International Journal of Applied Pharmaceutics

ISSN- 0975-7058 Vol 11, Issue 4, 2019

Original Article
HPLC QUANTIFICATION OF LACTOPEROXIDASE IN THERAPEUTIC DAIRY WASTE ENRICHED
BY BUBBLESEPARATION

PRABIR K. DATTAa*, GOUTAM MUKHOPADHAYAYb, AMITAVO GHOSHc

a*Bengal College of Pharmaceutical Sciences and Research, Durgapur 713212 India, bB. C. D. A. College of Pharmacy and Technology,
Barasat, Kolkata 700127, India, cResearch Assistant, Flinders University, Australia 5001
Email: pkdatta57@gmail.com
Received: 23 Jan 2019, Revised and Accepted: 11 Apr 2019
ABSTRACT
Objective: The objective of this study was to enrich therapeutic proteins and remove pollutants from dairy wastewater for establishing foam
fractionation as a lucrative unit operation.
Methods: Dairy wastewater collected from dairy industry was processed to fat-free dried protein waste mass diluted to 1-liter feed by distilled
water in different concentrations and foam fractionated by sodium dodecyl sulphate (surfactant) to enriched proteins extract (foamate) in a foam
fractionator. Foamate were analysed to quantify total proteins and lacto peroxidase respectively. The efficiency was evaluated by varying
parameters like pH, initial waste and ionic concentrations, the waste-surfactant mass ratio of feed and flow rate of gas (N2) through feed solution by
several experiments. Heat of desorption (λ) and mass transfer coefficient (K) were determined as indicators of adsorptive bubble separation to
foam phase governed by Gibb’s equation of adsorption isotherm.
Results: The process was optimized at pH 5.5, initial feed concentration 500μg/ml, waste–surfactant mass ratio (1.5:1), gas flow rate (350 ml/min)
and ionic concentration 0.1 gram-mole of NaCL per litre of feed with enrichment factor (49.09), percent recovery (98.18%) observed in foamate.
One natural preservative specifically lactoperoxidase was quantified by RP-HPLC analysis as 0.49% (w/w) of total proteins at optimal condition.
Heat of desorption(λ), mass transfer coefficient(K)were determined 3140cal/mol and 12.68* 10-9 mol/cal/cm2/s respectively at pH 8.5, initial feed
concentration 500μg/ml and gas flow rate 350 ml/min.
Conclusion: The method may be a useful unit operation for recovery of biomolecules and removal of toxic pollutants from industrial wastewater for
coming days.
Keywords: Foam fractionation, Sodium dodecyl sulphate, Enrichment, Lactoperoxidase, Medicinal proteins, RP-HPLC
© 2019 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open-access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijap.2019v11i4.32058

