Heat-moisture treatment of freshly harvested high-amylose maize kernels improves its starch thermal stability and enzymatic resistance.

The objective of this work was to study the effects of heat-moisture treatment (HMT) of freshly harvested mature high-amylose maize (HAM) kernels on its starch structure, properties, and digestibility. Freshly harvested HAM kernels were sealed in Pyrex glass bottles and treated at 80 °C, 100 °C, or 120 °C. HMT of HAM kernels had no impact on its starch X-ray diffraction pattern but increased the relative crystallinity. This result together with the increased starch gelatinization temperatures and enthalpy change indicated starch molecules reorganization forming long-chain double-helical crystalline structure during HMT of HAM kernels. The aggregation of starch granules were observed after HMT, indicating interaction of starch granules and other components. This interaction and the high-temperature crystalline structure led to reductions in the starch digestibility, swelling power, solubility, and pasting viscosity of the HAM flours. Some starch granules remained intact and showed strong birefringence after the HAM flours were precooked at 100 °C for 20 min and followed by enzymatic hydrolysis, and the amount of undigested starch granules increased with increasing HMT temperatures. This result further supported that HMT of HAM kernels with high moisture level could increase the starch thermal stability and enzymatic resistance.

Improvement of antibacterial activity of polysaccharides via chemical modification: A review.

Antibiotic residue and bacterial resistance induced by antibiotic abuse have seriously threatened food safety and human healthiness. Thus, the development and application of safe, high-efficiency, and environmentally friendly antibiotic alternatives are urgently necessary. Apart from antitumor, antivirus, anti-inflammatory, gut microbiota regulation, immunity improvement, and growth promotion activities, polysaccharides also have antibacterial activity, but such activity is relatively low, which cannot satisfy the requirements of food preservation, clinical sterilization, livestock feeding, and agricultural cultivation. Chemical modification not only provides polysaccharides with better antibacterial activity, but also promotes easy operation and large-scale production. Herein, the enhancement of the antibacterial activity of polysaccharides via acetylation, sulfation, phosphorylation, carboxymethylation, selenation, amination, acid graft, and other chemical modifications is reviewed. Meanwhile, a new trend on the application of loading chemically modified polysaccharides into nanostructures is discussed. Furthermore, possible limitations and future recommendations for the development and application of chemically modified polysaccharides with better antibacterial activity are suggested.

Dynamic rheological behavior of high-amylose wheat dough during various heating stages: Insight from its starch characteristics.

The objective of this study was to understand how the dynamic rheological behaviors of high-amylose wheat (HAW) dough during various heating stages measured using a mixolab were affected by the starch properties. At the heating stage of 30 °C - 90 °C, low minimum (C2) and peak (C3) torques were observed for HAW doughs, which resulted from their reduced starch granule swelling. During holding at 90 °C, HAW doughs had low minimum (C4) and C3 - C4 torques, indicating a good resistance to mechanical shear and endogenous enzyme degradation. HAW doughs also had low final (C5) and setback (C5 - C4) torques, consistent with their low starch swelling power and solubility. The increased amylose in HAW starch formed long-chain double-helical B-type polymorph and amylose-lipid complex, which resulted in high starch gelatinization-temperatures and enthalpy change, low swelling power and solubility, low pasting viscosity, and high resistance of swollen granules to mechanical shear and enzyme degradation. The overall patterns of dough-rheological behavior of HAW doughs during heating were similar to their respective starch pasting profiles, indicating that starch was the dominant contributor to the dough rheology during heating. This study provides useful information for food applications and manufacturing of HAW-based products, especially none-fermented products requiring firm texture and low viscosity.

Effect of Microwave Treatment on Protease Activity, Dough Properties and Protein Quality in Sprouted Wheat.

In this study, the effects of microwave treatment on protease activity, dough properties and protein quality in sprouted wheat were investigated. Microwave treatment led to a significant (p < 0.05) reduction in protease activity in sprouted wheat. Proteases with a pH optimum of 4.4 (cysteine proteinases) were more susceptible to microwave heating, which contributed mostly to protease inactivation. Significant improvements (p < 0.05) in the dough properties and gluten quality of sprouted wheat were observed, which are probably attributable to the synergistic effectiveness of protease inactivation and heat-induced gluten cross-linking. After microwave treatment, the decrease in the solubility and extractability of protein in sprouted wheat indicated protein polymerization, which was induced by intermolecular disulfide bond cross-linking. The changes in gliadin were less pronounced due to the relatively low temperature of the microwave treatment. The cross-linking in sprouted wheat that occurred after microwave treatment seemed to mainly involve glutenin, especially B/C low-molecular-weight glutenin subunits (B/C-LMW-GSs) in the range of 30-50 kD.

