Substrate utilization by Aspergillus flavus in inoculated whole corn kernels and isolated tissues

Utilization of the major corn (Zea mays) reserve materials (free saccharides, starch, triglycerides, and zein) was monitored during infection of detached kernels by Aspergillus flavus (A. flavus) over a 12-day period. Inoculated whole kernels were compared to noninoculated kernels. Concentrations of sucrose and raffinose in inoculated seed decreased to nearly zero at 6 days, whereas concentrations of these saccharides in noninoculated seed dropped at a considerably slower rate, and significant levels remained at the end of the incubation period. Triglyceride concentrations remained unchanged in the noninoculated seed but dropped continuously after 2 days in the inoculated seed. Starch and zein concentrations did not change during the 12-day incubation period. Aflatoxin B1 was first detected after 2 days and increased to about 20 microg/g (20,000 ppb) after 12 days. Very low aflatoxin concentrations were detected in the noninoculated seed. Significant concentrations of erythritol, arabitol, and mannitol were produced during infection, with peak concentrations occurring at 8 days. Whole seed and germ tissue appeared to support good fungal growth and aflatoxin production, whereas ground tissues and endosperm did not. A. flavus preferentially utilized saccharides as initial carbon substrates followed by triglycerides. When invading nonwounded corn kernels, the fungus selectively targets the germ tissue where these materials are localized in the highest concentrations.     

Influence of lipids with and without other cottonseed reserve materials on aflatoxin B(1) production by Aspergillus flavus

Cottonseed storage lipids (primarily triglycerides), in either crude or refined form, were found to support growth and aflatoxin B(1) production by Aspergillus flavus. When lipids were removed from ground whole cottonseed by petroleum ether extraction, aflatoxin production dropped by more than 800-fold. Reconstitution of the lipid-extracted ground whole seed with a crude preparation of cottonseed lipids restored aflatoxin production to the previous levels. Fungal utilization of the three major cottonseed reserve materials, raffinose, triglycerides (refined cottonseed oil), and cottonseed storage protein, was monitored in vitro over a 7 day fermentation period. The fermentation medium contained the reserve compounds in proportions approximating those found in mature cottonseed. A. flavus rapidly converted raffinose to fructose and melibiose, presumably by action of invertase, and then hydrolyzed the melibiose. These simple sugars apparently supported initial growth and aflatoxin B(1) production. Raffinose and the resulting melibiose were nearly exhausted by day 2. Fungal hydrolysis of triglycerides began as exhaustion of carbohydrate approached. After day 2, rapid catabolism of the released fatty acids began and coincided with glucose regeneration through gluconeogenesis, which peaked on day 6. The fungus did not preferentially utilize specific fatty acids. A. flavus also produced a number of storage metabolites, including arabitol, erythritol, mannitol, and trehalose. Mannitol was produced in much higher concentrations than the other storage metabolites. Selective use of simple carbohydrates by A. flavus to drive aflatoxin production may suggest strategies for reducing vulnerability of cottonseed to aflatoxin contamination.     

Inhibition of aflatoxin biosynthesis in Aspergillus flavus by diferuloylputrescine and p-coumaroylferuloylputrescine

A mixture of diferuloylputrescine/p-coumaroylferuloylputrescine (85:15, w/w) demonstrated inhibitory activity against aflatoxin B1 biosynthesis in Aspergillus flavus isolate AF13. Inhibition was concentration dependent, with a 50% effective dose of 30 microg of diferuloylputrescine/p-coumaroylferuloylputrescine per milliliter of medium. Aflatoxin inhibition levels of up to 93% were achieved using this conjugated polyamine material. This diconjugated polyamine mixture did not display inhibitory effects on A. flavus growth (mycelial weight) at any of the concentrations tested. A survey of hand-dissected corn (Zea mays) kernel tissues, including endosperm, germ, pericarp, and wax, revealed that the highest concentrations of these conjugated polyamines were localized in the pericarp of the seed. Analysis of a number of corn accessions did not reveal a correlation between diferuloylputrescine/ p-coumaroylferuloylputrescine concentration and resistance/susceptibility to A. flavus infection. The localization of these diconjugated polyamine components in the pericarp, which functions as a physical barrier and surrounds the internal food storage reserves, suggests a defensive role for these materials.     

