Preparation of whey and isolation of the protein from Milk

Whey
Whey is the watery liquid left after the primary milk protein, casein, is removed. Whey
contains most of the minerals/electrolytes, lactose (milk sugar) and an array of minor proteins.
Although it represents only ~ 20% of the total milk protein, whey protein is highly nutritious
and biologically active.

Whey is left over when milk coagulates, and contains everything that is soluble from milk. It is
a 5% solution of lactose in water, with some minerals and lactalbumin. It is removed after
cheese is processed. The fat is removed and then is processed for human foods.

Processing can be by simple drying, or the protein content can be increased by removing lipids
and other non-protein materials. For example, spray drying after membrane filtration separates
the proteins from whey.

Whey is essential in the bodybuilding world today because of its ability to be digested very
rapidly. This allows the protein to become available for muscle building very quickly. Most
commonly it is used after workouts to help increase levels of amino acids in the blood, which
are taken up by the muscles to ultimately increase mass.

In addition, during exercise, whey helps open up blood flow by inhibiting an angiotensin-
converting enzyme which originally constricts blood vessels; this allows better flow of
nutrients to necessary areas to help repair and rebuild muscle tissues.

Whey proteins
Whey protein is a mixture of globular proteins isolated from whey, the liquid material created
as a by-product of cheese production. Some preclinical studies in rodents have suggested that
whey protein may possess anti-inflammatory or anti-cancer properties; however, human data is
lacking. The effects of whey protein on human health are of great interest and are currently
being investigated as a way of reducing disease risk, as well as a possible supplementary
treatment for several diseases. Whey protein is commonly marketed and ingested as a dietary
supplement, and various health claims have been attributed to it in the alternative medicine
community. Although whey proteins are responsible for some milk allergies, the major
allergens in milk are the caseins.

Whey protein can be denatured by heat. High heat (such as the sustained high temperatures
above 72 °C associated with the pasteurization process) denatures whey proteins. While native
whey protein does not aggregate upon renneting or acidification of milk, denaturing the whey
protein triggers hydrophobic interactions with other proteins, and the formation of a protein gel.
Heat-denatured whey can still cause allergies in some people.

Whey protein Composition

Whey protein is the collection of globular proteins isolated from whey, a by-product of cheese
manufactured from cow's milk. Cow's milk is 20% whey protein and 80% casein
protein,whereas human milk is 60% whey and 40% casein. Whey protein is typically a mixture
of beta-lactoglobulin (~65%), alpha-lactalbumin (~25%), and serum albumin (~8%), which are
soluble in their native forms, independent of pH.

The protein fraction in whey (approximately 10% of the total dry solids within whey)
comprises four major protein fractions and six minor protein fractions. The major protein
fractions in whey are beta-lactoglobulin, alpha-lactalbumin, bovine serum albumin and
immunoglobulins.

Whey protein typically comes in three major forms: concentrate, isolate, and hydrolysate.

• Concentrates contain a low level of fat and cholesterol but, in general, have higher levels
of bioactive compounds, and carbohydrates in the form of lactose — they are 29%–89%
protein by weight.

• Isolates are processed to remove the fat, and lactose, but are usually lower in bioactivated
 compounds as well — they are 90%+ protein by weight. Both of these types are mild to
slightly milky in taste.

• Hydrolysates are predigested, partially hydrolyzed whey proteins that, as a consequence,

are more easily absorbed, but their cost is generally higher. Highly-hydrolysed whey may
be less allergenic than other forms of whey. They are very bitter in taste.

Advantages of whey protein

Whey proteins, in the form of concentrates, isolates and hydrolysates, are the best supplement
choice for most bodybuilders and sports people for the following reasons:

 Over a quarter of whey protein is made up of Branched Chain Amino Acids (BCAAs).
These make up one third of muscle protein. BCAAs are important because, unlike other
essential amino acids, they are metabolised directly into muscle tissue and are the first
amino acids used during periods of exercise. BCAAs exert an anti-catabolic effect by
reducing the breakdown of protein and protecting muscle tissue. If present in a plentiful
supply, they can be deaminated by the muscle cell and burned as energy. This conserves
glycogen reserves and helps in aiding stamina. BCAAs also aid nitrogen retention,
facilitating the maintenance of sufficient nitrogen levels to avoid catabolic muscle loss.

