西藏高原粮油作物曲霉菌污染及菌株产毒力研究

为探明西藏高原粮油作物曲霉菌污染状况及黄曲霉菌产毒能力,连续5年对西藏青稞、小麦、花生3种作物中曲霉菌污染情况进行分析,并对其分离到的黄曲霉菌株开展产毒力研究,结果表明,204份样品中,共分离出15种曲霉菌,曲霉菌污染率呈花生>青稞>小麦。青稞、小麦中曲霉属优势种均为黑曲霉(Aspergillus niger),真菌毒素主要为杂色曲霉毒素和赭曲霉毒素;花生优势种为黄曲霉(A.flavus);仅受黄曲霉毒素污染。来源于不同作物的黄曲霉菌,其产毒类型也有差异,麦类作物产毒菌株以产黄曲霉毒素B₁(AFB₁)、黄曲霉毒素B₂(AFB₂)为主;花生产毒菌株以产AFB₁、AFB₂、AFG₁、AFG₂为主。     

贵州苦荞饭中黄曲霉毒素B1物理降解技术对比

应用微波、紫外线、γ射线辐照三种物理技术降解苦荞饭中黄曲霉毒素B₁(AFB₁),采用高效液相色谱法测定AFB₁的含量。试验考察了微波功率、微波时间、紫外照射时间、紫外照射高度、γ射线辐照剂量对其降解效率的影响。结果表明,苦荞饭中AFB₁降解率依次为γ射线辐照 > 紫外降解 > 微波降解,γ射线在辐照剂量为20 kGy时,降解率分别为33.6%±1.5%(污染剂量7.5 μg/kg)、35.1%±1.7%(污染剂量30 μg/kg)、45.7%±1.3%(污染剂量60 μg/kg)。与单独降解技术相比,将任意两种或者三种降解技术联合后,其降解率均显著增加(P<0.05),微波+紫外线+γ射线辐照三种技术联合降解率可达51.9%±0.9%(污染剂量7.5 μg/kg)、66.5%±0.7%(污染剂量30 μg/kg)、71.5%±0.9%(污染剂量60 μg/kg)。三种物理降解技术对于苦荞饭中低剂量的AFB₁污染有一定的应用价值。     

湘派卤制品卤汁中黄曲霉毒素产毒菌的筛选鉴定及其产毒能力测定

以湘派卤制品卤汁为研究对象，对其贮存过程中产黄曲霉毒素的微生物(产毒菌)进行筛选鉴定并对产毒能力进行测定。采用平板稀释涂布法和紫外荧光法对湘派卤汁进行产毒菌的初筛；对初筛菌株的ITS序列进行PCR扩增、测序，测序结果经NCBI同源性对比、MEGA6.0软件构建系统发育树进行分子生物学鉴定；应用HPLC-MS/MS检测各产毒菌培养液中黄曲霉毒素(aflatoxin, AFs)含量，确定各产毒菌的产毒能力。经平板稀释涂布法、紫外荧光法和分子生物学共筛选鉴定出5株产毒菌株，其中有4株是黄曲霉(Aspergillus flavus),1株为寄生曲霉(Aspergillus parasiticus);经HPLC-MS/MS法确定4株黄曲霉均不产生AFs,寄生曲霉产生黄曲霉毒素B₁(AFB₁)和黄曲霉毒素G₁(AFG₁)。AFB₁在第3天和第7天的含量分别为6.92、8.08μg/L,AFG₁在第3天和第7天的含量分别为5.80、7.24μg/L;湘派卤汁由于贮存条件...     

