电化学生物传感器在真菌毒素检测中的研究进展

真菌毒素是真菌在食品或饲料中生长所产生的代谢产物,这些低分子质量化合物既是天然存在的,同时也是无法避免的。真菌通过两种途径进入食物链,一方面可以直接从受真菌毒素污染的植物性食品成分进入食物链;另一方面也可以通过食物中产毒真菌生长的间接污染进入食物链。真菌毒素广泛存在于成熟的玉米、谷物、大豆、高粱、花生和饲料作物中。食用受真菌毒素污染的食物或饲料会对人和动物造成急性或慢性毒性。真菌毒素除了有直接食用受霉菌毒素污染的食物和饲料造成的不良影响外,还有因为摄入动物源性食品,如肉类、牛奶或鸡蛋,含有真菌毒素的残留物或代谢物而引起的公众健康问题。目前虽然已经鉴定出超过400种真菌毒素,但食物中广泛存在的6种毒素：黄曲霉毒素、赭曲霉毒素、玉米赤霉烯酮、伏马菌素、呕吐毒素及T-2毒素,已在世界范围内引发了持续的食品安全问题。该论文总结了6种真菌毒素的毒性,重点分析了近年来电化学生物传感器在这几种真菌毒素检测中的研究进展,旨在通过一系列的总结分析去展望其在真菌毒素检测方面的发展前景。     

Major biological consequences of aflatoxicosis in animal production.

Aflatoxins, a family of closely related, biologically active mycotoxins, have been known as a prominent cause of animal disease for 30 yr. The toxins occur naturally on several key animal feeds, including corn, cottonseed, and peanuts. Occurrence of aflatoxin on some field crops tends to spike in years when drought and insect damage facilitate invasion by the causative organisms, Aspergillus flavus and A. parasiticus, which abound in the crop's environment. Acute aflatoxicosis causes a distinct overt clinical disease marked by hepatitis, icterus, hemorrhage, and death. More chronic aflatoxin poisoning produces very protean signs that may not be clinically obvious; reduced rate of gain in young animals is a sensitive clinical register of chronic aflatoxicosis. The immune system is also sensitive to aflatoxin, and suppression of cell-mediated immune responsiveness, reduced phagocytosis, and depressed complement and interferon production are produced. Acquired immunity from vaccination programs may be substantially suppressed in some disease models. In such cases the signs of disease observed are those of the infectious process rather than those of the aflatoxin that predisposed the animal to infection. Mixtures of aflatoxin with other mycotoxins can result in greatly augmented biological responses in terms of rate of gain, lethality, and immune reactivity. Because of its great biological activity, its wide-spread potential presence in areas where critical feed crops are grown, and its propensity to spike in problem years, aflatoxin promises to be a continuing problem in animal production.     

Fumonisin B1 concentrations in feeds from 45 confirmed equine leukoencephalomalacia cases

During the fall of 1989 and winter of 1990, numerous reports of equine leukoencephalomalacia (ELEM) occurred from many regions of the United States. Typically, horses were consuming feed partially or entirely composed of corn and/or corn screenings. From October 1989 through May 1990, samples from 55 confirmed or suspected ELEM cases were received at the National Veterinary Services Laboratories, Ames, Iowa, for fumonisin B1 analysis. Samples from 9 cases in 1984-1985 were also obtained. Fumonisin B1, a mycotoxin produced by Fusarium moniliforme, causes ELEM, but little is known of naturally occurring toxic or safe levels in feeds. To determine what levels of fumonisin B1 in feeds are associated with ELEM, 45 selected cases were studied. The fumonisin B1 concentrations ranged from less than 1 ppm to 126 ppm, with the majority of the samples above 10 ppm. All types of feeds were included: corn, screenings, sweet feeds, and commercially pelleted rations. The length of exposure varied from 7 to greater than 35 days. Horse feed samples not associated with ELEM were also collected and analyzed. None of the nonproblem feed samples contained fumonisin B1 levels greater than 8 ppm.     

