荔枝果实复合保鲜剂的筛选及其常温保鲜效果的研究

为了筛选出有利于荔枝贮藏的复合保鲜剂,以‘井岗红糯’荔枝果实为试验材料,通过正交试验研究了不同浓度的油菜素内酯（brassinolide, BL）和曲酸（kojic acid, KA）配比对采后荔枝果实的保鲜效果。结果表明,在25 ℃贮藏条件下,对荔枝果实的最佳保鲜配方为：油菜素内酯40 μmol/L、曲酸35 mmol/L,浸泡时间为3 min,该复合保鲜剂配方能较好地抑制荔枝果实褐变和腐烂,降低果皮相对电导率、果皮pH和果皮丙二醛（malonaldehyde, MDA）含量,延缓果肉总可溶性固形物（total soluble solids, TSS）和维生素C（vitamin C, VC）含量的下降,维持较高的果皮色度L ^*值、a ^*值、C ^*值和花色苷含量,抑制了多酚氧化酶（polyphenol oxidase, PPO）、过氧化物酶（peroxidase, POD）及漆酶（laccase, Lac）的活性。     

玉米根系分泌物对连作花生土壤酚酸类物质化感作用的影响

为探究玉米花生间作系统中玉米根系分泌物对连作花生土壤的酚酸类物质化感作用的影响机制，利用CH2Cl2提取了玉米抽雄期根系分泌物，通过室内模拟培养法，研究了玉米根系分泌物对含有不同浓度肉桂酸、邻苯二甲酸及对羟基苯甲酸3种酚酸类物质土壤的微生态环境的影响。结果表明，酚酸类物质均显著降低了土壤微生物量碳和氮的含量，抑制了土壤微生物活性，土壤酶（脲酶、酸性磷酸酶和蔗糖酶）活性和养分含量（碱解氮、有效磷和有效钾）亦显著降低，且浓度越高化感抑制作用（RI＜0）越强（P＜0.05）。玉米根系分泌物可降低酚酸类物质对土壤微生物活性、微生物量、酶活性及养分含量的化感指数，以低浓度处理的降幅较大，且在处理第5 d和10 d显著增加了3种酚酸类物质处理的土壤呼吸强度、酶活性、微生物量及养分含量（P＜0.05）；不同酚酸类物质中，以邻苯二甲酸所受影响最大。整个培养时期，玉米根系分泌物对酚酸类物质化感作用的影响以处理第5 d最强，随后呈减弱趋势。3次取样，土壤微生物量、微生物活性、酶活性和养分含量的化感指数分别降低10.03%~64.13%、9.72%~57.51%、13.16%~78.85%和5.88%~59.71%。玉米根系分泌物可通过提高土壤微生物量、微生物活性及养分含量，来降低肉桂酸、邻苯二甲酸和对羟基苯甲酸对土壤微生态环境的化感作用。结果为玉米花生间作缓解花生连作障碍技术提供一定的理论依据。     

外施苹果酸对紫色小白菜生长、生理与品质的影响

【目的】探究外施苹果酸对紫色小白菜生长、生理与品质的影响,筛选最适的外施苹果酸质量浓度,为紫色小白菜的优质培育以及高产栽培奠定理论基础。【方法】以‘京研'紫色小白菜为试验材料,采用水培技术,设置外施苹果酸0 mg/L(对照,CK)、0.5 mg/L(T1)、1.5 mg/L(T2)、3.0 mg/L(T3)、5.0 mg/L(T4)、10.0 mg/L(T5)共6个处理,研究不同质量浓度外施苹果酸对紫色小白菜叶片和根系形态学指标、根系显微结构及相关品质指标的影响。【结果】(1)随苹果酸质量浓度的增大,紫色小白菜叶片和根系形态指标均呈现先上升后下降的趋势,其中T1、T2处理显著优于对照,叶片表面积、体积较大,且可促进植株根系增长、面积增大、侧根增粗,而T5处理因苹果酸质量浓度过高阻碍了根系伸展。(2)根系显微结构观察发现,T1、T2、T3处理紫色小白菜新生侧根根尖1 cm处直径均显著高于对照,维管束和木质部区域直径增大,有助于运输更多的营养物质和水分。T5处理根系皮层细胞层数较少,细胞体积较大且排列疏松。(3)紫色小白菜叶片可溶性蛋白和维生素C含量随处理时间延长逐渐增高,随苹果酸质量浓度的增大呈先上升后下降的趋势,以T2处理最优。紫色小白菜叶片可溶性糖和总有机酸含量9 d时整体下降,且随苹果酸质量浓度的增大呈先上升后下降趋势,T1、T2处理显著高于对照。(4)苹果酸、丙二酸、琥珀酸和酒石酸含量随苹果酸质量浓度的增加呈先上升后下降的趋势,而柠檬酸含量呈先下降后上升的趋势。T1、T2、T3处理苹果酸、丙二酸、琥珀酸含量较高,对照、T4、T5处理柠檬酸含量较高。【结论】外施苹果酸能够影响紫色小白菜的生长及生理状况,施用适宜质量浓度(1.5 mg/L)的苹果酸可有效促进紫色小白菜地上部和根系生长,提高苹果酸、丙二酸、琥珀酸含量,提升植株营养品质。     

