Trends in Biosciences 8(3), Print : ISSN 0974-8, 611-619, 2015
Estimation of pH of Soymilk on the Properties of Soy Protein Lipid Film
SIVASHANKARI. M, AKASH PARE*, B. K. YADAV AND A. SANTHAKUMARAN
Indian Institute of Crop Processing Technology, Ministry of Food Processing Industry,
Govt. of India, Pudukkottai Road, Thanjavur (TN)-613 005
*email:akashpare@iicpt.edu.in
ABSTRACT
The effect of various concentration of sodium
hydroxide (alkali) on the pH of soymilk, protein-lipid
film formation yield, chemical composition and boiling
resistance was studied. Film yield increased as the
pH of the soymilk increased above its natural pH (6.5).
Film yield was found to be more at 0.4(v/v) and 0.5(v/v)
percent of NaOH concentration in the soymilk than
at 0.1, 0.2 and 0.3 per cent. Maximum protein
incorporation was found at 0.2, 0.3 and 0.4 per cent
levels of alkali incorporation than at 0.1 and 0.5 per
cent. Whereas, the fat incorporation was less at 0.2,
0.3 and 0.4 per cent than at 0.1 and 0.5 per cent due to
the less holding capacity of protein towards non-polar
lipids at higher pH. Carbohydrates and minerals were
also higher as the protein has the capacity to hold
more polar groups at higher pH. Boiling resistance
of protein-lipid films decreased as the percentage of
alkali incorporation into the soymilk increased.
Key words soymilk, alkali, boiling resistance, film
yield, protein incorporation.
Soybeans have been an important source of
protein, fat and flavour for oriental people for
thousands of years. Soybean protein is unique
among plant proteins by virtue of its relatively high
biological value (Schroder, et al., 1973; Liener,
1972). It has been the subject of extensive
investigation as a source of protein for the human
diet. Direct use of soybean products as human food
is a more efficient way of utilizing the highly
nutritional soy protein as compared to feeding
animals and then eating the animal products.
The use of soy protein as a food source is
increasing due to its functional, nutritional, and
nutraceutical properties (Liu, 2000). Soy protein
used in the food industry is classified as soy flour,
concentrate, or isolate based on the protein content.
Soy flour contains 50–59% protein and is obtained
by grinding defatted soy flakes. Soy protein
concentrate contains 65–72% protein and is
obtained by aqueous liquid extraction or acid
leaching process. Soy protein isolate contains more
than 90% protein and is obtained by aqueous or
mild alkali extraction followed by isoelectric
precipitation (Soy Protein Council, 1987). Soy
protein also is used in infant formulas (soy protein
formulas account for a quarter of infant formula
sales) and in baked, meat, and dairy products
(Witherly, 1990). However, despite its high protein
quality, low cost, and plentiful supply, currently
only a small percentage of soy protein is used as
food ingredient, and is thus an underutilized product.
Protein is a good source for edible film formation
and therefore the application of the film could be
suggested (Nemet et al., 2010).
Protein lipid film is one of the most important
food products made from soybean milk. The
protein-lipid film is formed during heating of the
soymilk (Wu and Bates, 1973). It is an endothermic
polymerization of protein or lipoprotein monomers
on the liquid surface promoted by surface
dehydration (Wu and Bates, 1972). It can be
produced in either of two ways: surface film
formation on heated soymilk or film formation from
solutions of soy protein isolate (SPI) (Gennadios
and Weller, 1992). It is a kind of traditional soybean
food which is delicious and of high nutritional value
(Liu, 1997). The protein digestion rate of protein-
lipid film is almost 100% (Han Z., et al., 2005).
Protein and lipids are the main components in
protein-lipid film with carbohydrates and ash being
present in smaller amounts. A typical composition
(w/w, dry basis) of protein lipid film consists of
50.5% protein, 28% lipids, 10.2% carbohydrates
and 0.2% ash (Wu and Bates, 1972).
Organoleptically, Soy protein lipid film itself
is rather bland. It is normally used as an ingredient
in many food dishes, as a wrapping film for other
materials such as meat, and as a starting material
for the fabrication of a wide range of oriental
simulated meat products or meat analogues with
improved texture and flavour (Wu and Bates, 1975).
Dehydrated soy protein-lipid film has to be
reconstituted into a pliable product for these
purposes. The application of these films to food
processing is not new. Among protein-based
materials, those from soybean have been used as:
612 Trends in Biosciences 8 (3), 2015

Fig. 1. Flow diagram for manufacturing of protein-lipid film. The pH of soymilk was adjusted by adding
different concentrations of 5N NaoH and the films are formed.

