Studies on the digestive enzymes of the cultivable grey mullet liza parsia (hamilton buchanan, 1822)

Culturing of fish in captivity demands a detailed knowledge on well balanced diet and adequate feeding. Formulation and production of nutritionally balanced diets for fish require research, quality control and biological evaluation. It is often assuemed that what is ingested is also digested, but this is not always be the case. Digestion depends upon both the physical state of the food and the kind and quantity of enzymes in the digestive tract. The ability of fish to digest a particular component of diet can be ascertained by investigating the complement of digestive enzymes present along the digestive tract. Investigations on the basic digestive physiology will not only enhance our present knowledge on nutrition and feed development, but will also contribute in understanding the digestive functions of lower vertebrates. It is against this background that the present topic of investigation "Studies on the digestive enzymes of the cultivable grey mullet Liza parsia Hamilton Buchanan, l822" has been selected. The thesis is arranged and presented in eight chapters.

“Studies on extracellular enzyme production during growth of Pleurotus sp. on lignocellulosic agriwaste and the utilization of spent mushroom substrate”

Commercially, Pleurotus spp. of mushroom are cultivated in bags. After mushroom cultivation, spent substrate remains as residual material. Proper recycling of spent substrate is beneficial for our economy. Spent substrate can be utilized for various other value added purposes through the proper knowledge of its components. Composition of various components depends on the activity of extracellular enzymes in the spent substrate. The present study was conducted to know the enzyme profile of some major extracellular enzymes - cellulase, hemicellulase (xylanase), pectinase and ligninase (lignin peroxidase and laccase) and to estimate cellulose, hemicellulose, pectin and lignin in the substrate. The use of spent substrate as a source of fibre and ethanol, and in the biodegradation of phenol by Pleurotus spp. was also investigated

Thermdstable alpha amylase production by bacillus coagulans

Microbial enzymes are in great demand owing to their importance in several industries such as brewing, baking, leather, laundry detergent, dairy. starch processing and textiles besides pharmaceuticals. About 80% of the enzymes produced through fermentation and sold in the industrial scale are hydrolytic enzymes. Due to recognition of new and new applications, an intensive screening of different kinds of enzymes with novel properties, from various microorganisms, is being pursued all over the world. Bacillus sp are largely known to produce a-amylase, among the different groups of microoganisms, at industrial level. They are known to produce both saccharifying and liquefying a-amylases (Fukumoto 1963; walker and Campbell, 1967a). which are distinguishable by their mechanisms of starch degradation by the fact that the saccharifying asamylases produce an increase in reducing power about twice that of the liquefying enzyme (Fukumoto, 1963; Pazur and Okada, 1966). Under this circumstances, the present study was undertaken, with a View to utilise a fast growing B.coagu1ans isolated from soil, for production of thermostable and alkaline oz-amylase under different fermentation processes

Halocin SH10 production by an extreme haloarchaeon Natrinema sp. BTSH10 isolated from salt pans of South India

Halobacteria, members of the domain Archaea that live under extremely halophilic conditions, are often considered as dependable source for deriving novel enzymes, novel genes, bioactive compounds and other industrially important molecules. Protein antibiotics have potential for application as preserving agents in food industry, leather industry and in control of infectious bacteria. Halocins are proteinaceous antibiotics synthesized and released into the environment by extreme halophiles, a universal characteristic of halophilic bacteria. Herein, we report the production of halocin (SH10) by an extremely halophilic archeon Natrinema sp. BTSH10 isolated from salt pan of Kanyakumari, Tamilnadu, India and optimization of medium for enhanced production of halocin. It was found that the optimal conditions for maximal halocin production were 42 C, pH 8.0, and 104 h of incubation at 200 rpm with 2% (V/V) inoculum concentration in Zobell’s medium containing 3 M NaCl, Galactose, beef extract, and calcium chloride as additional supplements. Results indicated scope for fermentation production of halocin for probable applications using halophilic archeon Natrinema sp. BTSH10