Effects of apelin-13 on oxidative stress induced by high uric acid in adipocytes

AIM: To study the effects of apelin-13 on oxidative stress induced by high uric acid in 3T3-L1 adipocytes and its underlying mechanisms. METHODS: 3T3-L1 adipocytes were stimulated with uric acid at 10 mg/dL for 48 h. Some of the adipocytes were administered with 1 μmol/L apelin-13 in the presence of uric acid at 10 mg/dL. The adipocytes stimulated with 100 μmol/L H₂O₂ were served as positive controls. The intracellular reactive oxygen species (ROS) concentrations were detected by flow cytometry. The biochemical kits were used to measure the activities of superotide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and NADPH oxidase (NOX) activity, and the content of malondialdehyde (MDA) in the cell lysate and the supernatant. The mRNA levels of renin-angiotensin system (RAS) components, including angiotensinogen (AGT), angiotensin-converting enzyrne1 (ACE1), angiotensin II type 1 receptor (AT1R) and AT2R, as well as angiotensin II receptor -like 1 (APJ) were measured by real-time PCR. The concentrations of angiotensin II (AngⅡ) in the cell lysate and the supernatant were measured by ELISA. RESULTS: Adipocytes stimulated with uric acid at 10 mg/dL had lower activities of antioxidant enzymes (SOD, GSH-PX and CAT) and higher levels of NOX activity and MDA content (P < 0.05). Accordingly, the intracellular ROS levels were found to be dramatically increased. However, apelin-13 administration attenuated uric acid-induced oxidative stress in the 3T3-L1 adipocytes. Uric acid at 10 mg/dL upregulated the mRNA expression of local RAS, enhanced AngⅡ concentrations both in the cell lysate and the supernatant, and down-regulated the mRNA level of APJ in the adipocytes (P < 0.05). Conversely, apelin-13 partially reversed these parameters. CONCLUSION: Apelin-13 attenuates oxidative stress induced by uric acid, may be via down-regulation of local RAS expression in the 3T3-L1 adipocytes.     

Roles of protein phosphatase 4 in down-regulation of eNOS Ser633 phosphorylation induced by palmitic acid in human umbilical vein endotheli?al cells

AIMTo investigate the roles of protein phosphatase 4 (PP4) in down-regulation of endothelial nitric oxide synthase (eNOS) Ser633 phosphorylation induced by palmitic acid (PA). METHODSHuman umbilical vein endothelial cells (HUVECs) were treated with PA at 25 μmol/L, 50 μmol/L, 100 μmol/L and 200μmol/L for 36 h, or treated with PA at 100 μmol/L for 12 h, 24 h, 36 h and 48 h. Protein phosphatase 2A (PP2A) family inhibitor fostriecin (FST, 20 nmol/L) or okadaic acid (OA, 5 nmol/L) was selected to pretreat the HUVECs for 30 min. Protein phosphatase 4 catalytic subunit (PP4c) siRNA or protein phosphatase 2A catalytic subunit (PP2Ac) siRNA was transfected into the HUVECs. The protein expression levels of of eNOS, PP4c and PP2Ac, as well as the level of eNOS Ser633 phosphorylation, were detected by Western blot. The intracellular nitric oxide (NO) content was measured by DAF-FM DA. RESULTS(1) Compared with control group, the levels of eNOS Ser633 phosphorylation were decreased in PA groups in which the HUVECs were treated with 25 μmol/L, 50 μmol/L, 100 μmol/L and 200 μmol/L PA for 36 h (P<0.05) and 100 μmol/L PA for 24 h, 36 h and 48 h (P<0.05). No significant difference in the level of total eNOS protein expression among all the groups was observed. (2) Compared with control group, both FST and OA pretreatment reversed the reduction of eNOS Ser633 phosphorylation (P<0.05) and the decrease in intracellular NO content (P<0.05) induced by PA. No significant difference in the level of total eNOS protein expression among all the groups was observed. (3) Compared with si-Control group, the PP4c protein expression was significantly reduced (P<0.05), while the level of eNOS Ser633 phosphorylation was significantly increased in si-PP4c group (P<0.05). Although the levels of PP2Ac protein expression declined significantly (P<0.05), the level of eNOS Ser633 phosphorylation remained unchanged in si-PP2Ac group. No significant differencein the level of total eNOS protein expression among all the groups was found. CONCLUSION PA significantly reduces the level of eNOS Ser633 phosphorylation and the content of NO in the HUVECs, which may be due to PA inducing the activation of the PP2A family member PP4 rather than PP2A.     

