Oxidative transformation of natural and synthetic phenolic mixtures by Trametes versicolor laccase

The efficiency of Trametes versicolor laccase in the transformation of phenols (caffeic acid, catechol, hydroxytyrosol, methylcatechol, protocatechuic acid, syringic acid, m-tyrosol, 3-hydroxybenzoic acid, 3-hydroxyphenylacetic acid, 2,6-dihydroxybenzoic acid, 4-hydroxybenzaldehyde) usually present in waste water, such as that derived from an olive oil factory, was investigated. According to their response to 24 h laccase action the 11 phenolic compounds were classified in three groups: reactive (88-100% transformation), intermediate reactive (transformation lower than 50%), and recalcitrant (not transformed at all). The enzyme was able to transform the 11 substrates even when they were present in a mixture and also toward a phenolic extract from a Moroccan olive oil mill waste water (OMW) sample. The disappearance of protocatechuic, 3-hydroxyphenylacetic, and 2,6-dihydroxybenzoic acids, and 4-hydroxybenzaldehyde was enhanced whereas that of caffeic acid and m-tyrosol was depressed when the phenols were present in the mixture. A reduction of enzyme activity occurred in single and/or complex phenolic mixtures after enzymatic oxidation. No correspondence between phenol transformation and disappearance of enzymatic activity was, however, observed. The overall results suggest that laccases are effective in the transformation of simple and complex phenolic mixtures.     

Hydroxytyrosol 4-beta-D-glucoside,an important phenolic compound in olive fruits and derived products

There is increasing interest in olive polyphenols because of their biological properties as well as their contribution to the color, taste, and shelf life of olive products. However, some of these compounds remain unidentified. It has been shown that hydroxytyrosol 4-beta-D-glucoside (4-beta-D-glucosyl-3-hydroxyphenylethanol) coeluted with hydroxytyrosol [(3,4-dihydroxyphenyl)ethanol] under reversed phase conditions in the phenolic chromatograms of olive pulp, vegetation water, and pomace of olive oil processing. A method to separate this compound from hydroxytyrosol by HPLC has been developed. The concentration of this glucoside increased in olive pulp with maturation and could be the main phenolic compound in mature olives. In contrast, the presence of this compound was not detected in olive oil by using HPLC-MS. The compound must be considered both in table olives and olive oil processing because of its glucose and hydroxytyrosol contribution to these products.     

Convenient synthesis of hydroxytyrosol and its lipophilic derivatives from tyrosol or homovanillyl alcohol

Hydroxytyrosol, a naturally occurred o-phenolic compound exhibiting antioxidant properties, was synthesized by a three-step high-yielding procedure from natural and low-cost compounds such as tyrosol or homovanillyl alcohol. First, the efficient chemoselective protection of the alcoholic group of these compounds was performed by using dimethyl carbonate (DMC) as reagent/solvent; second, the oxidation with 2-iodoxybenzoic acid (IBX) or Dess-Martin periodinane reagent (DMP) and in situ reduction with sodium dithionite (Na2S2O4) allowed the preparation of carboxymethylated hydroxytyrosol; finally, by a mild hydrolytic step, hydroxytyrosol was obtained in high yield and purity, as confirmed by NMR spectra and HPLC profile. By using a similar methodology, lipophilic hydroxytyrosol derivatives, utilized as additives in pharmaceutical, food, and cosmetic preparations, were prepared. In fact, at first the chemoselective protection of the alcoholic group of tyrosol and homovanillyl alcohol was performed by using acyl chlorides without any catalyst to obtain the corresponding lipophilic derivatives, and then these compounds were converted in good yield and high purity into the hydroxytyrosol derivatives by oxidative/reductive pathway with IBX or DMP and Na2S2O4.     

