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European Journal of Biophysics 2015 36: 51-58 Published online December 21 2015 http://www.sciencepublishinggroup.com/j/ejb doi: 10.11648/j.ejb.20150306.12 ISSN: 2329-1745 Print ISSN: 2329-1737 Online Physical Spectroscopic and Thermal Characterization of Biofield Treated Fish Peptone Mahendra Kumar Trivedi 1 Alice Branton 1 Dahryn Trivedi 1 Gopal Nayak 1 Ragini Singh 2 Snehasis Jana 2 1 Trivedi Global Inc. Henderson USA 2 Trivedi Science Research Laboratory Pvt. Ltd. Bhopal Madhya Pradesh India Email address: publicationtrivedisrl.com S. Jana To cite this article: Mahendra Kumar Trivedi Alice Branton Dahryn Trivedi Gopal Nayak Ragini Singh Snehasis Jana. Physical Spectroscopic and Thermal Characterization of Biofield Treated Fish Peptone. European Journal of Biophysics. Vol. 3 No. 6 2015 pp. 51-58. doi: 10.11648/j.ejb.20150306.12 Abstract: The by-products of industrially processed fish are enzymatically converted into fish protein isolates and hydrolysates having a wide biological activity and nutritional properties. However the heat processing may cause their thermal denaturation thereby causing the conformational changes in them. The present study utilized the strategy of biofield energy treatment and analysed its impact on various properties of the fish peptone as compared to the untreated control sample. The fish peptone sample was divided into two parts one part was subjected to Mr. Trivedi’s biofield treatment coded as the treated sample and another part was coded as the control. The impact of biofield treatment was analysed through various analytical techniques and results were compared with the control sample. The particle size data revealed 4.61 increase in the average particle size d 50 along with 2.66 reduction in the surface area of the treated sample as compared to the control. The X-ray diffraction studies revealed the amorphous nature of the fish peptone sample however no alteration was found in the diffractogram of the treated sample with respect to the control. The Fourier transform infrared studies showed the alterations in the frequency of peaks corresponding to N-H C-H CO C-N and C-OH functional groups in the treated sample as compared to the control. The differential scanning calorimetry data revealed the increase in transition enthalpy ∆H from -71.14 J/g control to -105.32 J/g in the treated sample. The thermal gravimetric analysis data showed the increase in maximum thermal degradation temperature T max from 213.31°C control to 221.38°C along with a reduction in the percent weight loss of the treated sample during the thermal degradation event. These data revealed the increase in thermal stability of the treated fish peptone and suggested that the biofield energy treatment may be used to improve the thermal stability of the heat sensitive compounds. Keywords: Fish Peptone Biofield Energy Treatment Protein Hydrolysate Differential Scanning Calorimetry Thermogravimetric Analysis 1. Introduction The fisheries industry is a major source of income for various countries worldwide. However the industrially processed fish that is utilized for human consumption yields more than 3.17 million tons by-products per year 1. These by-products require proper disposal and hence creates the huge revenue loss to the seafood industry 2. Therefore the emphasis was done to find the new uses for these waste by- products. In recent years several advancements in biotechnology field utilize the marine by-products and convert them into some product of interest 3. It includes their conversion in protein isolates and hydrolysates having functional food properties and natural food antioxidants 4. The protein converts into smaller peptides through enzymatic conversion and their breakdown products yield protein hydrolysates. Many researchers have reported the biological activity and nutritional values of protein hydrolysates through their bioactive peptides 5 6. The peptones are a mixture of polypeptides and amino acids that are used in several biotechnological applications. They are derived from the acid or enzymatic hydrolysis of natural products such as bovine or porcine meat milk yeasts and plants. The peptones are mainly used in the production of

