As a new type of protein resource that does not rely on traditional livestock or fishery production， NeoProtein is a core strategic direction for practicing the ｀Big Food Concept' and safeguarding national food supply security. It holds great significance for promoting the upgrading of the food industry and realizing global sustainable development. Although the NeoProtein industry is currently showing a trend of rapid development， it still faces many bottlenecks in aspects such as technology industrialization and product quality optimization. Based on the current development status of the industry and the 2025 technology trends， this paper sorts out and identifies the top 10 technical challenges in the field of NeoProtein， and systematically expounds the background， challenges and solutions for each challenge. It is expected to provide references for scientific research， industrial innovation and policy formulation in related fields， and contribute to the high-quality development of the NeoProtein industry.

Citrus is a globally significant economic crop with the largest cultivation area and highest production yield. Currently， its processed products are still dominated by juice， canned goods， and similar items， resulting in relatively low comprehensive utilization of citrus resources. Citrus peels， pomace， physiological fruit drops， and other by-products are rich in high-value bioactive components such as pectin， flavonoids， and essential oils， which have wide applications in food， pharmaceuticals， chemical engineering， and other fields. This article reviews the status and technological bottlenecks in the citrus processing industry， systematically outlines the properties， biological activities， and advancements and limitations of traditional extraction technologies for key functional components in citrus resources. It also summarizes the technical advantages of synthetic biology in the green manufacturing of natural products， with a focus on recent progress in microbial biosynthesis of citrus flavonoids. In line with the demands for resource recycling and green， low-carbon development， synthetic biology is expected to open new pathways for obtaining high-value active ingredients from citrus， thereby promoting the industry's transition toward higher added value and sustainable development.

Objectives: The efficient release of bioactive peptides (BPs) from bovine milk proteins is of great significance. This study aimed to explore the potential of using the soluble proteinase of lactic acid bacteria (LAB) for production of BPs. Methods: A LAB strain exhibiting high proteolytic activity was screened and identified. The gene encoding the proteinase in the selected LAB was amplified via PCR and then modified by removing the sequence encoding the cell-wall-associated region， enabling it to be a soluble protein. Subsequently， the modified gene prtB3 was cloned and expressed in the food-grade expression strain Lactococcus lactis NZ9000. After purification， a comprehensive evaluation was conducted on the enzymatic properties of the recombinant proteinase PrtB3 and the biological activities of the hydrolysates resulting from casein degradation. Results: The screened LAB strain was identified as Lactobacillus delbrueckii subsp. lactis. A soluble， secreted， C-terminally Myc and His-tagged derivative PrtB3 was constructed and expressed in Lactococcus lactis via the NICE■ Expression System. Compared with the wild-type proteinase， PrtB3 demonstrated a significant expansion in substrate specificity. It was proteolytically active in the hydrolysis of α-， β-， and κ-casein. Regarding biological activity， the casein hydrolysate produced by PrtB3 showed remarkable angiotensin-converting enzyme (ACE) inhibitory activity. The maximum ACE inhibition rate reached 78%， which was twice that of the hydrolysate produced by the wild-type proteinase. The half-maximal inhibitory concentration (IC50) was 1.69 mg/mL， and the ACE inhibitory activity remained stable over an 8-week period. Furthermore， the enzymatically produced hydrolysate showed antimicrobial activity against Staphylococcus aureus and Escherichia coli. Conclusions: In this study， the soluble proteinase PrtB3 was successfully cloned and expressed. It effectively enhanced the degradation of casein and significantly improved the biological activity and stability of the casein hydrolysate. PrtB3 demonstrated great potential in producing BPs by degrading casein， providing a novel approach for the preparation of bioactive peptides.

