1 1. Name: M.S. Thakur 2. Date of Birth: 20 th June 1954 3. Current position and address: Dr. M.S. Thakur, Chief Scientist (Retd) and former Head Fermentation Technology and Bioengineering Department Central Food Technological Research Institute, Mysore- 570020 [email protected]Presently : Hon. Visiting Professor Center of Material Science University of Mysore, Mysore -570006, INDIA. Tel: 0821-2515792, 0821-2546936 Fax: 0821-251723 Mob. No. 9449055108 For publication please open following link: http://scholar.google.com/citations?user=5gmRvK8AAAAJ&hl=en 4. Research Experience: R& D - Professional Experience
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Summary of Significant contributions to science and/or technology development based on
the work done in India with specific reference is to be made to research papers published
and patents taken in this context
Dr. M. S. Thakur has made fundamental contribution in bio-inspired, biomolecular electronics, nanobiotechnology, nanobiosensors and biophotonics by interfacing biochemical events in biological systems with opto-electronic systems making complicated expensive diagnostics/biosensing, affordable, simple, specific, rapid and ultrasensitive. He has developed several biosensing technologies for food, environmental and clinical monitoring. His significant contribution has been in increasing the potential of bio-sensors by conjugating nanoparticleswith biomolecules like antibodies and aptamers,making biosensing a high throughput technology. His work has given considerable insight into the understanding of opto-physical properties of nanoparticles and their biomolecular interactions such as Fluorescence Resonance Energy Transfer (FRET) and Bioluminescence Resonance Energy Transfer (BRET) phenomena's. Publications and patents on above concept: 1. Biosensors and Bioelectronics, 2012, 38(1): 411-415,IF= 5.4
5. Journal of Hazardous materials. 2012, 225–226, 114–123. IF=4.173
AFFORDABLE DIAGNOSTICS:
An advancement in cutting edge technology to detect vitamin B12 in food. Attempts
were made for visual analysis of vitamin B12 using Gold nanoparticles and aptamer. This
particular technology was awarded the "Gandhian Young Innovation Awards, 2013.
Awards and Honors: 1. Recipient of LaljeeGodhooSmarakNidhi Award for contribution in Food Science and Technology
by Association of Food Scientists and Technologists of India. 2000.
2. Recipient of CFTRI Foundation day award for best research paper 2003 published.
3. Recipient of CFTRI Foundation day award for Best applied Research Paper published
2007-08.
4. Recipient of CFTRI Foundation day Award for best research paper published in 2008-09 .
5. Recipient of CFTRI Foundation day Award for best Scientist in 2008-09
6. Recipient of CFTRI Foundation day Award for best research paper published in 2012 .
7. Long-Term DBT Overseas Associateship Award during 1988-1990.
8. Recipient of Long Term National Scholarship for study Abroadby Ministry of Human
Resource Development (Govt. of India) from 1988-1990.
Honors:
1. Member of Expert Advisory Committee of Molecular electronics and Biosensor, (DST-GOI).
2. Expert Member, Board of Directors Madhya Pradesh Biotechnology Council, Government of
Madhya Pradesh.
3. Member of Board of Governors, the Institution of Engineers (INDIA), National Design &
Research Forum.
4. I was expert advisory committee member DBT.
5. Member- Advisory committee of UGC-SAP programme. University of Madras.
6. Expert member of future R&D planning of Defence Food Research Laboratory, Mysore.
7. Member of board of studies in Alagappa University.
4
8. Member of the selection committee for SRF- DFRL, Mysore.
9. Selection committee of scientists- Bhabha Atomic Research Center, Mumbai.
10. Editorial Board member: Member of the editorial board of international journal “Journal of
Agriculture, Food and Environment” published by the International Society for Food,
Agriculture and Environment, Finland.
11. Referee for several journal of national and international repute, name a few are Biosensor
and Bioelectronics, Talanta, Sensors and Actuators, Analytical Chimica Acta and Analytical
and Bioanalytical Chemistry.
12. I have chaired President position of Association of Microbiologists of India (Mysore Chapter)
2005-06.
13. Several invited lectures and key note addresses delivered by me in national and International
conferences.
