Novel Essential Oil-Based Thiosemicarbazone Compounds as Potential Fungicides in Controlling Postharvest Diseases of Citrus Fruit.

To develop novel fungicides for controlling postharvest fungal diseases in citrus fruits, 12 essential oil (EO)-based thiosemicarbazones compounds, termed hydrazine-carbothioamide, were prepared according to the condensation method. In vitro assays showed that compound 13j exhibited the strongest antifungal activity (minimum inhibitory concentration [MIC] = minimum fungicidal concentration [MFC] = 0.0125 mg/mL) against Penicillium digitatum. An in vivo study revealed that 5 × MFC of compound 13j can effectively mitigate the green mold incidence of citrus fruit inoculated with P. digitatum, as well as fruit rot during natural storage, at a level comparable to that of the chemical fungicide prochloraz. Throughout this process, fruit quality was maintained. The hemolysis assay showed that these thiosemicarbazone compounds have good biocompatibility and that their safety is comparable to that of prochloraz. The antifungal activity of compound 13j was attributed to membrane damage, as confirmed using scanning electron microscopy (SEM), Calcofluor white (CFW) staining, propidium iodide (PI) staining, Fourier transform-infrared (FT-IR) spectroscopy, optical density (OD)260, and relative conductivity assays. Collectively, our results indicate that compound 13j can be used as an antifungal agent to control the postharvest decay of citrus fruits.

Combination of Cinnamaldehyde/ß-cyclodextrin inclusion complex and L-phenylalanine effectively reduces the postharvest green mold in citrus fruit.

The essential oil and ß-cyclodextrin inclusion complex was able to inhibit the growth of Penicillium digitatum, a damaging pathogen that causes green mold in citrus fruit. In this study, cinnamaldehyde-ß-cyclodextrin inclusion complex (ß-CDCA) for controlling citrus green mold was synthesized by the co-precipitation method. Characterization of ß-CDCA revealed that the aromatic ring skeleton of cinnamaldehyde (CA) was successfully embedded into the cavity of ß-CD to form the inclusion complex. ß-CDCA inhibited P. digitatum at a minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of 4.0 g/L. FT-IR spectroscopy analysis, calcofluor white staining, extracellular alkaline phosphatase (AKP) activity and propidium iodide (PI) staining of hyphae morphology showed that ß-CDCA may damage the cell ultrastructure and membrane permeability of P. digitatum. The study further demonstrated that hydrogen peroxide (H2O2), malondialdehyde (MDA), and reactive oxygen species (ROS) markedly accumulated in 1/2 MIC ß-CDCA treated hyphae. This implied that ß-CDCA inhibited growth of P. digitatum by the triggering oxidative stress, which may have caused cell death by altering cell membrane permeability. In addition, in vivo results showed that ß-CDCA alone or combined with L-phenylalanine (L-PHe) displayed a comparable level to that of prochloraz. Therefore, ß-CDCA combined with L-PHe can thus be used as an eco-friendly preservative for the control green mold in postharvest citrus fruit.

Linalool as a key component in strawberry volatile organic compounds (VOCs) modulates gut microbiota, systemic inflammation, and glucolipid metabolism.

Strawberries are rich in volatile organic compounds (VOCs), which are increasingly recognized as potential health-promoting factors. This study explored the health effects of intaking strawberry VOC extract and its dominant terpene, linalool. The results indicated that linalool and strawberry VOC extract significantly increased the abundance of beneficial bacteria like Lactobacillus, Bacillus, and Alistipes in mice. Moreover, mice treated with linalool and strawberry VOC extract exhibited notable reductions in serum pro-inflammatory cytokines; interleukin IL-6 decreased by 14.5% and 21.8%, respectively, while IL-1ß levels decreased by 9.6% and 13.4%, respectively. Triglyceride levels in the treated groups were reduced by 38.3% and 58.1%, respectively. Spearman's correlation analysis revealed that Bacillus negatively correlated with glucolipid indices, and Bifidobacterium and Dubosiella negatively correlated with inflammatory factors, indicating that alterations in glucolipid metabolism might be associated with the regulation of gut microbiota and systemic inflammation.

Citrus oil gland and cuticular wax inspired multifunctional gelatin film of OSA-starch nanoparticles-based nanoemulsions for preserving perishable fruit.

