analyze the expression of Immunoglobulin receptors (IgG & IgA) on colonocysts isolated from stool before and after intervention by flow cytometry. Network R&D programme on identification, screening and mass production of probiotic strains and placebos for clinical trials was initiated. A cell culture-based assay has been developed to evaluate attenuation of the inflammatory response by test probiotic bacteria.
Two human epithelial cell lines and a human macrophage cell line were grown in monolayers and the effects of pathogenic bacteria as well as individual bacterial components including lipopolysaccharide and muramyl dipeptide on chemokine production was tested. Interleukin-8 was identified as the most prominent inflammatory chemokine. Probiotic bacteria from various sources were tested to identify those which suppressed IL-8 production in response to stimuli. Eight different bacteria (4 Lactobacilli and 4 Bifidobacteria) were identified for inclusion in a probiotic cocktail. One billion of each of these bacteria were administered daily to Swiss albino mice before induction of experimental colitis. Compared to controls that did not receive the probiotic cocktail, treated mice showed significant reduction in ulceration and increase in healing suggesting that the probiotic modulated inflammation and repair in colitis.
Analysis of the cytokine and chemokine mRNA response in these mice using reverse transcriptase qPCR is underway. Pathway-targeted human inflammatory chemokine array, analysis revealed that enteropathogen upregulated neutrophil chemoattractants, while downregulating monocyte and macrophage chemoatractant proteins. The probiotic were observed to reduced neutrophil chemoattractant response without altering monocyte and macrophage chemoattractant response.
c) Probiotics for reproductive health:
Phase-I clinical trials on safety and capability of
probiotic selected strains of Lactobacilli to colonize in vagina for reproductive health of women have been initiated, to determine whether Lactobacilli can colonize in vagina of women depleted of such organisms. The safety, lack of side effects and efficacy of replenishment for preventing episodes of vaginosis are being determined.
d) Bacteriocins from Probiotics: Natural isolates of lactic acid bacteria isolated from the rhizosphere were screened for bacteriocin production and , strain LR/14 has been found to be potential bacteriocin producer. This strain was identified as Lactobacillus plantarum LR/14 by biochemical tests and 16SrDNA sequencing. The crude bacteriocin of strain LR/14 was heat and pH stable with shelf-life upto 2 years was checked. The mode of action was observed to be bactericidal with broad host range against related as well as some food-borne pathogens like Listeria monocytogenes, Staphylococcus and Salmonella.
e) Probiotics as Potential Source of Vitamin B12: Various Propionibacteria are being explored as potential source of Vitamin B12 and functional probiotic ingredient in a dairy based nutraceutical formulation. Twenty two cultures were identified as dairy Propionibacteria. Molecular Characterization with the help of genus-specific primers from the 16S-23S rRNA intergenic spacer regions for dairy propionibacteria, isolated organism are under process. The estimation of Vitamin B12 in milk has been standardized using immunosorbent method.
Using ELISA kit, the values of vitamin B12 in cow milk, buffalo milk and goat milk were observed to be
4.91 ± 0.40, 21.68 ± 2.69 and 3.91 4.91 ± 0.26 ppb, respectively. Further work on the validation of ELISA method with microbiological assay employing Lactobacillus leichmannii as an assay organism is in progress.
f) F o o d m i x t u r e s w i t h P r o b i o t i c s : Standardization and quality evaluation of banana based probiotic fermented food mixtures are being accomplished. Initial standardization of the 14 different combinations of food mixtures based on raw banana flour, defatted soya flour, green gram flour, tomato, papaya and mango have been carried out.
Banana flour was the main ingredient in the food mixture which ranged from 50 to 70% in all combinations. A total of 56 food mixtures with the above raw ingredients were developed and subjected to organoleptic evaluation and out of this, 14 combinations were selected based on their sensory qualities, for further studies.
