Screening Endophytic Fungi For Diacylglycerol Production And Preparation And Application Of Fermentable Sugar From Rice Husk

Diacylglycerol（DAG）is a component in natural oil,but its content is very low in natural animal and vegetable oils.As a structural lipid,its caloric value is lower than that of triglyceride（TAG）.DAG can be added to food as a fat substitute to reduce the calorie of food,and will not damage the original texture and sensory properties of the product.Traditional DAG production methods such as chemical method and enzymatic method have some disadvantages,including high energy consumption and many by-products through chemical method,high cost,low reaction rate,and poor stability through enzymatic method.All these disadvantages limit the commercial production of DAG to a certain extent.Therefore,it is urgent to develop a new DAG production method to solve the above problems.Producing oil using microorganisms has many advantages such as less land occupation,short production cycle and high oil yield.Furthermore,filamentous fungi have greater advantages compared with other oil producing microorganisms because they are easier to harvest（filamentous or small ball growth）.China is an important rice producing country in the world.Rice husk is one of the main by-products of rice processing,which contains hemicellulose and cellulose that can be used to prepare fermentable sugar.Therefore,using rice husk as raw material to prepare fermentable sugar and ferment other important chemical substances has important development prospects.This study focused on the isolation,screening,and identification of endophytic fungi for producing DAG.The effects of fermentation conditions on the growth of endophytic fungi,oil accumulation,and DAG yield were evaluated.The preparation of fermentable sugar from rice hull and the utilization of the prepared fermentable sugar for producing DAG through fermentation with endophytic fungi were discussed.The main contents and conclusions are as follows:Totally 43 strains of endophytic fungi were isolated from the plant tissue of Taxus chinensis var.mairei.From them,five strains capable of accumulating DAG were isolated.The morphology and molecular biology of the five endophytic fungi were identified.The results showed that they belonged to five different genera:Penicillium cataractum（strain No.MLGP11,P.cataractum MLGP11）,Fusarium annulatum（strain No.MLP41,F.annulatum MLP 41）,Pestalotiopsis scoparia（strain No.MLY31W,P.scoparia MLY31W）,Trichoderma dorotheae（strain No.MLG23,T.dorotheae MLG23）,and Colletotrichum aeschynomenes（strain No.MLY23,C.aeschynomenes MLY23）.The analysis of fatty acid composition showed that the main fatty acids of DAG from these five endophytic fungi were oleic acid（C18:1）,palmitic acid（C16:0）,linoleic acid（C18:2）,and stearic acid（C18:0）.Among of these fatty acids,oleic acid was the main unsaturated fatty acid.The content of unsaturated fatty acid on 1,2-DAG from P.scoparia MLY31W was the highest,reaching 68.98%.Taking the cell dry weight,oil yield,and DAG yield as evaluation indexes,the fermentation conditions for DAG production through fermentation with endophytic fungi were optimized.The maximum DAG yield was obtained after F.annulatum MLP41,T.dorotheae MLG23,and C.aeschynomenes MLY23 were cultured for 5days,the maximum DAG yield was obtained after P.scoparia MLY31W was cultured for 7 days,and the maximum DAG yield was obtained when P.cataractum MLGP11was cultured for 9 days.The maximum DAG yield was obtained when the inoculation amount was 3 bacterial blocks/100ml medium for all the five strains.The maximum DAG yield was obtained when the fermentation temperature was 28 ℃.The maximum DAG yield was obtained with stirring speed of 120 rpm.The maximum DAG yields from F.annulatum MLP41 and T.dorotheae MLG23 were obtained when the C/N ratio was 38:1（g/g）.The maximum DAG yields from C.aeschynomenes MLY23 and P.cataractum MLGP11 were obtained when the C/N ratio was 68:1（g/g）.The maximum DAG yield from P.scoparia MLY31W was obtained when the C/N ratio was 98:1（g/g）.Under the optimal fermentation conditions,the DAG yield from C.aeschynomenes MLY23 was the highest among those from these five endophytic fungi,reaching 189.87 mg/L.At the same time,and the oil yield,the DAG content of oil,and the cell dry weight reached 1.85 g/L,10.26%,and 10.03 g/L,respectively,under the optimal fermentation conditions for C.aeschynomenes MLY23.Two-stage dilute H₂SO₄ catalytic reaction was carried out in an autoclave to prepare fermentable sugar from rice husk,and the hydrolysis conditions were optimized.The results showed that at the first stage the highest yield of reducing sugar was 40.5mol%under the optimal conditions of H₂SO₄ concentration 1.5wt%,reaction temperature 140℃,and retention time 0 min.The highest yield of reducing sugar was 28.0mol%at the second stage under the optimal conditions of H₂SO₄concentration 4.5wt%,reaction temperature 160℃,and retention time 30 min.Finally,the highest yield of total reducing sugar was 68.5mol%under the optimal conditions both at the first stage and at the second stage.The by-products produced during the hydrolysis process were removed by combination detoxification processes.The removal rates of formic acid,acetic acid,levulinic acid,5-hydroxymethyl furfural,furfural,and total phenol reached 45.3%,48.0%,61.2%,75.4%,100.0%,and 99.3%,respectively.FT-IR and SEM analysis showed that the dilute H₂SO₄ catalyzed hydrolysis of rice husk in a high-pressure reactor with two-stage procedues could effectively degrade cellulose and hemicellulose in rice husk.Amorphous Si O₂ with purity up to 98.0%was obtained by calcining the residues of rice husk after hydrolysis.Finally,DAG was produced from C.aeschynomenes MLY23 using the detoxificated sugar obtained from rice husk as the carbon source,and the biomass,the oil yield,and the DAG yield was 9.21 g/L,1.51 g/L,and 143.70 mg/L,respectively.The data were similar with those when pure glucose was used as the carbon source alone.The results showed that rice husk hydrolysate had potential application in the production of DAG from endophytic fungi by fermentation.