ПРИКЛАДНАЯ БИОХИМИЯ И МИКРОБИОЛОГИЯ, 2014, том 50, № 2, с. 147-155
UDC 577.154.36
MICROBIAL DEGRADATION OF CHITIN WASTE FOR PRODUCTION OF CHITOSANASE AND FOOD RELATED BIOACTIVE COMPOUNDS
© 2014 S. Sinha*, **, S. Chand*, and P. Tripathi**
* Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, New Delhi-16, India **Schools of Sciences, Indira Gandhi National Open University, New Delhi-68, India e-mail: subhashc46@hotmail.com;ptripathi14g@gmail.com Received May 30, 2013
Ecological samples rich in microbial diversity like cow dung, legume rhizosphere, fish waste and garden soil were used for isolation of chitosan-degrading microorganisms. Selected isolates were used for production of chitosanaseand food related bioactive compounds by conversion of biowaste. Production of glucosamine (Gln), N-acetylglucosamine (NAG), chitooligosaccharides (COS), antioxidants, antibacterial compounds and prebiotics was carried out by microbial fermentation of biowaste. The highest chitosanase activity (8 U/mL) was observed in Aspergillus sp. isolated from fish market waste and it could produce Gln and NAG while Streptomyces sp. isolated from garden soil was able to produce COS along with Gln and NAG. Radical scavenging activity was observed in culture supernatants of 35% of studied isolates, and 20% isolates secreted compounds which showed positive effect on growth of Bifidobacterium. Antibacterial compounds were produced by 40% of selected isolates and culture supernatants of two microbial isolates, Streptomyces zaomyceti-cus C6 and one of garden soil isolates, were effective against both gram positive and negative bacteria.
DOI: 10.7868/S0555109914020172
Bioconversion of abundantly available chitinous wastes to make useful compounds is in vogue nowadays, due to cost, toxicity and purification problems associated with chemical methods. It is considered as one of the potential alternatives for disposal of hugely available marine waste along with production of industrially important compounds like, enzymes, anti-oxidants, oligosaccharides, sugars and so on. Chitin or chitosan, produced by demoralization and depro-teinization of marine waste possess various functional properties which made them possible to be used in various sectors such as food, cosmetics, agriculture and environmental protection. Chitosan is biodegradable and biocompatible in nature and its derivatives are having number of biological activities [1] are suitable candidates for exploitation in food sectors but, poor solubility and high viscosity makes them difficult to be used in unhydrolysed form. These problems can be removed by converting chitosan into its low molecular weight derivatives, chitooligosaccharides (COS) and monomers, either chemically or enzymatically. Enzymatic method is preferred due to higher specificity and consumer acceptability.The use of chitosan and its derivatives as an ingredient for active bio-based film in modern food packaging has already been established
[2]. Biofunctional COS prepared by enzymatic hydrolysis is used as food additives and pharmaceuticals
[3]. COS has been reported to have various health benefits like antioxidant, anti-cancer, anti-diabetic, antihypertensive [1] and growth promoting activities on gut microflora [4]. Glucosamine (Gln) and its acety-
lated derivatives are produced continuously from non-reducing end of chitosan and used as food supplements [5]. These are known to have therapeutic and osteoarthritis preventing abilities and are available commercially in form of hydrochloricand sulfate salts [6, 7]. Cost effective production of these molecules are desirable but is limited to non-availability of chitosan specific enzymes, i.e. chitosanases (EC 3.2.1.132), at commercial level.
The aim of the study was to screen for chitosanase activity of chitosan-degrading microorganisms isolated from different ecological samples. Potential isolates were used for production of chitosanase and food related compounds. Gln, N-acetylglucosamine (NAG) and COS were produced by growing isolates on treated food wastes (Fig. 1). 2,2-diphenyl-1-picrylhydrazil (DPPH), reducing power, radical scavenging, antimicrobial and prebiotic activities or growth promoting effects on pro-biotic microorganisms were tested in the fermented broth after growing these isolates on biowastes.
MATERIALS AND METHODS
Materials, reagents and microorganisms. Commercial chitosan from crab shell with an average molecular weight 290 kDa was gifted by Marine Chemicals, India. Chitosan from shrimp shell (>75% deacetylated) was purchased from Sigma-Aldrich, Germany. Chitin and Gln-HCl were purchased from Hi-Media Laboratories Pvt. Ltd., India. Deacetylated chitosan of different degrees of deacetylation was pur-
Crab shell/shrimp shell chitosan powder
I
Treatment with HCl and NaOH Bioconversion
Food supplement
Healing effect in
osteoarthritis
Glucosamine
N-acetyl glucosam:
Antitumour Antioxidant Antihypertensive Antidiabetic
Chitooligomers (dimer to hexamers)
Antioxidant
rebiotics
Antimicrobial
Protect oxidation of cellular biomolecule
» Promotes growth of gut microflora
Food
preservation
Fig. 1. Graphical presentation of production of food related bioactive compounds from microbial conversion of shrimp and crab shell powder.