Nutraceuticals
a) Reduced calorie fats: Various novel reduced calorie fats have been developed. The conditions for the transesterification were optimized at laboratory scale and further upscaled. In a typical reaction, sunflower oil was transesterified with ethyl behenate in presence of specific enzyme Lipozyme. Reaction products from five similar reactions were pooled and distilled. Various structural lipids have been prepared and the products have been analyzed for RP-HPLC and physico-chemical properties.
b) Tea Polyphenols: Theaflavins, (TF) is a high valued neutraceutical found in Black Tea in limited amounts, through the bioconversion of catechins from tea by the immobilized polyphenol oxidase (PPO) enzyme system native to tea.
Identification of suitable matrix for the enzyme immobilization, isolation of tea PPO, kinetic studies of the immobilized enzyme system, number of turnovers of immobilized enzyme system to Theaflavins, stability of the immobilized enzyme matrix, isolation of tea PPO in soluble form, alogwith characterization of Theaflavins product through UV, IR and mass spectrometery, were completed. 100%
conversion of tea catechins to Theaflavins were achieved with the system with repeated turnover of E-Test (VA & CIP) - probiotic [C]
recorded 50 times batch run without any loss of efficiency of conversion rate.
In another study, potential of black tea and its constituents in reversal of multidrug resistance and as bio-enhancer are being studied. The non-toxic concentrations of tea polyphenols were found to effectively reverse the drug resistance as evident by p-glycoprotein expression (through western blotting) and increased drug uptake of doxorubicin (through flow cytometry). Potential of tea polyphenols as bio-enhancer in cancer chemoprevention studies, experiments on hepato-carcinogenesis and skin carcinogenesis were undertaken revealed that polyphenolic constituents of both varieties of tea, green and black are providing significant protection against cancer induction in vivo models.
c) Enhanced Omega-3 fatty acid content in foods: Alpha linolenic acid (ALA) is an essential omega-3 fatty acid required in the diet that gets converted in the body to Eicosapentaenoic acid and Docosahexaenoic acid that form precursors respectively for eicosanoids and membrane components especially, brain and retina. Many microorganisms can desaturate the abundant plant linoleic acid (LA) to ALA that is poorly present in plant oils. Work to develop recombinant yeast strains capable of converting LA to ALA extracellularly was carried out. More than 100 yeast isolates were collected, mostly from the premises of plant oil mills from different parts of the country, and grouped into forty independent types based on colony and cellular characteristics. Genomic DNA was isolated from the forty different strains and used for screening of omega-3 desaturase gene by PCR. Primers were designed based on sequence homology of a published yeast omega-3 desaturase with those present in algae, fungi and plants. When compared across different phylogenetic groups there is over all poor sequence similarity (23%-29%), with the exception of cyanobacetria and Arabidopsis which showed 45%-50% similarity. Three aminoacid clusters, 16-22 aa long with 45%-50% similarity among all the known desatuarse, was used for designing primers. Seven out of forty samples yielded ~600 bp amplicon. These were further confirmed by PCR using a different set of primers and 2 DNA hybridization studies. The PCR positive strains were able convert LA to ALA.
d) Beta-carotene from Alga: A total of 272 salt pans seawater samples were collected along the coasts of Andhra Pradesh and Tamil Nadu. Most of the samples contained the quadriflagellate alga like Tetraselmis. Only a few samples known to contain a mixture of naked green algal species Dunaliella, thus indicating the rare occurrence of the species. The algal colonies appeared after 20 days were isolated and maintained. Dunaliella strains were investigated for their growth study and total carotenoids and potential strains have been identified for enhanced beta carotene production. In another study, carotenoids and fatty acid composition of some selected Indian brown and red seaweeds were analysed and phenolic content and antioxidant activity of extracts from seaweeds were also analysed. The brown seaweeds analysed included Sargassum marginatum, Padina tetrastromatic and Turbinaria connoides; while, the red seaweeds included Acanthophora spicifera, Euchema kappaphycus and Gracilaria folifera. The lipid content in various seaweeds varied between 1.0 and 3.0% (dwb) with P. tetrasromatica showing the highest content. Glycolipids were found to be a major lipid class, followed by neutral and phospholipids, in brown as well as red seaweeds. Brown seaweeds were found to contain fucoxanthin as the major pigment. Fucoxanthin content of fraction obtained by column chromatography was found to be >90%.
