The present invention relates to the field of food and dairy products. Specifically, the
invention relates to milk protein concentrate and method of producing said milk protein
concentrate.
BACKGROUND OF THE INVENTION
Globally, the production and consumption of Buffalo (Bubalus bubalis) milk is
increasing day-by-day due to its unmatched nutritional quality like significantly higher total
solids, energy, protein (both casein and whey proteins), larger casein micelles, availability of
only A2 variant of β-casein, better buffering capacity, minerals (calcium, magnesium, and
phosphates), etc.
However, there are huge technical challenges in preparing milk products, especially
high protein milk powders, using buffalo milk. The technological challenges are mostly
linked with the isolation, fractionation, purification, concentration and solubilization of
buffalo milk protein while keeping the protein’s functionality intact.
Thus, the production of second generation dairy industry powdered products like milk
protein concentrates and isolates from buffalo milk is a difficult task as compared to cow
(bovine) milk due to significant variation in their major and minor constituents.
Additionally, the altered and lowered functionality of buffalo milk based high protein
powders also offers the challenges thus, received the attention of both academia and industry.
There are many techniques widely used for isolation of milk proteins. One of them is
membrane processing which is a pressure driven separation technique that widens the spectra
of isolation, fractionation, purification and concentration of milk proteins in their native
forms. Membrane processing is mostly used for the production of MPC with better
functionality and varying levels of protein content. The superior solubility, hydration
property, emulsifying, and whipping ability also enhance the scope of value addition of MPC.
Parallelly, about 2.5 - 3% decreasing export scenario of skim milk powder (SMP) in recent
years (FAO, 2021) and its negative effect on several product formulations like sandiness in
condensed milk, higher acidity in yogurt & undesirable browning in pizza toppings mainly
due to its higher lactose content provides the strong base as an opportunity for MPC
producers to offer MPC as an excellent quality material for dairy and food industry.
Additionally, increasing production demand of MPC powders also creates favorable
conditions for it but, for fulfilling the need base demand of the industry, the MPC should
exhibit superior solubility, wettability, sinkability, dispersibility, emulsification, whipping
and heat stability. These properties are principally dominated by the solubility of powder
3
thus, it is considered as key functional property and the chief requirement of high protein
powder(s) by the dairy/ food industry that jeopardize potential application of MPC as a
functional ingredient for further food formulations.
Notably, the solubility of the high protein powder(s) is mostly affected by several
factors like composition and type of milk; production conditions which has direct influence
on minerals composition, concentration and structure of protein; pH of the solution, ionic
concentration, heating and drying of the solution, modification through chemical, physical,
enzymatic agents, temperature of the process, etc. No doubt, the composition of milk and
type of milk also plays a major role in the solubility of MPC which is obtained from buffalo
milk as claimed by Mahadev and Meena, 2020, where the difference in the compositional
make-up (especially with respect to higher casein & higher calcium contents) significantly
decreased the solubility of MPC at higher protein level. Further, a patent document
CN103783256A discloses MPC produced from cow’s milk comprising a protein content of
not less than 80% by mass and solubility at room temperature of not less than 90%. The
process of production comprises continuous variable volume diafiltration that is the steps of
performing ultrafiltration concentration on a skim milk solution to obtain a milk protein
concentrate by using a continuous variable volume percolation mode. The final product is
produced by vacuum concentration and spray drying of the milk protein concentrate.
Another prior art US7157108B2 discloses the method of preparing MPC and MPI
having solubility of 70-80% comprising removal of 30–100% of calcium ions from a low fat
milk solution using cation exchange on an ion exchange in the sodium form, the potassium
form or the sodium and potassium form and acidification to pH 4.6–6 with subsequent
neutralization following calcium removal and prior to drying. Then, concentrating the
solution by ultrafiltration or ultrafiltration with diafiltration, to form MPC or MPI having at
least 70% dry weight of protein; and drying to prepare a dried product.
Any of the above discussed prior arts do not disclose MPC produced from a buffalo
milk and having high functional properties in terms of solubility and protein content.
Thus, there is a need in the art to provide a universal industry ready scalable process
for the production of high-quality Buffalo Milk Protein Concentrate with a high protein
content.
There is also needed to develop a process which is independent to the composition
and type of buffalo milk which can deliver a highly soluble and high protein powder to the
industry.
4
Such a product (milk protein concentrate) produced will also explore various uses
worldwide without any limitation or restriction. Thus, the process of the current invention
overcomes the drawbacks of prior arts in terms of being simpler and produces milk protein
concentrate of superior functionality. Further, it contains both slow-acting casein and fastacting whey proteins therefore, considered as an ideal component for various food
formulations like processed cheese, ice-cream, beverages, protein fortified foods, nutritional
bars, sports foods, geriatric foods, yogurt, salad dressings etc.
OBJECTS OF THE INVENTION
An important object of the present invention is to provide a method of production of
milk protein concentrate of enhanced functionality from buffalo milk.
Another object of the present invention is to provide a milk protein concentrate of
enhanced solubility and protein content.
BRIEF DESCRIPTION OF FIGURES
The accompanying figures illustrate some of the embodiments of the present
invention and, together with the descriptions, serve to explain the invention. These figures
have been provided by way of illustration and not by way of limitation.
Figure 1 is a schematic diagram showing process (designated as ‘Sukhar-Di Process’) for
producing highly soluble and functional Buffalo Milk Protein Concentrate (BuMRate 60 and
70).
Figure 2 is showing expected changes in casein micelles during refrigeration storage of skim
milk.
Figure 3A is showing combined effect of refrigeration storage and addition of ionic
environment modifier in UFR in the production of highly soluble Buffalo Milk Protein
Concentrate.
Figure 3B is showing combined effect of refrigeration storage, alkalization and ultrasound
treatment in the production of highly soluble Buffalo Milk Protein Concentrate (BuMRate -
70).
Figure 4 shows rehydration behavior of Buffalo Milk Protein Concentrate (BuMRate - 70) in
cold water.
Figure 5 provides scanned electron microscope images confirming the porous nature of
Buffalo Milk Protein Concentrate (BuMRate - 70).
