Evaluation of the Impact of Biofield Treatment on Physical and Thermal Properties of Casein Enzyme Hydrolysate and Casein Yeast Peptone

In the present study, the influence of biofield treatment on physical and thermal properties of Casein Enzyme Hydrolysate (CEH) and Casein Yeast Peptone (CYP) were investigated. The control and treated samples were characterized by Fourier transform infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC), Thermo Gravimetric Analysis (TGA), particle size and surface area analysis. The FTIR results revealed that biofield treatment has caused reduction of amide group (amide-I and amide-II) stretching vibration peak that is associated with strong intermolecular hydrogen bonding in treated CEH as compared to control. However, no significant changes were observed in FTIR spectrum of treated CYP. The TGA analysis of treated CEH showed a substantial improvement in thermal stability which was confirmed by increase in maximum thermal decomposition temperature (217°C) as compared to control (209°C). Similarly, the treated CYP also showed enhanced thermal stability as compared to control. DSC showed increase in melting temperature of treated CYP as compared to control. However the melting peak was absent in DSC of treated CEH which was probably due to rigid chain of the protein. The surface area of treated CEH was increased by 83% as compared to control. However, a decrease (7.3%) in surface area was observed in treated CYP. The particle size analysis of treated CEH showed a significant increase in average particle size (d50) and d99 value (maximum particle size below which 99% of particles are present) as compared to control sample. Similarly, the treated CYP also showed a substantial increase in d50 and d99 values which was probably due to the agglomeration of the particles which led to formation of bigger microparticles. The result showed that the biofield treated CEH and CYP could be used as a matrix for pharmaceutical applications.


Introduction
Over the last few decades, there has been continuous interest in biodegradable polymers for pharmaceutical and biomaterial applications [1]. Biodegradable polymers can be either synthetic or natural polymers. The synthetic polymers are more popular than their natural counterparts due to their excellent mechanical properties which can be used for biomedical applications. However the synthetic polymers are associated with toxicity problems which may cause problems during their intended medical use. Natural polymers are generally regarded as safe compared to synthetic polymers. Hence the natural polymers have clear advantages as drug delivery systems (DDS) [2]. Recently, protein based therapeutics, due to their excellent properties such as emulsification, foaming, gelling, and water holding ability have gained significant attention as DDS [3][4][5][6]. Moreover, the food proteins have their inherent ability to interact with wide range of bioactive compounds via functional groups present on their polypeptide structure. Hence, this offers the reversible binding of active molecules and protects them until their safe release in the human body [7,8]. Additionally, proteins are metabolizable; hydrolysis of the proteins by digestive enzymes releases the bioactive peptides that may cause a number of beneficial effects such as cardiovascular, endocrine, immune and nervous system [9,10].
Milk proteins are natural vehicles and widely explored in food industries due to their inherent nutritional and functional properties.
Casein is a main structural component of milk, where it accounts for 80% of total proteins content. Casein has been utilized in the production of food, pharmaceutical formulations and cosmetics. The interesting structure and physicochemical properties allows it to be used in DDS [11]. The casein has fascinating properties such as binding of ions and small molecules, excellent emulsification, surface active, gelation and water binding capacities.
Hydrolysation of protein makes changes in the composition of potential groups; hydrophobic properties and functional characteristics [12]. For example CEH is a protein that is rapidly absorbed and digested similar to whey protein. Enzyme hydrolysis was recently used to modify the protein structure in order to enhance the functional properties of proteins. However, these chemical and enzymatic treatments might induce denaturation of protein which directly affects its functional properties.
Bioelectromagnetism is an area which studies the interaction of living biological cells and electromagnetic fields. Researchers have demonstrated that short lived electrical current or action potential exists in several mammalian cells such as neurons, endocrine cells and muscle cells as well as some plant cells. An Italian physicist Luigi Galvani first time observed this phenomenon in a frog where he had been working on static electricity [13]. Similarly it was believed that electromagnetic field exists around the human body and the evidence was found using some medical technologies such as electromyography, electrocardiography, and electroencephalogram. This field is known as biofield and the exposure of the said biofield has been referred hereinafter as Biofield treatment.
Recently, biofield treatment was used to modify the physical, atomic and thermal properties of various ceramic, metals and carbon allotropes [14][15][16][17][18][19][20][21]. Mr. Trivedi is known to transform these materials using his biofield. The biofield treatment has also improved the production and quality of various agricultural products [22][23][24][25]. Moreover, the biofield has resulted into altered antibiotic susceptibility patterns and the biochemical characteristics of various bacteria [26][27][28]. Exposure to the said biofield has caused an enhancement in growth and anatomical characteristics of herbs like Pogostemon cablin that is commonly used in perfumes, in incense/insect repellents, and alternative medicine [29]. In this study, the effects of biofield treatment on two protein based organic compounds (CEH and CYP) are studied and their physicochemical properties are evaluated.

