Effect of bovine serum albumin on the lubricant properties of ricebran oil: a biomimetic approach

ABHIJITH KUNNEPARAMBIL SUKUMARAN1, ANANTHAN D THAMPI1,3, E SNEHA1,3,

MUHAMMED ARIF2 and S RANI1,3,*

1Department of Mechanical Engineering, Advanced Tribology Research Centre, College of Engineering

Trivandrum, Thiruvananthapuram, Kerala, India2Department of Chemistry, College of Engineering Trivandrum, Thiruvananthapuram, Kerala, India3A.P.J. Abdul Kalam Technological University, Thiruvananthapuram, Kerala, India

e-mail: ranigiri2011@gmail.com

MS received 6 November 2020; revised 4 July 2021; accepted 12 August 2021

Abstract. The carcinogenic effects and poor biodegradability of petroleum-based oils have pressurized the

industry to develop eco-friendly lubricants. Nowadays vegetable oils are used as a potential base stock for

industrial lubricants because of their excellent lubricating properties. Among the vegetable oils, rice bran oil is

considered for this work due to its excellent frictional properties but the wear scar diameter generated was not

comparable with that of industrial lubricants. These problems can be effectively solved by using suitable ad-

ditives. Most of the commercially used additives are toxic and non-degradable. This research work focused to

experimentally investigate the effect of bovine serum albumin (BSA) as a bio-molecular additive on the

lubricant properties of rice bran oil (RBO). The lubrication mechanism of oil with bio-molecular protein

additives is through the formation of an oil-protein layer between the surfaces, in which hydrophobic aggregates

of the proteins adhere to the bounding surfaces and forms a secondary layer to improve the frictional and wear

characteristics. The tribological, Physico-chemical, oxidation stability, and thermal properties of the modified

rice bran oil were evaluated and compared with that of the base oil. The performance evaluation of the green

cutting fluid formulated using modified RBO was also conducted and compared with that of the commercially

available cutting fluid. The results obtained have indicated that the formulated oil-protein combination can be a

potential base stock for bio-lubricants and bio-cutting fluids.

Keywords. Rice bran oil; bovine serum albumin; protein lubrication; biomimetic; lubricant property analysis;

DSC analysis.

1. Introduction

The widespread use of mineral oil-based lubricants causes

environmental problems and health issues throughout its

life cycle. The spilling and throwing of these used and fresh

petroleum-based products are causing a real threat to plants,

fishes, and wildlife [1]. The presence of mineral oils in the

soil will drastically change the Physico-chemical properties

of sand [2]. Tribologists were searching for an alternative

replacement of these mineral oil-based lubricants and they

found an answer in the form of developing bio-lubricants.

Around the globe, companies have started developing

various bio-lubricants for different industrial applications.

Many research works on bio-lubricants have been reported

on the usage of edible and non-edible vegetable oils like

coconut oil, palm oil, jojoba oil, rapeseed oil, pongamia oil,

and rice bran oil [3–7]. Ozcelik et al formulated cutting

fluids using three different vegetable oils such as crude

sunflower oil, refined sunflower oil, and refined canola oil.

The performance analysis of these vegetable oil-based

cutting fluids was performed during the drilling operation

and compared with that of the commercial semi-synthetic

cutting fluid. It was reported that canola oil-based cutting

fluid gave better results when compared with the other

cutting fluids at the constant cutting conditions [8]. Few

countries like India, Japan are widely using RBO for

cooking purposes. RBO is considered an effective substi-

tute for mineral oil-based lubricants because of its excellent

lubricant characteristics. Among vegetable oils, rice bran

oil has better oxidative stability due to the presence of

gamma oryzanol [7, 9]. To use rice bran oil as a proper

industrial lubricant, its various lubricant properties need to

be improved. Recently, it was reported that the lubricant

properties of rice bran oil were improved by chemical*For correspondence

Sådhanå (2021) 46:207 � Indian Academy of Sciences

https://doi.org/10.1007/s12046-021-01717-xSadhana(0123456789().,-volV)FT3](0123456789().,-volV)

modification methods and by the addition of suitable eco-

friendly additives [10–13]. So in this work, refined RBO

was selected as base oil.

