Purification, characterization, and in vitro digestion of novel antioxidant peptides from chicken blood hemoglobin.

The study aimed to purify and characterize antioxidant peptides from chicken blood hemoglobin hydrolysate. The fraction M2  (< 3 KDa) with the strongest antioxidant activity was isolated by ultrafiltration, and its DPPH (1,1-diphenyl-2-picryl-hydrazyl radical) free radical scavenging rate, ABTS [2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate)] free radical scavenging rate, and iron ion chelation activity were 82.91%, 77.49%, and 80.99%, respectively. After in vitro digestion, the antioxidant capacity of chicken blood hydrolysate was significantly higher than that before digestion (p < 0.05). M2 exhibited the strongest antioxidant activity after stomach digestion, with a DPPH radical scavenging rate and iron ion chelating power of 82.91% and 79.61%, respectively. Component A was purified from M2 by Sephadex G-25 gel chromatography. The peptide sequences were identified by LC-MS/MS from fraction A, and four peptides, AEDKKLIQ (944.54 Da), APAPAAK (625.36 Da), LSDLHAHKL (1033.57 Da), and LSNLHAYNL (1044.54 Da) were synthesized using the solid-phase peptide method, among which APAPAAK was a novel antioxidant peptide. Molecular docking was used to simulate the binding of these four peptides to the key active site of Keap1 via hydrogen bonding. This study suggests that chicken blood may provide a new natural source of antioxidant peptides.

Deciphering the Mechanism by Which Carbon Dioxide Extends the Shelf Life of Raw Milk: A Microbiomics- and Metabolomics-Based Approach.

Microbial community succession in raw milk determines its quality and storage period. In this study, carbon dioxide (CO2) at 2000 ppm was used to treat raw milk to investigate the mechanism of extending the shelf life of raw milk by CO2 treatment from the viewpoint of microbial colonies and metabolites. The results showed that the shelf life of CO2-treated raw milk was extended to 16 days at 4 °C, while that of the control raw milk was only 6 days. Microbiomics analysis identified 221 amplicon sequence variants (ASVs) in raw milk, and the alpha diversity of microbial communities increased (p < 0.05) with the extension of storage time. Among them, Pseudomonas, Actinobacteria and Serratia were the major microbial genera responsible for the deterioration of raw milk, with a percentage of 85.7%. A combined metagenomics and metabolomics analysis revealed that microorganisms altered the levels of metabolites, such as pyruvic acid, glutamic acid, 5'-cmp, arginine, 2-propenoic acid and phenylalanine, in the raw milk through metabolic activities, such as ABC transporters, pyrimidine metabolism, arginine and proline metabolism and phenylalanine metabolism, and reduced the shelf life of raw milk. CO2 treatment prolonged the shelf life of raw milk by inhibiting the growth of Gram-negative aerobic bacteria, such as Acinetobacter guillouiae, Pseudomonas fluorescens, Serratia liquefaciens and Pseudomonas simiae.

iTRAQ-based proteomics identifies proteins associated with betaine accumulation in Lycium barbarum L.

In order to better understand the mechanism of betaine accumulation in Lycium barbarum L. (LBL), we used iTRAQ (Isotope relative and absolute quantitative labeling) proteomics to screen and identify differentially abundant proteins (DAPs) at five stages (S1-young fruit stage, S2-green fruit stage, S3-early yellowing stage, S4-late yellowing stage, S5-ripening stage). A total of 1799 DAPs and 171 betaine-related DAPs were identified, and phosphatidylethanolamine N-methyltransferase (NMT), choline monooxygenase (CMO), and betaine aldehyde dehydrogenase (BADH) were found to be the key enzymes related to betaine metabolism. These proteins are mainly involved in carbohydrates, amino acids and their derivatives, fatty acids, carboxylic acids, photosynthesis and photoprotection, isoquinoline alkaloid biosynthesis, peroxisomes, and glycine, serine, and threonine metabolism. Three of the key enzymes were also up- and down-regulated to different degrees at the mRNA level. The study provide new insights into the of mechanism of betaine accumulation in LBL. SIGNIFICANCE: Betaine, a class of naturally occurring, water-soluble alkaloids, has been found to be widespread in animals, higher plants, and microbes. In addition to being an osmotic agent, betaine has biological functions such as hepatoprotection, neuroprotection, and antioxidant activity. Betaine metabolism (synthesis and catabolism) is complexly regulated by developmental and environmental signals throughout the life cycle of plant fruit maturation. As a betaine-accumulating plant, little has been reported about the regulatory mechanisms of betaine metabolism during the growth and development of Lycium barbarum L. (LBL) fruit. Therefore, this study used iTRAQ quantitative proteomics technology to investigate the abundance changes of betaine-related proteins in LBL fruit, screen and analyze the differential abundance proteins related to betaine metabolism, and provide theoretical references for the in-depth study of the mechanism of betaine metabolism in LBL fruit.