分层破胚剥皮玉米不同部位营养成分富集特征

为明确玉米籽粒营养成分的分布差异及不同部位富集特征,应用快速缓苏、微量着水半湿法分层破胚剥皮技术,结合靶向代谢组学方法,对郑单958玉米不同部位的营养成分及基础代谢物质进行分析与比较。结果表明玉米籽粒不同部位的淀粉、脂肪、矿物元素和膳食纤维等营养物质含量存在显著差异（P<0.05）。该研究中的玉米内皮层可能主要由种皮、糊粉层及部分外胚乳构成,该部位营养成分的种类及含量均较为丰富,其中水溶性膳食纤维含量显著高于其他部位（P<0.05）,可作为玉米水溶性膳食纤维的提取分离来源。K、P和Mg元素是玉米中含量最高的矿物元素,主要存在于胚芽中,Fe、Zn、Mn和Cu元素在胚芽和玉米皮层中均有较多分布,精制加工会导致这些矿物元素的损失。玉米胚芽中水解氨基酸种类较其他部位丰富且含量较高（P<0.05）,甜味氨基酸占总游离氨基酸含量的24.49%,高于玉米皮层部位、显著高于胚乳部位。研究结果为玉米营养健康食品的创制、玉米精深加工及相关专用装备的研发提供参考。     

异硫氰酸苄酯对于黄曲霉生长速率和产毒情况的影响

为明确异硫氰酸苄酯(BITC)在不同条件下对黄曲霉的抑制效果,本研究以熏蒸法,在28℃培养条件下,分别以花生和玉米为培养基质,研究不同浓度(0、5、10、15、20 mg·L-1)异硫氰酸苄酯在不同水分活度(aw)(0.930、0.960、0.980、0.995)下对黄曲霉(Aspergillus flavus)生长和...     

植物乳杆菌抑制黄曲霉活性代谢物的初步研究

植物乳杆菌（Lactobacillus plantarum）能够抑制黄曲霉生长,但起主要抑菌作用的物质尚未明确。该研究采用非靶向代谢组学技术比较分析了8株抑菌活性较好（S组）和8株抑菌活性较差（W组）的L. plantarum发酵上清液。结果显示,两组L. plantarum发酵上清液的代谢组存在显著差异（P<0.05）。通过数据库比对鉴定得到咪唑乙酸、酪氨酸等30个显著差异代谢物（P<0.05）,其中有机酸、脂肪酸等酸性物质较多为22个。通过与已报道的乳酸菌产生的抗真菌物质相比较,找到十八烷酸、吲哚乙酸等结构一致或结构类似物,表明上清液中酸性物质起主要的抑菌作用,且其抑菌活性依赖于低 pH 值的酸性环境。在L. plantarum产生的主要有机酸中,乳酸、乙酸、丙酸的抑菌活性良好,其抑制黄曲霉活性从大到小依次为丙酸、乙酸、乳酸。当乙酸浓度为2.64g/L、丙酸浓度为1.76 g/L时,可完全抑制浓度为106个/mL的黄曲霉孢子生长。综合表明,植物乳杆菌代谢物中有机酸和脂肪酸为主要抑菌物质,且抑菌活性随酸性物质浓度增大而增强。     

Chemical composition, in vitro fermentation characteristics, and in vivo digestibility responses by dogs to select corn fibers

The objective was to examine the chemical composition, in vitro fermentation characteristics, and in vivo digestibility responses of fiber-rich corn coproducts resulting from corn wet milling. Native corn fibers, native corn fibers with fines, hydrolyzed corn fibers, and hydrolyzed extracted corn fibers were analyzed chemically and their capacity to produce short-chain fatty acids determined. Ash content was low (<1.2%), crude protein content varied little, but fat and fiber concentrations varied widely. Most fiber was in the insoluble form, with glucose being predominant followed by xylose. Total short-chain fatty acid production ranged from 211.6 to 699.52 micromol/g of dry matter, whereas branched-chain fatty acid production was low. Four corn fibers (native and processed) were included in a canine diet matrix at the 7% inclusion level. Nutrient digestibility, food intake, and fecal characteristics were not affected by corn fiber inclusion in canine diets, suggesting that they should be considered as potential dietary fiber sources in dog foods.     

Methyl jasmonate stimulates aflatoxin B1 biosynthesis by Aspergillus parasiticus.

Aflatoxin B1 (AFB1) is a highly toxic and carcinogenic metabolite produced by certain Aspergillus species on agricultural commodities. One factor promoting the production of aflatoxin is the presence of high levels of fatty acid hydroperoxides often found in plant material under stress. Jasmonic acid (JA) and its methyl ester (MeJA) are derived from linolenic acid, and their biosyntheses involve the production of lipid hydroperoxides. Exposure of aflatoxigenic mold to jasmonates is likely because the mold attacks plant material and possibly initiates the production of jasmonates. In this study the effect of MeJA on the growth of Aspergillus parasiticus and AFB1 biosynthesis is reported. MeJA, at a final concentration of 10(-4) M in yeast extract sucrose medium, did not have any apparent effect on mycelial growth during the 16 days of observation but did increase significantly the levels of AFB1 after the seventh day of growth. After the ninth day, AFB1 production was decreased in contrast to the control cultures, where the production was constantly increasing. AFB1 determination was performed by immunoaffinity and HPLC after derivatization to AFB2a.     

