oil. Salad oil is defined as a
clear oil which must not exhibit cloudiness when stored in the refrigerator at 40°F
(5°C) for several hours. Salad oils are types of vegetable oil suitable for salad
dressing. The major characteristic is that the Oil is often combined with other
substances to achieve desired flavour and consistency. “Salad” can be referred to any
oil that is commonly used in dressings, so that has a wide range.
The fry oil contains an anti-foaming agent. This agent’s purpose is to break the
surface tension on the top of the oil when it is in a fryer. Most often, the agent used
is Dimethylpolysiloxane, and is typically added at 5 ppm. With this anti-foaming
agent, if you put something with a high moisture content in the fryer, it won't spatter.
This makes it much safer frying in any kitchen.
The salad oil form of any vegetable oil is the resulting oil processed through a
series of steps to produce RBD oil: Degumming - removal of phosphatides,
Bleaching - removal of color bodies and suspended solids, Refining - neutralization
and removal of free fatty acids, with the final step being Deodorization - removal of
tocopherols, sterols, and phenols as well as minute traces of other compounds taken
up during plant growth. The resulting oil is called Salad oil.
 Salad oil can be further processed or Fractionated to improve specific
functionality by separating the short and medium chain triglycerides from longer and
heavier triglycerides. Salad oil is not a Culinary oil or oils that are blended with herbs,
spices and liquids due to the taste or flavor of the oil such as walnut, olive, sesame,
avocado and others. Salad oil has a very low viscosity with no characteristic flavor
or odor and as such is suitable for producing salad dressings, coatings, prepared and
snack food ingredients and similar preparation. Salad oil has a very short shelf life
and is susceptible to rancidity when exposed to heat or oxygen and if often stored
under nitrogen to avoid oxidation. Salad oil is not suitable for frying, baking or heat
related food preparation.
For salads, olive, sunflower, safflower, flaxseed, or walnut oils are excellent
choices. With the exception of extra-virgin olive oil, olive oil has a robust flavor,
while sunflower oil has a delicate taste and a lighter texture. If you opt for sunflower
oil, you can get either high-oleic sunflower oil, which has monounsaturated levels of
80 percent and above, or the more common linoleic sunflower oil, which is high in
polyunsaturated or linoleic acid, an essential fatty acid. It goes rancid quickly - high
or even room temperatures destroy its fragile structure. So, it should be kept it in the
refrigerator and used within a few months of opening. Walnut oil is best used
uncooked or in cold sauces because it becomes slightly bitter when heated.
11. Write short notes on: Winterization of oils and fats.
The process of winterization consists of fractional crystallization of oils and fats
followed by the separation of solids to make high quality salad oils. To design a
winterization process, the rate of cooling of oil, the temperature of crystallization and
the mobility of triglyceride molecules in the oil mass are crucial. These variables play
a significant role both in separating the solid fats as distinct crystals and facilitating
their filtration from the oil. Thus, the main emphasis in this paper is on the effect of
the above variables on the performance of the winterization process.
The term winterization implies that the oil is subjected to cold temperature in
order to separate some number of solids and then separate the solids from the liquid
fraction. The solids are removed via filtration process. The liquid fraction thus
obtained is used as cooking oil, salad oil. This can also be used for formulation of
margarine and shortening. Winterization process removes waxes from sunflower oil,
safflower oil, canola oil, and corn oil and it removes the small amounts of stearines
present in cottonseed oil. Without this process, the oil appears cloudy when stored in
the refrigerator. Winterization makes salad oil that remains clear under refrigeration
for several hours or even in some cases up to 24 hours.
Fractionation is similar to winterization in terms of chilling of the oils in order
to create a solid and a liquid phase in the oil. The solids phase in the cottonseed oil is
made of saturated triglycerides with high melting point. In case of partially
hydrogenated soybean or regular palm oil, the solid fraction is made of saturated
triglyceride, disaturated glycerides, and some monosaturated glyceride.