Exploring the mechanism of Lycium barbarum fruit cell wall polysaccharide remodeling reveals potential pectin accumulation contributors.

The level of polysaccharides in the mature Lycium barbarum fruit (LBF) cell wall depends on their metabolism, trafficking, and reassembly within the cell. In this study, we examined the composition, content, and ultrastructure of the cell wall polysaccharides of LBF during maturation, and further analyzed cell wall polysaccharide remodeling using isotope tagging with relative and absolute quantification (iTRAQ)-based proteomics. The results showed that the contents of cellulose and hemicellulose tended to increase in the pre-maturation stage and decrease in the later stage, while pectin level increased before fruit maturing. The differential expression of the 54 proteins involved in the metabolic pathways for glucose, fructose, galactose, galacturonic acid and arabinose was found to be responsible for these alterations. The work provides a biological framework for the reorganization of polysaccharides in the LBF cell wall, and supports the hypothesis that pectic polysaccharide glycosyl donors come from starch, cellulose, hemicellulose and isomorphic pectin.

Proteomic and metabolomic analysis of ageing beef exudate to determine that iron metabolism enhances muscle protein and lipid oxidation.

The study aimed to assess differences in proteomic and metabolite profiles in ageing (1, 2, 4, and 6 days at 4 °C) beef exudates and determine their relationship with beef muscle iron metabolism and oxidation. Proteomic and metabolomic analyses identified 877 metabolites and 1957 proteins. The joint analysis identified 24 differential metabolites (DMs) and 56 differentially expressed proteins (DEPs) involved in 15 shared pathways. Ferroptosis was identified as the only iron metabolic pathway, and 4 DMs (l-glutamic acid, arachidonic acid, glutathione and gamma-glutamylcysteine) and 5 DEPs (ferritin, phospholipid hydroperoxide glutathione peroxidase, heme oxygenase 1, major prion protein, and acyl-CoA synthetase long chain family member 4) were involved in iron metabolism by regulating heme and ferritin degradation, Fe2+ and Fe3+ conversion, arachidonic acid oxidation and inactivation of glutathione peroxidase (GPX) 4, leading to increased levels of free iron, ROS, protein and lipid oxidation (P < 0.05). Overall, abnormal iron metabolism during ageing induced oxidative stress in muscle tissue.

Characterization and Discrimination of Volatile Compounds in Chilled Tan Mutton Meat during Storage Using HiSorb-TD-GC-MS and E-Nose.

Chilled Tan mutton is currently the mainstream of Tan mutton production and consumption in China, but the reports on chilled meat quality evaluation and shelf-life discrimination by volatiles are limited. This study aimed to investigate the changes of volatile compounds in chilled Tan mutton at four storage stages (1d, 3d, 5d, 7d) in order to differentiate the various storage stages. An analysis protocol was established for the characterization and discrimination of the volatiles in chilled Tan mutton based on high capacity sorptive extraction-thermal desorption-gas coupled with chromatography-mass spectrometry (HiSorb-TD-GC-MS), electronic nose (E-nose), and multivariate statistical analysis. A total of 96 volatile compounds were identified by HiSorb-TD-GC-MS, in which six compounds with relative odor activity value >1 were screened as the key characteristic volatiles in chilled Tan mutton. Four storage stages were discriminated by partial least squares discriminant analysis, and nine differential volatile compounds showed a variable importance for the projection score >1, including octanoic acid, methyl ester, decanoic acid, methyl ester, acetic acid, heptanoic acid, methyl ester, propanoic acid, 2-hydroxy-, methyl ester, (ñ)-, hexanoic acid, propanoic acid, butanoic acid, and nonanoic acid. With the volcano plot analysis, hexadecanoic acid, methyl ester, was the common volatile marker candidate to discriminate chilled stages of Tan mutton. Meanwhile, E-nose could discriminate chilled Tan mutton at different storage stages rapidly and efficiently using linear discriminant analysis. Furthermore, E-nose sensors could obtain comprehensive volatile profile information, especially in esters, acids, and alcohols, which could confirm the potential of E-nose for meat odor recognition. Thus, this analysis protocol could characterize and discriminate the volatiles in chilled Tan mutton during storage.

