|Bile salt; Proteose peptone; Particle size; Brunauer-
Emmett-Teller analysis; X-ray diffraction; Differential scanning
calorimetry; Thermogravimetric analysis
|PSA: Particle Size Analyzer; BET: Brunauer-
Emmett-Teller analysis; DSC: Differential Scanning Calorimetry;
XRD: X-ray Diffraction; TGA: Thermogravimetric Analysis; DTA:
Differential Thermal Analyzer; DTG: Derivative Thermogravimetry;
BS: Bile Salt; PP: Proteose Peptone
|Bile salts (BS) are commonly known as bio-surfactants that plays
crucial physiological role in human gastro intestinal tract such as
fat digestion and absorption of nutrients and also serve as a mean
for removal of waste products from blood [1,2]. Briefly, BS acts as a
carrier for fat soluble products due to its ability of forming micelles
with phospholipids. Moreover, the BS plays an important role in
nutrition by improving solubility and transport of fat soluble nutrients
to the mucosa of small intestine. Based on chemical nature of BS are
flat molecules with both hydrophilic and hydrophobic faces . Many
literature reports provided interesting information about the selfassembly
nature of BS in solution suggesting the fascinating properties
of BS aggregates as compared to conventional surfactants [4-6]. Due to
this micellar nature of BS, which enables solubilization and transport
of lipid soluble compounds thus it helps in fat digestion. Therefore,
this same biological function can be exploited for pharmaceutical
application since most drugs currently in development have low water
solubility . Thus BS based carrier systems are promising for specific
targeting and absorption of non-soluble compounds. However, it
was shown that BS is a poor surface active-agent compared to other
commonly used surfactants such as dodecyl sulfate and sodium
dodecanoate. Hence, in order to improve these properties BS should be modified in order to confer better physicochemical properties.
|On the other hand proteose peptone (PP) is obtained from bovine
milk which is partially consist of a number of heat stable minor proteins,
glycoproteins, and largely of casein derived peptides [7,8]. These are
generated in human body by the action of proteinases (mainly plasmin)
of all the four main casein proteins [9-11]. These protein compounds
require proper modification in order to alleviate its properties which
can be utilized for further applications.
|Scientists have demonstrated that short lived electrical events or
action potential occurs in several types of mammalian cells such as
neurons, muscle cells, and endocrine cells . For example, the cells
in the nervous system communicate with each another by means of
electrical signals that travel along the nerve processes. Therefore,
it is hypothesized that biofield exists around the human body and
the evidence can be found using medical technologies such as
Electromyography, Electrocardiography and electroencephalogram
|Thus, human has the ability to harness the energy from
environment or universe and can transmit into any living or nonliving objects around the Globe. The objects always receive the energy and
responding into useful way that is called biofield energy and the process
is known as biofield treatment. Recently, biofield energy has shown
significant effect on structural, crystalline and thermal properties of
various metals, ceramics and carbon allotropes [14-17].
|Mr. Mahendra K. Trivedi is known to transform the properties
of various living and non-living things under controlled experiments
using his unique biofield energy. Biofield treatment had substantially
changed the atomic, crystalline, surface properties of various materials.
The biofield had significantly changed the overall productivity
and quality in the field of agriculture and biotechnology [18-21].
Additionally, biofield has shown excellent results in improving
antimicrobial susceptibility, and alteration of biochemical reactions, as
well as induced alterations in characteristics of pathogenic microbes [22-
24]. The biofield had also caused an increase in growth and anatomical
characteristics of an herb Pogostemon cablin that is commonly used in
perfumes, in incense/insect repellents, and alternative medicine .
|In the present study, the influence of biofield treatment on
physicochemical properties of BS and PP were studied with the aid of
different methods like particle size analyzer (PSA), Brunauer-Emmett-
Teller (BET) analysis, differential scanning calorimetry (DSC), X-ray
diffraction (XRD) studies, and thermogravimetric analysis (TGA).
|Materials and methods
|The Bile salt and Proteose peptone were procured from Hi Media
Laboratories Pvt. Ltd., Mumbai, India. Each material was divided
into two parts; one was kept as a control sample, while the other was
subjected to Mr. Trivedi’s biofield treatment and coded as treated
sample (T). The treatment group (T) was in sealed pack and handed
over to Mr. Trivedi for biofield treatment under laboratory condition.