12. Prolonged boiled milk forms good consistency curds, why?

Curd is obtained by coagulating milk in a process called curdling. It can be a
final dairy product or the first stage in cheese making. The coagulation can be caused
by adding rennet or any edible acidic substance such as lemon juice or vinegar, and
then allowing it to coagulate.
The biggest reason to heat milk to almost boiling before fermenting is that it
improves the texture of the curd. The proteins involved are primarily the casein
proteins. All of the albumin proteins are water soluble and will not add to the structure
of the curd. These albumin proteins denature when they are heated. For this reason,
recipes universally call for the milk to be heated to 1900C and then cooled. The
albumin is denatured and is able to tangle up with the casein during fermentation and
add to the yogurt structure. Skipping this step will make a very profound difference
to the structure of curd. Without its curd will be thinner and much more fragile. When
you scoop it there will be more whey and all that albumin will wash out in it.
These treatments improve the consistency of the curd by denaturing the whey
protein lactoglobulin, whose otherwise unreactive molecules then participate by
clustering on the surfaces of the casein particles. With the helpful interference of the
lactoglobulins, the casein particles can only bond to each other at a few spots, and so
gather not in clusters but in a fine matrix of chains that is much better at retaining
liquid in its small interstices.
When well set curd is disturbed, the whey flows through the channels in the
network and finally separates out. This is called syneresis. The partially denatured
proteins for the reasons discussed above holds more moisture or whey, so syneresis
is less in the curds prepared with high heat-treated milk (boiling). In case of raw milk,
the whey proteins are in native state so possess low water binding ability, hence more
syneresis is expected to take place in that curd. In boiled milk, the proteins held more
moisture and tightly too, so gave low syneresis values. Thus, firmer the curd, lower
the syneresis and vice versa.
Coagulation is essentially the formation of a gel by destabilizing the casein
micelles causing them to aggregate and form a network which partially immobilizes
the water and traps the fat globules in the newly formed matrix. This may be
accomplished with:
 Enzymes
 Acid treatment
  Heat-acid treatment
Chymosin or Rennet, is most often used for enzyme coagulation. It is a complex
set of enzymes produced in the stomachs of ruminant mammals. Chymosin, its key
component, is a protease enzyme that curdles the casein in milk. In addition to
chymosin, rennet contains other enzymes, such as pepsin and lipase. Rennet is used
to separate milk into solid curds (for cheese making) and liquid whey, and so it or a
substitute is used in the production of most cheeses.
One of the main actions of rennet is its protease chymosin cleaving the kappa
casein chain. Casein is the main protein of milk. Cleavage causes casein to stick to
other cleaved casein molecules and form a network. It can cluster better in the
presence of calcium and phosphate, which is why it is occasionally added in cheese
making especially from calcium phosphate-poor goat milk. The solid truncated casein
protein network traps other components of milk, such as fats and minerals, to create
cheese.
Many soft cheeses are produced without use of rennet, by coagulating milk with
acid, such as citric acid or vinegar or the lactic acid produced by soured milk. Cream
cheese, paneer, and rubbing are traditionally made this way. The acidification can
also come from bacterial fermentation such as in cultured milk.

Acid Treatment
Lowering the pH of the milk results in casein micelle destabilization or
aggregation. Acid curd is more fragile than rennet curd due to the loss of calcium.
Acid coagulation can be achieved naturally with the starter culture, or artificially with
the addition of Gluconodeltalactone. Acid coagulated fresh cheeses may include
Cottage cheese, Quark, and Cream cheese.

Heat-Acid Treatment
Heat causes denaturation of the whey proteins. The denatured proteins then
interact with the caseins. With the addition of acid, the caseins precipitate with the
whey proteins. In rennet coagulation, only 76-78% of the protein is recovered, while
in heat-acid coagulation, 90% of protein can be recovered.

13. Enzymes get deactivated at higher temperature, why?

Heating increases molecular motion. So, the molecules of enzyme and substrate
move more quickly and so the probability of occurring a reaction increase. The
temperature that promotes maximum activity is called Optimum Temperature. If
the temperature is increased above this level, then the rate of reaction decreases,
despite the increasing frequencies of collisions. This happens because the secondary
and tertiary structure of enzyme has been disrupted and the enzyme is said to
be denatured.
A protein has to achieve its quaternary structure through secondary and tertiary
structures. Primary structure of proteins contains no hydrogen or covalent bond. Then
it starts folding and different types of bond occurs along the polypeptide chains. The
folding is very important for the proteins for functioning. When proteins are exposed
to excess heat or temperature the internal hydrogen and the non-covalent bonds break
down. So, they lose their quaternary structure and become unable to function. The
enzyme molecules unfold and the precise structure of active site is lost. The bonds
which are most sensitive to temperature change are Hydrogen bonds. If the
temperature is reduced to or under the freezing point, then the enzyme is inactivated
but not denatured. They will regain their catalytic influence once higher temperature
is restored.
Proteins lose their native conformation (i.e., their functional structure) when the
temperature exceeds an optimum value (specific for each). This is due to the fact that
the forces (mostly hydrophobic interactions) that stabilize the native conformation
are disrupted once the temperature is elevated. For most enzymes in the human body,
this optimum temperature is ~37°C. Enzymes are protein and the effect of excess heat
or temperature on enzyme will be same as protein.