Biocompatible polyhydroxybutyrate (PHB) production by marine Vibrio azureus BTKB33 under submerged fermentation

Polyhydroxybutyrate (PHB) is known to have applications as medical implants and drug delivery carriers and is consequently in high demand. In the present study the possibilities of harnessing potential PHB-producing vibrios from marine sediments as a new source of PHB was investigated since marine environments are underexplored. Screening of polyhydroxyalkanoate (PHA)-producing vibrios from marine sediments was performed using a fluorescent plate assay followed by spectrophotometric analysis of liquid cultures. Out of 828 isolates, Vibrio sp. BTKB33 showed maximum PHA production of 0.21 g/L and PHA content of 193.33 mg/g of CDW. The strain was identified as Vibrio azureus based on phenotypic characterization and partial 16S rDNA sequence analysis. The strain also produced several industrial enzymes: amylase, caseinase, lipase, gelatinase, and DNase. The FTIR analysis of extracted PHA and its comparison with standard PHB indicated that the accumulated PHA is PHB. Bioprocess development studies for enhancing PHA production were carried out under submerged fermentation conditions. Optimal submerged fermentation conditions for enhanced intracellular accumulation of PHA production were found to be 35 °C, pH −7, 1.5 % NaCl concentration, agitation at 120 rpm, 12 h of inoculum age, 2.5 % initial inoculum concentration, and 36 h incubation along with supplementation of magnesium sulphate, glucose, and ammonium chloride. The PHA production after optimization was found to be increased to 0.48 g/L and PHA content to426.88 mg/g of CDW, indicating a 2.28-fold increase in production. Results indicated that V. azureus BTKB33 has potential for industrial production of PHB.

Ethanol Production From Spent Substrate Of Pleurotus Eous

Spent substrate, the residual material of mushroom cultivation, causes disposal problems for cultivators. Currently the spent substrate of different mushrooms is used mainly for composting. Edible mushrooms of Pleurotus sp. can grow on a wide range of lignocellulosic substrates. In the present study, Pleurotus eous was grown on paddy straw and the spent substrate was used for the production of ethanol. Lignocellulosic biomass cannot be saccharified by enzymes to high yield of ethanol without pretreatment. The root cause for the recalcitrance of lignocellulosic biomass such as paddy straw is the presence of lignin and hemicelluloses on the surface of cellulose. They form a barrier and prevent cellulase from accessing the cellulose in the substrate. In the untreated paddy straw, the amount of hemicelluloses and lignin (in % dry weight) were 20.30 and 20.34 respectively and the total reducing sugar was estimated to be 5.40 mg/g. Extracellular xylanase and ligninases of P. eous could reduce the amount of hemicelluloses and lignin to 16 and 11(% dry weight) respectively, by 21st day of cultivation. Growth of mushroom brought a seven fold increase in the total reducing sugar yield (39.20 mg/g) and six fold increase in the production of ethanol (6.48 g/L) after 48hrs of fermentation, when compared to untreated paddy straw

Pretreatment Of Agricultural Waste With Pleurotus Sp. For Ethanol Production

Bioethanol is a liquid fuel obtained from fermentation of sugar/starch crops. Lignocellulosic biomass being less expensive is considered a future alternative for the food crops. One of the main challenges for the use of lignocellulosics is the development of an efficient pre-treatment process. Pretreatments are classified into three - physical, chemical, and biological pretreatment. Chemical process has not been proven suitable so far, due to high costs and production of undesired by-products. Biologically, hydrolysis can be enhanced by microbial or enzymatic pretreatment. Studies show that the edible mushrooms of Pleurotus sp. produce several extracellular enzymes which reduce the structural and chemical complexity of fibre. In the present study, P. ostreatus and P. eous were cultivated on paddy straw. Spent substrate left after mushroom cultivation was powdered and used for ethanol production. Saccharomyces sp. was used for fermentation studies. Untreated paddy straw was used as control. Production of ethanol from P. ostreatus substrate was 5.5 times more when compared to untreated paddy straw, while the spent substrate of P. eous gave 5 times increase in ethanol yield. Assays showed the presence of several extracellular enzymes in the spent substrate of both species, which together contributed to the increase in ethanol yield

