黄曲霉菌株的分离、鉴定及产毒能力分析

对几株从发霉粮食中分离出的黄曲霉菌菌株进行形态学和分子生物学鉴定,并进行发酵培养和产毒能力的HPLC测定。结果表明:试验分离菌株均为黄曲霉菌株且含有黄曲霉毒素产生的关键基因aflR;黄曲霉菌株之间产毒能力差异巨大:黄曲霉菌株3.4408产毒量最高,黄曲霉菌株HDWS产毒量最低,黄曲霉菌株3.2572甚至不产生黄曲霉毒素;产生黄曲霉毒素菌株中部分黄曲霉菌株产生4种黄曲霉毒素AFB1、AFB2、AFG1、AFG2,黄曲霉菌株HDWH只产生黄曲霉毒素AFB1、AFB2。     

黄曲霉菌株的筛选和产毒能力鉴定

本研究从发霉粮食中分离出数株黄曲霉菌菌株,并进行了形态学和分子生物学鉴定。为了探究分离菌株与黄曲霉标准菌株之间产毒能力的差异,通过对分离菌株和黄曲霉标准菌株进行发酵培养和HPLC测定,分析确定产毒能力。结果表明黄曲霉菌株之间产毒能力差异巨大：黄曲霉菌株3.4408产毒量很高,黄曲霉菌株HDWH产毒量很低,黄曲霉菌株3.2572甚至不产生黄曲霉毒素;产生黄曲霉毒素菌株中部分黄曲霉菌株产生四种黄曲霉毒素AFB1、AFB2、AFG1、AFG2,黄曲霉菌株HDWS只产生黄曲霉毒素AFB1、AFB2。     

香菇纤维素衍生物抑制黄曲霉菌产毒的研究

黄曲霉毒素具有诱导突变、抑制免疫和致癌作用。控制黄曲霉毒素污染一直是世界性难题,也是近年来的研究热点之一。实验研究了香菇纤维素衍生物GPX抑制黄曲霉菌产毒活性,初步探究了GPX抑制黄曲霉菌产毒的作用机制。结果表明,10、50μg/mL的GPX对黄曲霉菌产毒的抑制率分别达到70%、95%,而100、250、500、750、1000μg/mL的GPX对黄曲霉菌产毒的抑制率均可达到100%,抑制活性极其明显。GPX离体抗氧化能力很低,但是能够缓解黄曲霉菌丝的氧化胁迫。此外GPX促进了黄曲霉细胞内囊泡过早与大液泡的融合,该现象能够抑制黄曲霉毒素的产生。      

茶多酚对黄曲霉生长及产毒能力的影响

基于氧化应激与黄曲霉毒素生物合成的持续相关性,研究了天然抗氧化剂茶多酚对黄曲霉生长及产毒的影响,并通过扫描电镜观察茶多酚对黄曲霉菌丝体形态的影响,旨在寻找一种安全、高效、环境友好型的抑菌剂来控制食品中黄曲霉毒素的污染。结果表明:茶多酚对黄曲霉菌落生长有一定抑制作用,在10 mg/m L浓度下,抑制率达到48.41%,使黄曲霉菌落生长速率为(5.57±0.16)mm/d,延滞期为(1.32±0.13)d;而茶多酚对黄曲霉产毒抑制效果显著,在1,5和10 mg/m L浓度下,茶多酚对AFB1生物合成的抑制率分别达到了60.15%,87.12%和97.48%;且茶多酚可严重破坏黄曲霉菌丝体的超微结构。因此,茶多酚可作为天然抑菌剂应用于食品与饲料中,控制黄曲霉毒素的污染。     

吲哚对黄曲霉生长及产毒的抑制作用

本文研究了吲哚对黄曲霉的生长以及产毒的抑制作用。通过96孔板微量法发现吲哚能够抑制黄曲霉的生长,其最小抑制浓度(MIC)为100μg/m L;进而采用差量法测定了吲哚对黄曲霉菌丝生长量的抑制作用,结果表明200μg/m L的吲哚处理可以完全抑制黄曲霉的菌丝生长。此外,通过高效液相色谱分析发现吲哚浓度达到50μg/m L时,尽管对菌丝生长量没有明显影响,但是可以有效地抑制黄曲霉毒素B1的产生。这些结果说明吲哚抑制毒素产生并不是通过抑制生长来实现的。为了进一步了解吲哚对毒素产生的抑制机制,本研究通过RT-PCR分析了吲哚对黄曲霉产毒相关基因表达的影响。结果显示吲哚对产毒调控基因afl R的影响与其对毒素的作用趋势相同,同时吲哚还能够下调其他一些产毒相关基因afl K和alf D的表达。我们的研究结果表明吲哚具有很高的开发价值应用于粮食和饲料中黄曲霉毒素污染的控制。     

