Investigating the Mechanism of Microwave-Assisted Enzymolysis Synergized with Magnetic Bead Adsorption for Reducing Ovalbumin Allergenicity through Biomass Spectrometry Analysis.

This study found that, after microwave treatment at 560 W for 30 s, alkaline protease enzymolysis significantly reduced the allergenicity of ovalbumin (OVA). Furthermore, specific adsorption of allergenic anti-enzyme hydrolyzed peptides in the enzymatic products by immunoglobulin G (IgG) bound to magnetic bead further decreased the allergenicity of OVA. The results indicated that microwave treatment disrupts the structure of OVA, increasing the accessibility of OVA to the alkaline protease. A comparison between 17 IgG-binding epitopes identified through high-performance liquid chromatography-higher energy collisional dissociation-tandem mass spectrometry and previously reported immunoglobulin E (IgE)-binding epitopes revealed a complete overlap in binding epitopes at amino acids (AA)125-135, AA151-158, AA357-366, and AA373-381. Additionally, partial overlap was observed at positions AA41-59, AA243-252, and AA320-340. Consequently, these binding epitopes were likely pivotal in eliciting the allergic reaction to OVA, warranting specific attention in future studies. In conclusion, microwave-assisted enzymolysis synergized with magnetic bead adsorption provides an effective method to reduce the allergenicity of OVA.

Mechanism of the Allergenicity Reduction of Ovalbumin by Microwave Pretreatment-Assisted Enzymolysis through Biological Mass Spectrometry.

Ovalbumin (OVA) is a major allergen in hen eggs. Enzymolysis has been demonstrated as an efficient method for reducing OVA allergenicity. This study demonstrates that microwave pretreatment (MP) at 400 W for 20 s assisting bromelain enzymolysis further decreases the allergenicity of OVA, which was attributed to the increase in the degree of hydrolysis and promoted the destruction of IgE-binding epitopes. The results showed that MP could promote OVA unfolding, expose hydrophobic domains, and disrupt tightly packed α-helical structures and disulfide bonds, which increased the degree of hydrolysis by 7.28% and the contents of peptides below 1 kDa from 43.55 to 85.06% in hydrolysates compared with that for untreated OVA. Biological mass spectrometry demonstrated that the number of intact IgE-binding epitope peptides in MP-assisted OVA hydrolysates decreased by 533 compared to that in hydrolysis without MP; consequently, their IgG/IgE binding rates decreased more significantly. Therefore, MP-assisted enzymolysis may provide an alternative method for decreasing the OVA allergenicity.

Construction of a multi-scale effectiveness evaluation system for ecological restoration and protection of territorial space.

The ecological restoration and protection of territorial space is a systematic project for the protection and restoration of ecosystems damaged or degraded by human disturbance. Effectiveness evaluation is of great significance to the optimization, adjustment, and sustainability of ecological restoration and protection. Current research and practices tend to focus on a single element and the site scale. Based on the study on the connotation of ecological restoration and protection of territorial space and the multi-scale characteristics of ecosystems, we constructed a multi-scale effectiveness evaluation system for ecological restoration and protection of territorial space and a full-cycle monitoring system for effectiveness evaluation. The multi-scale effectiveness evaluation system consisted of the regional/watershed scale, the protection and restoration unit scale, and the sub-project scale. The full-cycle monitoring system contained the basic information investigation system before construction, the construction monitoring system during construction, and the multi-scale effectiveness evaluation system after construction. At the regional/watershed scale, structure, quality, and services of ecosystem were concerned and remote sensing was used as the main method to capture data. At the protection and restoration unit scale, ecological stress factors, ecosystem quality and services were concerned, and the main methods were remote sensing combined with field survey. At the sub-project scale, engineering technology measures were concerned and the field survey was used as the main method. In the implementation of the multi-scale effectiveness evaluation, it would be necessary to focus on and solve the key issues including the spillover effect, transmission mechanism, and potential impact of ecological restoration.

Investigation of the Mechanism of 60Co Gamma-Ray Irradiation-Stimulated Oxidation Enhancing the Antigenicity of Ovalbumin by High-Resolution Mass Spectrometry.