The inhibitory effect of antimicrobial peptide LFcin(17-30) on methicillin-resistant Staphylococcus aureus (MRSA) biofilm was studied. Methods: MRSA in 24 strains of Staphylococcus aureus were screened by PCR and cefoxitin disk diffusion method. The biofilm-positive strains were identified by Congo red plate method and crystal violet staining method， and the biofilm-forming ability and biofilm growth curve of biofilm-positive strains were evaluated. The relationship between MRSA biofilm and drug resistance was verified by disk diffusion method. The minimal inhibitory concentration(MIC) of LFcin(17-30) against MRSA was determined by microdilution method. The inhibitory effect of LFcin (17-30) on MRSA biofilm was determined by crystal violet staining and scanning electron microscopy (SEM). The effect of LFcin (17-30) biofilm composition was determined. The results showed that 4 strains of MRSA were screened out from 24 strains of Staphylococcus aureus， which were DQ05， DQ07， DQ09 and DQ23， and the biofilm was identified as biofilm positive strains. The results of crystal violet staining showed that DQ05 had the strongest membrane production ability among the four MRSA strains， DQ09 had the weakest membrane production ability， and the drug resistance of DQ05 was stronger than that of DQ09. The biofilm growth curve showed that the amount of biofilm was the highest at 24 h. The MIC of LFcin (17-30) against MRSA was 150 μmol/L. LFcin (17-30) at 75 μmol/L can significantly inhibit the formation and destruction of biofilms. LFcin (17-30) inhibited the synthesis and secretion of extracellular polysaccharides and extracellular proteins in extracellular polymers， and the effect on extracellular DNA increased with the increase of LFcin (17-30) concentration. In summary， LFcin (17-30) can inhibit the prevalence of food-borne MRSA strains and biofilm formation， and is expected to be used as an adjuvant therapy for food-borne MRSA.

Objective: To explore the effect of Na+/K+ in the intestinal environment on the interaction between soy hull polysaccharide (SHP) and intestinal mucus， and its effect on the diversity of intestinal flora. Methods: Molecular docking and molecular dynamics simulation were used to analyze the main binding force and complex stability of the target protein and soybean seed coat polysaccharide after Na+/K+ treatment. The effects of SHP and mucin on the changes of OTU and species abundance of intestinal flora after Na+ treatment was detected by 16S rDNA genomics. Results: The main force of SHP binding to mucin after Na+/K+ treatment was hydrogen bond. Within 100 ns， the RMSD value， RSMF value， Rg value and the number of hydrogen bonds of the complex structure fluctuated slightly. The secondary structure β-sheet of mucin mainly existed in anti-parallel mode after Na+ treatment， while the secondary structure β-sheet of mucinmainly existed in parallel mode after K+ treatment. The binding free energy [(196.7±26.7) kJ/mol] and the contribution of amino acid residues (18.18 kJ/mol) of SHP to mucin after Na+ treatment were greater than those after K+ treatment [(158.5±26.7) kJ/mol and 10.63 kJ/mol， respectively]. In the presence of Na+， the diversity of intestinal flora was up to 284 OTUs when SHP was used as a substrate for 8 h， and the abundance of various dietary fiber degrading bacteria was increased， and the abundance of Escherichia coli-Shigella was decreased. At the same time， the abundance of Bifidobacterium and Lactobacillus was also decreased. Conclusion: After Na+ treatment， the binding ability and stability of SHP and mucin are higher， and the diversity and abundance of intestinal flora are affected. Na+ treatment has a potential effect on the whole intestinal function of dietary fiber.

