Optimization of simultaneous saccharification and fermentation for the production of ethanol from lignocellulosic biomass

Simultaneous saccharification and fermentation (SSF) of alkaline hydrogen peroxide pretreated Antigonum leptopus (Linn) leaves to ethanol was optimized using cellulase from Trichoderma reesei QM-9414 (Celluclast from Novo) and Saccharomyces cerevisiae NRRL-Y-132 cells. Response surface methodology (RSM) and a three-level four-variable design were employed to evaluate the effects of SSF process variables such as cellulase concentration (20-100 FPU/g of substrate), substrate concentration (5-15% w/v), incubation time (24-72 h), and temperature (35-45 degrees C) on ethanol production efficiency. Cellulase and substrate concentrations were found to be the most significant variables. The optimum conditions arrived at are as follows: cellulase = 100 FPU/g of substrate, substrate = 15% (w/v), incubation time = 57.2 h, and temperature = 38.5 degrees C. At these conditions, the predicted ethanol yield was 3.02% (w/v) and the actual experimental value was 3.0% (w/v).     

Continuous hot pressurized solvent extraction of 1,1-diphenyl-2-picrylhydrazyl free radical scavenging compounds from Taiwan yams (Dioscorea alata)

This study investigates a semicontinuous hot pressurized fluid extraction process and the scavenging activity on the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical of the extract from Taiwan yams (Dioscorea alata). Liquid-liquid extractions were preliminarily employed to generate six fractions, initially extracted by ethanol. Then, the aqueous solution of dried crude ethanol extract was sequentially fractionated by hexane, chloroform, ethyl acetate, and n-butanol. The EC50 value was defined as the UV absorption of DPPH concentrations sufficiently decreased to 50% of the original value. It was found that all peel portions have a better effect on scavenging of the DPPH free radical than meat portions, especially for the ethyl acetate partition of the peel portion of Tainung #2 yam. Its EC50 value (14.5 microg mL(-1)) was even lower than that of ascorbic acid (21.4 microg mL(-1)). Furthermore, semicontinuous hot pressurized ethanol was superior to hot pressurized water in extracting the compound scavenging the DPPH radical from the Purpurea-Roxb peel. The recovery of four unknown compounds corresponded to the scavenging ratio of DPPH free radical in the hot pressurized ethanol extract. Finally, three-level and four-factor experimental design revealed that ethanol ratio and temperature were the most effective factors in order. Conditions of 80% of aqueous ethanol, 20.0 kg/kg solid ratio, 180 psig (1.342 MPa), and 100 degrees C were preferred to extract those antioxidants from the yam peel.     

Development of a rapid method for the sequential extraction and subsequent quantification of fatty acids and sugars from avocado mesocarp tissue

Methods devised for oil extraction from avocado (Persea americana Mill.) mesocarp (e.g., Soxhlet) are usually lengthy and require operation at high temperature. Moreover, methods for extracting sugars from avocado tissue (e.g., 80% ethanol, v/v) do not allow for lipids to be easily measured from the same sample. This study describes a new simple method that enabled sequential extraction and subsequent quantification of both fatty acids and sugars from the same avocado mesocarp tissue sample. Freeze-dried mesocarp samples of avocado cv. Hass fruit of different ripening stages were extracted by homogenization with hexane and the oil extracts quantified for fatty acid composition by GC. The resulting filter residues were readily usable for sugar extraction with methanol (62.5%, v/v). For comparison, oil was also extracted using the standard Soxhlet technique and the resulting thimble residue extracted for sugars as before. An additional experiment was carried out whereby filter residues were also extracted using ethanol. Average oil yield using the Soxhlet technique was significantly (P < 0.05) higher than that obtained by homogenization with hexane, although the difference remained very slight, and fatty acid profiles of the oil extracts following both methods were very similar. Oil recovery improved with increasing ripeness of the fruit with minor differences observed in the fatty acid composition during postharvest ripening. After lipid removal, methanolic extraction was superior in recovering sucrose and perseitol as compared to 80% ethanol (v/v), whereas mannoheptulose recovery was not affected by solvent used. The method presented has the benefits of shorter extraction time, lower extraction temperature, and reduced amount of solvent and can be used for sequential extraction of fatty acids and sugars from the same sample.     

