Characterization of Enzyme Released Antioxidant Phenolic Acids and Xylooligosaccharides from Different Graminaceae or Poaceae Members

Xylooligosacchrides (XOS) and phenolic acids were simultaneously produced from hemicelluloses using crude enzyme mixture synthesized by a novel Bacillus subtilis KCX006. The strain synthesized XOS-forming endo-xylanase and de-branching α-L-arabinofuranosidase and esterase but not β-xylosidase. This enzyme mixture can improve the yield of XOS and phenolic acids from xylan due to the absence of hydrolysis of XOS by β-xylosidase and release of phenolic acids by esterase. Hence, the enzyme mixture was tested for simultaneous production of XOS and phenolic acids from xylan (28–35%)-rich Graminaceae or Poaceae plant biomass, such as wheat bran, sugarcane bagasse, bamboo, and rise husk. Hemicellulose fractions of the biomasses were extracted by alkaline treatment and hydrolyzed using crude xylanase mixture. The profiles of XOS and phenolic acids formed were analyzed by HPLC. The lyophilized hydrolytic products were further analysed by ¹H NMR to identify the substitutions in XOS. The observed profile of XOS and phenolic acids varied with the biomass sources. Maximum amounts of XOS (665.2 mg/g dwt) and phenolic acids (89.69 μg/g dwt) were produced from hemicellulose A fractions of sugarcane bagasse and bamboo bagasse, respectively. HPLC and ¹H NMR analysis of XOS revealed the formation of free- and arabino-XOS. Phenolic acids consisted of hydroxycinnamic and hydroxybenzoic acids and the antioxidant activity correlated well with hydroxycinnamic acids content. The crude enzyme of B. subtilis is useful to produce mixture of XOS and phenolic acids from biomass.     

Production of active lipase by Rhizopus oryzae from sugarcane bagasse: solid state fermentation in a tray bioreactor

This study deals with production of lipase in solid state fermentation by Rhizopus oryzae from sugarcane bagasse. A tray bioreactor was designed for the extracellular enzyme production. Daily, lipase production was evaluated at several incubation temperatures. Furthermore, the influence of temperature and humidity of the cabinet, depth of solid bed, particle size, initial moisture content and supplementary substrate (olive oil) as carbon source was investigated. The obtained results showed that bioreactor temperature of 45 °C, humidity of 80%, solid bed depth of 0.5 cm, particle size in the range of 0.335–1 mm, substrate initial moisture content of 80% for the top tray and 70% for the middle tray and supplementary substrate of 8% (v/w) olive oil led to maximum lipase production. Under optimum fermentation conditions after 72‐h incubation, maximum lipase activities for the top, middle and bottom trays were 215.16, 199.36 and 52.64 U gds^?1, respectively.     

利用皮状丝孢酵母对甘蔗渣酶解液产油脂的研究

本试验旨在研究碱和高温液态水2种预处理方式对发酵的影响。甘蔗渣分别利用碱和高温液态水(LHW)预处理,然后使用纤维素酶进行酶解。在LHW和碱预处理后的酶解液中,分别含22.06和39.28 g/L总糖。不经过浓缩,也不需要添加任何营养物质,酶解液被用于皮状丝孢酵母的油脂发酵试验。经过3 d的培养,在LHW预处理的酶解液中,皮状丝孢酵母的生物量达到了8.08 g/L,油脂质量分数为52.00%,油脂系数高达19.03%。在碱预处理的酶解液中,经过8 d的培养,皮状丝孢酵母的生物量为13.67 g/L,油脂质量分数为43.20%,油脂系数为15.03%。脂肪酸组成分析表明所产油脂与棕榈油的组成相似。试验结果表明,甘蔗渣是一种有前景的产油脂原料,LHW是一种良好的预处理方式。该研究为利用甘蔗渣发酵产油脂提供了新思路。     

