家蝇幼虫体内一种抗菌蛋白的分离纯化

30%H2O2诱导家蝇幼虫90min,继续饲喂24h后利用硫酸铵分级盐析、Sephadex G-25 和Sephadex G-75两步凝胶过滤、CM-Sepharose Fast Flow弱阳离子交换的纯化方法,得到一种电泳纯的抗菌蛋白,经过VDS凝胶扫描得到其分子量为28kDa,命名为AP28。抑菌活性分析表明,AP28对实验中涉及的大多数革兰氏阳性菌和阴性菌都有明显的抑制作用。     

家蝇幼虫抗菌活性物质相关基因表达情况

研究了家蝇幼虫体内抗菌活性物质相关基因诱导后的表达情况。选择30％H2O2诱导家蝇幼虫90min,应用半定量RT—PCR的方法,以组成型表达的β-actin基因作为内参照,对抗菌活性物质基因cecropin,defensin,dipteri．cin,attacin和溶菌酶基因lysozyme在诱导后的表达情况进行了初步探索。Cecropin、defensin和attacin基因在家蝇幼虫体内都是诱导表达型基因,分别在诱导后2h和6h达到活性高峰。而diptericin和lysozyme两个基因表达量一直较高,在诱导后6h表达量达到最大水平,随后开始下降,到诱导后36h时还能检测到基因有较高水平的表达。     

稀碱法提取食用蝇蛆蛋白

对蝇蛆粉充分脱脂后采用稀碱法提取蝇蛆蛋白。结果表明,液料比、浸提温度、碱浓度对提取得率有显著影响(P<0.01),提取时间对提取得率影响差异不显著。蛋白提取的最佳条件为:液料比20mL/g,浸提温度80℃,浸提时间2h,碱浓度1%(w/v)。在此条件下,所得蝇蛆蛋白粉蛋白氮含量为11.14%,蛋白得率为57.53%,提取效率为69.84%。对最优条件下得到的蝇蛆蛋白粉进行理化指标检测,并与速溶豆粉的企业标准以及脱脂乳粉的国家标准进行比较。结果表明,水分、灰分、脂肪、蛋白含量均已达到可食用级别的要求,重金属铅、汞、砷含量均低于限量要求,微生物检测不含致病菌和大肠杆菌,在最优提取条件下所得蝇蛆蛋白粉,综合指标已达可食用标准。     

不同细菌对家蝇幼虫抗菌蛋白/肽的诱导效应

利用抑菌圈测量法和毛细管电泳研究、比较不同细菌对家蝇Musca domesticaL.幼虫抗菌蛋白/肽的诱导效应。结果表明,家蝇幼虫受细菌诱导后抗菌活性比对照都有不同程度的增加,同时不同细菌诱导表达样品对于相应的诱导菌均表现很高的抗菌活性。毛细管电泳图谱表明鼠伤寒沙门氏菌Salmonella typhimurium50013诱导后抗菌蛋白/肽表达强度增加了50倍,其它细菌诱导后抗菌蛋白/肽表达强度增加了1～40倍。与G+细菌相比,G-细菌具有更强的诱导效应。结论:家蝇幼虫对不同的细菌刺激有特异性反应,即不同细菌诱导抗菌蛋白/肽的强度、种类和数量都不一致。     

Preliminary studies on the zinc-induced metallothionein protein with antibacterial activity in housefly larvae, Musca domestica

Three isoforms of metallothionein protein induced with Zinc were isolated and purified from housefly larvae, Musca domestica, by gel filtration on Sephadex G-75, G-25 and anion exchange on DEAE-52 chromatography. Among them, one was found to possess antibacterial activity, and was further characterized by SDS-polyacrylamide gel electrophoresis, sulphydryl group determination, enzyme hydrolysis, and spectra property. Our results showed that the novel protein has the characteristics of heat-stable, low-molecular weight (6 kDa), rich-cysteine (approximately 12 cysteine residues in one molecule), metal affinity, and antibacterial activity. This paper was the first to report that metallothionein had antibacterial activity. We expect that this characteristic would give some help to investigate definite physiological functions of metallothionein.     

槲蕨多糖的提取工艺优化及其抑菌性研究

以槲蕨块茎为材料,采用水提醇沉法提取槲蕨多糖.在单因素考察的基础上,以多糖的得率为响应值,料液比、提取温度、提取时间为自变量,建立数学模型,利用响应面法获得槲蕨多糖的最佳提取工艺.采用纸片扩散法比较了不同浓度多糖的抑菌效果.结果表明:槲蕨多糖最佳提取工艺条件为:料液比1:25,提取时间2.0h,提取温度为85℃,在此优化条件下多糖得率为7.57％,与响应面分析预测值7.61％基本相符.槲蕨多糖对杆菌有明显的抑制作用,对供试球菌无明显抑制效果,多糖最低抑菌浓度为10 mg/mL.     