14. Received several best poster awards in national and international symposia/conferences.
Under “Turning point “ programme Doordarshan (DD) national network have made documentary on Tea biosensor (developed by my group) which was short listed as one of the
5. R& D projects handled: 1 International Projects:
1. On-line monitoring of fermentation processes using Biosensors: Work was done in-collaboration with Biotechnology Process Engineering
Center, Massachusetts Institute of Technology (MIT) and University of
Maryland UMBC) USA during my Overseas Associateship awarded by
Department of Technology 1988-1990
Capacity: As a visiting Scientist
2. Cultivation of microorganisms and production of lactic acid by fermentation. Funded by DBT- SIDA: Indo-Swedish collaborative project. 1993-2001.In this project we have developed L-lactate biosensor and Technology has been transferred to Mr. F.K. Godrej, M/S Solid State Electronics, Pune . Capacity: Co- Principal Investigator
3. Development of Immuno-bioreactor based biosensors for the analysis
of pesticides and herbicides in water. Funded by INDO-SWISS Collaborative project (SDC-DBT) 2000-2005.Phase 1
Capacity: Principal Investigator
5
4. Development of Immuno-bioreactor based Biosensor for Detection of Pesticides in Water and Environment. Funded by Indo-Swiss 2005-2008.Phase 2. Capacity: Principal Investigator
We are interfacing with Industries (M/s Bigtech, Bangalore) for technology
transfer through DBT SIBIRI Project.
5. Biosensors based on the action of transport proteins Funded
Swedish Research Council, 2006-2009.
Capacity: Principal Investigator
6. Aptamer based biosensing for the detection of Food Toxins, Indo-
Spanish Joint Programme for Technological Co-operation in
Biotechnology.Funded by DBT-India, CIFGA-CSIR-CFTRI collaboration,
2013-2016.
Capacity: Principal Investigator
2National projects
1. Development of Biosensor for food and fermentation analysis,Funded by CFTRI, 1994-96.
2. Development of a biosensor with flow injection analysis system for on-line monitoring of glucose and sucrose. Funded by DST, 1999-2002. Capacity: Principal Investigator
3. Development of Biosensor for quality assessment in tea processing. Fundedby DST –2003-2005. Capacity: Principal Investigator
4. Microprocessor based biosensor instrument development for monitoring of food and fermentation processes. Funded by DST, 1996- 2000. Capacity: Principal Investigator
5. Biotechnological Research for food safety: Development of Biosensor for OP pesticides. Funded by DBT 1995-1998. Capacity: Co-Principal Investigator
6. Development of Biosensor for chlorinated pesticides Funded by DBT 2005-2008. Capacity: Principal Investigator
6
7. Development of Biosensor for assessing the microbiological quality of foods and monitoring of formaldehyde concentration in selected marine foods. Funded by National Programme on Micro and Smart Systems (NPMASS) -ADA-DRDO. 2010-2013. Capacity: Principal Investigator.
8. Purification of caffeine from waste tea leaves and their biotransformation to valuable/potent pharmaceutical molecules. A collaborative project with IIT-Guwahati under North –East Twinning programme funded by DBT India. Capacity: Principal coordinator, 2011 – 2014
9. Detection of Food borne Pathogens by Nanobiosensors, funded by DST 211-2014. Capacity: Principal Investigator
10. Development of new protocols for evaluation of traditional foods.In
house (CSIR-CFTRI) funded projectMLP-092, 2010-2012, Capacity: Principal Investigator.
11. Fermentative production and monitoring of biologically
importantmolecules. In house (CSIR-CFTRI) funded project, MLP-0102,
2012- 2014.Capacity, Principal Investigator.
3. Industrial Projects handled:
a) Studies on development of a biotechnological process for decaffeination of beverages. Project funded by M/s TATA TEA, 2002- 2003. Project Leader
b) Bio-decaffeination of dhool (tea paste): Application of microbial enzymes
Consultancy project with M/s AV Thomas and company, Chennai. Project Leader
11. A process for microbial biotransformation of caffeine totheophylline. (Ref.No.329/DEL/2006 A)
INDIA 3Feb.2006. 12-Feb
2010
SanjuktaPatra
M.S.Thakur
N.G.Karanth
12. A biosensor device for the determination of caffeine, (Ref. No.726/DEL/2005).
INDIA 2005 SarathBabu
SanjuktaPatra
M.S.Thakur
13. A process for preparation of an enzyme electrode for the determination of copper ions 363/DEL/2002 A
INDIA 27-Mar.-
2002
9-Mar-
2007
M.D.Gouda
M.S.Thakur
N.G. Karanth
M.A. Kumar
14 Development of process for stabilization of enzymes 2159/DEL/98
INDIA 1998 M.D.Gouda
M.S.Thakur
N.G. Karanth
15. A device useful for the
measurement of organic acids
and their derivatives
21/DEL/98
Commercialized.