Mr. Trivedi provided the treatment through his energy transmission
process to the treated group without touching the sample.
|Particle size analysis: The average particle size and particle
size distribution were analyzed by using Sympetac Helos-BF Laser
Particle Size Analyzer with a detection range of 0.1 micrometer to 875
micrometer. Average particle size d50 and d99 (size exhibited by 99% of
powder particles) were computed from laser diffraction data table. The
d50 and d99 values were calculated by the following formula:
|Percentage change in d50 size=100 × (d50 treated-d50 control)/d50
|Percentage change in d99 size=100 × (d99 treated-d99 control)/d99
|Surface area analysis: The surface area of BS and PP were
characterized using surface area analyzer, SMART SORB 90 BET,
which had a detection range of 0.1-100 m2/g.
|Differential scanning calorimetry (DSC) study: The control and
treated samples (BS and PP) were analyzed using a Pyris-6 Perkin
Elmer DSC on a heating rate of 10°C/min under air atmosphere.
|X-ray diffraction (XRD) study: XRD of BS and PP (control and
treated) powders were analyzed by using Phillips Holland PW 1710
X-ray diffractometer system. The wavelength of the radiation was
1.54056 angstrom. The data was obtained in the form of 2θ versus
intensity (a.u) chart. The obtained data was used for calculation of crystallite size using the following formula.
|Crystallite size=kλ/b Cos θ
|Where λ is the wavelength and k is the equipment constant (0.94).
|Thermogravimetric analysis-Differential thermal analysis
(TGA-DTA): Thermal stability of control and treated samples (BS
and PP) were analyzed using Mettler Toledo simultaneous TGA and
Differential thermal analyzer (DTA). The samples were heated from
room temperature to 400°C with a heating rate of 5°C/min under air
|Results and Discussion
|Particle size and surface area analysis
|The average particle size (d50) and particle size (d99) of the organic
products were computed from particle size distribution graph and the
data are presented in Figures 1 and 2. The average particle size (d50) of
treated BS (13.24 μm) was enhanced as compared to control sample
(12.13 μm) (Figure 1). Similarly, the d99 value of the treated BS (121.65
μm) showed increase as compared to control sample (88.77 μm). The
calculated percentage change in average particle size (d50) was 9.2% and
d99 value was 37%. This substantial increase in particle size of the treated
BS may be due to biofield treatment which may be caused fracture in
the particles hence the powder may not have specific boundaries that
can be led to particles agglomeration and increased particle size. It is
assumed that bigger treated microparticles may be useful in designing
drug delivery systems. Many reports suggested that higher water
uptake of organic products such as rice bran, mainly depends on its
particle size [26-28]. It was envisaged that larger size particles take up
more water as compared to smaller particles . Albers et al., in a
research study showed that water absorption decreased with decreasing
particle size . Hence, bigger microparticles are likely to show more
water uptake and this property can be used for controlled release of
drugs. The drugs can be released from swellable microparticle through
diffusion, degradation or both depending on the level of swelling and
solubility of the drug . Therefore, the treated BS could be interesting
choice for drug delivery systems.