Structural characterization of the metabolites of hydroxytyrosol, the principal phenolic component in olive oil, in rats

Hydroxytyrosol is quantitatively and qualitatively the principal phenolic antioxidant in olive oil. Recently it was shown that hydroxytyrosol and five metabolites were excreted in urine when hydroxytyrosol was dosed intravenously or orally in an olive oil solution to rats. The conclusive identification of three metabolites of hydroxytyrosol by MS/MS as a monosulfate conjugate, a 3-O-glucuronide conjugate, and 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid) has been established in this investigation. The structural configurations of the glucuronide conjugate and 4-hydroxy-3-methoxyphenylacetic acid were confirmed by (1)H NMR. The radical scavenging potencies of homovanillic acid, homovanillic alcohol, hydroxytyrosol, and the metabolites were examined with the radical 2,2-diphenyl-1-picrylhydrazyl. These studies showed them to be potent antioxidants with SC(50) values of 14.8 and 11.4 microM for homovanillic acid and homovanillic alcohol, respectively. The 3-O-glucuronide conjugate was more potent than hydroxytyrosol, with an SC(50) of 2.3 in comparison to 11.0 microM, and the monosulfate conjugate was almost devoid of radical scavenging activity.     

Colorimetric evaluation of phenolic content and GC-MS characterization of phenolic composition of alimentary and cosmetic argan oil and press cake

The global phenolic content of argan oil and press cake samples (alimentary and cosmetic) was evaluated using the Folin-Ciocalteu colorimetric method and the phenolic composition of argan oil (alimentary and cosmetic) and press cake (alimentary) samples were analyzed by GC-MS after extraction with 80:20 (v/v) methanol:water and silylation. Identification of chromatographic peaks was made by mass selective detection. Nineteen simple phenols were detected, 16 in press cake, 6 in the alimentary oil, and 7 in the cosmetic oil, among which 15 compounds [3-hydroxypyridine (3-pyridinol), 6-methyl-3-hydroxypyridine, catechol, resorcinol, 4-hydroxybenzyl alcohol, vanillin, 4-hydroxyphenylacetic acid, vanillyl alcohol, 3,4-dihydroxybenzyl alcohol, 4-hydroxy-3-methoxyphenethyl alcohol, methyl 3,4-dihydroxybenzoate, hydroxytyrosol, protocatechuic acid, epicatechin, and catechin] were identified for the first time in such materials.     

Quercetin-dependent inhibition of nitration induced by peroxidase/H2O2/nitrite systems in human saliva and characterization of an oxidation product of quercetin formed during the inhibition

Local pH in the oral cavity can decrease to below 7 at the site where acid-producing bacteria are proliferating. Effects of pH on nitration of 4-hydroxyphenylacetic acid were studied using dialyzed human saliva. Dialyzed saliva nitrated 4-hydroxyphenylacetic acid to 4-hydroxy-3-nitrophenylacetic acid in the presence of nitrite and H(2)O(2). The rate of the nitration was dependent on pH, and the maximal rate was observed between pH 5.5 and 7.2. The optimum pH seemed to reflect rates of formation of nitrogen dioxide and 4-hydroxyphenylacetic acid radicals. Quercetin inhibited the nitration. The quercetin-dependent inhibition might be due to scavenging of nitrogen dioxide and 4-hydroxyphenylacetic acid radicals, which were formed by salivary peroxidase-dependent oxidation of nitrite and 4-hydroxyphenylacetic acid, respectively, and competition with nitrite and 4-hydroxyphenylacetic acid for peroxidase in saliva. An oxidation product of quercetin was formed during inhibition of the nitration by quercetin. The oxidation product was identified as 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone. This component could also be oxidized by salivary peroxidase and nitrogen dioxide radicals. The oxidation products were 2,4,6-trihydroxyphenylglyoxylic and 3,4-dihydroxybenzoic acids. On the basis of the results, the significance of quercetin for inhibition of nitrogen dioxide formation and for scavenging of nitrogen dioxide radicals in the oral cavity is discussed.     