INDIA 1998 M.S. Thakur
Nanda Kumar
M.A. Kumar
N.G. Karanth
M.C. Misra
9. Details of R& D work done: Application of Nanotechnology in biosensors
Development of new age and upcoming bio-diagnostic techniques has revolutionized the field of analytical and bioanalytical chemistry. The coordinated efforts from various fields such as biochemistry, biotechnology and material science has led to the development of several robust and reliable biosensing tools applicable in numerous fields such as health sector, environmental safety, clinical diagnostics and food technology. The usage of nanoparticles such as quantum dots, silver and gold nanoparticles for their efficient tailoring to conjugate with numerous biosensing agents such as enzymes, antibodies, aptamers, cells and tissues which can be used in high throughput and multiplexed analysis of a variety of analytes has greatly improved and replaced the conventional analytical methods. Unique opto-physical properties, surface plasmon resonance and field confinement effects of
19
semiconductor nanoparticles have greatly enhanced the sensitivity and robustness of bio-diagnostics involved in the detection of pathogens/ toxins and other hazardous materials such as pesticides and heavy metals. We have tried to deal with the upcoming and novel bioassays for quick and multivariate analytical approach for monitoring pathogens, toxins and other hazardous analytes at ultrasensitive levels.
1. Development of Nanobiosensors for analysis of food contaminants:
With gained experience in electrochemical, optical, enzyme, tissues, antibodies and DNA, we started thinking to progress in application of nanotechnology for biosensor development. With a vision to miniaturize the biosensor devices with very high sensitivity and affordable cost possess high reliability.
i) Detection of pesticides and Toxins using Q-dots:
Efforts are on for the detection of pesticides and microorganisms and microbial
toxins using nanoparticles such as quantum dots (CdSe, CdTe, and CdS) and gold
(Au) nanoparticles. Studies have been conducted on conjugation of Atrazine with
CdTe quantum dots for highly sensitive detection based on fluorescence. Antibodies
raised against pesticides/toxins and work is in progress. We have successfully used
these nanoparticles for detection of pesticides. We have proved a novel
phenomenon called Fluorescence Resonance Energy Transfer (FRET) between
QD-Nanoparticles and Protein molecules (please see our publication).
ii) DNA nano probes:
Quantum dots are being used for the detection of food pathogens such as
Staphylococcusaureus and E.coli. The simple process of hybridization between
complementary strands of targeted ss DNA is being use for detection. For this
purpose the gene for the SEB/Ent B toxins are being used as the target sequence.
Biotinylated complementary probes will be conjugated with streptavidin coated
quantum dots. These DNA probes on binding with the target sequence will show a
20-fold increase in fluorescence compared to conventional dyes and hence very low
number of target sequence in sample solution can be traced.