Inspired by the citrus oil gland and cuticular wax, a multifunctional material that stably and continuously released the carvacrol and provided physical defenses was developed to address issues of fresh-cut fruits to microbial infestation and moisture loss. The results confirmed that low molecular weight and loose structure of starch nanoparticles prepared by the ultrasound-assisted Fenton system were preferable for octenyl succinic anhydride modification compared to native starch, achieving a higher degree of substitution (increased by 18.59 %), utilizing in preparing nanoemulsions (NEs) for encapsulating carvacrol (at 5 % level: 81.58 %). Furthermore, the NEs-based gelatin (G) film improved with surface hydrophobic modification by myristic acid (MA) successfully replicated the citrus oil gland and cuticular wax, providing superior antioxidant (enhanced by 3-4 times) and antimicrobial properties (95.99 % and 84.97 % against Staphylococcus aureus and Escherichia coli respectively), as well as the exceptional UV shielding (nearly 0 transmittance in the UV region), mechanical (72 % increase in tensile strength), and hydrophobic (WCA 133.63°). Moreover, the 5%NE-G@MA film inhibited foodborne microbial growth (reduced by 50 %) and water loss (controlled below 15 %), extending the shelf life of fresh-cut navel orange and kiwi. Thus, the multifunctional film was a potential shield for preserving perishable fresh-cut products.

The regulation of postharvest strawberry quality mediated by abscisic acid under elevated CO2 stress.

Elevated CO2 was a potential strategy for strawberry preservation. However, the regulatory mechanism remained unclear. In current study, transcriptome analysis showed that elevated CO2 played important roles in regulating strawberry fruit quality at the transcriptional level, and plant hormones metabolism at least partially involved in the regulatory process. Further, ABA was demonstrated to play important roles in the response to elevated CO2. Elevated CO2 inhibited the accumulation of ABA, which was 61% lower than that in control. Elevated CO2 repressed ABA synthesis by inhibiting NCED activity and the expression of FaNCED1/2, leading to the reduction of ABA accumulation as a result. Meanwhile, elevated CO2 also decreased ABA sensitivity by down-regulating FaSnRK2.4/2.6 and FaABI5 expression. The dual down-regulation of ABA signaling accounted for the regulation of fruit quality under elevated CO2 treatment. These results provide new insights into the mechanism of strawberry fruit response to elevated CO2.

Visible light-responsive TiO2-based hybrid nanofiller reinforced multifunctional chitosan film for effective fruit preservation.

In this study, we developed a multifunctional chitosan film with visible light-responsive photocatalytic properties by incorporating a novel nanofiller-a nanohybrid particle of poly(tannic acid) (PTA) and TiO2 (TP-NPs). Firstly, the hybridization of TiO2 with PTA not only improved its dispersion but also obtained TP-NPs with smaller band gaps (from 3.11 eV to 1.55 eV) and higher separation efficiency of photogenerated e--h+ (about 1.5-fold enhancement), thereby producing more reactive oxygen species and enhancing the antibacterial efficacy (compared with TiO2, the antibacterial effect of TP-NPs on Staphylococcus aureus and Escherichia coli was heightened by about 2 times under visible light for 1 h). Secondly, TP-NPs were hydrogen bonded with chitosan, strengthening its mechanical and barrier properties, while imparting exceptional antibacterial efficacy. Moreover, the multifunctional properties enabled the active film to effectively delay the quality deterioration of grapes and kiwifruit. Hence, this study presented a multifunctional active packaging film tailored for fruit preservation.

The combination of metabolome and transcriptome clarifies the inhibition of the Alternaria toxin accumulation by methyl ferulate.

As one of the most typical pathogens in fruit postharvest diseases, Alternaria alternata (A. alternata) can produce Alternaria toxins (ATs) aggravating fruit decay and harming human health. In this study, ATs (tenuazonic acid, alternariol monomethyl ether, and alternariol) production was inhibited effectively by 200 and 8000 mg/L MF (methyl ferulate) in vitro and in vivo. 1-Octen-3-ol and 3-octanol were the potential iconic volatile organic compounds of ATs (R2 > 0.99). MF induced oxidative stress, resulting in physiological and metabolic disorders, membrane lipid oxidation and cell damage. It decreased precursors and energy supply by disturbing amino acid metabolism, ABC transporters, citrate cycle, pentose and glucuronate interconversions to regulate ATs synthesis. MF down-regulated the genes related to ATs synthesis (PksJ, AaTAS1, and OmtI), transport (AaMFS1 and MFS), and pathogenicity to affect ATs production and virulence. This study provided a theoretical basis for the control of ATs production.

Curdlan-polyphenol complexes prepared by pH-driven effectively enhanced their physicochemical stability, antioxidant and prebiotic activities.