Among the various solvent fractions obtained, butanolic fraction of E. kappaphycus was found to contain the highest. The red seaweeds showed relatively higher antioxidant activity than brown seaweeds as indicated by radical scavenging activity.
e) Microbial Production of Nicotinamide:
Nicotinamide is one of the important vitamins B3, which is mainly used in pellagra and niacin deficiency. It also has an antioxidant and cytoprotective effect. The other form of vitamin B is3 nicotinic acid, which is equally important as nicotinamide. In this project a large number of microbes have been screened for both nicotinamide and nicotinic acid production. The nitrile hydratase activity of these strains was determined by assaying the enzyme activity. The HPLC method was developed to determine 3-cyanopyridine, nicotinamide and nicotinic acid. Good nitrile
hydratase activity was shown by eight strains among the different strains screened for this purpose.
Reactions conditions are being optimized with the isolated strains.
f) Folate Supplementation: The study i n v e s t i g a t e d t h e o p t i m a l l e v e l o f f o l a t e supplementation in the presence and absence of Vit.
B12 using rat model. In the first protocol, there were three treatment groups (12% protein with folate supplementation at 2, 4 & 8 mg/kg diet) and one control group (18% protein). There was no difference observed in brain weights of pups from different groups at birth. In contrast, males pups from both 4 and 8 mg folic acid supplemented groups had significantly lower (p<0.05) absolute and relative liver weights. Mean weights of male and females in folic acid supplemented groups were similar
t h
between groups till 12 week but started differentiating beyond 13 week. Higher folate th
supplementation viz. 4 and 8 mg group, seemed to show higher body weights.
g) Nutraceuticals from medicinal plants:
Roots of five Sida species known by the name 'Kurunthotti' by the local Vaidyas were collected.
Petroleum ether, chloroform and methanol extracts of all the five species were prepared. Analgesic, diuretic and antiinflammatory activities of all these extracts were studied using animal models.
Analgesic activity was studied using the acetic acid induced writhing method, diuretic activity was studied by measuring total urine volume and anti-inflammatory activity was studied by the carrageenan induced paw edema method. The results were statistically analyzed. It was found that the methanol extract of one of the studied species gave excellent analgesic, antiinflammatory and diuretic activities as compared to standard drugs.
Biofortification :
Potential siderophorgenic microorganisms were isolated from the rhizospheric region of the plants like spinach, brinjal, cotton and wheat etc. The isolated cultures (48) were subjected to growth in the iron deficient succinate medium and subsequently, tested for Siderophore synthesis. 19 cultures showed positive results. Effect of these cultures on germination of wheat seeds and vigor of germinated
plants was analyzed.Protocol for solid phase extraction and purification by gel-filtration chromatography of microbial siderophores was devised. The protocol for production and purification of phytosiderophores was standardized and trials are in progress. Three (microbial siderophores, phytosiderophores & synthetic)-Fe chelates were made and its comparative account on absorption in wheat plants have been assessed. Field trials were conducted to test efficacy of siderophorgenic pseudomonads wherein yield, quality and iron content in wheat grains was observed to be improved by almost 18-23%.
Enzymes from wastes:
a) Utilization of shrimp processing waste for fermentative production of chitinolytic enzymes and N-acetyl-D-glucosamin was undertaken. Isolation and screening of chitinolytic microorganisms from terrestrial and marine sources from different geographical locations were carried out. Sixty two marine samples (29 sediments, 33 water and 6 others) were collected from the west and east cost of India and analyzed for chitinolytic microbes after enrichment with de-proteinated shrimp waste. A total number of 204 isolate were picked using colloidal chitin artificial seawater agar medium. 12 samples from self-heating natural sources, where the on site temperature was above 50 C, were analyzed for o
thermostable chitinolytic microorganism and 36 isolate were collected using colloidal chitin mineral salt agar medium incubated at 55 C. All the isolates o
were repeatedly purified by streak plate method on respective medium and incubation conditions.