5
SUMMARY OF THE INVENTION
The present invention primarily provides a method of production of buffalo milk protein
concentrate that has enhanced functionality especially high solubility and high protein
content. The simple process for producing the Milk Protein Concentrate comprises following
steps:
a. Pre-heating of raw milk followed by cream/fat separation by sequential
heating, cooling and obtaining skim milk;
b. Storing of skim milk under refrigeration conditions 4-8°C for 24 - 96 hours;
c. Adjusting ionic environment of stored skim milk at desired pH conditions in
the range of 6.7-8.0;
d. Performing ultrafiltration of pH adjusted skim milk at a lower temperature (10
– 30˚C) to obtain ultrafiltered retentate (UFR);
e. Adjusting ionic environment of ultrafiltered retentate to achieve desired pH in
the range of 6.7-8.0 to obtain the stabilized concentrated UFR;
f. Giving ultrasonic treatment to the UFR obtained in step (e) for suitable time
and temperature combination for producing highly stabilized concentrated
UFR;
g. Spray drying of UFR;
h. Obtaining the final milk protein concentrate with superior functionality.
The milk protein concentrate of the present invention has a base for producing various
food/dairy products such as milk powders, high protein dairy powders, dairy whitener, and
several varieties of cheese from buffalo milk.
DETAILED DESCRIPTION OF INVENTION
The details of one or more embodiments of the invention are set forth in the
accompanying description below including specific details of the best mode contemplated by
the inventors for carrying out the invention, by way of example. It will be apparent to one
skilled in the art that the present invention may be practiced without limitation to these
specific details.
The present invention provides a milk protein concentrate produced using simple
scalable processes. The method of producing said buffalo milk protein concentrate
(BuMRate) comprises following steps:
a. Pre-heating of raw milk followed by cream/fat separation by sequential heating, cooling
and obtaining skim milk;
6
b. Storing of skim milk under refrigeration conditions for 24 - 96 hours;
c. Adjusting ionic environment of stored skim milk towards higher side of pH conditions
(6.7-8.0);
d. Performing ultrafiltration of skim milk at a lower temperature to obtain ultrafiltered
retentate (UFR);
e. Adjusting ionic environment of ultrafiltered retentate to achieve desired pH in the range
of 6.7-8.0 to obtain stabilized concentrated UFR;
f. Giving ultrasonic treatment to the UFR obtained in step (e) for suitable time and
temperature combination for producing highly stabilized concentrated UFR;
g. Spray drying of UFR;
h. Obtaining the final stabilised milk protein concentrate.
Unless defined otherwise, all technical and scientific terms used herein have the same
meaning as commonly understood by one of ordinary skill in the art to which the methods
belong. Although any composition, method, system or equivalent to those described herein
can also be used in the production of milk protein concentrates or testing of it, representative
illustrative process and compositions are now described.
As will be apparent to those of skill in the art upon reading this disclosure, each of the
individual embodiments described and illustrated herein has discrete components and features
which may be readily separated from or combined with the features of any of the other
embodiments without departing from the scope or spirit of the present methods. Any recited
method can be carried out in the order of events recited or in any other order that is logically
possible.
In the present invention, the term “milk protein concentrate” or “MPC” refers to a
powder formed from the concentration of buffalo milk, usually comprising 60-70% protein,
solubility (>96%) and manufactured via ultrafiltration and drying.
The term BuMRate as being used in the specification refers to Buffalo Milk Protein
Concentrate.
The term “buffalo milk” as used herein refers to the opaque liquid (milk) containing
proteins, fats, lactose, and vitamins and minerals that is produced by the mammary glands of
mature female buffalo (Bubalus bubalis), after the buffalo has given birth to provide
nourishment for their young. The term encompasses raw milk from other mammals having
similar ionic environment and protein configuration as that of milk produced by Bubalus
bubalis.
7
The term “buffalo skim milk” as used herein is intended to mean heat-treated buffalo
milk of which the fat content cannot exceed 0.50% by weight for 100 g of final product.
The term “ultrafiltration” as used herein encompasses a variety of membrane filtration
methods in which hydrostatic pressure forces a liquid against a semi-permeable membrane.
Suspended solids and solutes of high molecular weight are retained, while water and low
molecular weight solutes pass through the membrane. This separation process is often used
for purifying and concentrating macromolecular solutions, including solutions containing
milk proteins. A number of ultrafiltration membranes are available depending on the size of
the molecules they retain. Ultrafiltration is particularly useful for separating colloids like
proteins from small molecules like sugars and salts. Ultrafiltration may be performed in a
number of ways including dead-end filtration mode and tangential flow filtration (TFF)
mode. The term “ultrafiltered retentate” refers to the portion of the solution that has been
retained by the membrane after ultrafiltration as described herein.
The term “ultrasonic treatment” or “ultrasonication” refers to treatment by applying
ultrasonic waves having a frequency and for a time period as described herein.
The term “spray drying” is used conventionally and broadly refers to processes
involving breaking up liquid mixtures into small droplets (atomization) and rapidly removing
solvent from the mixture in a spray-drying apparatus (e.g., a nozzle) where there is a strong
driving force for evaporation of solvent from the droplets. In a typical spray drying process,
the feed liquid may be a solution, slurry, emulsion, gel or paste, provided it is pumpable and
capable of being atomized. Spray-drying processes and spray-drying equipment are described
generally in Perry's Chemical Engineers' Handbook, pages 20-54 to 20-57 (Sixth Edition
1984). The driving force for solvent elimination or evaporation is usually provided by
keeping the partial pressure of solvent in the spray-drying equipment substantially below the
vapor pressure of the solvent at the temperature of the drying droplets.
The term "food product" as used herein includes solid foods, liquid beverages and
other edible materials regardless of their specific form.
The term "comprising" as used in this specification and claims means "consisting at
least in part of'; that is to say when interpreting statements in this specification and claims
which include "comprising", the features prefaced by this term in each statement all need to
be present but other features can also be present. Related terms such as "comprise" and
"comprised" are to be interpreted in similar manner.