野猪粪便中乳酸菌的分离鉴定及特性研究

【目的】筛选安全的、具有优良特性的乳酸菌菌株,进一步研发益生制剂,为饲料添加剂等动物相关产品提供资源。【方法】从我国黑龙江省大兴安岭地区采集野猪粪便样品13份,编号后置于4℃保温箱迅速运回实验室,利用MRS培养基分离纯化乳酸菌。使用细菌基因组DNA提取试剂盒提取分离菌株的基因组DNA,利用细菌16S rDNA通用引物进行PCR鉴定,将扩增得到的序列测序后在NCBI上使用BLAST与GenBank数据库中序列进行对比分析,确定各菌株的分类学地位。将鉴定后的乳酸菌菌株分别接种于酸性（pH 3.0）和含胆盐（0.3%）的MRS培养基,在不同条件下评价乳酸菌菌株的耐酸、耐胆盐特性。将过夜培养的乳酸菌于室温条件下静置,在不同时间测定其OD_600nm,进行自凝集能力评价;过夜培养的菌株分别与致病性埃希氏大肠杆菌、金黄色葡萄球菌和鼠伤寒沙门氏菌3种致病菌混合后于室温静置,进行共凝集能力检测。在体外,分别进行乳酸菌菌株对Caco-2细胞和IPEC-J2细胞的黏附能力测定,评价不同菌株的黏附能力。通过测定乳酸菌菌株对致病性埃希氏大肠杆菌、金黄色葡萄球菌和鼠伤寒沙门氏菌3种致病菌的抑菌环直径,评价分离菌株的抑菌活性。通过体内外试验评价乳酸菌菌株的安全性。在体外,分别以模式菌株嗜酸乳杆菌和金黄色葡萄球菌作为阴性对照和阳性对照,将3株乳酸菌菌株和对照菌株在血平板上划线,37℃厌氧孵育18—24 h,观察细菌菌落周围是否形成溶菌环,评价分离菌株的溶血特性。使用文献中已报道的毒力因子引物对分离的乳酸菌菌株进行PCR扩增,检测是否存在毒力因子的编码基因,评估分离菌株的安全性。在体内,将过夜培养的乳酸菌连续饲喂7周龄的BALB/c小鼠21 d,分别测定小鼠的初始体重和最终体重,观察计算体重变化情况;饲喂21 d后,解剖获取小鼠的脾脏、肝脏和肾脏计算器官指数,评价分离乳酸菌菌株的体内安全性。【结果】从野猪粪便中分离得到3株对酸和胆盐具备一定耐受力的乳酸菌,经鉴定分别为蒙氏肠球菌（Enterococcus mundtii）、耐久肠球菌（Enterococcus durans）和黏膜乳杆菌（Lactobacillus mucosae）。3株乳酸菌菌株均表现出较强的自凝集能力和对致病菌的共凝集能力,同时对Caco-2细胞和IPEC-J2细胞均表现出较强的黏附能力,抑菌试验结果显示黏膜乳杆菌对3种致病菌均具备较强的抑菌活性。经体内外安全性评价,3株乳酸菌菌株无溶血性,且均未检测到毒力基因,经其连续饲喂的小鼠行为表现正常、状态良好,其中,与对照组相比,黏膜乳杆菌饲喂后小鼠增重显著。【结论】从大兴安岭野猪粪便中分离的3株乳酸菌（特别是黏膜乳杆菌）具有良好的特性和安全性,具备进一步开发益生菌制剂的潜力。     

Molecular cloning and functional characterization of the fatty acid delta 6 desaturase (FAD6) gene in the sea cucumber Apostichopus japonicus