Qualitative and quantitative screening of the isolated microbes for their ability to produce native chitin hydrolyzing enzyme is in progress.
b) Reverse-Micellar extraction technique for separation/purification of enzymes from natural sources was also undertaken during the period. The work on the selection of suitable reverse micellar system components (solvent, co-solvent, salt and surfactant) for the identified enzymes has been completed. The factors such as enzyme characteristics (stability and iso-electric point), surfactant properties , dielectric properties of solvent and co-solvent, and salt characteristics are considered for the selection of system components.
The conditions for the preparation of crude bromealin extract from pineapple wastes (peel, core, stem and crown) have been standardized. The storage studies under different conditions (25±2ºC and 4±2ºC) are carried out to examine the stability of enzyme. The crude extract preparation of β-galactosidase from plant sources such as tomato, radish seeds, cow pea and spinach are attempted. The work on standardizing the extraction conditions for β-galactosidase from other plant sources is under progress. The processing conditions for the forward and back extraction of bromelain from the crude extract (from pineapple wastes) are being varied to optimize the enzyme activity recovery and purification. A purification factor of 5.2 with nearly 130% activity recovery has been achieved with the crude extract obtained from pineapple core. The bromelain activity in the pineapple peel is found to be very less (~17%) as compared to that of core. The RME of peel extract resulted in an activity recovery of 50% with 2 fold purification. The processing conditions have been suitably modified to selectively separate the bromelain from polyphenol oxidase (PPO). The selective separation of enzyme has been achieved based on the iso-electric point of the two enzymes. The work on integration of RME with membrane processing has been taken up and preliminary results are found to be encouraging
Large Cardamom - Product Plan
Evaluation of the performance of tissue culture raised large cardamom (Amomum subulatum) vis-à-vis open pollinated (OP) seedlings in farmers' field over a total area of 50 ha has been continued in four districts Chamoli, Uttarkashi, Champawat and Pithoragarh of Uttarakhand state jointly by the Herbal Research and Development Institute, Gopeshwar and Regional Research Station of the Indian Cardamom Research Institute (Spices Board), Gangtok, Sikkim. About 1.00 lakh OP seedlings were raised in two secondary nurseries located at Dewalthal Pithoragarh and Kothiyalsain Chamoli. A total of 47, 898 tissue culture plantlets of large cardamom were supplied by M/S Sunglow Biotech, Coimbatore. These were hardened at Kothiyalsain nursery and field planted during the planting season 2006. A total of about 34.45 area has been planted during 2006 season involving 222 beneficiaries using 90,000 OP seedlings (22.50 ha) and 47,818
tissue culture plantlets (11.95 ha). Accordingly, a total area of 50.45 ha has been covered in the project so far with OP seedlings (38.50 ha) and tissue culture plantlets (11.95 ha) involving 276 beneficiaries.
Four training programmes for project personnel and farmers on scientific cultivation and management practices of large cardamom were organized during the year at Pithoragarh, Gopeshwar and Gangtok.
Microbial and Industrial Biotechnology Microbial Enzymes for Industrial Use
In an ongoing project at Himachal Pradesh University, Shimla, 11 fold purification of nitrile hydratase enzyme (NHase, EC 4.2.1.84) with a yield of 52 % has been achieved from cell free extract of Rhodococcus rhodochrous PA-34. The molecular weight of holoenzyme of NHase was found to be 86 kDa by native-PAGE. Studies also revealed that the enzyme consisted of two subunits of 25.04 kDa and 30.6 kDa. The Km and Vmax values were 167 mM and 250 µmole/min/mg respectively. A DNA sequence of 1.7 kb containing NHase gene was cloned and expressed in E. coli JM109 using specific primers based PCR techniques from genomic DNA of R. rhodochrous and the gene sequence had 99%
homology with low molecular weight NHase gene of R. rhodochrous J1 (Fig.5).
