PA28γ coordinates the cross-talk between cancer-associated fibroblasts and tumor cells to promote OSCC progression via HDAC1/E2F3/IGF2 signaling.

PA28γ overexpression is aberrant and accompanied by poor patient prognosis in various cancers, the precise regulatory mechanism of this crucial gene in the tumor microenvironment remains incompletely understood. In this study, using oral squamous cell carcinoma as a model, we demonstrated that PA28γ exhibits high expression in cancer-associated fibroblasts (CAFs), and its expression significantly correlates with the severity of clinical indicators of malignancy. Remarkably, we found that elevated levels of secreted IGF2 from PA28γ+ CAFs can enhance stemness maintenance and promote tumor cell aggressiveness through the activation of the MAPK/AKT pathway in a paracrine manner. Mechanistically, PA28γ upregulates IGF2 expression by stabilizing the E2F3 protein, a transcription factor of IGF2. Further mechanistic insights reveal that HDAC1 predominantly mediates the deacetylation and subsequent ubiquitination and degradation of E2F3. Notably, PA28γ interacts with HDAC1 and accelerates its degradation via a 20S proteasome-dependent pathway. Additionally, PA28γ+ CAFs exert an impact on the tumor immune microenvironment by secreting IGF2. Excitingly, our study suggests that targeting PA28γ+ CAFs or secreted IGF2 could increase the efficacy of PD-L1 therapy. Thus, our findings reveal the pivotal role of PA28γ in cell interactions in the tumor microenvironment and propose novel strategies for augmenting the effectiveness of immune checkpoint blockade in oral squamous cell carcinoma.

A ß-Primeverosidase-like Enzyme in Soybean [Glycine max (L.) Merr] Hypocotyls: Specificity toward 1-Octen-3-yl and 3-Octanyl ß-Primeverosides.

A novel ß-primeverosidase-like enzyme, originating from the hypocotyl of soybeans, was isolated and characterized. This enzyme, with an estimated molecular weight of 44 kDa, was identified as a monomer and exhibited peak activity at 55 °C and pH 5.5. It demonstrated a specific and efficient hydrolysis of 1-octen-3-yl ß-primeveroside (1-octen-3-yl prim) and 3-octanyl ß-primeveroside (3-octanyl prim) but did not act on glucopyranosides. Mn2+ significantly enhanced its activity, while Zn2+, Cu2+, and Hg2+ exerted inhibitory effects. Kinetic analysis revealed a higher hydrolytic capacity toward 1-octen-3-yl prim. Partial amino acid sequences were determined and the N-terminal amino acid sequence was determined to be AIVAYAL ALSKRAIAAQ. The binding energy and binding free energy between the ß-primeverosidase enzyme and its substrates were observed to be higher than that of ß-glucosidase, thus validating its superior hydrolysis efficiency. Hydrogen bonds and hydrophobic interactions were the main types of interactions between ß-primeverosidase enzyme and 1-octen-3-yl prim and 3-octanyl prim, involving amino acid residues such as GLU-470, TRP-463, GLU-416, TRP-471, GLN-53, and GLN-477 (hydrogen bonds) and PHE-389, TYR-345, LEU-216, and TYR-275 (hydrophobic interactions). This study contributes to the application of a ß-primeverosidase-like enzyme in improving the release efficiency of glycosidically conjugated flavor substances.

Reconstruction of Extensive Maxillary Defects Using Flow-Through Fibula Free Flap With Anterolateral Thigh Free Flap.

BACKGROUND: The maxillary defects left unreconstructed or inadequately reconstructed often result in significant functional and esthetic impairments. Adequate reconstruction of extensive maxillary defects requires a sufficient volume of hard and soft tissues. METHODS: A 48-year-old male presenting bilateral extensive maxillary defects underwent secondary reconstruction with a flow-through fibula free flap in combination with an anterolateral thigh free flap. RESULTS: The use of flow-through technique allowed minimizing the problem of limited recipient vessels and the length of free flap vascular pedicle usually encountered in secondary reconstruction. The bilateral maxillary defects were successfully reconstructed, and the postoperative outcomes were uneventful. The patient was satisfied with the treatment outcomes. He is being followed up and was referred to the implantology department for the placement of osseointegrated dental implants. CONCLUSIONS: The flow-through fibula free flap, in combination with the anterolateral thigh free flap, was found reliable and feasible for this case of secondary reconstruction of bilateral maxillary defects. This technique has provided satisfactory functional and esthetic outcomes and effectively improved the patient's self-esteem.