|The PP also showed increase in d50 (14.4 μm) and d99 (130.09 μm)
as compared to control PP sample (d50; 13.42, d99; 107.58) (Figure
1). The percentage change in d50 and d99 values was 7.3% and 20.9 %
respectively (Figure 2). It is assumed that aggregation due to biofield
treatment might cause the particles to come together and form bigger microparticles. It was previously described that proteins have stronger
tendency of forming aggregates. They can form self-aggregates in a
number of ways such as formation of structural complexes, multimeric
native states with metal complexation [31-33]. These proteins have
sufficiently strong inter-protein interactions  which induce
formation of bigger aggregates. It is postulated that biofield may be
interacted with protein assembly of PP and caused bigger microparticle
|Surface area of BS and PP was measured and results are presented
in Table 1. The surface area of control BS was 0.63 m2/g. However, after
treatment it was decreased slightly i.e. up to 0.62 m2/g. The percentage
decrease in surface area was by 1.59% in treated BS sample as compared
to control. The minimal decrease in surface area was due to increase
in particle size of treated BS [35,36]. Contrarily, the treated PP (1.08
m2/g) showed increase in surface area as compared to control (1.00
m2/g). The percentage increase in surface area was 8% in the treated PP
with respect to control. It is assumed that biofield energy might cause
formation of sharp edges or pore formation over particle surface which
increased the resultant surface area.
|DSC thermogram of control and treated BS are presented in Figure
3. The DSC thermogram of control BS showed broad endothermic peak
at 128°C which was due to melting temperature of the sample. DSC
of treated BS showed endothermic peak at 232°C which was probably
associated with melting and hydration of the hydrophilic head in BS
structure. The increase in melting temperature may be correlated with
high thermal stability of treated BS. In BS the increase in temperature
raises the critical micelle concentration; however further increase in
temperature decreases the critical micelle concentration. At a fixed
temperature the critical micelle concentration value of BS is controlled
by balanced interaction of two forces namely van der Waals forces
between the hydrophobic alkyl groups that stabilizes the micelles and
opposing hydration of hydrophilic group that deny the formation
of micelles. These two opposite factors are to be considered in order
to understand this behavior, viz increase in temperature elevates the
dehydration of head group (resulting in increased hydrophobic nature
of the molecules) and thermal stability of the BS molecules . Hence,
it is postulated that thermal along with biofield energy might be acted
at BS molecules which enhanced the hydrophobic nature and thermal
|DSC thermogram of control and treated PP are shown in Figure 4. The control PP showed an endothermic peak due to absorbed
water at 122°C and another endothermic inflexion was observed at
around 300°C which was due to melting temperature and thermal
denaturation of the control sample. DSC thermogram of treated
sample exhibited a broad endothermic peak at 175°C. Researchers have
found that the major endothermic peak observed (from 0 to 180°C) in
case of soy protein, gelatin, sodium casein and corn gluten meal has
been attributed to loss of residual water or hydrogen bond disruption
between protein molecules [38-41]. Another endothermic peak was
observed at 290°C in the treated PP which was probably due to thermal
denaturation and melting temperature of the protein. The control PP
showed an exothermic temperature peak at around 267°C. However,
the DSC thermogram of treated PP showed (Figure 4) an exothermic
transition at 278°C. Tang et al. during their studies on heat induced
aggregation and denaturation of soy proteins observed much lower
exothermic peaks (150°C) . It is presumed that exothermic peak
might be increased due to biofield treatment. The native confirmation
of proteins is mainly maintained by its inherent hydrogen bonding and
electrostatic interactions, whereas thermal stability is closely related
to hydrophobic interactions. If the hydrophilic interactions retaining
the tertiary structure of protein are ruptured by heating, hydrophobic
regions initially hidden inside the proteins will be exposed to the protein
surface and subordinate with other hydrophobic protein molecules to
form aggregates. Hence, in this study the exothermic peak could be
attributed to the protein aggregation [42,43]. Additionally, the increase
in exothermic peak may be due biofield energy which raised the treated
PP aggregation temperature.
|XRD diffractogram of control and treated BS are illustrated in
Figure 5. The XRD diffractogram of control BS showed presence of
broad as well as intense peak. The control sample showed crystalline
peaks at 2θ equals to 31.61°, 45.40°, 56.42°, 66.12° and 75.26°. The
control BS showed a broad peak at 2θ equals to 11.1° which was due
to amorphous nature. These XRD peaks showed presence of both
crystalline as well as amorphous regions in the control BS (Figure 5).