In this study, the curdlan-polyphenol complexes were constructed by a pH-driven method. The interaction between curdlan and various hydrophobic polyphenols (curcumin, quercetin, and chlorogenic acid) was investigated. Curdlan could self-assemble into particles for loading polyphenols through hydrogen bonding and hydrophobic interactions. The three polyphenols were embedded in curdlan in an amorphous state. The curdlan-curcumin complex showed the lowest viscoelasticity but exhibited the highest curcumin loading ability (34.04 ± 1.73 mg/g). However, the curdlan-chlorogenic acid complex emerged the opposite trend, indicating that the loading capacity was associated with the hydrophobicity of polyphenols. The antioxidant activity of curdlan significantly increased after combining with polyphenols, which could be maintained during in vitro simulated gastrointestinal digestion. In particular, the curdlan-quercetin complex exhibited the highest antioxidant activity and short-chain fatty acid concentration, which could influence gut microbiota composition by promoting the proliferation of Prevotella and inhibiting the growth of Escherichia_Shigella. In conclusion, the curdlan-polyphenol complexes prepared by an alcohol-free pH-driven method could effectively enhance the gastrointestinal stability of polyphenols as well as increase the antioxidant and prebiotic activities of curdlan, which could be applied as a functional ingredient to improve gut health.

1-MCP treatment improves the postharvest quality of Jinxiu yellow peach by regulating cuticular wax composition and gene expression during cold storage.

This study aimed to analyze the effect of 1-methylcyclopropene (1-MCP) treatment on the postharvest quality, epidermal wax morphology, composition, and gene expression of Jinxiu yellow peach during cold storage. The results showed that 1-MCP treatment could maintain the postharvest quality of peach fruit as compared to control (CK) during cold storage. The wax crystals of peach fruit were better retained by 1-MCP, and they still existed in 0.6 and 0.9 µL/L 1-MCP treated fruit at 36 days. The total wax content in all the fruit increased first and then decreased during cold storage. Meanwhile, n-alkanes and primary alcohols were the main wax components. Compared to CK, 1-MCP treatment could delay the reduction of wax content during cold storage. The correlation analysis indicated that the postharvest quality of yellow peach was mainly affected by the contents of fatty acids and triterpenoids in cuticular wax. The transcriptomics results revealed PpaCER1, PpaKCS, PpaKCR1, PpaCYP86B1, PpaFAR, PpaSS2, and PpaSQE1 played the important roles in the formation of peach fruit wax. 1-MCP treatment upregulated PpaCER1 (18785414, 18786441, and 18787644), PpaKCS (18774919, 18789438, and 18793503), PpaKCR1 (18790432), and PpaCYP86B1 (18789815) to deposit more n-alkanes and fatty acids during cold storage. This study could provide a new perspective for regulating the postharvest quality of yellow peach in view of the application of cuticular wax. PRACTICAL APPLICATION: 'Jinxiu' yellow peach fruit is favorable among consumers because of its high commercial value. However, it ripens and deteriorates rapidly during storage, leading to serious economic loss and consumer disappointment. The effect of 1-methylcyclopropene (1-MCP) treatment on the postharvest quality, epidermal wax morphology, composition, and genes regulation of 'Jinxiu' yellow peach during cold storage was assessed. Compared to control, 1-MCP treatment could retain the storage quality of yellow peach by affecting cuticular wax composition and gene expression. This study could provide new perspective for regulating the postharvest quality of yellow peach in view of the application of cuticular wax.

Application of L-Cysteine Hydrochloride Delays the Ripening of Harvested Tomato Fruit.

Fruit ripening is controlled by internal factors such as hormones and genetic regulators, as well as external environmental factors. However, the impact of redox regulation on fruit ripening remains elusive. Here, we explored the effects of L-cysteine hydrochloride (LCH), an antioxidant, on tomato fruit ripening and elucidated the underlying mechanism. The application of LCH effectively delayed tomato fruit ripening, leading to the suppression of carotenoid and lycopene biosynthesis and chlorophyll degradation, and a delayed respiration peak. Moreover, LCH-treated fruit exhibited reduced hydrogen peroxide (H2O2) accumulation and increased activities of superoxide dismutase (SOD), catalase (CAT), and monodehydroascorbate reductase (MDHAR), compared with control fruit. Furthermore, transcriptome analysis revealed that a substantial number of genes related to ethylene biosynthesis (ACS2, ACS4, ACO1, ACO3), carotenoid biosynthesis (PSY, PDS, ZDS, CRTISO), cell wall degradation (PG1/2, PL, TBG4, XTH4), and ripening-related regulators (RIN, NOR, AP2a, DML2) were downregulated by LCH, resulting in delayed ripening. These findings suggest that the application of LCH delays the ripening of harvested tomato fruit by modulating the redox balance and suppressing the expression of ripening-related genes.