Ultrafine grinding improves the nutritional, physicochemical, and antioxidant activities of two varieties of whole-grain highland barley.

Effects of ultrafine grinding on the nutritional profile, physicochemical properties, and antioxidant activities of whole-grain highland barley (HB) including white highland barley (WHB) and black highland barley (BHB) were studied. Whole-grain HB was regularly ground and sieved through 80 mesh get 80 M powder, and HB was ultrafine grounded and sieved through 80 mesh, 150 mesh, and 200 mesh get 80UMM, 150UMM, and 200UMM samples. Particle size of WHB and BHB reduced significantly after ultrafine grinding. As the particle size decreased, moisture content of WHB and BHB decreased significantly, whereas fat content increased significantly. Redistribution of fiber components in WHB and BHB from insoluble to soluble fractions was also observed. Wherein, content of soluble pentosan of WHB and BHB increased significantly from 0.56% and 0.78% (80 M) to 0.91% and 1.14% (200UMM), respectively. Damaged starch of WHB and BHB increased significantly from 8.16% and 8.21% (80 M) to 10.29% and 10.07% (200UMM), respectively. Content of phenolic acid and flavonoid of WHB and BHB and associated antioxidant capacity were increased after ultrafine grinding. Color of L* value increased significantly, a* and b* values decreased significantly, indicating the whiteness of WHB and BHB was increased after ultrafine grinding. Pasting temperature of WHB and BHB decreased, whereas peak viscosity increased. X-ray diffraction patterns of HB showed typical A- and V-style polymorphs and the relative crystallinity of HB decreased as the particle size decreased. Taken together, ultrafine grinding has shown great potential in improving the nutritional, physiochemical, and antioxidant properties of whole-grain HB. Our research findings could help better understand the ultrafine grinded whole grain HB in food industry.

Understanding resistant-starch formation during drying high-amylose maize kernels.

Interests in using high-amylose maize (HAM) flour and starch for low glycemic index foods continue to grow. The objective of this work was to understand resistant-starch formation during drying the HAM kernels. Freshly harvested HAM kernels with 28.2 % initial moisture were subjected to sun drying (~30 °C) or hot-air drying at 50 °C, 70 °C, 90 °C, or 110 °C. The enzymatic digestibility of HAM flour decreased from 63.6 % to 41.1 % as the drying temperature increased from 30 °C to 110 °C. The swelling power, solubility, and overall viscosity of HAM flours milled from kernels dried at 110 °C decreased, whereas the peak and conclusion gelatinization temperatures, enthalpy change, and relative crystallinity increased compared to those of flours from kernels dried at 30 °C, 50 °C, 70 °C, and 90 °C. Light microscopic and scanning electron microscopic images showed that starch granule aggregation in HAM flour increased with increasing drying-temperatures. The aggregates remained after 16 h enzymatic hydrolysis of cooked HAM flours. These results suggested that the increase of enzymatic resistance of HAM flour resulted from the formation of high temperature-resistant ordered structures in starch granules and the starch aggregates less accessible to enzymatic hydrolysis.

Mechanism of inactivation of Aspergillus flavus spores by dielectric barrier discharge plasma.

Dielectric barrier discharge plasma (DBDP) displays strong against fungal spores, while its precise mechanism of spore inactivation remains inadequately understood. In this study, we applied morphological, in vivo and in vitro experiments, transcriptomics, and physicochemical detection to unveil the potential molecular pathways underlying the inactivation of Aspergillus flavus spores by DBDP. Our findings suggested that mycelium growth was inhibited as observed by SEM after 30 s treatment at 70 kV, meanwhile spore germination ceased and clustering occurred. It led to the release of cellular contents and subsequent spore demise by disrupting the integrity of spore membrane. Additionally, based on the transcriptomic data, we hypothesized that the induction of spore inactivation by DBDP might be associated with downregulation of genes related to cell membranes, organelles (mitochondria), oxidative phosphorylation, and the tricarboxylic acid cycle. Subsequently, we validated our transcriptomic findings by measuring the levels of relevant enzymes in metabolic pathways, such as superoxide dismutase, acetyl-CoA, total dehydrogenase, and ATP. These physicochemical indicators revealed that DBDP treatment resulted in mitochondrial dysfunction, redox imbalance, and inhibited energy metabolism pathways. These findings were consistent with the transcriptomic results. Hence, we concluded that DBDP accelerated spore rupture and death via ROS-mediated mitochondrial dysfunction, which does not depend on cell membranes.

