DOWN STREAM PROCESSING

Sanjeet Kumar
Ravenshaw University


·        Downstream processing refers to the recovery and purification of biosynthetic products. The extraction and purification of a biotechnological product from fermentation is referred to as downstream processing (DSP) or product recovery.
·        DSP is as complex and important as fermentation process.
·        The methodology adopted for downstream processing depends on the nature of the end product, its concentration, stability and the degree of purification required, besides on the presence of other products.
·         The desired products for isolation by DSP are most frequently metabolites which may be present as follows:-
1.      Intracellular metabolites- Vitamins
2.      Extracellular metabolites- amino acids
3.      Both Intra- and Extra- cellular- Flavomycin
Stages in Downstream processing
Downstream processing of metabolites is a multistage operation and broadly divided into the following stages:
1.      Release of intracellular products
2.      Solid-liquid separation
3.      Concentration
4.      Purification
5.      Formulation
Solid-liquid Separation
The first step in product recovery is the separation ofwhole cells and other insoluble ingradients from the culture broth.
Several methods are useful for solid- liquid separation such as:
a)      Flotation
b)      Filtration
c)      Flocculation
Filtration
Filtration is the most commonly used technique for separating the biomass and culture filtrate. Several types of filters are in use.
1)      Depth Filters:- Filamentous fungi are removed
2)      Absolute Filters:- Removal of bacteria from culture medium
3)      Rotary Drum Vacuum Filters:- Filtration of yeast cells
4)      Membrane Filters:- Reduces Clogging
Ø  Static Flow
Ø  Cross Flow
In cross-flow filtration, the culture broth is pumped in a crosswise fashion across the membrane.
Ther are 3 types of filtration used:
a)      Microfiltration
b)      Ultrafiltration
c)      Reverse Osmosis

Type
Applications
a)      Microfiltration
Separation of cells or cell fractions, viruses
b)      Ultrafiltration
Separation of compounds with molecular weight greater than 1000 μg
c)      Reverse Osmosis
Separation of compounds with molecular weight less than 1000 μg


Release of Intracellular Product
·         There are several biotechnological products such as vitamins, enzymes etc. are located within the cells. Such compounds have to be first released for their further processing and final isolation.
·         The selection of a particular methods for released of intracellular products, depends on the nature of the cells.
·         Gram-negative bacteria and filamentous fungi can be more easily broken compared to Gram-positive bacteria and yeasts.
·         In order to increase the efficiency of cell disintegration in a cost-effective manner, a combination of physical, chemical and enzymatic methods are employed.
Concentration
The filtrate that is free from suspended particles usually contains 80-98 % of H2O. The desired product is a very minor constituent. The H2O has to be removed to achieve the product concentration. The commonly used techniques are:
a)      Evaporation
b)      Liquid-liquid extraction
c)      Membrane filtration
d)      Precipitation
e)      Adsorption
    Evaporation can be done by evaporated such as plate evaporators, Falling film evaporator, Forced film evaporators and Centrifugal forced film evaporators.
Liquid-liquid extraction is useful for partial purification of a product, which is categorized as:
a)      Extraction of low molecular weight products using Dissociation, supercritical fluid etc.
b)      Extraction of high molecular weight compounds using Aqueous 2-phase systems (ATPs), and reverse miceller systems.
Precipitation is the most commonly used technique for the concentration of macromolecules using neutral salts, organic solvents etc.
Purification
The biological products of fermentation are very effectively purified by chromatography. It is basically an analytical technique dealing with the separation of closely related compounds from a mixture.
The different types of chromatographic techniques are used for such as
a)      Gel-filtration chromatography
b)      Ion-exchange chromatography
c)      Affinity chromatography and
d)      Hydrophobic interaction chromatography

CHROMATOGRAPHY
PRINCIPLE
Gel-filtration
Size and Shape
Ion-exchange
Net charge
Affinity
Molecular recognition
Hydrophobic Interaction
Polarity
Immobilized metal-ion affinity
Metal ion-binding


Formulation
Formulation broadly refers to the maintenance of activity and stability of biotechnological products during storage and distribution.
·         The formulation of low molecular weight products can be achieved by concentration them with removal of most of the water.
·         For certain small molecules such as citric acid, antibiotic etc., formulation can be done by crystallization by adding salts.

LIGAND
TYPES OF PROTEIN
Antibody
Antigen
Cofactor
Enzyme
Hapten
Antibody
Lectins
Glycoproteins

                       (Ligands use for separation of proteins by Affinity Chromatography)
Drying is an essential component of product formulation. It basically involves the transfer of heat to a product for removal of moisture. Freeze-drying or Lyophilization is the most preferred method for drying and formulation of wide-range of products such as food stuffs, pharmaceuticals, bacteria, viruses. Lyophiization is based on the principle of sublimation of liquid from a frozen state. In this technique, the liquid containing the product is frozen and then dried in a freeze-dryer under vacuum. The vacuum released and the product containing vials are sealed.  Penicillin can be freeze dried directly in ampoules. For good output can integrate the fermentation and downstream processing to finally get the desired product.