Optimization of Lignin Peroxidase, Manganese Peroxidase, and Lac Production from Ganoderma lucidum Under Solid State Fermentation of Pineapple Leaf

This study was undertaken to isolate ligninase-producing white-rot fungi for use in the extraction of fibre from pineapple leaf agriwaste. Fifteen fungal strains were isolated from dead tree trunks and leaf litter. Ligninolytic enzymes (lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase (Lac)), were produced by solid-state fermentation (SSF) using pineapple leaves as the substrate. Of the isolated strains, the one showing maximum production of ligninolytic enzymes was identified to be Ganoderma lucidum by 18S ribotyping. Single parameter optimization and response surface methodology of different process variables were carried out for enzyme production. Incubation period, agitation, and Tween-80 were identified to be the most significant variables through Plackett-Burman design. These variables were further optimized by Box-Behnken design. The overall maximum yield of ligninolytic enzymes was achieved by experimental analysis under these optimal conditions. Quantitative lignin analysis of pineapple leaves by Klason lignin method showed significant degradation of lignin by Ganoderma lucidum under SSF

Protease Production by haloarchaea Natrinema sp. BTSH10 isolated from salt pan of South India

The present study led to the recognition of Natrinema sp. BTSH 10 isolated from saltern ponds, as an ideal candidate species for production of gelatinase, which was noted as a halozyme capable of showing enzyme activity in the presence of 15% NaCl. Results obtained during the course of the present study indicated potential for application of this enzyme in industrial catalysis that are performed in the presence of high concentrations of salt. The enzyme characteristics noted with this gelatinase also indicate the scope for probable applications in leather industry, meat tenderization, production of fish sauce and soy sauce. Since halophilic proteases are tolerant to organic solvents, they could be used in antifouling coating preparations used to prevent biofouling of submarine equipments. The gelatinase from haloarchaea could be considered as a probable candidate for peptide synthesis. However, further studies are warranted on this haloarcheal gelatinase particularly on structure elucidation and enzyme engineering to suit a wide range of applications. There is immense scope for developing this halozyme as an industrial enzyme once thorough biochemistry of this gelatinase is studied and a pilot scale study is conducted towards industrial production of this enzyme under fermentation is facilitated. Based on the present study it is concluded that haloarchaea Natrinema sp. that inhabit solar saltern ponds are ideal source for deriving industrially important halozymes and molecular studies on enzymes are prerequisite for their probable industrial applications. This is the first time this species of archaea is recognized as a source of gelatinase enzyme that has potential for industrial applications.

Influence of fibrolytic enzymes on the hydrolysis and fermentation of pure cellulose and xylan by mixed ruminal microorganisms in vitro

A series of in vitro studies was, conducted to determine the effects of adding a commercial enzyme product on the hydrolysis and fermentation of cellulose, xylan, and a mixture (1:1 wt/wt) of both. The enzyme product (Liquicell 2500, Specialty Enzymes and Biochemicals, Fresno, CA) was derived from Trichoderma reesei and contained mainly xylanase and cellulase activities. Addition of enzyme (0.5, 2.55 and 5.1 muL/g of DM) in the absence of ruminal fluid increased (P < 0.001) the release of reducing sugars from xylan and the mixture after 20 h of incubation at 20degreesC. Incubations with ruminal fluid showed that enzyme (0.5 and 2.55 muL/g of DM) increased (P < 0.05) the initial (up to 6 h) xylanase, endoglucanase, and beta-D-glucosidase activities in the liquid fraction by an average of 85%. Xylanase and endoglucanase activities in the solid fraction also were increased (P < 0.05) by enzyme addition, indicating an increase in fibrolytic activity due to ruminal microbes. Gas production over 96 h of incubation was determined using a gas pressure measurement technique. Incremental levels of enzyme increased (P < 0.05) the rate of gas production of all substrates, suggesting that fermentation of cellulose and xylan was enzyme-limited. However, adding the enzyme at levels higher than 2.55 muL/g of DM failed to further increase the rate of gas production, indicating that the maximal level of stimulation was already achieved at lower enzyme concentrations. It was concluded that enzymes enhanced the fermentation of cellulose and xylan by a combination of pre- and postincubation effects (i.e., an increase in the release of reducing sugars during the pretreatment phase and an increase in the hydrolytic activity of the liquid and solid fractions of the ruminal fluid), which was reflected in a higher rate of fermentation.