Inhibition of aflatoxin B(1) biosynthesis in Aspergillus flavus by anthocyanidins and related flavonoids.

Anthocyanidins and precursors or related flavonoids were tested at concentrations from 0.3 to 9.7 mM ( approximately 0.1-3.0 mg/mL) for activity against growth and aflatoxin B(1) biosynthesis by Aspergillus flavus Link:Fr. NRRL 3357. Aflatoxin B(1) production was inhibited by all anthocyanidins tested, and 3-hydroxy compounds were more active than 3-deoxy forms. Monoglycosides of cyanidin were 40% less inhibitory than the aglycon, whereas a monoglucoside and a diglucoside of pelargonidin were 80 and 5%, respectively, as active as the aglycon. Of eight flavonoids tested, only kaempferol was moderately active, whereas luteolin and catechin were weakly inhibitory. Binary combinations of delphinidin and three other aflatoxin inhibitors acted independently of each other. Results with an aflatoxin pathway mutant indicated that anthocyanidin inhibition occurred before norsolorinic acid synthesis.     

Aflatoxin variation among corn samples with varying ratios of Aspergillus flavus-inoculated/noninoculated kernels.

Aflatoxin levels and physical properties of corn kernels inoculated with Aspergillus flavus during development and noninoculated kernels were compared in samples with various proportions of the 2 kernel types. The relationship between mean toxin levels and associated standard deviations of 5 samples demonstrated a linear association from the lowest toxin in noninoculated corn through a mixture of 60% inoculated/40% noninoculated. However, at the highest toxin level in the 100% inoculated material, a reduction in sample variation was observed. Examination of individual kernal weights showed that inoculated kernels were distinctly lighter than noninoculated seed. A uniform grinding procedure of the samples yielded heterogeneous particle sizes based on the starting corn. The large particle fraction (greater than 500 micrometers) decreased from 100% noninoculated kernels through the mixtures to the 100% inoculated seed; particles below 150 micrometers were most abundant in the ground samples from inoculated kernels. In addition, the density of particles within a size category varied; lower densities were observed in samples obtained from A. flavus-inoculated kernels.     

Identification and effects of maize silk volatiles on cultures of Aspergillus flavus

Volatiles generated from corn silks of individual genotypes of maize were found to exhibit differences in biological activities when the volatiles were exposed to 5-day solid cultures of Aspergillus flavus. In inverted potato dextrose-agar Petri plate bioassays, it was found that volatiles emitted from silks of the different maize genotypes had a profound effect on the growth of the fungus and, consequently, aflatoxin production. To determine the underlying cause for this bioactivity, volatiles emitted from the maize silks were trapped on Tenax glass columns and were analyzed by GC-MS. Aflatoxin field-resistant maize genotypes exhibited a larger relative concentration of the antifungal aldehyde, furfural (2-furancarboxaldehyde), when compared to the relative concentrations of the field-susceptible varieties tested. In a closed-container 5-day study, it was observed that fresh 1- and 4-day-old corn silk samples of aflatoxin-resistant maize genotypes emitted higher concentrations of furfural compared to those from susceptible genotypes. This observation probably explains the reason for the bioactivity observed in the in vitro bioassays, and the presence of furfural appears to contribute to a defense mechanism for protecting the developing maize kernel from fungal attack.     

半纤维素分解菌群HMC的微生物群落结构及功能

为明确半纤维素分解菌群HMC（hemicellulolytic microbial consortium）的微生物群落结构及其在半纤维素降解中的潜在功能;该研究测定了其在降解半纤维素主链木聚糖及玉米芯的典型理化指标,并利用16S rDNA测序技术及生物信息学手段对其微生物群落结构及功能注释进行了宏基因组学分析。结果表明,HMC在7d内能高效降解木聚糖和玉米芯,降解率达80%,木聚糖降解产生的高值产物包括还原糖、甲酸、乳酸及乙酸,最高浓度分别为1.30、0.50、1.19与1.23 mg/mL,HMC降解玉米芯时可累积挥发性脂肪酸浓度高达3.54 mg/mL。HMC是由厚壁菌门Firmicutes的多个属的微生物组成的复合微生物菌群,宏基因组分析揭示了HMC在木聚糖降解过程中的关键碳水化合物活性酶系;HMC的半纤维素降解酶系完整,包括19个木聚糖分解及下游产物代谢相关的酶;同时涵盖了乙酸、乳酸等半纤维素关键分解产物代谢相关的通路。HMC蕴含半纤维素及玉米芯高值生物转化的潜力,该研究为深入探究半纤维素在生物降解应用中的分子机制提供了重要线索,有助于推动天然半纤维素生物降解技术的发展。     