14. What is the role of flour proteins during transformation of flour into dough?
Gluten is a protein found in wheat products and is very important in bread
making as it forms the miraculous net that holds bread together, helps dough rise by
trapping gas bubbles during fermentation and gives bread its unique texture.
Although bread begins with many of the same ingredients as cookies, pastries, cakes,
and even shortbreads, it has a completely different consistency. Gluten makes bread
airy and satisfyingly chewy.
Gluten is formed when two of wheat’s native proteins glutenin and gliadin come
into contact with water. The more gluten a flour can produce, the more able the dough
is to hold gas bubbles, and those gas bubbles are what gives bread an open crumb.
Adding water to flour starts a chemical process that can eventually lead to gluten
development. When we grind wheat flour, we destroy the structure of the seed (the
cells and organelles), preventing germination. But a cascade of chemical reactions
will still occur when the flour is hydrated because the materials that cause the
reactions are still present. Gluten development occurs when we add water to flour
and let the enzymes work as they were intended.
Gluten development begins during mixing. The basic point of mixing is to
hydrate flour. Mixing is essential because it speeds up the hydration process and
ensures that water is evenly dispersed throughout the flour. When hydrated, the
glutenin and gliadin proteins almost immediately bind and form gluten. The longer
glutenin pieces link up with each other via disulfide bonds to form strong, stretchy
units of molecules. More compact gliadin proteins allow the dough to flow like a
fluid, whereas glutenins contribute strength. Although hydration happens quickly, it
takes time to form the chemical attachments that knit gluten proteins together into a
strong network. Proteases (protein-snipping enzymes) begin cutting strands of gluten
into smaller pieces that are able to make additional connections. Protease is found in
very small amounts in wheat flour; an excess of it would cut gluten strands too much
and have the opposite effect on the gluten network.
As mixing continues and the ingredients transform into dough, the chains of
proteins become more numerous and elongated; they organize into a sort of webbing
that has both elasticity and extensibility. Without this little protein tango, bread would
be a very different thing: flatter, crumblier, denser, and less chewy.
The network of gluten will continue to develop, gradually becoming stronger
and more complex, up until the dough is fully proofed. Enzymes have even more time
to act while the dough rests and begins to ferment. Chains of gluten grow longer and
stronger as more and more molecules stick together. During bulk fermentation,
bakers periodically fold the resting dough to help align the gluten strands into an
even, organized structure, which gives the dough the integrity it needs to expand as
the carbon dioxide produced by the yeast and water vapor are introduced into the
bubbles. When the gluten network is strong enough, the dough can be shaped. A well-
developed dough can be stretched so thin that it’s translucent.
There are other factors that influence gluten development, such as the type of
flour used. Generally, bread bakers are shooting for an 11%–13% protein level, which
will give good volume and texture to a loaf. Protein content varies among flours, and
in most cases the higher the protein content, the more gluten the dough can typically
form.
The quantity of water present also plays into the gluten-forming process.
Adding too little water won’t work and the flour must be sufficiently hydrated to
activate the proteins that form gluten. Too much water also causes problems, resulting
in more of a batter than a dough, in which a gluten network will form but never
produce a cohesive mass.
Salt strengthens gluten bonding more than providing flavour. Although the
gluten proteins naturally repel one another, the chloride ions in salt help them
overcome that repulsion and stick together. As the salt mixes in and dissolves, the
tacky dough firms up.
Fats, such as butter and oils, slow down the gluten-forming process by coating
the protein strands resulting longer mixing times. The coating acts like a barrier that
prevents gluten proteins from sticking to one another, stunting the growth of long
chains. Fat hinders gluten formation and lead to a soft, tender crumb that is more like
that of a cake.
Certain inclusions can have the same weakening effect. Any inclusion that
contains lots of gluten-killing enzymes that includes raw papaya (rich in papain) and
pineapple (high in bromelain) is generally tough on dough. A workaround is to cook
these ingredients first; high heat destroys the enzymes.
Time serves as a general tool for controlling gluten development; the longer the
flour and water spend together during the hydration process, the more numerous the
gluten bonds will be, while a longer mixing time will speed up hydration by forcing
the water into the flour. Time also allows enzymes to assist in gluten development,
and most notably extensibility.
Mixing methods also matter. Hand-mixing techniques won’t hydrate the dough
and develop the gluten as fast as machines. Using an electric mixer can make many
breads feasible that would otherwise be difficult to mix by hand.
The addition of salt, sugar and fat should be delayed to reduce dough mixing
time. This helps gluten proteins hydrate and develop quickly and provides maximum
friction against mixer bowl.
Mixing is an intensive mechanical operation that produces heat from friction.
This is evidenced by the temperature increase in the mass being transformed into
dough. For proper machining during makeup, a final dough temperature should be
close to 76–82°F (25–28°C).
To assess if the dough is properly developed, perform the gluten film test. A small
portion of dough is stretched between the hands into a thin, smooth, translucent film
to test its extensibility and elasticity.