The milk protein concentrate of the present invention has great potential of being used
as functional ingredient in various food and dairy products such as milk powders, high
8
protein dairy powders, dairy whitener, several varieties of cheese, formula milk
powder/infant food, dietary supplements, concentrated & evaporated milk etc.
The method of preparation comprises raw milk being obtained from buffalo or any
other cattle having similar ionic environment and protein configuration as that of buffalo
milk. The milk so obtained is pre-heated and processed further for obtaining skim milk by
cream/fat separation and sequential heating & cooling. In an embodiment, the present
invention provides a skim milk so prepared is stored under refrigeration conditions, wherein
temperature in in the range of 4-8°C for 24-96 hours.
In another embodiment, the milk protein concentrate has an enhanced solubility
wherein said solubility is greater than 96%.
In a further embodiment, the milk protein concentrate comprises protein in the range
of 60-70%, calcium in the range of 420-520 mg/l, phosphorous in the range of 194-260 mg/l,
potassium in the range of 20-120 mg/l, and magnesium in the range of 20-40 mg/l.
In another embodiment, the milk protein concentrate (60-70%) comprises depleted
level of calcium, phosphorous, potassium, and magnesium ranges from 85-88%, 87-90%, 80-
97%, and 87-91% respectively.
In still another embodiment, the present invention provides a method of production of
buffalo milk protein concentrate comprising:
a. Pre-heating of raw milk at 40-50°C followed by cream/fat separation by
sequential heating, cooling and obtaining skim milk;
b. Storing of skim milk under refrigeration conditions at a temperature 4-8°C for
24 - 96 hours;
c. Adjusting ionic environment of stored skim milk at desired pH conditions in
the range of 6.7-8.0 using alkali;
d. Feeding of skim milk for ultrafiltration at a temperature of below 10°C;
e. Performing ultrafiltration of skim milk at a lower temperature in the range of
10-30°C and polymeric membrane to obtain ultrafiltered retentate (UFR);
f. Adjusting ionic environment of ultrafiltered retentate to achieve desired pH in
the range of 6.7-8.0 to obtain stabilized concentrated UFR using alkali;
g. Giving ultrasonic treatment to the UFR obtained in step (f) at temperature of
20-30°C for 2 - 7 minutes for producing highly stabilized concentrated UFR;
h. Spray drying of UFR;
i. Obtaining the final milk protein concentrate with superior functionality.
9
In another embodiment, the method of the present invention involves heating of skim
milk at 80-90°C for 5 min in step (a) followed by cooling to less than 10°C.
In still another embodiment, the method of the present invention in step (c) and (f)
comprises alkali selected from the group consisting of sodium hydroxide and potassium
hydroxide having concentration 1-5 N.
In still another embodiment, the method of the present invention involves usage of
polymeric membrane in step (e), wherein the membrane is Spiral Wound Polymeric Material
(PolyEtherSulphone) membrane.
In a further embodiment, the present invention involves spray drying performed at set
inlet and outlet air temperature of 170-185°C and 70-85°C, respectively. The conditions for
spray drying of the concentrate comprises feed flow rate between 3-5 ml/min, atomization
pressure between 1.17-1.21 kg/cm2 and feed temperature at 55-70°C.
The basic chemistry (shown in Figure 2, Figure 3A and Figure 3B) behind the
invention is that the maximum dissociation of β-casein occurs when milk is stored at
refrigeration temperature (4°C) for 24-96 hours. At lower temperature β-casein is available in
more soluble form, thus, acts as an excellent substrate for plasmin (PL). Further, the PL
mediated actions on β-casein results in the formation of more solubilize β-casein, which is
then present in the serum phase. The storage treatment of milk at 4°C increased about 42%
concentration of soluble casein out of the total casein. And even after storage for 48 hours,
the proportion of soluble β-casein increased upto 60%. Along with β-casein the other fraction
of casein micelles like αsl- and κ-casein also become available in soluble phase up to 30 and
40%, respectively. Depletion of β-casein and availability of more soluble components of
casein micelles could also be responsible for lesser extent of casein-casein interaction along
with a smaller number of calcium bridges formations thus, leading to higher solubility of
developed Buffalo Milk Protein Concentrate using the present process in the invention. The
calcium in milk is present as colloidal calcium-phosphate complex with colloidal casein
micelles. However, the depletion of casein micelles, as discussed above, and increase in the
proportion of soluble fractions of casein protein also results in free calcium. On the other side
the available form of calcium in raw milk fetches poor bridging agent for casein-casein
interaction. Further, the UF processing of skim milk at lower temperature also increases the
solubility of calcium towards higher side thus, greater extent of calcium goes to the permeate.
Further, in normal milk at native pH, minerals are mainly present in colloidal phase
(CaHPO4, Ca2+, H2PO4, and HPO4
-
) and partly present in soluble phase. In buffalo milk, 4/5th
of calcium and 2/3rd of phosphorous are present in micellar form. Calcium is present in two
10
major forms i.e. micellar calcium phosphate and bound form with phosphoseryl residues.
Modification of ionic environment (using Na+
ions towards alkali region) results in the
change of ionization state of phosphorous (HPO4
2-
to PO4
3-
), that increased the affinity of
phosphate group towards calcium ions. As the pH adjustment is not of a great degree, a part
of Ca3(PO4)2 also interacts with micellar casein via phosphoseryl residue, without
degradation of colloidal calcium, in addition to the natural interaction. The modified ionic
environment leads to increase in the concentration of free Na+
ions with higher activity
compared to Ca2+ ions, thereafter the Na+
ions got partly replaced the Ca2+ ions bound with
casein via phosphoseryl residue. This results in availability of free calcium ions in soluble
phase which further enhances the concentration of soluble Ca3(PO4)2 and free calcium ions in
the serum phase. As a result of saturation, it goes to permeate side and the final concentration
of calcium in UFR and resultantly that of Buffalo Milk Protein Concentrate decreases. This is
also confirmed by mineral analysis. Additionally, the partial pH alteration and modified ionic
environment also changes the ionizing state of proteins and results in further degradation of
casein micelles with partial fragmentation thus, increases the intensity of small-small casein
particles in the serum phase. This fragmentation and availability of numerous smaller
fractions of casein particles in serum phase again starts accumulating by the action of
association phenomenon and finally become available in the form of large aggregates.