Objective: Correlation between alkali treatment process and carrageenan gel quality and prediction model were studied. Method: Carrageenan was extracted from Eucheuma muricatum by alkali extraction method， and a reliable and measurable model was established to predict the gel properties and molecular characteristic structure of carrageenan under different alkali treatment process conditions using artificial neural network (ANN) model. Results: Artificial neural network model fit the total set with R2 = 0.991 better than the multivariate linear model (MLR)， with mean absolute percentage errors (MAPEgel strength = 1.71%， MAPEmolecule = 18.84%， and MAPEsulphate group = 1.91%， MAPE3，6-endoethergalactose = 3.68%) are lower than the MLR model. In addition， in actual production， when the key process parameters of alkali treatment (temperature， time， and concentration) were 65 ℃， 6 h， and 10%， the gel strength of carrageenan was measured to be 614.32 g/cm2， and the gel strength of carrageenan was predicted to be 617.13 g/cm2 by the constructed prediction model of the correlation between carrageenan processing alkali treatment and its gel quality. The results show that the predicted value of the model is close to that of the actual production. Conclusion: The prediction performance of ANN model is better than that of multivariate linear model， and ANN model is more accurate than MLR model in predicting carrageenan gel properties and molecular characteristic structure. This study provides a technical reference for the intelligent and precise manufacturing of carrageenan high-quality products， so as to expand the application of carrageenan in biomedicine and other fields.

To investigate the effect of NRF2 on ferroptosis and beef color stability during post-mortem maturation， the longest dorsal muscle of cattle was used as the research object in this experiment. It was treated with 0.9% physiological saline， 0.5 mmol/L ML385， and 1 mmol/L tBHQ injection， and matured at 4 ℃ for 0， 24， 72， 168 hours， respectively. The expression level of NRF2， changes in iron homeostasis， expression of ferroptosis-related proteins， lipid oxidation， mitochondrial membrane potential， beef color， and other related indicators were analyzed. The research results showed that during the maturation period， the expression and mRNA levels of NRF2 in the ML385 treatment group were significantly lower than those in the control group， while the opposite was true in the tBHQ group， indicating that ML385 and tBHQ had significant inhibitory and activating effects on NRF2. Compared with the control group， the ML385 group showed a significant increase in iron deposition levels (P<0.05)， and the protein expression levels of FPN1， SLC7A11， and PRDX6 were significantly downregulated (P<0.05). After maturation， the contents of Fe2+， MDA， and b* increased by 10.56%， 16.67%， and 53.78%， respectively， while the tBHQ group decreased by 8.39%， 21.76%， and 30.26% compared with the control group. The mitochondrial membrane potential， pH， Mb content， GPX4， MRA， NADH oxidase activity， and a* value in the ML385 group were significantly reduced by 5.31%， 3.62%， 7.45%， 28.32%， 24.10%， 2.76%， and 12.1% respectively compared with the control group， whereas those in the tBHQ group increased by 15.96%， 1.11%， 30.73%， 8.69%， 3.96%， 7.39%， and 5.68% respectively compared with the control group. For L* values， both the ML385 group and tBHQ group were higher than the control group in summary， during the post-mortem maturation period， NRF2 regulates ferroptosis by maintaining muscle tissue iron homeostasis， inhibiting lipid peroxidation， and affecting mitochondrial membrane potential， thereby affecting Mb content and MRA to improve beef color stability.