chased from M P Biomedicals Ltd. (India). All other chemicals were purchased from Sisco Research Lab (SRL) Ltd. (India) and were of analytical grade. Shrimp and crab shell powder (SCP) was prepared from food waste procured from local Thai food joints. Waste containing shrimp or/and crab shells and small amounts of squid pen was washed thoroughly with tap water and dried at 40°C. Dried materials were milled to powder (SCP) and treated with HCl/NaOH by method of Teli and Sheikh [8]. Production of all food related compounds was tested by growing on SCP. These wastes were also used for selective enrichment of chitosanase producer where it was taken in raw form by cutting into small pieces and adding wherever required. Standard microbial strains used in these experiments were procured from Hi-Media Laboratories Pvt. Ltd., (India) and MTCC.
Samples and media preparation. Samples taken for isolation and screening were fresh cow dung, legumes (Sorghum bicolor) rhizosphere soil, soil obtained from waste dumping areas in the fish market (Chittaranjan park, New Delhi-16, India) and from garden soil taken from the institute campus (IIT Delhi, Hauz Khas, New Delhi-16, India). All samples were suspended in tap water and suspensions were kept in flasks with constant air sparging. Food wastes taken in raw form were shredded into small pieces and added to the flasks. Air sparging was done using air cylinder in the flasks. Inoculated flasks were incubated at room temperature up to 15—20 days for enrichment degrading microbial population by chitosan. Small volumes of these samples were serially diluted and used for plating on colloidal chitosan minimal salt agar medium. Isolated medium contained (g/L): K2HPO4 - 0.35; KH2PO4 -0.15; MgSO4 • 7H2O - 0.25; FeSO4 • 7H2O - 0.005;
ZnSO4 - 0.0005; MnCl2 - 0.0005 and agar - 20.0, pH of the medium was adjusted to 7.0 before autoclav-ing. Composition of chitosan minimal salt agar medium was as follows (%): chitosan — 0.5; 0.5% yeast extract — 0.5; K2HPO4 - 0.2; KH2PO4 - 0.1; MgSO4 • 7H2O -0.07; NaCl - 0.05; KCl - 0.05; CaCl2 - 0.01 and bac-toagar - 2.0, with final pH 7.2. Chitosanase was also induced in 8 commercial microorganisms (details not given) by growing them on chitosan minimal medium; five of them showed chitosan degrading activity and were selected for further study. These were Streptomy-ces toxytricini, Corynebacterium glutamicum MTCC 1518, Saccharomyces cerevisiae, Bacillus polymyxa and Aspergillus terreus. These organisms were not reported to have chitosanase activity previously. Medium used for Aspergillus sp. contained (%): chitosan - 0.5; NaNO3 - 0.3; K2HPO4 - 0.1; MgSO4 • 7H2O - 0.05; KCl - 0.05; FeSO4 • 7H2O - 0.001 (pH 5.5). Aspergillus sp. was transferred to medium consisting of(%): chitosan -1.5; tryptone - 1.0; glucose - 0.5; K2HPO4 - 0.1;, MgSO4 • 7H2O - 0.05 and KCl - 0.05. Chitosan minimal salt agar was used for S. toxytricini, S. cerevisiae and C. glutamicum MTCC 1518. For B. polymyxa composition of medium was as follows (%): chitosan -1.0; yeast extract - 0.1; tryptone - 0.05; K2HPO4 -0.15 and KH2PO4 - 0.05 (pH 7.0).
Isolation and screening. The spread plating method was used to isolate chitosan degrading microorganisms from soil samples. 100 ^L of suitably diluted soil and cow dung samples from enrichment flasks were transferred to plates containing chitosan minimal salt agar medium. Plates were incubated at room temperature for 7-8 days and observed at regular intervals for microbial growth. Microbial colonies forming clear halo zone were taken for screening by transferring microbial iso-
lates on chitosan minimal salt broth. Flasks were incubated for 6—7 days and supernatants after centrifugation at 3000 g for 10 min were withdrawn at different time intervals for chitosanase assay. Microorganisms having detectable enzyme activity (>0.02 U/mL) were further inoculated into the same medium and enzyme assay was done repeatedly. All the cultures were maintained on chitosan minimal agar slants throughout the study.
Enzyme assay. Chitosanase assay was done using >90% deacetylated chitosan (DAC chitosan) (1%w/v) as substrate. Reaction mixture consisted of 300 ^L of the crude enzyme solution, 300 ^L of chitosan substrate and 400 ^L of 200 mM acetate buffer (pH 5.5) was incubated at 40°C for 30 min. Enzyme was deactivated by heating the mixture at 100° C for 2 min and glucosamine was estimated. The amount of reducing sugars in the supernatant was determined by modified dinitrosalicylic acid (DNS) method [9] with glucosamine as the standard. One unit of chitosanase was defined as the amount of enzyme that liberated 1 ^mol of glucosamin
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