INTRODUCTION This technique has also been applied for removal of toxic metals and
chemicals from industrial waste streams [9-11].
Worldwide milk production rising every year by more than 1% that
reached around 800 tons in the year of 2017. India will become the The dairy wastewater chiefly contains a mixture of medicinal
leading milk production country for the coming year, 2026. Under proteins namely bovine serum albumin (BSA), alpha-lactalbumin (α-
these circumstances, huge amount of dairy wastes will be generating LA), beta-lactoglobulin (β-LG), immunoglobulin (IG) [major
from various dairy products such as milk, yoghurt, desserts and proteins] as well as bovine lactoferrin (BLF) and bovine
custards, cheese, butter, milk powders etc. Dairy wastewater contains lactoperoxidase (BLP)[minor proteins]. Lactoperoxidase is a natural
a variety of therapeutic wastes along with other compounds [1-3]. preservative free from an untoward effect on the human body and
useful anticaries component in toothpaste formula [1, 12-15]. It may
In this context, application of the lucrative technique for co-product be used as a harmless preservative in pharmaceutical and food
recovery from dairy wastewater is very significant to serve the dual products. The proteins under study have isoelectric pH (pI) at
purpose for controlling environmental pollution as well as recovery of approximately 5.5(major) and 9.0(minor) respectively [16, 17]. In
pharmaceutical and nutritional dairy waste such as the variety of this study, we assessed the batch process of foam fractionation for
proteins and other molecules for the health of man and animal the enrichment of multicomponent proteins mixture from dilute
kingdom. The currently applied techniques like ultrafiltration, Nano dairy waste. In addition, RP-HPLC analysis was performed to
filtration, electrodialysis, ion-exchange, gel-filtration, precipitation and measure the quantity of single protein (lactoperoxidase) present in
coagulation are expensive. Hence, it is necessary to search substitute enriched protein mixture of formate by foam fractionation.
gainful techniques of therapeutic assistance for the coming days. Foam
MATERIALS AND METHODS
fractionation provides various benefit like easy scale-up, nonstop
operation, suitable for refinement of heat sensitive molecules in the Materials
biotechnological process pathway without application of heat, limited
space, low power consumption, no additional cost of solvent and high Dairy wastewater was collected from local dairy industry (Kolkata). BLP
yield for dilute feed. The technique is under the foam division of was obtained as a gift sample from S. A. Pharma Chem. Pvt. Ltd., India. AR
“Adsorptive Bubble Separation Method” proposed by Robert Lemlich grade Sodium dodecyl sulphate (SDS), sodium chloride (NaCL),
in his edited book [4]. The principle of separation of the technique is acetonitrile (ACN), methanol (CH3OH), trifluoroacetic acid (TFA)(all
based on physical or chemical adsorption of surface active molecules HPLC grade), concentrated hydrochloric acid (HCL) and sodium
on the gas-liquid interface (bubble’s surface). The amount of surface hydroxide (NaOH) were procured from E. Merk Ltd (Mumbai, India). All
other solvents used were of analytical grade, procured from E. Merk.
active molecules adsorbed can be quantitatively expressed by Gibb’s
Equation of Adsorption Isotherm [4, 5]. Methods
Numerous researchers applied this technique in the field of Initial processing of dairy wastewater
pharmaceutical biotechnology for enrichment, purification and
extraction of thermolabile medicinal proteins and a variety of Dairy wastewater was filtered through muslin cloth and centrifuged
natural pharmaceuticals from plant extracts and bio-sources [6-8]. (Remi, India) several times) for removal of fat from wastewater till
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constant absorbance(UV-1800,Shimadzu,Japan) was recorded and Determination of critical micelle concentration and isoelectric
250 ml of such processed dairy protein solution was evaporated at pH of total and target protein (BLP) by surface tension (γ)
45 °C for 4h by using Eyela rotary evaporator (Indosathi scientific method at operating temperature 20-25 °C
lab, India) to dried protein mass. The obtained mass (195g) was
preserved in a refrigerator at (-18 °C) until use. Surface tensions of different concentrations (50-900μg/ml) of processed
dairy protein waste in double distilled water were determined in a
Preparation of standard curve for total protein quantification tensiometer by interfacial surface tension method to find out critical
micelle concentration (CMC) [fig. 1]. The slope of surface tension (γ) vs
The protein mass was diluted in the concentration range of 50-900 concentration (c) plot will give the value of the molar quantity of
μg/ml in double distilled water and absorbance of each therapeutic proteins adsorbed on the surface of the bubble (τ value)
concentration was determined in a spectrophotometer(UV- which is calculated from Gibb’s equation. The isoelectric pH of BLP
1800,Shimadzu,Japan) at wavelength 280 nm to draw the standard (target protein), as well as dairy waste protein solution, were
curve of protein waste powder solution essential for the analysis of determined at different pH by using 0.1(N) HCL and 0.1(N) NaOH below
total protein extracted in foamate. CMC by tensiometer (Deeksha instrument corporation, India) [18].

Fig. 1: Plot of surface tension vs concentration of dairy protein waste solution

Table 1: Characteristics of medicinal proteins in dairy waste

Medicinal protein in dairy waste Mol. wt. Isoelectric pH(pI) [dγ/dc] Range of conc. (μg/ml) CMC
(Da*103) (dyne cm2/μg) of constant slope (μg/ml)
BSA(major) 69 5.1 ----- ------ -----
BLF(minor) 84 9.0 ------ ------ -----
BLP(minor) 89 9.6 0.059 5-175 175
α-LA(major) 14 5.3 ------ ------ ------
β-LG(major) 18.30 4.8 ------ ------ ------
IG(major) 100 5.5 ------ ------ ------
Dil. protein waste 25.60 5.2 0.329 50-750 750
Dil.(Protein waste+SDS) [1.5:1(w/w)] ------- ------- 0.301 85-800 800

Fig. 2: Experimental set up for foam fractionation

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Foam fractionation HPLC analysis of BLP in foamate extract