iii) Fluorescent Nano-probes for targeted monitoring
Cancer is still extremely difficult to treat, so effective diagnosis strategies in the early
stages of cancer are critical. In this respect, imaging can become an indispensable
tool in cancer clinical trials and medical practice. In vivo fluorescence imaging of
tumors may offer a possibility for the direct bio-imaging of tumors for precise
diagnosis of cancer and monitoring of the treatment process. In situ fluorescent bio-
imaging is also of great significance for visualizing the expression and activity of
particular molecules, cells, and biological processes that influence the behaviour of
20
tumors and/or their responsiveness to therapeutic drugs. In this connection,
fluorescent gold Nano clusters will be synthesized and tagged with marker
proteins/aptamers for in-vivo self-bio-imaging. Protein markers like p53
antibody/aptamers bio conjugated with fluorescent gold nanoclusters will be targeted
for site specific response studies. The vast majority of all agents used to directly kill
cancer cells (ionizing radiation, most chemotherapeutic agents and some targeted
therapies) work through either directly or indirectly generating reactive oxygen
species that block key steps in the cell cycle. The question thus arises whether most,
programmed cell death caused by anti-cancer therapies is also ROS induced. The
“two-face” character of ROS is substantiated by growing body of evidence that ROS
within cells act as secondary messengers in intracellular signalling cascades, which
induce and maintain the oncogenic phenotype of cancer cells, however, ROS can
also induce cellular senescence and apoptosis and can therefore function as anti-
tumorigenic species. In this context, it is essential to know the role of ROS, the levels
at which they can bring about cellular damage and apoptosis and/or help cancer
cells maintain their phenotype. For in-vitro detection of ROS, a variety of
electrochemical, spectroscopic, chemiluminescent, and fluorescent methods have
been developed. Among them, the fluorescence detection method has distinctive
advantages in terms of high sensitivity and experimental convenience. For example,
previously commercialized fluorescent probes such as 20,70-
dichlorodihydrofluorescein (DCFH) and 2-[6-(40-amino)phenoxy-3H-xanthen-3-on-9-
yl] benzoic acid (APF) are widely used for the optical detection of intracellular ROS.
However, these ROS fluorescent probes exhibit several limitations such as light-
induced auto-oxidation and insufficient ROS sensitivity. Therefore, the development
of an environmentally stable, species selective, and extremely sensitive fluorescent
probe is necessary for the quantitative analysis of intracellular ROS in biological
systems. In particular, present work will be aimed at developing a situ intracellular
ROS/anti-cancer drug fluorescent probe that could provide real-time physiological
information within live cells responding to various external stimuli.
iv) Aptamer based biosensing platforms
Aptamer based ultrasensitive biosensing systems were developed for vitamin B12,
Theophylline, Bisphenol A and p53. Present work was based on interaction of
aptamer with gold nanoparticles and differential binding of aptamer in presence and
absence of analytes. Currently, as a part of Indo-Spain project, we are working on
generation of aptamer for marine toxins and aflatoxins and its detection at food
samples at sensitive levels.
2. Protein/ biomolecule stabilization:
There is no biomolecules on the earth, which remain forever. To stabilize the biomolecules are extremely difficult task, which is very essential for biosensor development. Attempts were made and are being continued to stabilize biomolecules
21
for operational and thermal stability by using protein based stabilizing agents (PBSA) and other stabilizers. This study is very much essential to reduce the cost of analysis, and repeated biosensor use. Stabilization of biomolecules is also an important factor governing the enzymatic biotransformations on an industrial scale. My group came out with important breakthrough in stabilizing biomolecules such as enzymes and antibodies extreme conditions. We have several patents and publications in these regards.
3. Biosensor for Vitamin B12 Analysis:
Methods for the detection of Vitamin B12 remain still questionable due to its low sensitivity and complex chemical structure. In the present work, a simple, faster and sensitive technique based on Immuno-chemiluminescence (CL) is being developed to detect vitamin B12at very sensitive level (ppt).
4. Optical principle based detection system:
During my biosensors research during 20 years I have worked in core areas of optics including light scattering, absorbance, surface plasmon resonance, atomic force microscopy (cantilever optical system), refraction, reflection, fluorescence and luminescence (Bio and chemiluminescence) for biosensor development. I find chemiluminescence system based biosensors are more sensitive to detect any analyte at very low concentration.
10. Food Biosensor research at CFTRI:
Realizing the importance of biosensor research for food analysis early 90’s, now my lab on biosensor has been recognized nationally and internationally. During the last 10-15 years, I have handled several projects on Biosensor research, which were funded byCFTRI, DST, DBT, Indo-Swiss and Indo-Swedish agencies.
Currently I am working on the development of biosensors for food, environment (water) and
fermentation monitoring. Biosensors are being developed in my group, based on enzymes,
antibodies, tissues, cells etc. as biological recognition elements with optical or amperometric
detector systems as physical transducers. Biochemical signal processing for the biosensors
including, signal detection, transduction, amplification and processing are being done using
microprocessors and microcomputer.