The common additives used in most industrial lubricants

are another major reason for their toxic and non-

biodegradable nature. The replacement of such toxic addi-

tives with bio-molecules is based on the idea of biomimetic,

which simply refers to the idea of learning from nature and

applying it effectively to satisfy the needs of society. In

nature, there are different examples of effective water

lubrication, which contain proteins in the form of aqueous

solutions. Most of the tribological studies based on proteins

are related to the field of orthopedic implants and their

polymeric materials, but there are only limited works in the

field of industrial lubrication [14, 15]. Recently Yoneyama

et al reported that the use of this albumin reduced friction

and wear by enhancing the hydrodynamic effects of the

solution. The addition of albumin has also improved the

viscosity [16]. It was reported that the reason for enhanced

wear-resistant characteristics of bovine serum albumin

solution was due to the proteinaceous gel-like structure

formed by the solution [17]. Ahlroos et al mentioned that

the replacement of commercial additives with biomolecules

will be a potential forthcoming change in industrial lubri-

cation. The use of proteins such as hydrophobins and BSA

significantly enhanced water lubrication [18]. Li et alreported that lubricating film formed by the aqueous solu-

tion of BSA increases with increasing speeds and decreases

with increasing loads. The film thickness formed was

observed to be time-dependent [19]. Mavraki et al notedthat the bovine serum solution will form a lubricating film

with 4–50 nm thickness over the speed range. The thickness

of the film formed was fairly constant but tends to increase

at a slower speed. It was also reported that the lubricating

characteristics of bovine serum solution are not similar to

that of a simple Newtonian fluid [20]. Thus it was noted

that the addition of proteins like BSA into water has

enhanced the lubricity of water. This BSA is a protein that

is extracted from cows and purified by the Edwin Cohn

purification method. It mainly consists of 583 amino acid

residues. The significant disadvantage of water lubrication

is its low viscosity. Recent studies have highlighted veg-

etable oils as a potential base-stock for industrial lubricants.

Thus came the idea of adding bio-molecular additives like

BSA into vegetable oils. This may improve the lubricant

properties of vegetable oils and also help in developing bio-

lubricants.

This study aims to find out whether the lubricant prop-

erties of RBO can be improved by adding protein molecules

such as BSA. This work evaluated the tribological, Phy-

sico-chemical, oxidative stability, and thermal properties of

RBO with BSA as an additive. The experimental results

indicated that BSA is an effective lubricant additive for

vegetable oils. The green cutting fluid formulated using

modified RBO has also performed better compared to

commercial cutting fluid.

2. Materials and methods

2.1 Materials

The RBO used in this work was purchased from Kalady

Rice Millers Consortium Pvt. Ltd., Kerala, India, and BSA

which was used as a bio-molecular additive in this work

were purchased from Sigma-Aldrich Co. The chemical

reagents required for this work were purchased from Nice

chemicals, Kerala, India.

2.2 Preparation of RBO-BSA test samples

The BSA at different concentrations varying from 0.1 to 0.6

mg/ml were added to pure RBO. The mixture of RBO and

BSA (RBM) was then stirred for 6 hours using a magnetic

stirrer for proper dispersion of BSA into RBO. The RBM

samples were then visually inspected for sedimentation.

The BSA concentration in RBO was optimized based on the

tribological properties. The structure of BSA and prepared

RBM test samples are shown in figure 1.

2.3 Evaluation of tribological properties

The tribological properties of the samples are evaluated

using a standard four-ball tester. In this tribo-tester, three

balls are maintained stationary in a ball pot and the fourth

ball is fixed to a collet and attached to a spindle which is

made to rotate against the stationary balls. AISI 52100 steel

balls with 12.7 mm diameter were used in this work. The

ball pot and test balls were properly cleaned using acetone

before and after testing. The coefficient of friction (COF)

and wear scar diameter (WSD) were measured as per

ASTM D 4172. The variation of frictional torque with time

was also obtained with the help of WINDUCOM software.

