变形假单胞菌2-酮基葡萄糖酸差向异构酶基因的原核表达

采用降落聚合酶链式反应（touchdown polymerase chain reaction,TD-PCR）技术,从2-酮基葡萄糖酸工业生产菌株变形假单胞菌JUIM01中克隆了编码2-酮基葡萄糖酸差向异构酶的基因kguE。将目的基因与质粒pET-28a（＋）重组后转入宿主表达菌中,获得了重组菌株大肠杆菌BL21（DE3）/pET-28a（＋）-kguE。在异丙基-β-D-硫代半乳糖苷的诱导下,该重组菌株表达了一个分子质量约为30.5?ku的融合蛋白,且该蛋白的Western-blot鉴定结果显示为阳性。生物信息学分析结果表明,该蛋白是一种由256?个氨基酸残基组成的分子质量为28.5?ku的亲水性蛋白,与恶臭假单胞菌的2-酮基葡萄糖酸差向异构酶在氨基酸序列上的一致性达78%,定位于细胞质中,其二级结构中α-螺旋、延伸链和无规则卷曲所占的比例分别为40.62%、17.19%和42.19%。本研究结果为变形假单胞菌2-酮基葡萄糖酸差向异构酶的功能研究提供了一定理论支持。     

碘量法测定发酵液中2-酮基-D-葡萄糖酸

在优化样品处理条件的基础上,考察葡萄糖对2-酮基-D-葡萄糖酸测定的影响,改进发酵液中2-酮基-D-葡萄糖酸的碘量法测定技术。结果表明,滴定样品溶液所消耗的I2标准溶液的体积、样品溶液中2-酮基-D-葡萄糖酸质量浓度和葡萄糖质量浓度之间存在良好的线性关系,R2>0.9999;该方法精密度、稳定性和重现性良好,2-酮基-D-葡萄糖酸的平均回收率为95.68%,RSD为1.35%（n=5）。该方法准确、快速,操作简便,可用于发酵液中2-酮基-D-葡萄糖酸的定量检测。     

旋光法测定发酵液中2-酮基-D-葡萄糖酸

通过测定发酵液的旋光度及其葡萄糖质量浓度,计算发酵液中2- 酮基-D- 葡萄糖酸的质量浓度,并考察该方法实际应用的可行性。结果表明：对照品溶液的旋光度、2- 酮基-D- 葡萄糖酸质量浓度和葡萄糖质量浓度之间存在良好的线性关系,R2 ＞ 0.9999;该方法精密度、稳定性和重现性良好,2- 酮基-D- 葡萄糖酸的平均回收率为102.60%,RSD 为2.17%(n=5)。该方法可用于发酵液中2- 酮基-D- 葡萄糖酸的定量检测。     

变形假单胞菌2-酮基葡萄糖酸转运蛋白基因的克隆与分析

该研究旨在克隆变形假单胞菌JUIM01的2-酮基葡萄糖酸转运蛋白基因kgu T,并明确其基本的生物学信息。根据已报道的假单胞菌的基因组信息及其2-酮基葡萄糖酸操纵子的物理图谱设计简并引物,采用TD-PCR技术从变形假单胞菌中克隆到全长为1278 bp的kgu T,其核苷酸序列与Pseudomonas sp.CCOS191的编码2-酮基葡萄糖酸转运蛋白(Kgu T)的核苷酸序列的一致性为87%,编码一个由425个氨基酸残基组成的蛋白。该蛋白与Pseudomonas sp.M1的Kgu T在氨基酸序列上的一致性达90%,定位于细胞膜,是一个具有12个跨膜结构的疏水性的跨膜蛋白,无信号肽,其二级结构中α螺旋、延伸链和无规卷曲所占的比例分别为75.76%、2.12%和22.12%。本研究首次从2-酮基葡萄糖酸的工业生产用菌中克隆到基因kgu T,并对其进行了生物信息学分析,为变形假单胞菌的Kgu T的功能研究奠定了基础。     