At NIO, Goa, among the 14 marine fungi identified for laccase activity, a basidiomycetous fungus, NIOCC # 2a, isolated from mangrove wood was found to be the best producer of laccase enzyme when grown in seawater of 25-30 ppt salinity. The enzyme with optimum activity at 60ºC with pH 3 and 6 decolorized several synthetic dyes, effluents from textile mills, paper and pulp mills and alcohol distilleries. In an ongoing project at IIT, Delhi, on lipases from haloalkalophilic and organic solvent tolerant microbes for industrial applications, a solvent tolerant strain of Pseudomonas aeruginosa has been isolated which secrets very good protease and lipase. These enzymes were purified and it also exhibited stability in alkaline range and in presence of surfactant and detergents. The gene responsible for expression of protease was identified and characterized as lasB gene and the phylogenetic tree based on the gene sequence was deduced.
At Birla Institute for Scientific Research, Jaipur, studies have been carried out for development of thermostable nitrile metabolizing enzymes for enantio- and regio-selective biotransformation.
Streptomyces sp. MTCC 7546 was identified as the best micro organism with desired characteristics and the enzyme production from this strain is optimized.
The enantio-selectivity using mandelonitrile as substrate in different percentage of organic solvent was thoroughly investigated and an ee% of more than 98% was achieved. The immobilization of bacteria in agar-agar powder as an entrapment was attempted and it was observed that the immobilized whole cells could be used for more than 25 cycles without much decrease in activity.
At NCL, Pune, biochemical and structural investigations of pharmaceutically important enzymes are being pursued. Fermentation p a r a m e t e r s f o r m a x i m u m p r o d u c t i o n o f cephalosporin acylase from a new bacterial source, Alcaligenes xylosoxidans ATCC 14648, have been optimised. Chemical modification studies of the purified enzyme revealed that serine plays an important role in catalysis of the enzyme. Studies on the substrate specificity of the enzyme indicated that alpha amino substitution does not affect binding but prevents catalysis. Crystal structure of gamma Glutaryl trans peptidase from Bacillus subtilis has been determined.
Drug Development and Delivery Systems
In a DBT funded project, KEM Hospital, Mumbai in collaboration with Delhi University had developed a liposomal amphotericin (Fungisome), which has been investigated in animals and man, and shown to be safe and effective. The manufactured formulation is patented and currently available in the Indian market. A multi centric, prospective, open label, randomized trial comparing fungisome (1mg/kg/day or 3 mg/kg/day) and conventional amphotericin B is being conducted to investigate the use of liposomal amphotericin in empirical therapy for presumed fungal infection in febrile neutropenic patients. 6 out of 7 patients enrolled in the study are assessable.
Three patients are randomized to the dose of 1mg/kg/day (Fungisome), and all are assessable.
Similarly 02 patients are randomized to 3 mg/kg/day (Fungisome) dose, of which 01 is assessable. Two
patients randomized to 1mg/kg/day (conventional) are assessable.
In another multicentric, open, comparative, randomized study to optimize dose, duration, safety, efficacy, and cost of two treatment regimens with Liposomal amphotericin (Fungisome) in the treatment of systemic fungal infections in India, with a sample size of 60, a total of 26 patients has been enrolled so far at various centers of which 20 are assessable. The analytical method for the estimation of the Amphotericin B has been
In another multicentric, open, comparative, randomized study to optimize dose, duration, safety, efficacy, and cost of two treatment regimens with Liposomal amphotericin (Fungisome) in the treatment of systemic fungal infections in India, with a sample size of 60, a total of 26 patients has been enrolled so far at various centers of which 20 are assessable. The analytical method for the estimation of the Amphotericin B has been