Effects of Different Mill Types on Ethanol Production Using Uncooked Dry‐Grind Fermentation and Characteristics of Residual Starch in Distiller's Dried Grains (DDG)

This study aimed to investigate impacts of milling methods on ethanol production using an uncooked dry‐grind (cold fermentation) process and characterize residual starch in the distiller's dried grains (DDG) coproduct. Four corn lines with different chemical compositions were ground with cyclone, ultra‐centrifugal, or hammer mills equipped with a screen of 0.5 mm opening and used for the cold fermentation process. Greater starch hydrolysis and ethanol yield were obtained from cyclone‐milled corn, resulting from larger damaged starch contents and smaller particle sizes of the ground corn. Corn grains and ground corn after five‐month storage showed less starch hydrolysis than the freshly ground counterpart. Residual starch (2.8–8.0%) with large proportions of intact amylopectin contents (up to 42.5%) was found in the DDG from all types of milling. The results suggested that the entrapment of starch granules in ground corn and a low activity of amylolytic enzymes at a high ethanol concentration were accountable for the remaining of starch in the DDG.     

Dual analysis of triglycerides from certain common lipids and seed extracts

A number of reference oils, two commercial oils, and several oil extracts from seeds of Nicotiana species were analyzed for the fatty acid content and also for triglyceride composition. The seed oils were obtained using an accelerated solvent extraction procedure, which was proven to be very efficient and reproducible. The fatty acids were analyzed after the hydrolysis of the oils, using trimethylsilylation and gas chromatography/mass spectrometry (GC/MS) analysis. The levels of sixteen molecular species of triglycerides in the oils were measured after GC separation using MS for identification and flame ionization detection (FID) for quantitation. The results for the fatty acids and those for triglycerides were combined to generate uniform information regarding the composition of the analyzed oils. For a number of oils, the individual triglyceride quantitation and mass spectra were reported for the first time. The study showed that in some cases, oils with similar fatty acid content do not have the same triglycerides profile. The fatty acids and triglycerides profile for selected Nicotiana species were described for the first time in the literature.     

Mold occurrence and aflatoxin B(1) and fumonisin B(1) determination in corn samples in Venezuela

Fumonisins are mycotoxins produced mainly by Fusarium moniliforme and Fusarium proliferatum, which have been associated with several animal and human diseases. Aflatoxins are hepatotoxic, mutagenic, and teratogenic metabolites produced by Aspergillus flavus and Aspergillus parasiticus. Both have been reported at high levels in corn. This study was pursued to determine mold, aflatoxin B(1) (AFTB(1)), and fumonisin B(1) (FB(1)) levels in white and yellow corn. Mold levels were determined using potato dextrose agar and identification of the main genus of molds present in corn, AFTB(1) levels by immunoaffinity chromatography, and FB(1) levels by a Bond-Elut SAX cartridge and HPLC. AFTB(1) an     

Lipid fatty acid profile analyses in liver and serum in rats with nonalcoholic steatohepatitis using improved gas chromatography-mass spectrometry methodology

Fatty acids (FAs) are essential components of lipids and exhibit important biological functions. The analyses of FAs are routinely carried out by gas chromatography-mass spectrometry after multistep sample preparation. In this study, several key experimental factors were carefully examined, validated, and optimized to analyze free fatty acid (FFA) and FA profiles of triglycerides and phospholipids in serum or tissue samples. These factors included (1) methylation/transesterification reagents, (2) validation of internal standards, and (3) final step concentration of FA methyl esters. This new method was utilized to analyze FFAs and the FA profiles of triglycerides and phospholipids in the serum and liver from a recently established rat model of nonalcoholic steatohepatitis (NASH). In this model, rats were fed a 220 kcal kg (-3/4) day (-1) diet containing either 5 or 70% corn oil for 21 days using total enteral nutrition. FA compositions of the serum and liver were found to shift from a pattern dominated by saturated and monounsaturated FAs (C16:0/18:1) to one dominated by polyunsaturated C18:2 derived from dietary linoleic acid. Alteration of FA composition in liver after overfeeding of high polyunsaturated fat diets may contribute to the progression of pathological changes from steatosis to inflammation, necrosis, and fibrosis observed in NASH.     