Spectrophotometric determination of cyanoglucosides in cassava

A new method is reported for determination of cyanoglucosides in cassava. The method is simple, rapid, and sensitive. Ten g cassava tuber is homogenized with warm (65-70 degrees C) 80% ethanol (1 + 6, w/v) to extract cyanoglucosides (CNG). The ethanol is evaporated, and an aliquot of the extract (0.1-0.2 mL) is incubated with added linamarase in pH 6.0 phosphate buffer for 15 min at 30 degrees C. The reaction is stopped by adding 0.2 N sodium hydroxide, the solution is neutralized, and cyanide is estimated by adding chloramine T and barbituric acid-pyridine reagent and measuring the absorbance at 570 nm. Complete CNG extraction and rapid inactivation of endogenous linamarase is possible with 80% ethanol. There is no interference from extractives in the linamarase reaction or in the estimation of cyanide. Recovery of added linamarin (as cyanide) is 98% by this assay. The minimum detection limit of cyanide in the assay is 0.1 micrograms/mL.     

石榴皮提取物的大孔树脂纯化及其抗氧化性能

为开发石榴皮资源提供技术支持,从D101、AB-8和DA-2013种大孔吸附树脂中筛选出D101树脂,进一步研究D101对石榴皮提取物中多酚和总黄酮的纯化工艺。所得最佳纯化工艺为:上柱液浓度5mg/mL,pH5,上柱液体积180mL,上柱液流速80mL/h,以60%乙醇溶液为洗脱液,洗脱流速80mL/h,洗脱液体积为100mL。在此条件下,多酚和总黄酮的含量由30.43%和17.12%分别提高到53.55%和35.71%。同时对纯化前后的抗氧化活性进行了比较研究。结果表明,石榴皮提取物经纯化后清除二苯基三硝基苯肼自由基(DPPH·)和抗油脂过氧化能力都强于粗提物。     

Optimization of Fermentation Temperature and Mash Specific Gravity for Fuel Alcohol Production

The effects of fermentation temperature and dissolved solids concentration adjusted by changing mashing water-to-grain ratios on wheat fermentation efficiencies, fermentation times, final ethanol concentrations, and ethanol production rates were studied by using response surface methodology. Final ethanol concentrations in fermentors depended primarily on mash specific gravities. Predictably, increases in fermentation temperatures dramatically reduced fermentation times and thereby shortened fermentation cycles. The highest ethanol production rates were achieved with a high fermentation temperature of 30°C and a low water-to-grain ratio of 2.0. At these settings, an ethanol concentration of 13.6% (v/v) was attained with a fermentation time of 54 hr and an ethanol production rate of 2.45 mL of ethanol/L/hr. Optimization of operating conditions suggested in the current study will provide existing fuel alcohol plants with increased productivity without alteration of plant equipment or process flow.     

