为提高青春双歧杆菌培养基的菌体数量,添加莲子低聚糖作为培养基的单一碳源,采用部分因子设计法确定培养基中对菌体干质量影响较大的因子,快速登高法逼近最大响应区域.在此基础上,利用中心旋转组合设计法进行响应面优化,通过SAS软件回归分析确定培养基各营养成分的最佳质量浓度.研究结果表明:影响青春双歧杆菌生长的培养基主要因子依次为莲子低聚糖、胰蛋白胨和pH值.添加莲子低聚糖后,青春双歧杆菌增菌培养基的最佳发酵条件为:大豆蛋白胨5 g/L,莲子低聚糖7.91 g/L,胰蛋白胨3.66 g/L,酵母粉10 g/L,吐温-80 1 mL,L-半胱氨酸盐酸盐0.5 g/L,无机盐溶液40 mL,pH 7.04.优化后,培养基中短链脂肪酸含量显著提高,菌体干质量达到4.722 g/L,活菌数为(2.92±0.13)×109 CFU/mL.

Lotus seed resistant starch (LRS) is commonly known as resistant starch type 3 (LRS3). The objective of this study was to investigate the effect of different preparation methods on the structural characteristics and physicochemical properties of LRS3. The molar mass of LRS3 prepared by autoclaving method (GP-LRS3) and ultrasonic-autoclaving method (UP-LRS3) was mainly distributed in the range 1.0 × 104–2 × 104 g/mol while a decrease of LRS3 prepared by microwave-moisture method (MP-LRS3) was observed. The particle of MP-LRS3 was smaller and relatively smoother while UP-LRS3 was bigger and rougher compared to GP-LRS3. Among these samples, GP-LRS3 exhibited the highest degree of ordered structure and crystallinity, the amorphous region of MP-LRS3 was the biggest and UP-LRS3 displayed the highest degree of double helical structure. Additionally, MP-LRS3 displayed the strongest solubility and swelling power while UP-LRS3 exhibited the strongest iodine absorption ability and thermostability, which were affected by their structural characteristics.

Iron is considered as an essential element for all living organisms. Therefore, limiting iron availability may be key part of the host's innate immune response to various pathogens. Ferritin is a major iron storage protein in living cells and plays an important role in iron homeostasis. One way the host can transiently reduce iron bioavailability is by ferritin over expression. In invertebrates, ferritin was found to be up-regulated after pathogens challenge and is considered to be an important element in the innate immune system. This study was designed to investigate the involvement of ferritin in shrimp Litopenaeus vannamei defense against WSSV. We discovered that the viral load of shrimp injected with recombinant ferritin protein was lower than that of control group. The suppression of ferritin by dsRNA increased susceptibility to WSSV with 3-fold high viral copies. The present study documented that ferritin protected shrimp L. vannamei from WSSV by inhibiting virus replication. We presume that ferritin reduce iron availability, leading to inhibit the activity of ribonucleotide reductase and delay the replication of virus genome. This study provided new insights into the understanding of molecular responses and defense mechanisms in shrimp against WSSV.

Lotus seed native starch was applied to prepare lotus seed retrograded starch and used as raw material by microwave method. The process parameters of lotus seed resistant starch were optimized by using single factor and orthogonal optimization analysis. The results indicated that starch concentration had the greatest impact on the yield of lotus seed resistant starch, microwave treatment time and microwave power was followed.The best conditions for microwave preparation were starch concentration 15%, microwave treatment time 120 s, microwave power 640 W, the yield of resistant starch was 39.56%under these conditions, less than autoclaving and ultrasonic-autoclaving process. However,with microwaving process, lotus seed resistant starch was easy to acquire and needed shortest time,it can be used wildly in industrial applications or large quantities of preparation in the laboratory.

采用响应面分析法对莲子低聚糖热水浸提工艺参数进行优化,探讨浸提时间(min)、浸提温度(℃)和水料比(V/W)对热水浸提莲子低聚糖得率的影响,建立提取莲子低聚糖的二次项数学模型并验证其可靠性,得到最优提取工艺参数,并采用高效液相色谱法对莲子低聚糖组分进行初步分析.结果表明:热水浸提莲子低聚糖的最佳提取工艺条件是浸提时间为66 min,浸提温度为81℃,水料比为80∶1,在此工艺条件下莲子低聚糖得率为8.09％.影响低聚糖提取工艺的主次因素顺序为:水料比＞浸提温度＞浸提时间.高效液相色谱法测定莲子低聚糖,发现莲子低聚糖由四聚糖、三聚糖和二聚糖组成.

In order to study the structure of lotus（Nelumbo nucifera Gaertn） seed oligosaccharides and their effect on the proliferation of Bifidobacterium adolescentis, we extracted the oligosaccharides from seeds collected from Jianning County, China. We preliminarily characterized the groups, molecular weights, molecular formulae, component monosaccharides and glycosidic bonds using mass spectrometry（MS） and nuclear magnetic resonance（NMR） after isolation and purification. The lotus seed oligosaccharides contained glycosidic bonds Manp-（1→）, Galp-（1→）, α（1→6）-Glup and α（1→6）-Manp; and mannose was the chief component monosaccharide. NMR analyses showed that α-glycosidic bonds and pyranoid rings were predominant in the oligosaccharides. The MS analyses showed that lotus seed oligosaccharides consisted of three oligosaccharides of different polymerization degree, with relative molecular weights of 342, 504 and 666 Da, and corresponding molecular formulae C12H22O11, C18H32O16 and C24H42O21. Research on the effect of lotus seed oligosaccharides on the proliferation of B. adolescentis showed that they effectively promoted the production of acetic, propionic and butyric acids by B. adolescentis through fermentation, and their effect was stronger than that of fructo-, xylo- and isomalto-oligosaccharides. Lotus seed oligosaccharides have potential as a new functional probiotic and lotus seeds should be further explored and utilized as a source of oligosaccharides.

Lotus seed native starch was obtained by the wet milling method,and the ultrasonic pretreatment combining autoclaving method was applied to prepare resistant starch. The influence of starch concentration,ultrasonic power,ultrasonic time,autoclaving temperature,and autoclaving time on the yield of lotus seed resistant starch was explored. The technology parameters of preparing resistant starch were optimized by orthogonal optimization analysis. The results revealed that the best conditions for lotus seed resistant starch preparation by ultrasonic-autoclaving were starch concentration 45%,ultrasonic power300 W,ultrasonic treatment time 55 min,autoclaving time 15 min,and autoclaving temperature 115 ℃.Under the conditions,the yield of lotus seed resistant starch was approximated to 56. 12%.

The SOD gene was cloned by RT-PCR from colon carcinoma cell line Colo320. After digested by BamH I and Sma I, it was inserted into the plasmid pGEX-4T-2 with the right reading frame sequence to construct the expression vector, which can express the fusion protein with GST tag. The fusion protein was expressed after inducing with IPTG and purified for antibody preparation by Glutathione Sepharose affinity chromatography.

The Vp28 gene was cloned by PCR from White Spot Syndrome Virus.After digested by Not I and Sma I,it was inserted into the plasmid pGEX-4T-2 with the right reading frame sequence to construct the expression vector,which can express the fusion protein with GST tag.The fusion protein was expressed after inducing with IPTG at 18 ℃ for 24 h and purified for antibody preparation by Glutathione Sepharose affinity chromatography.