槲皮素对黄曲霉毒素B_1致大鼠肝细胞毒性的保护作用

黄曲霉毒素B_1(AFB_1)是由黄曲霉菌在特定条件下产生的毒性最强的次生代谢产物,广泛存在于玉米、花生等农产品中。槲皮素(Quercetin,QUE)是一种具有抗氧化等生理作用的黄酮类化合物。本文拟研究槲皮素对AFB_1致大鼠肝细胞毒性的保护作用及其机制。CCK-8实验结果表明,AFB_1对大鼠正常肝细胞(BRL细胞)的IC_(50)为23μmol/L,而QUE在低于15μmol/L时未对BRL细胞造成毒性损伤。利用QUE对细胞进行预处理24 h后,再将AFB_1作用于细胞24 h,通过细胞增殖力、活性氧(ROS)、丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽s-转移酶(GST)指标的检测,表明QUE能够增强细胞内SOD和GST酶活性,提高细胞自身的抗氧化性,降低由AFB_1引起的MDA和ROS对细胞的氧化损伤程度,进而保持细胞活力。此外,AFB_1可引起促凋亡基因Bax的mRNA表达(p0.05),显著降低AKT、Nrf2和HO-1的转录水平(p0.05),而QUE预处理能显著提高AKT、Nrf2、HO-1和Bcl-2基因的表达(p0.05),进而发挥抗氧化及抗凋亡的作用。因此,槲皮素对黄曲霉毒素B_1致大鼠肝细胞的毒性损伤具有一定的保护作用。     

植物源天然产物对黄曲霉和黄曲霉毒素B_1的抑制作用研究进展

黄曲霉毒素B1(aflatoxin B1,AFB1)是化学物质中致癌性最强的一种,对人和动物的健康具有极大的威胁,尤其是对肝脏的损害。AFB1是谷物和粮油中最为多见的真菌毒素,危害性也最强,长期低剂量的摄入AFB1除了会导致肝脏损伤和诱发肝癌之外,也会损害人体的其他器官。因此,能够有效防护人体免遭AFB1损伤的功能食品和新型保健药物已成为研究重点。近年来,天然产物因其具有生物活性显著、副作用小、多靶点等优点而备受关注,多种植物源天然产物已被报道能够抑制黄曲霉的生长产毒和AFB1的毒性作用。该文主要综述了植物源天然产物对黄曲霉生长产毒和AFB1毒性作用的抑制作用以及其相关抑制机制,为进一步开展AFB1毒性损伤的防护研究工作,开发能降低AFB1对人和动物的毒性损伤的新型保健药物和功能食品提供参考。     

酱腌菜生产技术问答

二十三、为什么必须重视酱菜中黄曲霉毒素污染的问题? 黄曲霉毒素是一种真菌毒素,具有很强的致癌能力,主要破坏动物的肝脏,引起肝癌。同时也可引起其它器官的肿瘤,对人类健康的威胁很大。在自然界中,并不是所有的黄曲霉菌都产生黄曲霉毒素,能产生黄曲霉毒素的菌种约占黄曲霉菌的60%左右。目前,我国在食品酿造中使用的黄曲霉菌种都是经过有关科研单位鉴定为不产黄曲霉毒     

山苍子精油抑制黄曲霉菌的生长和产毒作用研究

山苍子精油是一种纯天然植物精油,本文研究了其对黄曲霉生长、代谢和毒素产生的抑制作用,探讨了山苍子精油对黄曲霉菌的抑菌能力和作用机理。本研究将花生放置于自然环境染菌并分离纯化目标菌,采用形态学并结合ITS序列法进行菌株分类鉴定;结合抑菌圈、抑菌率和最低抑菌浓度(MIC)的测定探讨山苍子精油对黄曲霉菌的抑制能力;进行了山苍子精油影响黄曲霉孢子萌发率、生长曲线和黄曲霉毒素B1产生的实验研究;从细胞膜渗透性、细胞酶活性的变化探讨了山苍子精油抑制黄曲霉的作用机理。实验结果表明:从腐败花生中分离筛选出菌株HB₂,经ITS序列法鉴定为黄曲霉(Aspergillus flavus);黄曲霉素测定结果显示其含有黄曲霉素B1(AFB1),质量浓度为3.4×10³μg·kg^-1(纯湿菌体);抑菌圈随精油浓度的增大明显变大,对黄曲霉的最低抑菌体积分数(MIC)为0.800μL·mL^-1;孢子萌发率、牙管长度、黄曲霉菌体的生长量和AFB1的浓度随培养液中精油浓度的增大呈显著下降趋势,当山苍子精油浓度为0.100μL·mL     