Lipid, polyamide, and flavonol phagostimulants for adult western corn rootworm from sunflower (Helianthus annuus L.) pollen.

Adult Diabroticites including western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, consume pollen of corn, squash, sunflower, and other species. Short-chain neutral amino acids in methanol-water extracts of pollen have been previously identified in our laboratory as strong phagostimulants for Diabrotica. Bioassay-driven fractionation was used to characterize the interacting lipid and midpolarity phagostimulants for adult WCR in Giant Gray Stripe sunflower, Helianthus annuus L., pollen. Lipids rich in omega3-linolenic acid including triglycerides, free fatty acids, phosphatidylethanolamines, phosphatidic acids, and phosphatidylcholines were highly phagostimulatory. Other important phagostimulatory components included a hydroxycinnamic acid-polyamine amide, N(1),N(5),N(10)-tri[(E)-p-coumaroyl]spermidine, and a flavonol, quercetin beta-3-O-glucoside. The structural characteristics of these phagoactive compounds and their role in the pollinivory specialization of rootworm beetles are discussed.     

Aflatoxins and fumonisins in corn from the high-incidence area for human hepatocellular carcinoma in Guangxi, China

A comparative study on the natural occurrence of aflatoxins and Fusarium toxins was conducted with corn samples from high- and low-incidence areas for human primary hepatocellular carcinoma (PHC) in Guangxi, China. In samples from the high-risk area, aflatoxin B(1) was the predominant toxin detected in terms of quantity and frequency, with its concentration ranging between 9 and 2496 microg/kg and an 85% incidence of contamination. Among the samples, 13 (76%) exceeded the Chinese regulation of 20 microg/kg for aflatoxin B(1) in corn and corn-based products intended for human consumption. Significant differences in aflatoxin B(1), B(2), and G(1) and total aflatoxin concentrations in corn between the areas were found (P < 0.05). The average daily intake of aflatoxin B(1) from corn in the high-risk area was 184.1 microg, and the probable daily intake is estimated to be 3.68 microg/kg of body weight/day, 3.20 times the TD(50) in rats. Corn samples from both areas were simultaneously contaminated with fumonisins B(1), B(2), and B(3). Aflatoxin B(1) may play an important role in the development of PHC in Guangxi.     

Detoxification of corn stover and corn starch pyrolysis liquors by ligninolytic enzymes of Phanerochaete chrysosporium

Phanerochaete chrysosporium (ATCC 24725) shake flask culture with 3 mM veratryl alcohol addition on day 3 was able to grow and detoxify different concentrations of diluted corn stover (Dcs) and diluted corn starch (Dst) pyrolysis liquors [10, 25, and 50% (v/v)] in defined media. GC-MS analysis of reaction products showed a decrease and change in some compounds. In addition, the total phenolic assay with Dcs samples demonstrated a decrease in the phenolic compounds. A bioassay employing Lactobacillus casei growth and lactic acid production was developed to confirm the removal of toxic compounds from 10 and 25% (v/v) Dcs and Dst by the lignolytic enzymes, but not from 50% (v/v) Dcs and Dst. The removal did not occur when sodium azide or cycloheximide was added to Ph. chrysosporium culture media, confirming the participation of lignolytic enzymes in the detoxification process. A concentrated enzyme preparation decreased the phenolic compounds in 10% (v/v) corn stover and corn starch pyrolysis liquors to the same extent as the fungal cultures.     

Amylose-Lipid Complex Formation in Acetylated Pea Starch-Lipid Systems

Starch-lipid interactions involving native and acetylated pea starch were studied by differential scanning calorimetry (DSC) and measurements of iodine affinity. Lipids including lauric acid, monopalmitin, and butterfat were added to aqueous starch dispersions after the starch was gelatinized at 85°C. DSC thermal curves of gelatinized modified pea starch systems containing fatty acid or monoglyceride did not show DSC transitions indicative of amylose complexes with external lipids, whereas a DSC endotherm of amylose-lipid complexes was observed for the corresponding native starch-lipid systems. However, iodine binding studies revealed that acetylated pea starch amylose complexed with added fatty acid or monoglyceride in the modified pea starch-lipid composites. The failure of DSC detection of the complexes in these systems was attributed to the absence of crystalline structures of acetylated pea starch amylose complexes. Furthermore, acetylation of starch decreased the complexing ability of the pea starch amylose as revealed by a reduction in iodine affinity. Both DSC and iodine affinity studies showed that neither native nor modified pea starch interacted to a significant extent with butterfat that consisted mainly of triglycerides.