The experimental foam fractionator (fabricated by the local The HPLC system (Waters, MA, USA) was consisted of Symmetry 300
fabricator in Kolkata, India) consists of a glass column (100 cm C4 protein analysis column (50 mm × 4.6 mm; particle size 5 μm; pore
length) and internal diameter (8 cm) with the thickness of 0.5 cm size 300 Å) and equipped with a guard column. The temperature of the
which was attached with nitrogen gas cylinder as the source of gas column was kept at 25 °C. The analysis was consisted of a 600
supply for the formation of bubbles by a glass porous frit no. G3 (15- controller pump, a multiple-wavelength ultraviolet-visible (UV-Vis)
40micron porosity) fused at the base of the column (fig. 2). The detector equipped with 50 μl loop injector (Rheodyne±, Cotati, CA,
dilute feed (1L) was prepared from processed dairy waste by adding USA). The outputs were processed and recorded in a compatible
SDS with varying mass ratios and concentration of total proteins in integrator (model 486, Waters, MA, USA).
the foamate was determined by spectrophotometric analysis (UV- HPLC assays were performed using an isocratic system of 0.1%
1800, Shimadzu, Japan) at wavelength 280 nm. The separation trifluoroacetic acid (TFA) in water (A) and acetonitrile (ACN) (B) with
efficiency of the experiment was optimized at pH5.5 of feed solution, the ratio of 95:5 (v/v). The run time was set at 30 min. The Flow rate
gas flow rate 350 ml/min, waste-surfactant mass ratio (WSR=1.5:1 was set at 1.0 ml/min and the absorbance was detected at 220 nm.
w/w), initial dairy waste concentration (CI=500 µg/ml) and ionic
concentration of feed(IC) by adding 0.1g mole of NaCl per litre of feed Studies on an interfacial area of adsorption
solution. The pH of dilute feed was adjusted with 0.1(M) of HCL or
NaOH solution. The gas flow rate (GFR) in ml/min was kept under Determination of surface area of adsorption is important to determine
observation by the rotameter. The GFR, pH of the feed solution, IC, the mass transfer coefficient (K) of protein molecules to foam phase
initial feed concentration (CI) and WSR were varied to find an impact [19]. The interfacial area is calculated by the following in equation 1.
on the adsorptive separation efficiency of total protein in foamate. 6AC Hε
Gas bubbles generated by the sparger (bubbles distributor through As= − − − − − − − − − − − − − − − (1)
feed solution) ascend through the column and deposit as foam over d32
the dilute feed. The aggregated bubbles in the form of foam with
adsorbed surface-active molecules (total dairy proteins from dilute Where H is the height of liquid feed in the column, Ac is the column
waste solution as feed) by formation of complex with SDS cross-sectional area (in this case 50.25 cm2), AS is the interfacial area
(surfactant) as well as due to individual surface active properties of foam phase for adsorption, ε is the void fraction determined from
move upward by the pressure of gas passing through the feed % gas hold up and d32 is bubble sauter mean diameter for individual
solution in the column and finally collected as foamate from the top location determined by equation 2.
outlet of the column which is converted into concentrated extract by
foam breaker (Remi, India). The weight, volume of foamate and foam d 32 =
∑d 3

− − − − − − − − − − − − − (2)
breaking time were accurately calculated for analysis and ∑d 2

determination of foam strength respectively. Foamate samples were

withdrawn at different time intervals (5, 15, 25,35,45,55 min) and d32 is the bubble mean sauter diameter given by equation 3, where
foamate extract analysed to quantify total proteins by (k) is the number of locations (in this case 4) of the column where
spectrophotometer (UV-1800, Shimadzu, Japan) and by RP-HPLC bubbles photograph taken.
(Waters, MA, USA) for analysis of BLP. k
d 32 = − − − − − − − − − − − − − ( 3)
1
Evolution of performance ∑d
32
Performance of foam fractionation are indicated by three
parameters namely (i) enrichment ratio(ER) equal to the ratio of Gas was passed at varying GFR (250,300 and 350 ml/min respectively)
CS/CI, where CS is the concentration of protein in foamate and CI is through 1liter feed solution and bubbles were photographed at 4
the initial concentration in feed, (ii) percent recovery (%Rp) different locations namely 5, 15, 25 and 35 cm vertical distances from
calculated by the ratio of amount of protein in foamate (AFM) to the the sparger (fig. 3). The photograph was developed in the computer and
amount of protein in feed (AFD) multiplied by 100 [% Rp=(AFM/AFD) * bubbles diameter was determined manually by super pixel scale after
100] and (iii) separation ratio(SR) is equal to the ratio of CS/CR, enlargement. 160-165 such bubbles were measured for each plate and
where CR is expressed as concentration in residual solution. The mean sauter bubble diameter (d32) was calculated for respective flow
highest values indicate the optimum efficiency of separation of rate. Data were recorded in table 2 [19].
proteins to the foam phase.
The percentage gas hold up (ε*) was also calculated for different
Quantification of total protein mixture in foamate by UV superficial gas velocities through the liquid feed from the maximum
absorbance drop in liquid level (∆L) from initial level (L) by sudden stoppage of
the gas supply by equation (4). Data were tabulated in table 2 and
The total protein in foamate, feed and residue in foam fractionation relation with interfacial area represented by fig. 4.
experiments were quantified at wave length 280 nm by using
spectrophotometer (UV-1800, Shimadzu, Japan) [19]. ∆L
(ε*)= *100----------------------- (4)
L