1. Biosensors for pesticide monitoring in Food and Environmental samples: It must be mentioned that a highly sensitive immuno-sensor system was developed for the detection of ethyl and methyl parathions, 2,4D and atrazine at Picograms
22
concentration (ppt) based on the immuno-chemiluminescence principle. Antibodies against pesticides were raised in Chicken (IgY) and rabbit (IgG). An economical IgY was produced for highly sensitive detection system based on immuno-chemiluminescence biosensor. No existing system available, which can detect pesticides at ppt concentrations. The high sensitivities of pesticides detection achieved in the project show promise of excellent applications of our immunosensor for field application. International Scientific collaboration in Biotechnology funded this project under Indo-Swiss collaboration (SDC-DBT). International monitoring committee (JAC) of this project appreciated the progress of the project and recommended for industrial collaboration. In this connection we are now interfacing with the Industries (M/s Bigtech, Bangalore) for technology transfer through collaborative project, which was submitted to DBT under Small Business innovation research Initiative (SBIRI). Significant work has been done in terms of establishing the concept of detection of pesticides based on chemiluminescence (CL) wherein the required sensitivity at sub-nanogram level could be attained.
2. Biosensors for organophosphorous pesticides based on enzymes:
A variety of pesticides and herbicides have been extensively used in agricultural practices to increase productivity, leading to pesticide residues in soil, water and food. These contaminants create serious health hazards to human population. Following biosensor systems were developed for the detection of pesticides. i) Acetyl Choline Esterase ( AChE ) inhibition based biosensor: AchE based biosensor system was developed for monitoring of OP pesticide. Electrode was polarised at +410mV and signals were correlated with OP pesticide concentrations. While biosensors based on AchE inhibition have been known for monitoring of OP, pesticides, in food and water samples. However strong inhibition of the enzyme is a major drawback in practical application of the biosensor, this can be at least partially overcome by reactivation of the enzyme for repeated use. In our laboratory study on enzyme reactivation by oximes was explored. Two oximes viz., 1,1’-trimethylene bis 4 – formylpyridinium bromide dioxime (TMB-4) and pyridine 2-aldoxime methiodide (2-PAM) were compared for the reactivation of the immobilized
23
AChE. TMB-4 was found to be a more efficient reactivator under repeated use, retaining more than 60% of initial activity.
ii) Detection of OP pesticides using ascorbic acid oxidase: A laboratory biosensor has been constructed at CFTRI, Mysore for paraoxon with a sensitivity of 0.5 ppm. This sensitivity is not quite adequate for practical applications and efforts are in progress to improve the biosensor performance. Considerable research has been carried out at CFTRI; Mysore on the development of single and multi-enzyme based amperometric biosensors for organophosphorous(OP) pesticides detection. It is known that organophosphates exhibit their pesticide power through a strong inhibition of acetylcholine esterase (AChE) activity. This inhibition principle has been used to develop a biosensor for detection of OP pesticides. iii) Acid phosphatase inhibition-based detection.
An amperometry-based biosensor has also been developed to analyse the OP pesticide
using the dual enzyme system consists of acid phosphatase and glucose oxidase (GOD).
Using the above system, an amperometric biosensor consisting of a potato layer rich in acid phosphatase and on immobilized GOD membrane, when operated in conjunction with a Clark type electrode, detected the pesticide. A notable advantage of this biosensor is that the inhibition of acid phosphatase by the pesticide is reversible and thereby eliminates the serious problem of enzyme inactivation.
3. Construction of a prototype biosensor instrument for glucose and sucrose analysis for Food Industry application:
A prototype biosensor instrument has been constructed, and has undergone tests and troubleshooting in the laboratory for glucose and sucrose analyses. With a view to commercialisation of the instrument collaboration was established with an instrument manufacturing company. Later it was also been field tested in the sugar factories and confectionery industries.
4. Construction of a Lactate Monoxigenase (LMO) enzyme electrode:
24
A batch type L-lactate biosensor for analysis in the concentration range 50-800 mg/dl has
been constructed at CFTRI, Mysore and the technology has been transferred to industry
(M/s Solid State Electronics, Pune). This features an enhanced operating life of 60 days
for enzymes sensing element of the biosensor, which is covered by patent (2159/DEL/98).
This biosensor can be used for L-lactate detection in fruit pulp, fermented samples and dairy
products.
5. On-line Monitoring of fermentation process using biosensor:
A biosensor with Flow Injection Analysis (FIA) system (Fig. 3), useful for continuous monitoring and control of food and fermentation processes was developedOn-line data acquisition and real time control of Food and Fermentation processes is a difficult task and limits the use of a batch type of biosensor. Through FIA system it was possible todetect Glucose and L-lactate subsequently and details of the process is given in figure 3.