A novel edible solid fat substitute: Preparation of biphasic stabilized bigels based on glyceryl monolaurate and gellan gum.

Solid fats contribute to a delicate and pleasant flavor for food, but its excessive intake increases the risk of cardiovascular disease. Bigel is considered a promising solid fat substitute as it significantly reduces fat content while meeting consumer demands for food flavor and a balanced diet. In this study, bigels were prepared by mixing glyceryl monolaurate-based oleogel (10 wt%) and gellan gum-based hydrogel (0.8 wt%) at ratios of 1:3, 1:1, and 3:1. The microscopic results indicated that the oleogel/hydrogel ratios influenced the structure of bigels, forming oil-in-water, bi-continuous, and water-in-oil bigels with the increase of oleogel proportion, respectively. All bigels presented a semi-solid structure dominated by elasticity, and their hardness, gumminess, chewiness, and cohesiveness increased with the enhancement of hydrogel proportion. Among them, the bigels (S25:L75 and S25:H75) prepared with an oleogel/hydrogel ratio of 1:3 showed excellent freeze-thaw stability, maintaining an oil holding capacity of >95 % after three freeze-thaw cycles. Meanwhile, they also presented good oxidative stabilities, where the peroxide values and malondialdehyde contents were below 0.07 g/100 g and 1.5 mg MDA/kg at 12 d, respectively. Therefore, S25:L75 and S25:H75 are expected to be green, low-cost, healthy, and sustainable alternatives to solid fats.

Grafting chlorogenic acid enhanced the antioxidant activity of curdlan oligosaccharides and modulated gut microbiota.

In this study, the effects of grafting chlorogenic acid (CA) on the antioxidant and probiotic activities of curdlan oligosaccharides (CDOS) were investigated. CDOS with degrees of polymerization of 3-6 was first obtained by degradation of curdlan with hydrogen peroxide and then grafted with CA using a free radical-mediated method under an ultrasonication-assisted Fenton system. The thermal stability and antioxidant ability of CDOS were enhanced after grafting with CA. In vitro fermentation, supplementation of CDOS-CA stimulated the proliferation of Prevotella and Faecalibacterium while inhibiting the growth of harmful microbiota. Notably, the concentration of total short-chain fatty acids and the relative abundance of beneficial bacteria markedly increased after fermentation of CDOS-CA, indicating that CA grafting could improve the probiotic activity of CDOS. Overall, the covalent binding of CDOS and CA could enhance the antioxidant and probiotic activities of CDOS, suggesting potential improvements in gastrointestinal and colonic health.

Development of a chitosan/pectin-based multi-active food packaging with both UV and microbial defense functions for effectively preserving of strawberry.

Multi-active food packaging was prepared for strawberry fruit preservation where epigallocatechin gallate (EGCG)-containing pectin matrix and natamycin (NATA)-containing chitosan (CS) matrix were utilized to complete LBL electrostatic self-assembly. The results showed that the physicochemical properties of the multi-active packaging were closely related to the addition of NATA and EGCG. It was found that NATA and EGCG were embedded in the CS/pectin matrix through intermolecular hydrogen bonding interactions. The CN/PE 15 % multi-active films prepared based on the spectral stacking theory formed a barrier to UV light in the outer layer, exhibited excellent NATA protection under UV light exposure conditions at different times, and provided long-lasting and sustained bacterial inhibition in the inner layer. In addition, the CN/PE 15 % multi-active packaging extended the shelf life of strawberry at room temperature compared with the control samples. In conclusion, the developed CN/PE 15 % packaging provided potential applications for multi-active food packaging materials.

How to effectively and greenly prepare multi-scale structural starch nanoparticles for strengthening gelatin film (ultrasound-Fenton system).