Thereafter, the ultrasound treatment of treated UFR, dominated with highly active
fragmented casein micelles and solubilized ionic load, results in the reduction of intensity of
attraction force between casein-casein molecules thus, retards or hinders the casein micelle
aggregation phenomenon and also promotes the partial unfolding of proteins (mainly whey
proteins). As a result of this, conformational and structural change in 3-D network of milk
proteins occurs, which increases the overall charge density, exposure of buried hydrophilic
bonds, reduction in static interaction between NH2
+
and COOions due to greater dispersion
and decrease in the size of particles present in UFR, leading to the further stabilization of
UFR during further processing. Substantially, the achievements of ionic equilibrium
conditions also maintain the altered salt balance and improve the heat stability of UF
retentate. This overall improvement in heat stability of UF retentate protects the protein
components of high protein feed from thermal shock during spray drying. This thermal
stabilization and protection of heat sensitive component of UFR along with their salt
balancing is one of the remarkable reasons for the superior solubility of developed Buffalo
Milk Protein Concentrate. Therefore, the resultant Buffalo Milk Protein Concentrate
contained lesser casein aggregates with small particle size, also having lesser calcium
11
available for casein-casein interaction and larger concentration of exposed hydrophilic bonds
which finally due to these phenomenon’s resulted in the higher solubility of Buffalo Milk
Protein Concentrate.
Obtaining buffalo skim milk
In an embodiment, the skim milk is prepared from buffalo milk, or any other milk
obtained from other cattle that have similar ionic environment and protein configuration as
that of buffalo milk.
In another embodiment, skim milk preparation involves heating of buffalo milk at a
temperature ranging from 40-50°C followed by cream or fat separation.
In an embodiment, cream separation can be performed using open bowl type table top
cream separator.
In yet another embodiment, cream separation can be performed using any table top
cream separator or any appropriate cream separator.
In further embodiment, skim milk is stored at appropriate refrigeration conditions and
time duration.
In an embodiment, skim milk is stored at a temperature ranging from 4-8°C for 24 -
96 hours.
Adjusting ionic environment of skim milk
In an embodiment, ionic environment and pH level of stored skim milk is adjusted.
In an embodiment, ionic environment and pH level is adjusted using an alkali.
In another embodiment, adjusting of ionic environment of skim milk at a pH level in
the range of 6.7-8 at a temperature in a range from 20-30°C.
In an embodiment, said alkali is selected from NaOH and KOH.
Ultrafiltration
An embodiment comprises of an ultrafiltration of skim milk is performed for
obtaining ultrafiltered retentate (UFR).
In an embodiment, ultrafiltration is performed at a temperature in a range from 10-
30°C and skim milk was fed at a temperature below 10°C.
In an embodiment, ultrafiltration involves membrane filtration using polyethersulfone
membrane, regenerated cellulose, polyamide, polyvinylidene fluoride or inorganic
membrane.
Adjusting ionic environment of ultrafiltered retentate
In an embodiment, ionic environment and pH level of ultrafiltered retentate is
adjusted to obtain stabilized ultrafiltered retentate.
12
In an embodiment, ionic environment and pH level is adjusted using an alkali.
In another embodiment, adjusting of ionic environment of ultrafiltered retentate at a
pH level in the range of 6.7-8 at a temperature in a range from 20-30°C.
In an embodiment, said alkali is selected from NaOH and KOH.
Ultrasonication
In an embodiment stabilized ultrafiltered retentate is subjected to ultrasonic treatment
to obtain a stabilized concentrated ultrafiltered retentate.
In an embodiment ultrasonic treatment is performed at an intensity in the range from
18 to 22 KHz for 2-7 minutes and a temperature in a range from 20-30°C.
Spray drying
In an embodiment, spray drying of stabilized ultrafiltered retentate is performed to
obtain a stabilised, highly soluble milk protein concentrate.
In an embodiment, spray drying is performed at an inlet air temperature of 170-185°C
and outlet air temperature ranging from 70-85°C.
In an embodiment, spray drying is performed using lab spray drier (SPD-P-111
Technosearch Instruments Thane, India) or any lab spray drier.
EXAMPLES
The following examples particularly describe the manner in which the invention is to
be performed. But, the embodiments disclosed herein do not limit the scope of the invention
in any manner.
Example 1: Process for producing Buffalo Milk Protein Concentrate - 60 (BuMRate -
60)
Buffalo milk used for the experimentation was obtained from Livestock Farm of Guru
Angad Dev Veterinary & Animal Sciences University, Ludhiana. For the preparation of
buffalo milk protein concentrate - 60, the raw buffalo milk was pre-heated at a temperature of
45±2°C followed for cream separation. The fat of the buffalo milk, in the form of cream, was
separated using open bowl type table top cream separator of 10 L capacity (Paras Milk
Seperators, India) to obtain skim milk. The cream separation process was performed twice to
achieve the possible minimum fat content in the skim milk. The total solids and the fat
content of the skim milk were 10.69± 0.50% w/v and less than 0.10±0.05% w/v, respectively.
The skim milk was then subjected to indirect heating at a temperature of 85±2°C for 5
min followed by indirect cooling (<10°C).
13
The skim milk, after heating and cooling treatment was stored under refrigeration for
48 hours at a temperature 5±1°C.
The pH conditions and ionic environment of the skim milk after completion of storage
period was adjusted to 6.8 - 7.3 at 25°C using 2N NaOH.
The pH adjusted skim milk was further subjected to ultrafiltration using CleaNsep
India Pvt. Ltd. (LABSEP-C System P-19004) supplied with Spiral Wound Polymeric
Material (polyethersulfone) membrane and the polyethersulfone membrane had dimension as
12” length and 2” diameter. The skim milk was fed at a temperature of 5°C but, during
ultrafiltration processing the temperature of the retentate raised to about 21±2°C due to the
mechanical or frictional heat generated by the components of the membrane plant even, the
temperature of the water of the double jacketed balance tank of the membrane plant was
maintained 5°C.