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52 Mahendra Kumar Trivedi et al.: Physical Spectroscopic and Thermal Characterization of Biofield Treated Fish Peptone media for fermentation tissue culture and vaccine stabilizers 7. The main source of peptone is animal tissues however it faces the problem of bovine spongiform encephalopathy a neurodegenerative disease and commonly known as mad cow disease. The main cause of this disease is a specific type of misfolded protein prion and transmitted to the healthy animals through infected sheep and goats 8. The problems related to animal tissue peptones and their increased demand as raw material focuses the attention towards fish peptones due to their non-meat origin free from swine-flu and inexpensive as derived from fish by-products 9 10. Further researches proved fish peptone as a source of high protein and a balance of amino acids hence used as the main source of industrial peptones. The peptones as a source of nitrogen become the most expensive part of growth media in the fermentation industry 11. Besides during processing such kind of products are subjected to various thermal treatments to inactivate the antinutritional factors remove allergens and to obtain the required solubility and texture 12. However they may face the problem of some conformational changes due to their thermal denaturation during heating that might affect their solubility stability and shelf-life 13 14. Therefore it creates the need for some alternative strategies that may help to improve the stability related issues of this compound in a cost-effective manner. The biofield energy treatment was reported as a measure for increasing the thermal stability of some organic products 15. It is a putative form of energy that surrounds the body of all living organisms and can be exchanged with the environment 16 17. A human can harness the energy from the environment or universe and can transmit it to any living or non-living object. The objects will receive the energy and respond to useful way this process is termed as biofield treatment. It is reported for the efficacy and benefits in cancer and arthritis patients 18 19. Moreover Mr. Trivedi is also well known for his unique biofield energy treatment The Trivedi Effect ® . It was reported for its impact on the plants 20 microorganisms 21 and culture medium 22. Hence the present work was aimed to treat the fish peptone by Mr. Trivedi’s biofield energy and evaluate the impact on the physicochemical properties and stability of the fish peptone using various analytical techniques viz. particle size analyser surface area analyser X-ray diffraction Fourier transform infrared spectroscopy UV-visible spectroscopy differential scanning calorimetry and thermogravimetric analysis. 2. Materials and Methods The fish peptone was procured from HiMedia Laboratories India. In treatment methodology the fish peptone sample was divided into two equal parts in which one part was coded as the control and another part as the treated. The treated part was handed over in sealed pack to Mr. Trivedi under standard laboratory conditions. Mr. Trivedi provided the biofield energy treatment to this part treated through his unique energy transmission process without touching the sample. The control part was kept untreated. The impact of biofield treatment on the treated sample was subsequently analysed as compared to the control sample using various analytical techniques. 2.1. Particle Size Analysis The SYMPATEC HELOS-BF laser particle size analyser was used for the determination of particle size of the control and treated samples. The analyser was having a detection range of 0.1 µm to 875 µm. The parameters determined in the analysis were d 50 average particle size and d 99 size below which 99 of the particles are present. 2.2. Surface Area Analysis The Brunauer-Emmett-Teller BET surface area analyser Smart SORB 90 was used to calculate the surface area of the control and treated samples. 2.3. X-Ray Diffraction XRD Study The X-ray powder diffractograms were recorded using Phillips Holland PW 1710 X-ray diffractometer that uses 1.54056Å wavelength of radiation. The X-ray generator was operating at 35kV and 20mA and equipped with a copper anode with nickel filter. The diffractograms of control and treated samples were analysed to determine the nature of the sample i.e. crystalline or amorphous and XRD of treated sample was compared with the control to analyse any difference between them. 2.4. Fourier Transform-Infrared FT-IR Spectroscopic Characterization The Shimadzu’s Fourier transform infrared spectrometer Japan was used for recording the FT-IR spectra of the control and treated samples in the frequency range 4000-450 cm -1 . The spectra were obtained in the form of wavenumber 1/cm vs. percent transmittance T. The peaks obtained from the spectra of control and treated samples were assigned on the basis of functional groups present in the sample. The frequency of the peaks corresponding to the functional groups were compared in the control and treated samples for analysing the impact of biofield energy with respect to the bond length and bond angle of those functional groups. 2.5. UV-Visible UV-Vis Spectroscopic Characterization The UV-Vis spectral analysis was carried out using Shimadzu UV-2400 PC series spectrophotometer. The spectra of the control and treated samples were recorded using 1 cm quartz cell that has a slit width of 2.0 nm. 2.6. Differential Scanning Calorimetric DSC Analysis The DSC analysis of control and treated samples was carried out using model Perkin Elmer/Pyris-1. The samples were heated at a rate of 10°C/min under air atmosphere 5 mL/min. The thermograms were collected over the