Use of Phytases in Ethanol Production from E‐Mill Corn Processing

Effects of phytase addition, germ, and pericarp fiber recovery were evaluated for the E‐Mill dry grind corn process. In the E‐Mill process, corn was soaked in water followed by incubation with starch hydrolyzing enzymes. For each phytase treatment, an additional phytase incubation step was performed before incubation with starch hydrolyzing enzymes. Germ and pericarp fiber were recovered after incubation with starch hydrolyzing enzymes. Preliminary studies on phytase addition resulted in germ with higher oil (40.9%), protein (20.0%), and lower residual starch (12.2%) contents compared to oil (39.1%), protein (19.2%), and starch (18.1%) in germ from the E‐Mill process without phytase addition. Phytase treatment resulted in lower residual starch contents in pericarp fiber (19.9%) compared to pericarp fiber without phytase addition (27.4%). Results obtained led to further investigation of effects of phytase on final ethanol concentrations, germ, pericarp fiber, and DDGS recovery. Final ethanol concentrations were higher in E‐Mill processing with phytase addition (17.4% v/v) than without addition of phytase (16.6% v/v). Incubation with phytases resulted in germ with 4.3% higher oil and 2.5% lower residual starch content compared to control process. Phytase treatment also resulted in lower residual starch and higher protein contents (6.58 and 36.5%, respectively) in DDGS compared to DDGS without phytase incubations (8.14 and 34.2%, respectively). Phytase incubation in E‐Mill processing may assist in increasing coproduct values as well as lead to increased ethanol concentrations.     

Optimizing Extraction of Zein and Glutelin‐Rich Fraction During Sequential Extraction Processing of Corn

This study was conducted to improve yields and qualities of corn protein co‐products produced by the sequential extraction process (SEP), a process using ethanol to fractionate corn in producing fuel ethanol. A two‐stage extraction protocol was evaluated to recover zein and subsequently recover a glutelin‐rich fraction (GRF). After the simultaneous oil‐extraction and ethanol‐drying step of SEP, zein was extracted from the anhydrous‐ethanol‐defatted, flaked corn by using 70% (v/v) ethanol at 60°C for 1.5 hr in a shaking water bath. Zein was recovered by ultrafiltering and then drying in a vacuum‐oven. Zein yield was 65% of the available zein in the flaked corn. SDS‐PAGE band patterns of the recovered zein closely resembled that of commercial zein. After zein extraction, the GRF was extracted using 45% ethanol and 55% 0.1M NaOH at 55°C for 2 hr. The extract was concentrated by ultrafiltration and then freeze‐dried. GRF yield was ≈65% of the available protein. Freeze‐dried GRF contained 90% crude protein (db), which classified the protein as a protein isolate. As with the protein concentrate from the original SEP, the GRF isolate was highly soluble in water at pH ≥ 7, had good emulsifying and foaming properties, formed stable emulsions, and was heat‐stable.     

Investigation of Australian olive mill waste for recovery of biophenols

Olive mill waste is a potential source for the recovery of phytochemicals with a wide array of biological activities. Phytochemical screening of hexane, methanol, and water extracts revealed a diversity of compounds, perhaps overlooked in previous studies through intensive cleanup procedures. Methanol and water extracts contained large amounts of biophenols, and further testing of polar extraction solvents, including ethyl acetate, ethanol, propanol, acetone, acetonitrile, and water/methanol mixtures, highlighted the latter as the solvent of choice for extraction of the widest array of phenolic compounds. Stabilization of the resulting extract was best achieved by addition of 2% (w/w) sodium metabisulfite. Quantitative data are reported for nine biophenols extracted using 60% (v/v) methanol in water with 2% (w/w) sodium metabisulfite. Six compounds had recoveries of greater than 1 g/kg of freeze-dried waste: hydroxytyrosol glucoside, hydroxytyrosol, tyrosol, verbascoside, and a derivative of oleuropein.     

Comparison of Raw Starch Hydrolyzing Enzyme with Conventional Liquefaction and Saccharification Enzymes in Dry‐Grind Corn Processing