蔗渣堆场空气及渗滤液挥发性有机物成分分析

研究了甘蔗榨季和非榨季期间蔗渣堆场空气中挥发性有机物的种类和含量的变化,并对蔗渣堆场渗滤液的有机物种类进行了分析。结果表明,在榨季和非榨季堆场空气中可分别检出32种和24种有机污染物;榨季堆场空气中挥发性有机污染物的浓度比非榨季高。在蔗渣堆场渗滤液中共检出32种有机污染物,其中酸类和芳烃类化合物各占14种。     

Evaluation of Ammonia Pretreatment for Enzymatic Hydrolysis of Sugarcane Bagasse to Recover Xylooligosaccharides

Sugarcane bagasse is a useful biomass resource. In the present study, we examined the efficacy of ammonia pretreatment for selective release of hemicellulose from bagasse. Pretreatment of bagasse with aqueous ammonia resulted in significant loss of xylan. In contrast, pretreatment of bagasse with anhydrous ammonia resulted in almost no xylan loss. Aqueous ammonia or anhydrous ammonia-pretreated bagasse was then subjected to enzymatic digestion with a xylanase from the glycoside hydrolase (GH) family 10 or a xylanase from the GH family 11. The hydrolysis rate of xylan in bagasse pretreated with aqueous ammonia was approximately 50 %. In contrast, in the anhydrous ammonia-treated bagasse, xylan hydrolysis was > 80 %. These results suggested that anhydrous ammonia pretreatment would be an effective method for preparation of sugarcane bagasse for enzymatic hydrolysis to recover xylooligosaccharides.     

Optimisation of the microwave‐assisted extraction process for six phenolic compounds in Agaricus blazei murrill

An efficient microwave‐assisted extraction (MAE) technique was employed in the extraction of phenolic compounds from Agaricus blazei murrill, and phenolic compounds were quantified by High‐performance liquid chromatography (HPLC). The MAE procedure was optimised, validated and compared with other conventional extraction techniques. MAE gave the best result because of the highest extraction efficiency within the shortest extraction time. The optimal conditions of MAE were 60% ethanol, ratio of solid/liquid 1:30, temperature 110 °C, irradiation power 500 W and three extraction cycles, each 5 min. This is the first report on combining MAE with HPLC for the extraction and quantification of phenolic compounds in A. blazei murrill. The developed MAE method provided a good alternative for the extraction of phenolic compounds in A. blazei murrill as well as other materials.     

Bioethanol production from ball milled bagasse using an on-site produced fungal enzyme cocktail and xylose-fermenting Pichia stipitis

Sugarcane bagasse is one of the most promising agricultural by-products for conversion to biofuels. Here, ethanol fermentation from bagasse has been achieved using an integrated process combining mechanical pretreatment by ball milling, with enzymatic hydrolysis and fermentation. Ball milling for 2 h was sufficient for nearly complete cellulose structural transformation to an accessible amorphous form. The pretreated cellulosic residues were hydrolyzed by a crude enzyme preparation from Penicillium chrysogenum BCC4504 containing cellulase activity combined with Aspergillus flavus BCC7179 preparation containing complementary β-glucosidase activity. Saccharification yields of 84.0% and 70.4% for glucose and xylose, respectively, were obtained after hydrolysis at 45 °C, pH 5 for 72 h, which were slightly higher than those obtained with a commercial enzyme mixture containing Acremonium cellulase and Optimash BG. A high conversion yield of undetoxified pretreated bagasse (5%, w/v) hydrolysate to ethanol was attained by separate hydrolysis and fermentation processes using Pichia stipitis BCC15191, at pH 5.5, 30 °C for 24 h resulting in an ethanol concentration of 8.4 g/l, corresponding to a conversion yield of 0.29 g ethanol/g available fermentable sugars. Comparable ethanol conversion efficiency was obtained by a simultaneous saccharification and fermentation process which led to production of 8.0 g/l ethanol after 72 h fermentation under the same conditions. This study thus demonstrated the potential use of a simple integrated process with minimal environmental impact with the use of promising alternative on-site enzymes and yeast for the production of ethanol from this potent lignocellulosic biomass.     

