S-adenosyl-L-methionine（SAM）is an important metabolic intermediate in organism which is involved in many biochemical reaction.There are vast market for SAM as nutrient and drug.Our main task is carries on screening to yeast,and its fermentation conditions to do research.The separation and purification of them were preliminary studied.SAM can be obtained by three ways:chemosynthesis,enzymatic conversion technique,as well as microbial fermentation,of which fermentation is the primary way of industrial production.In our experiment one was choosen from four strains to produce SAM high saccharomycetes Sake,its output is the 1.71g/L.By the space-mutation Yeast Sake,was picked out 500 yeasts,then through refilter we obtained one to produce the SAM high strain,No.552 of the yeast than the originally and its production output increase 11.16%.By the fermentation,fermentation conditions Sake552 strain of did conduct a preliminary exploration.It were studied and identified the best initial pH is 5.0,while SAM accumulation phase is 6.0 in shake flask;best fermentation temperature is 28℃;Best medium are 20%distilled potatoes, glucose 7%,yeast extract 1%,Urea 1.4%,L-Met 1%,KH2PO4 0.4%,K2HPO40.2%,MgSO4·7H2O 0.5%, CaCl2·2H2O 0.2%.The culture conditions of the Yeast Sake 552 in fermentor 5L were optimized, including the prescription of cultural medium,pH,dissolved oxygen,agitation speed,aeration,feeding strategy of complementarity-cultural medium in different stages.The SAM content of the Yeast Sake 552 reached 3g/L after fermentation for 4 days under the optimized conditions.We also has researched the separation and purification process of SAM and has established a preliminary technics.The procedure followed with that the cell suspension of pH2 to be frozen for 24h then to disrupt it and then purify it with weak acid cation exchange resin,to crystal and deposit with methanol,to centrifugal deposition to freeze-dry it finally we got the crude SAM.
Post about "Separation"
As to problem of low utilization value and cost experience in lignin in the papermaking wastewater teatrment, Treatment of papermaking wastewater with precipitation was studied. According to the basement of the experiment, treatment of papermaking wastewater with precipitation was proved as an effective separation method in papermaking wastewater. And the modification of lignosulfonate was studied in the article.The effects of pH-value, amount of precipitation reagent(Cca2+), temperature（T）, and storing time（t） on lignosulfonates recovery from concentrated red liquor were discussed to determine the optimize conditions. And the mechanism of precipitation reaction was studied in the article. It was found that pH-value played an important role in complexing action between lignosulfonates and calcium. While the optimize conditions were obtained: pH=13, Cca2+=8g/L, T= 40℃, t=30min, and the precipitation capacity of lignosulfonate was 4.5g.According to thermogravimetic analysis and infrared spectrum analysis, the outlier and the lignosulfonate in sell were in similar structure. And the outliers were composed of a serial of molecular lignosulfonate. The sulfonation modification in lignosulfonate was studied in the article. The sulfonation process was determined by pre-oxidization and sulfonation process.The sulphoating agent was sodium and the oxidizing agent was potassium hypermanganate. In pre-oxidization, the effects of amount of the oxidizing agent, temperature （T）, reaction time （t） were discussed to determine the optimize conditions. While the optimize conditions were obtained: amount of the oxidizing agent was 5%,T=90c,t=2h.In the sulfonation process, the effects of amount of the sulphoating agent, pH value, temperature（T）, reaction time（t） were discussed to determine the optimize conditions. While the optimize conditions were obtained: amount of the sulphoating agent was 60%, pH=ll,T=90°C,t=3.5h.The corrosion inhibitor of lignosulfonate was studied in the article. According to binary-Multiplex experiment and orthogonal experiment, the formulation made. The ratio of Lignosulfonate: sodium: molybdate: sodium tungstate, zinc sulfate: thiocarbamide: Hexa-methylenetetraamine was. 1: 1: 0.6: 0.3: 0.8: 0.8: 0.4. According to Polarization behavior of steel in a simulated boiler solution experiment, the corrosion inhibitor was anode inhibitor compound. According to electrochemical impedance spectroscopy of steel in a simulated boiler solution, the results shown that the formulation inhibitor could resist corrosion.