In a conventional dry‐grind corn process, starch is converted into dextrins using liquefaction enzymes at high temperatures (90–120°C) during a liquefaction step. Dextrins are hydrolyzed into sugars using saccharification enzymes during a simultaneous saccharification and fermentation (SSF) step. Recently, a raw starch hydrolyzing enzyme (RSH), Stargen 001, was developed that converts starch into dextrins at low temperatures (<48°C) and hydrolyzes dextrins into sugars during SSF. In this study, a dry‐grind corn process using RSH enzyme was compared with two combinations (DG1 and DG2) of commercial liquefaction and saccharification enzymes. Dry‐grind corn processes for all enzyme treatments were performed at the same process conditions except for the liquefaction step. For RSH and DG1 and DG2 treatments, ethanol concentrations at 72 hr of fermentation were 14.1–14.2% (v/v). All three enzyme treatments resulted in comparable ethanol conversion efficiencies, ethanol yields, and DDGS yields. Sugar profiles for the RSH treatment were different from DG1 and DG2 treatments, especially for glucose. During SSF, the highest glucose concentration for RSH treatment was 7% (w/v), whereas for DG1 and DG2 treatments, glucose concentrations had maximum of 19% (w/v). Glycerol concentrations were 0.5% (w/v) for RSH treatment and 0.8% (w/v) for DG1 and DG2 treatments.     

Characterization of flavonoids and pectins from bergamot (Citrus bergamia Risso) peel, a major byproduct of essential oil extraction

Bergamot peel is an underutilized byproduct of the essential oil and juice-processing industry. As with other Citrus peels, it still contains exploitable components, such as pectins and flavonoids. Commercial glycoside hydrolases, specifically a combination of pectolytic and cellulolytic enzymes, solubilized a high percentage of the material (81.94%). The flavonoid profile of the peel consisted of characteristic Citrus species flavanone rutinosides and neohesperosides derived from naringenin, eriodictyol, and hesperetin. In addition, a number of minor flavanone and flavone glycosides, not found in orange and lemon peels, were identified. The majority of flavonoids were extracted in the two 70% v/v EtOH extractions. Processing this material clearly has economic potential leading to low environmental impact.     

Investigation on Soil Lipid Extraction with Different Organic Solvents

Soil lipid content is an important characteristic of the soil organic matter. One of the commonly used extractants for the determination of soil lipid is benzene/ethanol 1:1(v/v). In this study the toxic solvent mixture benzene/ethanol was substituted with the less harmful one toluene/ethanol 1:2.3 (v/v). Both solvent mixtures give a comparably high recovery of soil lipid and the composition of both lipid fractions are very similar according to their thin layer chromatography (TLC). Further investigations with NMR-spectroscopy confirmed that the extracted lipid fraction consists mainly (7gt;90%) of structure elements of aliphatic hydrocarbons.     

Ethanol-dependent heat-induced dissociation of casein micelles

The dissociation of casein micelles when heated to approximately 65 degrees C in the presence of ethanol [1:1 mixture (v/v) of milk and 65% (w/w) aqueous ethanol] was investigated using L* values and transmission measurements. Mixtures of milk and ethanol became transparent on heating, which suggests dissociation of casein micelles. Results of experiments using confocal laser scanning microscopy, light scattering (static and dynamic), and dialysis to examine the changes of milk during heating in the presence of ethanol supported the assertion that such treatments result in dissociation of casein micelles, as did studies of model beta-casein micellar systems.     

Sensitive and rapid reversed-phase liquid chromatography-fluorescence method for determining bisphenol A diglycidyl ether in aqueous-based food simulants.

A method has been developed for determination of bisphenol A diglycidyl ether (BADGE) in 3 aqueous-based food simulants: water, 15% (v/v) ethanol, and 3% (w/v) acetic acid. BADGE is extracted with C18 cartridges and the extract is concentrated under a stream of nitrogen. BADGE is quantitated by reversed-phase liquid chromatography with fluorescence detection. Relative precision at 200 micrograms/L was 3.4%, the detection limit of the method was 0.1 micrograms/L, and recoveries of spiking concentrations from 1 to 8 micrograms/L were nearly 100%. Relative standard deviations for the method ranged from 3.5 to 5.9%, depending on the identity of the spiked aqueous-based food simulant.     