Characterization of a fermented dairy, sour cream: Lipolysis and the release profile of flavor compounds.

Lipolysis and flavor development during fermentation of sour cream were studied by evaluating the physicochemical changes, sensory differences and volatile components. The fermentation caused significant changes in pH, viable count and sensory evaluation. The peroxide value (POV) decreased after reaching the maximum value of 1.07 meq/kg at 15 h, while thiobarbituric acid reactive substances (TBARS) increased continuously with the accumulation of secondary oxidation products. The Free fatty acids (FFAs) in sour cream were mainly myristic, palmitic and stearic. GC-IMS was used to identify the flavor properties. A total of 31 volatile compounds were identified, among which the contents of characteristic aromatic substances such as ethyl acetate, 1-octen-3-one and hexanoic acid were increased. The results suggest that lipid changes and flavor formation in sour cream are influenced by fermentation time. Furthermore, flavor compounds may be related to lipolysis such as 1-octen-3-one and 2- heptanol were also observed.

Reduced water-holding capacity of beef during refrigeration is associated within creased heme oxygenase 1 expression, oxidative stress and ferroptosis.

Low molecular weight iron (LMW-Fe)-mediated oxidative stress from heme degradation may reduce beef water-holding capacity (WHC). However, the underlying mechanism of heme degradation is still unknown. In the present study, we assessed the WHC, tissue morphology, reactive oxygen species (ROS), apoptosis, heme oxygenase(HMOX) 1 expression, and ferroptosis characteristics of beef chilled at 4 °C for 6 days. Results showed that water loss increased and WHC decreased during beef storage (P < 0.05). Increased protein and mRNA expression of HMOX1 promoted the decomposition of heme and facilitated the liberation of iron ions (P < 0.05), and excess LMW-Fe was associated with ROS formation, depletion of glutathione, and inhibition of glutathione peroxidase 4 activity (P < 0.05). Muscle tissue showed typical features of ferroptosis, including expression of ferroptosis-related genes, malondialdehyde accumulation, and structural damage to mitochondria (P < 0.05). It was also found that HMOX1 and the heme pathway-mediated ferroptosis were associated with structural changes in myofibrils and reduced WHC in chilled beef.

GC-MS and GC×GC-ToF-MS analysis of roasted / broth flavors produced by Maillard reaction system of cysteine-xylose-glutamate.

Maillard reaction products (MRPs) with roasted/broth flavors were prepared and analyzed for the resulting flavor differences. The identification of volatile compounds in MRPs was carried out by GC-MS and GC × GC-ToF-MS. A total of 88 compounds were identified by GC-MS; 130 compounds were identified by GC × GC-ToF-MS, especially acids and ketones were identified. Principal component analysis (PCA) was used to visualize the volatile compounds, and the roasted/broth flavors were differentiated. The contents and types of pyrazines were more in roasted flavors; thiol sulfides and thiophenes were more in broth flavors. All in all, the differences in volatile compounds producing roasted/broth flavors were studied through the cysteine-xylose-glutamate Maillard reaction system, which provided a theoretical basis for the future use of Maillard reaction to simulate meat flavor.

Ferritin-dependent cellular autophagy pathway promotes ferroptosis in beef during cold storage.

Ferroptosis plays a pivotal role in regulating various physiological processes and quality of post-mortem muscle. However, the molecular mechanisms underlying ferroptosis remain unclear. The study investigated how ferroptosis was induced in beef during cold storage. Results showed that the expression of autophagy-related genes, LC3, ATG5, ATG7, and NCOA4 in beef during cold storage promoted the degradation of ferritin heavy chains. Ferritin evoked ferroptosis by releasing free iron, inducing reactive oxygen species (ROS) accumulation and inhibiting the glutathione (GSH)-glutathione peroxidase 4 (GPX4) pathway. Furthermore, treatment of myoblasts with GSK 2656157 (autophagy inhibitor) showed that ferritin degradation was lower in the GSK 2656157-treated myoblasts than in the control, while GSH content and GPX4 activity were higher than the control (P < 0.05), and the contents of free iron, ROS and malondialdehyde, and apoptosis were lower than the control (P < 0.05). These results suggest that ferroptosis is induced by degradation of ferritin via the autophagic pathway.