Table 2: Effect of superficial gas velocity (SGV) on interfacial area

SGV (cm/s) GFR Sauter mean %gas hold up Interfacial area (cm2) Percent recovery Feed density Feed
(ml/min) diameter (ε*100) (% Rp) (g/cc) viscosity
d32 (cm) (Poise)
0.083 250 0.0621 0.90 845.09 90.98 1.235 0.0095
0.099 300 0.0705 1.19 1012.85 93.99
0.116 350 0.0821 1.38 1117.29 98.18

Table 3: Effect of molar ionic concentration(IC) on (ER) and (% Rp)

Molar ionic concentration (IC) Percent gas hold up (ε*) Enrichment ratio (ER) Percent recovery (%Rp)
0.0125 0.70 32.80 70.40
0.05 0.785 33.39 78.80
0.1 0.905 40.45 90.99
0.15 0.810 36.52 80.35

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V  1 C 
ln  O  = ln  O  − − − − − − − − − − − − − − − − − ( 5 )
VB  ( )
e λ / RT − 1  C B 

A θ
∫ dθ = K Asθ − − − − − − − − − − − (6)
CS d (Cs)
∫CSo  CS 
= K  S
 VS 0
Vs
λ − RT ln 
 CB 

Where, V0= initial bulk volume of liquid(ml) at zero time (θ=0),VB=

bulk volume of liquid (ml)after any time (θ min), VS= volume of
liquid of foam phase at any time (θ), C0 =concentration in bulk at
time (θ=0), CB= protein concentration in bulk(μg/ml) after any time
(θ), CS=protein concentration (μg/ml) in foam phase after any time
(θ min),T(K) = absolute operating temperature, R= gas molar
constant=8.3143*107ergs/degree/mol, AS= interfacial area in cm2,
Fig. 3: Bubble distribution at SGV 0.083 cm/s θ= residence time of foam phase obtained from the volumetric flow
rate of bubbles and the height of the column, λ, the latent heat of
desorption in cal/mol can be determined from the slope [1/(e λ/RT-
1)] of ln [V0/VB ] vs. [ln C0/CB] plot [ equation 5, fig. 6][4].
For determination of K, the left-hand side integral of equation
(6)was determined by graphical integration of [λ-RT ln (CS/CB)]-
1vs.[CS]plot initiating from CS0 to CS by determining different values of

area under curve those are integrals of [λ-RT ln (CS/CB)]-1at different

intervals of foamate collection i.e.(0-5), (5-15),(15-25),(25-35) mins
etc. The different values of integrals i.e. CS*[λ-RT ln (CS/CB)]-1* 107
were plotted against different collection times (θ=t) and the slope
(m) of the line was equal to the value of, m=[K(AS/VS)](fig. 7and
equation 6). The foam thickness [t]=[Volume of foam (VS)/area of
foam(AS)] was determined by Gibb’s equation: (e λ/RT-1) =(1/t RT)
*(-dγ/dC) and “t” value can be calculated from R(=8.317*107ergs/
°C/mole), T(=298K), (-dγ/dC)= 0.329 (from table1), λ and average
mol. wt.(25,600) of dairy proteins waste. The mass transfer
Fig. 4: Effect of superficial gas velocity on the interfacial area
coefficient (K) was determined from the measured value of t and
value of slope (m), K= [t*m][4]. In this study, the mass transfer
coefficient was determined on the basis of average molecular weight
Where ∆L is the maximum drop in liquid level and L is the initial of multicomponent dairy protein waste [25.6 Kilo Dalton (KDa) as
level of the liquid pool. Void fraction (ε) multiplied by 100 will give shown in table1] calculated from the respective molar mass fraction
the percentage gas hold up. of individual proteins (Bovine serum albumin-5%, β-lactoglobulin-
50%, α-Lactalbumin-12%, Immnoglobulin-10%, Bovine lactoferrin-
Studies of the effect of ionic concentration on gas hold up
1%, Bovine lactoperoxidase-0.5%.).
The effect of ionic concentration in feed on gas hold-up percent
(ε*),enrichment ratio (ER) and percent recovery(%Rp) was studied
at a fixed GFR(250 ml/min), CI (500μg/min), pH5.5 and WSR
1.5:1(w/w). Four different concentrations namely 0.0125, 0.05 0.10
and 0.15g mol of NaCL/l of feed were chosen for the study and data
recorded in (table 3 and shown by fig. 5) [20].