变形假单胞菌2-酮基葡萄糖酸操纵子的克隆与分析

从变形假单胞菌JUIM01中克隆2-酮基葡萄糖酸操纵子(kgu操纵子),明确其基因组成及生物学信息。根据报道的假单胞菌的基因组信息设计简并引物,采用LA-PCR技术克隆变形假单胞菌的kgu操纵子,对克隆的基因片段进行测序,在此基础上利用生物信息学方法进行分析。结果表明:克隆到的基因片段全长为6 130bp,其中包括基因ptx S、kgu E、kgu K、kgu T和kgu D,分别编码2-酮基葡萄糖酸代谢调控蛋白Ptx S、2-酮基葡萄糖酸差向异构酶、2-酮基葡萄糖酸激酶、2-酮基葡萄糖酸转运蛋白和2-酮基葡萄糖酸-6-磷酸还原酶;这些基因与铜绿假单胞菌PAO1的kgu操纵子中相应基因的序列一致性达56.15%~76.74%,然而变形假单胞菌的ptx S很可能并不位于kgu操纵子中。研究首次从变形假单胞菌中克隆到kgu操纵子,并对其进行生物信息学分析,为变形假单胞菌2-酮基葡萄糖酸代谢机理的研究奠定了基础。     

镉超标大米水解糖发酵生产2-酮基葡萄糖酸过程中镉的迁移规律

目的 揭示以镉超标大米为原料发酵生产2-酮基葡萄糖酸过程中镉的迁移规律,并获得符合食品抗氧化剂D-异抗坏血酸钠合成要求的2-酮基葡萄糖酸。方法 以正常大米、镉超标大米和混合大米为原料,系统分析大米糖化、种子培养、2-酮基葡萄糖酸发酵、发酵产物提取等过程中镉的含量和分布,确定镉的迁移规律及其对2-酮基葡萄糖酸发酵性能和D-异抗坏血酸钠产品品质的影响。结果 大米原料和辅料碳酸钙中的镉的含量对2-酮基葡萄糖酸发酵性能影响不显著;发酵产物的提取过程有效降低了2-酮基葡萄糖酸中的镉含量,提取过程中产生的酸化废渣（主要由硫酸钙和菌体组成）可富集90%以上的镉;以镉超标大米为原料制备的D-异抗坏血酸钠的质量符合国家标准要求,未检出镉的存在。结论 该研究为综合利用镉超标大米生产2-酮基葡萄糖酸、进而生产高附加值的食品添加剂D-异抗坏血酸钠提供了理论依据和技术支撑。     

荧光假单胞菌JD1202利用大米淀粉水解糖连续发酵生产2-酮基-D-葡萄糖酸

研发荧光假单胞菌JD1202转化大米淀粉水解糖为2-酮基-D-葡萄糖酸的连续发酵工艺.考察了连续发酵起点、稀释率和补料培养基中葡萄糖浓度对2-酮基-D-葡萄糖酸生产的影响.结果表明,稀释率和葡萄糖浓度对2-酮基-D-葡萄糖酸发酵具有显著影响;在稀释率和补料培养基成分保持不变的条件下,2-酮基-D-葡萄糖酸大量合成期的任何时段均可作为连续发酵的起点,其细胞浓度、残糖浓度和2-酮基-D-葡萄糖酸浓度均稳定在一定范围.较适的连续发酵操作条件为:稀释率0.065h-1,补料培养基中葡萄糖的质量浓度为170.0g/L.在此条件下,连续发酵达到稳态时发酵液中2-酮基-D-葡萄糖酸的质量浓度为130.34g/L,生产强度平均为8.47g/L·h,发酵转化率为89.46％.连续发酵工艺是一种高效的2-酮基-D-葡萄糖酸生产方法,有望应用于工业生产中.     

补料分批培养生产2-酮基-D-葡萄糖酸的研究

研究了补糖对荧光假单胞菌(Pseudomonas fluorescens)AR4生产2-酮基-D-葡萄糖酸的影响,并在50kL发酵罐上进行了补料分批培养工业应用性试验。研究结果表明,采用补料分批培养方法,能有效提高发酵液中的产物浓度;开始补糖时发酵液中的残糖浓度以3%～6%为宜;补料分批培养方法及AR4菌株可用于2-酮基-D-葡萄糖酸的工业化规模生产中。     

荧光假单胞菌AR4在2-酮基-D-葡萄糖酸工业生产中的应用研究

在噬菌体存在的情况下,采用抗噬菌体菌株荧光假单胞菌(Pseudomonasfluorescens)AR4在200m3发酵罐上进行了2-酮基-D-葡萄糖酸的发酵生产。结果表明,荧光假单胞菌AR4对生产环境中噬菌体的抗性稳定,发酵周期短,发酵转化率高;经过10次传代,荧光假单胞菌AR4对噬菌体的抗性及其发酵生产2-酮基-D-葡萄糖酸的能力没有改变;该菌株的发酵产物可以用于食品抗氧化剂D-异抗坏血酸钠的合成。     