Sequential high pressure extractions applied to recover piceatannol and scirpusin B from passion fruit bagasse

Passion fruit seeds are currently discarded on the pulp processing but are known for their high piceatannol and scirpusin B contents. Using pressurized liquid extraction (PLE), these highly valuable phenolic compounds were efficiently extracted from defatted passion fruit bagasse (DPFB). PLE was performed using mixtures of ethanol and water (50 to 100% ethanol, w/w) as solvent, temperatures from 50 to 70 °C and pressure at 10 MPa. The extraction methods were compared in terms of the global yield, total phenolic content (TPC), piceatannol content and the antioxidant capacity of the extracts. The DPFB extracts were also compared with those from non-defatted passion fruit bagasse (nDPFB). Identification and quantification of piceatannol were performed using UHPLC–MS/MS. The results showed that high TPC and piceatannol content were achieved for the extracts obtained from DPFB through PLE at 70 °C and using 50 and 75% ethanol as the solvent. The best PLE conditions for TPC (70 °C, 75% ethanol) resulted in 55.237 mg GAE/g dried and defatted bagasse, whereas PLE at 70 °C and 50% ethanol achieved 18.590 mg of piceatannol/g dried and defatted bagasse, and such yields were significantly higher than those obtained using conventional extraction techniques. The antioxidant capacity assays showed high correlation with the TPC (r > 0.886) and piceatannol (r > 0.772). The passion fruit bagasse has therefore proved to be a rich source of piceatannol and PLE showed high efficiency to recover phenolic compounds from defatted passion fruit bagasse.     

甘蔗不同组织中游离态和结合态酚酸的分布及抗氧化活性

为了探讨甘蔗不同组织中酚酸的存在形式,本文利用反相高效液相色谱法(RP-HPLC)对蔗汁、蔗叶及蔗渣三个组织中游离态和结合态的酚酸进行了定性和定量检测分析。结果表明,甘蔗组织中的酚酸主要以结合态形式存在,但其在甘蔗三个组织中的含量和存在形式有差别。甘蔗叶、蔗渣中游离的酚酸含量最高,蔗汁较低。蔗汁、蔗叶及蔗渣中总游离态酚酸含量分别为317.18 mg/kg(干固物),1568.37 mg/kg(干基)及1504.57 mg/kg(干基);总结合态酚酸含量分别为740.32 mg/kg(干固物),2725.41 mg/kg(干基),2452.13 mg/kg(干基)。在蔗汁中,咖啡酸、没食子酸主要以游离态存在,阿魏酸、香豆酸主要以结合态存在,而在蔗叶和蔗渣中,咖啡酸、阿魏酸、香豆酸主要以结合态形式存在。甘蔗不同组织中游离态酚酸的DPPH和ABTS自由基清除活性优于结合态酚酸,不同组织中的游离态酚酸和结合态酚酸含量分别与抗氧化活性呈正相关性。     

Production of coumaric acid from sugarcane bagasse

Phenolic acids were released from sugarcane bagasse by alkaline hydrolysis at 30 °C for 4 h; The alkaline hydrolysates were ultrafiltrated, the permeates purified with anion exchange resin. The phenolic acids bound by the resin were desorbed by a mixture of water–ethanol–HCl solution (36: 60: 4) after washing the resin with water, ethanol and dilute HCl respectively. The combined eluents were concentrated for crystalization, and the crystals filtered and washed using 1% (v/v) HCl. After this purification process, the purity of products reached 89.7% based on coumaric acid. Results of HPLC/MS, HPLC using standard coumaric acid and ferulic acid showed that the main component of the purified bagasse hydrolysate was p-coumaric acid rather than ferulic acid. The purified products showed the same antioxidant activity, reducing power and free radical scavenging capacity as the standard p-coumaric acid.The technology could be applied on industrial scale.Industrial relevanceThis research presents a technology to produce coumaric acids from sugarcane bagasse. The first step is to release coumaric acid by alkaline hydrolysis. The second step is to remove the viscous polysaccharides and protein by ultrafiltration. The third step is to purify coumaric acid from the permeate of ultrafiltration by anion chromatography, and the alkaline could be reused to hydrolyze the bagasse. The technology showed potential application on industrial scale.     