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European Journal of Biophysics 2015 36: 51-58 53 temperature range of 50°C to 300°C. Any differences in the transition temperature and transition enthalpy were recorded from the thermogram to determine the impact of biofield energy treatment on the treated sample with respect to the control. 2.7. Thermogravimetric Analysis / Derivative Thermogravimetry TGA/DTG The thermal stability profile of fish peptone was analysed using Mettler Toledo simultaneous thermogravimetric analyser TGA/DTG. The temperature range was selected from room temperature to 400ºC and a heating rate of 5ºC/min under air atmosphere. The impact of biofield treatment was analysed by comparing the pattern of degradation maximum degradation temperature and percent weight loss of the treated sample as compared to the control sample. 3. Results and Discussion 3.1. Particle Size Analysis Fig. 1. Percent change in particle size and surface area of the treated fish peptone as compared to the control. The particle size analysis data depicted that the d 50 and d 99 were 23.44 and 120.17 µm respectively in the control sample. However in treated sample the d 50 and d 99 were found as 24.52 and 124.11 µm respectively. The percent change observed in the particles sizes of the biofield treated sample with respect to the control is depicted in Fig. 1. It revealed that d 50 was increased by 4.61 and d 99 was increased by 3.28 in the treated sample as compared to the control. The protein molecules have a tendency to aggregate through the weak interactions 23. Moreover the temperature has a significant impact on the aggregation rate 24. Hence it is assumed that the biofield energy treatment might provide some energy to the sample that resulted in the slight increase in the particle size of the treated sample through the process of aggregation. 3.2. Surface Area Analysis The surface area of control and treated samples of fish peptone was investigated using BET method. The data reported that the control sample had a surface area of 0.188 m 2 /g however the treated sample had a surface area of 0.183 m 2 /g. It showed that the surface area was decreased by 2.66 Fig. 1 in the treated sample as compared to the control. The effective surface area is inversely related to the particle size of the compound 25. Thus the slight decrease in surface area might be attributed to the increase in the particle size of the treated sample after biofield treatment. 3.3. X-Ray Diffraction XRD The X-ray powder diffractograms of control and treated samples of fish peptone are shown in Fig. 2. The XRD pattern of the control sample did not contain any diffraction maxima which indicated that the molecules were internally disordered and glassy in nature. The glassy material showed one broad peak that resulted due to short range ordering of the molecules as compared to the long-range order in a crystal 26. The diffractogram of the treated sample also showed similar XRD pattern indicated that biofield energy treatment may not cause any alteration in the ordering of the molecules of fish peptone sample. Fig. 2. X-ray diffractograms of control and treated samples of fish peptone. 3.4. FT-IR Spectroscopic Analysis The FT-IR spectra of fish peptone control and treated samples are shown in Fig. 3. The fish peptone contains mainly proteins and carbohydrates 27. Hence the major vibration peaks observed Table 1 were assigned to the functional groups present in these ingredients. The peak at 3080 cm -1 in the control sample was assigned to NH 3 + antisymmetric stretching of amino acids however the peak -4 -3 -2 -1 0 1 2 3 4 5 Percent change d 50 d 99 Particle size Surface area

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54 Mahendra Kumar Trivedi et al.: Physical Spectroscopic and Thermal Characterization of Biofield Treated Fish Peptone might get merged with the C-H stretching peaks 28. Besides in the treated sample the peak was shifted to a lower frequency at 3066 cm -1 . Similarly the C-H stretching peaks of carbohydrates were appeared at 2993 and 2893 cm -1 in the control sample whereas in the treated sample the peaks were appeared at 2976 and 2885 cm -1 . Besides the C-H stretching peak of CH 2 group attached to -O- of lactone ring present in carbohydrate was observed at higher frequency i.e. at 2835 cm -1 as compared to the control 2815 cm -1 . The peak observed at 1753 cm -1 in the control sample was assigned to the CO stretching of the lactone ring that was not observed in the treated sample. Moreover the peak at 1635 cm -1 in the control was assigned to the NH 2 deformation and CO stretching of the amide present in the peptone. However the corresponding peak was shifted to 1643 cm -1 in the treated sample. Similarly the peak observed at 1589 cm -1 in the control sample was assigned to the NH 2 deformation of amino acids and