2006~2007年中国饲料及饲料原料霉菌毒素污染调查报告

作者评估了2006年和2007年上半年我国饲料原料和全价饲料中6种主要霉菌毒素的检出水平和分布特点,得出如下结论：①我国饲料原料和全价饲料中超标率和检出水平最高的霉菌毒素是玉米赤霉烯酮、烟曲霉毒素和呕吐毒素（田间型毒素）。人们一直重视的黄曲霉毒素（仓储型毒素）超标率和检出水平均比较低;②饲料原料和全价饲料中多种霉菌毒素往往同时存在,由于不同毒素间具有协同作用,将加剧毒素的毒性作用和中毒症状的严重程度;③副产品原料,特别是玉米副产品如干燥酒糟（DDGS）和玉米蛋白粉中霉菌毒素的污染非常严重;④与单一能量和蛋白质饲料原料相比,全价饲料中的多种霉菌毒素的检出水平才是相对比较准确和客观的控制指标;⑤控制霉菌毒素危害的最经济有效的措施是控制原料质量和使用有效的霉菌毒素吸附剂。     

黄曲霉毒素免疫学检测方法的研究进展

黄曲霉毒素毒性大,在食品及饲料中分布广,严重威胁人畜的生命健康,对它的检测十分必要,其中又以免疫学检测方法运用最广泛。作者总结了近年来国内外报道的常见免疫分析方法,评述了各方法的优劣,并对将来检测的发展趋势进行了展望。     

Occurrence of mycotoxins in Polish animal feed in years 2006-2009

We performed a 4‐year survey (2006–2009, 1255 samples) of fungal secondary metabolites in feed material (cereal and corn grains) and feedstuffs (silages, mixed feeds). Five major mycotoxin groups were studied, including aflatoxins (AF), ochratoxin A (OTA), trichothecenes [deoxynivalenol (DON), nivalenol (NIV), T‐2 toxin, HT‐2 toxin], zearalenone (ZEA) and fumonisins (FUM). The metabolites were identified using HPLC methods with fluorescent, UV and MS/MS detection. Both immunoaffinity and SPE columns were used for sample preparation. In eleven samples, the concentration of several mycotoxins exceeded the recommended guidelines for feedstuffs. DON was detected at the highest concentration in the majority of analysed samples (cereal grains, silages and mixed feeds, maximum values ranged from 409 to 14 470 ng/g). Corn grains also contained other Fusarium toxins (FUM) at maximum levels ranging from 435 to 9409 ng/g. The highest average_positive concentration of the other trichothecenes (NIV, T‐2 and HT‐2 toxins) was <5.0–139 ng/g. ZEA was found at the highest concentration in corn grains and silages (maximum values ranging from 292 to 603 ng/g and 116 to 1150 ng/g, respectively). The highest average_positive concentration and the maximum level of OTA were detected in cereal grains (33.0 ng/g in 2009 and 760 ng/g in 2007, respectively). Less than 7% of the 557 samples were contaminated with AF at low levels (maximum of 0.61 ng/g). Our results support the need for further monitoring of mycotoxins in Polish feedstuffs and their components.     

Mycobiota and aflatoxins in raw materials and pet food in Brazil

Commercial feedstuffs are a basic element in modern pet husbandry in the world. In dogs, the effect of mycotoxins is severe and can lead to death. Few reports on the influence of dietary mycotoxins were found in the scientific literature. The aims of this work were to isolate and identify the mycoflora and to determine the aflatoxins (AFs) natural occurrence in raw materials and ready dry pet food. Therefore, the aflatoxigenic capacity of Aspergillus flavus species was investigated. Aspergillus was the prevalent genera (65-89%) followed by Penicillium and Fusarium spp. Aspergillus flavus was the most prevalent species, followed by Aspergillus sydowii, Aspergillus fumigatus and Aspergillus versicolor. Aspergillus flavus frequencies ranged from 58% to 86% except in sorghum meal. All samples assayed (except corn grains and ready pet food) showed Fusarium spp. contamination. Corn meal and corn meal and gluten samples had 100% Fusarium verticillioides. Fusarium graminearum was isolated from sorghum meal. Aspergillus flavus strains (75%) isolated from raw materials and 57% from pet food were able to produce AFs. All samples showed AFs contamination percentages over 70%; corn and sorghum meal obtained the highest AFs levels. Ready pet food did not show quantitative levels of the tested toxins. This is the first report of the aflatoxigenic capacity by A. flavus from Brazilian pet food.     