Rice straw is one of the major farming wastes in China. A number of studies have been reported on the utilization of the straw waste. However, most of the works focused on making use of the straw cellulose for manufacturing synthetic plank or wood-plastic composite (WPC) materials, and little is achieved on using rice straw as an alternative to petroleum for chemical raw material resource. Therefore, the synthesis of environment-benign degradable thermoplastics using cellulose separated from rice straw, substituting petroleum-derived chemical raw materials, is of great significance in terms of coping with the petroleum crisis, protecting the environment and ecosystem, as well as promoting an sustainable agricultural development.In this paper, high boiling solvents (HBS) method was employed for high efficient separation of the lignin and cellulose from rice straw waste at a relatively low temperature of 160℃. Modified catalyst ABand mixed solvent of Glycerol and 1,4-Butanediol was also used in the experiment.The results of the experiment show that the method used in this paper is of much more efficiency and higher yield according to the method reported.And the separated lignin and cellulose is less degraded,beneficial for the further use.The benzylating modification of the resulting HBS cellulose was performed using benzyl chloride as the benzylation reagent and toluene as the diluent.The benzylation product was investigated through IR, XRD and DSC. The results show that the chemicalstructure of the cellulose has been changed, almost all of the–OH has been benzylized and the cellulose was of decrystallization,a glass transition temperature of 148.48°C has occurred, indicating that the benzylation cellulose has been transformed into a thermoplastic material.
Flos lonicerae,which is one of the Chinese traditional herbs,is the flower buds of Loniccra japonica Thunb,L hypoglauca Miq,L confusa DC and L dasystyla Rchd.It could be used to cure many diseases in China.To make the further use of the Flos lonicerae resource,Salvia miltiorrhiza Bunge was took as material and investigated its extraction process,separation,purification, composition using separation and analysis techniques.The antioxidant activity of the polysaccharides was also studied according to the modern way of evaluating function in this article, the main research results were as follows:1.The optimum conditions with the hot-water extraction method obtained by the orthogonai experiment design were as follows:the ratio of sample to water was 1:20,extraction times was 2, extraction temperature was 40℃and extraction time was 2 hours2.The results about polysaccharides with different molecule weights separated from Flos lonicerae by ultra-filtration show that over 50%molecule weight of polysaccharides was larger than 100KD.The polysaccharides with different molecule weights among 100-50KD,50-30KD and 30-10KD also could be found.3.Two fractions named respectively FL-C1 and FL-D1 were isolated and purified from the extracts by the DE-52 ion-exchange and Sephadex G-100 gel filtration chromatogram,the purity were identified by high pressure gel filtration chromatogram and the results showed two fractions were all homogeneous fractions.The molecule weight was 120000Da and 30000 Da,respectively.4.The physical and chemical determination showed FL-C1 and FL-D1 isolated and purified from the extracts contained 5 monosacharides.The spectrograms were obtained by UV and IR analysis.5.Antioxidant activity of polysaccharides with different molecule weights separated from Flos lonicerae by ultra-filtration was studied in this article.The reducing power of the polysaccharides has observed a direct correlation between antioxidant activity and concentration of certain plant extracts.The results showed that suspension after ultra-filtration has a significant inhibit effect on superoxide radicals generated in a PMS/NADH/NBT system.6.Crude polysaccharides extracts reduced lipid peroxidate malony dialdehyde(MDA) content in serum and tissue in rats,improved glutathione peroxidase(GSH-Px) and catalase(CAT)activity and medial and topmost enhanced significantly superoxide dismutase(SOD) activity in serum and tissue in mice.
