Identification of Hyperuricemia Alleviating Peptides from Yellow Tuna Thunnus albacares.

The development of food-derived antihyperuricemic substances is important for alleviating hyperuricemia (HUA) and associated inflammation. Here, novel peptides fromThunnus albacares (TAP) with strong antihyperuricemic activity were prepared. TAP was prepared by alkaline protease (molecular weight <1000 Da), with an IC50 value of xanthine oxidase inhibitory activity of 2.498 mg/mL, and 5 mg/mL TAP could reduce uric acid (UA) by 33.62% in human kidney-2 (HK-2) cells (P < 0.01). Mice were fed a high-purine diet and injected with potassium oxonate to induce HUA. Oral administration of TAP (600 mg/kg/d) reduced serum UA significantly by 42.22% and increased urine UA by 79.02% (P < 0.01) via regulating urate transporters GLUT9, organic anion transporter 1, and ATP-binding cassette subfamily G2. Meantime, TAP exhibited hepatoprotective and nephroprotective effects, according to histological analysis. Besides, HUA mice treated with TAP showed anti-inflammatory activity by decreasing the levels of toll-like receptor 4, nuclear factors-κB p65, NLRP3, ASC, and Caspase-1 in the kidneys (P < 0.01). According to serum non-targeted metabolomics, 91 differential metabolites between the MC and TAP groups were identified, and purine metabolism was considered to be the main pathway for TAP alleviating HUA. In a word, TAP exhibited strong antihyperuricemic activity both in vitro and in vivo.

Isolation and differential structure characteristics of calcium-binding peptides derived from Pacific cod bones by hydroxyapatite affinity.

Calcium-chelating peptides were found in Pacific cod bone, but their binding structure and properties have not been elucidated. Novel calcium-binding peptides were isolated by hydroxyapatite affinity chromatography (HAC), and their binding structure and properties were investigated by isothermal titration calorimetry (ITC), multispectral techniques, and mass spectrometry. Based on multiple purifications, the calcium binding capacity (CBC) of Pacific cod bone peptides (PBPs) was increased from 1.71 ± 0.15 µg/mg to 7.94 ± 1.56 µg/mg. Peptides with a molecular weight of 1-2 kDa are closely correlated with CBC. After binding to calcium, the secondary structure of peptides transitioned from random coil to ß-sheet, resulting in a loose and porous microstructure. Hydrogen bonds, electrostatic interaction, and hydrophobic interaction contribute to the formation of peptide­calcium complexes. The F21 contained 42 peptides, with repeated "GE" motif. Differential structure analysis provides a theoretical basis for the targeted preparation of high CBC peptides.

Collagen/gelatin and polysaccharide complexes enhance gastric retention and mucoadhesive properties.

This article has focused on collagen-gelatin, the gelation process, as well as blend interaction between collagen/gelatin with various polysaccharides to boost mucoadhesion and gastric retention. The interaction between mucoadhesive materials and mucin layers is of significant interest in the development of drug delivery systems and biomedical applications for effective targeting and prolonged time in the gastrointestinal tract. This paper reviews the current advancement and mucoadhesive properties of collagen/gelatin and different polysaccharide complexes concerning the mucin layer and interactions are briefly highlighted. Collagen/gelatin and polysaccharide blends biocompatible and biodegradable, the complex biomolecules have shown encouraging mucoadhesive properties due to their cationic nature and ability to form hydrogen bonds with mucin glycoproteins. The mucoadhesion mechanism was attributed to the electrostatic interactions between the positively charged amino (NH2) groups of blend biopolymers and the negatively charged sialic acid residues present in mucin glycoprotein. At the end of this article, the encouraging prospect of collagen/polysaccharide complex and mucin glycoprotein is highlighted.

Chitosan interaction with stomach mucin layer to enhances gastric retention and mucoadhesive properties.