Potential Aflatoxin and Ochratoxin A Production by <Emphasis Type="Italic">Aspergillus</Emphasis> Species in Poultry Feed Processing

Poultry feeds are prone to fungal growth and mycotoxin production during processing. The identification of biota with the ability to produce mycotoxins is essential. The aims of this study were (1) to monitor the mycobiota counts at different stages of poultry feed processing; (2) to determine the occurrence of Aspergillus species; (3) to evaluate the natural incidence of aflatoxins and ochratoxin A. The ability of Aspergillus spp. and its teleomorphs isolated here to produce these toxins was also investigated. Samples (144) were collected at random from a factory in Brazil. The occurrence of Aspergillus and Eurotium species was demonstrated on DRBC and DG18 media and the production of aflatoxins and ochratoxin A and their natural incidence were determined by TLC and HPLC methods. A. flavus and E. chevalieri were the most prevalent species isolated. Fungal contamination was not found after the pelleting process, though Aspergillus and Eurotium species were recovered from trough samples. High levels of aflatoxin and ochratoxin A producers were found at all stages of poultry feed processing. Also, high natural contamination with aflatoxins and ochratoxin A was found in the samples. Contact of feed with remainder poultry feed could lead to fungal contamination, so the risk of aflatoxin and/or ochratoxin A contamination of feed must be taken into account.     

Dietary origin of mycotoxins with estrogenic potential and possible health implications to female dogs

In Poland, occurrence of toxigenic fungi in cereals, foods, feeds and their components as well as mycotoxins accumulation in such material has been studied by numerous teams including our research group for over thirty years since 1969. Mostly cereal kernels and feeds have been examined for presence of toxigenic fungi, their toxigenic potential and natural contamination with mycotoxins. Ochratoxin A, deoxynivalenol, nivalenol and moniliformin were found to be significant contaminants of agricultural products in high percentage of cereal grain samples. The profile of toxic metabolites was similar but the concentration levels of the toxins were lower when compared to already published data of the same climate zone. Zearalenone (ZEA), a nonsteroidal mycotoxin with estrogen-like activity, is synthesized by molds (Fusarium) commonly contaminating poorly stored agricultural products and foodstuffs. Since in the course of examinations and during surgical procedures performed in dogs, ovarian cysts were detected and because frequently this is the first stage of the endometrica pyometra complex (EPC) found in approximately 30% of the females we assume that both factors, mycotoxins (ZEA) and pathological aberrations are possibly related in these animals. Similar activity of the toxin (possibly present in pelleted dog feed) and effects including infertility of female dogs is speculated with indication and suggestion on necessity of additional studies on the problem.     

Aflatoxin B_1, zearalenone and deoxynivalenol in feed ingredients and complete feed from different Province in China

Background: The current study was carried out to provide a reference for monitory of aflatoxin B_1(AFB_1),zearalenone(ZEN) and deoxynivalenol(DON) contamination in feed ingredients and complete feeds were collected from different Province in China from 2013 to 2015.Methods: A total of 443 feed ingredients, including 220 corn, 24 wheat, 24 domestic distillers dried grains with soluble(DDGS), 55 bran, 20 wheat shorts and red dog, 37 imported DDGS, 34 corn germ meal and 29 soybean meal as well as 127 complete feeds including 25 pig complete feed(powder), 90 pig complete feed(pellet), six duck complete feed and six cattle complete feed were randomly collected from different Province in China,respectively, by high-performance chromatography in combined with UV or fluorescence analysis.Results: The incidence rates of AFB_1, ZEN and DON contamination of feed ingredients and complete feeds were80.8, 92.3 and 93.9 %, respectively. The percentage of positive samples for DON ranged from 66.7 to 100 %.Domestic DDGS and imported DDGS presented the most serious contamination AFB_1, ZEN and DON contamination levels of feeds ranged from 61.5 to 100 %, indicated that serious contamination over the studied 3-year period.Conclusion: The current data provide clear evidence that AFB_1, ZEN and DON contamination of feed ingredients and complete feeds in different Province in China is serious and differs over past 3-year. The use of corn, domestic DDGS, imported DDGS and corn germ meal, which may be contaminated with these three mycotoxins, as animal feed may triggered a health risk for animal. Feeds are most contaminated with DON followed by ZEN and AFB_1.Mycotoxins contamination in feed ingredients and complete feeds should be monitored routinely in China.     

The Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) in animal feeding

The contamination of cereal grains with toxic secondary metabolites of fungi, mycotoxins, is a permanent challenge in animal nutrition as health and performance of the animals may be compromised as well as the quality of animal derived food. Therefore the present article reviews the issue of mycotoxins in animal nutrition. As the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) are of particular importance under the production conditions in central Europe and Germany, with respect to their frequent occurrence in toxicologically relevant concentrations, special emphasis is layed on those mycotoxins. The effects of DON and ZON on susceptible animals as well as management strategies to cope with the contamination of grain with those toxins are reviewed.     

The role of mycotoxins in the health and performance of dairy cows

In comparison to monogastric species, ruminant animals are generally considered to be less susceptible to the adverse effects caused by contamination of feeds with mycotoxins. This is based on the assumption that the rumen flora degrade and inactivate mycotoxins, thus protecting the animal. A number of mycotoxins, however, resist rumen degradation, causing distinct clinical signs of intoxication. Moreover, due to their complex diet, dairy cows may be exposed to a varying number of mycotoxins, originating from different feed materials such as roughage and concentrates. Exposure to these complex mixtures of mycotoxins may result in unexpected health risks. Due to a pre-existing negative energy balance, cows in the transition period are considered to be particularly sensitive to the exposure to feeds contaminated with moulds, fungal spores and mycotoxins.     

Mycotoxins in animal feedstuffs and tissues in Western Canada 1975 to 1979.

Results of analyses of specimens of plant or animal origin for various mycotoxins are presented. Analyses for aflatoxins, ochratoxins and zearalenone were most frequently requested. Aflatoxin B1 was found in one of 474 specimens at a level of 60 ppb in a sample of hay. Ochratoxin A was detected in four of 148 specimens of grains and two of 19 specimens of corn at levels up to 500 ppb. Trichothecenes were qualitatively found in two of 108 specimens of forage, three of 182 specimens of feeds and one of 148 specimens of grains. Ergot was detected qualitatively in three specimens of rye and one of forage. An overall detection rate of 3.8% of potent mycotoxins suggests that acute or chronic mycotoxicoses may occasionally occur in farm livestock or poultry.     

Recent progress on analytical techniques for mycotoxins in feedstuffs.

Analysis of mycotoxins in feedstuffs is a difficult task because only trace amounts of the toxins are present in the sample. However, rapid progress in the area of mycotoxin analysis has been made during the last few years. Simplified sample cleanup protocols and new chromatographic methods, especially HPLC, have been developed. New, more sensitive and versatile instruments such as high-resolution mass spectrometry (MS) and gas chromatography/tandem MS/MS are coming to the market. After 15 yr of laboratory research, immunoassay techniques have gained more acceptance as analytical tools for mycotoxins. Several immunoassay kits for mycotoxins are currently available. The development of these new techniques and their application for monitoring various mycotoxins in foods and feeds are described in this review.     

Implications of mycotoxins in animal disease

Mycotoxins are formed on animal feeds when conditions of moisture and temperature permit the growth of naturally occurring toxigenic fungi. In general, mycotoxins are low molecular weight, nonantigenic substances, many of which are relatively heat stable. Mycotoxins can cause acute disease episodes when animals consume critical quantities of them. Specific toxins affect specific organs or tissues such as the liver, kidney, oral and gastric mucosa, brain, or reproductive tract. In acute mycotoxicoses, the signs of disease often are marked and directly referable to the affected target organs. Most frequently, however, concentrations of mycotoxin in feeds are below those that cause acute disease. At lower concentrations, the effects of mycotoxins are more protean. They reduce the growth rate of young animals, and some interfere with native mechanisms of resistance and impair immunologic responsiveness, making the animals more susceptible to infection. These effects on immunity and resistance are difficult to recognize because the signs of disease are associated with the infection rather than with the mycotoxin that predisposed the animal to infection. Animals vary in their susceptibility to some mycotoxins, according to the species and age of animal; young growing animals are more susceptible to certain mycotoxins than are adults. The major effects, sources, and dose response relationships of mycotoxins important to the health of food-producing animals are presented in accompanying tables.     