After about 4.0 folds concentration of the feed (i.e. skim milk), an ultrafiltered
retentate having total solids of about 21% was obtained with a disturbed salt balance or
mineral composition as compared to the mineral composition of the feed.
Thereafter, the pH or the ionic environment of the ultrafiltered retentate was checked
or adjusted to 6.8 - 7.3 (same as that of the feed of the ultrafiltration) at 25°C using 2N NaOH
to get stabilized ultrafiltered retentate for further processing.
Further, the stabilized ultrafiltered retentate (UFR) was subjected to high intensity
sonication at 20 KHz with 13mm diameter probe fitted ultrasonic 1200W processor (CV 334,
Cole Parmer) at 20% amplitude with 2s ON and 2s OFF pulsation cycle at a temperature of
25°C for 2-7 minutes to provide ultrasonic treatment and a highly stabilized concentrated
UFR was produced. The total energy applied for this process during sonication was 1416
Joule.
Thereafter, the temperature of ultrafiltered retentate was raised up to 70°C using
indirect heating in thermostatic water bath.
The spray drying of the treated UF retentate was performed using Lab Spray Drier
(SPD-P-111, Technosearch Instruments Thane, India) wherein during processing the
temperature of inlet air was at 170°C and outlet air temperature was 70°C.
The other conditions for spray drying of the concentrate was feed flow rate between 4
ml/min, atomization pressure between 1.19 kg/cm2 and hot plate temperature 50°C with
stirrer speed around 290 rpm.
Finally, a stabilized, highly soluble and proteinaceous milk protein concentrate
(BuMRate-60 having protein content of 66%) was obtained.
14
Example 2: Process for producing Buffalo Milk Protein Concentrate - 70 (BuMRate -
70)
Buffalo milk used for the experimentation was obtained from Livestock Farm of Guru
Angad Dev Veterinary & Animal Sciences University, Ludhiana. For the preparation of
buffalo milk protein concentrate - 70, the raw buffalo milk was pre-heated at a temperature of
45±2°C followed for cream separation. The fat of the buffalo milk, in the form of cream, was
separated using open bowl type table top cream separator of 10 lt capacity (Paras Milk
Seperators, India) to obtain skim milk. The cream separation process was performed twice to
achieve the possible minimum fat content in the skim milk. The total solids and the fat
content of the skim milk were 10.69± 0.50% and less than 0.10± 0.05%, respectively.
The skim milk was then subjected to indirect heating at a temperature of 85±2°C for 5
min followed by indirect cooling (<10°C).
The skim milk, after heating and cooling treatment was stored under refrigeration for
48 hours at a temperature 5±1°C.
The pH conditions and ionic environment of the skim milk after completion of storage
period was adjusted to 6.8 – 7.3 at 23°C using 2N NaOH.
The pH adjusted skim milk was further subjected to ultrafiltration using CleaNsep
India Pvt. Ltd. (LABSEP-C System P-19004) supplied with Spiral Wound Polymeric
Material (polyethersulfone) membrane and the polyethersulfone membrane had dimension as
12” length and 2” diameter. The skim milk was fed at a temperature of 5°C but, during
ultrafiltration processing the temperature of the retentate raised to about 21±2°C due to the
mechanical or frictional heat generated by the components of the membrane plant even, the
temperature of the water of the double jacketed balance tank of the membrane plant was
maintained 5°C.
After about 4.5 folds concentration of the feed (i.e. skim milk), an ultrafiltered
retentate having total solids of about 24% was obtained with a disturbed salt balance or
mineral composition as compared to the mineral composition of the feed.
Thereafter, the pH or the ionic environment of the ultrafiltered retentate was checked
or adjusted to 6.8 – 7.3 (same as that of the feed of the ultrafiltration) at 21°C using 2N
NaOH to get stabilized ultrafiltered retentate for further processing.
Further, the stabilized ultrafiltered retentate (UFR) was subjected to high intensity
sonication at 20 KHz with 13 mm diameter probe fitted ultrasonic 1200W processor (CV
334, Cole Parmer) at 20% amplitude with 2 s ON and 2 s OFF pulsation cycle at a
temperature of 25°C for 1 - 4 minutes to provide ultrasonic treatment and a highly stabilized
15
concentrated UFR was produced. The total energy applied for this process during sonication
was 563 Joule.
Thereafter, the temperature of ultrafiltered retentate was raised up to 70°C using
indirect heating in thermostatic water bath.
The spray drying of the treated UF retentate was performed using Lab Spray Drier
(SPD-P-111, Technosearch Instruments Thane, India) wherein during processing the
temperature of inlet air was at 170°C and outlet air temperature was 70°C.
The other conditions for spray drying of the concentrate was feed flow rate between 4
ml/min, atomization pressure between 1.19 kg/cm2 and hot plate temperature 50°C with
stirrer speed around 290 rpm.
Finally, a stabilized, highly soluble and proteinaceous milk protein concentrate
(BuMRate-70 having protein content of 70%) was obtained.
Example 3: Physico-Chemical Analysis of Milk Protein Concentrate (BuMRate)
A physio-chemical analysis of the buffalo milk protein concentrate (BuMRate)
obtained in Example 2 was performed. The total solid concentration, protein concentration,
fat concentration, lactose concentration and mineral analysis was performed.
Total solid concentration:
Total solids of BuMRate were determined by using the gravimetric method as per IS:
SP: 18 (Part XI, 1981). About 1-2 gm of the sample was taken in clean and dried pre-weighed
aluminium dishes. Oven drying of samples was done using a hot air oven at 110 ±1°C for 3-4
hours. Aluminium dishes containing dried samples were placed in a desiccator for cooling
and the weight of cooled aluminium dishes was taken. The process was repeated till no
significant difference was observed in the dried weight of samples. The total solids content
was calculated as:
Total solids (%) =
W2 – W
 100
W1 – W
Where,
W = Weight of empty aluminium dish, in gram
W1 = Weight of dish with sample before drying, in gram
W2 = Weight of dish with the dried sample, in gram
Thus, the content of total solids came out to be approximately 97.5 ± 0.5%.
Protein concentration:
16
For measuring the protein concentration, about 0.5gm sample of BuMRate was taken
for estimation of protein content and the protein content was measured by using the
MicroKjeldahl method (IDF 020-3:2004).