Ultrasound (US) assisted with Fenton (US-Fenton) reaction was developed to efficiently and greenly prepare starch nanoparticles (SNPs) that were employed as nanofillers to enhance gelatin (G) film properties. Compared to Fenton reaction alone, US-Fenton reaction significantly improved preparation efficiency and dispersion of SNPs (p < 0.05). An optimal US-Fenton reaction parameter (300 mM H2O2, ascorbic acid 55 mM, US 45 min) was found to prepare SNPs with uniform sizes (50-90 nm) and low molecular weight (Mn 7.91 × 105 Da). The XRD, FT-IR, and SAXS analysis revealed that the US-Fenton reaction degraded the amorphous and crystalline zones of starch from top to down, leading to the collapse of the original layered structure starch and the progressive formation of SNPs. The different sizes of SNPs were selected to prepare the composite films. The G-SNP3 film (with 50-90 nm SNPs) showed the most outstanding UV blocking, tensile, and barrier properties. Especially, the tensile strength of G-5%SNP3 film (containing 5 % SNPs) increased by 156 % and 6 % over that of G film and G-5%SNP2 film (containing 5%SNPs with 100-180 nm), respectively. Therefore, the nanomaterial was promisingly prepared by the US-Fenton system and provided a strategy for designing and producing nanocomposite films.

Ultrasonication significantly enhances grafting efficiency of chitosan-ferulic acid conjugate and improves its film properties under Fenton system.

Ultrasonication (US)-assisted Fenton-system (US-Fenton) with different US time was developed for synthesizing chitosan (CS)-ferulic acid (FA) conjugates. The optimal US-Fenton for a suitable time was selected for preparing a film with CS-FA conjugate and its structural, functional, rheological, and physical properties were also investigated. Compared with Fenton-system, US-Fenton enhanced the grafting ratio of the conjugates, which increased firstly and then decreased as US time. The conjugate obtained by US-Fenton for 1 min (FUS1) possessed the highest grafting ratio (121.28 mg FA/g) and its grafting time was also shortened from 12 h to 1 min contrasted with Fenton grafted method. Structural characterization results showed that FA was conjugated on CS via ester and amide bonds with decreased crystallinity. Scanning electron microscopy and molecular weight analysis indicated that the degradation degree of CS-FA conjugates increased with US time. The DPPH and ABTS radical-scavenging activities of FUS1 were the closest to ascorbic acid, and it also showed the best antibacterial effect among the test conjugates. Accordingly, FUS1 was selected to obtain the film for contrasting with CS film. FUS1 film solution exhibited a decreased viscosity. In comparison to CS film, UV transmittance of FUS1 film approached zero, and its moisture, oxygen, and carbon dioxide permeabilities significantly decreased (P < 0.05). Moreover, its water solubility and tensile strength increased by 58.09% and 25.72% than those of CS film, respectively. Therefore, US-Fenton for 1 min could be a promising method for efficiently preparing active food package materials and FUS1 film possessed broad application prospects.

Transcriptomics and gas chromatography-mass spectrometry metabolomics reveal the mechanism of heat shock combined with 1-methylcyclopropene to regulate the cuticle wax of jujube fruit during storage.

Cuticle wax is closely related to fruit quality during storage. In this study, changes in epidermal wax morphology, composition, and genes regulation induced by heat shock (HT), 1-methylcyclopropene (1-MCP) or their combination (HT + 1-MCP) were investigated in jujube fruit during cold storage. HT, 1-MCP, or HT + 1-MCP caused a smoother wax layer and fewer micro-cracks compared to the control (CK) during cold storage. It was confirmed that acids and terpenoids were the main wax components by gas chromatography-mass spectrometry. HT + 1-MCP and 1-MCP treatments could significantly increase (p < 0.05) the wax content at 45 d of cold storage. The transcriptomics results indicated that HT + 1-MCP treatment up-regulated FATB, FATB, FAB2, FAD2 and CYP716A, and maintained the wax content of jujube fruit during cold storage. These results could provide new perspective for regulating the cuticle characteristics to extend the shelf life of jujube fruit.

Fabrication and Characterization of Eco-Friendly Polyelectrolyte Bilayer Films Based on Chitosan and Different Types of Edible Citrus Pectin.

The eco-friendly polyelectrolyte bilayer films were prepared by layer-by-layer (LBL) casting method using chitosan (CS) and four types of edible citrus pectin as film substrates. The results showed that the polyelectrolyte bilayer films exhibited excellent comprehensive properties. Furthermore, the interaction between CS and pectin was closely related to the degree of methyl-esterification (DM), molecular weight (Mw), and zeta potential of pectin. The low DM, Mw, and high zeta potential of the low methyl-esterified pectin (LM) resulted in a denser internal structure of the bilayer film, stronger UV shielding performance, and stronger gas barrier ability. The high DM and Mw of the high methyl-esterified pectin (HM) endow the bilayer film with stronger mechanical properties, thermal stability, and antifogging property. The microstructural and spectroscopic analysis showed that there are hydrogen bonds and electrostatic interactions between the layers. Overall, the developed CS-pectin polyelectrolyte bilayer films provided potential applications for food bioactive packaging.