(i) coatings of fruits, vegetables, cheese, precooked
meats, fried foods, and other food products, (ii) as
carriers of antioxidant agents, flavours and
antimicrobial compounds, and (iii) for encapsulation
and stabilization of flavours and medicines
(Gennadios and Weller, 1991; Petersen, et al., 1999;
Park, et al., 2002).
Protein film formation is pH dependent, (Park,
et al., 2002). Soy protein films can be formed at
both alkaline and acidic conditions. A soymilk of
pH value around nine maximized the film yield and
protein incorporation into films (Wu and Bates,
1972; Sian and Ishak, 1990). Dispersion pH is one
of many variables in film formation, and also has a
considerable influence on the denaturation process.
Soy protein film formation has been achieved within
pH 1-3 and 6-12. No film formation occurred near
the protein isoelectric point (pH 4.5), also the effect
of pH of the film solutions were investigated on
solubility, molecular and structural properties of soy
protein films (Mauriand María, 2006). Chemical
treatments with acid, alkali or cross-linking agents
have been extensively used to improve the
properties of films (Bourtoom, 2009) .For film to
form, the proteins must be dissolved completely
by adjusting pH or by selecting suitable solvents
(Okamato, 1978). Traditional methods of film
formation as performed in the orient consist of
heating soymilk in shallow pans and periodically
removing the films manually when their strength
so warrants, (Wu and Bates, 1975). The
conventional heating method of producing protein-
lipid film is water bath heating, in which it is difficult
to control the heating temperature. Ohmic heating
 MEHRA, et al., New Approaches in Insect Pest Management 613

Fig. 2. Details of the ohmic heating trough: 1, Teflon tray; 2, Stainless steel electrodes all along the width;
3, Thermocouple ports; 4, Data logger; 5, Computer.

of food products is regarded as a potential alternative MATERIALS AND METHODS

to conventional heating, (Lei Long, et al., 2007).
Also ohmic heating holds advantages in producing
safe, wholesome and nutritious food for consumers
(Manvell, 1997) and recently ohmic heating
technology has been applied in food production
successfully. Ohmic heating is a new method in
the production of protein-lipid film of higher quality
from soymilk, (Lei Long, et al., 2007). Thus, it
seemed desirable to study the effect of varying
levels of alkali on the pH of soymilk, protein-lipid
film formation yield, chemical composition and
boiling resistance of the films produced by ohmic
heating technology.
Materials
Soybeans (Glycine max) were obtained from
the local market in Coimbatore, Tamil Nadu and
same variety of soybean was used throughout the
study. They were stored at refrigerated temperature
before protein lipid film production. A batch type
ohmic heating setupdeveloped at IICPT, Thanjavur
was used for the study.
Extraction of soymilk
Extraction of soybean (Glycine max) milk with
a bean-to-water ratio of 1:10 was carried out.
Soybeans were washed and soaked in tap water