蒸汽爆破甘蔗渣中抗氧化物质的提取及评价

以蒸汽爆破甘蔗渣(SESB)为原料,乙醇、甲醇、水及乙酸乙酯为溶剂,对其中的多酚和糖进行提取并评价了提取液的抗氧化能力。其中甲醇提取液中酚类物质含量最高((1.5174±0.0140)mg/mL),水次之((1.2382±0.0025)mg/mL),乙酸乙酯最低。提取液中的总糖含量以水中最多,其次为甲醇,乙酸乙酯中最低。四种提取液对DPPH自由基均具有很强的清除能力。水、乙醇和甲醇提取液具有较强的还原力,呈明显的量效关系。采用溶剂对SESB中抗氧化物质提取是可行的。综合经济效益、环境问题及水提取液中酚类含量、总糖含量以及抗氧化能力,认为以水作为溶剂最佳。     

ULTRASONICALLY ASSISTED EXTRACTION OF PHENOLIC COMPOUNDS FROM AROMATIC PLANTS: COMPARISON WITH CONVENTIONAL EXTRACTION TECHNICS

Extracts of aromatic plants contain bioactive substances such as phenolic compounds, which could be used as natural antioxidants. Conventional, as well as ultrasonically assisted extractions (UAEs ) of phenolic compounds from aromatic plants using different solvents, have been studied. Reversed phase high performance liquid chromatography (RP‐HPLC ) with ultraviolet detection was employed for the analysis of phenolic compounds. Total phenolic compounds were determined by the Folin–Ciocalteu assay. The amount of extractable phenolic substances for this method decreased with decreasing polarity of the solvent in the order water, 60% methanol, 60% acetone, and ethyl acetate/water (60:30, v/v). The HPLC results indicate that UAE is a more effective technic compared to the conventional method but was dependent on the solvent and the temperature employed. Increasing the temperature of sonication, the efficiency of extraction of phenolic compounds for all solvents is enhanced. However, water extraction was found to give reduced amounts of phenolic compounds when compared with the conventional method.     

Biochemical characterisation and application of lipases produced by Aspergillus sp. on solid‐state fermentation using three substrates

Lipase from Aspergillus sp. obtained by solid‐state fermentation (SSF) on wheat bran (LWB), soybean bran (LSB) and soybean bran combined with sugarcane bagasse (LSBBC) were 67.5, 58 and 57.3 U of crude lipase per gram substrate, respectively. The optimum pH of activity and stability of the LWB was between 8 and 9, and the optimum temperature of activity and stability was 50 °C and up to 60 °C, respectively. The LSB and LSBBC showed two peaks of optimum pH (4 and 6) and optimal values of temperature and stability at 50 °C. The LSB was stable in the pH range of 6–7, while LSBBC in the range of pH 4–7. All the enzymes show activities on p‐nitrophenyl esters (butyrate, laurate and palmitate). LWB stood out either on the hydrolysis of sunflower oil, presenting 66.1% of the activity over commercial lipase and on the esterification of oleic acid and ethanol, surpassing the activities of the commercial lipases studied. The thin layer chromatography showed that LWB and LSB have produced ethyl esters from corn oil, while LWB produced it from sunflower oil.     