For the digestion, a 0.5g BuMRate was taken in the 300ml of the Kjeldahl flask and
digestion mixture containing 0.2g of copper sulfate and 1 g of potassium sulfate were added.
25ml of concentrated sulphuric acid was carefully added to the flask rinsing any residue of
the sampled sticking to the neck of the flask into the bulb. The samples were heated till the
digest clear and the light blue-green color is obtained. Then heating was continued at the
maximum setting for 1 to 1.5 hours. The flask was removed from the heater and allowed to
cool. The sides of the flask were washed with the jet of approximately 20ml of distilled
water. Again, the flask was heated on the heater for 1 hour and heating was stopped when the
clear sample was obtained. Volume of digested samples were made to 100ml using distilled
water in volumetric flask.
For distillation, the 10ml of the diluted digested sample from the volumetric flask was
transferred to the distillation assembly by using the measuring cylinder. The cylinder was
rinsed with the distilled water and the washing was also transferred to the assembly.
10ml of a boric acid solution of strength 4% was taken in 150ml of the conical flask
and it was placed below the condenser of the distillation unit. The tip of the condenser must
be remaining immersed in the boric acid solution so that liberated ammonia could not escape
from it. 20ml of 50% NaOH was added to the distillation assembly following the addition of
5-10ml distilled water to rinse the wall of the distillation unit.
Immediately after that, the heater was started to liberate the ammonia from the
digested sample. The liberated ammonia was trapped in the boric acid due to which the
purple color will change to green. 60-70ml of distillate was collected in the 150ml conical
flask and it was titrated against 0.02N Hydrochloric acid solution. The endpoint of the
titration was detected by the change from green to purple color. The volume of the standard
hydrochloric acid used in the titration was noted as V ml. The blank was prepared by taking
one gram of sucrose instead of the sample and the digestion and distillation were performed
as above. The titration volume obtained was considered as B. The protein content was
calculated by using the following formulae.
𝑇𝑜𝑡𝑎𝑙 𝑛𝑖𝑡𝑟𝑜𝑔𝑒𝑛 % 𝑖𝑛 𝑠𝑎𝑚𝑝𝑙𝑒 =
14.007 × (𝑉 − 𝐵) × 𝑁 × 100
1000 × 10 × 𝑊
𝑇𝑜𝑡𝑎𝑙 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 % 𝑖𝑛 𝑡ℎ𝑒 𝑚𝑖𝑙𝑘 =
14.007 × (𝑉 − 𝐵) × 𝑁 × 100 × 100
1000 × 10 × 𝑊
× 6.38
17
Where,
N= Normality of the standard hydrochloric acid
V= Volume in ml of standard hydrochloric acid used for milk sample
B = Volume in ml of HCL used for titration of the blank
W = Weight in gram of sample taken
Therefore, the final protein content of BuMRate was found to be nearly 70.1 ± 0.30%.
Fat content:
The fat content of BuMRate was determined by the Mojonnier fat extraction method
as specified in Indian Standards (IS: 1986). 1 gm of BuMRate was taken in Mojonnier tube
and then 1.25ml ammonia, 10 ml ethanol, and 25 ml diethyl ether (DEE) (sp. gr. 0.721) were
added sequentially and shaked properly for 1 min. After the formation of a clear layer, 10 ml
petroleum ether (PE) (40-60°C BP) was added and shaken properly and allow to stand for 15
min. After the formation of two layers, the upper layer was taken into the beaker and then 15
ml of DEE and PE was added and shaked properly. This process was repeated 2-3 times and
the upper layer was collected in the beaker. After that, the beaker was heated in hot water so
that the most of solvent got evaporated. Beaker was put into the oven for oven drying at 98-
100°C and cooled in a desiccator till constant reading was obtained.
Fat (%) =
W2 – W1
 100
W
Where,
W1 = Weight of empty beaker (in gm)
W2 = Weight of extracted fat with beaker (in gm)
W = Weight of sample (in gm)
Therein, the overall fat content was found to be 1.10± 0.05%.
Ash content:
The ash content of BuMRate was estimated gravimetrically by using BIS (2001a)
method with slight modifications. In a pre-weighed silica crucible, about 1 gm of BuMRate
sample was taken and heated for 1-1.5 hr in hot air oven at 100°C.
In a muffle furnace, the contents of the crucible were ignited at a temperature of not
more than 550°C for 3-4 hours, until the ash was carbon-free. The residue was weighed after
cooling in a desiccator. The ash content was calculated as
Ash (%) =
W3 – W1
 100
W2 – W1
Where,
18
W1 = Weight of empty silica crucible (in gram)
W2 = Weight of sample with silica crucible (in gram)
W3 = Weight of dried sample with silica crucible (in gram)
The ash content was about 7.75 ± 0.35%.
Lactose content:
The lactose content was determined by subtracting protein, fat, and ash from total
solids.
Lactose (%) = %TS - (%Protein + %Fat + %Ash)
The final lactose content after subtracting protein, fat and ash from total solids was 18.44±
0.25%.
Mineral analysis:
Mineral analysis was performed by adopting the method given by BorkowskaBarnecka et al. (1996) with some modifications. 0.5 gm of BuMRate was taken in the
digestion flask. A mixture of acid (HClO4 + HNO3 in 2:1) was used to digest the sample
using direct heating of the mixture. Heating was stopped when the mixture was fumes-free.
After that distilled water was added to make up the final volume of digested sample to 25ml,
the same process was used for the preparation of blank, and analysis was done using optical
emission spectroscopy.
Therefore, the mineral analysis resulted in concluding that the content of calcium was
468.1 mg/l, magnesium was 25.38 mg/l, potassium was 22.81 mg/l and phosphorous was
215.24 mg/l in BuMRate-70.