Fig.3. Effect of pH on the soy protein-lipid film yield

614 Trends in Biosciences 8 (3), 2015

Fig. 4. Effect of pH on the protein content of protein-lipid film

for 12 h at refrigerated temperature (4-8 °C). The
hydrated beans were drained and ground with tap
water (ten times bean weight), using a soymilk
grinder/extractor plant (Pristine Plants India (Pvt.)
Limited, Faridabad, India) installed at IICPT,
Thanjavur. The grinder/extractor, equipped with
grinder, de-odorizer and filter, could separate
soymilk from the residue okara. The volume and
solids content of the collected soymilk were
measured and maintained uniform throughout the
study. It was recommended that the concentration
of total solids in soymilk be less than 9% as higher
concentrations caused gelation and thickening of
the system upon heating (Wu and Bates, 1973).
The flowchart (Figure 1) for the whole process is
given.
Heating of soymilk
The percentages of alkali (5 N NaoH) used
for the film formation were 0.1 (v/v), 0.2 (v/v),
0.3 (v/v), 0.4 (v/v) and 0.5 (v/v) per cent, where
the soybean milk did not curdle and films could
form on the surface during heating. In each case,
soybean milk was added with desired quantity of
alkali and the pH change was noted down. The pH
change of soymilk was found to be 7.6, 8.2, 9.5,
9.7 and 10.6, respectively. The soymilk with the
varied pH was then used for the film formation
using ohmic heating setup developed at IICPT,
Thanjavur, (Figure 2). Triplicate 500 ml samples
of soymilk after varying the pH were poured into
the acrylic ohmic heating tray of dimension
(15Lx15Wx5H cm) with the two stainless steel
electrodes placed at two opposite ends and was
attached to a control panel where the voltage was
fixed to be 150 V. A data acquisition system for
temperature showing was used, from which the
heating temperature of soymilk could be read
directly. The temperature was monitored using a
T-type thermocouple, which connected with the
stainless steel electrodes and data acquisition
system. The soymilk was heated using a low
frequency alternating current from the public utility
supply (50Hz, 200 V). The milk was heated to and
maintained at a temperature of about 95°C.

Fig. 5. Effect of pH on the fat content of protein-lipid film

 MEHRA, et al., New Approaches in Insect Pest Management 615

Fig. 6. Effect of pH on the carbohydrates content of protein-lipid film

Separation of film determination of protein, fat, moisture and ash

contents according to (AOAC., 1975, method no.
During heating, the films formed on the milk
47.021). The carbohydrate content was calculated
surface were loosened, picked up with a plastic L-
by difference. Boiling resistance of the film formed
rod at 10 mins interval on an average. The separated
at different level of pH was expressed as the amount
films were numbered according to the sequence
of protein leached into boiling solution. Yield referred
of removal and were air dried for 48 h at 25 °C,
to the protein-lipid film yield of every 100 mL
and the weight of films were recorded as film yield.
soymilk, while film formation rate referred to the
Film formation was continued until film could not
protein-lipid film yield every 10 min. They were
fully form at that voltage. The dried films from
calculated as follows:
each batch were ground using mortar &pestle and
thoroughly mixed. All chemical analyses and (1)
experiments were carried out using the ground
samples. (2)
Estimation of film properties
Where Y and V were the yield (g/100 ml) and
Films obtained from each interval for every film formation rate (g/10 min) of protein-lipid film
different percentage of alkali were ground and respectively; M is the total protein-lipid film obtained
stored (after determination of color values) for (g); G is the volume of soybean milk for film

Fig. 7.Effect of pH on the carbohydrates content of protein-lipid film

616 Trends in Biosciences 8 (3), 2015

Fig. 8.Effect of pH on the ash content of protein-lipid film

formation (ml); T is the total time used before
ending film recollection (min).
RESULTS AND DISCUSSION
Film yield
Protein concentration of soymilk have some
effects on protein-lipid film yield and film formation
rate. Some factors other than protein concentration
of soymilk were also important for the yield of
protein-lipid film, (Shen, et al., 2009).
The effect of incorporation of different
concentrations of NaOH to the soymilk on the
properties of protein-lipid film formed were
investigated. The film yield was found to be more
at 0.3 (v/v) and 0.5 (v/v) per cent levels of alkali
incorporation to the soymilk, whereas it was less
in 0.1 (v/v), 0.2 (v/v) and 0.4 (v/v) levels. It can
be said that alkaline pH is favorable for film
formation as at this pH the incorporation of protein
will be more in the film thereby increasing the film
weight. It was the highest at 0.3 (v/v) percent
NaOH incorporation, followed by 0.5 (v/v) and 0.1
(v/v) percent alkali incorporation to the soymilk.
Since there was more protein available at pH 9.5
and 10.6 for protein lipid interaction and
carbohydrate binding, films formed at these two
pH were heavier than those at pH below 9. The
film yield increased by increasing the pH of soymilk
from 6.0 to 9.0. (Wu and Bates, 1973).
By adding 0.1% 10 N HCl to the soymilk
approaching the isoelectric range lowered its pH to
5, no film was formed and the protein coagulated.
Also at soymilk of pHs higher than 10.6, protein
tends to be solubilized or dissociated into its
molecular subunits (Kelley and Pressey, 1966). The
film yield was higher at pH 9.5 and 10.6. However,
the films darkened at an alkali incorporation of 0.6(v/
v) percent to the soymilk as the pH raised to 11.
This may be due to the acceleration of destruction
of sulphur-containing amino acids at alkaline pH’s
(Badenhop and Hackler, 1970) that caused the
weights of the soy protein lipid film to decrease,