Occurrence of on-farm risk factors and health effects of mycotoxins in dairy farms in Jordan

The aim of this study was to determine the occurrence of on-farm risk factors and health effects associated with contamination of dairy feeds with aflatoxins (AFs), zearalenone (ZEN), trichothecenes (T-2), deoxynivalenol (DON), and fumonisins (FB) in Jordan. A pre-tested and validated questionnaire was used to determine on-farm practices and health effects associated with high levels of mycotoxins. A total of 88 feed samples were collected from the 37 farms participating in the study and analyzed using commercially available ELISA kits. The mean total AF concentration exceeded the European Union (EU) limit in alfalfa (4%) and total mixed ration (TMR) (3%) samples. Similarly, levels exceeding EU limits were observed for T-2 in alfalfa (29%), TMR (30%), and corn silage (4%). The average concentrations of ZEN and FB were 300 ppb and 11,638 ppb, respectively, which were below the EU maximum limits in all feed samples examined. Intensive management system (OR?=?7.70), imported feed (OR?=?3.40), feed storage on the farm for more than 1-month duration (OR?=?7.90), and not using antitoxins (OR?=?2.30) were significantly (P?<?0.05) associated with high levels of mycotoxins in feed samples. A significant correlation (P?<?0.05) was evident between the presence of mycotoxins in dairy feed and feed refusal (R?=?0.70), low milk production (R?=?0.50), diarrhea problems (R?=?0.60), infertility (R?=?0.50), and repeated breeder problems (R?=?0.80). Results show that mycotoxin contamination in dairy feeds is a problem in Jordan, and appropriate measures need to be undertaken to reduce risks to human and animal health and improve production.

     

Occurrence of mycotoxins in feed ingredients and complete feeds obtained from the Beijing region of China

Background

The current study was carried out to provide a reference for the control of mycotoxin contamination in feed ingredients and complete feeds for swine.

Methods

A total of 55 feed ingredients, including 14 corn, 13 wheat bran, 11 soybean meal and 17 dried distillers grains with solubles (DDGS) as well as 76 complete swine feeds including 7 creep feeds, 14 starter feeds, 14 grower feeds, 18 grower-finisher feeds, 10 gestating sow feeds, and 13 lactating sow feeds were randomly collected from 15 swine farms located in the Beijing region of China from July to August 2011. Immunoaffinity clean-up, using High-Performance Liquid Chromatography (HPLC) in combination with UV or Fluorescence Detection, was used for quantitative analysis of aflatoxin B₁ (AFB₁), deoxynivalenol (DON), zearalenone (ZEA) and ochratoxin A (OTA) in the ingredients and complete feeds.

Results

DON and ZEA were the most prevalent mycotoxins found. DON was detected at percentages of 93, 92, 54, 100 and 97% with a mean level of 1.01, 0.44, 0.05, 1.36 and 0.65 ppm in the samples of corn, wheat bran, soybean meal, DDGS and complete feeds, respectively. The detected percentages of ZEA were 100, 100, 54, 100 and 100 with mean levels of 109.1, 14.9, 9.2, 882.7 and 58.9 ppb in the same samples. In the wheat bran and soybean meal samples, the content of all four mycotoxins were below the maximum limits set by Chinese regulations while the percentage of samples that exceeded regulatory limits were 7, 57 and 7% for corn, and 7, 14 and 3% for the complete feeds for AFB₁, DON and OTA respectively. DDGS showed the most serious mycotoxin contamination and the percentage of samples that exceeded regulatory limits were 6, 88 and 41%, for AFB₁, DON and ZEA, respectively.

Conclusions

This paper is the first to present data on the natural occurrence of AFB₁, DON, ZEA and OTA in ingredients and complete feeds obtained from swine farms in China’s Beijing region. The data shows that feed ingredients and complete swine feeds obtained from these farms are most often contaminated with DON followed by contamination with AFB₁ and ZEA.     