Materials and Methods
The casein enzyme hydrolysate and casein yeast peptone were procured from HiMedia Laboratories Pvt. Ltd. India. The samples were grouped into two parts; one was kept as a control sample, while the remaining sample was subjected to Mr. Trivedi's biofield treatment and coded as treated sample. After that, all the samples (control and treated) were characterized with respect to FTIR, DSC, TGA, particle size and surface area analysis.

Characterization
Fourier Transform Infrared (FTIR) spectroscopy: The infrared spectra of CEH and CYP (control and treated) were recorded on FT-IR spectrometer, (Perkin Elmer, USA).
The IR spectrum was recorded in the range of 4000-500 cm -1 .
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 (d 50 ) and d 99 (maximum particle size below which 99% of particles) were computed from laser diffraction data table. The d 50 and d 99 value were calculated using the following formula.

Results and Discussion
FTIR spectroscopy: Figure 1a and 1b showed the FTIR spectrum of control and treated CEH, respectively. The FTIR spectrum of control CEH showed ( Figure  1a) an important absorption peaks at 3215 cm -1 , 2974 cm -1 which were attributed to -OH and -CH stretching vibration peaks respectively. Other absorption peaks were observed at 1654 cm -1 and 1596 cm -1 due to amide-I and amide-II stretching vibration peaks. The spectrum showed peaks at 1078 cm -1 which was responsible to -OH bending vibration peaks. The treated CEH showed (Figure 1b) shifting of the -OH/-NH stretching and amide (amide-I and amide-II) peaks toward lower wavenumbers. The -OH stretching vibration peak was shifted to 3199 cm -1 and amide group peaks were shifted to lower wavenumber 1633 cm -1 and 1587 cm -1 respectively. This showed that biofield treatment has induced strong intermolecular hydrogen bonding in treated CEH structure. It was previously shown that hydrogen bonding lowered the frequency of stretching vibrations in proteins, since it lowers the restoring force, however increases the frequency of bending vibrations since it produces an additional restoring force [30,31]. Additionally it was shown that hydrogen bonding in -NH group lowers the stretching vibration by 10 to 20 cm -1 [32]. Hence in treated CEH, the amide-I band lowered by 21 cm -1 and amide-II lowered by 13 cm -1 provided a strong proof of hydrogen bonding in the treated sample.
The Figure 2a and 2b shows the FTIR spectrum of control and treated CYP powder. FTIR spectrum of treated and control powder shows (Figure 2a) slight reduction in the hydrogen bonded -OH stretching of treated sample as compared to control. The control CYP sample showed 1635 and 1589 cm -1 which were due to amide-I and amide-II stretching vibration peaks. The treated sample showed (Figure 2b) minimal changes in wavenumber of -OH (3060 cm -1 ) , amide-I (1687 cm -1 ) and amide-II (1585 cm -1 ). The results confirmed that biofield treatment has induced the structural changes in the treated samples.

