A protective mechanism of heat inactivation to enhance Levilactobacillus brevis PDD-2 against alcohol-induced chronic liver disease based on proteomic analysis.

A proteomics-based analysis of the effect of heat inactivation on the alleviation of alcoholic liver disease (ALD) using Levilactobacillus brevis PDD-2 is presented, aimed at exploring the potential and mechanisms of postbiotic elements prepared through heat inactivation in the treatment of ALD. It was found that L. brevis PDD-2 and its postbiotic (heat-inactivated L. brevis PDD-2) alleviate chronic ALD via the gut-liver axis. In particular, heat-inactivated L. brevis PDD-2 significantly increased the relative abundance of Erysipelotrichaceae and better facilitated the oxidative stress balance in the liver. The tandem mass tag (TMT)-based quantitative proteomics technique analyses revealed that heat-inactivated L. brevis PDD-2 was associated with up-regulated expression levels of proteins related to the redox system, cellular metabolism, amino acid and oligopeptide transport, and surface proteins with immunomodulatory capacity. These findings provide a theoretical basis for developing novel therapeutic strategies and lay a solid foundation for further revealing its exhaustive mechanisms.

Characterization of the Effects of Low-Sodium Salt Substitution on Sensory Quality, Protein Oxidation, and Hydrolysis of Air-Dried Chicken Meat and Its Molecular Mechanisms Based on Tandem Mass Tagging-Labeled Quantitative Proteomics.

The effects of low-sodium salt mixture substitution on the sensory quality, protein oxidation, and hydrolysis of air-dried chicken and its molecular mechanisms were investigated based on tandem mass tagging (TMT) quantitative proteomics. The composite salt formulated with 1.6% KCl, 0.8% MgCl2, and 5.6% NaCl was found to improve the freshness and texture quality scores. Low-sodium salt mixture substitution significantly decreased the carbonyl content (1.52 nmol/mg), surface hydrophobicity (102.58 µg), and dimeric tyrosine content (2.69 A.U.), and significantly increased the sulfhydryl content (74.46 nmol/mg) and tryptophan fluorescence intensity, suggesting that protein oxidation was inhibited. Furthermore, low-sodium salt mixture substitution significantly increased the protein hydrolysis index (0.067), and cathepsin B and L activities (102.13 U/g and 349.25 U/g), suggesting that protein hydrolysis was facilitated. The correlation results showed that changes in the degree of protein hydrolysis and protein oxidation were closely related to sensory quality. TMT quantitative proteomics indicated that the degradation of myosin and titin as well as changes in the activities of the enzymes, CNDP2, DPP7, ABHD12B, FADH2A, and AASS, were responsible for the changes in the taste quality. In addition, CNDP2, ALDH1A1, and NMNAT1 are key enzymes that reduce protein oxidation. Overall, KCl and MgCl2 composite salt substitution is an effective method for producing low-sodium air-dried chicken.

Binary probiotic fermentation promotes signal (cyclic AMP) exchange to increases the number of viable probiotics, anthocyanins and polyphenol content, and the odor scores of wolfberry fermented beverages.

The effects and underlying molecular mechanisms of binary probiotics (Lactiplantibacillus plantarum subsp. plantarum CGMCC 1.5953 and Lacticaseibacillus casei CGMCC 1.5956) on the quality of wolfberry fermented beverages (WFB) were investigated. The results indicated that binary probiotics increased the number of probiotics, anthocyanin (89.92 ± 1.64 mg/L), polyphenol content (283.04 ± 3.81 µg/mL), and odor score (24.19) in WFB. Metabolomics found that they could enhance signal exchange (cyclic AMP) between binary probiotics and improve the utilization of citrulline, d-proline, d-glucose, and d-galactose through galactose metabolism and amino acid biosynthesis pathway to promote probiotics growth. Furthermore, HS-SPME-GC-MS and GS-IMS revealed that the improvement in flavor was mainly due to an increase in the content of the aromatic flavor substances 3-heptanol, glutaraldehyde, and 2-heptanone, and a decrease in the content of the off-flavor substances methyl isobutyl ketone-D and 2-undecanone. This is strategically important for the development of WFB with high probiotic content and unique flavor.