Fig. 9. Effect of pH on the Film formation rate of protein-lipid film

 MEHRA, et al., New Approaches in Insect Pest Management
Fig. 10. Effect of pH on the Boiling Resistance
and the yield was very low. At lower pH, some of
the proteins coagulated and could not function in
protein matrix, resulting in low film weight and
yield. Therefore, the percentage alkali incorporation
of choice for the maximum protein lipid film yield
is 0.3 (v/v) and 0.5 (v/v) percent levels (Figure 3).
Protein content
Protein is the vital component in soymilk for
film formation. Maximum protein incorporation in
the film was found at 0.2(v/v), 0.3(v/v) and 0.4(v/
v) percent levels of alkali incorporation whereas
less at 0.1(v/v) and 0.5(v/v) percent (Figure 4).
This shows that an optimum pH range is required
for the maximum protein incorporation into the film.
This is because the pH affects the protein charge
and the degree of protein denaturation. Proteins in
films maintains their native conformation at pH 8.0,
and were extensively and partially denatured at pH
2 and 11, respectively (Mauri, et al., 2008).
At very high pH, protein content of the films
decreased. This may be due to the intermolecular
repulsion of the highly negative charges of proteins.
Similarly at lower pH, some of the proteins
coagulated and could not function in protein matrix
formation resulting in low film weight and yield
(Okamato, 1978).At the extreme acidic or alkaline
pH values, strong electrostatic repulsion of ionized
group occurs in the film-forming solution, which
leads to the solubilization of proteins (Hamaguchi,
et al., 2007). This is because the unfolded proteins
obtained using either acidic or alkaline solubilizing
process underwent the aggregation through
hydrogen, ionic, hydrophobic and covalent
617
bondings, particularly when the water was
removed (Damodaran and Paraf (Eds.), 1997). 
Fat content
Several researchers have reported
improvements of functional and physicochemical
properties of proteins resulting from their
interaction with lipids include improving the
properties of soy films (Bates and Wu, 1975;
Farnum, et al., 1976). Protein-lipid complexes are
known to occur in wheat grains, peanuts, and in
the oil bodies of oil seeds such as soybean,
sunflower and rapeseed and in some fruits and
vegetables. Soy film consists of structure of
continuous protein matrix, in which lipid droplets
are dispersed. Temperature, pH and concentration
are important in producing films from soymilk
(Farnum, et al., 1976; Beckwith, 1984). Soy films
and soymilk are examples of complexes of protein
and lipid; the main soybean proteins (7S and 11S)
are involved in the film formation and in protein-
lipid complexes (Farnum, et al., 1976; Beckwith,
1984). It was observed that at 0.2 (v/v), 0.3 (v/v),
and 0.4 (v/v) percent of alkali incorporation where
the pH of soymilk was quite high (than the native
pH of 6.5) protein solubility was high causes an
increase in emulsifying capacity of proteins which
tends to disperse fat globules more evenly in the
milk, resulting in less fat at the surface and hence
less fat got incorporated into the films. Fat content
incorporation in the films showed a decreasing trend
because of these reasons.
Also, the higher incorporation of negatively
charged proteins at higher pH probably increased
618 Trends in Biosciences 8 (3), 2015
its capacity to hold more polar groups such as
minerals and carbohydrates but less tendency to
hold the non-polar lipids (Figure 5).
At 0.5 (v/v) percent of alkali incorporation
where the pH is very high, the protein carries a net
negative charge and there is electrostatic repulsion
and a reduced tendency to interact.
Carbohydrate and minerals
In all cases, soymilk protein and fat were
incorporated into the films more effectively than
carbohydrates (Figure 6 and Figure 7). Therefore,
the majority of protein and lipid are incorporated
into the film, and the carbohydrate and ash
(minerals) are increasingly concentrated in the
residual whey as well as in the sub sequentially
removed films.