15. What is dough mixing?

Dough mixing is a process in which flour and water are mixed until gluten is
developed, a result of the enhanced interaction between dispersed and hydrated
gluten-forming proteins. It’s quite different from batter mixing due to differences in
their respective formulations specifically, the proportion between dry and liquid
ingredients.
The goal is to:
1. Incorporate air
2. Hydrate dry ingredients
3. Homogenize the dough by evenly distributing all the ingredients
4. Knead the dough
5. Develop the gluten
Dough mixing can be viewed as a simple reaction in which the reactants transform
into a homogeneous and aerated dough:
 Flour + Water + Air + Energy (Work) → Dough
The mixed dough consists of continuous (gluten) and discontinuous or dispersed
(air cells) phases. Ideally, this mechanical process creates a visco-elastic mass that
has optimum dough handling properties and gas retention capacity, essential for
product expansion during proofing and oven spring.

16. Boiled egg is more nutritious than raw egg, why?

 Poor digestibility and absorption of protein:
Eggs are one of the best sources of protein. It contains all the 9 essential amino
acids which your body needs. But eating them raw, reduces the absorption of this
high-quality protein. Digestibility of cooked egg is 90% whereas that of raw egg is
only 50% being a 40% difference in protein digestibility in favor of cooked eggs. The
higher digestibility of protein in cooked eggs is due to structural changes caused in
egg on cooking.
 Raw eggs may block the absorption of biotin:
Biotin is a water-soluble vitamin and also known as vitamin B7. This vitamin helps
our body in producing fatty acids and glucose. In contrast to egg yolks, that contain
a good dietary source of biotin, raw egg whites contain avidin. Avidin is a protein
that may block the absorption of biotin. It binds with the biotin in the small intestine
which prevents its absorption. Raw egg whites contain avidin which can prevent the
absorption of biotin. Cooking eggs resolves this issue.
Unlike other egg dishes, boiled eggs don’t contain any extra ingredients like oil
or spices. Hence, boiled eggs give pure nutrients with minimum calories. In addition,
boiled egg whites are one of the best protein sources in a post-workout meal. Boiled
eggs are also preferred on a fat loss diet, as there are no extra calories in them. Though
in both forms, an egg contains the same amount of protein, but the digestibility and
absorption of cooked eggs are about 91% whereas raw egg is only 50%. So, cooked
eggs are always healthier as eating cooked eggs are better for protein intake.
Cooking reduces the risk of bacterial infection and increases the absorption of
protein and biotin. However, overcooking eggs can destroy some micro-nutrients,
cooking should be done on a low to medium flame.

17. Roasted soybean is more nutritious than raw soybeans, why?

As for minerals, roasted soybeans include high levels of calcium (supports
bone health) and potassium (perfect for heart health). They also are great source of
magnesium, which promotes a healthy heart, immune system, muscles and nerve
function. Roasted soybeans are the perfect snack. They’re delicious, healthy and can
be thrown into a variety of other tasty snack mixes.
 The best part is that roasted soybeans, also called soy nuts in some circles, are
very easy to prepare. Then, after you are done roasting your soybeans, you can dream
up a bunch of other snack combinations and create your very own trail mix.
Roasted soybeans have several health benefits. Good source of fuel for our
body. High in protein, dietary fiber and carbohydrates. Dietary fiber helps manage
the body’s blood sugar and cholesterol levels. It also helps maintain a healthy
digestive system. Protein is an essential component of any healthy diet. It helps to
repair and grow cells and tissue. Rich in folate and minerals Eating dry roasted
soybeans as a snack adds great levels of folate and minerals to our diet. Folate is a
vitamin helpful for pregnant women and nursing mothers. It can also promote proper
development of new cells.
Soybeans and other soy foods contain phytochemicals called isoflavones. These
are plant-based compounds that could help to lower blood pressure and cholesterol.
Some studies have shown successful results in lowering blood pressure and
cholesterol, specifically in women with high blood pressure, by introducing half a
cup of roasted soybeans daily for eight weeks. The isoflavones found in soy could
also help women overcome adverse symptoms commonly experienced during
menopause and act similar to estrogen in the body.