共培养体系下米曲霉与产毒黄曲霉的相互影响

研究了在共培养体系中,产毒黄曲霉对米曲霉产蛋白酶、淀粉酶的影响及米曲霉对黄曲霉产黄曲霉毒素B₁（AFB₁）的影响,还分析了米曲霉降解AFB₁的能力。结果发现黄曲霉对米曲霉产蛋白酶和α-淀粉酶有明显的抑制作用,而对β-淀粉酶的分泌无影响,培养10 d后,酸性蛋白酶活性、中性蛋白酶活性、碱性蛋白酶活性和α-淀粉酶活性分别降低了3.1、3.2、4.1和6.9倍;同时发现在整个培养过程中,米曲霉能有效抑制黄曲霉合成AFB₁的能力,抑制率达75～90.91%,而对AFB₁无降解作用。     

生物技术对黄曲霉毒素B1的脱除及机制研究进展

黄曲霉毒素是危害最大的真菌毒素之一,而黄曲霉毒素B₁(AFB₁)是黄曲霉毒素中毒性最大的一种,具有高毒性、致癌、致畸和致突变的作用,对动物和人类健康造成危害。为了有效脱除AFB₁,综述了近年来研究的具有解毒作用的微生物,介绍了采用生物技术脱除AFB₁的方法,重点探讨了生物脱除AFB₁机制。用于脱除AFB₁的菌株有芽孢杆菌、假单胞菌、乳酸菌、非产毒曲霉等,可采用单菌种发酵、多菌种协同发酵和菌-酶协同作用脱除AFB₁。生物脱除AFB₁机制主要为降解脱除和吸附脱除。可进一步筛选具有高优良能力的菌株以及更深层次地研究微生物脱毒的机制,探索微生物制剂对AFB₁的降解作用。     

低温等离子体对黄曲霉毒素B1的降解效能

目的:确定低温等离子体降解黄曲霉毒素B₁（Aflatoxin B₁，AFB₁）最佳工艺条件，并探究其在农产品中应用的可行性。方法:选取低温等离子体不同激发条件（峰值电压、工作频率、作用时间），研究其对溶液中AFB₁的降解效果。通过Center Composite Design（CCD）法进行响应面试验，获取最优降解组合及各因素交互作用机制，并考察此条件下玉米中AFB₁降解效果。结果:当AFB₁浓度为1000 μg/L时，其降解率随峰值电压及作用时间（除90~120 s）的增加，工作频率的下降而极显著升高（P<0.01）。响应面优化后最佳工艺条件为峰值电压160 kV、工作频率50 Hz、作用时间165 s，此时AFB₁降解率为99.62%。此外，将优化后的降解条件在受AFB₁污染的玉米（23.18±0.06 μg/kg）中进行应用，发现180 s处理时间下，其降解率可达39.29%。结论:通过CCD法确定了低温等离子体技术降解AFB₁最优工艺，证实了其在玉米中的降解效果。表明低温等离子体技术在降低谷物黄曲霉毒素污染方面具有巨大潜力。     

酶联免疫法测定苦荞制品中的黄曲霉毒素B1

用酶联免疫法（ELISA）测定不同苦荞制品中的黄曲霉毒素B1,对如何防控苦荞制品中的黄曲霉毒素B1进行探讨。在分析的4类产品中,苦荞营养粉的黄曲霉毒素B1含量最低,其次是苦荞速溶片和苦荞醋,苦荞米茶中最高。方法的检测灵敏度为0.1 μg/kg,平均回收率在84.70~84.95%,相对标准偏差小于5%。结果表明,酶联免疫法测定准确,稳定性和重现性好,可广泛应用于苦荞及其制品中黄曲霉毒素B1的检测。     

ELISA法检测五类食品中黄曲霉毒素B1前处理方法的改进研究

用酶联免疫吸附法（ELISA）检测五类食品（酱油、醋、面粉、大米、食用油）中黄曲霉毒素B₁（AFB₁）含量,关键在于样品的前处理。本文针对五类食品中AFB₁的前处理作了方法学研究。结果表明,改进后的前处理使检测灵敏度达0.1μg/kg,在0.1～10μg/kg之间呈良好线性关系,平均回收率在75%～170%之间。      

中高温大曲中黄曲霉的分离鉴定及其安全性初步研究

采用传统微生物分离手段从中高温大曲中分离纯化得到黄曲霉菌株NJSYS-15。通过菌落观察、镜检以及分子生物学手段得知其为黄曲霉属;利用AFPA培养基验证其产毒能力,采用酶联免疫法对其产黄曲霉毒素B1进行定量检测,结果表明,其在液态发酵进行到9 d时,黄曲霉毒素B1达到最高值13.6μg/kg。在进行固态模拟发酵时,黄曲霉菌株的接种量加大至10‰,黄曲霉毒素B1含量为3.2μg/kg,仍在国家标准限制以内。     

