Study On Key Technology Of Efficient Preparation Of Trehalose From Beer Yeast Cells

Study on Key Technology of Efficient Preparation of Trehalose from Beer Yeast Cells is attached to the key agricultural program of technology hell in Jilin province(item number: 20070201)。Trehalose is one of the studying focuses about functionality oligosaccharide for scientist in the recent years. Now, the studying focus the trehalose has two directions in internal and abroad. The one is mechanism of action to protect biology, other is the efficiently preparation technology of cheap trehalose.The studying,study on extracting trehalose from beer waste yeast cells, this method can be to efficiently solve the problem of pollution in beer Industry.The main conclusions obtained are as follows:1. Study on the detection method of trehaloseThe detection method of trehalose must be the premise of trehalose production and application.(1)The result of methodology were RSD<5% in precision test and the stability / reproducibility test and the recovery rate test when using enthrone-sulfuric acid method to detect trehalose. Indicating that the method is accurate can be used for routine testing and process of trehalose.(2)HPLC method is based on the retention time to analyze the sugar content, different kinds of sugar have different retention time, and HPLC method can accurately detect the true content of trehalose.The result of methodology were RSD<2% in precision test and the stability / reproducibility test and the recovery rate test when using HPLC to detect trehalose.Through analyze of two detect trehalose methods, anthrone-sulfuric acid method can be used for routine testing and process of trehalose. HPLC can be used to test purity and quality of trehalose.2. Researches on the methods of beer waste yeast cells wall breaking Researches on the yield of trehalose from beer yeast cell and the six methods of ce ll wall breaking tell us that:(1)Ultrasonic cell wall breaking methodThe optimum parameters of technology of Ultrasonic cell wall breaking are: ultrasonic time 55min, ultrasonic power 300W, liquid-solid ratio 30:1, yield of trehalose 4.64%(2)Ultrasonic assistant cell wall breaking methodThe optimum conditions of ultrasonic assistant cell wall breaking method are: water extraction temperature 100℃, Water extraction time 6h,ultrasonic power 600W, ultrasonic time 20min,the yield of trehalose is 7.72%.(3)Microwave cell wall breaking methodwe have found that three dimensions orthogonal polynomial regressive equation of microwave cell wall breaking method is y=3.672+0.00142Z12-0.0297Z1+0.00187Z3(where Z1- microwave time/min; Z2-liquid-solid ratio1/mL:g; Z3-microwave power/W)The optimum conditions of microwave cell wall breaking method are: microwave power: 500W,microwave time 25min,microwave temperature 80℃, the yield of trehalose is 5.07%.(4)Microwave assistant cell wall breaking methodwe can construct the four dimension linear regressive equation: y=4.660+0.001Z1-0.484Z2+0.092Z3+1.494Z4; (where Z1- microwave power/W; Z2-microwave time/min; Z3-extraction temperature/℃; Z4- extraction time/min). The optimum conditions for the microwave assistant cell wall breaking are: liquid- solid ratio 30:1,microwave time 3min, microwave power 600w, extraction temperature 100℃, Extraction time 3.5h,and the yield of trehalose is 9.24%.(5)Microwave assisted extraction of TCAThe optimum parameters that extract trehalose from beer yeast are: microwave inactivation 4 min, extraction temperature 65℃, liquid- solid ratio 22:1, extraction time 120min, under this process parameters, the yield of waste beer yeast trehalose reaches 11.07%. We obtains a high yield of trehalose.(6)PEF cell wall breaking methodThe best parameters of the high voltage pulse field cell wall breaking method are: electrical field strength 45kv/cm, liquid-solid ratio 40:1, number of pulses 6, and the yield of waste beer yeast trehalose reaches 4.4%.(7)Through analyze of 6 cell wall breaking methods, we found that only considerin g the yield of trehalose,microwave-assisted method can be taken; if considering the extraction efficiency and energy conservation, PEF method expend the shortest time, so extraction efficiency is the highest.3. Study on separation and purification of trehalose extracting solutionStudy on separation and purification of trehalose extracting solution by these experiments that are include removal of proteins in trehalose extracting solution by The isoelectric point associated with thermal denaturation ,and decolorization by activated carbon, and decolorization of desalination by Ion exchange, and concentration, and crystallization by ethanol, and quality analysis of trehalose product by HPLC and optical activity method.(1)Study on the removal of proteins in trehalose extracting solutionThe design of trehalose extraction method for removal of proteins: The isoelectric point associated with thermal denaturation, laid the foundation t for the extraction of trehalose from beer yeast. When using thermal denaturation and isoelectric point to removal proteins: thermal denaturation of 15min, settling time 7h, precipitation PH value 4.2, rotation speed 4200r/min, centrifugation time of 10min, under this process parameter , the removal rate of protein is 36.15%, the retention rate of trehalose is 93.46 %.(2)Study on activated carbon experimentalThrough single factor analysis and four-linear regression experimental design, ultimately determine the regression model works by the activated carbon for decolorization of trehalose extracts of: = 89.951 +0.295 Z1-5.024Z2 +1.778 Z3-0.128Z4. Z1 is: bleaching temperature (℃), Z2 is the pH value, Z3 is carbon content (%), Z4 is the decolorization time (min). Determined the optimal parameters of the decolorization process: bleaching temperature is 65℃, pH value is 3.5, activated carbon dosage is 1.5%, marking time is 25min.Under this condition, the ratio of decolorization reaches 82.88%, the retention ratio of sugar reaches 95.05%.(3)The study on decolorization and desalination by Ion exchangeThe tandem type ion exchange column is 001x7(30mL)+ D301(60mL)+001 x7(10mL), Under this condition, the through put of ion exchange column is 4.8 times than trehalose extracting solution, ratio of desalinization reaches 98.18%, the ratio of decolonization reaches 90.9%. (4) The optimization result of crystallization conditions of trehaloseThe crystallization conditions of trehalose were concentration temperature60℃, rotation speed 60 r?min-1,trehalose density 40%~50%,the ratio of alcohol 5 times, crystallization time 24h, drying temperature 60℃, drying time12h. Under this condition, Crystallization rate reaches 90%.(5) Quality analysis of trehalose productThe purity of trehalose product is 97.35% by HPLC detection; the optical purity of trehalose product is 99.2% by polarimeter detection.