Investigation of a multienzyme based amperometric biosensor for determination of sucrose in fruit juices

Determination of sucrose content is one of the most important routinely performed tests for quality control in the beverage industry. Thus, it is rather important to have a fast, simple and sensitive method that can be used for determination of sucrose in sweet-drink processing. The biosensor investigated was based on a thin layer enzyme cell in which glucose oxidase, invertase and mutarotase were immobilized on a protein membrane. Hydrogen peroxide as the product of the consecutive enzyme reaction was detected by amperometric technique. The optimum enzyme composition of invertase, mutarotase, and glucose oxidase for immobilisation was found to be 8498, 672, and 120 U using 2.2 mg BSA as protective agent dissolved in 200 μL phosphate buffer solution. To achieve the best measuring technique, changes in several parameters such as pH value, temperature and flow rate were studied. Having found the optimum conditions, the statistical parameters of the analysis were established. The linear range of the determination was between 1.0 × 10⁻⁴ and 2.5 × 10⁻³ mol/L with a correlation coefficient of 0.99. The sucrose content of several fruit juices was determined with this sensor, and the results were compared with an enzymatic reference method. The correlation between the two sucrose determination methods was found to be 0.97.     

大豆多酚氧化酶、过氧化物酶的酶学特性研究

对大豆氧化还原酶系中的多酚氧化酶和过氧化物酶的酶学特性进行了较深入系统地研究,其中包括酶活力、酶热稳定性、pH值的影响,以及大豆卵磷脂、二巯基丙醇对酶活力的抑制作用.结果表明多酚氧化酶在120℃时,酶活力值已达到不可逆失活状态,pH6～7时酶活力最大,当二巯基丙醇的浓度为1.5mmol/L时其酶活力降至6%以下.过氧化物酶具有较高的耐热性,酶活力最适的pH值为5.5左右,大豆卵磷脂浓度达到80mg/L时其酶活力可降至20%左右.     

Hydrolysis of sucrose by invertase immobilized on nylon-6 microbeads

A commercial extracellular invertase (EC 3.2.1.26) from Saccharomyces cerevisiae has been inmobilized by covalent bonding on novel microbeads of nylon-6 using glutaraldehyde. The enzyme was strongly bound on the support, immobilized with an efficiency factor of 0.93. The biocatalyst showed a maximum enzyme activity when immobilized at pH 5.0, but optimum pH activity for both immobilized and free invertases was 5.5. The optimum temperatures for immobilized and free enzymes were 60 and 65 °C, respectively. Kinetic parameters were determined for immobilized and free invertases: V_max values were 1.37 and 1.06 mmol min⁻¹ mg⁻¹, respectively. The K_m and K_i values were 0.029 and 0.71 M for immobilized invertase and 0.024 and 0.69 M for free invertase. It was found that the thermal stability of the immobilized invertase with regard to the free one increased by 25% at 50 °C, 38% at 60 °C and 750% at 70 °C. The immobilized biocatalyst was tested in a tubular fixed-bed reactor to investigate its possible application for continuous sucrose hydrolysis. The effects of two different sugar concentrations and three flow rates on the productivity of the reactor and on the specific productivity of the biocatalyst were studied. The system demonstrated a very good productivity up to 2.0 M sugar concentration, with conversion factors of 0.95 and 0.97, depending on sucrose concentration in the feeding. This approach may serve as a simple technique and can be a feasible alternative to continuous sucrose hydrolysis in a fixed bed reactor for the preparation of fructose-rich syrup.     

Glass transition and water effects on sucrose inversion in noncrystalline carbohydrate food systems

The effects of water and glass transition on the hydrolysis of sucrose by invertase in noncrystalline carbohydrate systems were investigated. Maltodextrin/sucrose (2:1) and maltodextrin/lactose/sucrose (1:1:1) were dissolved in distilled water. Invertase (10 mg/17.2 g) was added. Amorphous samples were produced by freeze-drying the solutions. Sorption isotherms were determined gravimetrically at 24 °C over the 0.113–0.763 a_w, and over 0.239–0.764 a_w, the glass transition, T_g was determined using differential scanning calorimetry (DSC). DSC and water sorption results suggested that samples remained noncrystalline. Sucrose inversion was analysed by monitoring glucose content during storage. Sucrose hydrolysis occurred at significant rates at 0.662 and 0.764 a_w. The rate increase was not related to the apparent glass transition of the systems.     