含水量对新疆鲜食核桃贮藏期黄曲霉生长及黄曲霉毒素M1积累的影响

目的:提高新疆鲜食核桃的贮藏安全性。方法:以新疆“新2”薄皮核桃为材料，无菌水接种为对照组，黄曲霉菌接种为试验组，将从自然霉变核桃上分离纯化出的黄曲霉菌人工接种至不同含水量(10%,15%,20%,25%,30%)的新疆薄皮鲜食核桃上，探究黄曲霉菌生长量及产毒变化情况。结果:最适宜黄曲霉菌生长繁殖并分泌黄曲霉毒素M₁的核桃含水量为15%;随着核桃含水量的升高，黄曲霉菌生长量呈先上升后下降趋势，但各含水量之间的生长量各不相同，且黄曲霉菌生长量与产生黄曲霉毒素M₁的量成正比。结论:原料的含水量与黄曲霉菌生长量及产生黄曲霉毒素M₁的量有着密切的关系。     

多功能净化柱-高效液相色谱法同时检测食用油中黄曲霉毒素和赭曲霉毒素A

目的:研究并建立高效液相色谱法同时检测20批食用油中黄曲霉毒素B₁、B₂、G₁、G₂和赭曲霉毒素A的定量方法。方法:样品经涡旋、离心提取后,采用多功能净化柱对目标毒素净化富集,采用SHIMADZU Inertsil ODS-C₁₈色谱柱（4.6 mm×150 mm,5μm）,FLD检测器,以甲醇-0.5%乙酸水为流动相进行梯度洗脱,进样量为10μL,流速为0.8 m L/min,柱温为35℃,改变波长荧光检测并对方法学进行考察。结果:AFB₁在0.25⁴0.6 ng/m L（r=0.9999）,AFG1在0.5⁴0.4 ng/m L（r=0.9997）,AFB₂在0.06¹0.06 ng/m L（r=0.9999）,AFG₂在0.06¹0.08 ng/m L（r=0.9999）,OTA在1.01⁵0.5 ng/m L（r=1.00）范围内呈良好线性关系,5种毒素的加标平均回收率为91.40%¹09.36%。样品检测结果表明,20批食用油样品中有4批样品检测结果呈阳性,均受到黄曲霉毒素G₁、G₂的污染。结论:该方法准确、快速,能够广泛用于食用油中黄曲霉毒素B₁、B₂、G₁、G₂和赭曲霉毒素A的定量检测。      

Aflatoxins B1 in different grades of chillies (Capsicum annum L.) in India as determined by indirect competitive-ELISA

Samples of the three grades of chilli pod (grades 1 to 3) were collected during surveys in 1998 and 1999 from the principal market yards and cold storage facilities of the major chilli-growing areas of Andhra Pradesh (AP), India. Chilli powders were collected from different supermarkets in Hyderabad, AP. They were analysed for aflatoxin B₁ (AFB₁) content by an indirect competitive ELISA. To avoid the influence of interfering substances present in chilli extracts, it was necessary to prepare the aflatoxin standards in methanol extracts of chillies free from aflatoxins. For nine representative samples there was good agreement between ELISA and HPLC estimations of AFB₁ and the results suggested that the ELISA procedure adopted was dependable. Of the 182 chilli samples tested, 59% of the samples were contaminated with AFB₁ and 18% contained the toxin at non-permissible levels. The highest AFB₁ concentration of 969 µg/kg was found in one sample representing grade 3. Overall the maximum percentage of chilli pods showing AFB₁ levels higher than 30 µg/kg (non-permissible levels) was in grade 3. Chilli pods stored in refrigerated rooms showed the lowest proportion of samples containing aflatoxin. Nearly 9% of the chilli powders sold in supermarkets contained non-permissible aflatoxin levels. This report highlights the importance of using grade 1 chilli pods to minimize aflatoxin contamination.     