Use of fibrolytic enzymes to improve the nutritive value of ruminant diets - A biochemical and in vitro rumen degradation assessment

Two commercial enzyme products, Depol 40 (D) and Liquicell 2500 (L), were characterised from a biochemical standpoint and their potential to improve rumen degradation of forages was evaluated in vitro. Enzyme activities were determined at pH 5.5 and 39 degreesC. Analysis of the enzyme activities indicated that L contained higher xylanase and endoglucanase, but lower exoglucanase, pectinase and alpha-amylase activities than D. The Reading Pressure Technique (RPT) was used to investigate the effect of enzyme addition on the in vitro gas production (GP) and organic matter degradation (OMD) of alfalfa (Medicago sativa L.) stems and leaves. A completely randomised design with factorial arrangement of treatments was used. Both alfalfa fractions were untreated or treated with each enzyme at four levels, 20 h before incubation with rumen fluid. Each level of enzyme provided similar amounts of filter paper (D1, L1), endoglucanase (D2, L2), alpha-L-arabinofuranosidase (D3, L3) and xylanase units (D4, L4) per gram forage DM. Enzymes increased the initial OMD in both fractions, with improvements of up to 15% in leaves (D4) and 8% in stems (L2) after 12 h incubation. All enzyme treatments increased the extent of degradation (96 h incubation) in the leaf fractions, but only L2 increased final OMD in the stems. Direct hydrolysis of forage fractions during the pre-treatment period did not fully account for the magnitude of the increases in OMD, suggesting that the increase in rate of degradation was achieved through a combined effect of direct enzyme hydrolysis and synergistic action between the exogenous (applied) and endogenous (rumen) enzymes. (C) 2003 Elsevier Science B.V. All rights reserved.

In vitro evaluation of fibrolytic enzymes as additives for maize (Zea mays L.) silage - I. Effects of ensiling temperature, enzyme source and addition level

A series of experiments was completed to investigate the impact of addition of enzymes at ensiling on in vitro rumen degradation of maize silage. Two commercial products, Depot 40 (D, Biocatalysts Ltd., Pontypridd, UK) and Liquicell 2500 (L, Specialty Enzymes and Biochemicals, Fresno, CA, USA), were used. In experiment 1, the pH optima over a pH range 4.0-6.8 and the stability of D and L under changing pH (4.0, 5.6, 6.8) and temperature (15 and 39 degreesC) conditions were determined. In experiment 2, D and L were applied at three levels to whole crop maize at ensiling, using triplicate 0.5 kg capacity laboratory minisilos. A completely randomized design with a factorial arrangement of treatments was used. One set of treatments was stored at room temperature, whereas another set was stored at 40 degreesC during the first 3 weeks of fermentation, and then stored at room temperature. Silages were opened after 120 days. Results from experiment I indicated that the xylanase activity of both products showed an optimal pH of about 5.6, but the response differed according to the enzyme, whereas the endoglucanase activity was inversely related to pH. Both products retained at least 70% of their xylanase activity after 48 h incubation at 15 or 39 degreesC. In experiment 2, enzymes reduced (P < 0.05) silage pH, regardless of storage temperature and enzyme level. Depol 40 reduced (P < 0.05) the starch contents of the silages, due to its high alpha-amylase activity. This effect was more noticeable in the silages stored at room temperature. Addition of L reduced (P < 0.05) neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents. In vitro rumen degradation, assessed using the Reading Pressure Technique (RPT), showed that L increased (P < 0.05) the initial 6 h gas production (GP) and organic matter degradability (OMD), but did not affect (P > 0.05) the final extent of OMD, indicating that this preparation acted on the rumen degradable material. In contrast, silages treated with D had reduced (P < 0.05) rates of gas production and OMD. These enzymes, regardless of ensiling temperature, can be effective in improving the nutritive quality of maize silage when applied at ensiling. However, the biochemical properties of enzymes (i.e., enzymic activities, optimum pH) may have a crucial role in dictating the nature of the responses. (C) 2003 Elsevier B.V. All rights reserved.