Glutathione metabolism-mediated ferroptosis reduces water-holding capacity in beef during cold storage.

To investigate the potential mechanisms by which cold storage affects the water-holding capacity (WHC) of beef through analysis of exudates using an untargeted metabolomics strategy. A total of 877 metabolites were detected in four groups of beef exudates that have been frozen for 1, 2, 4, and 6 days, of which, 278 were identified as differential metabolites (DMs). The metabolic pathways of the DMs analysed by KEGG pathway enrichment included ABC transporters, purine metabolism, biosynthesis of cofactors, protein digestion and absorption, and ferroptosis. Ferroptosis was identified during storage of beef, and the reduction in WHC of beef was accompanied by a ferroptosis process. In addition, six DMs were identified in the KEGG pathway of ferroptosis, and the process of cellular ferroptosis was dependent on the inhibition of glutathione metabolic processes. Overall, the ferroptosis of cells during beef storage had a negative impact on WHC, and the finding of ferroptosis complemented the post-slaughter apoptosis.

Footprint analysis of CO2 in microbial community succession of raw milk and assessment of its quality.

With the growing production of raw milk, interest has been increasing in its quality control. CO2, as a cold processing additive, has been studied to extend the cold storage period and improve the quality of raw milk. However, it is yet uncertain how representative microbial species and biomarkers can succeed one another at distinct critical periods during refrigeration. Therefore, the effects of CO2 treatment on the succession footprint of the microbial community and changes in quality during the period of raw milk chilling were examined by 16S rRNA analysis combined with electronic nose, and electronic tongue techniques. The results indicated that, the refrigeration time was shown to be prolonged by CO2 in a concentration-dependent way. And CO2 treatment was linked to substantial variations in beta and alpha diversity as well as the relative abundances of various microbial taxa (p < 0.01). The dominant bacterial phylum Proteobacteria was replaced with Firmicutes, while the major bacterial genera Acinetobacter and Pseudomonas were replaced with lactic acid bacteria (LAB), including Leuconostoc, Lactococcus, and Lactobacillus. From the perspective of biomarkers enriched in CO2-treated sample, almost all of them belong to LAB, no introduction of harmful toxins has been found. The assessment of the quality of raw milk revealed that CO2 improved the quality of raw milk by lowering the acidity and the rate of protein and fat breakdown, and improved the flavor by reducing the generation of volatiles, and increasing umami, richness, milk flavor and sweetness, but reducing sourness. These findings offer a new theoretical foundation for the industrial use of CO2 in raw milk.

Assessment of the Bioaccessibility of Carotenoids in Goji Berry (Lycium barbarum L.) in Three Forms: In Vitro Digestion Model and Metabolomics Approach.

Goji berry (Lycium barbarum L., LBL) is a good source of carotenoids, while the bioaccessibility of various types of LBL carotenoids has not been explored. In the study, eight carotenoids, three carotenoid esters and two carotenoid glycosylated derivatives were identified by a non−targeted metabolomics approach. The dried LBL (DRI), LBL in water (WAT), and LBL in "Baijiu" (WIN) were used to recreate the three regularly chosen types of utilization, and the in vitro digestion model showed that the bioaccessibility of the carotenoids increased significantly from the oral to the gastric and intestinal phase (p < 0.05). The bioaccessibility of LBL carotenoids was the most elevated for DRI (at 28.2%), followed by WIN and WAT (at 24.9% and 20.3%, respectively). Among the three carotenoids, zeaxanthin dipalmitate showed the highest bioaccessibility (51.8−57.1%), followed by ß−carotene (51.1−55.6%) and zeaxanthin (45.2−56.3%). However, the zeaxanthin from DRI exhibited significantly higher bioaccessibility (up to 58.3%) than WAT and WIN in both the gastric and intestinal phases (p < 0.05). Results of antioxidant activity tests based on DPPH, FRAP, and ABTS showed that the addition of lipids improved the bioaccessibility of the carotenoids. (p < 0.05).