Production of triacetylhydroxytyrosol from olive mill waste waters for use as stabilized bioantioxidant

A hydroxytyrosol triacetyl derivative was very efficiently produced as a highly pure stabilized antioxidant compound by a short treatment of olive mill waste water (OMWW) organic extracts, rich in hydroxytyrosol, with an acetylating mixture composed of HClO4-SiO2 and Ac2O (Chakborti and Gulhane reaction), in mild and safe conditions. A successive single step of middle pressure liquid chromatography (MPLC) purification of the reaction product was performed, with an overall yield of 35.6%. (This process, including both the Chakborti and Gulhane reaction and the MPLC purification, is protected by an international patent under PCT/IT2005/000781.) The o-diphenol triacetyl derivative was also produced by direct reaction of hydroxytyrosol, previously purified by MPLC, with HClO4-SiO2 and Ac2O, with an overall yield of 29.5%. A further procedure for the production of the hydroxytyrosol triacetyl derivative was consistent with the direct treatment of raw OMWW with the acetylating agent and a single step of MPLC purification, with an overall yield of 27.6%. The purified natural triacetylhydroxytyrosol confirmed the same strong protective effects against the oxidative stress in human cells as the corresponding synthetic compound, likely because of the biochemical activation of the acetyl derivative into the active parent hydroxytyrosol by esterases. We therefore propose the utilization of OMWW for recovering hydroxytyrosol as a natural antioxidant in a chemically stabilized form, with a good yield, which can be potentially used as a nontoxic functional component in nutritional, pharmaceutical, and cosmetic preparations.     

卷枝毛霉重组菌株Mc-MT-2培养条件优化及产油特性

前期工作构建了苹果酸转运蛋白基因过表达的卷枝毛霉重组菌株Mc-MT-2,该菌株的脂质含量大幅度提高。该研究从培养基成分筛选、微生物生长控制以及发酵模式等方面深入探讨Mc-MT-2菌株发酵产油脂的调控策略。结果表明,Mc-MT-2菌株最佳生长及产孢培养条件是以葡萄糖与苹果酸为复合碳源且复合配比为9∶1,氮源为胰蛋白胨,其他成分同Kendrick培养基,初始pH值为5。经分批培养获得生物量、脂质含量和脂质产量分别为11.2 g/L,24%和2.6 g/L。在3 L发酵罐扩大培养中,补料培养Mc-MT-2菌株获得生物量、脂质含量和脂质产量最大值为15.4 g/L、28.6%和4.4 g/L,比分批培养分别提高1.38、1.19和1.69倍。该研究为卷枝毛霉重组菌株Mc-MT-2在脂质生产中的进一步应用奠定理论基础。     

Production of a novel 9,12-dihydroxy-10(E)-eicosenoic acid from eicosenoic acid by Pseudomonas aeruginosa PR3

Microbial conversions of unsaturated fatty acids often generate polyhydroxy fatty acids, giving them new properties such as higher viscosity and reactivity. A bacterial strain Pseudomonas aeruginosa (PR3) has been intensively studied to produce mono-, di-, and trihydroxy fatty acids from different 9-cis-monoenoic fatty acids such as oleic acid, ricinoleic acid, and palmitoleic acid. However, from the results and the postulated similar metabolic pathways involved in these transformations, it was assumed that the enzyme system involved in transformation of the monoenoic fatty acid by strain PR3 could utilize fatty acids with different chain lengths and locations of the double bond. In this study was used as a substrate for bioconversion by strain PR3 eicosenoic acid (C20:1, ω-9) containing a singular cis double bond at different positions from the carboxyl end as oleic acid, and it was confirmed that PR3 could produce a novel 9,12-dihydroxy-10(E)-eicosenoic acid (DED) with 6.2% yield from eicosenoic acid. The structure of DED was confirmed using GC-MS, FTIR, and NMR analyses. DED production was maximized at 72 h after the substrate was added to the 24 h culture. Some other nutritional factors were also studied for optimal production of DED.     