6. Biosensor for Ascorbic acid analysis:
Work has been carried out at CFTRI, Mysore on the development of a tissue based
biosensor for L-ascorbic acid analysis in food and pharmaceutical samples. An immobilized
Ascorbic acid oxidase enzyme was used fordetection of ascorbic acid oxidase obtained from
cucumber peels. We found that Ascorbic acid oxidase was suitable enzyme for the
development of several biosensor systems for detection of pesticides (Pl. see Annexure I,
section 2, ii), Vitamin C (as above) and Copper ions (see below).
7. Detection of Copper ions by biosensor:
An ascorbic acid oxidase based system was used for the detection of Cu ions. This enzyme contains Cu++ in its active site. Based on its folding and unfolding (Biomolecular phenomenon) activity a biosensor was constructed. It was able to detect Cu ions in water sample.
8. Tea Biosensor:
India is exporting a large quantity of black tea all over the world. Tea polyphenols play a crucial role in determining quality of black and green tea. Major quality attributes such as colour and astringency directly linked with polyphenol contents. Therefore, it is necessary to know quantity of Polyphenols in tea. Also, tea polyphenols are gaining importance due to their strong antioxidant properties for nutrition and health. In this context in our lab, we have successfully developed an enzyme based amperometric biosensor (Fig
25
4) for the determination of total polyphenol content in tea infusions. Both in lab and industry trials were satisfactory for tea polyphenols detection and tea biosensor technology is going to be transferred to M/s Ti Industries Calcutta (MoU signed).
Food Microbiology and Fermentation Technology:
1979 to 2000:
1. Microbial Production of Glucose Isomerase enzyme for high Fructose Syrup (Large scale trial) : In this project Streptomyces fradie culture was cultivated in 10 to 200L capacity fermentors for the production of Glucose Isomerase which was very important enzyme for production of High Fructose Syrup (HFS) for beverages industries. This enzyme was isolated and immobilised for the production of HFS. A process was developed.
2. Microbial production of rennet on solid state and submerged fermentation: Rennet enzyme is very important enzyme for cheese making and in early 80’s this
enzyme was obtained through slaughtering the calf, keeping this in view a process
for production, extraction and purification of microbial rennet for vegetarian cheese
using Mucormuhei and Rhizopus was developed using solid state fermentations.
Cheese made out of this rennet was very suitable.
Several papers on this aspect were published.
3. Studies on the engineering aspects of solid state fermentation ( SSF): Bioengineering and microbiological studies on the growth of microorganisms and
production of many food enzymes were done using SSF. Several papers were
published and our contribution on SSF through this project was unique.
4. Cultivation of mammalian cells in fermentation for Human growth hormones and hybridoma cells ( MIT, USA ).
5. Microbial production of Single cell oil in submerged fermentation: Rhodotorulagracilis(Rg) was used to produce single cell oil. It was observed that Rg
could able to accumulate oil 75-80% intracellularly. Now it is an important area for
biofuel.
6. Cultivation of microorganisms and production of lactic acid by fermentation. Funded by DBT- SIDA: Indo-Swedish collaborative project. 1993-2001:
26
7. Ecofriendlybiodegradable packing material: 1999-2000. Several strains of microorganisms were isolated and biodegradable
Biopolymers PHA, PHB, PHV etc were isolated, extracted purified and identified. It
was found that Pseudomonas and RhizobacteriumSp. were potent to produce these
biopolymers. Research papers published.
2000 –continued:
8. Microbial degradation of Caffeine and polyphenols to produce value added products.
9. Microbial Production of Caffeine degrading enzymes in submerged and solid state fermentation
10. Biotransformation of caffeine to theophylline using whole microbial cell, Enzymes Funded by CFTRI.
11. Immobilization of enzymes/cells for decaffeination of coffee and tea.
I ) Biodecaffeination of Tea and coffee:
Caffeine when taken in excess exhibits various deleterious effects. It stimulates the central nervous system, shows toxicity when fed in excess and is even mutagenic in vitro. Excessive consumption of caffeine through beverages is associated with a number of health problems like adrenal stimulation, irregular muscular activity, cardiac arrhythmias and increased heart output. Excess caffeine is reported to cause mutation, inhibition of DNA repairs and inhibition of adenosine monophosphodiesterase and during pregnancy causes malformation of fetus and may reduce central nervous system, shows toxicity when fed in excess and is even mutagenic in vitro. It also causes osteoporosis, i.e. decrease in mineral density.