Effects of enzymatic modification of soybean protein on the pasting and rheological profile of starch–protein systems

Reformulation of traditional food systems to introduce new ingredients may change their structure and perceived texture. Interactions between proteins and starch during processing can markedly influence starch gel network structure and rheological profile. The present work aimed to study the effects of soybean protein and the products of enzymatic modification on the pasting and rheological profile of corn and cassava starch. The behavior of those protein‐enriched gels during storage was also assessed. Soybean protein isolate (SPI) was incubated with endopeptidase (AL) or food grade microbial transglutaminase (TG). Pasting and rheological behavior, water retention capacity, and structure of protein– and hydrolyzed protein–starch gels were analyzed. Protein incorporation increased the viscosity of starch suspension during and after heating. SPI‐modified proteins increased peak viscosity. Only the structural modifications brought by TG on SPI increased the final viscosity during starch pasting and the storage modulus (G′). This modulus (G′) of the gelled systems decreased with the addition of AL‐treated protein isolate. Light and fluorescence microscopy showed that SPI formed a continuous phase, like a network, in the gelled system. Different network structures and rheological properties can be obtained when SPI are modified by protease and TG enzymes, which may be very useful for designing new food products.     

大豆油酶法脱胶与吸附脱酸工艺的优化研究

本研究以大豆毛油为原料,采用酶法脱胶和物理脱酸法对其进行精炼处理。通过单因素实验和正交实验进行工艺优化,以获得合适的大豆油精炼条件。结果表明：采用磷脂酶C酶法脱胶的最佳工艺条件为pH 5.4,酶添加量10.0 μL/kg,酶解温度45.0 ℃。在此条件下,大豆毛油磷含量可降低至7.3 mg/kg;最佳的脱酸工艺条件为保温时间40.0 min、加水温度90.0 ℃、加水量3.5%、絮凝剂量0.7%,此条件下大豆油酸值为0.08 mg/g,磷含量为5.5 mg/kg。大豆毛油经酶法脱胶和物理脱酸处理后的磷含量和酸价显著降低,品质提高。实验结果为大豆油的非化学精炼提供了依据。     

Development of monoclonal antibodies and a competitive ELISA detection method for glycinin,an allergen in soybean

Soybean glycinin is a major food allergen causing anaphylaxis. A sensitive detection method for glycinin is needed to evaluate soybean allergies in food and feed products. In the present study, monoclonal antibodies (Mabs) against glycinin were prepared using purified glycinin as the immunogen. The generated Mabs, named 3B2 and 4B2, were identified as being IgG_2b and IgG_2a iso-types respectively, and exhibited high specificity to glycinin. Then we developed a competitive ELISA based on Mab 4B2 to measure glycinin which showed an IC₅₀ value of 1.7 ng/mL with a detection limit of 0.3 ng/mL, and the linear portion of the curve was 0.3–11.2 ng/mL. Recovery tests indicated that the competitive ELISA based on Mab 4B2 gave reliable reproducibility. The produced Mab 4B2 and the developed ELISA could provide a valuable tool for sensitive determination of glycinin and for future studies conducted to reveal the mechanism of how glycinin functions in anaphylaxis.     

响应面法优化大豆油脂肪酸乙酯合成工艺

以大豆油为原料,KOH作催化剂,通过大豆油与乙醇的酯交换反应合成了大豆油脂肪酸乙酯。应用响应曲面分析法中的Box-behnken模型对影响大豆油脂肪酸乙酯转化率的四个主要因素（催化剂用量、醇油摩尔比、反应温度、反应时间）进行了优化。研究表明大豆油脂肪酸乙酯的最佳合成工艺条件为：KOH用量1.3%,醇油比8.3∶1,反应温度74.8℃,反应时间130min。在此条件下,酯转化率达98.93%。     

Reducing cracking and breakage of soybean grains under combined near-infrared radiation and fluidized-bed drying