ring stretching of the benzene ring. The corresponding peak was appeared at 1579 cm -1 in the treated sample. The peaks due to aliphatic CH 3 scissoring and bending were observed at 1438 and 1340 cm -1 in the control sample however the corresponding peaks were observed at 1458 and 1342 cm -1 in the treated sample. The O- H in-plane bend and C-N stretching of amide group present in amino acids showed a peak at 1400 cm -1 in the control and 1402 cm -1 in the treated sample. Similarly the -C-O-C- stretching peak of the lactone ring was observed at 1245 and 1247 cm -1 in the control and treated samples respectively 28. Besides the N-H bending peak of pyrrole ring C-N stretching peak of pyrazole ring present in proteins was appeared at 1151 cm -1 in the control sample 29 30 whereas at 1118 cm -1 in the treated sample. The C-OH stretching peak of carbohydrates was observed at 1072 cm -1 in the control and 1085 cm -1 in the treated sample. The C-H out of plane bending peak of pyrazole ring in was observed at the same frequency in both the control and treated samples i.e. at 921 cm -1 . Similarly the peaks due to OC-O bend of carboxylic acid and -N-CO bend of amide group present in amino acids were observed at nearly same frequencies in both the samples i.e. at 617 and 536 cm -1 in the control and 619 and 536 cm -1 in the treated sample respectively. Fig. 3. FT-IR spectra of control and treated samples of fish peptone.

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European Journal of Biophysics 2015 36: 51-58 55 Table 1. Vibration modes observed in fish peptone. S. No. Functional group Wavenumber cm -1 Control Treated 1 NH 3 + stretching 3080 3066 2 C-H stretching 2993 2893 2976 2885 3 C-H stretching 2815 2835 4 CO stretching lactone 1753 ND 5 NH 2 deformation CO stretching 1635 1643 6 NH 2 deformation Ring stretching benzene 1589 1579 7 CH 3 bending 1438 1340 1458 1342 8 O-H in plane bend C-N stretching amide 1400 1402 9 C-O-C stretching lactone 1245 1247 10 N-H bending C-N stretching heterocyclic ring 1151 1118 11 C-OH stretching 1072 1085 12 C-H out of plane bend heterocyclic ring 921 921 13 OC-O bending 617 619 14 N-CO bending amide 536 536 The observation showed the alterations in the frequency of several peaks in the treated sample such as N-H C-H CO C-N C-OH etc. as compared to the control. It revealed that the biofield energy treatment might have an impact on the bond length bond angle or dipole moment of the corresponding functional groups in the treated sample. However further analysis is required to determine the effect of biofield energy on the particular functional group and its impact on the properties of fish peptone sample. 3.5. UV-Vis Spectroscopic Analysis The UV spectrum of control sample showed the absorption peak at λ max equal to 258 nm. The biofield treated sample also showed absorption peak at similar wavelength i.e. 259 nm. It showed that the biofield energy treatment had not affected the HOMO→LUMO transition within the components of fish peptone sample. 3.6. DSC Analysis The DSC thermograms of control and treated samples of fish peptone are presented in Fig. 4. The thermogram of control sample showed a broad endothermic peak in the range of 193°C-197°C. The broadness of peak confirmed the amorphous nature of the sample as evident from the XRD studies. Moreover the DSC can be used in the evaluation of stability of the sample by determining the temperature at the maximum point of the endothermic curve T m 31. The DSC thermogram of the control sample showed T m at 197.14°C. This temperature can be considered as the denaturation temperature of the control sample 32. Furthermore the treated sample showed the endothermic curve in the range of 189°C-200°C and the T m was reported at 197.20°C. The results of control and treated samples do not reveal any significant alteration in the denaturation temperature T m however the major difference was observed in the transition enthalpy ∆H during this event. The control sample showed the ∆H of -71.14 J/g whereas the treated sample showed ∆H of -105.32 J/g. It revealed that the biofield treated sample required 31.14 J/g more energy to undergo the process of denaturation that might be related to the increased thermal stability of the treated sample as compared to the control. Fig. 4. DSC thermograms of control and treated samples of fish peptone. 3.7. TGA/DTG Analysis The TGA/DTG studies analyse the change in the mass of the sample and thereby measure the physical or chemical changes that may occur within the sample during the heat treatment. This method is also used as a complementary to the DSC technique 33. These techniques were used to determine the thermal stability of the sample. The TGA/DTG thermograms of the control and treated samples of fish peptone are reported in Fig. 5. The TGA thermograms of both samples showed the presence of two-step degradation. In the control sample the first step showed an onset temperature of 195°C and an endset of about 240°C which involved a weight loss of 16.76 of the sample. On the other hand the treated sample showed an onset temperature of 193°C and the endset of 250°C with a weight loss of 12.96

