Screening Of Bacterial Cellulose Strains,Fermentation Optimization And Studying On The Technology Of Producing Cellobiose

Cellulose mainly exists in plants.It is one of the most abundant bio-polymer with the greatest economic potential on the earth.In addition,some bacteria also have the ability to synthesize cellulose.Generally speaking,the cellulose obtained by bio-synthesis is called"bacterial cellulose"（Bacterial cellulose,BC）.BC is a highly pure porous reticulated nanoscale bio-polymer synthesized by microbial fermentation.Compared with plant cellulose,it has its unique properties:structural uniformity,high hydrophily and high bio-compatibility,as well as good biodegradability.Therefore,BC is regarded as the best cellulose in the world,which can be widely used in food processing,biological medicine,paper-making,textile and other fields.So the research and development of BC will produce great economic benefits.Cello-oligosaccharides is an important soluble dietary fiber,which is composed of D-glucopyranose byβ-1,4 glycoside bond polymerization.Cellobiose is one of the important components of Cello-oligosaccharides,which is widely used in food,feed,medicine and other fields.In addition to some common characteristics of carbohydrates,cellobiose can also promote intestinal peristalsis and is not absorbed by human intestinal tract.It can be used as a proliferation factor for Bifidobacterium,which can reduce blood pressure,improve lipid metabolism and enhance the immunity of the body.In the process of preparation of cellobiose,due to many impurities in raw materials of biological method and many by-products of chemical method,the production of cellobiose has not been achieved in a large scale.In this study,a new idea of preparing cellobiose by enzymatic hydrolysis high purity bacterial cellulose was put forward for the first time,which not only expand the application field of bacterial cellulose,but also greatly improve the yield of cellobiose.It provides a theoretical basis for the research and production of cellobiose in the future.This paper focuses on BC and cellobiose,and carries out four aspects of research.The content and results are as follows:（1）Mutation breeding of BC-producing bacteria:Using JR-02（Gluconacetobacter hansenii JR-02）,which is preserved in our laboratory,as the starting strain,after activation and UV mutagenesis,the genetically stable strain J2-1 with high yield of BC was selected,and the static fermentation yield was4.52 g/L.Compared with the original strain C40,the yield increased by 37.39%.（2）Fermentation process optimization of BC-producing strain:The mutant strain J2-1 of Acetobacter gluconicum was used as the experimental strain,the fermentation process of BC was optimized by single factor and orthogonal optimization,and the optimum fermentation medium composition was determined as follows:glucose 8%,yeast extract powder 1.8%,ethyl alcohol2%（v/v）,Mg SO₄·7H₂O 0.04%,lactic acid 0.2%,Na₂HPO₄·12H₂O 0.3%;the optimum fermentation conditions were as follows:inoculum size 2%,optimal liquid loading amount 50 m L（250 m L conical bottle）,and under the condition of initial p H 5.8,culture temperature 30℃,7 days fermentation cycle,the yield of bacterial cellulose reached 9.34 g/L,which was 1.89 times of that before optimization.（3）Optimization of solid state fermentation process of cellulase producing strain:The mutant strain of Trichoderma koningii F-6 was used as the starting strain.The single factor and orthogonal optimization experiments were carried out to optimize the enzyme production process.The optimum fermentation medium was obtained as follows:rice straw powder 8g bran 4g,Mandels nutrient solution 20 m L containing 2%glucose and 2%Tween-80.The optimum fermentation conditions were as follows:fermentation cycle 60 h,inoculum size3 m L,initial p H 4.3（natural）,temperature 37℃.Under this medium and fermentation conditions,the activity of FPA and CMC reached 17.32 U/g and71.08 U/g respectively.（4）Studying on the preparation of cellobiose by enzymatic method:Using bacterial cellulose as raw material,β-glucanase was used for enzyme hydrolysis,and the hydrolyzed products were quantitatively detected by high performance liquid chromatography（HPLC）.Through single factor and orthogonal optimization experiments,the effects of p H value,enzymatic hydrolysis temperature,substrate concentration,enzyme dosage and enzymatic hydrolysis time on components and content of enzymatic hydrolysis products were investigated.The results showed that only cellobiose and glucose were contained in the hydrolyzed product,and the yield of cellobiose could reach 6.89g/L when the p H value,temperature,substrate concentration,enzyme dosage,time and buffer solution were 6.0,50℃,4%,180 U/m L,10 h and 20 m L,respectively.Therefore,this condition is determined to be the best enzyme hydrolysis condition for the preparation of cellobiose.