A Novel and Highly Efficient Microextraction Method for the Determination of Aflatoxin Precursor Averantin in Fatty Grain Samples.

Averantin (AVN) is an important aflatoxin biosynthetic precursor and has been listed in the screening range of mycotoxins. Herein, a novel ionic liquid-based one-, two-, and three-phase transition microextraction (IL-OTTPTME) method was combined with high-performance liquid chromatography for the extraction and determination of AVN in fatty grain samples. The formation of a homogeneous solution and three-phase system during the IL-OTTPTME process allowed both efficient extraction and coextracted lipid cleanup. Density functional theory calculations and distribution coefficient determination results demonstrated that AVN extraction by IL mainly occurred through hydrogen-bond and π-π interactions. Under optimized conditions, the LOD and LOQ of the proposed method were 0.5 and 1.5 ng/g, respectively. Finally, the method was used to determine AVN in several grains with different fat contents, achieving satisfactory relative recoveries (86.0-107.8%) and RSDs (1.2-6.2%, n = 3).

Pt@AuNF nanozyme and horseradish peroxidase-based lateral flow immunoassay dual enzymes signal amplification strategy for sensitive detection of zearalenone.

Lateral flow immunoassay (LFIA) has been employed extensively for the rapid, accurate, and portable detection of foodborne toxins. Here, the platinum gold nanoflower core-shell (Pt@AuNF) nanozyme with excellent optical properties, good catalytic ability and controllable reaction conditions were prepared to effectively improve the performance of lateral flow immunoassay (LFIA) strips. The Pt@AuNF nanozyme and horseradish peroxidase (HRP) combined with monoclonal antibody were used as signal probes based on the dual enzymes catalytic signal amplification strategy to detect Zearalenone sensitively. Dual enzymes catalyze the decomposition of hydrogen peroxide into hydroxyl radicals, and under the influence of hydroxyl radicals, colorless 3,3',5,5' -tetramethylbenzidine (TMB) is oxidized to blue ox-TMB, which is superimposed on the strips for signal amplification to broaden the detection range. The limit of detection (LOD) of the Pt@AuNF-HRP labeled LFIA strips after signal amplification was 0.052 ng/mL, and the detection range was 0.052-7.21 ng/mL. Compared with the Pt@AuNF labeled strips, while reducing the probes amount by half to achieve antibody conservation, the detection range was expanded by 5-fold based on achieving improved sensitivity. The study provided a meaningful reference for expanding the detection range based on immunoassay.

Optimizing the degradation of aflatoxin B1 in corn by Trametes versicolor and improving the nutritional composition of corn.

BACKGROUND: Corn, being an important grain, is prone to contamination by aflatoxin B1 (AFB1 ), and AFB1 -contaminated corn severely endangers the health of humans and livestock. Trametes versicolor, a fungus that can grow in corn, possesses the ability to directly degrade AFB1 through its laccase. This study aimed to optimize the fermentation conditions for T. versicolor to degrade AFB1 in corn and investigate the effect of T. versicolor fermentation on the nutritional composition of corn. AFB1 -contaminated corn was used as the culture substrate for T. versicolor. A combination of single-factor experiments and response surface methodology was employed to identify the optimal conditions of AFB1 degradation. RESULTS: The optimal conditions of AFB1 degradation were as follows: 9 days of fermentation, a fermentation temperature of 26.7 °C, a moisture content of 70.5% and an inoculation amount of 4.9 mL (containing 51.99 mg of T. versicolor mycelia). With the optimal conditions, the degradation rate of AFB1 in corn could reach 93.01%, and the dry basis content of protein and dietary fiber in the fermented corn was significantly increased. More importantly, the lysine content in the fermented corn was also significantly increased. CONCLUSION: This is the first report that direct fermentation of AFB1 -contaminated corn by T. versicolor not only efficiently degrades AFB1 but also improves the nutritional composition of corn. These findings suggest that the fermentation of corn by T. versicolor is a promising, environmentally friendly and efficient approach to degrade AFB1 and improve the nutritional value of corn. © 2023 Society of Chemical Industry.

