Process development of continuous glycerolysis in an immobilized enzyme-packed reactor for industrial monoacylglycerol production

Continuous and easily operated glycerolysis was studied in different lipase-packed columns to evaluate the most potential process set-ups for industrial monoacylglycerol (MAG) production. Practical design-related issues such as enzyme-filling degree, required reaction time, mass transfer investigations, and capacity and stability of the enzyme were evaluated. A commercially available immobilized Candida antarctica lipase B was used to catalyze the glycerolysis reaction between glycerol and sunflower oil dissolved in a binary tert-butanol: tert-pentanol medium. Considering easy handling of the enzyme and measured expansion when wetted with a reaction mixture, a filling degree of 52 vol % dry enzymes particles per column volume seemed appropriate. Twenty minutes was required to reach equilibrium conditions with a MAG content of 50-55 wt %. Only insignificant indications of mass transfer limitations were observed. Hence, the commercial lipase seemed adequate to use in its available particle size distribution ranging from 300 to 900 microm. A column length-to-diameter ratio of less than 25 did not interfere with the transfer of the fluid mixture through the column. Under the tested conditions, the enzyme could be active for approximately 92 days before enzyme renewal was needed. This corresponds to a very high enzyme capacity with approximately 2000 L pure MAG produced per kg enzyme.     

Enzymatic production of monoacylglycerols containing polyunsaturated fatty acids through an efficient glycerolysis system

The aim of the study was to develop an efficient glycerolysis system for the enzymatic production of monoacylglycerols (MAGs) containing polyunsaturated fatty acids. Glycerolysis has been widely applied in industry for the chemical production of food MAGs under high temperature. The enzymatic glycerolysis system at 40-70 degrees C is unfortunately a multiphase system, which leads to the lower reaction efficiency. A tert-butyl alcohol system was developed after careful evaluation and more than 20-fold of the reaction efficiency from this system was obtained compared to the solvent-free system. Novozym 435 was employed as a catalyst in the glycerolysis from the screening. In the batch reaction system with tert-butyl alcohol, temperature higher than 40 degrees C was favored. The glycerol/oil ratio was best in the study with 4.5 while the solvent weight ratio from 1 to 3 had little effect. In general, 60-70% yield can be obtained at 2 h in the stirred tank reactor. The continuous glycerolysis was conducted in a packed bed reactor. MAG yield up to 70% was reached at 30-40 min residence time. The continuous glycerolysis was more sensitive to the amount of tert-butyl alcohol, and in the weight ratio to oil more than 2 was favored. The continuous process was optimized with the assistance of response surface methodology. Optimal conditions for the packed bed reactor after all considerations were recommended as glycerol/oil 4:1 (mol/mol), temperature 40 degrees C, and residence time 45 min. The operation stability study showed that there was no slight reduction of reaction performance at more than 30 days, implying a high feasibility in practical applications.     

Process optimization using response surface design and pilot plant production of dietary diacylglycerols by lipase-catalyzed glycerolysis

Two approaches to shift the acylglycerol equilibrium were tested as follows: addition of monoacylglycerols and lowering of the temperature. None of these approaches were able to shift the equilibrium toward higher diacylglycerol (DAG) contents. The glycerolysis reaction was optimized with five factors using response surface methodology. Evaluation of the resulting model enabled the determination of optimal reaction conditions for glycerolysis aiming at high DAG yield. However, verification of the model showed that the model was unable to take the molecular equilibrium into account but it provided good insight in how process settings can be chosen to, for example, minimize production costs. Optimal conditions were found to be the following: no extra water, low content of glycerol (molar ratio of 2), temperature of 60-65 degrees C, 4-5 h reaction time, and only 5 wt % lipases. Up scaling of the glycerolysis process was performed and revealed that scale-up to a 20 kg production in a pilot plant batch reactor was possible with a similar DAG yield (60 wt %) as in lab scale. Purification of DAG oil using batch deodorization and short path distillation yielded 93 wt % pure DAG oil.     

Spectrophotometric analysis of binary mixtures of sulfonamides.

Different methods for analyzing binary mixtures by using 2 wavelengths are reviewed. The absorbance ratio calculated at 2 wavelengths, not including the isoabsorptive point, was a quadratic function of relative concentration. The curve-fitting process using orthogonal polynomials was applied to obtain the quadratic equation. An absorbance ratio can be used as a rapid purity index for sulfacetamide sodium in the presence of sulfanilamide. Sulfacetamide sodium has been determined in eye drop preparations.     