Fig. 6: ln (V0/VB) vs. ln (C0/CB) Curve for (λ) determination

Fig. 5: Effect of ionic concentration on ER and % Rp

Theory of molar mass transfer to the foam phase

By application of material balance for surface active proteins in the
two-phase system (liquid and foam), we can determine
experimentally the latent heat of desorption (λ) by equation (5) and
mass transfer coefficient (K) by equation (6) respectively from the
known value of λ. These two parameters are the markers for the Fig. 7: CS* [1/(λ-RTlnC0/CB)]*107 vs. time (t) plot for K
separation of protein molecules to the foam phase [4]. determination

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RESULTS Foam thickness (t) = (VS/AS) was calculated by the equation:

[1/(eλRT-1)] = (1/t RT) (-dγ/dc).
Determination of λ and K values
Effect of pH at a fixed GFR of 350 ml/min
From fig. 6, [1/(eλRT-1)] =0.0059(slope) and λ=3140cal/mol
approximately. Effect of pH of feed solution at a fixed GFR was recorded in table 5
and represented in fig. 9. The maximum values were recorded with
From fig. 7, K [AS/VS] = (K/t) = 0.021*10-7(slope). So, K= [t]* enrichment ratio (ER=49.09) and percent recovery (%Rp=98.18) for
[0.0218*10-7] =6.041*0.0218*107=12.68*10-9 mol/cal/cm2/s,t= foam total protein as well as BLP of 0.49% (w/w) in enriched protein
thickness=6.014 cm(calculated from R, λ,T(298K) and (dγ/dc)= (- extract (foamate) at pH 5.5. All data were found in the order of
0.329) from table 1. pH5.5˃2.5˃8.5 respectively.

Table 4: Experimental results showing the effect of changing GFR at pH5.5

Lot Exp pH Feed conc. Gas flow Concentration in Enrichment %Rp (BLP) Heat of
No. No. (µg/ml) rate foamate (CS) ratio (ER) (Total %(w/w) desorption
(CI) (ml/min) (μg/ml) protein) λ (cal/mol)
1 2 5.5 400 250 13478 33.70 77.50 0.38 2907
2 2 5.5 500 250 20898 34.84 91.95 0.44 2833
3 2 5.5 600 250 22295 37.16 81.75 0.39 2991
4 2 5.5 400 300 15087 37.72 86.75 0.41 3420
5 2 5.5 500 300 20207 40.41 92.95 0.46 2878
6 2 5.5 600 300 22595 37.65 82.85 0.40 3204
7 2 5.5 400 350 16718 41.80 91.85 0.45 3127
8 2 5.5 500 350 24545 49.09 98.18 0.49 3360
9 2 5.5 600 350 24585 40.98 83.75 0.42 3326
Feed volume=1liter; foamate collection time =55 min

(A) (B)

(C)
Fig. 8: Effect of different GFR: (A) [250 ml/min], (B) [300 ml/min], (C) [350 ml/min] on ER and %Rp at pH 5.5 of feed solution

Table 5: Experimental results showing the effect of pH at GFR 350 ml/min

Lot Exp pH Feed conc. Gas flow Concentration in Enrichment %Rp (BLP) Heat of
No. No (μg/ml)(CI) rate foamate (CS) ratio(ER) (Total %(w/w) desorption
(ml/min) (μg/ml) protein) λ (cal/mol)
8 1 2.5 500 350 20440 40.90 79.91 0.40 3330
8 2 5.5 500 350 24545 49.09 98.18 0.49 3360
8 3 8.5 500 350 19950 39.90 77.85 0.39 3140

Effect of changing GFR at a fixed pH 5.5 and CI 500 μg/ml of feed foamate enhanced with the increase of GFR at fixed pH 5.5 and CI500
solution μg/ml. From experimental results recorded in table 4, it was observed
that the enrichment ratio (ER), percent recovery (% Rp) and mg % of
From table 4 and fig. 8, 10, it was found that enrichment ratio (ER) and BLP in extract increased as a whole when GFR changed gradually from
percent recovery (%Rp) of total protein as well as mass % of BLP in the values of 250, 300 and 350 ml/min respectively.