Physicochemical properties and molecular structure of starches from different wheat varieties and their influence on Chinese steamed bread.

Starch is one of the key factors for the texture of Chinese steamed bread (CSB). In this study, the molecular structures and physicochemical properties of starches from 11 wheat varieties with amylose content (AC) of 1.75%-28.79% were investigated. Northern style CSB was made using these wheat varieties to explore the structure-property-quality relationship of starches. AC was negatively correlated with the pasting and gelatinization properties. The relative crystallinity (RC) had a negative correlation with AC but a positive correlation with gelatinization. The molecular structure results from the fluorophore-assisted capillary electrophoresis spectrophotometer indicated that the length of short amylopectin chains (ßAp,i ) was positively correlated with hot paste and cool paste viscosities. The amount of medium amylopectin chains (hAp,iii ) was positively correlated with peak and breakdown viscosities but negatively correlated with setback viscosity. The hAp,iii had positive correlations with gelatinization temperatures and RC. The amount of long amylopectin chains (hAp,v ) had a positive correlation with peak temperature. For the CSB texture, ßAp,i had negative correlations with hardness and chewiness, whereas had a positive correlation with resilience. The hAp,iii was negatively correlated with springiness and resilience. The hAp,v was negatively associated with resilience. PRACTICAL APPLICATION: Starch has a vital role in wheat flour products. Clarifying the structure-property-quality relationship of starches will help illuminate the role of starch molecular structure in CSB production and provide valuable information for the control of CSB quality. It also provides a significant reference for wheat breeding.

AgPdNFs and AuNOs@GO nanocomposites for T-2 toxin detection by catalytic hairpin assembly.

T-2 toxin is the most potent and toxic mycotoxin, produced by various Fusarium species that can potentially affect human health, and widely exists in field crops and stored grain. In this work, an electrochemical aptasensor with nonenzymatic signal amplification strategy for the detection of T-2 toxin is presented, using noble metal nanocomposites and catalytic hairpin assembly as signal amplification strategy. Silver palladium nanoflowers and gold octahedron nanoparticles@graphene oxide nanocomposites are used for synergistic amplification of electrical signals. Simultaneously, the catalytic hairpin assembly strategy based on artificial molecular technology was introduced to further amplify the signal. Under optimal conditions, T-2 toxin was measured within a linear concentration range 1 × 10-2 ~ 1 × 104 pg·mL-1 with an extremely low detection limit of 6.71 fg·mL-1. The aptasensor exhibited high sensitivity, good selectivity, satisfactory stability, and excellent reproducibility. Moreover, this method had high accuracy in detecting T-2 toxin in beer sample. The encouraging results show the potential application in foodstuff analysis. A dual signal amplification electrochemical biosensor for the detection of T-2 toxins was constructed, through the signal amplification of noble metal nanomaterials and CHA strategy.

Degradation of Aflatoxin B1 in Moldy Maize by Pseudomonas aeruginosa and Safety Evaluation of the Degradation Products.

Aflatoxin B1 (AFB1) is the most harmful mycotoxin commonly found in food and feed. Pollution from AFB1 causes serious economic and health issues worldwide because it causes strong mutagenicity and carcinogenicity in humans and animals. In this study, Pseudomonas aeruginosa was used to degrade AFB1 in moldy maize, and the safety of this biological method was investigated using genotoxicity and cytotoxicity tests. Using response surface methodology, we established the optimal conditions for degrading AFB1 by the fermentation supernatant of P. aeruginosa. Under these conditions, the degradation rate of AFB1 reached 99.67%. Furthermore, the Ames mutagenicity test showed that AFB1 treated with P. aeruginosa fermentation supernatant for 72 h was not mutagenic. CCK-8 cell assay showed that AFB1 cytotoxicity was significantly reduced after degradation. Overall, our findings show that the fermentation supernatant of P. aeruginosa may be a good candidate for biodegradation of AFB1.

Edible fungi efficiently degrade aflatoxin B1 in cereals and improve their nutritional composition by solid-state fermentation.