响应面法优化重组AhPR-1蛋白抑制黄曲霉菌侵染花生的条件

基于大肠杆菌重组花生Ah PR-1蛋白具有抑菌的作用功能,利用响应面法优化蛋白抑制黄曲霉菌在花生上生长的条件因素。以表观评价法测评的黄曲霉生长程度和HPLC法检测的毒素合成的含量为评价标准,考察Ah PR-1蛋白质量浓度、作用温度和水分活度对蛋白抑制黄曲霉菌侵染花生及黄曲霉毒素产生的影响。结果显示,在花生染黄曲霉菌浓度为4×106CFU/m L的条件下,随着蛋白质量浓度升高,抑菌效果明显增强,蛋白质量浓度为0.32 ng/μL和0.40 ng/μL,效果最好且差别不是很明显;在温度28℃,水分活度0.3~0.4的条件下,AhPR-1蛋白的抑制效果最显著。在一定的环境条件下,Ah PR-1蛋白能够一定程度上抑制黄曲霉菌在花生表面的繁殖生长和毒素的合成。     

Effects of flavonoids on the expression of the pro-inflammatory response in human monocytes induced by ligation of the receptor for AGEs

Increasing evidence has shown advanced glycation end products (AGEs) receptor ligation (RAGE) to be an important part of complex interactions of the oxidative stress and pro-inflammatory responses. In this study, flavonoids were used to monitor the protective effects against the oxidative damage and inflammation mediated by AGEs in human monocytes. S100B (RAGE ligand) treatment in human THP-1 monocytic cells (THP-1) significantly increased gene expression of the pro-inflammatory cytokines TNF-alpha and IL-1beta; chemokines MCP-1 and IP-10; adhesion factors platelet endothelial cell adhesion molecule (PECAM-1) and beta2-integrin; and pro-inflammatory cyclooxygenase-2 (COX-2). S100B treatment with quercetin and catechin in THP-1 cells had inhibitory effects on the expression of pro-inflammatory genes and protein levels. Quercetin and catechin could regulate S100B-activated oxidant stress-sensitive pathways through blocking p47phox protein expression. Treatment with quercetin and catechin could eliminate reactive oxygen species (ROS) to reduce oxidative stress stimulated by S100B in THP-1 cells. Quercetin and catechin also showed different regulatory abilities on mitogen-activated protein kinase (MAPK) signaling pathways by inhibiting protein expression in S100B-stimulated inflammatory responses in THP-1 cells. This study suggests that quercetin and catechin may be of benefit for diabetic vascular complications due to its antioxidant abilities against AGE-mediated oxidative stress through oxidative stress-sensitive and oxidative stress-responsive signaling pathways, which lead to inflammation in human monocytes.     

Antioxidant treatment with quercetin ameliorates erectile dysfunction in streptozotocin-induced diabetic rats

Oxidative stress is demonstrated to be involved in the pathophysiological mechanism of erectile dysfunction (ED). Quercetin, a potent bioflavonoid, has been reported to have the antioxidant role. In the present study, we examined the effect of quercetin on ED and oxidative stress in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in Sprague–Dawley rats with a single intravenous injection of STZ. The diabetic rats were then randomized to diabetic group and quercetin therapy groups which were treated with quercetin at different doses of 5, 20 and 50 mg/kg per day respectively. At the end of the 8th week, erectile function was assessed by measuring the rise in intracavernous pressure (ICP) following cavernous nerve electrostimulation. Superoxide dismutase (SOD) activity, thiobarbituric acid-reacting substance (TBARS) and nitrite and nitrate (NOx) levels were measured in cavernosum tissue. Endothelial NO synthase (eNOS) expression was determined using Western blot method. ICP in diabetic rats was significantly decreased than that in controls. After treatment with quercetin at the doses of 20 and 50 mg/kg, ICP was significantly increased compared to that in untreated diabetic rats. Decreased levels of SOD activity, NOx and eNOS expression, as well as elevated levels of TBARS were found in diabetic group compared with control group. Treatment with 20 and 50 mg/kg quercetin improved SOD activity, NOx and TBARS levels in corpus cavernosum of diabetic rats. Decreased expression of eNOS in diabetic rats was only ameliorated by 50 mg/kg quercetin treatment. Quercetin could ameliorate ED in diabetic rats by inhibiting oxidative stress.     