Particle size and surface area analysis
The particle size analysis results of CEH and CEP (control and treated) are depicted in Figures 3 and 4. The average particle size  (Figure 3). The control CEH showed d 50 value of 11.88 μm and it has found increased to 12.6 μm in treated sample. The d 99 value was also increased in treated sample (136.74 μm) as compared to control (115.16 μm). The percentage average particle size, d 50 and d 99 of the treated CEH were increased (6.1 and 18.7%) substantially as compared to control (Figure 4).
Whereas, in treated sample of casein yeast peptone (12.61 μm), the d 50 value has been found increased in comparison with control (10.86 μm). Nonetheless, most significant result was observed in d 99 value of treated sample of CYP which was found to be 317.52 μm as compared to 143.4 μm in control sample. In treated, the average particle sizes, d 50 and d 99 of CYP were found increased by 16.1% and 121.4% respectively ( Figure 4).
The surface area was analyzed by BET analysis and the results are presented in Table 1. The surface area of treated CEH (1.004 m 2 /g) showed significant improvement as compared to control sample (0.5459 m 2 /g). After calculation, the percentage change in surface area was found to be increased by 83.9% in the treated sample of CEH. Contrarily the treated CYP (1.12 m 2 /g) showed a decrease in surface area by 7.3% as compared to control sample (1.21 m 2 /g). This result can be correlated with increased particle size results of CYP. The surface area and particle size changes are usually opposite to each other, i.e. smaller the particles size, larger the surface area and vice versa [33][34][35]. Hence, we conclude that increase in particle size substantially reduced the surface area of treated CYP as compared to control sample.

Differential Scanning Calorimetry (DSC)
DSC is a popular technique for investigating the glass transition, melting nature and change in specific heat capacity of materials. The DSC thermogram of control and treated CEH is presented in Figure 5a and 5b. The control CEH sample showed (Figure 5a) an endothermic peak at 140°C which was probably due to bound water with the protein sample. The thermogram also showed a very broad endothermic inflexion at 198°C which was responsible for its melting temperature. The broad peak was probably due to associated water with the sample. DSC of treated CEH showed (Figure 5b) no thermal transition in its thermogram. This was probably due to the highly rigid nature of the treated protein network which was not melted even on the higher temperature. It may be correlated with good thermal stability of the treated sample. Based on the results, we postulate that biofield may have acted directly upon amorphous regions of protein hydrolysate and induced the atoms to come together that led to the formation of a long range order. This may have caused higher crystallinity and ordered regions which probably required more energy in order to break the chains.
The DSC thermogram of CYP (control and treated) powders are presented in Figure 6a and 6b. The control sample showed ( Figure  6a) an endothermic peak at 144°C. However the DSC thermogram of treated CYP showed (Figure 6b) a broad endothermic peak at 191°C which was associated with its melting temperature. This showed the increased thermal stability of the CYP after biofield treatment.

Thermo Gravimetric Analysis (TGA)
TGA analysis provides information about thermal stability of the sample. TGA thermogram of control and treated samples of CEH is presented in Figure 7a and 7b respectively. The control CEH sample showed (Figure 7a) single step thermal degradation which started at 170°C and stopped at 240°C. The derivative thermogravimetry (DTG) showed maximum thermal decomposition temperature at 209°C in control sample. TGA thermogram of treated sample (Figure 7b) showed two step thermal decomposition pattern. In the first step, the sample started to degrade at 190 o C and ended at 240°C. During this event the sample lost 12.4% of its original weight. The second step commenced at 260°C and ended at 380°C. The DTG analysis showed a maximum thermal decomposition peak at 217°C in treated sample. The increase in maximum thermal decomposition peak in treated sample probably enhanced the thermal stability as compared to control. It is presumed that biofield treatment has probably induced strong hydrogen bonds in treated CEH sample which raised the decomposition temperature of the sample. It is worthwhile to note here that the FTIR spectrum ( Figure 1b) of treated CEH showed hydrogen bonding in the sample. This is also well supported by DSC results.
TGA thermogram of CYP (control and treated) sample is presented in Figure 8a and 8b. The thermal decomposition of the control CYP ( Figure 8a) started at 180°C and ended at 228°C. The sample has showed maximum thermal decomposition at 202°C. During this thermal process sample lost 11.26% of its original weight. The comparative evaluation of DTG peaks showed that after biofield treatment the thermal stability of the treated CYP (216°C) (Figure 8b) is found to be increased as compared to control (202°C). This shows the enhanced thermal stability of the treated CYP sample.

Conclusion
This study showed the influence of biofield treatment on the physical and thermal properties of the CEH and CYP. Biofield treatment did cause a significant change in structure characterization, along with an increase in particle size, melting temperature and maximum decomposition temperature as compared to control sample, which were analyzed by standard techniques. Hence we postulate that the biofield treated organic protein products (CEH and CYP) could be used either as an interesting matrix for drug delivery or as a medium for cell culture research.