A Review on Pharmacological aspects of Amorphophallus species

A. Behera & Sanjeet
sanjeet.biotech@gmail.com

Introduction
Millions of people in many developing countries do not get enough food to meet their daily requirement and many more suffer deficiency of micronutrients. The rural and tribal communities even today depend on wild resources including wild edible tuber(s) to meet their daily food requirement and more particularly during the period of food crisis. The value of wild edible plants in food security has not been given sufficient attention in India and particularly in Odisha. During last few years some attempt has been made to document the rich indigenous knowledge on the medicinal use of wild plants but socio-economic, traditional nutritional aspect of wild food plants still lack adequate attention. There are about 1532 edible wild food species available in India mostly in Eastern Ghats, Western Ghats and Himalayan Hill ranges (Reddy et al., 2007). Tubers play a major role in supplementing staples foods with micronutrients and can constitute a “Safety Net” during the period of food shortage (Chattopadhyay et al., 2010). Knowledge on wild tubers is gradually declining and even disappearing with increased modernization and migration to urban area due to poverty and other socio-economic factors. Documentation and revalorisation indigenous knowledge on wild tubers is urgently needed to promote nutritional health of the local inhabitants and to conserve genetic and cultural diversity as well. Almost all tribal groups of Odisha have been using Amorphophallus species (Oluā) as a food that are generally taken as vegetables and used as medicine in different formulations. Amorphophallus tubers (corms) constitute an economically important staple food and medicine for millions of people in the world (Sen et al.,1995).
Ethnobotanical Values
Amorphophallus are long been used in China, Japan and South East Asia as a food source and traditional medicine. In traditional Chinese medicine (Tem), a gel prepared from the flower has been used for detoxification, tumour- suppression, blood stasis alleviation and phlegm liquefaction and for treatment of asthma, cough, hernia, breast pain, burns as well as haematological and skin disorders (Chua et al., 2010). Amorphophallus campanulatus tuber is used for the treatment of enlarged spleen, rheumatism and tumour (Tripathi et al., 2010). The tubers are known to treat stomach ailments, has medicinal properties against fever, swelling, diarrhoea, used in piles and given as restorative in dyspepsia enlargement, asthma and rheumatism. Rhizomes are used for boils and opthalmia, also as an emmenagogue. Petioles used in scorpion bites, dysmenorrhea and are applied externally as an irritant to treat rheumatism (Hasan et al., 2009). Corms are also used in case of ear aches, swelling of throat, pimples, intereostal neuralagia, fever and also useful in cases of elephantiasis, tumours, inflammations, constipation, seminal weakness, fatigue, anaemia and general disability (Ravi et al., 2009).