Estimation of the daily exposure of Koreans to aflatoxin B1 through food consumption

A survey for aflatoxin B₁ (AFB₁) was conducted on 88 food-grade rice samples randomly collected during July and August 2002 in Seoul, Korea. The presence of AFB₁ was determined by enzyme-linked immunosorbent assay, and the positive samples from enzyme-linked immunosorbent assay were confirmed using high-performance liquid chromatography. Besides this, from the surveying data from the literature published since 1997, the intake of AFB₁ from food in Korea was calculated and compared with the provisional maximum tolerable daily intakes. Naturally occurring AFB₁ was found in 5/88 (6%) samples of rice with an average of 4.8 ng g^-1. A calculated probable daily intake of AFB₁ for Koreans fell into the range 1.19-5.79 ng kg^-1 bw day^-1, hence exceeding the estimated provisional maximum tolerable daily intakes. In conclusion, the exposure of Koreans to AFB₁ could bring about health concerns. This is the first report discovering that rice is the major contributor to the dietary intake of AFB₁ in Korea.     

Natural occurrence of aflatoxins in Korean meju

Aflatoxin B₁ (AFB₁) was found in 35 of 60 (58.3%) meju samples with an average concentration of 7.3ng/g by ELISA. Contamination of AFB₁ was confirmed in 25 of 60 samples (41.6%) using HPLC, with an average concentration of 6.9ng/g. Mean recoveries from meju ranged from 107% to 170% for AFB₁ using ELISA at a spiking range of 1 to 50ng/g. Over the same range, recoveries using HPLC were from 70% to 83%. The levels of AFB₁ determined by ELISA and by HPLC demonstrated a close relationship between the two methods (r2 = 0.9324) employed in this study. In order to evaluate the potential health risks of AFB₁ on Koreans consuming meju, we calculated the estimated probable daily intake (PDI) based on the average contamination levels and compared it with the estimated tolerable daily intake (TDI). The PDIs of AFB₁ from kanjang and dwenjang were determined to be 0.04 and 0.21ng/kg bw/day, respectively, and were higher than TDIs.     

Evaluation and validation of two fluorometric HPLC methods for the determination of aflatoxin B1 in olive oil

Two methods for the determination of aflatoxin B₁(AFB₁) in olive oil were tested and compared. In method A the oil sample was mixed with methanol + water (60 + 40), extracted with hexane and then with chloroform. Chloroform was evaporated and the residue was dissolved with dichloromethane which was then transferred for clean-up onto a silica 'Sep-Pak' cartridge. The cartridge was pre-washed with hexane, ethyl ether and dichloromethane. AFB₁ was eluted with chloroform + acetone (9 + 1) and evaporated to dryness. In method B, the oil sample was mixed with methanol + water (80 + 20), shaken and centrifuged. The supernatant was diluted 1:10 with water and 10ml of the diluted mixture transferred to an 'Aflaprep' immunoaffinity column for the clean-up step. AFB₁ was eluted with acetonitrile and evaporated to dryness. AFB₁ from both methods was derivatized to its hemiacetal (AFB_2a ) and then quantitated by HPLC using a C₁₈ (60 A 4.6 x 250 mm) column with fluorescence detection. Both methods are simple, reliable and efficient, but method A showed a lower detection limit (2.8 ng/kg) than method B (56 ng/kg). With a 95% confidence level there was no significant difference in recovery between the two methods, which was 87.2% for method A and 84.8% for method B. In addition, application of a two-tailed F-test to the variances within spiked samples at concentrations 1, 2, 5 and 10 mu g/kg separately showed that there was no significant difference in the precisions of the two methods. Fifty samples of olive oil of Greek origin produced between 1995 and 1998 were examined with both methods for the presence of AFB₁. When analysing the samples with method B, the presence of AFB₁ was not detected. The use of method A revealed the presence of AFB₁ in 72% of the samples. The range of contamination was generally found to be very low (2.8-15.7 ng/kg), however one sample was contaminated with 46.3 ng/kg.     