柚子皮基可食性膜的制备及性能研究

为了促进果蔬加工副产物的高附加值利用,以柚子皮干粉为材料,海藻酸钠为增稠剂,甘油为增塑剂,采用流延法制备了柚子皮基可食性膜,分析柚子皮干粉质量分数、海藻酸钠、甘油质量分数对柚子皮基膜力学性能、阻湿性能和透光率等的影响,并对柚子皮基膜的制备配方进行优化。结果表明,柚子皮基膜的最佳制备配方为:柚子皮干粉质量分数3%、海藻酸钠质量分数0.15%(以柚子皮粉浆料质量为基础,下同)、甘油质量分数0.5%,此配方制备的柚子皮基膜外观平滑、颜色淡黄,具有较好的综合性能,其拉伸强度为17.53±0.35 MPa,断裂伸长率为19.46%±0.43%,水蒸气透过系数为(2.327±0.128)×10^-12g·cm^-1·s^-1·Pa^-1,透光率为63.2%±0.15%。本研究结果为柚子皮基可食性膜在食品包装等领域的应用提供了理论参考。     

Fourier transform infrared spectroscopic analysis of the polymethoxylated flavone content of orange oil residues

Preliminary studies have shown that orange peel polymethoxylated flavones (PMFs) exhibit beneficial biological properties in animals. These properties have increased the demands for these compounds as candidate nutraceuticals and specialty food ingredients. Orange oil residues are a likely commercial source of the PMFs, and a rapid, solvent-free method for the analysis of the PMFs in orange oil residues has been developed based on Fourier transform infrared (FTIR) spectroscopy. The intensities of the FTIR vibrations of the phenyl ring nu(C=C) stretch at 1515 cm(-1) of the PMFs can be used, relative to the intensity of the carbonyl stretch at 1733 cm(-1) of the non-PMF orange oil residue components, to measure PMF content. Excellent correlations for the ratios of the intensities of these vibrations and the total PMF content were observed irrespective of the source, viscosity, and presence of particulate material. The detection limit by this method is approximately 0.1% PMF.     

乙醇水溶液提取玉米胚芽油的工艺优化

为了解决水酶法提取玉米胚芽油生产成本高、提取时间长的缺点,该文采用乙醇水溶液作为提取剂提取玉米胚芽油。通过对粒径、料液比、温度、乙醇体积分数、p H值和时间等条件对油在油相、水相和渣相中分布的研究发现,物料粒径和乙醇体积分数对提高清油得率具有显著(P0.05)的影响,而提取时间对清油得率的影响最小(P0.05)。在单因素试验的基础上通过正交试验,得出乙醇水溶液提取玉米胚芽油的最佳工艺参数为:物料细粉4次(此时粒径为49.18μm)、料液比1∶7 g/m L、温度70℃、乙醇体积分数30%、p H值9.0、提取时间2 h。在该条件下,清油的得率为94.05%±0.32%,水相含油量为3.49%±0.77%,渣相含油量为2.55%±0.82%。分析乙醇水溶液提取的玉米胚芽毛油酸价、过氧化值和含水率等指标发现,该毛油的质量优于国标规定的玉米原油,并且和压榨一级成品油指标接近,只需要经过简单精炼就可以达到食用油要求。研究结果为乙醇水溶液工业化生产玉米胚芽油提供参考。     

Experimental study on the removal of dioxins and coplanar polychlorinated biphenyls (PCBS) from fish oil

Recently, it has been found that fish oils contain a high proportion of contaminants, namely, polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and coplanar polychlorinated biphenyls (cPCBs). In this study, the removal of contaminants from fish oil by supercritical CO2 extraction (SCE) and by using adsorbents (0.13 wt % of oil) was investigated. Dioxins and cPCBs were extracted from fish oil by SCE at a temperature of 60 degrees C and a pressure of 28 MPa, and the removal efficiencies for PCDDs and PCDFs were in the range of 15-90% and those for cPCBs were in the range of 70-90%. However, 40% of the oil was extracted simultaneously with contaminants. On the adsorbent treatment, activated carbon showed high efficiency, and the removal efficiencies were >90% for PCDDs and PCDFs, but below 30% for cPCBs. A combination of both of these methods is more effective, and almost 100% of the total toxicity equivalence quantity value could be reduced.     