Pharmacological Activity
Anti-Inflammatory Activity
Antioxidant activity of methanolic and aqueous extract of Amorphophallus campanulatus tuber was studied for its free radical scavenging property (Sahu et al., 2009). The anti-inflammatory activity of the hydroalcohol extract of Amorphophallus  bulbifer (whole plant) was studied in Wistar rats and mice at dose levels of 100 and 200 mg/kg p.o. with a plethysmograph  paw volume difference of the animals pre- and post-treatment. Ibuprofen (10 mg/kg) was employed as reference standard. The extract exhibited anti-inflammatory activity of 56.5 (p< 0.001) and 57.1 % (p< 0.001) inhibition compared to the control group in the carrageenan and histamine-induced inflammation model at a dose of 200 mg/kg (Reddy et al., 2012).
Analgesic Activity
Analgesic activity in Amorphophallus bulbifer (whole plant) was evaluated using tail flick and tail immersion techniques by measuring the reaction time of the animals (rats) treated with either standard or extract. Pentazocin (30 mg/kg) was used as reference which showed significant anti-inflammatory at the two test dose levels at the 4th hour (p< 0.001). The extract showed reaction times of 7.33 (p< 0.001) and 7.83 (p< 0.001) min in the tail flick and tail immersion models at a dose of 200 mg/kg while the normal and reference groups exhibited reaction times of 2.16, 2.66 and 8.16 (p< 0.001) and 8.5 (p< 0.001) in the tail flick and tail immersion methods, respectively, this lends some support for its use in traditional medical practice (Reddy et al., 2012).
Anti-Cancer Activities
Epidemiological evidence suggests that dietary factors play an important role in human health and in the treatment of certain chronic diseases including cancer (Trichopoulos et al., 1996). Some dietary sources of Amorphophallus species contain anti - tumour compounds and such compound are candidates for chemo preventive agents against cancer development (Dorai et al., 2004). The anticancer  property of nutrients derived from plants as well as non - nutritive plant derived constituents have been proved indifferent in vitro and in vivo models , which has led to an increased emphasis on cancer prevention strategies in which these dietary factors are utilised (Barnes et al.,1995). Epidemiological evidence suggests that dietary factors play an important role in human health and in the treatment of certain chronic diseases including cancer (Madhurima et al., 2012). Amorphophallus campanulatus has anticancer properties (Madhuri et al., 2008). Antioxidant   and anti- tumor activity of ethanolic extract of Amorphophallus paeonifolius tubers against 7,12- dimethyl benz (a) anthracene (DMBA) induced mammary tumour in rats were studied in-vitro and in-vivo methods and the plant extract was evaluated for mammary tumour activity by chemical induced tumour. The results showed total flavanoids compound in ethanolic tuber extract of Amorphophallus paeonifolius was found to be 8.8 g/100g calculated as Quercetin equivalent, effect of ethanolic extract on RBC, WBC, Hb & Neutrophils as a-P< 0.001,b-P<0.01,c-P<0.05, ns-non significant, effect of AP-extract on tumor latency and tumour burden were found as extremely significant at P<0.001. One-way ANOVA Tukey method was followed for statistics. It was concluded that ethanolic extract of Amorphophallus paeonifolius has shown significant antitumor and antioxidant effect in animals. The flavonoid in tuber extract of Amorphophallus paeoniifolius showed significant antitumour and antioxidant effect (Jagatheesh et al., 2010).
Hepatoprotective Properties
The pretreatment with dried tuber of Amorphophallus campanulatus (MAC) reduced the biochemical markers of hepatic injury like serum glutamate pyruvate transaminase (SGPT), serum oxaloacetate transaminase (SGOT), alkaline phosphatase (ALP), bilirubin (BRN) and total protein shows hepatoprotective activity. There is increase in the levels of superoxide dismutase (SOD), Catalase (CAT), and glutathion peroxidise (GPx) shows that the plant may possess hepatoprotective and antioxidant property (Singh et al., 2011). The hepatoprotective activity of methanol and aqueous extracts of Amorphophallus paeoniifolius tubers was confirmed against paracetamol induced liver damage in rats which was induced by paracetamol. The phytochemical investigation of the extracts showed presence of carbohydrates, proteins, steroids and flavonoids. Pre-treatment of the rats with methanol and aqueous extract prior to paracetamol administration caused a significant reduction in the values of sGOT, sGPT, sALP and sB (P<0.01) almost comparable to the silymarin and Liv-52. Thus was confirmed by histopathological examination of the liver tissue of control and treated animals (Hurkadale et al., 2012).
Anti-bacterial Activity
The flavonoid, 3,5-diacetyltambulin isolated from Amorphophallus campanulatus was studied for  in-vitro antibacterial, antifungal and cytotoxic activities which showed significant antibacterial activites against four gram positive bacteria   (Bacillus subtilis, Bacillus megaterium, Staphylloccous aureus, Streptococcus β – hemolyticus ) and six gram negative bacteria (Escherichia coli, Shigella dysenteria, Shigella sonnei, Shigella flerneri, Pseudomonas aeruginosa, Salmonella typhii). The MIC values against these bacteria ranged from 8 to 64 μg/ml but had weak antifungal activity against a number of fungi. In cytotoxicity determination, LC50 of the compound against brine shrimp nauplii was 10.02 μg/ml (Khan et al., 2009). The aqueous and methanolic extracts of spinach (Spinacea oleracia), pumpkin (Cucurbita pepo), suran (Amorphophalus campanulatus) and ghuiya (Colocasia esculenta) were evaluated for antimicrobial activity against bacterial strains (Bacillus cereus, Bacillus subtilis, Escherichia coli, Enterobacter aerogenes, Enterobacter agglomerans, Salmonella enteritidis, Salmonella cholerasius, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Penicillium chrysogenum, Enterobacter faecalis, Klebsiella pneumonia, B.sphericus, B.thruengiensis and Cryptococcus meningitis and was performed by Agar well diffusion method on Nutrient Agar medium and Muller Hinton Agar medium. The methanolic extracts of all the vegetables showed moderate to high activity against all the investigated microbial strains (Dubey et al., 2010). The Amorphophallus species showed remarkable antibacterial activity against gram positive (Bacillus subtilis, Staphylococcus aureus) gram negative (E. coli, P. aeruginosa ) bacteria and antifungal test showed significant activity of Candida albicans (Maharajan et al.,2012).