Association between COVID 19 exposure and expression of malignant pathological features in oral squamous cell carcinoma: A retrospective cohort study.

OBJECTIVES: To analyze the relationship between the clinical and pathological characters of OSCC and COVID 19 exposure. MATERIALS AND METHODS: A retrospective cohort study in patients with OSCC with or without COVID 19 was performed. A total of 200 OSCC patients treated with surgery from 2019 to 2023 were included. Clinical and pathological features were analysed between two groups. Characters with statistical difference were further analysed by performing univariate analysis and logistic regression analysis. RESULTS: The expression of Ki67 (n = 57, 71.3 %, P < 0.001) and CyclinD1 (n = 64, 80 %, P < 0.001) in OSCC with the exposure history of COVID 19 is higher than that in patients never exposed to COVID 19. COVID 19 exposure history is an independent influencing factor for higher expression of Ki67 (OR = 4.04, 95 % CI: 1.87-8.72, P < 0.001) and CyclinD1 (OR = 5.45, 95 % CI: 2.56-11.60, P < 0.001). CONCLUSION: COVID 19 may suggest more invasive malignant biological behavior of cancer cells in OSCC.

Identification, rapid screening, docking mechanism and in vitro digestion stability of novel DPP-4 inhibitory peptides from wheat gluten with ginger protease.

To better understand the hypoglycemic potential of wheat gluten (WG), we screened dipeptidyl peptidase IV (DPP-4) inhibitory active peptides from WG hydrolysates. WG hydrolysates prepared by ginger protease were found to have the highest DPP-4 inhibitory activity among the five enzymatic hydrolysates, from which a 1-3 kDa fraction was isolated by ultrafiltration. Further characterization of the fraction with nano-HPLC-MS/MS revealed 1133 peptides. Among them, peptides with P'2 (the second position of the N-terminal) and P2 (the second position of the C-terminal) as proline residues (Pro) accounted for 12.44% and 43.69%, respectively. The peptides including Pro-Pro-Phe-Ser (PPFS), Ala-Pro-Phe-Gly-Leu (APFGL), and Pro-Pro-Phe-Trp (PPFW) exhibited the most potent DPP-4 inhibitory activity with IC50 values of 56.63, 79.45, and 199.82 µM, respectively. The high inhibitory activity of PPFS, APFGL, and PPFW could be mainly attributed to their interaction with the S2 pocket (Glu205 and Glu206) and the catalytic triad (Ser630 and His740) of DPP-4, which adopted competitive, mixed, and mixed inhibitory modes, respectively. After comparative analysis of PPFS, PPFW, and PPF, Ser was found to be more conducive to enhancing the DPP-4 inhibitory activity. Interestingly, peptides with P2 as Pro also exhibited good DPP-4 inhibitory activity. Meanwhile, DPP-4 inhibitory peptides from WG showed excellent stability, suggesting a potential application in type 2 diabetes (T2DM) therapy or in the food industry as functional components.

Fermentation Characteristics, Antinutritional Factor Level and Flavor Compounds of Soybean Whey Yogurt.