Optimum Steeping Process for Wet Milling of Sorghum

Pioneer 8500, a red hard sorghum hybrid, was steeped batchwise using three steeping solutions at 50°C: SO₂ solution; SO₂ solution containing 1.25% (w/w) of a commercial multiple‐enzyme preparation (Novo SP249); and SO₂ solution with the addition of 0.5% (w/w) lactic acid. Novo SP249 contained pectolytic, cellulolytic, hemicellulolytic, and proteolytic activities and small amounts of saccharolytic activities. Three SO₂ concentrations (0.1, 0.2, and 0.3% w/v) prepared by dissolving sodium bisulfite in distilled water and three steeping times (24, 36, and 48 hr) were used. Incorporation of multiple enzymes into the SO₂ resulted in an increase in starch yield with reduced protein content compared with the SO₂ solution alone. The best wet‐milling performance for sorghum resulted from the SO₂ solution containing 0.5% lactic acid; it produced the whitest starch with the highest yield and the lowest protein content. Both higher SO₂ concentration of the steeping solution and longer steeping time led to higher starch yield, lower protein content in starch, and whiter starch. However, no significant differences in starch yield, protein content in starch, and starch color occurred between SO₂ concentrations of 0.2 and 0.3% for all three steeping solutions. The optimum steeping process for wet milling of sorghum was using a 0.2% SO₂ solution with 0.5% lactic acid for 36 hr at 50°C. Under these conditions, the starch yield, protein content in starch, and L value of starch color were 60.2% (db), 0.49% (db), and 92.7, respectively, which were not significantly different from the best values from the 48‐hr steeping using the solution with 0.3% SO₂ and 0.5% lactic acid.     

Oil goldenberry (Physalis peruviana L.)

Whole berries, seeds, and pulp/peel of goldenberry (Physalis peruviana L.) were compared in terms of fatty acids, lipid classes, triacylglyerols, phytosterols, fat-soluble vitamins, and beta-carotene. The total lipid contents in the whole berries, seeds, and seedless parts were 2.0, 1.8, and 0.2% (on a fresh weight basis), respectively. Linoleic acid was the dominating fatty acid followed by oleic acid as the second major fatty acid. Palmitic and stearic acids were the major saturates. In pulp/peel oil, the fatty acid profile was characterized by higher amounts of saturates, monoenes, and trienes than in whole berry and seed oils. Neutral lipids comprised >95% of total lipids in whole berry oil and seed oil, while neutral lipids separated in lower level in pulp/peel oil. Triacylglycerols were the predominant neutral lipid subclass and constituted ca. 81.6, 86.6, and 65.1% of total neutral lipids in whole berry, seed, and pulp/peel oils, respectively. Nine triacylglycerol molecular species were detected, wherein three species, C54:3, C52:2, and C54:6, were presented to the extent of approximately 91% or above. The highest level of phytosterols was estimated in pulp/peel oil that contained the highest level of unsaponifiables. In both whole berry and seed oils, campesterol and beta-sitosterol were the sterol markers, whereas Delta5-avenasterol and campesterol were the main 4-desmethylsterols in pulp/peel oil. The tocopherols level was much higher in pulp/peel oil than in whole berry and seed oils. beta- and gamma-tocopherols were the major components in whole berry and seed oils, whereas gamma- and alpha-tocopherols were the main constituents in pulp/peel oil. beta-Carotene and vitamin K(1) were also measured in markedly high levels in pulp/peel oil followed by whole berry oil and seed oil, respectively. Information provided by the present work is of importance for further chemical investigation of goldenberry oil and industrial utilization of the berries as a raw material of oils and functional foods.