Antifungal Activity
The complete genome sequence of Bacillus subtilis strain BSn5, isolated from Amorphophallus species, calli tissue show strong inhibitory activity to Erwinia carotovora subsps- carotovora, which causes Amorphophallus soft rot disease and affects the industry development of this organism (Deng et al., 2011). Amorphophallus campanulatus showed good antifungal activity against Aspergillus flavus, Aspergillus nizer and Rhizopus aryzae (Khan et al.,2008).
Anti-Diabetic Activities  
The Amorphophallus species can be used as anti-diabetic agent (Li et al., 2004). The effect of the acetone extract of elephant-foot yam (Amorphophallus paeoniifolious Dennst.) at 0.1 and 0.25% in the diet of streptozotocin-induced male Wistar diabetic rats was studied which involved a comparison between control and diabetic groups: starch-fed control/diabetic (SFC/SFD), 0.1% acetone extract fed control/diabetic (AEFC0.1/AEFD0.1), 0.25% acetone extract fed control/diabetic (AEFC0.25/AEFD0.25) and aminoguanidine fed control/diabetic (AFC/AFD). The rats were examined for water intake, diet intake, urine output, gain in body weight, urine sugar, fasting blood sugar (FBS) and glomerular filtration rate (GFR). A concentration-dependent amelioration of the diabetic status was observed with respect to all the above studied parameters. FBS of AEFD0.1 and AEFD0.25 groups showed a 23% and 37% reduction, respectively whereas the AFD group showed a 45% reduction relative to the SFD group. The GFR of experimental rats in AEFD0.1 and AEFD0.25 groups showed a 28% and 41% reduction, respectively whereas the AFD group showed a 54% reduction compared to the SFD group which clearly indicate that the acetone extract of elephant foot yam is an effective for streptozotocin-induced diabetic rats (Arva et al., 2012).
Immunomodulatory Activity
Amorphophallus campanulatus tuber is used for the treatment of enlarged spleen, rheumatism and tumour. The effect of the methanol extract (ME) of Amorphophallus campanulatus tuber on immunological function in mice was studied using charcoal clearance, spleen index and delayed-type hypersensitivity (DTH) response models. The extract was administered orally at doses of 250 and 500 mg/kg, thus exhibited immunomodulatory activity by causing a significant decrease in charcoal clearance, spleen index and delayed-type hypersensitivity (DTH) response (Tripathi et al., 2010).

Anti-helminthic Activity
Corm was extracted with petroleum ether, chloroform and methanol. Crude tannins were isolated from methanol extract. The extracts and crude tannins were evaluated for anthelmintic activity. Chloroform, methanol extracts and crude tannins showed very good anti-helmintic activity (Ramalingham et al., 2010).

Toxicity
The cytotoxic property of different solvent extracts of Amorphophallus paeoniifolius tuber using Allium cepa L. root tip cells and HEp-2 cell line as two model in vitro systems. The seven extracts of Amorphophallus tuber were tested; the mitotic index and cytotic index were found to be high in petroleum ether and ethanol fractions. The magnitude of cytotoxicity displayed a dose dependent antiproliferative activity on HEp-2 cells which confirms the cytotoxic property (Angayarkanni et al., 2007).

Pita Aalu (Dioscorea bulbifera L.) of Simlipal Biosphere Reserve Forest: Diversity and ethnobotanical values with its role in Health Care

This is abstract of  accepted article , going to publish in National Institute of Technology

Sanjeet Kumar1, Shakti Kant Rath2 and Padan Kumar Jena1
1.        Department of Botany, Ravenshaw University, Cuttack-75 3003, Odisha
2.        School of Life Sciences, Ravenshaw University, Cuttack-75 3003, Odisha

Simlipal Biosphere Reserve (SBR) forest is located in the district Mayurbhanj of the state, Odisha.It has a mixed type of vegetation such as semi-evergreen, tropical moist broadleaf and tropical moist deciduous forest. It is the hub of different tribal communities such as Mankardia, Kharia, Kolho, Santhal, Munda etc. They do agriculture for short periods and depend on wild plants for their food and medicine. Among their wild foods Dioscorea bulbifera (Pita Aalu) is important as per availability and consumption rate. The details of the plant in terms of ethnobotanical values, diversity at SBR and its use in maintaining health care has been documented through field survey using passport data and from literature. The availability of this valuable wild tuber crop is declining due to various anthropogenic activities. Therefore an attempt was made to document the therapeutic values, diversity and food values of the plant parts. The implications of this study in terms of sustainable use by the tribal communities, conservation and environmental education have been discussed.
Key Words: Dioscorea bulbifera, Environmental Education, Genetic diversity, Health care, Simlipal Biosphere Reserve Forest


*Corresponding Author
  Sanjeet Kumar


 

Floral wealth of Mahanadi River