一株高效还原亚硒酸盐的粘质沙雷氏菌的分离鉴定及特性

从广西钦州市钦北区的富硒土壤中筛选出一株可高效还原亚硒酸盐的细菌QZB-1。16S rDNA基因序列分析确定菌株为粘质沙雷氏菌(Serratia marcescens)。研究发现菌株QZB-1可耐高达180 mmol/L的亚硒酸盐。进一步研究发现菌株QZB-1在36 h之内对1 mmol/L亚硒酸盐的还原率为95%，随着硒浓度升高还原率有所下降。正交实验表明，对菌株还原亚硒酸盐的影响程度依次是初始亚硒酸盐浓度>培养时间>接种量，最佳还原条件是初始亚硒酸盐浓度为1 mmol/L，接种量为10%，培养时间为36 h，此时菌株QZB-1对亚硒酸盐的还原率大于98.55%。研究结果表明新型亚硒酸盐还原菌粘质沙雷氏菌QZB-1可高效还原亚硒酸盐为单质纳米硒，可高效应用于亚硒酸盐污染水体和土壤的治理。     

Lycopene production using Blakeslea trispora in the presence of 2-methyl imidazole: yield, selectivity, and safety aspects

The potential role of 2-methyl imidazole in improving lycopene production by Blakeslea trispora with regards to yield, selectivity, and safety aspects was investigated in batch culture. Optimization of the bioprocess conditions in terms of (a) (+) and (-) strain ratio in the inoculum, (b) initial crude soybean oil (CSO) addition level, and (c) the amount of 2-methyl imidazole was based on response surface methodology to achieve maximum lycopene production. The dependence of growth kinetics, lycopene yield, and selectivity of the bioprocess on the above factors was clear. 2-Methyl imidazole at 50 mg/L was found equally active in terms of lycopene cyclase inhibition with that at 200 or 100 mg/L; in all cases, lycopene accounted for 94% of the total carotenoids. The highest yield was observed at a 50 mg/L level of addition (24 mg/g of biomass dry weight,) in a substrate supplemented with CSO (48 g/L of culture medium) and inoculated with 1(+)/7(-) strain ratio.     

High-yielding preparation of a stable precursor of hydroxytyrosol by total synthesis and from the natural glycoside oleuropein

The unprecedented acetonide of the antioxidant hydroxytyrosol has been synthesized by a two-step high-yielding procedure and found to be both purifiable by chromatography and stable over a wide pH range. The protection stabilizes hydroxytyrosol against oxidation, thereby allowing long-term storage. The protection can quantitatively be removed, under nonaqueous conditions, to afford pure hydroxytyrosol suitable for use as an additive in food and cosmetic preparations. Extension of the same methodology to the natural and easily accessible glycoside oleuropein, followed by saponification of the resulting complex mixture of acetonides, allowed hydroxytyrosol acetonide to be recovered in high yield. This constitutes a new interesting methodology to obtain the antioxidant hydroxytyrosol.     

Synthesis of tritium-labeled hydroxytyrosol, a phenolic compound found in olive Oil

(3,4-Dihydroxyphenyl)ethanol, commonly known as hydroxytyrosol (1), is the major phenolic antioxidant compound in olive oil, and it contributes to the beneficial properties of olive oil. Bioavailability and metabolism studies of this compound are extremely limited, in part, related to unavailability of radiolabeled compound. Studies with radiolabeled compounds enable use of sensitive radiometric analytical methods as well as aiding elucidation of metabolic and elimination pathways. In the present study a route for the formation of hydroxytyrosol (1), by reduction of the corresponding acid 2 with tetrabutylammonium boronate, was found. Methods for the incorporation of a tritium label in 1 were investigated and successfully accomplished. Tritiated hydroxytyrosol (1t) was synthesized with a specific activity of 66 Ci/mol. The stability of unlabeled and labeled hydroxytyrosol was also investigated.     