Soybean whey contains high levels of off-flavors and anti-nutritional factors and is generally considered unsuitable for direct application in the food industry. In this work, to reduce beany off-flavors and anti-nutritional factors, and to improve its fermentation characteristics, soybean whey was treated with electrodialysis desalination, vacuum concentration and lactic acid bacteria (LAB) fermentation. The results showed that electrodialysis desalination increased the fermentation rate and the number of viable lactic acid bacteria of soybean whey yogurt. More than 90% of the antinutritional factor level (urease and trypsin inhibitory activity) was removed due to high-temperature denaturation inactivation and LAB degradation. Concentrated desalted soybean whey yogurt (CDSWY) possessed larger values for firmness and consistency, and a denser network microstructure compared with undesalted yogurt. Over 90% of off-flavors including hexanal, 1-octen-3-ol and 1-octen-3-one were removed after electrodialysis desalination and concentration treatment. Meanwhile, the newly generated ß-damascenone through carotenoid degradation and 2,3-butanedione improved the pleasant flavor and sensory quality of CDSWY, while the salty taste of CSWY lowered its sensory quality. This study provided a theoretical basis for better utilization of soybean whey to develop a plant-based yogurt like dairy yogurt.

Characterization of mung bean endogenous proteases and globulins and their effects on the production of mung bean protein.

Mung bean protein possesses several health benefits, and aqueous processing methods are used for its production. However, mung bean protein yields are different with different methods, which are actually different in conditions (e.g., pH, temperature, and time). Herein, liquid chromatography tandem mass spectrometry identified 28 endopeptidases and exopeptidases in mung bean protein extract, and the positions of 8S and 11S globulins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel were confirmed in our experimental conditions. The SDS-PAGE, trichloroacetic acid-nitrogen solubility index, and free amino acid analysis revealed that (1) 8S globulins showed strong resistance to the endopeptidases (optimal at pH 5 and 50 °C) at pH 3-9, and 11S globulin exhibit strong resistance expect at pH 3-3.5; (2) the exopeptidases (optimal at pH 6 and 50 °C) preferred to liberate methionine and tryptophan. These proteases negatively affected protein yield, and short production time and low temperature were recommended.

New insights into the off-flavor improvement of soymilk by three grinding processing: Dry-blanching grinding, wet-blanching grinding, and wet-anaerobic grinding.

Advances in grinding strategies have been beneficial to eliminating the off-flavor of soymilk and improving the quality soy products. Herein, four grinding processing, dry-blanching grinding (D-BG), wet-blanching grinding (W-BG), wet-anaerobic grinding (W-AG) and traditional grinding (TG) were employed and found to impose a significant impact on off-flavor components, accompanied by changes of hydroperoxides and free radicals. The results showed that all three methods could significantly hinder the formation of C6 aldehydes. C8 Alcohols and (E)-2-heptenal could be removed by D-BG, but lipids in dehulled soybean were prefer to be oxidized during storage, resulting in the accumulation of hydroperoxides and radicals. W-BG and W-AG have higher levels of 1-octen-3-ol, and soaking at an alkaline pH and increasing the number of rinses is beneficial for its removal. Gas chromatography-olfaction-mass spectrometry (GC-O-MS) combined with sensory evaluation showed that off-flavor profile of d-BGS, W-BGS and W-AGS was different. D-BG and W-AG possessed better flavor quality.

An epulis-like camrelizumab related reactive cutaneous capillary endothelial proliferation (RCCEP) in the oral cavity: A case report.

BACKGROUND: Immunotherapy, especially anti-PD-1 and anti-PD-L1 antibodies, have observably improved the overall survival of patients with advanced solid tumors following the unavoidable immune-related adverse events (irAEs). Camrelizumab is a novel anti-PD-1 agent with the reported most common irAEs of reactive cutaneous capillary endothelial proliferation (RCCEP). Despite it is widely occurred in the skin, oral RCCEP is rarely reported. CASE SUMMARY: A 59-year-old man complained about a painless nodule on left mandibular gingiva for two weeks. He started to inject Camrelizumab because of the recurrence of esophageal squamous cell carcinoma two month ago. An 8 mm lesion was observed on his mucosa. Several disseminated bright purple red papules were then found on his skins. The oral lesion and one lesion on his face was removed by surgery. After the final diagnosis of reactive cutaneous capillary endothelial proliferation was confirmed by histological examination. Other operable lesions on his face were removed by ligation. All the removed lesions had a good prognosis without recurrence within the follow-up visit. CONCLUSION: With the widespread use of Camrelizumab in other solid tumors, the occurrence of oral RCCEP will increase. Surgery and ligation are both effective treatment for RCCEP with a good prognosis.