Study on Bioconversion of Lovastatin into Wuxistatin by Amycolatopsis sp.ST2710 and Preliminary Purification of Wuxistatin
Statins, which were inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A(HMG-CoA) reductase, were the prime drugs of cardiovascular diseases. They were the focus of most research at home and abroad. Wuxistatin, discovered by our research grounp, was a novel inhibitor of 3-hydroxy-3-methyl-glutaryl(HMG)-CoA reductase, which was a rate-limiting enzyme of cholesterol biosynthesis. Amycolatopsis sp.ST2710 could convert lovastatin into wuxistatin. In this paper, we made search about the optimization of the fermentation conditions of lovastatin bioconverting into wuxistatin by Amycolatopsis sp.ST2710, and purification of the bioconversion product (wuxistatin) by macroperous adsorption resins.The effects of all kinds of fermention conditions on its myceliuim morphology in shake flask were studied. In the flask conditions, the initial pH of the medium, medium volume, inoculating mode, the inoculation concentration, the shear rate were the factors affecting myceliuim morphology. The result showed that uniform mycelium pellets(pellet diameter of 0.5 to 0.7 mm)enhanced the transformation of lovastatin. And we obtained that the shear was the key factor affecting the bioconversion rate of lovastatin.The concentration of lovastatin for Amycolatopsis sp.ST2710 was studied, the critical inhibitory concentration of lovastatin for Amycolatopsis sp.ST2710 was 1.5 g/L, and the suitable concentration of lovastatin was 1 g/L. The best temperature for the period of transforming lovastatin was 28℃, consisting with the optimum temperature of myceliuim growing. Based on the myceliuim morphology, the best optimized fermentation conditions were as follows: initial pH 7.0~8.0, medium volume 25 mL/250 mL shake flask, the inoculum in the form of spore, inoculum 10%, shaking rate 120 r/min. On the best optimized flask conditions, the yield could reach 0.48 g/L.To enhance the yield of wuxistatin by Amycolatopsis sp.ST2710, several fermentation parameters of batch fermentation at different agitation rates from 100 to 400 r/min in the 5 L fermentor were investigated. It was found that the dissolved oxygen increased and the lag stage of cell growth could be shortened with controlling at the agitation rate of 300 r/min. On the other hand, at the lower agitation rate of 200 r/min, the foam was decreased at the stage of bioconversion and the bioconversion rate of lovastatin was promoted. Based on the parameters analysis, a two-stage agitation rate controlling strategy, in which agitation rate was controlled at 300 r/min in the stage of cell growth and then shifted to 200 r/min, was suggested and experimentally verified. Following this strategy, the following results were obtained that the biomass was 4.02 g/L at the end of the stage of cell growth, the concentration of wuxistatin reached 0.50 g/L in culture filtrate and the whole fermentation time in 5 L fermentor was 80 h, which was shorter 16 h than in shake flask. In fed-batch culture, we effectively affected mycelial shape and increase yields of wuxistatin by controlling the concentration of lovastatin. While using fed-batch process with lovastatin, the yield could be increased to 1.2 g/L, which was up to 2.4 times higher than that of normal batch mode.Five kinds of macroporous adsorption resins were used to extract wuxistatin from fermented broth. The static experiments showed that HP20 macroporous resin had the highest adsorption capacity of 89.89 mg(wuxistatin)/g(dry resin), the optimal pH of adsorption was 7.0, and the optimum adsorption time was 3 h. Dynamic experiments showed that in the optimal flow rate of fermented broth of 1 BV/h, the adsorption ability of HP20 was 60 mg wuxistatin/ml wet resin, and the suitable concentration was 0.9 to 1.1 g/L. By acetone concentration gradient, we got colorless bioconvension products, which recovery and concentration could reach 75% and 95%.