一株耐盐溶磷真菌的筛选、鉴定及其生物肥料的应用效果

【目的】 从内蒙古种植向日葵的盐碱地中筛选高效溶磷真菌，为农业生产中增产节肥，开发耐盐、溶磷微生物肥料提供菌种资源。 【方法】 利用形态特征和ITS rDNA序列鉴定菌株；LC-MS技术测定菌株M2在液体培养基中分泌有机酸和植物激素含量，明确菌株M2的溶磷和促生机理。采用液体摇床培养试验测定了鉴定菌株的溶磷能力。试验处理包括:在磷酸三钙、磷酸铝和5个磷矿的磷矿粉制备的100 mL难溶磷磷源 (含5 g/L难溶磷) 中，接入1 mL灭菌培养液对照，和分别接种1 mL斜卧青霉菌P83和草酸青霉菌M2共15个处理。置于28℃、160 r/min摇床培养，分别于3、6和9 d，取菌液5 mL，在12000 r/min、4℃离心5 min，取上清液测定有效磷含量。采用含NaCl的固体培养基测定菌株的耐盐性。NaCl含量分别为0%、5%、7.5%、10%和12.5%的PDA平板中接入溶磷菌，置于28℃恒温培养箱中5 d，观察并记录菌丝的生长状况。采用盆栽试验方法检验了菌株的溶磷能力。以玉米种子 (郑单958) 为供试作物，以水稻土、黏性潮土、盐潮土和石灰性潮土为供试土壤，以Ca₃(PO₄)₂、AlPO₄ 和昆阳磷矿粉 (RP) 为供试磷源 (磷源用量为1.0 g/kg土壤)。设置只加入灭菌草炭和Pikovskaya培养液对照，分别接种溶磷菌P83、M2，共计38个处理，144盆。玉米播种40天后收获，测定植株鲜重、干重和玉米根际土壤有效磷含量。田间试验以花生为供试作物，设置只加灭菌草炭和Pikovskaya培养液对照和分别接种ATCC20851、P83、M2溶磷菌剂三个处理。花生生长155 d后收获，称量花生植株鲜重和干重、花生果实鲜重和干重，同时采集花生根部土壤测定有效磷含量。 【结果】 溶磷菌株M2鉴定为草酸青霉 (Penicillium oxalicum)。液体培养基摇床培养6 d后，接种菌株M2，以Ca₃(PO₄)₂为磷源的上清液中有效磷含量达972 mg/L，Ca₃(PO₄)₂溶解率为59.2%；以AlPO₄为磷源的有效磷含量达988 mg/L，溶解率为48.2%；以江苏锦屏、贵州开阳、云南晋宁、河北钒山和云南昆阳磷矿粉为磷源的有效磷释放量达21.0～556 mg/L。菌株M2在7.5%NaCl培养基中正常生长。盆栽试验结果发现，菌株M2对玉米植株促生效果显著，玉米植株鲜重比不接种菌剂 (CK) 提高26.4%～99.2%、干重增加20.0%～262.9%，土壤有效磷提高19.2～25.3 mg/kg。菌株M2与4种土壤的适配性均高于对照菌株P83。田间小区花生产量结果显示，接种溶磷菌剂M2增产效果最好，花生果实产量达4.50 t/hm2，比CK增加0.85 t/hm2，增产23.29%。菌株M2 在含有磷酸三钙、磷酸铝和开阳磷矿粉3种难溶磷培养液中经过6 d培养，均产生7种有机酸，其中草酸和柠檬酸含量最高，分别为653.46 mg/L和269.61 mg/L；培养液中均能检测到吲哚乙酸 (IAA) 和玉米素，IAA含量为32.38～66.17 mg/L，玉米素浓度为0.05～0.07 mg/L。 【结论】 获得了一株耐盐、高效溶解多种难溶磷的草酸青霉菌M2，可显著增加土壤有效磷，促进玉米生长和花生增产，与4种典型土壤适配性好，具有良好的农业应用前景。      