As mentioned earlier, the incorporation of
carbohydrates and minerals are also high as the
protein has the capacity to hold more polar groups
such as carbohydrates and minerals. Carbohydrates
accounts for almost one third of the weight of dry
soy film. The nature of the protein-carbohydrate
interactions is unknown, however, in general, these
constituents associate through ionic and hydrogen
bonding rather than covalent or hydrophobic forces.
So films with higher protein content also had high
levels of carbohydrates and minerals (Figure 7 and
Figure 8).
The differences in the incorporation
efficiencies of the major components (percentage
in films based on total amount in soymilk) among
five soymilks was probably due to pH. In the
soymilk with extreme alkaline or acidic pH, protein
content of the films decreased slightly more than
films at higher pH. This decrease is due to the
solubilisation or dissociation of soybean protein into
its molecular subunits.
Also the presence of highly charged protein
molecules caused less hydrophobic but more
hydrophilic and ionic interactions; thus fat content
decreased, while ash and carbohydrate content
increased. At extremely acidic pH, protein contents
of the films were the lowest, probably due to the
precipitation of the acid-sensitive protein and
formation of insoluble complexes past the
isoelectric point. Thus less proteins were present
at the surface, and less fat globules are trapped.
On the other hand, the percentage of ash and
carbohydrates increased relatively (Sian and Ishak,
1990).
Film formation rate
The natural pH of soymilk is about 6.5. Film
formation is favoured in alkaline pH. At pH values
lower than 6.5 and near the isoelectric range of
protein fractions in soymilk, film formation is
impaired due to protein coagulation. On the other
hand, in extremely alkaline environments, negative
charges along the protein chains create repulsive
forces that inhibit formation of the film protein
matrix (Gennadios and Weller, 1991).
Film formation rate was maximum at 0.2(v/
v) percent followed by 0.1(v/v), 0.3(v/v), 0.4 (v/
v) and 0.5(v/v) percent of alkali incorporation in
soymilk (Figure 9). This was well correlated with
the results observed by Wu and Bates, 1972 and
Sian and Ishak, 1990, where they suggested an
optimum soymilk pH range for film formation
between 7.0 and 9.5.
Boiling resistance
Boiling resistance of the film formed at every
pH was expressed as the amount of protein leached
into the boiling solution. It was found to decrease
as the percentage of alkali incorporation into the
soymilk increased (Figure 10).
As the alkali incorporation to soymilk
increased to 0.3 (v/v), 0.4 (v/v) and 0.5 (v/v)
percent, the number of negative charges increased
due to the increase in pH and as a result weaker
films are formed as the protein molecules tend to
repel each other. This was well correlated with the
results observed by Sian and Ishak (1990). Thus
the boiling resistance of protein-lipid films at high
pH was found to be lower than the natural pH (6.5).
The studies performed allow us to correlate
the chemical composition, yield, film formation rate
and boiling resistance of soy protein-lipid film with
the pH of soymilk. Increasing the soymilk pH by
incorporating alkali in the range of 0.2 (v/v) to 0.4
(v/v) percentage is suggested for film formation
with better protein incorporation and boiling
resistance. The films formed at the pHs above 10.6
are not recommended due to the lower protein
incorporation than the required and decreasing
boiling resistance which may have possible ill effects
on human health. It was observed that at extreme
acidic and alkaline conditions, film formation is
inhibited because of the strong repulsive forces of
highly negative (pH > 10.6) or positive (pH < 6.5)
charges along protein chains, which prevent protein
molecules from associating and forming films.
 MEHRA, et al., New Approaches in Insect Pest Management
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Received on 09-01-2015 Accepted on 13-01-2015