球形节杆菌C224分批发酵2-酮基-D-葡萄糖酸的条件优化及其动力学模型的构建

通过优化发酵条件,提高球形节杆菌C224菌株利用大米淀粉水解糖分批发酵2-酮基-D-葡萄糖酸的生产强度,并在此基础上构建其发酵动力学模型。在50 L的发酵罐上考察了通气量与葡萄糖浓度对2-酮基-D-葡萄糖酸发酵的影响,并基于Logistic方程、Luedeking-Piret方程和物料平衡计算分别构建了菌体生长、产物形成和底物消耗的动力学模型。研究结果表明,通气量低于1.5 v.v-1.m-1时,发酵生产强度明显减小,糖酸转化率有所降低;发酵培养基中的葡萄糖浓度以120.0～180.0 g/L为宜,过高或过低对生产强度和糖酸转化率均有不利影响。在适宜的发酵条件下,球形节杆菌C224菌株分批发酵生产2-酮基-D-葡萄糖酸的生产强度达6.36～6.54 g/(L.h),糖酸转化率为0.96～0.97 g/g。所构建动力学模型的计算值与实验值拟合效果良好,各项模型的相关系数R2均大于0.95,说明其能够揭示球形节杆菌C224分批发酵2-酮基-D-葡萄糖酸代谢基本特征。     

气调贮藏对腐败菌引起的鲜切黄瓜品质、滋味和挥发性物质变化的影响

本研究采用电子舌、电子鼻和气相色谱-质谱等手段,探讨3%（体积分数,下同）O2＋7% CO2气调贮藏对变形假单胞菌导致的鲜切黄瓜营养品质、滋味和挥发性物质变化的影响。结果表明,变形假单胞菌生长影响了鲜切黄瓜的营养品质,气调贮藏抑制了鲜切黄瓜的呼吸作用,延缓了丙二醛含量和相对电导率的增加,维持了可溶性固形物质量分数和色泽,保持了鲜切黄瓜硬度。电子舌分析结果表明变形假单胞菌对鲜切黄瓜鲜味和鲜味丰度影响最为明显,气调贮藏抑制了变形假单胞菌导致的各滋味变化。不同处理鲜切黄瓜中挥发性物质存在差异。与接菌空气贮藏组相比,气调贮藏延缓了由变形假单胞菌引起的黄瓜特征香气正己醛、(E,Z)-2,6-壬二烯醛和(Z)-6-壬烯醛相对含量的下降,抑制了2,3-二甲基辛烷、6-甲基-3-庚酮、2-正丙基呋喃和二丙基二硫醚这些特征代谢物的产生。本实验结果可为研究微生物腐败引起的黄瓜营养与风味变化提供理论参考。     

气调箱贮藏对鲜切黄瓜品质的影响及对假单胞菌的抑制作用

鲜切黄瓜在国内外具有很大的消费市场,但在贮藏期间极易受微生物污染而腐败变质。变形假单胞菌是低温贮藏条件下导致鲜切黄瓜腐败的主要微生物之一。该研究以变形假单胞菌为实验菌株,接种至鲜切黄瓜表面后,探讨3%O2、7%CO2气调箱贮藏对菌株生长和侵染情况,对黄瓜细胞壁降解和细胞壁降解引起的生理生化变化的影响。结果表明,与对照组相比,3%O2、7%CO2气调贮藏抑制了变形假单胞菌的生长,保持了细胞壁的完整性,延缓了原果胶的降解,降低了纤维素酶活性,抑制了β-半乳糖苷酶活性的增加和可溶性果胶的产生,从而保持了硬度,延缓了丙二醛含量和相对电导率的增加,维持了鲜切黄瓜的外观和色泽。总之,3%O2、7%CO2气调箱贮藏抑制了由变形假单胞菌导致的鲜切黄瓜细胞壁的降解,延缓了腐败变质。     