家蝇幼虫营养保健果冻的研制

家蝇Musca domestica L幼虫营养丰富,体内含有多种具有特异生物活性物质,有很好的开发利用价值。以家蝇幼虫为原料,运用正交试验设计对家蝇幼虫营养保健果冻的加工工艺进行研究。结果表明:35%的家蝇幼虫粗提液,15%的白砂糖,0.18%的柠檬酸和1.4%的卡拉胶的工艺参数,可生产具有良好感官和食用品质的家蝇幼虫营养保健果冻;家蝇幼虫粗提液的护色剂使用0.02%抗坏血酸与0.02%柠檬酸,果冻防腐剂使用0.04%山梨酸钾。     

Studies on the mechanisms of the antibacterial action of ortho-phthalaldehyde

The reaction of ortho-phthalaldehyde (OPA) with amino acids and proteins was investigated as a possible mode of action. Bacterial pellets (obtained by centrifugation) changed colour after exposure to OPA. These colours were more intense at alkaline than acidic pH. Acidic and alkaline OPA reacted with primary amino acids to form coloured products. The reaction rate accelerated with increasing pH. OPA increased the optical density of bacterial cell suspensions (an indication of protein coagulation or microbial surface or other changes in the opacity of cell constituents). The inhibition of ethylenediaminetetraacetic acid- and sodium lauryl sulphate-induced lysis was not as great as for glutaraldehyde (GTA), possibly indicating less cross-linking of amines. Interactions with primary amino groups of the outer envelope or cell wall probably play a part in the action of OPA but the level of cross-linking associated with the outer membrane does not appear to be as extensive as that of GTA. The aromatic component might allow OPA to penetrate the outer layers of cells, thus helping to explain the very high activity of OPA against Gram-negative vegetative organisms even though the degree of cross-linking seems to be less than that seen with GTA. Thus, OPA reacts strongly with primary amines and stabilizes, to some extent, the outer membrane and cell walls of vegetative organisms and this probably accounts for part, but not necessarily all, of its lethal action.     

家蝇幼虫壳聚糖的抑菌活性及影响因子

为研究昆虫壳聚糖的抑菌活性及影响因子, 由家蝇Musca domestica幼虫制备了10个不同分子量的壳聚糖,在不同条件下分别对6种细菌作抑菌实验, 并通过测定细菌细胞膜和细胞壁的透性初步探讨了壳聚糖的抑菌机理。结果表明,分子量在21～251 kD的壳聚糖有很强的抑菌活性,抑菌活性呈现随pH的降低而增加的趋势,pH 5.5时最低抑菌浓度在0.03％～0.06%之间,Ca²⁺和Mg²⁺能够显著降低壳聚糖的抑菌作用。通过对实验结果的方差分析表明,壳聚糖的不同分子量、pH值和金属离子等外界因素都是壳聚糖抑菌活性的极显著影响因素,而菌株本身也是极显著影响因素之一。壳聚糖能够增加细胞膜通透性,造成细胞内容物的外泄。     

不同提取工艺对家蝇幼虫蛋白粗提液抗菌活性的影响

利用不同条件提取诱导过的家蝇Musca domestica 3龄幼虫的总蛋白,并用平板扩散法测定所得蛋白粗提液的抗菌活性。结果表明,收集幼虫时的处死温度、沸水浴时间及提取液的pH值均对家蝇幼虫粗提液的抗菌活性有明显的影响。     

Cu2+胁迫对黑水虻幼虫抗菌肽组分及抑菌活性的影响

研究不同浓度Cu~(2+)胁迫对黑水虻5龄幼虫抗菌肽分离纯化组分及抑菌活性影响,为黑水虻无害化处理粪便技术的有效实施提供有力的理论依据,为其副产品在饲料、食品及医药研发中的应用提供有价值的实验数据。本文在人工饲料中添加不同浓度Cu~(2+)(0、150、1 200 mg/kg)以饲喂黑水虻幼虫,采用金黄色葡萄球菌针刺法诱导5龄幼虫,断头收集血淋巴,高速冷冻离心结合超滤离心制备抗菌肽粗提物;利用RP-HPLC对三组抗菌肽粗提物进行分离纯化,收集各纯化峰对应组分,采用纸片琼脂扩散法测定各纯化峰对应组分的抑菌效果,以阐述Cu~(2+)胁迫对黑水虻抗菌肽的影响。结果表明,经Cu~(2+)胁迫,抗菌肽粗提物分离纯化后各组分的出峰时间及峰面积所占比例均不同,不同处理浓度下各分离组分第2峰面积均大于第1峰面积,其中150-2峰面积最大,为73.31%;分离纯化后所得6个组分对金黄色葡萄球菌、大肠杆菌及白色念珠菌均有抑菌活性,对金黄色葡萄球菌、白色念珠菌的抑菌活性显著高于对大肠杆菌抑菌活性,但其对金黄色葡萄球菌及白色念珠菌的抑菌活性未见显著差异;与其他5个组分相比,组分150-2对金黄色葡萄球菌、白色念珠菌及大肠杆菌抑菌活性最强,抑菌直径分别为27.85±0.74 mm、28.34±0.76 mm、21.97±0.54 mm。由此可见,不同浓度的Cu~(2+)胁迫对黑水虻幼虫抗菌肽组分及抑菌活性均产生显著影响,其中组分150-2抑菌活性最强,具有很好的开发潜能。     