Biodecaffeination is defined as the complete removal of caffeine and related methyl
xanthines form caffeine-containing materials like coffee, tea, cocoa etc., by the use of
enzymes/cells capable of degrading caffeine. We report the development of a
biodecaffeination process for coffee and tea using enzymes isolated from
Pseudomonas alcaligenes MTCC 5264. The enzymes involved in biodecaffeination
were caffeine 1N-Demethylase, 7N-Demethylase, xanthine dehydrogenase, xanthine
oxidase, uricase, allantoinase, allantoicase, glyoxylatede-hyrogenase and urease.
These enzymes could be stabilized for 90 days under cold storage in the presence of
lysozymeand sorbose.
Biodecaffeination of green coffee beans (Fig 5 item 7) was carried out using the
multienzyme system immobilized in calcium alginate beads ( Fig 5, item 3) and 70%
of the caffeine could be completely degraded which was present in the beans and tea
leaves. Inhibitory factors like caffeine- polyphenol complexes and polyphenol- protein
27
complexes, which were inhibitory for biodecaffeination were prevented by the use of
glycine in the dhool. Biodecafeeianted samples of Tea and coffee were supplied to
M/s AVT, Natural products Pvt, Ltd. And company is interested to explore the
possibility of biodecaffienated tea and coffee in world market using our technology.
This is the first report on the stabilization of enzymes involved in biodecaffeination
and their application in the successful development of a biodecaffeination process for
coffee and tea.
ii) Molecular studies on biodecaffeinating Enzyme- Caffeine Demethylase.
Molecular charecterization of decaffeinating enzyme was done. The Decaffeinating
enzyme was found to have less than 31% similarity with existing proteins, which
indicates that the enzyme is novel. The region of similarity is in the Rieske Fe-S
cluster, which is a dioxygenase subunit protein iron-sulfur oxidoreductase
ferredoxin electron transport, which has a length of 116aa.
iii) Biotransformation of caffeine to Theophylline:
Keeping in view the deleterious effects of caffeine and its easy availability, it can be biotransformed to potent therapeuticmolecules as theophylline. The structural resemblance of theophylline to adenine is being made use of to venture into the possibilities of its use as anti viral (HIV), anti cancerous, anti tumourous activity. Production of Theophylline was carried out with an isolated strain of Penicilliumcitrinum MTCC 5215 in a 5-liter fermentor in a designed media. The fermentation parameters for theophylline production were optimised. Under Optimised conditions 80% conversion of caffeine to Theophylline was achieved. The fermentation broth was extracted using ethyl acetate and then acetone precipitated to separate theophylline from the rest of the crude extract. Theophylline was further crystallised using ethyl alcohol and confirmed as theophylline by HPLC, FTIR and NMR.
iv) Production of Theaflavin (TF)
Theaflavins in tea has a high health significance and medicinal properties. The content of TF in tea is much higher than that of many popular herbal extracts, such as dry standardized extracts of Ginkgo biloba, bilberry, grape seed extract and so on.Theaflavin, and theaflavindigallate induced apoptosis. Theaflavinspresent in black tea possess at least the same antioxidant potencyas catechins present in green tea. Specifically theaflavin-3, 3’-digallate (TF3)has strong antioxidant activity similar to (-)-
epigallocatechingallate (EGCG), a major antioxidant in green tea.
Crude enzyme was isolated from fungi and immobilized in suitable matrix. Sodium
alginate was found to be the best matrix. Production of Theaflavins was carried out
with an immobilized enzyme based reactor. Polyphenols extracted from waste tea
leaves (fallen at time of pruning having no commercial value) were taken for
28
biotransformation to TF and TR. The parameters for theaflavin production were
optimised and a fluidised bed reactor was developed. Under Optimized conditions
60% conversion of Theaflavins from green tea catechins was achieved. The product
was purified, spray dried and contained 20% w/w of theaflavins.