60Co gamma-ray irradiation-induced antigenicity changes in ovalbumin (OVA) were investigated, and the molecular mechanism was analyzed. Irradiation treatment at 0-100 kGy could significantly enhance the IgG/IgE binding ability of OVA in a dose-dependent paradigm by concomitant oxidative modification, which exhibited color browning and an increase in carbonyl content caused by high-penetrable rays. More allergenic epitopes of OVA were exposed after irradiation treatment reflected by structural changes including the unfolding of tertiary structure, the conversion of α-helix structures to ß-sheet and random coil structures, and the cleavage of several peptide bonds. Meanwhile, three oxidation sites of K46, T49, and N260 located in key linear epitopes were observed, which might increase the allergenic ability of OVA via the disaggregation of noncovalent bonds and the unwinding of α-helix structures. Conclusively, irradiation may enhance the potential allergenicity of OVA by oxidative modification, which provides theoretical guidance for effectively controlling the oxidation of proteins in the irradiation process.

Isolation and allergenicity evaluation of glycated α-lactalbumin digestive products and identification of allergenic peptides.

This study aimed to isolate and evaluate the allergenicity of glycated α-lactalbumin (ALA) digestive products and identify its allergenic peptides. The digestive products of native-, alone glycated- and ultrasound-assisted glycated ALA (ALA-D, ALA-gal-D, 100ALA-gal-D) were isolated into three fractions (F1, F2 and F3). High-resolution mass spectrometry showed that the digestion-resistant peptides of F2 and F3 mainly distributed in amino acid sequence (AA) 25-31, AA32-53, AA40-53, AA54-60, AA80-90, AA94-104. The allergenicity of the three fractions of glycated ALA was lower than that in ALA-D, indicating glycation of ALA could indeed reduce its allergenicity after digestion. Furthermore, most fractions isolated from high glycation-degree ALA had the lowest allergenicity. The IgG/IgE binding abilities of synthesized peptides indicated that AA94-104 firstly identified by us embodied the strongest allergenicity and might be the potential allergenic peptide. This will provide a theory for preparing hypoallergenic products based on the identified allergenic peptides.

Effects of Superheated Steam Treatment on the Allergenicity and Structure of Chicken Egg Ovomucoid.

The aim of this study was to explore the effects of an emerging and efficient heating technology, superheated steam (SS), on the allergenicity and molecular structure of ovomucoid (OVM). OVM was treated with 120-200 °C of SS for 2 to 10 min. The allergenicity (IgG/IgE binding abilities and cell degranulation assay) and molecular structure (main functional groups and amino acids modification) changes were investigated. The IgG-binding ability of OVM decreased and the releases of ß-hex and TNF-γ were inhibited after SS treatment, indicating that the protein allergenicity was reduced. Significant increases in oxidation degree, free SH content and surface hydrophobicity were observed in SS-treated OVM. The protein dimer and trimer appeared after SS treatment. Meanwhile, obvious changes occurred in the primary structure. Specifically, serine can be readily modified by obtaining functional groups from other modification sites during SS treatment. Moreover, the natural OVM structure which showed resistance to trypsin digestion was disrupted, leading to increased protein digestibility. In conclusion, SS-induced OVM aggregation, functional groups and amino acids modifications as well as protein structure alteration led to reduced allergenicity and increased digestibility.

Protective effect of antioxidant peptides from grass carp scale gelatin on the H2O2-mediated oxidative injured HepG2 cells.

The protective effect of antioxidant peptides from grass carp scale gelatin on hydrogen peroxide (H2O2)-mediated oxidative injured HepG2 cells was investigated, and the protective mechanism as well as peptide structure were studied. Pretreated with grass carp scale gelatin hydrolysates (GSGH) for 24 h significantly increased the survival rates and decreased the apoptosis rates of H2O2-mediated oxidative injured HepG2 cells. The increase in SOD, CAT and GPX activities, reduce in ROS level and MDA content, and weaken in damage on cell membrane and DNA could be responsible for the protective effect of GSGH on H2O2-mediated oxidative injured HepG2 cells. Peptide sequences of GSGH were analyzed by LC-ESI-Q-Orbitrap-MS/MS, and results showed that most of them were low molecular weight peptide at 358-986 Da. Synergistic effect existed among antioxidant peptides and contributed to the strong antioxidant activities of GSGH.

Insight into the mechanism of d-allose in reducing the allergenicity and digestibility of ultrasound-pretreated α-lactalbumin by high-resolution mass spectrometry.