In vitro evaluation of fibrolytic enzymes as additives for maize (Zea mays L.) silage - II. Effects on rate of acidification, fibre degradation during ensiling and rumen fermentation

A study was carried out to determine the influence of fibrolytic enzymes derived from mesophilic or thermophilic fungal sources, added at ensiling, on time-course fermentation characteristics and in vitro rumen degradation of maize silage. The mesophilic enzyme was a commercial product derived from Trichodenna reesei (L), whereas the thermophilic enzyme was a crude extract produced from Thermoascus aurantiacus (Ta) in this laboratory. The fungus was cultured using maize cobs as a carbon source. The resulting fermentation extract was deionised to remove sugars and characterised for its protein concentration, main and side enzymic activities, optimal pH, protein molecular mass and isoelectric point. In an additional study, both enzymes were added to maize forage (333.5 g DM/kg, 70.0, 469.8, 227.1 and 307.5 g/kg DM of CP, NDF, ADF and starch, respectively) at two levels each, normalized according to xylanase activity, and ensiled in 0.5 kg capacity laboratory minisilos. Duplicate silos were opened at 2, 4, 8, 15, and 60 days after ensiling, and analysed for chemical characteristics. Silages from 60 days were bulked and in vitro gas production (GP) and organic matter degradability (OMD) profiles evaluated using the Reading Pressure Technique (RPT), in a completely randomised design. The crude enzyme extract contained mainly xylanase and endoglucanase activities, with very low levels of exoglucanase, which probably limited hydrolysis of filter paper. The extract contained three major protein bands of between 29 and 55 kDa, with mainly acidic isoelectric points. Ensiling maize with enzymes lowered (P < 0.05) the final silage pH, with this effect being observed throughout the ensiling process. All enzyme treatments reduced (P < 0.05) ADF contents. Treatments including Ta produced more gas (P < 0.05) than the controls after 24 h incubation in vitro, whereas end point gas production at 96 h was not affected. Addition of Ta increased (P < 0.01) OMD after 12 h (410 and 416 g/kg versus 373 g/kg), whereas both L and Ta increased (P < 0.05) OMD after 24 h. Addition of enzymes from mesophilic or thermophilic sources to maize forage at ensiling increased the rate of acidification of the silages and improved in vitro degradation kinetics, suggesting an improvement in the nutritive quality. (C) 2003 Elsevier B.V All rights reserved.

In vitro evaluation of fibrolytic enzymes as additives for maize (Zea mays L.) silage - III. Comparison of enzymes derived from psychrophilic, mesophilic or thermophilic sources