蔗渣木质素的分离提取及应用研究进展

蔗渣木质素是蔗渣的主要成分之一,是一种重要的可再生生物质资源。本文综述了蔗渣木质素在分离提取、结构表征、改性制备新材料、催化液化和抗氧化性方面的研究进展情况,以期为蔗渣木质素的资源化高效利用提供参考。     

In Vitro Hypoglycemic Activity of the Phenolic Compounds in Longan Fruit (Dimocarpus longan var. Fen Ke) Shell Against α-Glucosidase and β-Galactosidase

The in vitro hypoglycemic effect of longan fruit (Dimocarpus longan var. Fen Ke) shell extracts was evaluated by inhibiting α-glucosidase and β-galactosidase activities. The IC₅₀ values of hot water and 50% ethanol ultrasonic extracts against the α-glucosidase were 9.2 and 13.4 mg/mL, and those against the β-galactosidase were 12.9 and 19.7 mg/mL, respectively. The hot water extracts (20 mg/mL) with aid of ultrasound-assisted extraction may enhance the inhibitory rates of α-glucosidase and β-galactosidase by 10.6 and 12.0% as compared with conventional extraction, respectively. Two phenolic compounds, gallic acid and ellagic acid, were identified as the major phenolic in hot water extracts from longan fruit shell against α-glucosidase and β-galactosidase. Inhibition of α-glucosidase and β-galactosidase were proven to be as a therapeutic approach for decreasing postprandial hyperglycemia. As a result, the longan fruit shell extracts may develop to be as a potential strategy for early treatment of postprandial hyperglycemia.     

Xylitol production on sugarcane biomass hydrolysate by newly identified Candida tropicalis JA2 strain

Xylitol is a building block for a variety of chemical commodities, besides being widely used as a sugar substitute in the food and pharmaceutical industries. The aim of this work was to develop a microbial process for xylitol production using sugarcane bagasse hydrolysate as substrate. In this context, 218 non-Saccharomyces yeast strains were screened by growth on steam-exploded sugarcane bagasse hydrolysate containing a high concentration of acetic acid (8.0 g/L). Seven new Candida tropicalis strains were selected and identified, and their ability to produce xylitol on hydrolysate at low pH (4.6) under aerobic conditions was evaluated. The most efficient strain, designated C. tropicalis JA2, was capable of producing xylitol with a yield of 0.47 g/g of consumed xylose. To improve xylitol production by C. tropicalis JA2, a series of experimental procedures were employed to optimize pH and temperature conditions, as well as nutrient source, and initial xylose and inoculum concentrations. C. tropicalis JA2 was able to produce 109.5 g/L of xylitol with a yield of 0.86 g/g of consumed xylose, and with a productivity of 2.81 g·L·h, on sugarcane bagasse hydrolysate containing 8.0 g/L acetic acid and177 g/L xylose, supplemented with 2.0 g/L yeast nitrogen base and 4.0 g/L urea. Thus, it was possible to identify a new C. tropicalis strain and to optimize the xylitol production process using sugarcane bagasse hydrolysate as a substrate. The xylitol yield on biomass hydrolysate containing a high concentration of acetic acidobtained in here is among the best reported in the literature.     

The Use of an Extracellular Ferulic Acid Esterase from Lactobacillus acidophilus K1 for the Release of Phenolic Acids During Mashing

A purified extracellular ferulic acid esterase from Lactobacillus acidophilus K1 was successfully used during mashing for the release of free phenolic acids into sweet wort. The enzyme was produced in bioreactors and partially purified to obtain the monoenzyme preparation. Release of free ferulic and vanillic acid into the wort at 52°C (with the use of 4.09–14.60 enzyme activity units/L of the mash) and ferulic acid at 62°C (14.60 units/L) was observed. Free p‐OH‐benzoic and syringic acids were effectively released at 26°C at each enzyme concentration used. Free p‐OH‐benzoic acid was also released by the enzyme (14.60 U/L) at 52°C‐74°C. Free protocatechuic acid was effectively hydrolyzed by the enzyme preparation (8.75 U/L and 14.60 U/L) at 26°C‐52°C. Free caffeic acid (effectively released at 26°C‐62°C) originated from chlorogenic acid. No p‐coumaric acid was released due to the action of bacterial esterase during mashing. Ferulic acid esterase from L. acidophilus K1 exhibited no ability to release free phenolic acids during mashing at 62°C or at 74°C due to its low thermostability. In conclusion, L. acidophilus K1 is an attractive source for the production of ferulic acid esterase, which can be useful for the release of antioxidant phenolic acids in the early stages of mashing.     