The mechanism of d-allose in reducing the allergenicity and digestibility of ultrasound-pretreated α-lactalbumin (α-LA) was studied. The intensity reduction and peak red shift occurred in fluorescence spectra of glycated samples. Enzyme-linked immunosorbent assay and basophil degranulation analysis showed that d-allose significantly reduced the allergenicity of α-LA, and ultrasound-pretreated α-LA showed the lowest allergenicity after glycation. Compared with α-LA, the degree of hydrolysis decreased in glycated samples. Size-exclusion high-performance liquid chromatography showed that the glycated α-LA was resistant to digestive enzymes. The glycated sites and average degree of substitution per peptide molecule were determined using LC Orbitrap MS/MS. These results suggested that the masking of linear allergenic epitopes by glycation could reduce the allergenicity. Therefore, the combination of ultrasound pretreatment and glycation is a potential method to reduce protein allergenicity in food processing and provides a useful approach for the application of rare sugars in food processing.

The Transcriptome Characteristics of Severe Asthma From the Prospect of Co-Expressed Gene Modules.

Rationale: Severe asthma is a heterogeneous disease with multiple molecular mechanisms. Gene expression studies of asthmatic bronchial epithelial cells have provided biological insights and underscored possible pathological mechanisms; however, the molecular basis in severe asthma is still poorly understood. Objective: The objective of this study was to identify the features of asthma and uncover the molecular basis of severe asthma in distinct molecular phenotype. Methods: The k-means clustering and differentially expressed genes (DEGs) were performed in 129 asthma individuals in the Severe Asthma Research Program. The DEG profiles were analyzed by weighted gene co-expression network analysis (WGCNA), and the expression value of each gene module in each individual was annotated by gene set variation analysis (GSVA). Results: Expression analysis defined five stable asthma subtype (AS): 1) Phagocytosis-Th2, 2) Normal-like, 3) Neutrophils, 4) Mucin-Th2, and 5) Interferon-Th1 and 15 co-expressed gene modules. "Phagocytosis-Th2" enriched for receptor-mediated endocytosis, upregulation of Toll-like receptor signal, and myeloid leukocyte activation. "Normal-like" is most similar to normal samples. "Mucin-Th2" preferentially expressed genes involved in O-glycan biosynthesis and unfolded protein response. "Interferon-Th1" displayed upregulation of genes that regulate networks involved in cell cycle, IFN gamma response, and CD8 TCR. The dysregulation of neural signal, REDOX, apoptosis, and O-glycan process were related to the severity of asthma. In non-TH2 subtype (Neutrophils and Interferon-Th1) with severe asthma individuals, the neural signals and IL26-related co-expression module were dysregulated more significantly compared to that in non-severe asthma. These data infer differences in the molecular evolution of asthma subtypes and identify opportunities for therapeutic development. Conclusions: Asthma is a heterogeneous disease. The co-expression analysis provides new insights into the biological mechanisms related to its phenotypes and the severity.

Mechanism on the Allergenicity Changes of α-Lactalbumin Treated by Sonication-Assisted Glycation during In Vitro Gastroduodenal Digestion.

Physical-assisted chemical modification is effective to reduce the allergenicity of α-lactalbumin (ALA). However, there are few in-depth studies on the allergenicity changes of physical-assisted chemical-modified ALA during digestion. The effect of gastroduodenal digestion on the allergenicity changes of ALA treated by sonication-assisted glycation was assessed. Digestion of both ALA and its glycated forms generated peptide fractions, and intact undigested glycated ALA in the hydrolysates still covalently bound to d-galactose. High-resolution mass spectrometry revealed that a higher glycation degree was discovered in sonication-preprocessed ALA compared to native ALA. Enzyme-linked immunosorbent assay and basophil degranulation showed that sonication-assisted glycation could significantly reduce ALA allergenicity. The allergenicity of both gastric and gastroduodenal hydrolysates was further increased, and the hydrolysates of sonication-assisted glycated ALA showed the lowest allergenicity. The reason could be the shielding effect of the linear epitope found to be caused by a higher glycation degree; although linear epitopes were exposed, d-galactose covalently bound to intact undigested glycated ALA in the hydrolysates retained its masking role. These results indicated that sonication-assisted glycation could be a promising method to prepare immunotherapeutic agents for allergen immunotherapy to achieve the purpose of allergy desensitization.

Mechanism of the Reduced IgG/IgE Binding Abilities of Glycated ß-Lactoglobulin and Its Digests through High-Resolution Mass Spectrometry.