This paper focused on studying the skin-whitening agent from radix puerariae and its inhibitory capability against tyrosinase. The active agents were extracted from radix puerariae and the processing conditions were optimized with the orthogonal design test. After the process of separation and purifying, the skin-whitening agent was obtained, and the inhibitory effect of puerarin on tyrosinase was studied, using enzymological kinetic method.The water and ethanol extracts of radix puerariae, Lonicera japonica Thunb., Artemisia anomala S. Moore and Dispyrosl kaki Lf. were obtained by refluxing with both water and 75%(v/v)ethanol, concentration and drying, respectively. The inhibitory capability of water and 75%(v/v)ethanol extracts against tyrosinase were investigated. The results showed that extract of radix puerariae has the stronger inhibition than others.The optimum reaction conditions of skin-whitening agent’s inhibition on tyrosinase determined by the single factor experiment method were as follows: the pH value of Phosphate Buffer Solution(PBS) was 6.85, the volume of L-Tyrosine was 1.0mL, the volume of tyrosinase was 0.4mL,time in water bath before tyrosinase added was 10 minutes and reacting time was 5 minutes.The processing conditions of ethanol extract of radix puerariae were optimized with the orthogonal design test. The optimum extracting condition was obtained as the concentration of ethanol was 75%; time for refluxing was 4h, the ratio of material and solvent was l:8 and repeat the process for 2 times. The amount of flavonoid from extract of radix puerariae was investigated, results showed that the largest amount of flavonoid was13.74%.The ethanol extract was further separated with solvents of different polar such as petroleum ether, trichloromethane, ethyl acetate and n-butylalcohol and their inhibitory capability against tyrosinase were compared. It has been found that the inhibitory capability of ethyl acetate-extracted fraction against tyrosinase was the strongest. The extract of ethyl acetate was purified by silica gel column chromatography and tracked by TLC. 10 eluants were obtained and the inhibitory capability of each eluant against tyrosinase was investigated. The results showed that the inhibition of B was 65.7% and the inhibition of D was 79.2%. B and D was further purified by silica gel column chromatography and tracked by TLC. It has been found that the inhibition of B3 obtained from B was 92.3%, and the inhibition of D4 obtained from D was 98.5%.To identify the skin-whitening agent from radix puerariae, HPLC, UV and LCMS were used. It has been confirmed that daidzein and puerarin were the two main skin-whitening agents obtained from radix puerariae.The mechanism of puerarin’s inhibition on tyrosinase was studied, using enzymological kinetic method. The results showed that puerarin was a noncompetitive inhibitor proved by its Lineweaver-Burk plots.Puerarin was applied in cosmetic. The results confirmed that puerarin has skin-lightening effect after 3 months of trial by 15 people.The antimicrobial effect of radix puerariae extract and puerarin were studied. Results showed that radix puerariae extract has better antimicrobial effect than puerarin.
Currently, there are two methods to synthesize physterol esters: chemical synthesis and enzymic synthesis. But the enzymic synthesis could not be developed into the large scale production in industry due to its high cost. The chemical methods are simple, economical and high in yields. However, they have their own drawbacks such as high temperature and prolonged reaction time. Thus, it is desired to find out catalysis with high efficiency for the synthesis of physterol esters.Cupric dodecyl sulfate as an efficient micelle-catalyst was utilized in the synthesis of phytosterol ester. The synthetic procedure of phytosteryl laurate representative of phytosterol saturated fatty acid ester was studied. The effect of the ratio of FA to phytosterol, amount of catalyst, reaction temperature and reaction time on reaction was investigated. By single factor test and orthogonal design experiment, the optimum parameters of preparation of phytosteryl laurate were obtained. The optimal reaction conditions were as follows: ratio of FA to phytosterol 1.3 : 1, amount of catalyst 1.0 mol % of phytosterol, reaction temperature 120℃, and reaction time 4 hours under N2. And the 92.1% of rate of esterification was acquired.The synthesis of phytosteryl unsaturated fatty acid ester was studied using conjugated linoleic acid. Sadium dodecyl sulfate was sorted out as catalyst under comprehensive consideration of the rate of esterification and the degree of oxidization evaluate by p-anisidine value of the system. Similarly, the influence of the ratio of CLA to phytosterol, amount of catalyst, reaction temperature and reaction time on reaction was investigated. The optimal conditions were: the ratio of CLA to phytosterol 1.2 : 1, amount of catalyst 2.0 mol %, temperature 110℃, and time 3 hours under N2, and the rate of esterification was 77.1% in optimum conditions.Silica gel column was applied to separate phytosteryl esters. And cyclohexane: diethyl ether （19:1, v/v） was used as mobile phase. The flow rate was 25 mL/h. The separated product was identified as phytosteryl ester by TLC, HPLC, IR and GC – MS.The solubility of phytosterol, phytosteryl laurate and phytosteryl palmitate in various solvents were determined by gravimetric analysis. Their curves were plotted. And the separation and purification methods of physterly ester from the reaction mixture were studied.The reaction kinetics ofβ-sitosterol with lauric acid using SDS-HCl as a catalyst were studied. The reaction met the second 2 order reaction. And the activitation energy of the reaction was about 88.03 kJ/mol. And kinetic constants at different temperatures were calculated. The experimental results show that rate equation of esterification forβ-sitosterol with lauric acid could be expressed as r = 1.1595×1012 exp[88.03/（RT）]cAcB.The solubility of phytosterol, phytosteryl laurate, phytosteryl palmitate, phytosteryl oleate, and phytosteryl conjugated linoleate in fish oil at 50℃were 1.0 %, 8.7 %, 4.9 %, 42.0 % and 46.2 %, respectively.The antioxidation activity of phytosteryl esters in fish oil at 50℃was studied by the determination of POV of the fish oil system. Their optimum concentration was 0.05‰. Under this condition the order of antioxidation ability from high to low was: phytosterly laurate, phytosteryl oleate, phytosteryl conjugated linoleate and phytosterly palmitate. And there were synergy effects on antioxidant ability between physteryl ester and Vitamin E.