Effects of Thoracic Paravertebral Blockon Inflammatory Response, Stress Response, Hemodynamics and Anesthesia Resuscitation inGallbladder Carcinoma.

The study aimed to explore the effects of the ultrasound-guided thoracic paravertebral block (TPVB) on the inflammatory response, stress response, hemodynamics and anesthesia resuscitation in gallbladder carcinoma. Eighty gallbladder carcinoma patients undergoing open cholecystectomy in Heilongjiang Provincial Hospital from February 2016 to April 2019 were selected and divided into observation group (n=40) and control group (n=40) using a random number table. All patients underwent open cholecystectomy under general anesthesia and tracheal intubation. Patient-controlled intravenous analgesia was adopted after the operation in the control group, while right TPVB was performed before general anesthesia in the observation group. The changes in inflammatory factors and oxidative stress factors were compared between the two groups, the anesthesia resuscitation indexes, and the changes in the bispectral index (BIS) and Ramsay score during anesthesia resuscitation were recorded, and the changes in the hemodynamic indexes in perianesthesia and anesthesia resuscitation-related complications were analyzed. At 15 min after anesthesia, the observation group had lower levels of inflammatory factors high-sensitivity C-reactive protein (hs-CRP) and interleukin-6 (IL-6) (p<0.05), malondialdehyde (MDA) (p<0.05) and a higher level of superoxide dismutase (SOD) (p<0.05) than the control group. The anesthesia resuscitation time was shorter in the observation group than that in the control group (p<0.05). At 10 min, 20 min and 30 min after anesthesia, both BIS and Ramsay scores were significantly higher in the observation group than those in the control group (p<0.05). Moreover, the proportion of circulatory function-related complications and anesthesia resuscitation-related complications were lower in the observation group than that in the control group (p<0.05). The NRS score in the observation group was lower than that in the control group after anesthesia (p<0.05). TPVB in perianesthesia for gallbladder carcinoma patients can effectively lower the body's inflammatory and stress responses, promote anesthesia resuscitation, reduce complications in perianesthesia, and relieve postoperative pain.

Physicochemical properties of a new structural lipid from the enzymatical incorporation of flaxseed oil into mutton tallow.

This study evaluated the physio-chemical properties of a structural lipid (SL) obtained by the enzymatical incorporation of flaxseed oil into mutton tallow (MT). By measuring the melting point, colour, safety, fatty acids, apparent viscosity, shear stress and volatile compounds of the samples, the results showed that compared to MT, SL exhibited lower L∗(lightness) value, melting point, apparent viscosity and shear stress (p < 0.05). Noteworthy, the Saturated fatty acids (SFA)content of MT was reduced from 61.46% to 25.49% (p < 0.05), although SL was found to be more prone to oxidation during storage. The clearest discrepancy in volatile compounds was the increase of heterocyclic compounds in SL. In summary, improving the edible properties of animal fats by adding vegetable oils is an effective solution, and SL may have a great potential to be developed into a high-quality product with improved nutritional composition of animal fat.

Identification of the cell wall proteins associated with the softening of Lycium barbarum L. fruit by using iTRAQ technology.

Excessive softening of Lycium barbarum L. (LBL) fruit can limit the storage and transportation of fresh fruit. To better understand the underlying molecular mechanisms of fruit softening in LBL, changes in the pre-climacteric (S1) and post-climacteric (S2) proteomes were investigated by iTRAQ methods. The 14-fold reduction in S2 fruit firmness compared to S1 was accompanied by increased espiratory intensity and degradation of cell wall components. A total of 258 differentially expressed proteins (DEPs) were identified, which were mainly associated with photosynthesis, carbohydrate, amino acids and fatty acids metabolism. From the functional proteomic analysis, enhanced energy metabolisms, such as glycolysis/gluconeogenesis and citrate cycle (TCA cycle) contributed to cell wall degradation and conversion to substrates for respiratory metabolism, leading to fruit softening. These findings have provided new insights into the molecular pathways associated with fruit softening in LBL and the bioinformatics analyses provided insightful information for further transcriptional studies.

Haem-mediated protein oxidation affects water-holding capacity of beef during refrigerated storage.