The sea cucumber (Apostichopus japonicus), an important echinodermata, had high value in nutrition and medicine for its rich collagen, sulphated polysaccharide, glycosides and polyunsaturated fatty acids (PUFA). The cDNA of the fatty acid desaturase gene in A. japonicus (AJFAD6) was cloned and was found to encode a desaturase with delta 6 FAD activity. Sequence analysis indicated that AJFAD6 included an open reading frame of 1392 bp, encoding 463 amino acids. AJFAD6 has all the conserved motifs found in other members of the FAD6 family, including an N‐terminal cytochrome b5 domain and three histidine‐rich regions. qRT‐PCR showed that AJFAD6 was expressed in all tissues tested during juvenile development and was mainly expressed in the respiratory tree at 150 days after adherence (150 days) and in the intestine at 100 days. Furthermore, AJFAD6 mRNA was also detected in the analysed adult tissues, with higher expression in the intestine and testis. Functional characterization of AJFAD6 in a recombinant yeast, Pichia pastoris, showed that AJFAD6 could catalyse exogenous linoleic acid (LA) and α‐linolenic acid (ALA) to produce γ‐linoleic acid (GLA) and stearidonic acid (STA), respectively, at conversion rates of 11.1% for LA to GLA and 3.4% for ALA to STA. Our results suggested that the biosynthetic pathway of PUFA existed in the sea cucumber, but endogenous production of eicosapentaenoic acid, arachidonic acid and docosahexaenoic acid from either LA or ALA precursor appeared to be limited.     

Dietary α‐linolenic acid affects lipid metabolism and tissue fatty acid profile and induces apoptosis in intraperitoneal adipose tissue of juvenile grass carp (Ctenopharyngodon idella)

A 56‐day feeding trial was conducted to elucidate the effects and mechanism action of dietary α‐linolenic acid (ALA, 18:3n‐3) on lipid accumulation and fatty acid profile of muscle, hepatopancreas and intraperitoneal fat (IPF) in juvenile grass carp using three isonitrogenous and isoenergetic semi‐purified diets containing 0.0% (control group), 1.0% and 2.0% ALA, respectively. The lowest intraperitoneal fat (IPF) ratio was found in 2.0% group. In the muscle, hepatopancreas and IPF, docosahexaenoic acid (DHA, 22:6n‐3) and eicosapentaenoic acid (EPA, 20:5n‐3) contents increased with the increase in dietary ALA. In the IPF, caspase 3, caspase 8 and caspase 9 showed the highest activities in 2.0% group, while the value of Bcl‐2/Bax (B‐cell leukaemia 2/Bcl‐2‐associated X protein) reached the lowest. Meanwhile, swelling of the IPF mitochondria was observed in 2.0% group. The gene expressions of fatty acid desaturase (FAD) and fatty acid elongase (ELO) in the hepatopancreas and muscle showed significantly higher levels in the treatment groups, whereas an opposite trend was existed in the IPF. Fatty acid synthase (FAS), sterol regulatory element binding protein‐1c (SREBP‐1c) in the IPF and hepatopancreas reached the lowest in 2.0% group. Overall, dietary ALA could promote n‐3 highly unsaturated fatty acids (HUFAs) synthesis and suppress the accumulation of lipid by decreasing the expression of related genes and promoting the apoptosis in IPF.     

Antioxidant activity and lipid peroxidation in Artemia nauplii enriched with DHA‐rich oil emulsion and the effect of adding an external antioxidant based on hydroxytyrosol

Artemia nauplii catabolize polyunsaturated fatty acids (PUFA); in particular, they retroconvert docosahexaenoic acid (DHA, 22:6n‐3), so enrichment is a continuous quest towards increasing PUFA through the use of PUFA‐rich enrichment products. However, optimal conditions during enrichment (aeration, illumination and temperatures around 28°C) tend to accelerate autoxidation of PUFA and the formation of potentially toxic oxidation products. Water‐soluble antioxidants like the polyphenolic compound hydroxytyrosol (3,4‐dihydroxyphenylethanol), a polar molecule found in the water fraction resulting after the milling process of olives, arise as promising compounds to prevent oxidation during Artemia enrichments. We investigated the antioxidant activity and lipid peroxidation in Artemia nauplii during enrichment and the effect of adding an external antioxidant based on hydroxytyrosol during the enrichment with a PUFA‐rich emulsion (M70). For this purpose, the activity of antioxidant enzymes (catalase, superoxide dismutase, glutathione‐S‐transferase, glutathione peroxidase), as well as lipid peroxidation, was determined in enriched and unenriched Artemia nauplii. To validate antioxidant activity and lipid peroxidation, in a first experiment, nauplii were enriched with microalgae (Tetraselmis suecica), yeast (Saccharomyces cerevisiae) and M70 emulsion. In a second experiment, enrichment with a commercial emulsion (DC Super Selco), M70, and a combination of M70 and hydroxytyrosol (Hytolive, HYT) added as an external antioxidant were performed. The combination of M70 with HYT produced the best results, in terms of activity of antioxidant enzymes. The analysis of the fatty acids from total lipids showed that the addition of hydroxytyrosol preserved the DHA percentage of enriched nauplii.