Plant flavonol quercetin and isoflavone biochanin A differentially induce protection against oxidative stress and inflammation in ARPE-19 cells

In this work, differential ability of plant flavonol quercetin and plant isoflavone biochanin A to modulate oxidative stress and inhibit inflammation-related responses was investigated using human retinal pigment epithelial cells (RPE) at gene expression level. Quercetin protected cells from oxidative stress-induced cell death, whereas biochanin A had no statistically significant protective effects. Quercetin reduced the expression of cytokines IL-6 and IL-1?? in cells treated with H₂O₂, and expression levels of Nrf2 and HO-1 were increased by quercetin treatment suggesting protective function against oxidative stress. Our data indicate that quercetin may protect cells by inhibiting the production of pro-inflammatory factors such as IL-6, and by inducing the expression of ROS-catalyzing phase II proteins such as HO-1. Therefore, plant extracts rich in flavonol quercetin may be an interesting resource for functional food products and other foods targeted for reduced risks of age-related macular degeneration.     

ANTI-OXIDANT EFFECT OF QUERCETIN ON TYPE 2 DIABETIC ERYTHROCYTES

There is much evidence that oxidative stress is involved in the etiology of several diabetic complications. Many in vitro and in vivo studies have demonstrated that several parameters of red blood cell function and integrity are negatively affected by increased oxidative stress. Quercetin (3,3', 4',5,7-pentahydroxyflavone) is one of the most abundant bioflavonoids commonly present in most edible fruits and vegetables. Quercetin has been reported to prevent oxidant damage and cell death by scavenging free radicals. The present study was undertaken to evaluate the antioxidant effect of quercetin on markers of oxidative stress in erythrocytes from type 2 diabetic patients. Quercetin (at micromolar concentration) shows significant antioxidant effect in protecting erythrocytes from tert- butylhydroperoxide induced oxidative changes. These results suggest that a diet rich in quercetin may provide protection to diabetic patients against some late complications.

PRACTICAL APPLICATIONS

In recent years, there has been heightened interest in the possible therapeutic role of plant polyphenols for large number of human diseases. Among other known biological effects, plant polyphenols are known to have strong antioxidant properties. Out of more than 4,000 different types of plant polyphenols, relatively few are known to be effectively absorbed in the human digestive system. Among this group is the flavonoid quercetin that is present in many fruits and vegetables. We present here experimental proof of the protecting effect of this bioflavonoid on erythrocytes subjected to increased oxidative stress. Since diabetic patients are known to have increased oxidative stress, our results add to the scientific knowledge that a higher intake of diet rich in quercetin may protect diabetic patients from some late complications arising due to oxidative stress.     

Saccharomyces cerevisiae strains and the reduction of Aspergillus parasiticus growth and aflatoxin B(1) production at different interacting environmental conditions, in vitro

The effect of Saccharomyces cerevisiae RC008 and RC016, previously selected based on their aflatoxin B(1) binding ability and beneficial properties, against Aspergillus parasiticus under different interacting environmental conditions was evaluated. Studies concerning the lag phase, growth rate and aflatoxin B(1) production were carried out in?vitro under different regimes of a (w) (0.95 and 0.99), pH (4 and 6), temperature (25 and 37°C), and oxygen availability (normal and reduced). Both yeast strains showed great antagonistic activity at pH 4, decreasing growth rate compared with the control. The RC008 strain showed the greatest inhibitory activity at all assayed conditions. A. parasiticus produced large amounts of AFB(1) in?vitro. A significant decrease of AFB(1) levels in comparison with the control were observed with yeast interaction. Differences between control and treatment values ranged from 130 to 5400?ng?ml(-1). S. cerevisiae RC008 and RC016 could be considered as effective agents in reducing growth and AFB(1) production at different interacting environmental conditions, related to that found in stored feedstuff. The importance of the present work lies in the search for live strains with both probiotic and biocontrol properties able to prolong the safe storage of feedstuff and exert beneficial properties after animal consumption and which could be included in a novel product for animal feed.     

Modelling the Probability of Growth and Aflatoxin B1 Production of Aspergillus Flavus under Changing Temperature Conditions in Pistachio Nuts

The aim of this work was to use probability models for the prediction of growth and aflatoxin production by Aspergillus flavus as a strategy to mitigate the aflatoxin presence in pistachio nuts during postharvest. Logistic models, with temperature and time as explanatory variables, were fitted to the probability of growth and aflatoxin B₁ (AFB1) production under constant temperature levels, afterwards they were used to predict probabilities under non-isothermal scenarios. The models obtained showed levels of concordance from 80 to 100% in most of the cases. Moreover, the presence of AFB1 in pistachio nuts could be correctly predicted through AFB1 models developed in agar medium or through growth models in pistachio nuts. These findings can support decision making, at transport and storage level, and could be used by producers and processors to predict the time for AFB1 production by A. flavus in pistachio nuts in postharvest.