酶法制备玉米缓慢消化淀粉的工艺条件优化

缓慢消化淀粉是一种新型功能性淀粉,具有多种生理功能。该文以蜡质玉米淀粉为原料,采用酶控制降解技术及湿热-冷却技术制备缓慢消化淀粉,确定其优化工艺条件。研究结果表明：普鲁兰酶用量9 U/g,酶作用时间7 h,淀粉乳浓度12.5%,热处理时间50 min,热处理温度110℃,储藏温度1℃,储藏时间2 d,烘干温度60℃,并在此条件下进行3次重复试验,缓慢消化淀粉得率为27.33%。     

Comparison of different extraction solvent mixtures for characterization of phenolic compounds in strawberries

Eight different solvent mixtures containing acetone or methanol pure or combined with an acid (acetic, formic, hydrochloric) were tested for their efficiency for extraction of phenolic compounds from strawberries belonging to five groups of polyphenols: anthocyanins, flavonols, flavan-3-ols, hydroxycinnamic acid derivatives and conjugated forms of ellagic acid. Twenty-eight compounds from these five groups have been detected and quantified using HPLC-DAD-ESI-MS(n). The yield of each phenolic compound and group was evaluated with regard to the extraction solvent composition. Acetone containing extraction mixtures were superior to the ones containing methanol for extraction yield of total phenolic compounds, which was especially pronounced for the groups of flavan-3-ols and conjugated forms of ellagic acid. The mixture acetone/acetic acid (99:1, v/v) gave the best results for the qualitative and quantitative assay of the polyphenols present in strawberries since all 28 compounds were detected only in these extracts in quantities higher or comparable to the other extraction solvents tested.     

Modifications of stearidonic acid soybean oil by enzymatic acidolysis for the production of human milk fat analogues

Structured lipids (SLs) from stearidonic acid (SDA) soybean oil pre-enriched with palmitic acid (PA) at the sn-2 position with Novozym 435 (NSL) or Lipozyme TL IM (LSL) from previous research were further enriched with γ-linolenic acid (GLA) or docosahexaenoic acid (DHA). Small-scale acidolysis reactions with Lipozyme TL IM were performed to determine the optimal reaction conditions as 1:1 substrate mole ratio of NSL or LSL to free DHA at 65 °C for 24 h and a 1:0.5 substrate mole ratio of NSL or LSL to free GLA at 65 °C for 12 h. Optimized SL products were scaled up in a 1 L stir-batch reactor, and the resulting SLs of NSL:DHA (NDHA), LSL:DHA (LDHA), NSL:GLA (NGLA), and LSL:GLA (LGLA) were chemically and physically characterized. The SLs contained >54% PA at the sn-2 position with GLA >8% for the GLA SLs and DHA >10% for the DHA SLs. The oxidative stabilities of the SLs were increased by the addition of 200 ppm TBHQ, with NGLA being more stable due to higher tocopherol content than the other SLs. The melting and crystallization profiles did not differ between the DHA SLs or the GLA SLs. The triacylglycerol (TAG) species were similar for the GLA SLs but differed between the DHA SLs, with tripalmitin being the major TAG species in all SLs.     

A continuous method for enzymatic assay of sucrose synthase in the synthetic direction.

An appropriate method was developed for the continuous assay of sucrose synthase (SS) (EC 2.4.1.13) by spectrophotometry. The uridine 5'-diphosphate derived from sucrose synthesis was stoichiometrically coupled to oxidation of beta-nicotinamide adenine dinucleotide by the enzymes nucleoside-5'-diphosphate kinase (NDPK), pyruvate kinase, and lactate dehydrogenase. Utilization of crude extracts led to a complete masking of SS assay by adenylate kinase, adenosine 5'-triphosphatase (ATPase), and phosphoenolpyruvate phosphatase found in the crude extracts. These interfering enzymes were mostly removed from the crude extracts by using a combination of gel filtration, centrifugation through a selectively permeable membrane (Biomax-100 Ultrafree centrifugal device), and inhibition by the addition of K(2)HPO(4) to the assay buffer. Sensitivity of the SS assay was significantly increased by the inclusion of NDPK and ATP, which are essential to the reaction in the coupling system.     