12. An enzyme assisted process for the preparation of natural vanilla extract:
The major aim of the present study was to prepare vanilla flavor extract from green beans with enzymatic process without going through the elaborate and time consuming conventional curing process (4-6 weeks). We have successfully produced better quality natural vanilla extract using our tea enzyme andvanillin content was found to be three times higher when compared to the control. Also it had higher intensity of vanilla flavor, sweet and floral notes compared to conventionally cured bean extract in the sensory analysis.
14. Cultivation and whole cell immobilization of marine bioluminescent
bacteria for environmental monitoring: Highly luminescent and potent strain of bioluminescent bacteria was isolated from marine mussels. These bioluminescent bacterial strains were immobilized using physical entrapment methods and were used for the prescreening/monitoring of heavy metals and pesticides at ppm levels within a period of thirty minutes.
11. Dissertations supervised:
a. Ph.D. : 8 Nos 4 Nos. Students submitted Ph.D. Theses
4 Nos. (Working)
b. Post Graduate : 65 students
18. Technology / Process / Product development:
Technology Transferred and being commercialized:
Technology Transferred :
1. Biosensor for L-Lactate:
29
A batch type L-lactate biosensor device for the analysis of L-lactate in food samples
within the concentration range of 50-800 mg/dL was constructed at CFTRI, Mysore
and the technology has been transferred to industry (M/s Solid State Electronics,
Pune). This features an enhanced operating life of 60 days for enzymes sensing
element of the biosensor, which is covered by patent (2159/DEL/98). This
biosensor can be used for L-lactate detection in fruit pulp, fermented samples and
dairy products.
2. Biosensor for Sugars:
An amperometric Biosensor system based on enzyme membrane for the detection
of Sugars in food, beverages and clinical samples has been of transferred to
M/s Innovate Software Solutions Pvt. LTD, 333,18th G main Road, 6th Block,
Kormangala, Bangalore.
3. Biosensor for tea quality assessment:
Tea Biosensor :Patent No. 653 DEL/ 2009
India is exporting a large quantity of black tea all over the world. Tea polyphenols play a crucial role in determining the quality of black and green tea. Major quality attributes such as colour and astringency are directly linked with polyphenol contents. Therefore, it is necessary to know quantity of Polyphenols in tea. Also, tea polyphenols are gaining importance due to their strong antioxidant properties for nutrition and health. In this context in our lab, we have successfully developed an enzyme based amperometric biosensor for the determination of total polyphenol content in tea infusions. Both in lab and industry trials were satisfactory for tea polyphenols detection and tea biosensor technology is going to be transferred to M/s TiIndustries Calcutta (MoU signed) and M/s Innovate Software Solutions Pvt LTD, 333,18th G main Road, 6th Block, Kormangala, Bangalore (MoU signed).
4. Biosensors for pesticide monitoring in Food and Environmental samples:
a) A Rapid Test kit for pesticide analysis based on charge coupled device (Indian Patent Ref. No. 502 NF/2003). b) A Rapid Test kit for pesticide analysis based on charge coupled device International Patent application Ref. No. PCT/IN 03/00446 ( Dated 31st Dec. 2003). It must be mentioned that a highly sensitive Immunobiosensor system was developed for the detection of ethyl and methyl parathions, 2,4-D and atrazine at Pico gram concentration (ppt=parts per trillion) based on the Immuno-chemiluminescence principle. Antibodies against pesticides were raised in Chicken (IgY) and rabbit (IgG). An economical IgY was produced
30
for highly sensitive detection system based on Immuno-chemiluminescence biosensor. No existing system is available, which can detect pesticides at ppt concentrations. International monitoring committee (JAC) of this project appreciated the progress of the project and recommended for industrial collaboration. In this connection we are now interfacing with the Industries (M/s Bigtech, Bangalore) for technology transfer through collaborative project, which was submitted to DBT under Small Business innovation research Initiative (SBIRI).
19. Overseas visits:
Sl No.
Organization and country
Period
Purpose
1
Massachusetts Institute of
Technology , Cambridge, USA
April, 89- April, 90 DBT Long term Associateship
2 University of Maryland Baltimore, USA
May, 90- Sep., 90
Continuation of above Associateship
3 University of Lund, Sweden April, 94 - June, 94 Indo-Swedish collaborative project