精氨酸脱亚胺酶基因在大肠杆菌中的克隆、表达及纯化

通过PCR扩增得到菌株编码ADI的arcA基因,并构建表达载体pET24a-ADI。将该载体导入大肠杆菌BL21(DE3),获得高效表达ADI基因的重组菌。对ADI诱导表达条件进行优化,结果发现宿主菌在A600达到1.0时加入0.2 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG),在30℃诱导4 h酶活最高,为2.04 U/mL发酵液。经超声波破碎、HiPrep DEAE FF阴离子交换层析、SuperdexTM200凝胶过滤层析,可获得SDS-PAGE电泳纯重组精氨酸脱亚胺酶(rADI)。rADI相对分子质量大小为92 600,由两个相同亚基组成,纯化后的rADI比酶活达20.9 U/mg。     

变形假单胞菌吡咯喹啉醌合成基因簇的克隆与分析

从变形假单胞菌JUIM01中克隆到吡咯喹啉醌(PQQ)合成基因簇,阐明了其基因组成和生物学信息。根据已报道的假单胞菌的基因组进行简并引物设计,采用LA-PCR技术克隆变形假单胞菌的PQQ合成基因簇,对克隆的基因片段进行测序并使用生物信息学方法进行综合分析。结果表明:克隆到的基因片段全长为11 659 bp,其中包括pqqF、pqqA、pqqB、pqqC、pqqD、pqqE、pqqM、pqqH和pqqI共9个基因,编码PQQ生物合成的前体短肽PqqA和合成途径的相关酶;这些基因与荧光假单胞菌Pf0-1的PQQ合成基因簇的基因组成类似,相应基因的序列一致性达41%～94%。本研究中首次从变形假单胞菌中克隆到PQQ合成基因簇,并对其进行生物信息学分析,为变形假单胞菌的PQQ生物合成途径和胞内再生机制的研究奠定了基础,进而为提高2KGA的生产强度提供了理论支撑。     

Development of a Single Kernel NIR Barley Protein Calibration and Assessment of Variation in Protein on Grain Quality

In this paper, we describe results from a preliminary experiment on the development of a near infrared reflectance (NIR) calibration for single kernel (SK) % protein content in barley. The SKNIR calibration was developed using kernels from breeding lines and commercial barley varieties with a range in protein content of 7.3% to 16.6% “as is”. The calibration model produced an R² = 0.903, while the validation set had a R² = 0.837 with a standard error of cross validation and a standard error of prediction of 0.8% for both the calibration and validation sets respectively. The calibration was then used to estimate the variation in % protein of 4,000 single kernels from a commercial variety (Gairdner at 9.3% protein) by segregating kernels into six sub‐groups (<7.8%, 7.9–8.3%, 8.4–9.0%, 9.1–9.7%, 9.8–10.4%, >10.5% “as is”). These sub‐groups then had additional grain quality tests carried out including grain size, thousand kernel weight and NIR estimates of % protein, starch, hardness and barley hot water extract (HWE). The results showed an increase in grain size, and a decrease in HWE from the low to high % protein sub‐groups. While only a single variety was used in the SKNIR protein segregation study, the results suggested SKNIR could be used to screen for the variation in grain quality traits based on variation in protein content.     

Viscosity changes and gel formation during storage of liquid micellar casein concentrates