The extreme pressure (EP) test was carried out as per

ASTM D 2783. The worn surface image was analyzed with

the help of a Leica optical microscope.

2.4 Evaluation of physico-chemical properties

The physicochemical properties such as density, viscosity,

acid value, peroxide value, anisidine value, and total oxi-

dation value were evaluated in this work. The density of the

oil samples was measured using a specific gravity bottle.

The viscosity of the samples can be measured as kinematic

or dynamic viscosity. It was reported in studies related to

proteins that the behavior of protein under external force

decides the characteristics of final products [21]. Thus in

the present study, the dynamic viscosity of the oil samples,

which provides a better understanding of the shearing

action of the lubricant under the external force applied, was

measured using an Anton Paar modular compact rheometer

(MCR 102) at 75�C. The acid value (AV) indicates the

207 Page 2 of 10 Sådhanå (2021) 46:207

number of fatty acids separated from the molecule. This

determines the oxidation stability and corrosion resistance

of the oil. The lower the acid value, the better will be the

shelf life of the sample. The peroxide value (PV) indicates

the degree of primary oxidation of the oil and therefore its

likeliness of becoming putrid. These chemical properties

were evaluated based on IS: 548 (Part 1) –1964. The ani-

sidine value (ANV) indicates the secondary oxidation

products of oil which produce a bad odor during storage.

The ANV is determined by using equation (1). The

absorbance of contents at a wavelength of 350nm was

measured using Spectrophotometer.

ANV ¼ 25 1:2Eb � Eað Þð Þ �W ð1ÞWhere, Ea- net absorbance of the lubricant solution, Eb-net

absorbance of the lubricant solution with anisidine solution,

W- the weight of the sample.

The total oxidation value (TOTOX) gives the oxidation

value of both the primary and secondary oxidation prod-

ucts. The lesser the TOTOX value, the better the stability of

the sample. The TOTOX value is determined by equation

(2).

TOTOX Value ¼ ANVþ 2PV ð2ÞWhere, ANV = Anisidine value, PV = Peroxide value.

2.5 Evaluation of oxidation stability

Oxidative stability of the oil is defined as the resistance of

oxidation during processing and storage. Oxidation

destroys fatty acids and produces toxic components. The

primary oxidation products are hydrogen-peroxides and the

secondary oxidation products are aldehydes and ketones.

The oxidative stability of oil was evaluated by Hot Oil

Oxidation Test (HOOT) as per American oil chemists’

society (AOCS Cd-12-57) standards. The sample will be

undergone accelerated aging by storing in a dark hot air

oven at 75C for 18 hours. Then the samples are checked for

the acid and peroxide values at regular time intervals.

2.6 Evaluation of thermal properties

The flash point indicates the temperature at which the oil

will be heated to give off sufficient vapors to form an

inflammable mixture with air that ignites momentarily

when exposed to a flame or an electric spark. The fire point

is the temperature to which oil is heated to produce a vapor

air mixture that burns continuously at least for 5 seconds

once it is ignited The flash and fire point of the samples

were measured by using the Pensky Marten apparatus. The

temperature at which oil becomes hazy when it is cooled at

a specified rate is called the cloud point. The pour point is

the corresponding temperature at which oil loses its

flowability. The pour point and cloud point of the oil

samples were evaluated as per ASTM D97 and ASTM

D2500, respectively. The pour point was also measured by

the Differential scanning calorimetry (DSC) method. The

DSC method computes the variation of heat flows associ-

ated with transitions in materials as a function of temper-

ature and time in a controlled atmosphere. The DSC

experiments were performed on Universal V4.5A TA

Instruments.

2.7 Formulation and performance evaluationof the green cutting fluid

The green cutting fluid (GCF) was prepared using rice bran

oil with optimum BSA concentration. The emulsifier used

in this study was span 80. The oil-in-water emulsion was

prepared at a ratio of 1:20 using a magnetic stirrer.