Haem is considered to be a potential producer of meat oxidation and the effect of its mediated oxidation on the water holding capacity (WHC) of beef is not yet clear. This work investigated the interrelationships between haem, protein and lipid oxidation, and WHC in beef during refrigerated storage. The increase in juice loss during storage (p < 0.05) indicates a reduction in WHC. Haem was oxidised, resulting in its structural disruption and an increase in the proportion of random coil in the protein secondary structures (p < 0.05). Extractable haem iron content was decreased and non-haem iron content was increased (p < 0.05), indicating the degradation of haem and the release of iron during storage. The levels of lipid and protein oxidation products significantly increased throughout the storage time (p < 0.05). Furthermore, Spearman analysis verified significant correlations between these changes. In conclusion, these processes are mutually reinforcing and may exacerbate muscle juice loss.

TMT Quantitative Proteomics Analysis Reveals the Effects of Transport Stress on Iron Metabolism in the Liver of Chicken.

Abnormal iron metabolism can cause oxidative stress in broilers, and transport stress (TS) may potentially influence iron metabolism. However, the mechanisms by which TS affects iron metabolism are unclear. This study used quantitative proteome analysis based on tandem mass tag (TMT) to investigate the effects of TS on liver iron metabolism in broilers. Broilers (n = 24) reared under the same conditions were selected randomly into the transported group for 4 h (T2) and non-transported group (T1). Results showed that the serum iron level and total iron-binding capacity of broilers in the T2 were significantly higher than those in the T1 (p < 0.05). The liver iron content of broilers in the T2 (0.498 ± 0.058 mg·gprot-1) was significantly higher than that in the T1 (0.357 ± 0.035 mg·gprot-1), and the iron-stained sections showed that TS caused the enrichment of iron in the liver. We identified 1139 differentially expressed proteins (DEPs). Twelve DEPs associated with iron metabolism were identified, of which eight were up-regulated, and four were down-regulated in T2 compared with T1. Prediction of the protein interaction network for DEPs showed that FTH1, IREB2, and HEPH play vital roles in this network. The results provide new insights into the effects of TS on broilers' liver iron metabolism.

Quality changes in high pressure processed tan mutton during storage.

The current study investigated the effects of high pressure (HP) treatment at 200 MPa and 500 MPa on quality characteristics of post-rigor tan mutton stored for 7 days at 4 °C, and textural changes were monitored during storage by means of the stress relaxation test. Application of 500 MPa high pressure significantly increased the elasticity and stiffness of meat after 7 days of storage (P < 0.05), accompanied by a lighter and less red appearance and markedly enhanced centrifugal loss during storage campared to untreated (P < 0.05). High pressure treatment at 200 MPa also substantially increased the lightness of samples throughout storage (P < 0.05), and showed a significant increase in stiffness at the end of storage (P < 0.05). Immunoblotting and electrophoresis (SDS-PAGE) analysis of key structural proteins revealed that myosin heavy chain denaturation began at 200 MPa, while actin denaturation occurred at 500 MPa. Troponin-T was continuously degraded in different treatments as storage progressed, and 200 MPa treatment and untreated represented similar degradation patterns, while 500 MPa treatment displayed more intense intact troponin-T at 38 kDa degradation. Results suggest that HP induced changes in cytoskeleton proteins, thereby affecting texture, water holding properties and lightness.

Enzymatic hydrolysis of flaxseed (Linum usitatissimum L.) protein and sensory characterization of Maillard reaction products.

We aimed to simplify the enzymolysis process for flaxseed protein hydrolysates production as Maillard reaction products (MRPs) to generate different flavor characteristics. More than 50% activity of immobilized enzymes (Alcalase and Flavourzyme) was retained after repeated use. Subsequently, after five weeks, the activities of the immobilized enzymes were also observed to be higher after storage at 4 °C. The optimum conditions for production of flaxseed protein hydrolysates using sequential enzymatic hydrolysis were as follow: 3,000 U/g of Alcalase at 60 °C and pH 8.0 for 2 h and 120 U/g of Flavourzyme at 50 °C and pH 6.5 for 2 h. Partial least squares regression analysis revealed that resulting peptides with the molecular weight (MW) higher than 1,000 Da could improve the mouthfulness and stability in umami soup; whereas, peptides with MW of 128-1,000 Da mainly contributed to the generation of meat-like flavor compounds with a significant effect on umami taste and bitterness.