Demetalation of Fe, Mn, and Cu chelates and complexes: application to the NMR analysis of micronutrient fertilizers

The application of nuclear magnetic resonance (NMR) for the quality control of fertilizers based on Fe(3+), Mn(2+), and Cu(2+) chelates and complexes is precluded by the strong paramagnetism of metals. Recently, a method based on the use of ferrocyanide has been described to remove iron from commercial iron chelates based on the o,o-EDDHA [ethylenediamine-N,N'bis(2-hydroxyphenylacetic)acid] chelating agent for their analysis and quantification by NMR. The present work extended that procedure to other paramagnetic ions, manganese and copper, and other chelating, EDTA (ethylenediaminetetraacetic acid), IDHA [N-(1,2-dicarboxyethyl)-d,l-aspartic acid], and complexing agents, gluconate and heptagluconate. Results showed that the removal of the paramagnetic ions was complete, allowing us to obtain (1)H NMR spectra characterized by narrow peaks. The quantification of the ligands by NMR and high-performance liquid chromatography showed that their complete recovery was granted. The NMR analysis enabled detection and quantification of unknown impurities without the need of pure compounds as internal standards.     

Development of a sensitive and specific solid phase extraction--gas chromatography-tandem mass spectrometry method for the determination of elenolic acid, hydroxytyrosol, and tyrosol in rat urine

A novel gas chromatography-tandem mass spectrometry (GC-MS/MS) method was developed, using an ion trap mass spectrometer, for the simultaneous determination of olive oil bioactive components, elenolic acid, hydroxytyrosol, and tyrosol, in rat urine. Samples were analyzed by GC-MS/MS prior to and after enzymatic treatment. A solid phase extraction sample pretreatment step with greater than 80% analytical recoveries for all compounds was performed followed by a derivatization reaction prior to GC-MS/MS analysis. The calibration curves were linear for all compounds studied for a dynamic range between 1 and 500 ng. The limit of detection was in the mid picogram level for tyrosol and elenolic acid (300 pg) and in the low picogram level for hydroxytyrosol (2.5 pg). The method was applied to the analysis of rat urine samples after sustained oral intake of oleuropein or extra virgin olive oil as a diet supplement.     

Biodegradation of buprofezin by Rhodococcus sp. strain YL-1 isolated from rice field soil

A buprofezin-degrading bacterium, YL-1, was isolated from rice field soil. YL-1 was identified as Rhodococcus sp. on the basis of the comparative analysis of 16S rDNA sequences. The strain could use buprofezin as the sole source of carbon and nitrogen for growth and was able to degrade 92.4% of 50 mg L(-1) buprofezin within 48 h in liquid culture. During the degradation of buprofezin, four possible metabolites, 2-tert-butylimino-3-isopropyl-1,3,5-thiadiazinan-4-one, N-tert-butyl-thioformimidic acid formylaminomethyl ester, 2-isothiocyanato-2-methyl-propane, and 2-isothiocyanato-propane, were identified using gas chromatography-mass spectrometry (GC-MS) analysis. The catechol 2,3-dioxygenase activity was strongly induced during the degradation of buprofezin. A novel microbial biodegradation pathway for buprofezin was proposed on the basis of these metabolites. The inoculation of soils treated with buprofezin with strain YL-1 resulted in a higher degradation rate than that observed in noninoculated soils, indicating that strain YL-1 has the potential to be used in the bioremediation of buprofezin-contaminated environments.     