The interaction between mucoadhesive materials and mucin layers is of significant interest in the development of drug delivery systems and biomedical applications for effective targeting and prolonged stay in the gastrointestinal tract. In this article, the current advancement and mucoadhesive properties of chitosan concerning the stomach mucin layer and its interactions have been briefly addressed. Chitosan a biocompatible polysaccharide exhibited promising mucoadhesive properties attributed to its cationic nature and ability to establish bonds with mucin glycoproteins. The mucoadhesion mechanism is ascribed to the electrostatic interactions between the positively charged amino (NH2) groups of chitosan and the sialic acid residues in mucin glycoprotein which carry a negative charge. The article provides a succinct overview of prior uses, current trends, and recent advancements in chitosan-based gastric-targeted delivery systems. We look forward to further innovations and emerging research related to chitosan-based methods of delivery that may increase the chitosan suitability for use in novel therapeutic approaches.

Fatty acid esters of hydroxy fatty acids: A potential treatment for obesity-related diseases.

Obesity, a burgeoning worldwide health system challenge, is associated with multiple chronic diseases, including diabetes and chronic inflammation. Fatty acid esters of hydroxy fatty acids (FAHFAs) are newly identified lipids with mitigating and anti-inflammatory effects in diabetes. Increasing work has shown that FAHFAs exert antioxidant activity and enhance autophagy in neuronal cells and cardiomyocytes. We systematically summarized the biological activities of FAHFAs, including their regulatory effects on diabetes and inflammation, antioxidant activity, and autophagy augmentation. Notably, the structure-activity relationships and potential biosynthesis of FAHFAs are thoroughly discussed. FAHFAs also showed potential roles as diagnostic biomarkers. FAHFAs are a class of resources with promising applications in the biomedical field that require in-depth research and hotspot development, as their structure has not been fully resolved and their biological activity has not been fully revealed.

A comparative investigation of anionic polysaccharides on the structure and gastrointestinal digestion of collagen fibrils.

Collagen, the most abundant and widely distributed functional protein in mammals, typically assembles into collagen fibrils through side-by-side packing. The purpose of this study was to comparatively investigate the fate of sea cucumber collagen fibrils in the gastrointestinal tract when interacting with different anionic polysaccharides (fucoidan (FUC), Kappa-carrageenan (K-car), sodium alginate (SA)). Results revealed that the gel properties and viscosity values of collagen fibrils were notably enhanced, and the rate of structural alteration in collagen fibrils was reduced when K-car and SA were introduced. Conversely, in the presence of FUC, collagen fibril viscosity decreased, and the secondary structure of collagen fibrils underwent changes. FUC was found to diminish the structural stability of collagen fibrils and accelerate the gastric digestion rate, which was further exacerbated by thermal treatment. All these anionic polysaccharides were observed to facilitate the formation of collagen peptide aggregates by binding to polysaccharides during intestinal digestion. This study bridged the knowledge gap regarding the impact of anionic polysaccharides on the gastrointestinal digestion of collagen fibrils, potentially paving the way for broader applications of collagen in the food industry.

Novel synergistic cross-linking ameliorate ready-to-eat sea cucumber deterioration and its quantum chemical analysis.

Synergistic cross-linkers could improve the taste acceptability of ready-to-eat sea cucumber (RSC). Besides, the hardness of RSC was increased by 331.00% and 266.87% after synergistic cross-linking. Synergistic cross-linking treatment could ameliorate the non-enzymatic degradation of RSC collagen and polysaccharides. Gaussian calculations results showed that dipeptides containing asparagine residues may have different reaction pathways. The main cleavage pathways of CH3CO-Asn-Gly-NHCH3 (NG) might be water-assisted side chain cyclization, stepwise cyclamide hydrolysis via a Gemdiol Intermediate, deamination, and peptide bond breakage. The relative free energy of cyclamide hydrolysis process of NG was increased by 8.2 kcal/mol after synergistic cross-linking. The mass spectrometry results showed that typical peptides could cleavage at NG, CH3CO-Asn-Lys-NHCH3 (NK) and CH3CO-Asn-Leu-NHCH3 (NL) sites after heating, which justified the breakage pattern of peptides in Gaussian calculations. It can offer a comprehensive theoretical basis for the processing of the ready-to-eat sea cucumber with storage stability.

Insights into the molecular mechanism of dextran sulfate inhibiting DNA digestion by pepsin: a crucial role of sulfate group on the binding to DNA.