Figure 1. Pictorial demonstration of (a) BSA Structure and (b) prepared RBM sample.

Sådhanå (2021) 46:207 Page 3 of 10 207

Similarly, the commercial cutting fluid (CCF) was prepared

by mixing commercially available oil (COM) with water.

Servocut S was the COM used in the study. The stirring

performed during the formulation procedure and the final

developed green cutting fluid is shown in figure 2.

The performance evaluation was conducted using a pin

on the disc tribometer as per the ASTM G99 standard. Cast

iron and EN31 steel are used as materials for the pin and

disc, respectively. The pin is loaded against the rotating

disc and the load was maintained constantly at 100N

throughout the test duration of 15 min with the help of a

deadweight loading system. The dimensions of the pin used

are 40 mm in length and 6 mm in diameter. Whereas, the

wear track diameter was kept constant throughout the tests

at 60 mm. The performance evaluation was conducted at 4

different speeds, i.e., 400, 600, 800, and 1000 RPM. The

cutting fluid flow was maintained at 25 ml/sec. The coef-

ficient of friction and weight loss of the pin are noted after

each test.

3. Results and discussion

3.1 Tribological properties of RBM

The four-ball tribological test was initially conducted as per

ASTM D 4172 to optimize the concentration of BSA in

RBO based on the COF and WSD results. The COF and

WSD results obtained for RBO and RBM with different

concentrations are shown in table 1.

From table 1, it was observed that the COF and WSD

value was minimum for RBM with 0.4 mg/ml BSA con-

centration. The WSD value of RBM with different BSA

concentrations was noted to be lower than that of RBO. The

COF value of RBM with 0.3 to 0.6 mg/ml BSA

concentration was observed to be lower than that of RBO.

The tribological results indicate that RBM with 0.4 mg/ml

BSA concentration formed a stable colloid. Further, the

addition of BSA has increased the COF and WSD value.

This may due to the suspension of the excess amount of

BSA which resulted in three-body abrasion. The optimum

concentration of BSA in RBO is noted to be 0.4 mg/ml. The

mixture of RBO with 0.4 mg/ml BSA concentration is

called modified RBO (MRBO). The worn surface image

obtained from Leica optical microscope for RBO and

MRBO at different magnifications is shown in figure 3.

From figure 3, it is clear that the wear scar of MRBO is

smaller than that of the RBO. It was also observed that the

grooves on the worn surface caused by MRBO were noted

to be very smooth compared to those caused by RBO. The

variation of frictional torque with time for RBO and MRBO

is shown in figure 4.

From figure 4, it was noted that the frictional torque

throughout the experiment was stable for RBO and MRBO.

The frictional torque was noted to be lower for MRBO

compared to RBO. The results indicated that the wear and

frictional characteristics of RBO have been improved by

the addition of BSA. This may be due to the presence of an

aromatic chain of amino acids in BSA. The extreme pres-

sure test results of RBO and MRBO are shown in table 2.

From table 2, it was noted that the EP test result obtained

for MRBO is better than that of RBO. The load-carrying

capacity of RBO has slightly improved with the addition of

BSA. It was noted that the melting temperature of BSA is

about 63�C [22]. The heat transfer at temperatures above

the melting point will break the intramolecular bonds,

which causes the unfolding and exposure of hydrophobic

patches. This, in turn, will form b-sheet rich aggregates

[23]. These hydrophobic aggregates may prevent the metal

to metal contact. It was reported that a boundary lubrication

layer will be formed by proteins, which may adsorb to the

contact surface [24–26]. The improvement in the tribolog-

ical properties of RBO after adding BSA may be due to the

secondary layer of lubricant formed by these hydrophobic

aggregates.Figure 2. (a) Stirring performed during GCF formulation and

(b) Finally developed green cutting fluid.

Table 1. COF and WSD of RBO with different BSA

concentration.