Optimization of solid-state enzymatic hydrolysis of chestnut using mixtures of alpha-amylase and glucoamylase

Solid-state hydrolysis of starch present in chestnut was assayed in a single step with a mixture of a thermostable alpha-amylase and glucoamylase at three temperatures: 17 and 30 degrees C, for simultaneous hydrolysis and ethanol fermentation, and 70 degrees C, the optimal temperature for these enzymes. Total hydrolysis was only reached at the highest temperature, leading to a more concentrated hydrolysate than in submerged hydrolysis. Mass transfer limitations and starch retrogradation appear as the main causes for the incomplete hydrolysis of chestnut starch in solid-state operation at 17 and 30 degrees C. Even accepting that this limitation causes a 15% reduction of the yield of the hydrolysis with respect to the submerged process or the solid process at high temperature, solid-state hydrolysis at low temperatures seems to be adequate for simultaneous solid-state hydrolysis and fermentation processes.     

Evaluation of a selective medium for hydrogen sulfide production by salmonella.

An evaluation was made of Padron-Dock-Stader sulfide (PDS) agar for the rapid detection of Salmonella and Arizona microorganisms in foods. Analysts from 9 Food and Drug Administration District laboratories determined the reaction of 638 Salmonella cultures, 30 Arizona cultures, and 1754 non-Salmonella and non-Arizona cultures isolated from 157 food samples in PDS agar. The degree of positive reactions of these cultures in this agar was scored on a scale of "-" to "+++". The highest percentage of Salmonella isolates (64.1%) was in the category of +++ reactions. Progressively lower percentages of Salmonella isolates occurred in the more negative reaction categories. The highest percentage of Arizona isolates (66.7%) occurred in the intermediate ++ category. The majority of non-Salmonella and non-Arizona isolates occurred in the minus (41.0%) and plus or minus (44.8%) categories. Advantages of using the PDS agar are that it is simple to use, requires no specialized training or equipment, and alerts analysts to those Salmonella cultures which produce atypical reactions in triple sugar iron agar particularly the lactosepositive Salmonella. However, in this study the occurrence of false-negative reactions ranged from 4.3 to 13.9% for Salmonella and from 0 to 6.7% for Arizona, according to the criteria used to interpret the reactions in PDS agar.     

猪粪中温半干法连续厌氧发酵产气性能

为改善猪粪在连续型沼气工程中的容积产气效率和降低其进出料过程的热损失,该研究拟采用高浓度和小体积喂料方式进行,将新鲜猪粪分别稀释成总固体质量分数(total solid,TS)为10%、12%和14%3个水平,通过逐级缩短水力停留时间(hydraulic retention time,HRT)(HRT:25 d→20 d→18.5 d)的方式来改变各组反应器的负荷。试验结果表明,当HRT:25 d时,各组平均日产气量均表现最高,约为460 m L/g,此阶段可获得85%以上的沼气转化效率;当HRT:20d时,各组均获得最大容积产气率,最高达到2.29 L/(L·d)(TS:14%);当HRT下降至18.5 d时,各组产气量均呈下降趋势,表明有机负荷已超出反应器的最大转化能力。通过综合原料产气转化效率和容积产气效率2个指标,发现进料TS为14%和HRT为25 d为较优组合条件。该研究可为在实际沼气工程中如何协调进料浓度和HRT的关系提供参考。     

玉米秸秆酶解上清液厌氧发酵产氢工艺优化

该文主要以粒度小于0.088 mm秸秆粉的酶解上清液为底物与热预处理后的活性污泥进行厌氧发酵产氢试验,以累积产氢量为考察指标,基于响应面Box-Behnken模型研究不同影响因素对玉米秸秆酶解上清液厌氧发酵产氢的影响,对玉米秸秆酶解上清液厌氧发酵产氢工艺进行优化。结果表明:温度、初始p H值和还原糖浓度三因素中,温度和还原糖浓度对玉米秸秆酶解上清液厌氧发酵产氢的影响最大。采用Box-Behnken模型获得的最佳产氢条件为:温度38.32℃,初始p H值4.93,还原糖浓度20.70 mg/m L,最大产氢量685.59 m L,此时最大产氢率为57.13 m L/g(玉米秸秆)。通过试验验证,实际最大产氢量为659.24 m L,产氢率为54.94 m L/g(玉米秸秆),与模型预测值相比,相对误差为3.84%,说明该模型具有较好的拟合性。该优化工艺可为后期连续流状态下的生物制氢系统提供参考。     