The treated BS also displayed similar XRD peaks at 2θ equals to 27.2°,
31.63°, 45.39°, 56.45°, 66.14° and 75.10°. The result showed a minimal
reduction in the intensity of the XRD peaks in the treated BS which
may be due to decrease in crystallinity of the sample.
|XRD diffractogram of control and treated PP are shown in Figure
6. The XRD of control sample showed an amorphous nature which
was confirmed by a broad peak at around 2θ equals to 14.48° and 23°.
However, the treated PP showed (Figure 6) XRD peaks at around 2θ
equals to 28.39° and 32.71° which showed no significant change in
amorphous nature of the treated PP after biofield treatment.
|TGA was used to get further insights about the thermal stability of
the control and treated samples (BS and PP). The TGA thermogram
of BS (control and treated) are presented in Figures 7 and 8. The TGA
thermogram of control BS exhibited one step thermal degradation.
The thermal degradation commenced at 80°C and continued up
to 130°C. The sample had lost 3.28% of its total original weight
during this process. This thermal event was probably associated with elimination of water or dehydration of the control sample (Figure
7). The derivative thermogravimetry (DTG) thermogram of control
sample exhibited maximum thermal degradation at 106°C. Whereas
the treated BS sample showed two step thermal degradation process.
The first step commenced at around 190°C and terminated at around
240°C. The second thermal degradation event was commenced at
320°C and terminated at around 390°C. Major sample weight loss was
observed during this process (15.87%). DTG thermogram of treated
BS showed (Figure 8) maximum thermal decomposition step at 221°C
which was higher as compared to control sample (106°C). The DTA
thermogram of control and treated BS did not show any changes in
the respective thermograms. However, the comparison of the DTG
peaks confirmed that thermal stability of treated BS was enhanced after
biofield treatment as compared to control sample .
|TGA thermogram of control and treated PP are depicted in Figures
9 and 10. TGA thermogram of control PP showed two step thermal
degradation. The first step thermal degradation started at 190°C
and terminated at 230°C. However the second thermal degradation
commenced at 260°C and terminated at 310°C (Figure 9). The control
sample lost -9.15% and -9.84% weight respectively from the sample.
DTG thermogram showed two peaks which were mainly due to
initial decomposition temperature (205°C) and maximum thermal
decomposition temperature (283°C) of the control sample. The TGA
thermogram of treated PP also showed two step thermal degradation
pattern. The first step thermal degradation started at 172°C and
terminated at 259°C. Thereafter the second step was observed at
273°C and thermal degradation stopped at 371°C. During this thermal
process the treated sample lost -18.28% and -21.70% of its weight. The DTG of treated PP showed (Figure 10) a decrease in maximum
thermal decomposition temperature and it was observed at 217°C. This
decrease in DTG peak could be due to decrease in thermal stability of
treated PP as compared to control. DTA showed no changes in the
thermal pattern of control and treated PP.
|This research study has evaluated the influence of biofield
treatment on thermal and physical properties of BS and PP. The treated
BS showed increase in particle size (d50 and d99) as compared to control
which might be due to fracturing of internal boundaries in the particles
caused by biofield treatment. The treated PP also showed increase in
particle size with respect to control; possibly due to biofield treatment
and protein aggregation. Additionally, biofield treatment showed
significant alteration in thermal nature of the treated samples. Based on
the results the biofield treated BS and PP could be used in drug delivery
systems. Further, Circular dichroism spectroscopic and Scanning
electron microscopy/Transmission electron microscopy studies could
be carried out to get further depth insights about thermal stability and
aggregation nature of these treated organic products.
|The authors would like to thank all the laboratory staff of MGV Pharmacy
College, Nashik for their assistance during the various instrument characterizations.