Comparison of physicochemical properties and volatile flavor compounds of plant-based yoghurt and dairy yoghurt.

The aim of this study was to investigate and compare the physicochemical characteristics and volatile flavor compounds of three kinds of yoghurt made from reconstituted milk, soy drink and oat drink. The results showed that with the same fermentation ending pH of 4.5, reconstituted yoghurt had the highest titratable acidity mainly due to the highest buffering capacity and microbial counts (LAB). The textural and water holding capacity (WHC) parameters revealed that soy-based yoghurt had the highest firmness, consistency and WHC, indicating more rigid gel was formed. Meanwhile, rheological analysis showed soy-based yoghurt owned higher G' and G'' values and higher stability against external stress, demonstrating that more and stronger interactions between soy proteins were built during fermentation. The confocal laser scanning microscopy (CLSM) image witnessed that soy-based yoghurt had the densest and finest network, while oat-based yoghurt had much coarser and looser structure, which was consistent with the lowest firmness and G' value for oat-based yoghurt. In terms of color, reconstituted yoghurt was the lightest and oat-based yoghurt showed more reddish and yellowish. The main volatile flavor compounds in all yoghurts were ketones, while aldehydes contributed more in soy and oat yoghurt. PCA plot showed that volatile flavor compounds of reconstituted yoghurt and oat-based yoghurt were relatively similar, while soy-based yoghurt was much more different with high OAVs of hexanal, 1-octen-3-one, 1-octen-3-ol and 2-octenal. This study supplied a theoretical basis and an improvement direction for the better development of healthier plant-based yoghurt similar to dairy yoghurt.

Phenolic compounds in walnut pellicle improve walnut (Juglans regia L.) protein solubility under pH-shifting condition.

This study focused on the interaction of walnut protein with phenolic extracts of walnut pellicle (PEWP) under alkaline condition, leading to enhancement of protein solubility under neutral condition. First, the change of PEWP under alkaline condition was determined by RP-HPLC and mass spectrometry, and the results showed that most ellagitannins in PEWP could be retained under alkaline condition within 3 h. Interaction between PEWP and walnut protein under pH-shifting condition resulted in the remarkable increase of protein solubility (above 90%) at neutral pH. The results from SDS-PAGE and SEC showed that the improved solubility lied in the formation of large and soluble protein aggregates due to the covalent interaction among walnut protein and polyphenols. A significant change in tertiary structure of protein-phenolic complex was witnessed by fluorescence spectrum and near-UV circular dichroism. Meanwhile, walnut protein-polyphenol interaction led to a slight increase in ß-turn while a slight decrease in ß-sheet. Combined with amino acid composition, it could be illustrated that the covalent bonding for walnut protein with polyphenol mainly occurred at Lysine residues.

Growing of fungi on the stored low denatured defatted soybean meals and the hydrolysis of proteins and isoflavone glycosides by fungal enzymes.

Recently, more and more attention has been paid to the effects of fungal contamination and fungal enzymes secreted in raw grain on product quality. As the starting material of protein and active components, the quality of low denatured defatted soybean meals (LDSM) directly determines the qualities of subsequent products. In previous studies, we have revealed that infection with Aspergillus ochraceus protease causes significant hydrolysis of proteins. In this study, growing of fungi on the stored low denatured defatted soybean meals (LDSM) was analyzed by high-throughput sequencing and real-time PCR, which revealed that the abundance of Aspergillus increased significantly after storage. Twenty fungal proteases and 9 fungal glucosidases were found in stored LDSM and zymography showed that the proteases were of serine-type with some cysteine and aspartic activities. Proteolysis of the soybean storage proteins mainly occurred after the hydration of LDSM and the average molecular weight of soy proteins decreased from 57.9 kDa to 30.7 kDa after 60 min's of hydrolysis. Two-dimensional electrophoresis (2-DE) analysis found the polypeptide fragments from soybean 7S and 11S proteins with molecular weight around 10-25 kDa in the hydrated LDSM. Glycosylated isoflavones were hydrolyzed in both dry and hydrated stored LDSM which resulted in significant (p < 0.05) increase in the contents of isoflavone aglycones. This study suggested that fungi contamination be a new factor affecting the properties of LDSM derived soy protein products.

