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SECTION II
Nutraceuticals
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CHAPTER 2
Isolation and Screening for Bioactive Compounds
Rama Bhadekar, Anuradha Mulik, and Sonali Ambulkar
CONTENTS
2.1 Introduction |
2.2 Sample Collection and Isolation |
2.2.1 Isolation of Extremophiles |
2.2.2 Media |
2.3 Screening for Industrially Important Bioactive Compounds |
2.3.1 Enzymes |
2.3.2 Enzyme Inhibitors |
2.3.3 Exopolysaccharides |
2.3.4 Biosurfactants |
2.3.5 Polyunsaturated Fatty Acids |
2.3.6 Pigments |
2.3.7 Marine Probiotics |
2.4 Screening for Biomedically Important Compounds |
2.4.1 Antibacterial Activity |
2.4.2 Antifungal Activity |
2.4.3 Anti-HIV Activity |
2.4.4 Anticancer Activity |
2.4.5 Antioxidant Activity |
2.5 Conclusion |
References |
2.1 INTRODUCTION
Today, biotechnological procedures are gaining importance in various sectors, such as pharmaceuticals, food, cosmetics, agriculture, and so on, due to their sustainability, cost-effectiveness, and eco-friendly nature. These methods are studied and optimized to achieve superior quality and quantity of the product as compared to chemical methods. Chemical synthesis strategies have certain limitations, such as environmental pollution, excessive use of chemicals, adverse effects on human health, and high cost. Therefore, plants, animals, and microorganisms are being scrutinized as natural resources of bioactive compounds and are being harvested in the last few years. Among these, microorganisms are of tremendous value because of their fast growth rate, ubiquitous presence, and ease of optimization. Bacteria, fungi, and actinomycetes are widely explored for the range of bioactive compounds they offer that have various industrial as well as biomedical applications. These microorganisms may be sourced from various ecosystems, such as terrestrial, freshwater, and marine. They are found to adapt to the environmental conditions in their respective habitats. Of these three ecosystems, marine ecosystem is the largest since water accounts for more than 70% of the earth’s surface and marine environment accounts for more than 97% of that (Foucher, 2009). The peculiarity of marine environment can be attributed to the following properties of marine water: (1) higher density and viscosity than air, (2) better ability to transmit sound, (3) low electrical resistivity, (4) ability to absorb light, (5) variation in oxygen concentration with temperature and salinity, and (6) very low oxygen diffusion rate (Nybakken and Bertne, 2004).
This chapter elaborates bioprospecting of marine microorganisms with respect to their isolation and screening for bioactive compounds. For the isolation of microorganisms, various samples from the marine environment prove to be useful sources. They include seawater at different depths, sea bottom, deep sea hydrothermal vent, microorganisms present in symbiotic association with marine plants and animals, fouling ship hulls, and so on. Usually, seawater contains 1 million microorganisms/mL. Marine microorganisms survive under conditions of high pressure, low temperature, and high salinity. It may be one of the reasons for the isolation of a majority of Gram-negative bacteria from marine environment—their cell envelope is well suited to such surroundings. As a result, bioactive compounds obtained from marine microorganisms are found to withstand harsh conditions such as extreme pH and temperature, high salt concentration, and high pressure.
2.2 SAMPLE COLLECTION AND ISOLATION
The isolation of marine microorganisms is usually aimed at the selection of better strains which have an ability to produce novel bioactive metabolites with unique structural characteristics. Common isolation procedures involve sample collection, transport to the laboratory, enrichment, and serial dilution, followed by plating on respective media. However, the method cannot be generalized as it can vary with the sample and may or may not require enrichment. Soil, marine sediments, water, sea animals, mangroves, and seaweed are starting materials reported thus far. Next, we describe methods to collect these samples.
The collection of marine sediments is carried out by an alcohol-rinsed Peterson grab followed by a transfer of samples into ziplock bags using a sterile spatula (Dhevendaran and Anithakumarai, 2002). Samples from deep sea sediments are also collected aseptically using trawls or push core samplers (da Silva et al., 2013), which are transferred to falcon tubes and stored at 4°C. The filter-sterilized seawater can be used as a diluent for serial dilution. For bacterial isolation from soil samples, soil suspension is prepared by diluting 10 g of sample in 90 mL sterile water followed by shaking to get a homogenized suspension. However, for microbial isolation from a mangrove soil sample (rhizosphere and non rhizosphere), the samples are pretreated with dry heat or chemicals such as phenol or wet heat in sterile seawater at 50°C for 15 min. The pretreatment of soil is followed by 1:10 dilution (V/V) with sterile ¼ strength Ringer’s solution and serial dilution (Hong et al., 2009).