We thank Dr. Cheng Dong of NLSC, institute of physics, and Chinese academy of
sciences for permitting us to use PowderX software for analyzing XRD results.
- Mukhopadhyay S, Maitra U (2004) Chemistry and biology of bile acids. Curr Sci 87: 1666-1683.
- Bauer E, Jakob S, Mosenthin R (2005) Principles of physiology of lipid digestion. Asian Australas J Anim Sci 18: 282-295.
- Cabral DJ, Small DM (1989) Physical chemistry of bile: Handbook of Physiology. American Physiology Society, Bethesda, USA.
- Hofmann AF, Small DM (1967) Detergent properties of bile salts: correlation with physiological function. Annu Rev Med 18: 333-376.
- Madenci D, Egelhaaf SU (2010) Self-assembly in aqueous bile salt solutions. Curr Opin Colloid Interface Sci 15: 109-115.
- Holm R, Müllertz A, Mu H (2013) Bile salts and their importance for drug absorption. Int J Pharm 453: 44-55.
- Andrews AT, Alichanidis E (1983) Proteolysis of caseins and the proteose-peptone fraction of bovine milk. J Dairy Res 50: 275-290.
- Paquet D (1989) Review: the proteose-peptone fraction of milk. Lait 69: 1-21.
- Andrews AT (1983) Proteinases in normal bovine milk and their action on caseins. J Dairy Res 50: 45-55.
- Eigel WN, Butler JE, Ernstrom CA, Farrell Jr HM, Harwalker VR, et al. (1984) Nomenclature of proteins of cows’ milk: fifth revision. J Dairy Sci 67: 1599-1631.
- Kaminogawa S, Mizobuchi H, Yamauchi K (1972) Comparison of bovine milk protease with plasmin. Agric Biol Chem 36: 2163-2167.
- Myers R (2003) The basics of chemistry. Greenwood Press, Westport, Connecticut, USA.
- Movaffaghi Z, Farsi M (2009) Biofield therapies: biophysical basis and biological regulations? Complement Ther Clin Pract 15: 35-37.
- Trivedi MK, Patil S, Tallapragada RM (2013) Effect of biofield treatment on the physical and thermal characteristics of vanadium pentoxide powders. J Material Sci Eng S11: 001.
- Trivedi MK, Patil S, Tallapragada RM (2013) Effect of biofield treatment on the physical and thermal characteristics of silicon, tin and lead powders. J Material Sci Eng 2: 125.
- Trivedi MK, Patil S, Tallapragada RM (2014) Atomic, crystalline and powder characteristics of treated zirconia and silica powders. J Material Sci Eng 3: 144.
- Trivedi MK, Patil S, Tallapragada RMR (2015) Effect of biofield treatment on the physical and thermal characteristics of aluminium powders. Ind Eng Manag 4: 151.
- Shinde V, Sances F, Patil S, Spence A (2012) Impact of biofield treatment on growth and yield of lettuce and tomato. Aust J Basic Appl Sci 6: 100-105.
- Sances F, Flora E, Patil S, Spence A, Shinde V (2013) Impact of biofield treatment on ginseng and organic blueberry yield. Agrivita J Agric Sci 35: 22-29.
- Lenssen AW (2013) Biofield and fungicide seed treatment influences on soybean productivity, seed quality and weed community. Agricultural Journal 8: 138-143.
- Altekar N, Nayak G (2015) Effect of biofield treatment on plant growth and adaptation. J Environ Health Sci 1: 1-9.
- Trivedi MK, Patil S (2008) Impact of an external energy on Staphylococcus epidermis [ATCC - 13518] in relation to antibiotic susceptibility and biochemical reactions - An experimental study. J Accord Integr Med 4: 230-235.
- Trivedi MK, Patil S (2008) Impact of an external energy on Yersinia enterocolitica [ATCC - 23715] in relation to antibiotic susceptibility and biochemical reactions: An experimental study. Internet J Alternative Med 6: 2.