Near-infrared radiation was combined with fluidized-bed drying to reduce the cracking and breakage of soybean grains because of its advantages such as a decrease in the moisture gradient and the stresses development within the grain kernel, leading to high quality of product. Physical qualities, i.e. cracking, breakage and colour, were investigated together with microstructure of soybean grains. Protein solubility and urease activity were also determined. The parameters studied were near-infrared radiation powers of 4, 6 and 8 kW, air velocity of 4.5 m/s, air temperature of 40 °C and grain bed depth of 6 cm. Results showed that the drying rate was increased with the increase of near-infrared radiation power. The moisture content was reduced from an initial moisture content of 20% d.b. to the final moisture contents of 13.5% d.b. (4 kW), 12.8% d.b. (6 kW) and 12.5% d.b. (8 kW). The cracking and breakage of soybean grains occurred negligible, which was lower than 4.4% and 5.3% for cracking and breakage, respectively. At near-infrared radiation powers of 4 and 6 kW was accepted for both soybean grains trade and animal feed industries in Thailand. The total colour difference changes (ΔE) were varied in the range of to 2.9-4.2. The protein solubility of final product was in an acceptable range (77-82%) for feed meal. The reduction of residual urease was varied in the range of to 39-70%. This study has demonstrated that the soybean grains under combined near-infrared radiation and fluidized-bed drying was properly treated.     

大豆品种抗旱性早期鉴定方法

为探讨鉴定大豆品种抗旱性的简易方法,依据吸水后48h的发芽率对109个大豆种质材料进行抗旱性分级。继而在萌发期、营养生长期（V3）和生殖生长期（R4）进行盆栽控水试验,根据抗旱等级验证大豆植株的抗旱性。结果发现,大豆品种间抗旱性存在显著差异。种子吸水量与抗旱性无关,吸水后48h发芽率与抗旱性密切相关。幼苗侧根数量、主根长度和根系干重与抗旱性呈显著（P〈0.05）相关。对萌发后幼苗供水24h后,随即停水24h,反复干旱处理,抗旱品种存活率显著高于普通品种。在V3期对盆栽植株进行暂时萎蔫（土壤含水量为9%～11%）的水分胁迫处理,发现停水后普通品种叶片首先发生萎蔫,且株高、茎直径及叶面积下降的程度均高于抗旱品种。水分胁迫下,抗旱品种株高、茎直径和叶片面积均显著（P〈0.05）高于普通品种,其中茎直径和叶片面积达到极显著水平（P〈0.01）。在R4期暂时萎蔫的水分胁迫下,抗旱品种根的生物量和分枝的生物量显著（P〈0.05）高于普通品种。鼓粒期（R6）普通品种发生永久萎蔫、植株死亡,而抗旱品种仍能保持生长能力。研究结果表明,大豆种子48h发芽率可作为大豆品种抗旱性的鉴定和筛选方法,鼓粒期大豆对水分胁迫最敏感。     

Structure characterization and IgE-binding of soybean 7S globulin after enzymatic deglycosylation

This study investigated the effects of enzymatic deglycosylation following ultrasound pretreatment on structure and immunoreactivity of soybean 7S globulin. Soybean 7S globulin was pretreated by ultrasound (40 kHz, 300 W) and enzymatically deglycosylated by peptide-N-glycosidase F (PNGase F). Changes in structure of processed soybean 7S globulin were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, reversed-phase high-performance liquid chromatography, ultraviolet absorption spectrum, circular dichroism spectrum, and surface hydrophobicity analysis. Enzyme-linked immunoabsorbent assay was used to evaluate IgE-binding ability. The results showed that the glycan moieties of soybean 7S globulin were effectively removed by PNGase F, which significantly modified protein structures including the secondary and tertiary structures of 7S globulin. Individual enzymatic deglycosylation could reduce IgE-binding capacity of 7S globulin, whereas enzymatic deglycosylation following ultrasound pretreatment enhanced its IgE-binding capacity. In conclusion, soybean 7S globulin treated by single enzymatic deglycosylation can reduce potential allergenicity and may be employed in hypoallergenic food preparation.     

大豆抗SCN机制及抗病相关基因研究进展

摘要：大豆胞囊线虫（soybeancystnematode，SCN）引起的病害能给大豆生产造成极大损失。目前在SCN抗性机制研究方面的结果表明，大豆通过分泌一些小分子物质，如防御酶系、酚类代谢物质、植保素等来抑制SCN的发育，是大豆对SCN产生抗病性的一种抗性机制；在抗病相关基因研究方面，大豆中的r恬1和rhg4位点是SCN病最重要的抗性位点，SCN中的果胶酸裂解酶（pel）、抗甜菜线虫同系物GmHs^Ipro-1、效应蛋白30C02、受体激酶GmR—LK18—1是SCN病的抗性的关键基因和蛋白。本文针对大豆抗SCN机制研究及抗病相关基因克隆研究进展进行综述，以期为SCN的抗病分子育种研究提供参考。     