Gas Chromatography-Mass Spectrometry Metabolite Analysis Combined with Transcriptomic and Proteomic Provide New Insights into Revealing Cuticle Formation during Pepper Development.

The cuticle plays an important role for the quality of pepper fruit. However, the molecular mechanism of cuticle formation in pepper fruit remains unclear. Our results showed that the wax was continuously accumulated during pepper development, while the cutin monomer first increased and then decreased. Hexadecanoic acid and 10,16-hydroxyhexadecanoic acid were the main components of wax and cutin, respectively. Combined with transcriptome and proteome, the formation patterns of wax and cutin polyester network for pepper cuticle was proposed. The 18 pairs of consistent expression genes and proteins involved in cuticle formation were revealed. Meanwhile, 12 key genes were screened from fatty acid biosynthesis, biosynthesis of unsaturated fatty acids, fatty acid elongation, cutin, suberine, and wax biosynthesis, glycerolipid metabolism, and transport pathway. This study would provide important candidate genes and theoretical basis for the molecular mechanism of cuticle formation, which is essential for the breeding of peppers.

Fabrication and characterization of pullulan-based composite films incorporated with bacterial cellulose and ferulic acid.

Pullulan-based composite films incorporated with bacterial cellulose (BC) and ferulic acid (FA) were prepared by solution casting method. The rheological, morphological, barrier, optical, anti-fogging, and antioxidant properties of pullulan-based composite films doped with BC and FA were investigated. The rheological results showed that all film-forming solution was pseudoplastic fluid and its viscosity increased with the increase of BC content. An appropriate BC (2 %) and FA were uniformly dispersed in pullulan to form uniform and dense composite films. With the increase of BC content, the roughness and opacity of composite films increased while their UV-vis barrier performance was improved by incorporating BC and FA. Fourier transform infrared spectrometer analysis demonstrated that hydrogen bond interactions among pullulan, BC, and FA were found, and incorporating BC could increase the crystallinity of the composite films, thus enhancing their mechanical, barrier, hydrophobic, and thermal stability properties. Pullulan-based composite films incorporated with 2 % BC and FA (P-BC2-FA) showed better mechanical properties, water, oxygen, and carbon dioxide barrier performances, and its water contact angle value also increased compared with control, respectively. P-BC2-FA film showed superior anti-fogging and antioxidant activities. These results indicate that the P-BC2-FA film are expected to be a potential target of bioactive packaging.

Substrates and Loaded Iron Ions Relative Position Influence the Catalytic Characteristics of the Metalloenzymes Angelica archangelica Flavone Synthase I and Camellia sinensis Flavonol Synthase.

Metalloenzymes are a class of enzymes that catalyze through the metal ions they load. Angelica archangelica flavone synthase I (AnFNS I) and Camellia sinensis flavonol synthase (CaFLS), both of which belong to metalloenzymes, have highly similar structures and metal catalytic cores. However, these two enzymes catalyze the same substrate to produce significantly different products. To identify the cause for the differences in the catalytic characteristics of AnFNS I and CaFLS, their protein models were constructed using homology modeling. Structural alignment and molecular docking was also used to elucidate the molecular basis of the differences observed. To analyze and verify the cause for the differences in the catalytic characteristics of AnFNS I and CaFLS, partial fragments of AnFNS I were used to replace the corresponding fragments on CaFLS, and the catalytic characteristics of the mutants were determined by bioconversion assay in E. coli and in vitro catalytic test. The results suggest that the difference in catalytic characteristics between AnFNS I and CaFLS is caused by the depth of the active pockets and the relative position of the substrate. Mutant 10 which present similar dock result with AnFNS I increased the proportion of diosmetin (a flavone) from 2.54 to 16.68% and decreased the proportion of 4'-O-methyl taxifolin (a flavanol) from 47.28 to 2.88%. It was also indicated that the atoms in the substrate molecule that determine the catalytic outcome may be H-2 and H-3, rather than C-2 and C-3. Moreover, it is speculated that the change in the catalytic characteristics at the changes relative spatial position of H-2/H-3 of hesperetin and the loaded carbonyl iron, caused by charged residues at the entrance of the active pocket, is the key factor for the biosynthesis of flavone from flavanone.