Sl. No. Samples COF WSD (mm)

1 RBO ? 0 mg/ml BSA 0.090 ± 0.003 0.565 ± 0.010

2 RBO ? 0.1 mg/ml BSA 0.091 ± 0.003 0.522 ± 0.015

3 RBO ? 0.2 mg/ml BSA 0.090 ± 0.002 0.503 ± 0.012

4 RBO ? 0.3 mg/ml BSA 0.080 ± 0.002 0.485 ± 0.008

5 RBO ? 0.4 mg/ml BSA 0.073 ± 0.003 0.472 ± 0.008

6 RBO ? 0.5 mg/ml BSA 0.082 ± 0.004 0.489 ± 0.015

7 RBO ? 0.6 mg/ml BSA 0.085 ± 0.003 0.496 ± 0.013

207 Page 4 of 10 Sådhanå (2021) 46:207

3.2 Physico-chemical properties of MRBO

The Physico-chemical properties such as viscosity, density,

acid value, peroxide value, anisidine value, and TOTOX

values are evaluated and shown in table 3.

From table 3, it was observed that the evaluated chemical

properties such as acid value, peroxide value, anisidine

Figure 3. Microscopic images of the worn surface caused by RBO and MRBO.

Figure 4. Variation of frictional torque with time for RBO and MRBO.

Table 2. Extreme pressure test results of RBO and MRBO.

Sl. No. Samples Load (N)

1 RBO 1126 ± 10

2 MRBO 1165 ± 12

Sådhanå (2021) 46:207 Page 5 of 10 207

value, and TOTOX value of RBO have reduced after the

addition of BSA. This shows that the shelf life and oxida-

tive stability of MRBO are better than that of RBO. It was

reported that the BSA structure is free from fatty acids and

can bind fatty acids [27]. This may have caused a slight

reduction in the acid value after adding BSA. The peroxide,

anisidine, and TOTOX value which indicates the oxidative

stability has also improved after adding BSA due to the

presence of tryptophan in BSA [28]. It was also noted that

the evaluated physical properties such as viscosity and

density of RBO have increased after the addition of BSA.

The physical properties such as viscosity and density of

RBM with different BSA concentrations were evaluated

and shown in table 4.

From table 4, it was noted that the viscosity and density

of all the RBM samples were higher than that of the pure

RBO. The results indicated that the threshold viscosity was

obtained for RBM samples with 0.4 mg/ml BSA. The vis-

cosity kept on increasing with an increase in BSA con-

centration. The change in viscosity was considered to be

negligible for RBM samples with more than 0.4 mg/ml

BSA concentration. The density of RBM samples was

noted to be increasing with an increase in BSA concen-

tration. The density has shown a significant jump from

0.942 to 0.979 gm/cm3 when the BSA concentration varied

from 0.1 mg/ml to 0.4 mg/ml. The results of viscosity and

density obtained for RBM samples with more than 0.4 mg/

ml BSA concentration indicates the chances of uneven

suspension of BSA. It is evident that the addition of BSA

after a certain limit will cause three-body abrasion, which

in turn affects the tribological properties.

3.3 Oxidative stability of MRBO

The oxidative stability of MRBO was evaluated by the

HOOT method. The test was conducted in a hot air oven at

75�C for 18 hours. The acid values (AV) in mg KOH /gm

and peroxide value (PV) in Meq /kg were measured at a

regular interval of 6 hours. The experimental results

obtained for MRBO are compared with that of the RBO.

The HOOT test results are shown in figure 5.

From figure 5, it was observed that the acid value and

peroxide value of MRBO at every stage of heating showed

a lower value when compared to that of the RBO. It was

noted that after heating for 18 hours, the difference in

peroxide value for MRBO, RBO was 22.45, 24 respec-

tively. Whereas, the difference in acid value for MRBO,

RBO was 1.06, 2.01 respectively. Thus the variation in acid

value and peroxide value of MRBO was noted to be lower

than that of the RBO. The HOOT results have indicated that

the oxidative stability of MRBO is better than that of RBO.