酶水解法提高大豆蛋白水解度的研究

用蛋白酶水解方法提高大豆分离蛋白水解度.通过对多种蛋白酶的对比分析可知,风味蛋白酶的水解效果好于其它蛋白酶,但正交试验结果表明,即使在优化条件下水解,单一的风味蛋白酶水解所得大豆分离蛋白水解度最高只达到43.12%.若先用胃蛋白酶水解再用风味蛋白酶水解则水解度最高可达68.81%,而风味蛋白酶与其它酶的联合应用效果略差;若先使用风味蛋白酶后使用胃酶则水解度只有47.89%.表明不同酶对大豆蛋白分子具有不同的水解特点.     

Structural property of soybean lunasin and development of a method to quantify lunasin in plasma using an optimized immunoassay protocol

Lunasin is a 43-amino acid naturally occurring chemopreventive peptide with demonstrated anti-cancer and anti-inflammatory properties. The objectives of this study were to determine the effect of temperature on the secondary structure of lunasin, to develop a method of isolating lunasin from human plasma using an ion-exchange microspin column and to quantify the amount of lunasin using an optimized enzyme-linked immunosorbent assay. Lunasin was purified using a combination of ion-exchange chromatography, ultrafiltration and gel filtration chromatography. Circular dichroism showed that increased in temperature from 25 to 100 °C resulted in changes on the secondary structure of lunasin and its capability to interact with rabbit polyclonal antibody. Enzyme linked immunosorbent assay showed that lunasin rabbit polyclonal antibody has a titer of 250 and a specific activity of 0.05 mL/μg. A linear response was detected between 16 to 48 ng lunasin per mL (y = 0.03x − 0.38, R² = 0.96). The use of diethylaminoethyl microspin column to isolate spiked lunasin in human plasma showed that most lunasin (37.8–46.5%) bound to the column eluted with Tris–HCl buffer, pH 7.5 with a yield up to 76.6%. In conclusion, lunasin can be isolated from human plasma by a simple DEAE microspin column technique and can be quantified using a validated and optimized immunoassay procedure. This method can be used directly to quantify lunasin from plasma in different human and animal studies aiming to determine its bioavailability.     

大豆EST—SSR标记开发及与Genomic—SSR的比较研究

对458220条大豆EST序列进行SSR搜索,共检测出EST—SSR序列39989条,经拼接得到无冗余EST—SSR序列8190条,包括357种重复基元。其中二、三核苷酸重复基元类型居多,分别占无冗余EST总数的11．13％和16％,统计得到二核苷酸重复类型12种,三核苷酸重复类型60种。以含有简单重复序列的元冗余EST序列设计200对引物,其中148对引物有清晰且单一条带扩增产物,以30份大豆品种资源进行引物筛选,获得多态性引物31对。以21份大豆不同基因型的基因组DNA为模板选取30对显示多态性的大豆EST—SSR引物和30对大豆基因组SSR引物进行扩增,带型统计结果显示：大豆EST—SSR与基因组SSR在供试基因型间多态性指数均值分别为0．55和0．44,二者揭示的多态性水平差异不大。从而说明利用生物信息学方法基于大豆EST开发SSR标记是切实可行的,大豆EST—SSR可以用于大豆遗传多样性分析,是大豆DNA分子标记体系的一个重要补充。     

Measurement of glucose,sucrose and lactose in food samples with enzyme‐immobilised packed‐bed column reactors integrated to an amperometric enzyme electrode

An amperometric enzyme electrode integrated with packed‐bed column reactors containing immobilised enzymes was improved to measure glucose, sucrose and lactose content of food samples with only one sampling. Performance parameters of the system were investigated on the bases of linearity, sensitivity and response time. Those values were found to be 20 mM , 24.9 nA/mM and 20 s for glucose; 20 mM , 12.0 nA/mM and 3 min for sucrose; 12 mM , 10.8 nA/mM and 4 min for lactose, respectively. Overall analysis time for three analytes with one sampling was less than 8 min without pretreatment and interference of electroactive compounds and substrates. Glucose, sucrose and lactose content of the model and food samples were measured with both the improved system and commercial enzyme kits. There was high correlation between the results. This gives us the opportunity to use the improved electrode system for determination of glucose, sucrose and lactose in food samples. Also this result was found to be very promising in extending the spectrum of detectable analytes simply by addition of new columns containing immobilised enzymes that are specific to target analytes.