黄曲霉毒素B1降解菌的筛选鉴定及有效降解组分初探

黄曲霉毒素B₁(AFB₁)是具有强毒性、强致癌性的一种真菌毒素。为了筛选出AFB₁降解菌,将实验室保藏的真菌菌株与AFB₁共培养,选取降解率最高的菌株,经形态学和ITS rDNA分析确定其种属,分离菌株不同组分(菌悬液、菌体、孢子液、发酵液)后探究其降解AFB₁的有效组分,并将有效组分菌体破碎后作进一步探究。结果表明:培养72 h后的郝克氏青霉MD1菌悬液和未破碎菌体对AFB₁降解率分别为98.15%和28.20%,菌体破碎后对AFB₁的降解效果更好,培养24 h后对AFB₁的降解率为55.40%。因此,郝克氏青霉MD1的菌悬液和菌体内的活性组分能有效降解AFB₁。本研究筛选出的郝克氏青霉MD1扩大了AFB₁降解菌的菌种库,为AFB₁的生物降解提供了一定的参考价值。     

各食用米中活性成分及其抗氧化活性

本研究以黑米、白糯米、大米、黄米、糙米、香米、玉米、薏米、西米、高粱米10种常见食用米为研究对象,测定其总黄酮、总酚含量及其体外抗氧化活性。结果显示供试食用米品种间的总酚和总黄酮含量分别在2.61~287.61 mg/g和3.79~175.43 mg/100 g之间,黑米总酚、总黄酮含量最高(分别为287.61±18.38 mg/g和175.43±3.25 mg/100 g),西米的含量均最低(分别为2.61±0.04 mg/g和3.79±0.06 mg/100 g);黑米乙醇提取液的总抗氧化能力(354.18 U/g)和总还原力(吸光度为2.91)最强,大米提取液的总抗氧化能力(4.62 U/g)最弱,而西米的总还原力(5倍稀释时吸光度为0.11)最弱;黑米对DPPH·的清除能力最高(当加入量为2 μL时清除率为50%,IC₅₀=2 μL),西米的清除能力最弱(当加入体积大于10⁵ μL时清除率为50%,IC₅₀>1×10⁵ μL);黑米对·OH的清除能力最高(IC₅₀=250倍稀释),西米的最低(IC₅₀=1.71倍稀释);黑米对NO₂^-的清除能力最高(IC₅₀=500倍稀释),玉米为最低(IC₅₀=2.97倍稀释);西米和糯米对O₂^-·的清除能力最高(IC₅₀>10⁵倍稀释),玉米最低(IC₅₀=2.467倍稀释)。综合比较发现黑米具有较丰富的生物活性物质,抗氧化活性相对较好,本研究为进一步开发功能性米类产品提供一定的理论基础。     

不同苦荞茶总黄酮冲泡特性及其与抗氧化能力相关性的研究

目的:研究不同苦荞茶总黄酮冲泡特性与其抗氧化能力的相关性。方法:以市售的12份米茶型和11份造粒型苦荞茶为研究对象,分析其总黄酮的冲泡特性、清除DPPH·和总抗氧化能力。结果:米茶型苦荞茶总黄酮均值为1.37%,造粒型苦荞茶均值为3.04%,造粒型苦荞茶总黄酮均值比米茶型苦荞茶高出121.9%;米茶型苦荞茶三次冲泡总黄酮溶出率平均值为47.53%,造粒型苦荞茶平均值为15.07%。造粒型苦荞茶清除DPPH·的IC₅₀为0.59 mg,总抗氧化能力FRAP值为0.549 mmol FeSO₄/g;米茶型苦荞茶清除DPPH·的IC₅₀为1.50 mg,FRAP值为0.152 mmol FeSO₄/g。造粒型苦荞茶总黄酮含量极显著高于米茶型苦荞茶的总黄酮含量(p<0.01);米茶型苦荞茶总黄酮冲泡溶出率极显著高于造粒型苦荞茶(p<0.01);苦荞茶总黄酮含量和DPPH·(IC₅₀)呈极显著负相关关系(p<0.01),与总抗氧化能力呈极显著正相关关系(p<0.01),造粒型苦荞茶抗氧化能力极显著高于米茶型苦荞茶(p<0.01)。结论:在饮用米茶型苦荞茶产品时,同时将茶渣一起食用,更能发挥苦荞茶的保健功效;造粒型苦荞茶因其耐泡性更适合餐饮行业选用。