 Studies have shown that poor quality or imbalanced dietary proteins can increase
nitrogen losses and limit muscle protein synthesis, due to inefficient use of essential
amino acids.

 Whey proteins are more rapidly absorbed by the digestive system. The absorption of
other proteins,such as casein, can be slowed by acid coagulation in the stomach .

 Whey proteins provide a taste advantage. They can be easily flavoured and sweetened.
  Instantised agglomerated whey powders are highly soluble and easy to reconstitute. This
makes them perfect for ready-to-mix (RTM) beverages.

 There are no documented side effects or danger of overdose, provided that a person does
not have an allergy to dairy proteins and is not lactose intolerant.
Disadvantages of whey protein
Although whey protein is one of the most excellent forms of protein, it can have dangerous side
effects if taken excessively and even in normal situations like for people who are lactose
intolerant.

• Many people cannot tolerate milk or other dairy products that contain lactose. These
people have what is called lactose intolerance. Such lactose intolerant people may
develop allergic reactions after having whey protein which is made from milk. However,
two forms of this protein- whey protein isolates and hydrolysates- are processed to
remove the fat, and lactose and therefore they might not cause allergy to such people who
can't tolerate milk products.
• Whey protein consumed in very high quantities can affect kidney functions negatively.
• Extremely high doses of whey protein exerts unbearable pressure on liver and may lead
to liver damage.
• Again, if whey protein is taken excessively, it can lead to an imbalance of minerals in the
bones causing loss of bone mineral density. This can result into osteoporosis.

Thus, excessive whey protein intake should be avoided to keep its side effects at bay.
Generally, it is recommended to take about 1 gram of protein (or a little less) per pound
of one's body weight. Training athletes may consume 25 grams of whey protein per day.
Bodybuilders who want some serious body muscle gains may consume 150 grams per
day but only if they exercise heavily.
Available forms and usage
There are many forms of whey protein available. Some contain colostrum, others contain
glutamine and some are primarily lactose free. The removal of lactose may be a particular
advantage for PHAs, who are often lactose intolerant. No conclusive evidence exists to help
identify the best brand at the moment, although DAAIR produces a chart comparing various
whey protein supplements, particularly those most widely used or studied. PHAs using whey
protein generally dissolve 20 to 40 grams of the powder a day in juice, milk or water. It should
not be dissolved in a heated liquid because heat may destroy some of the proteins. This dose
can be divided into several small doses per day.

Isolation and Purification of Casein from milk

Make a dilute solution (approx. 10%) of acetic acid by adding 1 mL glacial (100%) acetic acid
to 10 ml distilled water in 10-mL-Erlenmeyer flask. Mix thoroughly and set aside.

Place 4.0 g of powdered nonfat milk and 10 mL of water into a 50- or 100-mL beaker. Heat on
a sand bath to about 40°C (top of sand bath at about 50°C). Monitor the temperature of the milk
solution with a thermometer.

When the mixture has reached 40°C, add the dilute acetic acid dropwise to the warm milk. Do
not add all of the dilute acetic acid at one time! Maintain the solution at about 40°C and after
every 5 drops, stir the solution gently using a small spatula.

Using the spatula, push the precipitated casein onto the side of the beaker so that most of the
liquid drains from the solid.

Then transfer the congealed casein to a 20-mL vial in small portions. The casein will stick
together and be hard to transfer if you use large pieces. If any liquid separated from the casein
in the vial, use a Pasteur pipet to transfer the liquid back into the beaker.
Slowly continue the dropwise addition of the 1 mL of dilute acetic acid solution to the beaker to
complete the casein precipitation. Remove as much casein as possible from the beaker and
transfer it to the vial.

Avoid adding an excess of acetic acid to the milk solution, as this will cause the lactose in the
milk to hydrolyze to glucose and galactose.

When most of the casein has been removed from the milk solution, add 0.2 g of calcium
carbonate to the milk in the beaker. Stir this mixture for a few minutes and save it for use in the
isolation of lactose below.