Glycation between proteins and reducing sugars is the common chemical modification in food protein, and many studies have focused on the allergenicity of the glycated protein. However, a systemic study on the allergenicity change of its digests is lacking. In this work, we explored the change rule of the digestibility and allergenicity of glycated ß-Lg during in vitro gastrointestinal digestion and interpreted the mechanism using high-resolution mass spectrometry. Glycation with arabinose increased the resistance of ß-Lg to digestive enzyme, with a low hydrolysis value. Indirect competitive ELISA showed that the IgG/IgE binding rates of ß-Lg were reduced after glycation and further reduced after digestion, in comparison with the digests of unglycated ß-Lg. There are two reasons for this phenomenon. On the one hand, 11 glycated sites were determined in the lowest allergenicity arabinose-ß-Lg conjugation (Ara-ß-Lg), which was distributed in the IgG and IgE linear allergic epitopes of ß-Lg. On the other hand, glycation masking linear allergenic epitopes had a more significant effect on reducing allergenicity in comparison to digestive enzyme hydrolysis. These results indicated that the allergenicity of Ara-ß-Lg in the human body might be lower than that of unglycated ß-Lg.

The IgE/IgG binding capacity and structural changes of Alaska Pollock parvalbumin glycated with different reducing sugars.

Parvalbumin (PV) is one of the major allergens in fish. The aim of our present work was to research the influence mechanism of glycation with different reducing sugars (glucose, fructose, ribose, lactose, and galactose) on the immunoglobulin E (IgE) and immunoglobulin G (IgG) binding capacity and structure changes of PV in Alaska Pollock. PV glycated with glucose or fructose (PV-Glu/ PV-Fru) exhibited the higher IgE/IgG binding capacities than that of ribose, galactose, or lactose. During glycation, the lysine (Lyr), tyrosine (Tyr), and phenylalanine (Phe) of PV were gradually embed into core area of three-dimensional structure of protein, which reflected in the ultraviolet (UV) spectrum and fluorescence spectra. Moreover, the increase of surface hydrophobicity had confirmed the conformation alteration of glycated PV. These results suggest that there is a specific association among the change of PV in glycation and in potential allergenicity. The types and conformation of reducing sugar greatly influenced the IgE/IgG binding capacity of PV, and glycation with ribose and galactose was a promising approach for reducing the IgE/IgG binding capacity of PV from Alaska Pollock. PRACTICAL APPLICATIONS: Parvalbumin (PV), the major allergen of fish, it can not only maintain the physiological activity of cells, but also cross react with human amyloid protein to alleviate Alzheimer's disease and Parkinson's syndrome. This study revealed that the IgE/IgG binding capacity and structural changes of PV from Alaska Pollock modified by glycation with different reducing sugars. This will help us to understand the sensitization and structural change of the glycated products after the reaction of PV with different reducing sugars. It provides an effective carbonyl source for the preparation of low antigenicity PV based on glycation and lays a foundation for glycation modification of other food allergens.

Investigation of the effect of oxidation on the structure of ß-lactoglobulin by high resolution mass spectrometry.

In this work, high-resolution mass spectrometry was used to identify the oxidation sites and forms of ß-lactoglobulin (ß-Lg) induced by hydrogen peroxide with 1.5% concentration, and the influence of oxidation sites on the structure of ß-Lg was discussed from the molecular level. Twelve kinds of oxidation products and 36 oxidation sites were identified, including sulfoxidation in sulfur-containing amino acid residue, hydroxylation in aromatic group residue, deamination in amino-containing amino acid etc. The destruction of hydrogen bonds and disulfide bonds in ß-Lg caused by oxidation is the main factor causing its structural changes, which were manifested in the decrease of ß-sheet component and increase of ß-turns and random coil contents, intrinsic fluorescence intensity and surface hydrophobicity. In addition, several peptides as potential oxidative markers were found to be capable of monitoring the degree of oxidation of ß-Lg. In short, this work provided insights into structural changes of ß-Lg by oxidation.

Insight into the mechanism of urea inhibit ovalbumin-glucose glycation by conventional spectrometry and liquid chromatography-high resolution mass spectrometry.

The inhibition effect of urea on ovalbumin (OVA) glycation was investigated, and the mechanism was evaluated through the changes in protein structure as well as glycation sites and average degree of substitution per peptide molecule (DSP) by conventional spectrometry and liquid chromatography-high resolution mass spectrometry (LC-HRMS). A urea concentration of 3 M was chosen as the optimum condition. Ultraviolet and fluorescence spectra suggested that both glycation and urea treatment could unfold the OVA, but urea inhibited the glycation-induced protein unfolding. Circular dichroism spectra showed that urea treatment could increase the ß-sheet content and reduce the α-helix content of OVA. LC-HRMS indicated that the number of glycation sites was reduced from 15 to 3, and DSP values decreased with urea treatment. In conclusion, urea could significantly inhibit the OVA-glucose glycation, and the sites competition as well as structure unfolding inhibition resulted from urea could be the main factors.