Sulfate polysaccharides can inhibit DNA digestion in simulated gastric juice in vitro, which is important for regulating dietary nucleic acids metabolism, but the mechanism of inhibition is unclear. This study used dextran sulfate (DS) with different sulfate groups and molecular weights to explore the effect of DS on DNA digestion. Molecular interactions between DS and DNA were investigated by biolayer interferometry (BLI), isothermal titration calorimetry (ITC) and molecular dynamics simulations. Results indicated that DS with higher molecular weight and sulfate group content showed stronger inhibitory effect of DNA digestion. ITC results showed that the combined Kd value of DNA and DS was about 2.53 mM. The main reason for inhibition of DNA digestion is that the formation of hydrogen bonds between the sulfate group of DS and DNA bases hinders the binding of DNA to pepsin. This finding will facilitate new strategies for nucleic acid metabolism and oral drug delivery.Communicated by Ramaswamy H. Sarma.

Physicochemical properties and cell proliferation and adhesive bioactivity of collagen-hyaluronate composite gradient membrane.

Membrane materials were widely used in guided tissue regeneration (GTR) to prevent fibroblast invasion and form a confined area for preferentially growing of osteoblast. A novel collagen-hyaluronate composite gradient membrane was prepared by Tilapia (Oreochromis mossambicus) skin collagen and sodium hyaluronate for potential GTR applications and their bioactivities were investigated by cellular viability. SEM results indicated the membrane showed a dense outer and a porous inner surface for effectively guiding the growth of bone tissue. Physicochemical and biosafety experiments showed the tensile strength of membrane was 466.57 ± 44.31 KPa and contact angle was 74.11°, and the membrane showed perfect biocompatibility and cytocompatibility as well, which met the requirements of GTR material. Cell morphology revealed that the membrane could facilitate the adherence and proliferation of fibroblast and osteoblast. The results of qRT-PCR and ELISA demonstrated that the membrane could effectively activate TGF-ß/Smad pathway in fibroblast, and promote the expressions of TGF-ß1, FN1 and VEGF. Remarkably, RUNX2 was stimulated in BMP2 pathway by the membrane to regulate osteoblast differentiation. In summary, the collagen-hyaluronate composite gradient membrane not only fulfills the prerequisites for use as a GTR material but also demonstrates substantial potential for practical applications in the field.

Distribution, Typical Structure and Self-Assembly Properties of Collagen from Fish Skin and Bone.

The source and type of collagen are crucial to its application, and both play a decisive role. Collagen was prepared from both tilapia skin and bone and skate skin and cartilage, named as CI-TI-s, CI-TI-b, CI-SK-s, and CII-SK-c, respectively. Types, distributions, structures, and self-assembly of collagen were studied. It showed that yellow collagen fibers from skin arranged longitudinally, while collagen fibers from skate cartilages displayed varying colors. CI-TI-s, CI-TI-b, CI-SK-s, and CII-SK-c showed the typical amide A (3316-3336 cm-1) and amide B (2929-2948 cm-1) in FTIR spectra. CI-TI-b and CII-SK-c showed 218-229 nm of UV absorption, 11.56-12.20 Å of d values in XRD, and 0.12-0.14 of Rpn values in CD. The thermal denaturation temperatures of CI-TI-s and CI-SK-s were 30.7 and 20.6 °C, respectively. The self-assembly of CI-TI-s and CII-SK-c were maximum at pH 7.2 and 7.4-7.6, respectively. The unique collagen peptides of tilapia and skate were GPSGPQGAVGATGPK, PAMPVPGPMGPMGPR, SPAMPVPGPMGPMGPR, GESGPSGPAGPAGPAGVR, SSGPPVPGPIGPMGPR, GLTGPIGVPGPPGAQGEK, GLAGPQGPR, and GLSGDPGVQGIK, respectively. The unique peptides of type I and type II collagen were GPTGEIGATGLAGAR, GVLGLTGMR, LGLTGMR, GEPGAAGPAGPSGPMGPR, SSGPPVPGPIGPMGPR, and GLSGDPGVQGIK, respectively.

Preventive effect of salmon sperm DNA on acute carbon tetrachloride-induced liver injury in mice through Nrf2/ARE and mitochondrial apoptosis pathway.