Characterization of proteases from Irpex lacteus grown on minimally denatured soybean meal.

BACKGROUND: Acid and thermal stabilities are important properties for the preparation of acidic protein beverage. It is an important method for enzymatic modification to improve the functional properties of protein. Irpex lacteus protease showed a selective hydrolysis to soy proteins. The purpose of this study was to investigate the mechanism of enzymatic hydrolysis and its effects on acid and thermal stabilities of soy proteins. RESULTS: The I. lacteus protease selectively hydrolyzed the α and α' subunits of the native soybean ß-conglycinin (7S globulin) to produce products that presented as the 55 kDa band upon sodium dodecyl sulfate polyacrylamide gel electrophoresis. The amino acid sequences of 55 kDa polypeptides were analyzed in gel multi-enzyme digestion followed by liquid chromatography-mass spectrometry. By matching the multi-enzyme digestion peptides with the published polypeptide chain sequences of the α and α' subunits, it was confirmed that the 55 kDa polypeptides were formed by eliminating amino acid residues on both sides of the N- and C-terminals. From the published protein structure database (https://www.uniprot.org/), it is known that the cleaved peptide bonds were in extension regions. Non-selective enzyme hydrolysis of both ß-conglycinin (7S globulin) and glycinin (11S globulin), with corresponding drastic increases in the degree of hydrolysis, was observed when the substrates were preheated to the denaturation degree of 40% and above. However, 55 kDa hydrolyzed products and B polypeptides showed some extent of resistance to the proteolysis by I. lacteus protease even if denaturation degree was 100%. Both selective and non-selective hydrolysis of soy proteins by I. lacteus protease improved the acid and heat stabilities under the same hydrolysis conditions (enzyme/substrate ratio, time, and temperature). CONCLUSION: Enzymatic hydrolysis of soybean proteins by the I. lacteus protease can effectively improve the acid and thermal stabilities of proteins. This discovery is significant to avoid aggregation during processing in the beverage industry. In the near future, the protease has potential application value for modification of other proteins. © 2022 Society of Chemical Industry.

Recovery and Utilization of Pea Albumins as Acidic Emulsion Stabilizer by Complexation with Dextran Sulfate.

In this work, pea albumins (PAs) were efficiently recovered by complexation with dextran sulfate (DS), and the emulsifying ability and stability of PA/DS complexes were studied. The largest amounts of PAs (81.25%) were recovered at r = 5:1 and pHmax (pH 3.41) by forming insoluble complexes; and only soluble complexes were formed at r = 2:1 and over the whole pH range (2.0-7.0). The emulsions stabilized by PA/DS soluble complexes remained stable under acidic conditions due to the highly negatively charge (from -45.10 ± 0.40 to -57.23 ± 0.66 mV) and small particle size (0.168 ± 0.010-0.448 ± 0.004 µm), while emulsions stabilized by PAs alone generated a strong creaming and serum separation at pH 5 and 6. In terms of emulsifying stability, all PA emulsions and unheated PA/DS emulsions became unstable with different creaming index after 14 days storage. SDS-PAGE results showed that the interface adsorption proteins of unheated emulsions mainly consisted of PA1a, which was unfavorable to the stability of the interface. On the contrary, heat treatment (95 °C, 30 min) and complexation (PA/DS = 2:1) enhanced the adsorption of PA2 and lectin at the interface, inhibiting the aggregation of PA2 and lectin. This resulted in long-term stability of the PA/DS emulsions under acidic conditions.

Formation and characterization of solid fat mimetic based on pea protein isolate/polysaccharide emulsion gels.