Transfer the casein from the 20 mL vial to a Hirsch suction filter funnel. Draw a vacuum on the
casein for about 5 min to remove as much liquid as possible, pressing the casein with a spatula
during this time. (The liquid contains the albumins and lactose--so a great loss of liquid at this
point will result in decreased yields of these other two components.)

Transfer the casein to a 7- to 10-cm piece of filter paper, fold this over onto the casein, and
press gently to absorb any remaining liquid. Place the solid on a tared watch glass, let air dry
until the next lab period, and weigh.

Casein is used to make white glue, so it is important that you don’t leave it on the filter paper or
it will become glued to it.

Whey Protein Isolation

To produce whey isolate the protein goes through the following steps:

• Fresh milk is tested by Quality Assurance experts.

 • The curd portion of the milk fat is separated out for cheese.
• The watery liquid portion that remains is filtered through a process called microfiltration
process; to remove lactose and ash.
• The concentrated liquid goes through the cross flow microfiltration process. Which is
where the whey is isolated by being driven through extremely fine ceramic membranes.
This process eliminates the need for harsh chemicals used in other processes.
• The whey is dried and water is removed.
• Finally, pure whey protein isolate is packaged in its container.

Step 1 – Milk

All of Reflex Nutrition’s protein starts from dairy herds that have be raised and reared in the
EU without the use of banned anti-biotics, steroids or growth hormone. These cattle are also
certified to be free from BSE, the cattle graze on rich pastures to obtain the best possible milk.

The milk is collected on a daily basis by the farmers using milking machinery. Prior to milking
each cow important checks are made to ensure that the cow’s milk meets the required standard.
The cow is then milked using computerised machinery that mimics the action of a suckling
calf, the whole process is designed to be a stress free as possible for the cow.

The milk is stored in refridgerated vats whilst it awaits collection by the dairy company. Milk is
collected daily by large refridgerated lorries where it is then taken to the dairy plant to be
pasteurised. The milk at this stage can either be sold either as full fat milk or used to make
cheese which is what we are interested in.
Step 2 – Cheese and Sweet Dairy Whey.

After the milk has been pasteurised a bacteriological starter is added to 'sour' and thicken the
milk. A renneting agent is added to the milk to form curds. Nowadays, most EU cheese
produced uses a non-animal renneting agent, making the cheese suitable for vegetarians. The
curd is left to set.

The curds are cut so that the whey is released. The curds are either 'cooked' or are piled on top
of each other, and further cut to expel whey. The whey that is expelled during this process used
to be quite literally thrown away or fed to pigs. This whey or sweet dairy whey is the raw
material used to make your favourite protein powder. Nowadays the sweet dairy is carefully
collected and used in the next process.

Step 3 – Ultra Filtration of Sweet Dairy Whey

Sweet dairy whey is passed through vast numbers of ultra filtration units at room temperature.
As the "whey" passes through the filters fat and lactose are removed to leave a concentrated
whey protein in liquid suspension. The liquid concentrate is then dried and instantised for easy
mixing. The whole process is carried out without the use of excess heat which helps prevent
any damage occurring to the product.

There are two primary processes for purifying whey proteins: ion-exchange and ultrafiltration.
These are very different techniques that yield somewhat different results.
The ion-exchange process separates proteins by charge. It involves adjusting the pH of a
solution, so the proteins of interest have a net positive or negative charge.

When the solution is passed over an oppositely charged matrix, these proteins "stick," while the
other components pass through. The "stuck" proteins can then be removed simply by changing
the conditions and washing them out.

There are pluses and minuses to this technique. On the one hand, it can produce a highly
purified protein that is virtually carb and fat-free. On the other, critics charge that it selects for
the major—and less interesting—proteins like beta-lactoglobulin, and depletes the lesser, more
bioactive fractions.

This is why the best quality whey protein supplements are made using the second process:
ultrafiltration. Semi-permeable membranes are used, that permit water and very small
molecules (such as electrolytes and sugars) through, but retain larger ones. This technique is
perfect for producing a supplement with the full range of whey proteins. "Microfiltration" and
"cross-flow microfiltration" are variations on the same theme.