Liver injury refers to the damage of liver function, which will seriously harm the body's health if it is not prevented and treated in time. Sporadic researches have reported that ingestion of DNA has a hepatoprotective effect, but its effect and mechanism were not clarified. The purpose of this study was to explore the preventive effect and mechanism of salmon sperm DNA on acute liver injury in mice induced by carbon tetrachloride (CCl4). Six-week-old ICR (Institute of Cancer Research) male mice were used to establish a liver injury model by injecting with 4% CCl4, silymarin, and three different concentrations of DNA solutions were given to mice by gavage for 14 days. The histological and pathological changes in the liver were observed. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum and the levels of oxidative and antioxidant markers such as malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and glutathione (GSH) in liver tissue were determined. The levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA), and hepatic oxidative stress and apoptosis-related markers were determined by western blotting. The results showed that compared with the model group, the DNA test group significantly improved the liver pathological changes and the level of liver function, regulated liver oxidative stress, reduced hepatocyte apoptosis, and decreased the levels of inflammatory factors such as TNF-α and IL-6. Compared with the silymarin group, the high dose of DNA was even more effective in preventing liver injury. In conclusion, salmon sperm DNA has a potential protective effect against acute liver injury induced by CCl4, which is achieved by regulating the Nrf2/ARE (nuclear factor erythroid 2 (NF-E2)-related factor 2/antioxidant responsive element) oxidative stress pathway and mitochondrial apoptosis pathway.

A comprehensive review of calcium and ferrous ions chelating peptides: Preparation, structure and transport pathways.

Calcium and iron play crucial roles in human health, deficiencies of which have globally generated public health risks. The poor solubility, low bioavailability and gastrointestinal irritation of existing commercial mineral supplements limit their further application. As an emerging type of mineral supplement, mineral chelating peptides have drawn plenty of attention due to their advantages in stability, absorptivity and safety. A majority of calcium and ferrous ions chelating peptides have been isolated from food processing by-products. Enzymatic hydrolysis combined with affinity chromatography, gel filtration and other efficient separation techniques is the predominant method to obtain peptides with high calcium and ferrous affinity. Peptides with small molecular weight are more likely to chelate metals, and carboxyl, amino groups and nitrogen, oxygen, sulfur atoms in the side chain, which can provide lone-pair electrons to combine with metallic ions. Unidentate, bidentate, tridentate, bridging and α mode are regarded as common chelating modes. Moreover, the stability of peptide-mineral complexes in the gastrointestinal tract and possible transport pathways were summarized. This review is to present an overview of the latest research progress, existing problems and research prospects in the field of peptide-mineral complexes and to provide a more comprehensive theoretical basis for their exploitation in food industry.

Salmon sperm DNA prevents acute liver injury by regulating alcohol-induced steatosis and restores chronic hepatosis via alleviating inflammation and apoptosis.

Current medications used to treat alcoholic liver injury (ALD) can cause secondary damage to the liver. Therefore, it is important to improve alcoholic liver injury from the perspective of dietary and nutritional supplementation. Nucleic acids, as functional biomolecules, are present in almost all foods, especially in aquatic products, but their edible research has been neglected for a long time. Hence, the effects of a typical aquatic nucleic acid, namely, salmon sperm DNA, in acute, and chronic alcoholic liver injury model of male ICR mice were studied. The results showed that salmon sperm DNA significantly attenuated the accumulation of cholesterol (TC) and triglycerides (TG) in acute alcoholic liver injury, and it was further demonstrated to mainly regulate lipid metabolism by fluorescent quantitative PCR and immunoblotting experiments. In addition, nucleic acid intervention alleviated inflammation and apoptosis in mice with chronic alcoholic liver injury. PRACTICAL APPLICATIONS: These results suggest that salmon sperm DNA can prevent and ameliorate alcoholic liver injury and can be used as an effective dietary and nutritional supplement for the prevention and treatment of ALD. Moreover, this study provided some new ideas for the development and utilization of large aquatic nucleic acid resources, promoted the comprehensive use of fish processing waste, such as fish sperm, and provided new directions for reducing emissions.

Altered fecal microbial and metabolic profile reveals potential mechanisms underlying iron deficiency anemia in pregnant women in China.