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Effect of superficial gas velocity (SGV) on% gas hold up (ε*)

The effect of superficial gas velocity (SGV) on % gas hold and % Rp were
represented in fig. 4. Gas hold up enhances linearly up to the superficial
gas velocity (SGV) of 0.199 cm/s. In the present study, SGV was
maintained in the range of 0.0829–0.116 cm/s as shown in table 2.
Percent recovery (% Rp) is enhanced with the increase of the interfacial
area.
Effect of ionic concentration (IC) on % gas hold up at a fixed GFR
250 ml/min
Fig. 9: Effect of varying pH (2.5; 5.5 and 8.5) on ER and %Rp
From table 3 and fig. 5, it was observed that values of ER and %Rp
increased from 0.0125 gmol/l to 0.1 gmol/l and after reduced at
0.15 gram-mole/l. The maximum % gas hold up (ε*) was noted
0.905 at 0.1 gmol of NaCL/l of feed solution as ionic concentration
(IC) of feed [20].
Quantification of BLP by HPLC method
The mean Rt was observed for lactoperoxidase at 18.01±0.03 min
by comparing between standard fig. 11 (A) and dairy waste
protein chromatogram fig. 11 (B). The calibration range of BLP
was found to be 100-800 μg/ml, with the linear equation Y=
184006.75X+44218, with the coefficient of determinants (r2) of
Fig. 10: Effect of GFR on mass % (w/w) of Lactoperoxidase at pH 0.994. The amount of BLP was found 0.49% (w/w) of enriched
5.5 and feed concentration 500μg/ml protein extract of foamate.

(A) (B)
Fig. 11: RP-HPLC chromatogram of BLP in (A) Standard; (B) in enriched foamate

DISCUSSION pH 2.5. So, foam at pH 8.5 is quite wet and less dense than at pH 8.5.
Minor proteins being heavy find difficulty for adsorption at pH8.5
From table 1, it was noted [dγ/dc] value of protein waste solution (adjacent to their pI=9.0) due to weak hydrophobicity of raw protein
without SDS (-0.0329 dynes/cm2/μg) more than that of SDS and protein than protein-SDS complex at pH 5.5 and 2.5 [22].
waste solution mixture, so the addition of SDS have no effect on CMC.
“Ekichi et al. (2005) have also remarked that optimum foaming process From (fig. 4, 9 and 10) and (table 2 and 4), increase of ER, %Rp of
is attained below 750 μg/ml with a proper gas flow rate which will have total proteins and % (w/w) of BLP in foamate were found to
a positive outcome on protein enrichment” (fig. 1) [21]. increase due to the steady enhancement of SGV and GFR. This can be
elucidated by the fact that the gradual increase of both the values
In foam fractionation experiment, pH of feed solution plays a vital role generate an additional number of bubbles followed by an increase of
in controlling the adsorption of protein at the gas-liquid interface the interfacial area of adsorption. From fig. 5 and table3, it was
(bubble’s surface). Isoelectric pH (pI) of all the major and minor dairy observed that the effect of inorganic ions (NaCL) enhanced the gas
waste proteins are nearly 5.5 and 9.0 respectively as mentioned in hold up volume, ER and %Rp at the maximum of 0.1(M) of ionic
table1 [16, 17]. From table 5 and fig. 10, it was noted that enrichment concentration which is below critical concentration of NaCL [0.145
factor (ER) and percent recovery (%Rp) were in the order of (M)] due to inhibition of coalescence between the bubbles and
pH5.5˃pH2.5˃8.5. This is because at pH 5.5, all the major proteins increase of interfacial area of adsorption by formation of
prefer hydrophobic adsorption at their isoelectric pH. Minor proteins microbubbles [20]. The SDS–protein complex enhances the foam
such as BLF and BLP having pI approximately 9.0˃5.5 will behave properties like width, flexibility, and sturdiness of foam membranes
cationic and form a strong hydrophobic complex with anionic and foamability of proteins for the enrichment of adsorption.
surfactant SDS to adsorb maximum on the surface of the bubble [22,
23]. At pH2.5˂pI, major proteins(more than 95% of total protein) get In fig. 12, linearity indicates the theory of material balance of
cationic and fastened by columbic attraction with anionic SDS to form proteins of total mass (MT) in feed equal to the sum of masses in
surfactant links between lamella (intra space of foam) by enhancing foamate and residue (MT=MS+MR) indicating minimum loss of
the foam’s tackiness and rigidity which will resist the rising flow of material. Rate of separation and time for 50% separation (t50%) can
liquid causing reduction of enrichment and recovery. At pH 8.5(>pI of be determined from the slope and point of intersection of curves.
major proteins), major proteins become anionic and columbic Based on the experimental condition of exp no 2 of lot no 9, λ and K
repulsion between proteins and SDS-protein complex molecules will values were determined from fig. 6,7 and equations 5,6 at the values
repel each other reducing thickness and viscosity of film than that at of 3140 cal/mol and 12.686 * 10-9 gm mol cal-1 cm-2 s respectively.