Whey protein powders also differ in their protein percentages. Many commercial supplements
are based on whey protein concentrates, which typically average 75%–85% protein and contain
some residual carbs (from lactose) and fat. These are less expensive than isolates, which
contain > 90% protein, but may also be less suitable for people who are lactose-intolerant, or
are on tightly carb-restricted diets.

Higher purity doesn't make isolates automatically "better" than concentrates, though. Some
products contain more additives than others, which can significantly reduce the amount of
protein you get per serving—even if a highly purified isolate is the primary ingredient. It's
important to read labels (both nutrition AND ingredient labels) to make sure you're not paying
"protein dollars" for inexpensive fillers.
Step 4 – Reflex Nutrition manufacture Instant Whey

Using strict IS09001 protocols Reflex Nutrition manufacture Instant Whey using whey protein
exactly the same as described above. The tricky part is blending the material with the correct
flavourings, colours and sweeteners. However, this process is very carefully controlled to
ensure that every batch we make matches up with our claims for protein content on the label.

Standardizing to target protein

Standardization normally means adding skim milk or skim milk solids, or removing cream to
increase the ratio of protein to fat (P/F). Several practical points are relevant.
• Multiple component pricing makes it possible to cost milk components as individual
ingredients. P/F can then be optimized according to relative costs of protein and fat,
transfer rates of protein and fat from milk to cheese, and the value of fat in the cheese
relative to its value as cream.
• Component yield economies must be balanced against quality.

• Calculation of P/F to produce cheese with required moisture and fat depends on retention
of fat, casein and serum solids in the cheese, where serum solids refers to recovery of the
soluble components of milk, namely, sugars, whey proteins, nonprotein nitrogen and
some minerals. Specifically the important principles with respect to serum solids are:

• Higher serum solids recovery means that a lower P/F is required (that is more fat
or less protein) in the cheese milk to achieve the target FDM in the cheese.
• Serum solids recovery is increased in high moisture cheese because the moisture
retained includes dissolved solids.
 • Serum solids recovery is reduced by curd washing treatments.
• Serum protein (whey protein) recovery is increased by milk pasteurization (there is
more discussion on heat treatments in the next section).

Standardizing to casein/fat ratios

casein number (CN), which we will use in the standardization procedures given below, but
which is important to understand. Total protein content of cows' milk is about 3.3Kg/hL of
which about 2.6 /Kg/hL is casein. The remainder is whey protein (about .7 Kg/hL) including
about .1 Kg/hL of some nitrogenous compounds which are not true protein and are referred to
collectively as non-protein nitrogen (NPN).

Casein is mostly recovered in cheese (i.e., transferred from milk to cheese during cheese
manufacture). Whey proteins remain soluble in whey so that only small amounts are recovered
depending on how much whey is retained in the cheese. Casein content is, therefore, most
relevant to cheese yield, so when cheese makers standardize milk on the basis of protein
content, they are using total protein as an index of casein content.

Direct measurement of casein would be better because the proportion of casein in total protein
varies with breed, season, region and other factors. However, wet chemical analysis of casein is
not feasible for most plants and rapid instrumental methods are still under development.

The percentage proportion of casein in total protein is referred to as the casein number (CN).

Methods of Standardizing
There are three methods of standardizing milk, namely:

1. Addition of concentrated non-fat milk solids (i.e., skim milk powder or condensed skim).

2. Addition of skim milk.

3. Removal of cream.

These methods are based on the assumption that the milk has a high fat content relative to the
protein content. This is normally the case, so that cows' milk usually has excess fat over that
required to produce a legal cheese. The exceptions are high fat cheese such as cream cheese or
double cream blue cheese.

It is not always economical to standardize milk. But the objective of standardization is to

maximize the total return from all milk components while meeting regulations and without
compromising quality. If the value of butter fat is low relative to protein, it is more economical
to sell the fat as cheese rather than as cream provided that the extra fat can be retained in the
cheese without compromising quality.

Protein Standardisation
Micro Protein Determination kit (Sigma Diagnostics, USA) was used to determine protein
concentration in whey. The kit contained biuret, folin and Ciocalteu’s Phenal reagents and a
protein standard. The calibration procedure consisted of the following steps.