Glycerol Monocaprylate is a metabolism product of the fat which is one of the three major nutritions, it is a high secure material with no adverse accumulation and reaction in persons’body. Glycerol Monocaprylate can be used to improve the food quality as a surfactant and to extend the food storage period as a preservative. In this paper, Glycerol Monocaprylate was synthesized by glycerol and octanoic acid in a solvent free system.In all enzymes, the immobilized lipase-Novo435 was found the most effective for esterification. The optimum conditions of synthesis were obtained through single factor test and response surface method successively. When the ratio of glycerol to octanoic acid 1:1, the lipase dosage 0.88% (W/W) based on the reactant was employed in the reacting system, the conversion rate of octanoic acid could reach 93.53% at 66.8℃for 11.5h reaction. The content of glycerol monocaprylate was about 47.69% in the reacting production .There were monoester, diester and triester in the product when analysed by LC-MS. The analysis condition for the Thin Layer Chromatography (TLC) was confirmed, chloroform: acetone: formyl (96:4:0.5) was used as the mobile phase, and the product was detected by iodine steam. The retention factor (Rf) of the monoester, diester and triester was 0.15, 0.5 and 0.65, 0.94, respectively. Then, the conditions of HPLC was applied to the Lichrospher C18 column chromatography for separating and purifying the Glycerol Monocaprylate, methanol: water (60%, 70%, 80%, 90%) was used as mobile phase, every mobile phase volume was 3 times of the C18 column volume. The LC-MS and IR were used to analyze the production, the result showed that the content of Glycerol Monocaprylate was more than 90%.The surfactant properties of Glycerol Monocaprylate were also studied, the HLB value was 4.7, the CMC was 0.8g/L, and the surface tension was 28.4mN/m which the mass concentration was 0.3%. The emulsion activity of Glycerol Monocaprylate was good and had excellent emulsion stability when the concentration was more than the CMC. The foam activity of Glycerol Monocaprylate was not good and had no foam stability.The results of antimicrobial activity of Glycerol Monocaprylate showed that its minimum inhibition concentration was 0.8g/L for Eschericheae coli, 0.7g/L for Staphylococcus aureus, 0.6g/L for Saccharomyces cerivisiae, 0.8g/L for Saccharomyces cerevlsiae, 0.8g/L for Aspergillus niger and 0.9g/L for Penicillium citrinum. It suggested that Glycerol Monocaprylate had a wide antimicrobial spectra. The Glycerol Monocaprylate could effectively prolong adaptive and decrease growth quantity of the Eschericheae coli, Saccharomyces cerivisiae, Aspergillus niger and milk spoilage microorganisms, and the effect was superior than the potassium sorbate which was in commom use. Furthermore it had better heat stability and acid and alkali stability when was used as preservative.