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Fig. 1: Plot of surface tension vs concentration of dairy protein waste solution

Fig. 2: Experimental set up for foam fractionation

Fig. 3: Bubble distribution at SGV 0.083 cm/s

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Fig. 4: Effect of superficial gas velocity on the interfacial area

Fig. 6: ln (V0/VB) vs. ln (C0/CB) Curve for (λ) determination

Fig. 7: CS* [1/(λ-RTlnC0/CB)]*107 vs. time (t) plot for K

Fig. 5: Effect of ionic concentration on ER and % Rp determination

(A) (B)

(C)
Fig. 8: Effect of (A) [GFR 250 ml/min], (B) [GFR300 ml/min], (C) [GFR300 ml/min] on ER and%Rp

Fig. 10: Effect of GFR on mass %(w/w) of Lactoperoxidase at pH

Fig. 9: Effect of varying pH (2.5; 5.5 and 8.5) on ER and %Rp 5.5 and feed concentration 500μg/ml

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(A) (B)
Fig. 11: RP-HPLC chromatogram of BLP in (A) Standard; (B) Enriched foamate

Fig. 12: Material balance diagram of experiment no 2 of lot 8

Table 1: Characteristics of medicinal proteins in dairy waste

Medicinal protein in dairy waste Mol. wt. Isoelectric pH(pI) [dγ/dc] Range of conc. (μg/ml) CMC
(Da*103) (dyne cm2/μg) of constant slope (μg/ml)
BSA(major) 69 5.1 ----- ------ -----
BLF(minor) 84 9.0 0.082 5-150 150
BLP(minor) 89 9.6 ------ ------ ------
α-LA(major) 14 5.3 ------ ------ ------
β-LG(major) 18.30 4.8 ------ ------ ------
IG(major) 100 5.5 ------ ------ ------
Dil. protein waste 25.60 5.2 0.329 50-750 750
Dil.(Protein waste+SDS) [1.5:1(w/w)] ------- ------- 0.301 85-800 800

Table 2: Effect of superficial gas velocity on the interfacial area

SGV (cm/s) Gas flow rate Sauter mean % gas hold up Interfacial area (cm2) Percent recovery Feed density Feed
(ml/min) diameter (ε*100) (%Rp) (g/cc) viscosity
d32 (cm) (Poise)
0.083 250 0.0621 0.90 845.09 90.98 1.235 0.0095
0.099 300 0.0705 1.19 1012.85 93.99
0.116 350 0.0821 1.38 1117.29 98.18

Table 3: Effect of ionic concentration on (ER) and (%Rp)

Molar Ionic concentration Percent gas hold up (ε*100) Enrichment ratio (ER) Percent recovery (%Rp)
0.0125 0.70 32.80 70.40
0.05 0.785 33.39 78.80
0.1 0.905 40.45 90.99
0.15 0.810 36.52 80.35

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Table 4: Experimental results showing the effect of changing GFR at pH5.5

Lot Exp pH Feed conc. Gas flow Concentration in Enrichment %Rp (BLP) Heat of
No. No. (µg/ml) (CI) Rate foamate ratio (ER) (Total %(w/w) desorption
(ml/min) (CS)(μg/ml) protein) λ (cal/mol)
1 2 5.5 400 250 13478 33.70 77.50 0.38 2907
2 2 5.5 500 250 20898 34.84 91.95 0.44 2833
3 2 5.5 600 250 22295 37.16 81.75 0.39 2991
4 2 5.5 400 300 15087 37.72 86.75 0.41 3420
5 2 5.5 500 300 20207 40.41 92.95 0.46 2878
6 2 5.5 600 300 22595 37.65 82.85 0.40 3204
7 2 5.5 400 350 16718 41.80 91.85 0.45 3127
8 2 5.5 500 350 24545 49.09 98.18 0.49 3360
9 2 5.5 600 350 24585 40.98 83.75 0.42 3326
Feed Volume=1liter; foamate collection time =55 min

