Screening And Identification Of A High Amylase-producing Strain And Its Application

In recent years,China's aquaculture industry has developed rapidly.The agricultural scale and intensive farming models are largely developed to increase the agricultural economy.In addition,a lot of farming problems are also exposed and increasing.Such as,organic pollutants,water over-nutrient,animal diseases,and the decline in the quality of aquatic products.It is reported that microorganisms are widely used in aquaculture due to their advantages of green,non-polluted,and non-residual.According to the reports,microorganisms can produce a large number of highly active extracellular enzymes?e.g.,amylase,protease,lipase,dextran,pectinase,and cellulase,etc.?when microorganisms was added to the aquaculture environment.These extracellular enzymes can degrade residual organic foods,such as baits and feces,and improve water quality.It can also inhibit the growth of pathogens and improve the immunity of aquatic animals.And that,application of the microorganism in aquaculture ponds can not only efficiently reduce the use of chemicals?e.g.,disinfectant and antibiotics?,but also improve the quality of aquatic product and protect human health.And that,the healthy,stable and sustainable development of aquaculture ponds will be promoted.In this study,the extracellular enzymes was used to screen a high Amylase-producing strain from grass carp aquaculture pond in water and sediment samples.Further exploring its optimum fermentation conditions on enzyme production by orthogonal experiments,and applying it to simulate fish farming.Finally,its practical application effect was also analyzed,including the water quality and fish blood biochemical indexes.And we hope to provide theory basis for the research and development of aquatic probiotics.The main results of this study are listed as follows:???Screening of Amylase-Producing StrainsIn this study,four amylase production strains?named S458-1,G,H,B?were screened from grass carp culture ponds.Primary screening use dilution-plate method and re-screening with punching method.Based on further comparing the size of the hydrolytic circle,the strain S458-1 was showed the highest ability of the enzyme-producing.Finally,according to the morphological,physiological and biochemical characteristics,as well as the 16S rDNA sequence analysis,identified strain S458-1 as Bacillus cereus.???Optimization of fermentation conditions for strain S458-1Based on the liquid fermentation of shake flasks in the laboratory,single-factor and orthogonal experiments were used to optimize the fermentation conditions of the strain S458-1.The optimum medium were 1.0%soluble starch,1%peptone,0.2 g/L MgSO₄·7H₂O,0.8 g/L CaCl₂,0.4 g/L MnSO₄·H₂O,0.6 g/L FeSO₄·7H₂O and 0.2 g/L Al₂?SO₄?₃·18H₂O.The optimal fermentation conditions of amylase were initial pH 9,culture temperature 37?,fermentation time 24 h,medium volumes 10%,and inoculation quantity 2%.Under these optimal condition,amylase activity can reach up to 173.014U/mL.???Effect of strain S458-1 on water quality of aquaculture ponds and biochemical indicators of fish serum?a?The strain S458-1 was added at the concentration of 0,1×10⁶,1×10⁷,and 1×10⁸CFU/mL to the aquaculture water.The results indicated that,the ammonia in the water was reduced at the concentration of 1×10⁷ CFU/mL.On the contrary,it also resulted in the accumulation of nitrogenous nitrogen.In addition,adding strain S458-1 can significantly increase the concentration of active phosphorus,and the addition of sediment to the culture pond has a strong buffer effect on the phosphorus.?b?When the strain S458-1 was added into the farming system,there was no significant effect on the activities of CAT,SOD,and GSH-Px in carp serum.While,it decreased the activities of GOT and GPT in carp serum to some extent.Thus,it was acknowledged that the strain S458-1 was not hurt to fish liver.In addtion,the activity of MDA was significantly reduced with the addition of strain S458-1,which indicated that the strain S458-1 was beneficial to the organism and can enhance the antioxidant capacity of fish.To sum up,a high Amylase-producing strain S458-1 was suitable for aquaculture production according to its enzyme production conditions.And that,it is beneficial to fish and has the potential for further research and development.Therefore,the strain S458-1has practical application value.