The gut microbiome and its metabolism may provide crucial insight into the cause of iron deficiency anemia (IDA) in pregnant women. This study aimed to investigate the effect of the gut microbiome and its related metabolites on pregnant women with iron deficiency (ID) and IDA. Maternal cubital venous blood and stool samples were collected from healthy control pregnant women (HC, non-anemic, n=10), pregnant women with ID non-anemia (ID, n=10), and IDA (n=10). All groups were subjected to fecal metagenomics and metabolomics. The composition and function of the gut microbiome were then compared in pregnant women with ID and IDA with HC after excluding the possibility of inflammation and insufficient iron absorption capacity. Whole-genome shotgun libraries were prepared by quantifying metagenomic DNA samples with Quant-iT PicoGreen dsDNA Assay. The levels of 41 microbial species, including 21 Streptococci and ten metabolites (catechol), which could serve as siderophores, were increased. In contrast, 3 Bacteroides and six metabolites were decreased in pregnant women with IDA (p<0.05). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the bio-pathways, including biosynthesis of siderophore group non-ribosomal peptides (p<0.01), ABC transporters (p<0.05) and membrane transport of the gut microbiota (p<0.01) in IDA patients were expressed differently compared with HC. Correlation analysis also indicates that these increased bacteria formed strong co-occurring relationships with metabolites in the occurrence and development of IDA in pregnant women. The current study identified that streptococci and catechol (fecal metabolite) were significantly increased in pregnant women with IDA. Therefore, adjusting the intestinal homeostasis using long-term living and eating habits on oral Streptococcus in pregnant women with IDA before iron supplementation may be more conducive to iron supplementation, thus providing novel therapies for IDA.

Development of a high-throughput SNP array for sea cucumber (Apostichopus japonicus) and its application in genomic selection with MCP regularized deep neural networks.

High-throughput single nucleotide polymorphism (SNP) genotyping assays are powerful tools for genetic studies and genomic breeding applications for many species. Though large numbers of SNPs have been identified in sea cucumber (Apostichopus japonicus), but, as yet, no high-throughput genotyping platform is available for this species. In this study, we designed and developed a high-throughput 24 K SNP genotyping array named HaishenSNP24K for A. japonicus, based on the multi-objective-local optimization (MOLO) algorithm and HD-Marker genotyping method. The SNP array exhibited a relatively high genotyping call rate (> 96%), genotyping accuracy (>95%) and exhibited highly polymorphic in sea cucumber populations. In addition, we also assessed its application in genomic selection (GS). Deep neural networks (DNN) that can capture the complicated interactions of genes have been proposed as a promising tool in GS for SNP-based genomic prediction of complex traits in animal breeding. To overcome the problem of over-fitting when using the HaishenSNP24K array as high-dimensional DNN input, we developed minmax concave penalty (MCP) regularization for sparse deep neural networks (DNN-MCP) that finds an optimal sparse structure of a DNN by minimizing the square error subject to the non-convex penalty MCP on the parameters (weights and biases). Compared to two linear models, namely RR-GBLUP and Bayes B, and the nonlinear model DNN, DNN-MCP has greatly improved the genomic prediction ability for three quantitative traits (e.g., wet weight, dry weight and survival time) in the sea cucumber population. To the best of our knowledge, this is the first work to develop a high-throughput SNP array for A. japonicus and a new model DNN-MCP for genomic prediction of complex traits in GS. The present results provide evidence that supports the HaishenSNP24K array with DNN-MCP will be valuable for genetic studies and molecular breeding in A. japonicus.

Intervention mechanism of self-degradation of ready-to-eat sea cucumber by adding green tea extract and gallic acid.

Gallic acid (GA) and green tea extract (GT) could retard the self-degradation of ready-to-eat sea cucumber (RSC). The physical and chemical properties of RSC were changed after cross-linking. Cross-linkers could retard the conversion of α-helix like structure to random coil. Besides, the peptides of cross-linked RSC were easily broken at the sites of G, E, A, L, S, H, Y, V and I after stored for 30 d. The self-degradation rate of RSC before and after cross-linking was determined by synthetic typical peptides. After cross-linked by GA, the relative peak heights of NQ, NL and GLQ increased by 20.59%, 11.14% and 31.49%, indicating that GA could effectively retard the degradation of the peptides during storage. Moreover, hydrogen bond was confirmed as the main force to maintain the stability of RSC body wall before and after cross-linking.

Contribution of secondary bonds to the storage stability of ready-to-eat sea cucumber.