A completely randomised study was completed to examine the influence of fibrolytic enzymes derived from psychrophilic, (F), mesophilic, (L) or thermophilic (Ta) sources, applied at ensiling, on the chemical characteristics and in vitro rumen fermentation of maize silage, assessed using the Reading Pressure Technique (RPT). Treatments, all in triplicate, consisted of untreated maize forage or treated with preparations F, L, Ta or a mixture (1: 1, v/v) of F and L (FL), at two levels each, and ensiled for 210 days in plastic mini-silos. Addition of enzymes L decreased (P < 0.05) silage pH relative to the control, whereas enzyme Ta tended (P < 0.10) to reduce it. Preparations F, L and Ta tended to reduce (P < 0.10) the fibre contents of the silages, with effects being attributable to a decrease in the cellulose fraction. Starch contents were reduced (P < 0.05) in the treatments including enzyme F. End-point (96 h) gas production (GP) values did not differ among treatments, suggesting that enzymes did not change the total amount of fermentable substrate. However, consistent with the decrease in starch contents, adding enzyme F reduced (P < 0.05) GP at most incubation times. Addition of enzymes increased (P < 0.05) the initial (6 h) organic matter degradation (OMD) levels in all but one treatment (F), with increases of 14, 19, and 26% for preparations L, Ta, and FL, respectively, averaged across levels. Furthermore, the addition of enzymes increased (P < 0.05) the soluble OM losses, however, these increases did not fully account for the initial increase in OMD. The latter suggests that enzymes increased solubility and also altered silage structure, making it more amenable to degradation by ruminal microorganisms. As a result of the increase in OMD, without a concomitant increase in GP, the fermentation efficiency was greatly increased (P < 0.05) in enzyme treatments. Addition of enzymes to maize at ensiling, particularly those from the mesophilic and thermophilic sources used here, have the potential to increase the initial rate of silage OMD. (C) 2003 Elsevier B.V. All rights reserved.

In vitro cumulative gas production techniques: History, methodological considerations and challenges

Methodology used to measure in vitro gas production is reviewed to determine impacts of sources of variation on resultant gas production profiles (GPP). Current methods include measurement of gas production at constant pressure (e.g., use of gas tight syringes), a system that is inexpensive, but may be less sensitive than others thereby affecting its suitability in some situations. Automated systems that measure gas production at constant volume allow pressure to accumulate in the bottle, which is recorded at different times to produce a GPP, and may result in sufficiently high pressure that solubility of evolved gases in the medium is affected, thereby resulting in a recorded volume of gas that is lower than that predicted from stoichiometric calculations. Several other methods measure gas production at constant pressure and volume with either pressure transducers or sensors, and these may be manual, semi-automated or fully automated in operation. In these systems, gas is released as pressure increases, and vented gas is recorded. Agitating the medium does not consistently produce more gas with automated systems, and little or no effect of agitation was observed with manual systems. The apparatus affects GPP, but mathematical manipulation may enable effects of apparatus to be removed. The amount of substrate affects the volume of gas produced, but not rate of gas production, provided there is sufficient buffering capacity in the medium. Systems that use a very small amount of substrate are prone to experimental error in sample weighing. Effect of sample preparation on GPP has been found to be important, but further research is required to determine the optimum preparation that mimics animal chewing. Inoculum is the single largest source of variation in measuring GPP, as rumen fluid is variable and sampling schedules, diets fed to donor animals and ratios of rumen fluid/medium must be selected such that microbial activity is sufficiently high that it does not affect rate and extent of fermentation. Species of donor animal may also cause differences in GPP. End point measures can be mathematically manipulated to account for species differences, but rates of fermentation are not related. Other sources of inocula that have been used include caecal fluid (primarily for investigating hindgut fermentation in monogastrics), effluent from simulated rumen fermentation (e.g., 'Rusitec', which was as variable as rumen fluid), faeces, and frozen or freeze-dried rumen fluid (which were both less active than fresh rumen fluid). Use of mixtures of cell-free enzymes, or pure cultures of bacteria, may be a way of increasing GPP reproducibility, while reducing reliance on surgically modified animals. However, more research is required to develop these inocula. A number of media have been developed which buffer the incubation and provide relevant micro-nutrients to the microorganisms. To date, little research has been completed on relationships between the composition of the medium and measured GPP. However, comparing GPP from media either rich in N or N-free, allows assessment of contributions of N containing compounds in the sample. (c) 2005 Published by Elsevier B.V.