The following tables provide a conclusive chemical composition and mineral content
in Buffalo Milk Protein Concentrate-70 (BuMRate – 70):
Table 1. Chemical composition, HCT, and pH of skim milk, ultrafiltered retentate, and
BuMRate 70
Sample Total
Solids
(%)
Protein
(%)
Lactose
(%)
Ash
(%)
Fat (%) HCT (min)
Buffalo skim
milk
10.69 ±
0.50
4.38 ±
0.45
5.52 ±
0.10
0.64 ±
0.09
0.15 ±
0.05
-
Ultrafiltered
retentate
24.0 ±
0.48
17.26 ±
0.60
4.54 ±
0.15
1.90 ±
0.1
0.27 ±
0.07
23.50 ± 2.0
Milk protein 97.5 ± 0.5 70.1 ± 18.44± 7.75 ± 1.10± 42.0 ± 4.0
19
concentrate
(BuMRate 70)
0.30 0.25 0.35 0.05
Values are presented as Mean ± Standard Deviation (n = 3)
Table 2. Mineral content in BuMRate-70
Calcium (mg/l) Magnesium (mg/l) Potassium (mg/l) Phosphorous (mg/l)
468.1 25.38 22.81 215.24
Example 4: Assessing of functional properties of Buffalo Milk Protein Concentrate
Heat coagulation time (HCT):
A dispersion containing 3.5% BuMRate was prepared by adding 1.75gm of milk
protein concentrate in 50ml distilled water. The dispersion was stirred magnetically using a
magnetic stirrer (Tarsons Digital Spinot) for 3 hrs at 20°C. Stored the prepared dispersion for
18 hrs at 4°C for complete rehydration of BuMRate. After complete rehydration, heat the
dispersion at 70-80°C thereafter, 2ml of heated dispersion was poured into the Pyrex tubes.
HCT was determined by the same method as described by Khatkar & Gupta (2014).
The HCT observed for BuMRate-70 was 42.0 ± 4.0 minutes.
Solubility:
The solubility of BuMRate was determined by using the method as described by
Marella et al. (2015) with some modifications. Five percent of protein solutions were
prepared by adding BuMRate in distilled water at room temperature (25±1°C). Solutions
were stirred magnetically using a magnetic stirrer (Tarsons Digital Spinot) at 300 rpm for 30
min so that proper dissolution took place. For complete rehydration, samples were stored
under refrigeration conditions for 18-24 hours. Rehydrated samples were subjected for
mixing using magnetic stirrer at 300rpm for 30 min at 25±1°C and then pH of dispersion was
adjusted, if required, using 1N NaOH to pH 7 and samples were allowed to undisturbed at
specified temperature for 1 hour for pH equilibrium. Then the dispersion was subjected for
centrifugation process at 700g for 10 min at 20°C. The samples of dispersion and centrifuged
supernatant were analyzed for the determination of total solids content using oven drying at
110°C for 18-20hr. The solubility of milk protein concentrate was calculated as the total
solids in the supernatant to the total solids in the dispersion.
Thus, the solubility achieved in the final BuMRate - 70 was 97.09 ± 0.66%.
20
Emulsion activity and stability:
Emulsification in terms of emulsion activity (EA) and stability (ES) was determined
by the method given by Pedroche et al. (2004). The samples were homogenized at 10,000
rpm for 2 minutes using a high-speed ultra-turax homogenizer (Ika). Then, added 15 ml of
soyabean oil and again homogenized for 3 minutes. After that, the twice homogenized
samples were centrifuged at 2000 rpm for 5 minutes and evaluated for emulsion activity. For
the evaluation of ES, the same tubes were, then, heated in the water bath at 85°C for 30
minutes and followed by centrifugation at 3000 rpm for 10 minutes. The formula for EA and
ES was kept the same as described by Pedroche et al. (2004). Emulsion activity was
calculated by dividing the emulsified layer volume by the total volume of emulsion before
centrifugation. Emulsion stability was calculated by dividing the volume of emulsified layer
after heating by the volume before heating.
The emulsion activity and stability of BuMRate – 70 were nearly 58.33 ± 0.18% and
85.71 ± 0.50%, respectively.
Water activity index and Water solubility index:
Hydration capacity in terms of water activity index (WAI) and water solubility index
(WSI) were determined by using the centrifugation method as described by COBB III &
Hyder, 1972. 1 gm of BuMRate sample was placed in a pre-weighed centrifugal tube and
hydrated with 10 ml of Milli Q water. Mix the solution properly using a vortex shaker
(Tarsons, 220V) at the highest speed for 1 min and allow to stand for 30 min and centrifuged
at 3000g (CPR 30, Remi Elektrotechnik Limited, Vasi, India) for 20 min at 20°C. Decant the
supernatant in a pre-weighed aluminum dish and dried at 105°C until constant weight
recorded. The weight of the gel that remained in the centrifuge tube was noted.
WAI (g/g)=
Weight of gel
Weight of sample
WSI (%)=
Weight of dry solids in the supernatant
 100
Weight of sample
The final water activity index and water solubility index was found to be 0.370 ± 0.001% and
69.00 ± 0.07%, respectively.
Oil binding capacity:
21
OBC of BuMRate was determined by using the method given by Saha and Deka
(2017). 1 gm of BuMRate sample was placed in a pre-weighed centrifugal tube, added 5 ml
of soyabean oil (obtained from local market Ludhiana), and mixed at high-speed using vortex
mixture for 5 min. After thorough wetting, the mixture was allowed to stand for 30 min at
room temperature. Then centrifugeed the mixture at 3000g (CPR 30, Remi Elektrotechnik
Limited, Vasi, India) for 20 min for 20 °C. Decanted the supernatant and centrifugal tube
containing sediment were weighed.
OBC (g/g) =
Weight of tube with sediment
Weight of tube with sample
Therefore, the oil binding capacity of BuMRate - 70 was found out to be 2.260 ± 0.004 g/g.
Foaming capacity and stability:
The foaming capacity and stability were determined using the method given by
Shilpashree et al. (2015b) with some modifications. 0.3 gm sample was taken in 10 ml of
phosphate buffer (pH 7), and mixed properly. Homogenize the samples using a high-speed
ultra-turax homogenizer at 10000 rpm for 1 minute.
Foaming capacity (FC %) =
Volume of liquid immediately after whipping
- volume of sample before whipping
 100
Volume of the sample before whipping
Foaming stability (FS) was calculated by the volume of remaining foams after 30 minutes at
30±2 °C and expressed in terms of initial foams volume.