利用菜籽油酶法生产生物柴油的初步研究

生物柴油作为可再生能源，对环境友好，引起了世界范围内的广泛关注。该文利用固定化脂肪酶－Novo435，在无有机溶剂存在的情况下，催化菜籽油与甲醇酯交换反应制取生物柴油，对影响酯交换反应过程的因素：甲醇与油脂的摩尔比、反应温度、反应时间、脂肪酶用量、转速、水分等进行深入研究，得到了菜籽油间歇酯交换反应的适宜工艺条件：转速200 r/min、醇油摩尔比1.5∶1、反应温度50℃、酶用量10%(与油脂的质量比)、反应10 h后菜籽油的酯交换率达到47％。水分的存在不利于固定化酶在无有机溶剂系统下催化菜籽油的酯交换反应，使酯交换率降低到30％。反应所需理论甲醇量分两次加入，反应26 h后，油脂的酯交换率达到80％。     

Acidification of mixtures of granite powder and compost for reuse in plant production

We evaluated the utilization of granite powder—a residue of stone cutting and polishing resulting from its preparation for construction and ornamental use—in the preparation of nursery potting mixtures, within a general objective of waste recycling and reuse. Granite powder was blended with two composts of different origins and properties: one elaborated from the organic fraction of municipal solid waste (MSW), and another from pine bark. Since all materials presented pH values over 8, which are excessive for growing most vegetal species, the use of iron sulphate to acidify them has been assessed. Three doses of iron sulphate (14, 69, and 97 g kg^?1, equivalent to 0.1, 0.5, and 0.7 meq H⁺ g^?1) were added to the granitic powder and mixtures with 25 or 75% (v/v) of each compost, which were incubated in the laboratory during 30 days. Doses of iron sulphate above 0.1 meq H⁺ g^?1 resulted in excessive electrical conductivity (>2 dS m^?1) in all the samples, and too low pH values (<5) in most of them. The productivity of granite powder/compost mixtures acidified with 0.1 meq H⁺ g^?1 was tested in a greenhouse experiment with ryegrass, where it was observed that plant productivity increased with respect to a control commercial substrate based on peat, in particular when the MSW compost was employed. On the basis of the physicochemical properties of the mixtures and the results of the greenhouse experiment, the use of granite powder mixed with 75% of MSW compost (v/v), acidified with 14 g kg^?1 of iron sulphate (0.1 meq H⁺ g^?1) is recommended.     

Beet sugar syrup and molasses as low-cost feedstock for the enzymatic production of fructo-oligosaccharides

Sugar syrup and molasses from beet processing containing 620 and 570 mg/mL sucrose, respectively, were assayed as low-cost and available substrates for the enzymatic synthesis of fructo-oligosaccharides (FOSs). A commercial pectinase (Pectinex Ultra SP-L, from Aspergillus aculeatus) characterized by the presence of a transfructosylating activity was used as a biocatalyst. The FOS production increased when lowering the initial pH value of syrup (7.5) and molasses (8.9) to 5.5. Sugar syrup and molasses were diluted in order to reduce substrate viscosity; interestingly, the percentage of FOS with regards to total sugars remained almost constant, which indicated a high transferase-to-hydrolase ratio for this enzyme. Kinetics of FOS production was analyzed. Using approximately 10 U transfructosylating activity per g sucrose, the FOS concentration reached a maximum of 388 mg/mL after 30 h using syrup and 235 mg/mL in 65 h with molasses. These values corresponded to approximately 56 and 49% (w/w), respectively, of the total amount of carbohydrates in the mixture. The enzyme was also covalently immobilized on an epoxy-activated polymethacrylate-based polymer (Sepabeads EC-EP5). We found that immobilized Pectinex Ultra SP-L can be efficiently applied to the synthesis of FOS using syrup and molasses as substrates.     

中性蛋白酶水解鸡骨泥制备短肽工艺优化

为了探究酶法制备鸡骨泥短肽的最佳工艺,该文研究了以中性蛋白酶酶解鸡骨泥时各因素对短肽得率和羟自由基清除率的影响,以及鸡骨泥肽清除超氧阴离子自由基和1,1-二苯基-2-三硝基苯肼自由基(2,2-diphenyl-1-picrylhydrazyl)的能力。试验探究了酶解过程中不同底物浓度、酶浓度、反应温度和反应时间对短肽得率和羟自由基清除率的影响,采用响应曲面优化得到了以短肽得率为目标的中性蛋白酶酶解鸡骨泥的最优工艺,当反应液p H值为7.2,反应温度42℃,反应时间2 h,底物质量分数5%,酶添加量200 mg/g,该条件下制备鸡骨泥肽,测得其短肽得率为56.16%,羟自由基清除率为54.12%。采用该酶解工艺制备鸡骨泥肽,测得其对超氧阴离子自由基最高清除率为56.01%,对DPPH自由基最高清除率为81.57%,说明鸡骨泥肽具有一定的抗氧化活性,研究结果为酶法制备鸡骨泥肽提供参考。     