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56 Mahendra Kumar Trivedi et al.: Physical Spectroscopic and Thermal Characterization of Biofield Treated Fish Peptone in the first step of degradation. The TGA results were similar to the data revealed by DSC studies and suggested that the onset temperature of weight loss in control and treated samples were due to the thermal degradation of the samples. However the treated sample lost less weight as compared to the control sample that might be due to increase in the thermal stability of the treated sample after biofield treatment. Moreover the second step of degradation in the control sample commenced at 260°C is also delayed by 8°C and observed at 268°C in the treated sample. Besides DTG thermogram data showed that T max was observed at 213.31°C in the control sample while 221.38°C in the treated fish peptone. It indicated that T max was increased in the treated sample as compared to the control. Furthermore the reduction in percent weight loss and the increase in T max in the treated sample of fish peptone with respect to the control sample may be correlated with the increased thermal stability. The biofield treatment was also reported for increasing the thermal stability in casein enzyme hydrolysate and casein yeast peptone 34. Hence it is assumed that the biofield energy treatment might induce the aggregation of the molecules of treated sample through the weak intermolecular interactions that resulted in increased thermal stability. Besides as reported earlier during processing the thermal treatment can cause the denaturation of such type of compounds and result in the conformational changes. These changes might affect the solubility and stability of compound along with their long term storage 13 14. Hence the biofield treatment might be used as an effective measure to increase the thermal stability thereby increasing the stability efficacy and shelf-life of these compounds. Fig. 5. TGA/DTG thermogram of control and treated samples of fish peptone. 4. Conclusions The biofield treated fish peptone reported the increased particle sizes d 50 and d 99 suggesting the aggregation of molecules that might occur due to the impact of biofield energy. The slight reduction in surface area was also revealed in the treated sample as compared to the control that supported the impact of biofield energy on the particle size. The XRD studies revealed the amorphous nature of fish peptone sample however no significant alteration was observed in the diffractogram of treated sample as compared to the control. The FT-IR spectroscopy results suggested some alteration in the frequency of peaks of various functional groups in the treated sample such as N-H C-H CO C-N C-OH etc. that

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European Journal of Biophysics 2015 36: 51-58 57 may be due to the impact of biofield energy treatment on the bond length bond angle or the dipole moment corresponding to these groups. Moreover the DSC analysis revealed the increase in transition enthalpy during degradation of the treated sample that suggested the increased need for energy by the treated sample to undergone the degradation process as compared to the control. The TGA/DTG studies depicted the increase in T max and reduced percent weight loss of the treated sample as compared to the control. Hence the DSC and TGA/DTG studies showed the increased thermal stability of the treated sample. Thus it can be concluded that the biofield treated fish peptone sample may be more thermally stable as compared to the control and the biofield energy treatment could be used as an alternative strategy for improving the thermal stability of different compounds. Acknowledgements The authors would like to acknowledge the whole team from the Sophisticated Analytical Instrument Facility SAIF Nagpur and MGV Pharmacy College Nashik for providing the instrumental facility. Authors also greatly acknowledge the support of Trivedi Science Trivedi Master Wellness and Trivedi Testimonials in this research work. References 1 Ferraro V Cruz IB Jorge RF Malcata FX Pintado ME et al. 2010 Valorisation of natural extracts from marine source focused on marine by-products: A review. Food Res Int 43: 2221-2233. 2 Dekkers E Raghavan S Kristinsson HG Marshall MR 2011 Oxidative stability of mahi mahi red muscle dipped in tilapia protein hydrolysates. Food Chem 124: 640-645. 3 Galvez RP Berge JP 2013 By-products from fish processing: Focus on French industry. Utilization of fish waste. CRC Press Taylor Francis Group New York. 4 Kristinsson HG Rasco BA 2000 Fish protein hydrolysates: Production biochemical and functional properties. 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