Mechanism of Selenium Nanoparticles Inhibiting Advanced Glycation End Products.

Selenium nanoparticles (SeNPs) have been applied in fields of nanobiosensors, environment, nanomedicine, etc. as a result of their excellent characteristics. Early studies had shown that SeNPs have certain inhibition ability against glycation, but the inhibition mechanism, especially for the influence of SeNPs on the reaction activity of glycation sites, remains unclear. The aim of the presented research was to reveal the effects of SeNPs on the ß-lactoglobulin (ß-Lg)/d-ribose glycation system at the molecular level and explore the possible inhibitory mechanism of SeNPs on the formation of advanced glycation end products (AGEs) by analyzing the glycation sites via high-performance liquid chromatography (HPLC)-Orbitrap-tandem mass spectrometry (MS/MS). Changes in contents of AGE formation and free amino acid contents had indicated that SeNPs could significantly slow the glycation process, thus attenuating the formation of AGEs. HPLC-Orbitrap-MS/MS analysis revealed that, at 6, 12, and 24 h, the number of glycation sites of glycated ß-Lg decreased from 7, 7, and 9 to 5, 5, and 6 after the intervention of SeNPs, respectively. The glycation extent of each glycation site was controlled, and the dual-glycation ability of K8, K14, K47, K91, and K101 was changed. All of these results confirmed that SeNPs could indeed slow the process of protein glycation at the molecular level. This may be the reason for SeNPs reducing the formation of AGEs during glycation. Therefore, this study shed light on the insight of how SeNPs reduce the formation of AGEs.

Mechanism of the effect of 2, 2'-azobis (2-amidinopropane) dihydrochloride simulated lipid oxidation on the IgG/IgE binding ability of ovalbumin.

In this work, the mechanism of the effect of lipid oxidation on the IgG/IgE binding ability of ovalbumin (OVA) was investigated via the peroxyl radicals produced by 2, 2'-azobis (2-amidinopropane) dihydrochloride to simulate lipid oxidation. Results showed that the structure of OVA unfolded partially with an increase in oxidation degree, leading to the exposure of the allergenic epitopes and increasing the IgG/IgE binding ability of OVA. Nine oxidation sites were found on the α-helix, and these sites may unwind the α-helix and expose the allergenic epitopes on the OVA surface, leading to antibody recognition and combination. Consequently, the IgG/IgE binding ability of OVA was increased. In conclusion, the allergenic capacity of OVA can be promoted by modifying peroxyl radical oxidation in processing egg products.

Conformational alteration and the glycated sites in ovalbumin during vacuum freeze-drying induced glycation: A study using conventional spectrometry and liquid chromatography-high resolution mass spectrometry.

Ovalbumin (Oval)-ribose glycation induced by vacuum freeze-drying (VFD) was studied. The protein conformational changes based on fluorescence, ultraviolet and circular dichroism spectra were evident with the increase in VFD time. The glycated sites and the average degree of substitution per peptide molecule (DSP) were determined using LC-HRMS. Lysine was shown to be the sole glycated site. Two glycated sites and the minimum DSP values were found during the first 6 h of VFD and increased to nine and the maximum DSP values after 48 h of VFD. The glycated sites located on the protein surface were mostly more active than those in the folded or helical regions, and the hydrophilic/hydrophobic environment could also influence DSP values. This study gave relationships between VFD time and the conformational structure and glycated sites of VFD-treated Oval-ribose system, providing a theoretical basis for VFD technique-based protein food and drug industries.

Predicting Spatial Variations in Soil Nutrients with Hyperspectral Remote Sensing at Regional Scale.