Our objective was to determine the effects of temperature and protein concentration on viscosity increase and gelation of liquid micellar casein concentrate (MCC) at protein concentrations from 6 to 20% during refrigerated storage. Skim milk (∼350 kg) was pasteurized (72°C for 16 s) and filtered through a ceramic microfiltration system to make MCC and replicated 3 times. The liquid MCC was immediately concentrated via a plate ultrafiltration system to 18% protein (wt/wt). The MCC was then diluted to various protein concentrations (6–18%, wt/wt). The highest protein concentrations of MCC formed gels almost immediately on cooling to 4°C, whereas lower concentrations of MCC were viscous liquids. Apparent viscosity (AV) determination using a rotational viscometer, gel strength using a compression test, and protein analysis of supernatants from ultracentrifugation by the Kjeldahl method were performed. The AV data were collected from MCC (6.54, 8.75, 10.66, and 13.21% protein) at 4, 20, and 37°C, and compression force test data were collected for MCC (15.6, 17.9, and 20.3% protein) over a period of 2-wk storage at 4°C. The maximum compressive load was compared at each time point to determine the changes in gel strength over time. Supernatants from MCC of 6.96 and 11.61% protein were collected after ultracentrifugation (100,605 × g for 2 h at 4, 20, and 37°C) and the nitrogen distributions (total, noncasein, casein, and nonprotein nitrogen) were determined. The protein and casein as a percent of true protein concentration in the liquid phase around casein micelles in MCC increased with increasing total MCC protein concentration and with decreasing temperature. Casein as a percent of true protein at 4°C in the liquid phase around casein micelles increased from about 16% for skim milk to about 78% for an MCC containing 11.6% protein. This increase was larger than expected, and this may promote increased viscosity. The AV of MCC solutions in the range of 6 to 13% casein increased with increasing casein concentration and decreasing temperature. We observed a temperature by protein concentration interaction, with AV increasing more rapidly with decreasing temperature at high protein concentration. The increase in AV with decreasing temperature may be due to the increase in protein concentration in the aqueous phase around the casein micelles. The MCC containing about 16 and 18% casein gelled upon cooling to form a gel that was likely a particle jamming gel. These gels increased in strength over 10 d of storage at 4°C, likely due either to the migration of casein (CN) out of the micelles and interaction of the nonmicellar CN to form a network that further strengthened the random loose jamming gel structure or to a gradual increase in voluminosity of the casein micelles during storage at 4°C.     

Production of a high‐protein meal and fermentable sugars from defatted soybean meal,a co‐product of the soybean oil industry

The goal of this research was to produce a high‐protein meal by treating defatted soybean meal, a by‐product of soybean oil production, with dilute acid. Treatments were a mild hydrolysis at 80 °C with sulphuric acid at concentrations ranging from 0.5% to 2.0% (w/v) and times varying from 1 to 16 h that were arranged according to a central composite rotatable experimental design. The end products were an enhanced‐protein meal and a carbohydrate concentrate of fermentable and nonfermentable sugars. The highest protein content rise, from 48% to 58%, was for treatments with concentrations of acid ranging between 1.2% and 1.7% and times between 1.0 and 2.6 h. The maximum yield of fermentable sugar was 21.0% d.b. at 2.0% H₂SO₄ and treatments of at least 6 h. The conditions that provide a highest protein and sugar contents were the treatments with concentrations of sulphuric acid ranging from 0.9 to 1.9% H₂SO₄ for 1–4 h.     

Extrusion-enzyme liquefaction as a method for producing sorghum protein concentrates

A novel method was developed for concentrating proteins from sorghum flour utilizing a combination of extrusion and α-amylase treatment for starch liquefaction. A central composite design was used to optimize in-barrel moisture content (MC), enzyme concentration during extrusion (E1) and post-extrusion enzyme concentration (E2) in order to produce sorghum protein concentrates with high protein content (PC) and in vitro protein digestibility (D). Extrusion-enzyme liquefaction yielded concentrates with higher protein yield (82 db%) and digestibility (66%) than batch liquefaction alone because extrusion promoted starch and protein degradation. The optimum conditions for developing a sorghum protein concentrate with both high yield and digestibility were 32% MC, no E1, and 2.5% E2. The sorghum protein concentrate developed in this study can augment the nutritional value of gluten-free foods for individuals suffering from celiac disease and other forms of gluten and wheat intolerance.     

Effect of frozen storage on thermal stability of sarcoplasmic protein and myofibrillar protein from common carp (Cyprinus carpio) muscle

Thermal stability of sarcoplasmic protein and myofibrillar protein extracted from fresh and frozen common carp was comparatively studied. Total sulphydryl content (SH) in sarcoplasmic protein solution from 5‐month frozen carp decreased by 19.43% compared with fresh sample. The SDS‐PAGE patterns showed that all the bands of sarcoplasmic protein from frozen‐stored samples were almost invisible at 80 °C. Myofibrillar protein from fresh sample exhibited lower turbidity and surface hydrophobicity and higher Ca²⁺‐ATPase activity and SH content than frozen‐stored sample when heated from 20 to 80 °C. The Ca²⁺‐ATPase activity from fresh (M0), 2 (M2)‐ and 5 (M5)‐month frozen‐stored carp was completely lost at 48, 46 and 46 °C, respectively. When heated to 80 °C, the SH content of myofibrillar solutions in M0, M2 and M5 decreased by 26%, 60% and 70%, respectively. Sarcoplasmic and myofibrillar proteins from frozen carp were more susceptible to aggregate during heating treatment.