The emulsion gels that can be used as solid fat replacers were produced with different polysaccharides (κ-carrageenan, κC; high-acyl gellan, HA; konjac glucomannanon, and KGM), pea protein isolate (PPI) and sunflower seed oil. The effect of polysaccharide concentration on the texture, rheological property, microstructure, and water holding capacity of the mixed emulsion gels were investigated. Rheological results showed that the presence of polysaccharides enhanced the hardness, storage modulus and resistance against deformation of emulsion gel, where PPI/κC system exhibited superior hardness with a similar level of pig back fat, due to the self-gelation behavior of κC. CLSM and SEM results showed that the presence of κC, HA, and KGM broke the uniform structure of gel network and formed irregular, threadlike, and oval shaped inclusions respectively, resulting in the broken and coalescence of oil droplets. The α-helix content of emulsion gels decreased, while ß-sheet, ß-turn and random coils slightly increased due to the unfolding of protein during gel formation. This study may offer a valuable strategy for the development of solid fat mimetic with the characteristics closing to the pig back fat.

Effects of heat treatments on the properties of soymilks and glucono- δ - Lactone induced tofu gels.

Optimal heat treatment of the soymilks is important to the production of tofu. In this study, soymilk with protein concentration of 40 mg/mL were heated at different temperatures for the fixed 50 s and were characterized by surface hydrophobicity, disulfide linked protein species determined by non-reducing SDS-PAGE and protein structural elements determined by the circular dichroism (CD). Tofu gels were prepared by acidifying the heated soymilks at 60 ℃ and 80 ℃ respectively and gelation time, gel mechanical properties as well as gel viscoelastic properties were determined by rheological analysis. The results showed that most soymilks' properties except surface hydrophobicity changed rapidly when heating temperature was higher than 80 ℃. Gelation time, storage modulus (G') at the end of acidifying and cooling processes as well as retardation time (λ) and recovery rate of tofu gels were affected by the heat treatments of soymilks. The distances between the standardized data describing heated soymilks and tofu gels respectively were calculated and compared. It was found that gelation time, G' and λ were most closely related to disulfide bond linked polymer, [CD]222 and surface hydrophobicity respectively. This study will provide useful information to the improvement of tofu processing.

Identification of a BRAF/PA28γ/MEK1 signaling axis and its role in epithelial-mesenchymal transition in oral submucous fibrosis.

Oral submucous fibrosis (OSF) is a chronic and insidious oral potentially malignant disorder associated with a 4-17% risk of oral squamous cell carcinoma (OSCC). Our previous study found that proteasomal activator 28 gamma (PA28γ) is frequently overexpressed in oral squamous cell carcinoma and negatively correlated with poor patient prognosis. However, the role of PA28γ in the occurrence and development of OSF remains unclear. Here, we screened PA28γ-related genes and investigated their function in OSF. We demonstrated that the expression of PA28γ was positively associated with MEK1 and gradually elevated from normal to progressive stages of OSF tissue. Arecoline, a pathogenic component of OSF, could upregulate the protein levels of PA28γ and phosphorylated MEK1 and contribute to epithelial to mesenchymal transition (EMT) in epithelial cells. Notably, PA28γ could interact with MEK1 and upregulate its phosphorylation level. Furthermore, arecoline upregulated BRAF, which can interact with PA28γ and upregulate its protein level. Additionally, BRAF, PA28γ, and MEK1 could form protein complexes and then enhance the MEK1/ERK signaling pathways. The concrete mechanism of the protein stability of PA28γ is that BRAF mediates its degradation by inhibiting its ubiquitination. These findings underscore the instrumental role of PA28γ in the BRAF/MEK1 pathway and enhanced EMT through MEK1/ERK activation in OSF.

Hydrolyzing behaviors of endogenous proteases on proteins in sesame milk and application for producing low-phytate sesame protein hydrolysate.