Aflatoxin B1 (AFB1) is extremely harmful to human and livestock. Laccase, a green catalyst, has been shown to effectively degrade AFB1 and can be obtained from edible fungi. The objective of this study was to screen edible fungi with high laccase activity and determine their effects on the degradation of AFB1 in cereals and the nutritional composition of the cereals through solid-state fermentation. Results from plate assays confirmed that 51 of the 55 tested edible fungi could secrete laccase. Submerged fermentation results showed that 17 of the 51 edible fungi had maximum laccase activity exceeding 100 U/L. The growth of different edible fungi varied significantly in corn, rice and wheat. More importantly, 6 edible fungi with high laccase activity and good growth could efficiently degrade AFB1 in cereals. We found for the first time that Ganoderma sinense could not only secrete highly active laccase and efficiently degrade AFB1 in corn by 92.91%, but also improve the nutritional quality of corn. These findings reveal that solid-state fermentation of cereals with edible fungi is an environmentally friendly and efficient approach for degrading AFB1 in cereals and improving the nutritional composition of cereals.

Chinese traditional sourdough steamed bread made by retarded sponge-dough method: Microbial dynamics, metabolites changes and bread quality during continuous propagation.

Continuous propagation of Chinese traditional sourdough (CTS) was adopted to simulate the industrial production of sourdough steamed bread made by retarded sponge-dough method (SSB). Establishment of a stable microbial ecosystem occurred in mature sourdough within four days of continuous propagation, as revealed by both microbial and metabolic analyses. Lactobacillus sanfranciscensis and Kazachstania humilis were the predominant bacterial and fungal species in mature sourdoughs. Their relative abundances changed significantly from the first to third day of continuous propagation while exhibited relatively constant from the fourth day onwards despite the use of flour/water for each back-slopping step. Major changes in the metabolites and fermentative characteristics were observed during the initial three days and dough samples showed little temporal metabolic and fermentative variations from the fourth days onwards. Consequently, volumetric and textural properties as well as the volatile flavor compounds of SSB displayed rather high stability from the fourth day onwards.

FeMOF-based nanostructured platforms for T-2 toxin detection in beer by a "fence-type" aptasensing principle.

In this work, a label-free electrochemical aptasensor for the detection of T-2 toxin was constructed, using gold nanoparticles/Fe-based metal organic framework@graphene oxide (AuNPs/FeMOF@GO) nanocomposites, exonuclease (RecJf), and the "fence-type" structure of aptamer-single stranded DNA (Apt-sDNA) complex as the signal amplification element. Wherein, AuNPs/FeMOF@GO nanocomposite effectively improves the aptasensor performance by improving the electron transfer capacity of the electrode and providing a larger specific surface area to load a large number of Apt-sDNA structures. Meanwhile, the shear effect of RecJf, which was induced by T-2 toxin, further improves the analytical performance of the aptasensor. Under the optimum conditions, the constructed aptasensor shown excellent performance for T-2 toxin detection, with a wide linear range (5.0 × 10-1 pg·mL-1-5.0 × 106 pg·mL-1) and a low limit of detection of 0.19 pg mL-1. Also, the aptasensor showed high specificity, excellent stability, and available repeatability. What's more, the prepared aptasensor was explored for its application in actual samples and showed good detection results, which provided a new strategy for detecting T-2 toxin in the food and feed. A label-free electrochemical aptasensor for the detection of T-2 toxin was constructed using gold nanoparticles/Fe-metal organic framework@graphene oxide (AuNPs/FeMOF@GO) nanocomposites, exonuclease (RecJf), and the "fence-type" structure of aptamer-single stranded DNA (Apt-sDNA) complex as the signal amplification element.

Colloidal Au sphere and nanoflower-based immunochromatographic strips for sensitive detection of zearalenone in cereals.

Zearalenone (ZEN), also known as an F-2 toxin, is a secondary metabolite in the toxic Fusarium species with estrogen properties. ZEN and its derivatives can cause developmental and reproductive disorders in humans and other mammals. In this study, colloidal Au spheres (AuSPs) and Au nanoflowers (AuNFs) were used as signal labels to detect ZEN in cereals, and the critical factors affecting the sensitivity of the immunochromatographic strip (ICS), namely the volume of antigen, antibody, and probe quantities were optimized and compared in detail. Since the large specific surface area of AuNFs reduces the steric hindrance of proteins, it is more conducive to improving the fixation rate of antibodies and proteins. Compared with the traditional colloidal AuSP immunochromatographic strip (AuSP-ICS), the volume of the antibody used in the AuNF immunochromatographic strip (AuNF-ICS) was 0.6 times that in the AuSPs-ICS. At the same antigen volume, a lower amount of probe can achieve the desired visual detection effect and higher sensitivity. For the AuNF-ICS, the limit of detection (LOD) was as low as 0.08 ng mL-1. ZEN could be detected quickly and accurately from 0.08-10.2 ng mL-1. And the AuNF-ICS had a high degree of specificity and sensitivity to ZEN. In summary, the AuNF-ICS serves as a valuable tool in large-scale on-site detection of ZEN.