The BSA consists of tryptophan along with 583 amino

acids. This tryptophan was reported to be present in the

surface and hydrophobic cavities of BSA [28–30]. The

antioxidant activities of tryptophan, which is an amino acid

with an aromatic side chain and an indole ring have also

been reported [31, 32]. The presence of tryptophan in BSA

has improved the stability of RBO against oxidation.

Table 3. Physico-Chemical properties of RBO and MRBO.

Evaluated properties RBO MRBO

Physical properties Viscosity (Pa-s) 0.01330 ± 0.00010 0.01435 ± 0.00008

Density(gm/cm3) 0.920 ± 0.004 0.979 ± 0.003

Chemical properties Acid value 0.56 ± 0.05 0.39 ± 0.03

Peroxide value 5.52 ± 0.50 4.35 ± 0.25

Anisidine value 16.02 ± 0.74 13.80 ± 0.50

TOTOX value 27.02 ± 1.74 22.50 ± 1.00

Table 4. Viscosity and density of different RBM samples.

Sl. No. Sample Viscosity (Pa-s) Density(gm/cm3)

1 RBO?0 mg/ml BSA 0.01330 ± 0.00010 0.920 ± 0.004

2 RBO?0.1 mg/ml BSA 0.01350 ± 0.00015 0.942 ± 0.005

3 RBO?0.2 mg/ml BSA 0.01370 ± 0.00012 0.964 ± 0.004

4 RBO?0.3 mg/ml BSA 0.01400 ± 0.00010 0.972 ± 0.003

5 RBO?0.4 mg/ml BSA 0.01435 ± 0.00008 0.979 ± 0.003

6 RBO?0.5 mg/ml BSA 0.01437 ± 0.00008 0.981 ± 0.004

7 RBO?0.6 mg/ml BSA 0.01439 ± 0.00006 0.983 ± 0.003

207 Page 6 of 10 Sådhanå (2021) 46:207

3.4 Thermal properties of MRBO

The thermal properties such as flash point, fire point, cloud

point and pour point of RBO and MRBO are evaluated and

shown in table 5.

From table 5, it was observed that the flash and fire point

of MRBO is almost the same and comparable with that of

RBO. The low-temperature characteristics such as the

cloud and pour point of RBO have slightly improved after

the addition of 0.4 mg/ml of BSA. Thus it can be noted that

the low-temperature properties of MRBO are better than

that of RBO. The DSC results of RBO and MRBO are

shown in figure 6.

When the heat flow (W/g) is noted to be constant, the

DSC curve will be a straight line. These vegetable oils

which consist of different fatty acids will solidify over a

certain temperature range. Hence during the cooling of

these vegetable oils, the DSC curves will show certain

peaks. The low temperature at which the DSC peak occurs

is noted to be almost comparable with that of the pour point

results obtained as per ASTM D 97. The DSC results

revealed that the pour point of MRBO has improved by

about 2�C when compared to that of the RBO. From fig-

ure 6, it was noted that the DSC curve of MRBO has shown

Figure 5. Acid and Peroxide value of RBO and MRBO during different heating stages in HOOT.

Table 5. Thermal properties of RBO and MRBO.

Thermal properties RBO MRBO

Flash point (�C) 315 ± 3 308 ± 4

Fire point (�C) 320 ± 2 314 ± 2

Cloud point (�C) - 6 ± 1 - 8 ± 2

Pour point (�C) - 9 ± 1 - 11 ± 2

Figure 6. DSC results of RBO and MRBO.

Sådhanå (2021) 46:207 Page 7 of 10 207

a slight variation in heat flow after 150�C. This may be due

to the thermal instability of the BSA structure after 150�C.Whereas, the heat flow of RBO is constant throughout the

experiment from room temperature. BSA is reported to

denature at a higher temperature above the melting tem-

perature, which initially proceeds through a reversible

transformation and then further heating causes an irre-

versible transformation to a hydrophobic conformation and

finally results in the aggregation [33, 34].