Based on the brewing characteristics of wine, With the brewing characteristics of MuSaiLaiSi in The southern border, Three yeasts are isolated from the fermented strong wood in The southern border, On this basis, and improve the fermentation process, so that the flavor of the products can be improved .The results are as follows.The different temperatures, pH value, culture media and concentration of alchol were choosed as the enrichments. Twenty-eight yeasts had been isolated from the brewing characteristics of MuSaiLaiSi in The southern border, The fermentation capacity, tolerance to SO2 and alcohol were used as selective conditions to select the yeast. During the selection, the sensory remark was emphasized, According to the above requirement, three stains of yeast were selected. The three yeasts were identified as Saccharomyces cerevisiae of Saccharomyces, and No. 2, No. 3 and on the 8th. Since all three of a kind of yeast, Therefore, selected on the 2nd of biological characteristics to do research.The biological properties of selected yeasts were analyzed in the paper,By comparison, The optimum growth temperatures of 2 was detemined as 36℃,The optimum pH values of 2 was 4.5. Through analysis of different factors on the growth curve we can see that temperature had been more influential on the growth curve and the fementation curve than the any other factors. Different fungal culture temperature of the final has been significantly affected, and the final volume of the initial inoculation of Mycology basic implications, and the optimum fermentation temperatures of it were all 30℃. the optimum fermentation pH values of 2 was 4.5. Similarly, In relation to pH value and addition of yeast, the influence of Fermentation of the temperature was more obvious.Selection of Table Grape, With the 2nd fermentation bacteria, Through sensory analysis to determine the best fermentation conditions, the temperature of fermentation was 25℃, The addition of yeast was 1% , primitive pH value ,4.0, the primitive sugar content of 150 g/L , Click conditions under fermentation brewing wine, liquor clarify wines soft and elegant, the better to maintain the original grape-fruit.
Study on Technique of Extraction, Separation and Purification of Polysaccharide in Tricholoma Mongolicum Imai
Tricholoma mongolicum Imai is a kind of valuable and nutritive edible fungus which is rich in polysaccharide and protein.Although it’s good to human’s health,the study on the bioactive substance of fruiting body was rarely reported.The Polysaccharide is one of the most substances in Tricholoma mongolicum Imai,and it is of important research value.In this dissertation,the extraction,separation and purification of polysaccharide in Tricholoma mongolicum Imai were systematic studied,and prepared the polysaccharide flour with high purity.This dissertation may provide a theoretical basis for the large scale industrial production and it also can lay a scientific basis for the exploitation of Tricholoma mongolicum Imai.The results are mainly as follow:1.The raw materials were defatted by Supercritical CO2 extraction technology first,and the optimum conditions were determined through orthogonal test:extraction pressure was 30MPa,extraction temperature was 35℃,extraction time was 160min,CO2 flow was 30L/h。The supercritical CO2 extraction technology plays a good auxiliary role to the extraction of polysaccharide because it can remove fat,coloring material and abnormal flavour.2.The optimum conditions of extraction were determined through orthogonal test.The optimum conditions of hot water extracting were as follow:solid-liquid ratio was 1:40,extraction temperature was 90℃,extraction time was 3h,material grade size was 0.147mm.The optimum conditions of microwave extracting were as follow:microwave power was 300W,microwave processing time was 4min,solid-liquid ratio was 1:30,material grade size was 0.147mm.By comparing,the extraction rate was better than microwave extraction,but it waste time and energy.3.Studied on the volume of ethanol and the time of precipitating by ethanol.and the optimum conditions was determined as follow:precipitate with 3 times 95%ethanol for 6h.After precipitating and drying,we can get 21.34g crude polysaccharide per100g raw materials.4.The protein in polysaccharide was removed by Sevag method,trichloroacetic acid（TCA） method and anion exchange resin chromatography method,then the optimum conditions of each method was determined.The remove rate of protein by three method as follow:52.05%、41.29%and 56.25%,and the loss rate of polysaccharide by three method as follow:14.68%、24.67%and 14.91%.By comparing,the anion exchange resin chromatography method was the best mothod.5.Two gel column chromatography include Sephadex G-100 and Sephadex G-75 were used to separate and purify the polysaccharide after removing protein.Finally finding that there were two composition at least in polysaccharide,the ratio of high molecular composition was 16.77%,the ratio of low molecular composition which composed by three sections was 63.92%.The ratio of these three sections in the low composition was 28.35%、7.87%and 23.96%.Every composition can be gained in different conditions.