- Trivedi MK, Bhardwaj Y, Patil S, Shettigar H, Bulbule A (2009) Impact of an external energy on Enterococcus faecalis [ATCC - 51299] in relation to antibiotic susceptibility and biochemical reactions – An experimental study. J Accord Integr Med 5: 119-130.
- Patil SA, Nayak GB, Barve SS, Tembe RP, Khan RR (2012) Impact of biofield treatment on growth and anatomical characteristics of Pogostemon cablin (Benth.). Biotechnology 11: 154-162.
- Albers S, Muchova Z, Fikselova M (2009) The effects of different treated brans additions on bread quality. Scientia Agriculturae Bohemica 40: 67-72.
- Auffret A, Ralet MC, Guillon F, Barry JL, Thibault JF (1994) Effect of grinding and experimental conditions on the measurement of hydration properties of dietary fibers. LWT-Food Sci Technol 27: 166-172.
- Zhang DC, Moore WR (1997) Effect of wheat bran particle size on dough rheological properties. J Sci Food Agr 74: 490-496.
- Robertson JA, Eastwood MA (1981) An investigation of the experimental conditions which could affect water-holding capacity of dietary fiber. J Sci Food Agr 32: 819-825.
- Omidian H, Park K (2008) Swelling agents and devices in oral drug delivery. J Drug Del Sci Tech 18: 83-93.
- Midelfort KS, Wittrup KD (2006) Context-dependent mutations predominate in an engineered high-affinity single chain antibody fragment. Protein Sci 15: 324-334.
- Hooper NM (1994) Families of zinc metalloproteases. FEBS Lett 354: 1-6.
- Wiseman RL, Powers ET, Kelly JW (2005) Partitioning conformational intermediates between competing refolding and aggregation pathways: insights into transthyretin amyloid disease. Biochemistry 44: 16612-16623.
- Amin S, Barnett GV, Pathak JA, Roberts CJ, Sarangapani PS (2014) Protein aggregation, particle formation, characterization & rheology. Curr Opin Colloid Interface Sci 19: 438-449.
- Mennucci B, Martinez JM (2005) How to model solvation of peptides? Insights from a quantum-mechanical and molecular dynamics study of N-methylacetamide. I. Geometries, infrared, and ultraviolet spectra in water. J Phys Chem B 109: 9818-9829.
- Bendz D, Tuchsen PL, Christensen TH (2007) The dissolution kinetics of major elements in municipal solid waste incineration bottom ash particles. J Contam Hydrol 94: 178-194.
- Rub MA, Sheikh MS, Asiri AM, Azum N, Khan A, et al. (2013) Aggregation behaviour of amphiphilic drug and bile salt mixtures at different compositions and temperatures. J Chem Thermodynamics 64: 28-39.
- Tang CH, Chen Z, Li L, Yang XQ (2006) Effects of transglutaminase treatment on the thermal properties of soy protein isolates. Food Res Int 39: 704-711.
- Bell LN, Touma DE (1996) Glass transition temperatures determined using a temperature cycling differential scanning calorimeter. Food Sci 61: 807-810.
- Di Gioia L, Cuq B, Guilbert S (1999) Thermal properties of corn gluten meal and its proteic components. Int J Biol Macromol 24: 341-350.
- Tang CH, Yang XQ, Chen Z, Wu H, Peng ZY (2005) Physicochemical and structural characteristics of sodium caseinate biopolymers induced by microbial transglutaminase. J Food Biochem 29: 402-421.
- Arntfield SD, Murray ED (1981) The influence of processing parameters on food protein functionality I. Differential scanning calorimetry as an indicator of protein denaturation. Can Inst Food Sci Technol J 14: 289-294.
- Privalov PL (1982) Stability of proteins. Proteins which do not present a single cooperative system. Adv Protein Chem 35: 1-104.