Antioxidant and antibacterial activities of eugenol and carvacrol‐grafted chitosan nanoparticles

Essential oils are known to possess antimicrobial and antioxidant activity while chitosan is a biocompatible polymer with antibacterial activity against a broad spectrum of bacteria. In this work, nanoparticles with both antioxidant and antibacterial properties were prepared by grafting eugenol and carvacrol (two components of essential oils) on chitosan nanoparticles. Aldehyde groups were first introduced in eugenol and carvacrol, and the grafting of these oils to chitosan nanoparticles was carried out via the Schiff base reaction. The surface concentration of the grafted essential oil components was determined by X‐ray photoelectron spectroscopy (XPS). The antioxidant activities of the carvacrol‐grafted chitosan nanoparticles (CHCA NPs) and the eugenol‐grafted chitosan nanoparticles (CHEU NPs) were assayed with diphenylpicrylhydrazyl (DPPH). Antibacterial assays were carried out with a representative gram‐negative bacterium, Escherichia coli (E. coli) and a gram‐positive bacterium, Staphylococcus aureus (S. aureus). The grafted eugenol and carvacrol conferred antioxidant activity to the chitosan nanoparticles, and the essential oil component‐grafted chitosan nanoparticles achieved an antibacterial activity equivalent to or better than that of the unmodified chitosan nanoparticles. Cytotoxicity assays using 3T3 mouse fibroblast showed that the cytotoxicity of CHEU NPs and CHCA NPs were significant lower than those of the pure essential oils. Biotechnol. Bioeng. 2009; 104: 30–39 © 2009 Wiley Periodicals, Inc.     

蝇蛆几丁低聚糖咀嚼片的调节血脂作用

研究和评价了蝇蛆几丁低聚糖咀嚼片的调节血脂作用及其安全性。将50只实验动物（大鼠）随机分为普通饲料对照组、高脂饲料对照组和3个实验组,实验组分别喂以不同剂量蝇蛆几丁低聚糖咀嚼片和高脂饲料。饲养28天后,分别测定大鼠血清总胆固醇（total cholesterol, TC）、血清总甘油三酯（triglyceride, TG）、血清高密度脂蛋白胆固醇（high density lipoprotein cholesterol, HDLC）的含量。用Horn's法对蝇蛆几丁低聚糖咀嚼片的急性毒性进行研究,连续观察7天,记录各组动物的中毒反应情况和死亡只数,计算咀嚼片对小鼠的半数致死剂量。同时对几丁低聚糖咀嚼片作了调节血脂的动物试验,研究结果表明,蝇蛆几丁低聚糖咀嚼片对大鼠血清总胆固醇、总甘油三酯有明显降低和对血清高密度脂蛋白胆固醇有明显稳定作用,具有辅助降血脂作用;对咀嚼片进行急性毒性实验表明,蝇蛆几丁低聚糖咀嚼片对小鼠的半数致死剂量大于10 g/kg·bw,提示其基本无毒。
     

Chemical changes in cutin obtained from cuticles isolated by the zinc chloride-hydrochloric Acid method

The composition of Malus pumila Mill. cutin from cuticles isolated using ZnCl₂-HCl reagent has been compared to that isolated by an oxalate-enzymic method. Molecular changes were observed and included the loss of unsaturated components and hydroxyl groups. Such changes may alter the results of physiochemical investigations of cuticles.     

Extraction of chitin and chitosan from housefly,Musca domestica,pupa shells

Chitins and chitosans are some of the most abundant natural polysaccharide materials, and are used to increase innate immune response and disease resistance in humans and animals. In this work, chitin and chitosan from housefly, Musca domestica, pupa shells were obtained by treatment with HCl and NaOH. For chitin extraction, 2 N HCl and 1.25 N NaOH solutions were used to achieve decalcification and deproteinization, respectively. For chitosan extraction, 50% NaOH solution was used to achieve deacetylation. The yields of chitin and chitosan from pupa shells of M. domestica were 8.02% and 5.87%, respectively. The deacetylations of chitosan (from chitin C1 and C2) were 89.76% and 92.39%, respectively, after the first alkali treatment with 50% NaOH (w/w) solution at 105 °C for 3 h and 5 h, respectively. The viscosities of the chitosans (from chitin C1 and C2) were 33.6 and 19.2 cP, respectively.