Table 5: Experimental results showing the effect of pH at GFR 350 ml/min

Lot Exp pH Feed conc. Gas flow Concentration in Enrichment %Rp (BLP) Heat of
No. No (mcg/ml) rate foamate (CS) ratio(ER) (Total %(w/w) desorption
(CI) (ml/min) (mcg/ml) protein) λ (cal/mol)
8 1 2.5 500 350 20440 40.90 79.91 0.40 3330
8 2 5.5 500 350 24545 49.09 98.18 0.49 3360
8 3 8.5 500 350 19950 39.90 77.85 0.39 3140

CONCLUSION 6. Datta PK, Ghosh A, Chakraborty P, Gangopadhyay A. Foam

fractionation in the separation of pharmaceutical biomolecules:
It is noted that the foam fractionation is a useful unit operation to a promising unit operation for industrial process and waste
enhance the concentration of medicinal proteins from dilute dairy waste control. J Fundamental Pharm Res 2015;3:33-41.
as well as to eliminate pollutant proteins to certain level from dairy 7. Backeh M, Leupoid G, Ekichi P, Parlar H. Rapid quantitative
wastewater to control environmental pollution. The method was found extraction of carnosic acid from rosemary (Rosemarinous
to have optimal effectiveness at initial concentration of 500mcg/ml, gas officinalis) by isoelectric focused adsorptive bubble
flow rate 350 ml/min, waste surfactant mass ratio 1.5:1 and ionic chromatography. J Agric Food Chem 2003b;51:1297-301.
concentration 0.1 gm-mole of NaCL per liter of feed at pH5.5. Superficial 8. Holmstorm B. Foam fractionation of streptokinase from crude
gas velocity and ionic concentration enhance the interfacial area for culture filtrates. J Biotechnol Bioeng 1968;10:551-2.
adsorption by increasing the size and number of bubbles. Mass transfer 9. Rubio J, Tessele F. Removal of heavy metal ions by an
coefficient inspected in this study was to some extent high than that of adsorptive particulate floatation. J Min Eng 1997;10:671-9.
earlier studies. The difference in value may be due to the impact of SDS 10. Kim YS, Choi YS, Lee YL. Determination of zinc and lead by
and inorganic electrolyte (NaCL) resulting in adsorption of high solvent sublation using ion-pair. Bull Korean Chemical Society
molecular weight proteins such as lactoferrin and lactoperoxidase. 2001;22:821-6.
Evaluation of the performance of experiment number 2 of lot 8 was 11. Qu YH. Recovery of surfactant SDS and Cd2+from permeate in
found acceptable. The study focused foam fractionation as the lucrative MEUF using a continuous foam fractionator. J Hazard Mater
unit operation to concentrate thermo labile medicinal proteins as well as 2008;155:32-8.
eliminate pollutant proteins from dairy wastewater for coming days 12. Durham RJ, Hourigan JA, Sleigh RW, Johnson RL. Whey
ACKNOWLEDGMENT fractionation: wheying up the consequences. Food Australia
1997;49:460-5.
Authors would like to acknowledge all the researcher workers who 13. Jam MS, Van Hooijdonk ACM. Occurrence, structure,
are engaged to develop this novel and promising unit operation biochemical properties and technological characteristics of
through their untiring effort for technological development so that it lactoferrin. Br J Nutr 2000;84 Suppl 1:11–7.
can be applied in the practical field. 14. Giansanti F, Panella G, Labolfe L, Antonini G. Lactoferrin from
milk: nutraceutical and pharmacological properties. J Pharm
AUTHORS CONTRIBUTIONS
2016;9:1-15.
All authors contributed uniformly to this research work and 15. Kussendrager KD, Van Hooijdonk ACM. Lactoperoxidase:
preparation of this manuscript. physicochemical properties, occurrence, mechanism of action
and applications. Br J Nutr 2000;84 Suppl 1:19-25.
CONFLICT OF INTERESTS 16. Michel MJ, Pouliot Y, Britten M, Noel I, Lebrun R. Study on the
molecular mass changes of bovine k-casein glycomacropeptide
Authors state no conflict of interest
and on its separation in ultra-filtration with and without
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