Henceforth, the foaming capacity was found to be 183.33 ± 0.25 % and foaming stability to
be 133.33 ± 0.50%.
The following table-3 briefly provides all the functional properties of the final product
BuMRate-70:
Functional property Value in BuMRate-70
Solubility (%) 97.09± 0.66
Emulsion activity (%) 58.33± 0.18
Emulsion stability (%) 85.71 ± 0.50
Water activity index (g/g) 0.370± 0.001
Water solubility index (%) 69.00± 0.07
Oil binding capacity (g/g) 2.260± 0.004
22
Foaming capacity (%) 183.33± 0.25
Foaming stability (%) 133.33 ± 0.50
Values are presented as Mean ± Standard Deviation (n = 3)
ADVANTAGES
1. The process of the present invention is a simple mechanical and easy to reproducible
process.
2. Buffalo milk protein concentrate (BuMRate) of the present invention comprises
enhanced solubility of greater than 96% which is a key functional property.
3. The product has high protein content ranging from 60-70% along with high solubility
leading to overall higher functionality.
4. BuMRate can be used for further production of products of high protein and solubility
simultaneously.
5. Valorisation of Buffalo milk so that the available milk can be effectively utilized for the
production of high protein and highly soluble milk concentrate as a good quality protein
source for various food or dairy products such as formulation of several variety of cheese
without nugget formation.
6. The BuMRate has high foaming capacity with high foaming stability due to which it can
be used in formulation of products like cold coffee and ice-cream where foaming is the
pre-requisite requirement and also in production of nano-bubble that to be incorporated
in the products like special types of beverages and other where stability of foams can
provide the desirable characteristics in the product.
7. The product can be effectively incorporated in the formulation of beverages without
flocculation phenomenon due to higher depletion of calcium overcoming the drawback
of high calcium content of high protein dairy powders that creates flocculation in
beverages.
8. BuMRate can also be effectively incorporated in the formulation of cultured dairy
products with desirable acidity (due to lower lactose content compared to skim milk
powder), protein standardization (due to higher solubility), growth of starter culture (due
to high calcium depletion as calcium is the excellent substrate for the bacteriophage) and
high heat treatment products (adaptable heat stability).
9. BuMRate has an enhanced adaptable range of oil binding capacity due to which can be
incorporated in the formulation of dietetic foods also as flavor enhancer.

We Claim:
1. A method of production of a buffalo milk protein concentrate (BuMRate), comprising
the steps of:
a. heating buffalo milk at a temperature in a range from 40-50°C followed by
cream separation to obtain buffalo skim milk and
b. heating of skim milk at 85±2°C for 5 min followed by cooling at a
temperature of less than 10°C;
c. storing the skim milk at a temperature in a range from 4-8°C for 24 - 96 hours;
d. adjusting the ionic environment of stored skim milk at a pH in the range of
6.7-8.0 at a temperature in a range from 20-30°C;
e. subjecting the pH adjusted skim milk to ultrafiltration, wherein the skim milk
was fed at a temperature below 10°C and the ultrafiltration was performed at a
temperature in a range from 10-30°C to obtain ultrafiltered retentate;
f. adjusting the ionic environment of ultrafiltered retentate to achieve at a pH in
the range of 6.7-8.0 to obtain stabilized concentrated ultrafiltered retentate;
g. subjecting the ultrafiltered retentate to ultrasonic treatment at an intensity in
the range from 18 to 22 KHz for 2-7 minutes and a temperature in a range
from 20-30°C; and
h. spray drying the ultrafiltered retentate to obtain buffalo milk protein
concentrate.
2. The method as claimed in claim 1, wherein the skim milk contains less than 12% w/v
total solids and less than 0.5% w/v fat.
3. The method as claimed in claim 1, wherein, the pH is adjusted in step (c) using an
alkali selected from sodium or potassium ions having concentration in a range from 1-
5 N.
4. The method as claimed in claim 1, wherein, the pH is adjusted in step (e) using an
alkali selected from sodium or potassium ions having concentration in a range from 1-
5 N.
5. The method as claimed in claim 3 or claim 4, wherein the alkali is NaOH or KOH.
6. The method as claimed in claim 1, wherein ultrafiltration is performed using a
membrane selected from group comprising polyethersulphone, ceramic and polymeric
membranes.
24
7. The method as claimed in claim 1, wherein the ultrafiltration step (d) reduces the
volume of skim milk by 3.0 to 5.0 folds to achieve a protein content in a range from
60-70% w/v in final product.
8. The method as claimed in claim 1, wherein spray drying is preceded by prior heating
of the ultrafiltered retentate at a temperature in a range from 50 - 70°C.
9. The method as claimed in claim 1, wherein the spray drying of ultrafiltered retentate
is performed at an inlet air temperature of 165-185°C and outlet air temperature
ranging from 70-85°C.
10. The method as claimed in claim 1, the spray drying is performed at feed flow rate
between 3-5 ml/min, atomization pressure between 1.17-1.21 kg/cm2 and feed
temperature ranges from 55-70°C.
11. A buffalo milk protein concentrate obtained by the method as claimed in claim 1.
12. The buffalo milk protein concentrate as claimed in claim 11, wherein the concentrate
has a solubility greater than 96%, and comprises protein in the range of 60-70%,
calcium in the range of 420-520 mg/l, phosphorous in the range of 194-260 mg/l,
potassium in the range of 20-120 mg/l and magnesium in the range of 20-40 mg/l.
13. A buffalo milk protein concentrate comprising protein in the range of 60-70% w/v,
calcium in the range of 420-520 mg/l, phosphorous in the range of 194-260 mg/l,
potassium in the range of 20-120 mg/l and magnesium in the range of 20-40 mg/l.
14. A composition comprising the buffalo milk protein concentrate as claimed in claim 11
or claim 13.
15. An article of manufacture comprising the buffalo milk protein concentrate as claimed
in claim 11 or claim 13, wherein the article of manufacture is a food or dairy products
selected from the group consisting of milk powders, high protein dairy powders, dairy
whitener, and several varieties of cheese