Endogenous proteases with high activity have been identified in sesame seeds. However, the hydrolyzing behaviors of endogenous proteases on proteins in sesame milk are not well understood. In this study, the endogenous proteases optimally hydrolyzed proteins at pH 4.5 and 50 °C for 6 h. Tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis and liquid chromatography tandem mass spectrometry analyses revealed that endogenous proteases randomly cleaved the cleavable peptide bonds on 11S globulins, and amino acid analysis indicated that serine carboxypeptidases preferentially cleaved tryptophan, phenylalanine, methionine, tyrosine, and leucine. The hydrolyzed sesame milk was separated into cream, transparent skim, and precipitate fractions by centrifugation (3000g, 5 min). The major protein components in skim were 42% peptides (<1500 Da) and 35% free amino acids. The phytate in skim was greatly reduced by adjusting to neutral and alkaline pH. This study is meaningful at supplying a strategy for producing low-phyate sesame protein hydrolysate.

Formation Mechanism of Hexanal and (E)-2-Hexenal during Soybean [Glycine max (L.) Merr] Processing Based on the Subcellular and Molecular Levels.

Hexanal and (E)-2-hexenal in soymilk mainly form during the soaking and grinding of soybeans. In this study, freshly dehulled soybeans were soaked or ground in the presence or absence of different enzyme inhibitors. The results showed that (1) 1-palmitoyl-2-linoleoyl-sn-3-phosphatidylcholine, 1-stearoyl-2-linoleoyl-sn-3-phosphatidylcholine, 1-palmitoyl-2-linolenoyl-sn-3-phosphatidylcholine, and 1-stearoyl-2-linolenoyl-sn-3-phosphatidylcholine were preferentially acted upon by lipoxygenases (LOXs) and made predominant contributions to hexanal/(E)-2-hexenal formation. Phospholipase A2 (PLA2) is one of the key enzymes for hexanal/(E)-2-hexenal formation. (2) The ratio of net increase in hexanal/(E)-2-hexenal and net decrease in linoleic acid/linolenic acid was close to 100% during soaking, but it was only 60% during grinding. Only 13-hydroperoxy octadecad(tr)ienoic acid (13-HPOD/T) was formed for the membrane LOX, but both 13- and 9-hydroperoxy octadecad(tr)ienoic acid (9-HPOD/T) were produced for the cytoplasm LOX. Thus, only the membrane LOX was involved during soaking, while both membrane- and cytoplasm-bound LOXs worked during grinding. (3) Hydroperoxides and hexanal/(E)-2-hexenal during soybean grinding were studied. PC hydroperoxides formed almost instantly and reached a maximum in 10 s, while fatty acid hydroperoxides and hexanal/(E)-2-hexenal formed relatively slowly and reached a maximum in 50 s. The experimental data were fitted to the integrated form of the Michaelis-Menten equation, and Km, Vmax, and kcat for the LOX, PLA2, and hydroperoxide lyase were obtained, respectively.

Raw walnut kernel: A natural source for dietary proteases and bioactive proteins.

Walnut kernels are health-promoting nuts, which are mainly attributed to polyunsaturated fatty acids, phenolics, and phytosterols. However, the information concerning benefits of walnut proteins are limited. In this study, endopeptidases, aminopeptidases, carboxypeptidases, superoxide dismutases, catalases, and phospholipases with respective relative abundance of 2.730, 1.728, 0.477, 3.148, 0.743, and 0.173‰ were identified by liquid chromatography tandem mass spectrometry. These endogenous proteases exhibited activity in a broad pH range of 2-6.5, and optimal at pH 4.5 and 50 °C. Aspartic endopeptidases were predominant endopeptidases, followed by cysteine ones. There were two types of aspartic endopeptidases, one (not inhibited by pepstatin A) exerted activity at pH 2-3 and the other (inhibited by pepstatin A) optimal at pH 4.5. Carboxypeptidases were optimal at pH 4.5, and aminopeptidases exerted activity at pH near 6.5. These endogenous proteases assisted the digestion of walnut proteins, and soaking, especially peeling, greatly improved the in vitro digestibility.