Electrochemical aptasensor based on the target-induced strand displacement strategy-driven for T-2 toxin detection.

Herein, an aptasensor based on target-induced strand displacement (TISD) strategy was developed for sensitive detection of T-2 toxin. Gold nanoparticles@ aminated manganese dioxide (AuNPs@NH2-MnO2) exhibited excellent electrical conductivity and provided more binding sites for aptamer (Apt). Besides, polyethyleneimine-reduced graphene oxide/gold­platinum core-shell nanorods composites (PEI-rGO/Pt@Au NRs) were used to be carriers for signaling tags, as their sufficiently large specific surface area improved the loading capacity for signal molecules. In the presence of T-2, the Apt sequence was more inclined to form an Apt-T-2 complex, and the cDNA was displaced from the Apt-cDNA duplex, while the signal tag was released, resulting in a weakened MB signal, differential pulse voltammetry (DPV) was used to record the signal change. Under optimal conditions, the signal response of the constructed electrochemical aptasensor exhibited a good linear relationship with the concentration of T-2. The detection limit was 8.74 × 10-7 ng mL-1over a wide range of concentration from 5 × 10-6 ng mL-1 to 5 ng mL-1. Furthermore, the proposed aptasensor had excellent specificity, good stability and can be well applied to the detection of real samples. It provided a new avenue for the research and development of sensitive aptasensors in food detection and analysis.

Electrochemical aptasensor based on Ce3NbO7/CeO2@Au hollow nanospheres by using Nb.BbvCI-triggered and bipedal DNA walker amplification strategy for zearalenone detection.

Herein, an electrochemical aptasensor combining Nb.BbvCI-triggered bipedal DNA walking strategy was constructed for ultrasensitive assay of zearalenone (ZEN). The aptasensor used Ce3NbO7/CeO2 @Au hollow nanospheres as electrode modification material and PdNi@MnO2/MB as the signal label. Importantly, the Ce3NbO7/CeO2 synthesized by hydrothermal method were combined with Au nanoparticles and applied to the electrode surface. The as-prepared Ce3NbO7/CeO2 @Au possessed a large surface area, excellent electrical conductivity, stability and more binding sites. PdNi@MnO2 with high specific surface area and porosity combined with molecule methylene blue (MB) was introduced into electrodes as the signal label. The proposed aptasensor utilized the advantages of specific recognition of aptamers and target molecules to release bipedal DNA walker (w-DNA), and then the w-DNA was triggered by Nb.BbvCI and entered the cycle to release more signal probes. The feasibility of this strategy was recorded by the differential pulse voltammetry (DPV) method. Under the optimized conditions, the electrochemical aptasensor exhibited a wide linear dynamic range from 1 × 10-4 to 1 × 103 ng mL-1 with a low detection limit of 4.57 × 10-6 ng mL-1. Moreover, the aptasensor had high selectivity, good stability, excellent repeatability and provided an effective method for the trace detection of ZEN in real samples.

In-depth genetic analysis reveals conditioning of polyphenol oxidase activity in wheat grains by cis regulation of TaPPO2A-1 expression level.

Time-dependent darkening and discoloration of wheat product caused by high polyphenol oxidase enzymes (PPO) activity is the most undesirable character in wheat processing industry. We performed GWAS of PPO activity in wheat grains utilizing an association panel and identified 22 significant SNPs. The most significant GWAS peak on chromosome 2A was verified by QTL analysis of PPO activity. The candidate gene for this GWAS peak was identified as TaPPO2A-1, which was the highest expressed PPO gene in wheat grains. The expression level of TaPPO2A-1 was significantly correlated with PPO activity. The most significant association signal for GWAS of the expression values of TaPPO2A-1 pinpointed to the genomic region containing TaPPO2A-1. The results suggested that cis regulation of TaPPO2A-1 expression is the key factor in regulation of PPO activity in wheat grains. The conclusion was further enhanced by haplotype analysis of seven SNPs in the promoter of TaPPO2A-1.