3.5 Performance evaluation of cutting fluid

The performance test of the GCF was conducted and the

results obtained were compared with that of the CCF. The

COF obtained after the test with different speeds of rotation

is shown in table 6.

From table 6, it was observed that at different disc speeds

the COF value of GCF formulated using MRBO was lower

than that of CCF formulated using COM. The weight loss

of the pin after the test with different speeds of rotation is

shown in figure 7.

From figure 7, it was noted that the weight loss of the pin

was lower while using GCF compared to CCF. Based on

the performance analysis conducted, GCF formulated using

MRBO was observed to be better than that of CCF for-

mulated using COM.

4. Conclusions

In this research work, the base oil used is rice bran oil

and bovine serum albumin as a biomolecular additive.

The experimental study investigated the effect of this

eco-friendly additive on the lubricant properties of rice

bran oil. The following conclusions are made from this

study:

• The optimum BSA concentration in RBO is noted to be

0.4 mg/ml based on the COF and WSD readings. The

evaluated lubricant properties of MRBO are noted to

be better than that of RBO. Thus, BSA can be

considered as an effective lubricant additive.

• The tribological properties such as COF, WSD,

frictional torque, and EP of MRBO are noted to be

better than that of the RBO. The worn surface caused

by MRBO is observed to have smooth grooves when

compared to that of the RBO. This may be because of

the presence of aromatic amino acid chains in BSA and

Table 6. COF obtained for GCF and CCF at different speeds.

Sample

Disc speed (RPM)

400 600 800 1000

Coefficient of friction

CCF 0.126 ± 0.005 0.151 ± 0.007 0.176 ± 0.008 0.183 ± 0.008

GCF 0.117 ± 0.004 0.142 ± 0.005 0.160 ± 0.005 0.171 ± 0.007

Figure 7. Weight loss of Pin for CCF and GCF at different speeds.

207 Page 8 of 10 Sådhanå (2021) 46:207

the secondary support layer formed by hydrophobic b-sheet rich aggregates.

• The viscosity and density of MRBO are found to be

higher when compared to that of RBO. The physical

properties such as viscosity of RBM seemed to be

achieving threshold values at 0.4 mg/ml BSA concen-

tration. Whereas, the density of RBM increased at a

negligible rate after 0.4 mg/ml BSA concentration.

• The chemical properties such as acid value, peroxide

value, anisidine value, and TOTOX value of MRBO

are found to be lower than that of the RBO. The HOOT

result also revealed that after each successive heating

of 6 hours, the acid and peroxide value of MRBO are

noted to be lesser compared to RBO which means that

the oxidation stability of the MRBO is better than that

of RBO. This may be due to the presence of tryptophan

in BSA along with natural anti-oxidant such as gamma

oryzanol in RBO.

• The flash and fire point of MRBO are found to be

comparable with that of the RBO. Whereas, the low-

temperature characteristics such as cloud and pour

point of MRBO are noted to be better than that of the

RBO.

• The DSC curve revealed that the MRBO is unsta-

ble after 150�C. This may be because of the thermal

instability of the BSA structure at higher temperatures.

The change in protein structures at high temperatures

needs to be investigated further.

• The green cutting fluid formulated using MRBO has

performed better compared with that of the commercial

cutting fluid in terms of COF and wear loss. This is

mainly because of the excellent lubricant properties of

the developed MRBO.

Acknowledgements

The authors sincerely acknowledge the financial assistance

from Kerala State Council for Science, Technology and

Environment [KSCSTE] to carry out the research. The

authors would also like to thank the technical assistance

provided by CSIR – NIIST, Trivandrum for viscosity

evaluation using a rheometer and NIT, Calicut for DSC

results. The second and third authors would like to

acknowledge the CERD of APJ Abdul Kalam Technolog-

ical university, Thiruvananthapuram, Kerala and NDF of

AICTE, New Delhi for providing Ph.D. fellowship.

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