To explore the contribution of secondary bonds on storage stability of RSC, urea, n-propanol and sodium nitrite (NaNO2) were used for the breakdown of hydrogen bonds, hydrophobic bonds, side-chain groups, respectively. The impact of the breakdown of secondary bonds before and after storage on texture properties and microstructure were investigated. The breakdown of hydrogen bonds, especially before storage, significantly reduced the hardness (from 3.12 to 2.21 N), chewiness (from 1.82 to 1.05 mJ), and springiness (from 2.57 to 1.57 mJ) of RSC, while the breakdown of hydrophobic bonds and side-chain groups had a slight effect. Similarly, degradation of collagen fibers by breaking hydrogen bonds before storage was more serious than other groups. Furthermore, moisture content and water activity decreased and the degradation degree of collagen in RSC body wall increased with hydrogen bonds breaking. The results showed that hydrogen bonds were essential for the storage stability of RSC.

Rheological properties, thermal stability and conformational changes of collagen from sea cucumber (Apostichopus japonicas).

Sea cucumber collagen (SCC) properties affected the thermal processing of sea cucumber. SCC showed the shear-thinning and pseudo-plastic properties, and the viscosity and frequency of viscoelastic crossover were decreased gradually with the temperature from 15 to 30 °C. Differential scanning calorimetry of SCC confirmed that it was thermolabile with the increase of temperatures, acid or NaCl concentrations. As the temperature increasing, the triple helix of SCC disappeared with the decrease of the relative proportion of P2 structures by circular dichroism spectrometry and Fourier transform infrared spectroscopy, and shearing could accelerate the change. Intramolecular changes investigated by molecular dynamics simulation showed the average number of hydrogen bonds decreased from 47 (20 °C) to 42 (80 °C), indicating triple helix of SCC was triggered to uncoil within 250 ns. These results could provide a scientific basis for processing of sea cucumbers.

Structure of type II collagen from sturgeon cartilage and its effect on adjuvant-induced rheumatoid arthritis in rats.

The purpose of this paper was to extract and characterize type II collagen of sturgeon cartilage (SC-CII), and to explore the effects of taking SC-CII orally on rheumatoid arthritis (RA) in rats. SC-CII showed a triple-helix structure (RPN = 0.12), with d1 of 11.82 Å and d2 of 4.08 Å, which was analyzed by FT-IR, CD, XRD, and MS. It was constructed of the repeating tripeptide unit Gly-X-Y, where X and Y are generally Pro or Hyp, proved by amino acid composition and peptide mass fingerprinting. Furthermore, the effects of SC-CII on RA were evaluated. Ankle thickness was significantly decreased in SC-CII groups, with changes in lymphocyte proliferation also observed. Compared with the model control group, there was an evident decrease in TNF-α, IL-1ß, COX-2, MCP-1, and TLR-4 mRNA levels, but no remarkable differences in APF, MMP-3, and MyD88 mRNA levels in the SC-CII groups. In addition, TNF-α, IL-1ß, RF, Anti-CII Ab were significantly reduced in the SC-CII groups, proved by ELISA. Therefore, SC-CII showed alleviating effects on RA through the TLR4/MyD88-NFκB pathway.

Characteristic flavor of Antarctic krill (Euphausia superba) and white shrimp (Penaeus vannamei) induced by thermal treatment.

For the good acceptance and preference of heated shrimp, characteristic flavor composition analysis was necessary. The sensory evaluation, electronic tongue, electronic nose, and gas chromatography-ion mobility spectrometry were employed in this study. After steaming or cooking, the sensory scores of Antarctic krill (KM) and white shrimp (PM) were significantly increased, and five basic tastes were remarkably changed by electronic tongue analysis. Free glycine level increased from 86.48 to 687.12 mg/100 g in PM after steaming, but no significant changes in KM. 5'-nucleotides in heated PM were higher than those in heated KM. In two kinds of shrimp, inorganic ions and lactic acids contents exhibited the decrease trends after cooking, and the response intensities of S4, S5, and S6 showed increase trends after steaming. Nonanal, benzaldehyde, (Z)-3-hexen-1-ol, 1-8-cineol and limonene were produced by thermal treatment. Therefore, characteristic flavor formation was related to thermal treatment.