Screening And Identification Of Chlorogenic Acid Producing Strains And Optimization Of Fermentation Conditions

As cafeylquinic acid,chlorogenic acid（CGA）is a phenolic acid synthesized by the condensation of cafeic acid and quinic acid.It widely exists in various kinds of phenylc compounds in plants,and is one of the main active ingredients in many Chinese herbal medicines such as honeysuckle,eucommia ulmoides,coffee and Wormwood.Studies have found that CGA has a variety of functions such as scavenging free radicals,antibacterial,antiviral,anti-tumor and prevention of diabetes,hyperlipidemia and hepatitis.Therefore,CGA is widely used in food,medicine,daily chemical industry and other fields.At present,CGA production is mainly extracted from the cells of traditional Chinese medicine plants.However,due to the long growth cycle of traditional Chinese medicine plants,lack of resources,low CGA content,complex extraction process and high cost,CGA production efficiency is low.Using microbial fermentation technology to produce CGA efficiently can not only reduce the dependence on traditional Chinese medicine plants,but also reduce the production cost of CGA,which is a feasible scheme to realize the efficient production of CGA.In this study,endophytic bacteria with synthesizing CGA were isolated from honeysuckle leaves,sweet potato leaves,mint leaves,dandelion leaves and eucommia ulmoides leaves.Morphological analysis,molecular identification and fermentation process optimization of the endophytic bacteria were investigated.The study would lay a foundation for the development of CGA producing engineering strains and the production of CGA by microbial fermentation.The main research contents are as follows:1.Screening of CGA-producing strains:Plant samples such as honeysuckle leaves,sweet potato leaves,mint leaves,dandelion leaves and eucommia ulmoides leaves were cultured in beef extract peptone modified medium and potato dextrose modified medium culture,CGA is a phenolic hydroxyl organic carboxylic acid,Al³⁺modified medium can be combined with CGA hydroxyl,form the relatively stable CGA-Al³⁺complex,In other words,the color producing strains in the modified medium were selected for fermentation and culture,and then the fermentation products were analyzed by high performance liquid chromatography（HPLC）.Finally,seven endophytic bacteria with synthesizing CGA were obtained,including five bacteria and two fungi.Among the seven strains,endophytic bacteria XJ-1 exhibited the highest CGA production,and reached 9.9μg/m L.2.Morphological analysis and molecular identification of endophytic bacteria:the morphology of endophytic bacteria XJ-1 with high CGA production was observed by scanning electron microscope and light microscope respectively.The results showed that the strain was rod-shaped Gram-positive bacteria.PCR amplification of 16S r DNA gene of the strain and sequence comparison of National Center for Biotechnology Information（NCBI）showed that the endophytic strain is Bacillus subtilis.Meanwhile,the phylogenetic tree was constructed by MEGA7.0 software.3.Influence fermentation conditions on yield of CGA were investigated by single factor method.These factors include the carbon source（sucrose,glucose,fructose,maltose,lactose）,nitrogen source（peptone and beef extract,corn starch,ammonium sulphate,ammonium chloride,citric acid）,carbon and nitrogen ratio,inorganic salts（potassium dihydrogen phosphate,potassium hydrogen phosphate,sodium chloride,copper sulfate,ferrous sulfate,calcium chloride,magnesium sulfate）,temperature,p H,rotational speed,and fluid volume.The results showed that the best carbon source for CGA synthesis was fructose.When fructose was 30 g/L,CGA yield was high,and reached 7.39μg/m L,but the strain had poor ability to synthesize CGA using other carbon sources.Different organic and inorganic nitrogen sources had a great influence on CGA synthesis.The yield of CGA reached the highest at peptone 6 g/L,beef extract 6 g/L,corn pulp 5 g/L,ammonium sulfate4 g/L,ammonium chloride 6 g/L and ammonium citrate 5 g/L,and reached 11.02μg/m L、10.7μg/m L、14.9μg/m L、2.35μg/m L、2.02μg/m L、19.32μg/m L,respectively.The optimal organic nitrogen source was peptone（6 g/L）,and the optimal inorganic nitrogen source was ammonium citrate（5 g/L）.The yield of CGA was 38.21μg/m L when the C/N ratio was 1:4.Besides,the yield of CGA reached the highest at potassium dihydrogen phosphate 1.5 g/L,potassium dihydrogen phosphate 1.5 g/L,sodium chloride 3 g/L,copper sulfate 0.03 g/L,ferrous sulfate 0.15 g/L,calcium chloride 0.3 g/L,magnesium sulfate 0.2 g/L,and reached9.08μg/m L,9.81μg/m L,6.39μg/m L,10.89μg/m L,6.26μg/m L,6.12μg/m L,and 6.05μg/m L,respectively.Furthermore,optimization results of the initial p H of fermentation medium,culture temperature,filling volume,rotating speed and inoculating volume showed that the yield of CGA was high at p H 5.0,37℃,filling volume 20 m L,rotating speed 220 rpm,inoculating volume 10%,and reached 16.22μg/m L,18.36μg/m L,20.35μg/m L,11.37μg/m L and 10.83μg/m L,respectively.Orthogonal and response surface experiments were used to optimize the fermentation conditions.The results showed that CGA yield reached 24.02μg/m L when fructose,corn pulp,and ammonium citrate was at 30 g/L,6 g/L and 5 g/L,respectively.The results of the steepest climb showed that the established model was significant（P=0.0144）,fructose and p H（P<0.05）had significant effect on CGA synthesis;Ammonium citrate,corn pulp,dipotassium hydrogen phosphate,magnesium sulfate,copper sulfate,calcium chloride（P>0.05）had no significant effect on CGA synthesis;The experimental results of response surface design showed that the established model was significant（P<0.0001）.Furthermore,fructose and p H had interaction on CGA yield（P=0.0015）.The optimal fermentation conditions were determined as follows:fructose 40 g/L,ammonium citrate 5 g/L,corn pulp6 g/L,dipotassium hydrogen phosphate 1 g/L,magnesium sulfate 0.15 g/L,copper sulfate0.03 g/L,calcium chloride 0.4 g/L,sodium chloride 1 g/L,and initial fermentation p H6.0.Under optimal conditions,the CGA yield predicted by response surface model was79.03μg/m L,and the CGA yield verified by shaking was 75.82μg/m L,which was similar to the predicted result,indicating that the mathematical model established was accurate.The yield of CGA after optimization was 7.66 times higher than that before optimization.