Seawater samples are normally collected from the depth of 5 to 25 m. This is again followed by serial dilution and plating. Filtered autoclaved seawater and/or synthetic seawater medium are used for subculturing. Marine nutrient agar and Zobell Marine Agar can be used for spreading and isolation (Pimpliskar and Jadhav, 2014).
For the isolation of microorganisms from mangrove ecosystems, different plant parts, such as the root, stem, leaf, fruit, and flower, are used and samples are stored at 4°C until processed. The plant tissue surface is cleaned using sterile water and air-dried in laminar air flow, followed by 70% ethanol treatment for 5 min, 0.1% HgCl2 treatment for 15 min, and 5 times washing with 1% Tween® 80 each for 5 min. Then, the small pieces are cut and ground using mortar and pestle in 50% sterile seawater, and this preparation can be used for plating on selective media. The preference is to collect the samples in clean polythene bags, transport them to the laboratory, and process within 3 h (Hong et al., 2009). Artificial seawater or sterile seawater can also be used for repeated washings of mangrove leaf samples or seaweed tissue (Bonugli-Santos et al., 2015). Prior to the isolation of endophytic bacteria, the samples are kept in plastic bags, washed with water, and surface disinfected, and the cut pieces are used to prepare suspension in phosphate-buffered saline by shaking for 1 h (da Silva et al., 2013). This is followed by serial dilution and plating on tryptic soy agar. Incubation is carried out at 28°C until growth.
Surface sterilization with HgCl2 or ethanol followed by washing with sterile seawater is suggested for marine invertebrate samples (Newel, 1976). However, better microbial recovery is observed if HgCl2 is eliminated. Marine microorganisms have also been isolated from animals such as fish, sponges, corals, and so on. Sponge samples are collected in an ethyl polythene bag by SCUBA diving from the depth of 5 to 10 m and transferred to the laboratory ascetically in ice (Devi et al., 2010). Isolation involves surface sterilization of the sponge and removal of the sponge tissue, followed by maceration and homogenization and plating on Zobell Marine Agar after dilution. Incubation temperature is 27°C–30°C. For the isolation of microbes associated with coral reefs, the coral tissue is placed in sterile plastic bags, placed in ice, and brought to the laboratory. Further procedures involve washing with sterile seawater and the preparation of a suspension of the coral sample in sterile seawater. As stated, serial dilution and plating on artificial seawater nutrient agar is followed by incubation at 28°C for the isolation of mesophilic organisms (Babuet al., 2004).
2.2.1 Isolation of Extremophiles
For the isolation of marine thermopiles, muddy soil samples are collected from different sites of hot springs in sterile polybags and are brought immediately to the laboratory (Pandey et al., 2013). Further procedures are similar to that used for soil samples except with incubation at 50°C for 12 h in a nutrient broth. This can be continued with streaking on a nutrient agar and repeated testing of the isolates for their ability to withstand high temperatures. Hot springs, undersea hydrothermal vents, and volcanic lava can act as starting material for the isolation of hyperthermophiles such as Pyrolobus fumarii and Methanopyrus kandleri (Vieille and Zeikus, 2001). In contrast, true psychrophiles (at an optimum temperature 15°C or less) can be isolated from the Arctic or Antarctic environment and sea ice (Borriss et al., 2003).
The isolation of halophiles requires salt enrichment (10%–20% NaCl), which is followed by serial dilution and plating on a complete medium containing organic nutrients along with different salts, such as sulfates, phosphates, and chlorides, and trace metals (Kumar and Karan, 2012). Samples are incubated at 30°C for 96 h. Repeated streaking results in isolation of pure cultures. They can be maintained by subculturing every 15 days and stored at 4°C. Halophilic organisms moderately exhibit a 5% salt requirement (Kushner, 1985). Hypersaline environments such as the Dead Sea, the Great Salt Lake, and solar salt evaporation ponds are found to be useful habitats for isolation of extreme halophilic microorganisms (salt requirement >25%) (Ventosa et al., 1998).
TABLE 2.1 Isolation of Extremophiles
Extremophile | Growth Conditions | Reference |
Halophiles | Require at least 1 M salt for growth | Horikoshi and Bull (2011) |
Thermophiles | Grow at temperatures between 50°C and 80°C | Vieille and Zeikus (2001) |
Hyperthermophiles | Optimum growth at temperatures above 80°C | Vieille and Zeikus (2001) |
Psychrophiles | Grow at temperatures between 10°C and 20°C | Junge et al. (2011) |
Psychrotolerant | Grow at temperatures above 25°C, but also grow below 15°C | Junge et al. (2011) |
Barophiles | Optimal growth pressure is more than 40 MPa | Li et al. (1998) |
Sterilized mud samplers are used at a depth of more than 10,000 m for the isolation of barophilic microorganisms (Kato et al., 1998). The isolation procedure involves incubation under pressure of 100 MPa in plastic bags using marine broth, for example, strain MT41 (an ext...