Antioxidant effect of phenolic compounds, alpha-tocopherol, and other minor components in virgin olive oil

The effect of acidity, squalene, hydroxytyrosol, aldehydic form of oleuropein aglycon, hydroxytyrosyl acetate, tyrosol, homovanillic acid, luteolin, apigenin, alpha-tocopherol, and the mixtures hydroxytyrosol/hydroxytyrosyl acetate, hydroxytyrosol/tyrosol, and hydroxytyrosol/alpha-tocopherol on the oxidative stability of an olive oil matrix was evaluated. A purified olive oil was spiked with several concentrations of these compounds and, then, subjected to an accelerated oxidation in a Rancimat apparatus at 100 degrees C. Acidity, squalene, homovanillic acid, and apigenin showed negligible effect. At the same millimolar concentrations, the different o-diphenolic compounds yielded similar and significant increases of the induction time, alpha-tocopherol a lesser increase, and tyrosol a scarce one. At low concentrations of o-diphenols and alpha-tocopherol, a linear relationship between induction time and concentration was found, but at high concentrations the induction time tended toward constant values. To explain this behavior, a kinetic model was applied. The effect of the mixtures hydroxytyrosol/hydroxytyrosyl acetate was similar to that of a single o-diphenol at millimolar concentration equal to the sum of millimolar concentrations of both compounds. Concentrations of tyrosol >0.3 mmol/kg increase the induction time by 3 h. The mixtures hydroxytyrosol/alpha-tocopherol showed opposite effects depending on the relative concentrations of both antioxidants; so, at hydroxytyrosol concentrations <0.2 mmol/kg, the addition of alpha-tocopherol increased the induction time, whereas at higher hydroxytyrosol concentrations, the alpha-tocopherol diminished the stability.     

多轮次筛选选育高酚酸耐受性丁醇生产菌

纤维原料预处理过程中会产生酚酸等抑制菌株生长的物质,为选育出高丁醇产量及高耐受酚酸胁迫丁醇生产菌株,该研究利用多因子复合筛选策略筛选出一株能够合成足够还原力与对丁醇耐受性较好的菌株Clostridium beijerinckii W6。通过丁醇胁迫适应性进化获得丁醇耐受菌W6-1,其丁醇和总溶剂产量相较于菌株W6分别提高了14.01%和16.85%。通过紫外诱变处理菌株W6-1并结合理性筛选模型最终获得丁醇产量较高菌株W6-2,其丁醇及总溶剂产量分别可达到（9.51±0.06）和（15.32±0.11）g/L。最后将菌株W6-2通过酚酸胁迫适应性进化得到突变菌W6-3,其能耐受1.0 g/L酚酸胁迫环境,且丁醇和总溶剂产量相较于菌株W6-2分别提高了18.17%和17.49%。当以葛渣水解液为底物进行丙酮丁醇发酵时,突变菌W6-3的丁醇产量达（8.54±0.31）g/L,相较于菌株W6-2提高了26.71%。经多轮次诱变及适应性进化处理获得的突变菌的酚酸耐受性及发酵性能均有较大提高,该文所采用的多轮次筛选方法可以为其他快速筛选优良生产菌提供可靠的理论参考。     

Toward a high yield recovery of antioxidants and purified hydroxytyrosol from olive mill wastewaters

We investigated to develop effective procedures to recover the potentially high-added-value phenolic compounds contained in the discontinuous three-phase olive processing wastewaters (OMW). Particular emphasis was made to extract and purify hydroxytyrosol, one of the major compounds occurring in OMW. Batch optimization experiments showed that ethyl acetate is the most efficient solvent for the recovery of phenolic monomers from OMW. The latter was used with an optimal pH equal to 2. Furthermore, the percentage of each monomer, and particularly hydroxytyrosol, in the extract was maximum for a solvent ratio and a theoretical extraction stage number equal to 2 and 3, respectively. High yield (85.46%) recovery of hydroxytyrosol was achieved from OMW using a three-staged continuous counter-current liquid-liquid extraction unit. Hydroxytyrosol (1.225 g) were extracted per liter of OMW. One gram of hydroxytyrosol per liter of OMW was then purified by means of a chromatographic system which could be adapted to a large scale production process.