For calibration purposes, 0.5 mL of protein standard was diluted to 50 mL in a volumetric flask
with a standard sodium chloride solution. Subsequently, five test tubes are prepared according
to the specifications and 2.2 mL biuret reagent was added to each test tube.

The contents in each tube were mixed well and allowed to stand at room temperature for 10
minutes. In the next stage, 0.1 mL of folin and Ciocalteu’s reagents were added to each tube
and the tubes were shaken well after addition. The tubes were allowed to stand at room
temperature (20°C) for 30 minutes.

The solution from each test tube was transferred to a cuvette and the absorbance was read at
750 nm using test tube 1 as the reference.

The absorbance vs. protein concentration data were plotted and was used as the standard plot
for determining the protein concentration in unknown samples.
Concentration of whey

Two different concentration processes can be used to increase the protein concentration of
whey by the selective removal of water, namely evaporation and membrane filtration.

Concentration of liquid whey at a low temperature can be carried out by vacuum

evaporation, which lowers the boiling point of the liquid so that whey proteins are not
denatured.

Evaporation is commonly used to concentrate whey to 50-60% total solids before

further processing, such as lactose crystallization.

Membrane filtration process can be used to concentrate whey economically between 20-25%.
Further concentration is difficult due to membrane fouling. In this work, however, a ceramic
ultra filtration system was used to concentrate the protein from 0.37 % to 1.4% w/v. Some trials
showed that this concentration was suitable for uniform mixing with the agar.

Protein purification
Protein purification is a series of processes intended to isolateg a single type of protein from a
complex mixture. Protein purification is vital for the characterisation of the function, structure
and interactions of the protein of interest. The starting material is usually a biological tissue or a
microbial culture. The various steps in the purification process may free the protein from a
matrix that confines it, separate the protein and non-protein parts of the mixture, and finally
separate the desired protein from all other proteins. Separation of one protein from all others is
typically the most laborious aspect of protein purification. Separation steps exploit differences
in protein size, physico-chemical properties and binding affinity.
Methods of protein purification

Extraction
Depending on the source, the protein has to be brought into solution by breaking the tissue or
cells containing it. There are several methods to achieve this: Repeated freezing and thawing,
sonication, homogenization by high pressure, filtration (either via cellulose-based depth filters
or cross-flow filtration[1]), or permeabilization by organic solvents. The method of choice
depends on how fragile the protein is and how sturdy the cells are. After this extraction process
soluble proteins will be in the solvent, and can be separated from cell membranes, DNA etc. by
centrifugation. The extraction process also extracts proteases, which will start digesting the
proteins in the solution. If the protein is sensitive to proteolysis, it is usually desirable to
proceed quickly, and keep the extract cooled, to slow down proteolysis.

Precipitation and differential solubilization

In bulk protein purification, a common first step to isolate proteins is precipitation with
ammonium sulfate (NH4)2SO4.

This is performed by adding increasing amounts of ammonium sulfate and collecting the
different fractions of precipitate protein. One advantage of this method is that it can be
performed inexpensively with very large volumes.

The first proteins to be purified are water-soluble proteins. Purification of integral membrane
proteins requires disruption of the cell membrane in order to isolate any one particular protein
from others that are in the same membrane compartment.

Sometimes a particular membrane fraction can be isolated first, such as isolating mitochondria
from cells before purifying a protein located in a mitochondrial membrane. A detergent such as
sodium dodecyl sulfate (SDS) can be used to dissolve cell membranes and keep membrane
proteins in solution during purification; however, because SDS causes denaturation, milder
detergents such as Triton X-100 or CHAPS can be used to retain the protein's native
conformation during complete purification.

Future potential of whey protein

The natural ability of the milk protein, casein, to form nano-sized micelles offers huge potential
to nano-encapsulate sensitive ingredients. Indeed, using fat-soluble vitamin D2 as a model
hydrophobic ingredient, the researchers reported that the micelles could be "useful as nano-
vehicles for entrapment, protection and delivery"of sensitive ingredients. "Such nano-capsules
may be incorporated in dairy products without modifying their sensory properties. This study
introduces new possibilities for encapsulation and delivery of sensitive health-promoting
substances using natural GRAS (generally regarded as safe) ingredients.