Rapid acquisition of the spatial distribution of soil nutrients holds great implications for farmland soil productivity safety, food security and agricultural management. To this end, we collected 1297 soil samples and measured the content of soil total nitrogen (TN), soil available phosphorus (AP) and soil available potassium (AK) in Zengcheng, north of the Pearl River Delta, China. Hyperspectral remote sensing images (115 bands) of the Chinese Environmental 1A satellite were used as auxiliary variables and dimensionality reduction was performed using Pearson correlation analysis and principal component analysis. The TN, AP and AK of soil were predicted in the study area based on auxiliary variables after dimensionality reduction, along with stepwise linear regression (SLR), support vector machine (SVM), random forest (RF) and back-propagation neural network (BPNN) models; 324 independent points were used to verify the predictive performance. The BPNN model, which demonstrated the best predictive accuracy among all methods, combined ordinary kriging (OK) with mapping the spatial variations of soil nutrients. Results show that the BPNN model with double hidden layers had better predictive accuracy for soil TN (root mean square error (RMSE) = 0.409 mg kg-1, R² = 44.24%), soil AP (RMSE = 40.808 mg kg-1, R² = 42.91%) and soil AK (RMSE = 67.464 mg kg-1, R² = 48.53%) compared with the SLR, SVM and RF models. The back propagation neural network-ordinary kriging (BPNNOK) model showed the best predictive results of soil TN (RMSE = 0.292 mg kg-1, R² = 68.51%), soil AP (RMSE = 29.62 mg kg-1, R² = 69.30%) and soil AK (RMSE = 49.67 mg kg-1 and R² = 70.55%), indicating the best fitting ability between hyperspectral remote sensing bands and soil nutrients. According to the spatial mapping results of the BPNNOK model, concentrations of soil TN (north-central), soil AP (central and southwest) and soil AK (central and southeast) were respectively higher in the study area. The most important bands (464⁻517 nm) for soil TN (b10, b14, b20 and b21), soil AP (b3, b19 and b22) and soil AK (b4, b11, b12 and b25) exhibited the best response and sensitivity according to the SLR, SVM, RF and BPNN models. It was concluded that the application of hyperspectral images (visible-near-infrared data) with BPNNOK model was found to be an efficient method for mapping and monitoring soil nutrients at the regional scale.

The Mechanism of Decreased IgG/IgE-Binding of Ovalbumin by Preheating Treatment Combined with Glycation Identified by Liquid Chromatography and High-Resolution Mass Spectrometry.

Ovalbumin is one of the most important sensitizing ingredients in allergens of egg albumin, which restricts the application of egg in the field of food processing. Previous research has indicated that glycation could cause the protein to partially expand, which may bring about the destruction of the structural IgG and IgE epitopes and induce the decline of the IgG- and IgE-binding ability of ovalbumin. In this research, the effect of a preheating treatment integrated with glycation on the IgG- and IgE-binding capability and the conformation changes of ovalbumin was studied by detecting the glycated sites and the values of degree of substitution per peptide (DSP) by liquid chromatography and high-resolution mass spectrometry (LC-HRMS). Interestingly, we found that a glycation site (K227) attached by two ribose molecules was detected in glycated ovalbumin with preheating treatment. In addition, a new glycation site (K323) appeared in G-60. The results displayed that preheating treament could strengthen the changes in the secondary and tertiary structure of ovalbumin by enhancing glycation and further reduce the IgG/IgE-binding ability by integrating with glycation because of the cover of IgG and IgE epitopes. Therefore, preheating treatment integrated with glycation may offer a way for ovalbumin to reduce sensitization.

[Using sequential indicator simulation method to define risk areas of soil heavy metals in farmland.] / 基于序贯指示模拟的农田土壤重金属风险区域识别.

The heavy metals in soil have serious impacts on safety, ecological environment and human health due to their toxicity and accumulation. It is necessary to efficiently identify the risk area of heavy metals in farmland soil, which is of important significance for environment protection, pollution warning and farmland risk control. We collected 204 samples and analyzed the contents of seven kinds of heavy metals (Cu, Zn, Pb, Cd, Cr, As, Hg) in Zengcheng District of Guangzhou, China. In order to overcame the problems of the data, including the limitation of abnormal values and skewness distribution and the smooth effect with the traditional kriging methods, we used sequential indicator simulation method (SISIM) to define the spatial distribution of heavy metals, and combined Hakanson index method to identify potential ecological risk area of heavy metals in farmland. The results showed that: (1) Based on the similar accuracy of spatial prediction of soil heavy metals, the SISIM had a better expression of detail rebuild than ordinary kriging in small scale area. Compared to indicator kriging, the SISIM had less error rate (4.9%-17.1%) in uncertainty evaluation of heavy-metal risk identification. The SISIM had less smooth effect and was more applicable to simulate the spatial uncertainty assessment of soil heavy metals and risk identification. (2) There was no pollution in Zengcheng's farmland. Moderate potential ecological risk was found in the southern part of study area due to enterprise production, human activities, and river sediments. This study combined the sequential indicator simulation with Hakanson risk index method, and effectively overcame the outlier information loss and smooth effect of traditional kriging method. It provided a new way to identify the soil heavy metal risk area of farmland in uneven sampling.