Some fungi, zearalenone and other mycotoxins in chicken rations, stock feedstuffs, lucerne and pasture grasses in the communal farming area of Rhenosterkop in South Africa

Mycotoxins may be present in feeds without any visible signs of mould contamination. There is a need for rapid and accurate measurement of mycotoxins for purposes of continual monitoring and identification of high risk commodities. Samples from commercial chicken feed (maize kernels), cattle feed (lucerne, grass and hay) and milk were analysed for the presence of certain mycotoxins and cultured for fungi. Results of fungal profiles showed that most samples were contaminated by moulds belonging to the genera, Alternaria, Aspergillus, Fusarium, Penicillium, Mucor, Phoma and Rhizopus. All the chicken feed samples tested contained mycotoxins either below the recommended safe levels for poultry or below the detection limits of the ELISA tests. However, samples of grass contained levels of zearalenone greater than the allowable concentration for dairy and beef cattle (250 ppm), which may be linked to the presence of Fusarium scirpi identified in the fungal profiles. The levels of AFMI were below the detection limits of the ELISA tests, which may be attributed to the low levels of aflatoxins found in the feed (grass) samples. The presence of the fungus in samples analysed is not evidence for the presence of mycotoxins.     

Fusarium crookwellense-produced zearalenone in maize stubble in the field

Waste grain and vegetative material (stems and leaves) collected from a maize field several months after harvest was analysed by bi-dimensional thin layer chromatography for the presence of aflatoxins, deoxynivalenol, ochratoxin A, sterigmatocystin, T-2 toxin and zearalenone. Deoxinivalenol (0.7 mg/kg) and T-2 toxin (4.1 mg/kg) were found in the grain and zearalenone (3.0 mg/ kg) was found in the stem and leaf. No other toxins were detected. The stubble was examined for the presence of potentially toxigenic Fusarium species, and F. poae, F. moniliforme var. subglutinans and F. crookwellense were isolated and identified. When these isolates were cultured on cracked corn, only F. crookwellense was found to produce micotoxins and then only zearalenone was detected. As corn stubble is commonly grazed in Argentina and in other countries, these findings identify a further source of mycotoxins that may adversely affect animal health and productivity.     

中国青贮玉米中霉菌毒素的污染情况分析与动物健康风险评估

为了探明中国青贮玉米霉菌毒素的污染状况,评价霉菌毒素对动物健康的风险隐患,分别在东北地区(黑龙江)、西北地区(内蒙古、山西)、黄淮海地区(河南、河北)、华中地区(江苏)、西南地区(贵州、四川)和西藏地区9省(自治区)28县主要生产基地采集全株玉米(90份)和青贮玉米样品(161份),共计251份,基于固相分散萃取-液相色谱串联质谱法,测定样品中黄曲霉毒素(AFs)、呕吐毒素(DON)、伏马毒素(FB)、玉米赤霉烯酮(ZEN)等12种霉菌毒素含量,并利用暴露评估进行动物健康风险评估。结果表明:①参照中国饲料卫生标准,测定样品ZEN超标率为1.19%。当青贮玉米作为泌乳期奶牛饲粮的主要粗饲料时,参照美国食品药品监督管理局(FDA)和欧洲食品安全局(EC)饲料安全标准,FB(FDA)和AFs(EC)的超标率分别为23.90%和13.94%。②全株玉米在青贮前和青贮后,霉菌毒素含量变化差异不显著(P>0.05)。③在黄淮海、华中和西南地区,青贮玉米霉菌毒素检出种类和含量相对较高,而在西藏地区含量相对较低。④当动物食用被霉菌毒素污染的青贮玉米时,动物的健康风险排序为:羔羊>犊牛>奶牛;在羔羊中,霉菌毒素的风险排序为:AFs>ZEN>DON>FB。综上所述,中国青贮玉米中霉菌毒素的超标率较低,污染情况相对严重的地区是黄淮海、华中和西南地区。在这些地区,如果青贮玉米作为泌乳期奶牛饲粮中主要粗饲料时,AFs和FB的超标风险较高;如果青贮玉米作为羔羊和犊牛饲粮中主要粗饲料时,AFs污染可能引起动物健康风险。