Immobilization Of Inulinase And Research On Product Of Fructooligosaccharides

Jerusalem artichoke(Helianthus tuberoses L.),is commonly known as Guizi ginger, Yang ginger.It belongs the department of Asteraceae.It is a perennial herb.It can resistant drought,barren,windy and so on.The growth of state is strong,also it has a high output rate.Nowadays,there is a large number of cultivation in the north-south of China.Jerusalem artichoke is an ideal source of Inulin,High fructose syrup and Fructooligosaccharides.However, this resource is not well used,resulting in a great waste.Inulin is the polysaccharide chain withβ(2-1) glycosidic bond linking,and the reductive end is a glucopyranosyl.Inulin is a non-digestible carbohydrate.In the colon,it can selectively promote the growth of bacteria and improve the health status of the host.In the food industrial field,Inulin is a functional food.It can improve food qualities and nutritional characteristics.As natural functional food ingredients,Inulin is widely used in Europe and the United States.Inulinase(EC3.2.1.7) is hydrolytic enzyme of Inulin,also it can be mainly hydrolyzed to be High fructose corn syrup and Fructooligosaccharides.The Fructooligosaccharides with low calories,and it can discharge toxin;improve the structure of intestinal flora;promote the absorption of calcium,magnesium,iron and other mineral elements;enhance the human immune system.At the same time,it is used as a healthy dessert for patients with diabetes and hypertensions.Another hydrolytic product is High fructose syrup.It has high sweetness,high moisture,fine permeability,good fermentation performance,preservation performance and excellent anti-corrosion effect,therefore it is used in a wide range of the food industry.Fructooligosaccharides and High fructose syrup are good carbohydrates with physiological functions.As healthy carbohydrates source,they can be understood and concerned by more and more people.Inulinase can hydrolyze Inulin to be High fructose corn syrup and Fructooligosaccharides,however,the free Inulinase hydrolysis usually causes some trouble.For example,hardly separated by products and substrates,insecurity,the use of free Inulinase is not repeated,hardly control the reaction.Therefore Inulinase should be immobilized in order to achieve the repeat use of continuous operation and the purpose of separation and purification,besides simplify the process and enhance substrate convert rate.Consequently reduce costs and raise profits for the people and industrialization.In this paper,there are some studies on the selection of immobilized vectors and materials,the immobilized Inulinase properties,the immobilized Inulinase reaction and the product of Fructooligosaccharides.Hope from the above mentioned efforts to find a practical way, which with low-costs and simple process,high convert rate and high utilization rate of repeat operation.Provide some useful information or reference for enzymatic product of Fructooligosaccharides.1.Select the vectors and methods for immobilized Inulinase,the paper uses PVA-CAgelatin embed method,chitosan crosslinking method,D₂₀₁ anion macroporous resin adsorb-crosslinking method and cotton cloth couple-linking method to immobilize Inulinase. Comparing four methods,ultimately determine the D₂₀₁ anion macroporous resin adsorb-crossinglinking method is the best method for immobilization.At same time,using orthogonal test on D₂₀₁ anion macroporous resin adsorb-crosslinking method to optimize the react conditions of immobilized Inulinase.In this system,the ratio of carrier and enzyme is 1:4,the concentration of glutaraldehyde is 0.5%,the immobilized pH is 5.8,crosslinking time is 3h,the immobilized Inulinase activity can reach the highest recovery rate 42.89%.Under the above conditions,the mechanical strength and operational stability of immobilized Inulinase are good,also the method can be easily operated.2.Purify crude enzymatic solution of the Inulinase,through decolorization of the activated carbon,fractional precipitation of ammonium sulfate and dialysis,the purification is 4.29,furthermore enzymatic activity increases from 5.50 U·mg^-1to 23.58 U·mg^-1and enzymatic recovery rate reaches 87.82%.On this basis,immobilized Inulinase obtained kinetic constants Km is 175.68 mg·mL^-1(Inulin),and the fastest react speed Vm is 1.33 mg·(mL·min)^-1.3.By studying on immobilized Inulinase,it shows enzymatic properties:The optimum pH is 5.8,migrating 0.8 units toward alkaline direction compared with free enzyme;the optimum temperature is 60℃,is high 10℃than the free enzyme;pH stability and temperature stability of immobilized Inulinase variously increase than free enzyme. After Inulinase immobilized,the original enzymatic activity as the benchmark,in Ca²⁺, Fe²⁺,Na⁺,Zn²⁺ existed solution,under the same react conditions,measures immobilized Inulinase activity is higher than free Inulinase activity.Immobilized Inulinase is placed in buffer solution for three weeks and the activity still maintains about 85%.4.Product identification of immobilizated Inulinase reaction with the silica gel G as the stationary phase,using TLC to detect the product of immobilized enzyme reaction.The results show that N-butanol:glacial acetic acid:water(8:2:1) for the start agent, aniline-diphenylamine-phosphoric acid as chromogenic reagent,2μL/0.5μL dropping-sample volume,dropping-sample piont controls in 3mm below,TLC separation is better.And after the initial detection and identification,Inulinase enzymatic product is composited by Fructose,Glucose,Sucrose,and small amounts of Fructooligosaccharides.5.Use the continuous cycle reactor to optimize process conditions of Fructooligosaccharie. Respectively tests on different factors:the cycle reaction time,substrate concentration, feed rate,experiments show that:when the cycle reaction time is 38h,the product rate of Fructooligosaccharides reaches a maximum 26.44%;when the substrate concentration is 30g·L^-1,the Fructooligosaccharides content reaches the maximum 30.75%;when the feed rate is 1.2 mL·min^-1,the Fructooligosaccharides content reaches a maximum 27.89%.Orthogonal test further notes:the impact factors for product of Fructooligosaccharides as follows:reaction time＞substrate concentration＞feed speed. That is,the best conditions for product of Fructooligosaccharides is:the feed speed is 1.4mL·min^-1,the reaction time is 38h,substrate concentration is 30g·L^-1,the product rate of Fructooligosaccharides is up to 30.26%.