Evidence for gluconic acid production by Enterobacter intermedium as an efficient strategy to avoid protozoan grazing

The effect of pure gluconic acid and of gluconic-acid-producing bacteria on the activity of three protozoan species, Colpoda steinii (a ciliate), Vahlkampfia sp. (an amoeba) and Neobodo designis (a flagellate), was determined in vitro and in soil microcosms. Pure gluconic acid was shown to mediate disappearance of active cells, due to encystment and/or death of protozoa, at 0.15 mM in saline medium. Similarly, the presence of gluconic acid inhibited excystment of the three protozoa tested. Enterobacter intermedium 60-2G (Wt), a gluconic acid-producing rhizobacterium, elicited the same effects on protozoa when co-cultured in the presence of 5 g L⁻¹ glucose. However, the effect was not observed when glucose was omitted from the medium. Similarly, a pqqA isogenic mutant strain, unable to produce gluconic acid from glucose, exhibited a reduced effect on protozoan activity. Rhizosphere-microcosm studies performed with wheat (Triticum aestivum L.) confirmed the reduced ability of the pqqA mutant to limit protozoa reproduction compared to the Wt strain. Since the sodium salt of gluconic acid did not cause any significant stress to protozoa and considering that addition of 50 mM Tris-Cl (pH 7.2) abolished the deleterious effect of gluconic acid, acidification of the medium appeared as the key factor that induced encystment/death of protozoa. We propose that production and excretion of gluconic acid should be considered an efficient mechanism evolved by bacteria to escape, tolerate or defend themselves against protozoan grazing in rhizosphere environments.     

批式与连续两相发酵的果蔬废弃物厌氧产气性能

该研究采用批式试验与连续发酵试验探讨了苹果与蔬菜废物的厌氧发酵产气性能,结果表明:38℃下苹果与蔬菜废物混合进行厌氧发酵发生了协同促进作用,显著提高了混合原料的产气能力。在苹果废物与蔬菜废物的混合比例F:V=4:1时,混合原料的产沼气潜力达到了914.6 L/kg。利用修正的Gompertz模型能够很好的模拟果蔬废物批式厌氧发酵产气的累积产气过程,拟合结果的R2在0.983~0.999之间。果蔬废物连续进出料产气过程研究结果表明在HRT=24 d、35℃中温发酵条件下,果蔬废物为原料的一体化两相(combined two phase,CTP)反应器有机负荷OLR可达2.5 g/(L·d)时,此时CTP反应器的产气量最高可达12.9 L/d,反应器内物料比产气量为390 L/kg。这表明CTP反应器可以作为果蔬厌氧发酵产气的反应器。与全混式反应器相比,CTP反应器结构简单、成本低,但对果蔬废物的发酵效率低于全混式反应器。     

Evaluation of different zinc sources for lowland rice production

 ZnSO₄, Zn-enriched farmyard manure (Zn-FYM), Zn-tetraammonia complex sorbed on FYM [Zn(NH₃)₄-FYM] and Zn-ethylenediaminetetraacetate (Zn-EDTA) were compared as Zn sources for rice production under lowland conditions. The amount of Zn supplied by Zn-EDTA was one-tenth of that supplied by the other Zn sources. Zn application to a Zn-deficient soil corrected the visual symptoms of Zn deficiency and significantly increased the total biomass, grain yields and the harvest index of rice, as well as the Zn concentration in the grain and the uptake of Zn by the straw and the grains. Even with lower rates of application (0.25 and 0.5 mg Zn kg^–1 soil), Zn-EDTA treatments gave comparable values for these parameters, and the highest "Zn-mobilization efficiency" compared to the other Zn sources. The content of diethylenetriaminepentaacetate (DTPA)-extractable Zn in the soil of the different treatments after the harvest of rice was in the order; ZnSO₄=Zn-FYM>Zn(NH₃)₄-FYM=Zn-EDTA. The application of Zn also significantly increased the number of panicles that emerged between 80 to 93 days after transplanting, though the total number of panicles at harvest remained unaffected. The calculated panicle-emergence index had a positive correlation with the grain yield of rice. The Z_n-EDTA treatment, inspite of supplying the lowest amount of Zn, as well as leading to the lowest rate of Zn uptake, produced the highest yields. Therefore, we concluded Zn-EDTA to be the most efficient source of Zn for lowland rice production. Received: 20 October 1998