Effect of pulsed electric fields and high pressure on improved recovery of high-added-value compounds from olive pomace

Olive pomace is considered a solid by-product and a rich source of valuable compounds such as polyphenols, flavonoids with antioxidant properties, and proteins. Nonthermal technologies, which cause alterations to cell permeability, are being explored to assist conventional recovery techniques. The aim of this study was to assess the effect of pulsed electric fields (PEF) and high pressure (HP) on improved recovery yield of the high-added-value compounds or to shorten the extraction time of these compounds. Olive pomace (Tsounati cv) was pretreated with PEF (1.0 to 6.5 kV/cm, 0.9 to 51.1 kJ/kg, and 15 µs pulse width) or HP (200 to 600 MPa and 0 to 40 min). Evaluation of the intracellular compounds extracted via solid–liquid extraction (50% ethanol–water solution) was performed. More intense PEF and HP conditions resulted in a significant increase of the phenolic concentration up to 91.6% and 71.8%, respectively. The increased antioxidant capacity of each extract was correlated to phenolic compound concentration. The protein concentration that was achieved with PEF pretreatment was doubled; however, HP-pretreated extracts reached 88.1% higher yield than untreated for pressures up to 200 MPa. HP and PEF pretreatment decreased extraction completion time t₉₈ (needed time to recover the equal amount of phenolics and proteins of untreated after 60 min of conventional extraction) to 12 min and lower than 1 min, respectively. To conclude, both pretreatments are effective in improving the conventional extraction process for increased yield recovery of high-added-value compounds from olive pomace.     

Comparison of extraction conditions for phenolic,flavonoid content and determination of rosmarinic acid from Perilla frutescens var. acuta

Perilla frutescens var. acuta has traditionally been used to treat disease including, depression, inflammation and tumors. Especially, the presence of rosmarinic acid can induce beneficial and health promoting effects. According to, this work was conducted to comparison of various extraction conditions (extraction process, solvent and time) of total phenolic, floavonoid and rosmainic acid contents from the leaves of Perilla frutescens var. acuta. Also, rosmarinic acid was determined in leaves of Perilla frutescens var. acuta by high‐performance liquid chromatography (HPLC) and the described method was ideally suited for rapid routine analysis. The highest yield of total phenolic, flavonoid and rosmarinic acid was shown 12.15 mg gallic acid equivalents g^?1 dry weight (DW), 7.23 mg rutin equivalents g^?1 DW and 3.76 mg g^?1 DW, respectively. The best condition for extraction efficiency of total phenolic, flovonoid and rosmarinic were found to be: ethanol concentration of 70%, extraction time of 24 h and extraction process of refluxed extraction.     

Chemical composition of alkaline-pretreated sugarcane bagasse and its effects on the physicochemical characteristics of fat-replaced sausage

The potential of cellulose from sugarcane bagasse used for pork sausage was investigated. The purity level of cellulose fibre from sugarcane bagasse and the functional characteristics in pork sausage were studied. The differences in the sugarcane fibre and sausage characteristics were strongly related to the cellulose/lignin content of the extracted cellulose under different amounts of bleaching. Bleached sugarcane fibres showed better water (3.55) and oil (2.34) holding capacity in comparison with untreated fibre (2.92 and 2.38, respectively). The sugarcane fibres in each treatment stage showed different effects on sausage properties. Sausages with 1% cellulose fibres added had an improved texture profile, but the addition of 3% cellulose fibres to sausages resulted in a higher cooking yield. Consumers considered the sensory characteristics of all sausages as acceptable. These results indicated that low levels of cellulose extracted from sugarcane bagasse could be added and maintain the inherent characteristics of meat products.