Objective: To investigate the mechanism of synergistic inhibition of α-amylase (AMY) by three flavonoids [baicalein (Bai)， genistein (Gen)， isoliquiritigenin (Isl)] combined with acarbose (Aca). Through methods such as inhibition rate experiments， combination index (CI) analysis， fluorescence spectroscopy， ultraviolet spectroscopy， infrared spectroscopy， and molecular dynamics simulation， the inhibitory effects and action mechanisms of the combined inhibition on AMY were systematically studied. The results show that the inhibitory effects of Bai/Gen/Isl combined with Aca on AMY were significantly enhanced at molar ratios of 2∶1， 3∶1， and 4∶1. Among them， Bai-Aca and Isl-Aca exhibited a strong synergistic effect at a molar ratio of 3∶1(CI < 1)， while Gen-Aca showed synergism only at a molar ratio of 3∶1 and under the specific condition of 0.3 < Fa < 0.9(fraction affected). Fluorescence spectroscopy analysis showed that the combined inhibitors bind to AMY via a static quenching mechanism. Among them， Isl-Aca exhibits the highest binding constant (Ka) with AMY [K = (4.826±0.206)×105 L/mol at 298 K； Ka = (35.172±0.255)×105 L/mol at 300 K]， and its affinity is significantly enhanced with increasing temperature (P < 0.05)， indicating that the complex formed by Isl-Aca and AMY has the optimal stability. Thermodynamic parameters indicated that hydrophobic interactions were the main driving force for Bai-Aca and Isl-Aca， while Gen-Aca relied on hydrogen-bond binding. Synchronous fluorescence and ultraviolet spectroscopy showed that the combined inhibitors regulated the activity of AMY by changing the microenvironment of tryptophan (Trp) and tyrosine (Tyr) residues， with the inhibitory effect of Trp residues being dominant. Infrared spectroscopy and molecular dynamics simulation further revealed that Bai-Aca and Isl-Aca promoted the transformation of the secondary structure of AMY into a more stable β-turn， while Gen-Aca induced the formation of α-helix. However， the free-energy landscape of the latter complex presented a dispersed energy cluster， indicating weaker stability. In conclusion， the Bai-Aca and Isl-Aca combined inhibition systems significantly reduce the dosage of inhibitors through multi-target synergistic effects， while maintaining the high stability of the AMY complex. This provides a theoretical basis for the development of novel diabetes treatment strategies and functional foods based on flavonoids.

Objectives: Curcumin has good biological activity， but its application in food is limited because of its poor solubility， stability and bioaccessibility. Methods: Curcumin loaded Pickering emulsion was prepared by using whey protein isolate (WPI) and soy protein isolate (SPI) as solid particles. The rheological properties， microstructure and secondary structure of the emulsions were characterized and simulated digestion in vitro. Results: The Pickering emulsion stabilized by WPI / SPI had a good embedding rate of curcumin， with a maximum of 74.33%， and had good storage stability. The rheological properties show that the Pickering emulsion exhibits good gel properties under low shear force. The microstructure showed that the curcumin-loaded Pickering emulsion was oil-in-water type. The Fourier results showed that the relative content of protein secondary structure in Pickering emulsion was changed by hydrogen bonding after embedding curcumin. In vitro digestion results showed that WPI/SPI stabilized Pickering emulsion significantly improved the bioaccessibility (from 12.60% to 34.31%) and antioxidant activity of curcumin， and the release of curcumin in vitro conformed to the zero-order kinetic model. Conclusions: The Pickering emulsion stabilized by composite protein particles has good stability and can be used as a delivery carrier of curcumin to improve its bioaccessibility and further provide a reference basis for the stabilization， development and application of curcumin and other bioactive components.

Exploring the inhibitory effect of small molecules on pancreatic lipase (PL) has become an important method for screening active ingredients with anti-obesity effect in vitro. The binding reaction and mechanism of genistein (Gen) and naringenin (Nar) with PL were studied by fluorescence spectroscopy， non-radiative energy transfer theory and high performance liquid chromatography. At the same time， three-dimensional fluorescence， surface hydrophobicity， X-ray diffraction， infrared spectroscopy， particle size， differential scanning calorimetry， and scanning electron microscopy were used to characterize the structure of the composites from different angles， and their influence on the structure and properties of PL were comprehensively clarified. The results showed that the quenching of PL by Gen and Nar was static quenching， and the binding constants were 42.39 L/mg and 22.57 L/mg， respectively. The addition of both reduced the α-helix content of PL， increased the content of β-turn， β-sheet and random coil， and increased the crystallinity from 25.60% to 32.77% and 38.00%， respectively. The surface hydrophobicity decreased and the thermal stability decreased (the denaturation temperature decreased from 76.04 ℃ to 65.00 ℃ and 65.05 ℃， respectively)， and the PL changed from a smooth sheet structure to a rod-like particle. These results indicate that Gen and Nar significantly affect the structure and properties of PL， providing new ideas for the development of new PL inhibitors.