Effects of Semen Ziziphi Spinosae extract and binary probiotics co-fermentation on the quality of yogurt and their underlying molecular mechanisms.

The study aimed to investigate the impact of water-soluble extract from Semen Ziziphi Spinosae (SZSE) on yogurt quality and understand the underlying mechanism. The results demonstrated that adding 0.5% (w/v) SZSE had a significant effect on reducing yogurt syneresis and resulted in a more compact and uniform casein gel. Notably, the co-fermented yogurt with binary probiotics (Lacticaseibacillus casei CGMCC1.5956 and Levilactobacillus brevis CGMCC1.5954) along with SZSE led to increased viable probiotics and a higher odor score (23.23). This effect might be attributed to the increased amino acid utilization by binary probiotics through biosynthesis of valine, leucine and isoleucine, metabolic pathways, and amino acid biosynthesis to produce amino acid derivatives such as N5-(l-1-carboxyethyl)-l-ornithine and diaminopyrimidine acid. The yogurt contained 79 volatile flavor compounds, with hexanoic acid, 2-heptanone, and 2-nonanone potentially contributing to the high odor scores. These findings have strategic implications for developing yogurt with high gel characteristics and distinctive flavor.

Soy protein isolate-guar gum-goose liver oil O/W Pickering emulsions that remain stable under accelerated oxidation at high temperatures.

BACKGROUND: Goose liver oil (GLO) is a solid-liquid mixture, rich in polyunsaturated fatty acids and high in nutritional value, but poor in fluidity and easily oxidized. Therefore, oil-in-water (O/W) Pickering emulsions of three polysaccharides and soy protein isolate (SPI) with GLO were prepared to improve the stability of it. RESULTS: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Fourier-transform infrared spectroscopy, and zeta potential revealed that the SPI and complexes with konjac glucomannan, pectin, and guar gum (GG) ranged from 17 to 75 kDa, with the site of action being the -OH stretch and the amide group, and bound by hydrogen bonding. Adding konjac glucomannan and GG significantly increased the water contact angle of the SPI to 74.1° and 59.0°, respectively. Therefore, the protein-polysaccharide complexes could enhance the emulsion stability. In addition, the O/W Pickering emulsions with GLO had near-Newtonian fluid rheological properties with a significant increase in apparent viscosity and viscoelasticity, forming a dual network structure consisting of a ductile and flexible protein network and a rigid and brittle polysaccharide network. The microstructure observation indicated that the O/W emulsions were spherical and homogeneous. The highest emulsification activity was observed for the SPI-GG-GLO emulsions, without significant delamination or flocculation and high oxidative stability after 7 days in storage. CONCLUSION: These results demonstrate that the construction of SPI-GG-GLO O/W Pickering emulsions can stabilize GLO even at high temperatures that promote oxidation. © 2023 Society of Chemical Industry.

Screening of an efficient cholesterol-lowering strain of Lactiplantibacillus plantarum 54-1 and investigation of its degradation molecular mechanism.

In this study, an efficient cholesterol-lowering strain of Lactiplantibacillus plantarum 54-1 was screened and its degradation molecular mechanism was investigated. Furthermore, a novel practical MRS medium for screening cholesterol-lowering lactic acid bacteria (LAB) was developed based on ultrasound treatment. L. plantarum 54-1 was found to have the highest ability to eliminate cholesterol (340.69 ± 5.87 µg/mL). According to SEM and the count of viable LAB results, the morphology of LAB in the cholesterol-containing medium developed in this experiment was close to the normal (full and smooth), and it can grow normally. Metabolomics revealed that L. plantarum 54-1 initially converted a portion of cholesterol to 7α-hydroxy-cholesterol and then to the key metabolite taurine, via the phosphotransferase system. These metabolites were further transformed into L-alanine, L-lysine, N6-Acetyl-L-lysine, (R)-b-aminoisobutyric acid, and 2-oxoarginine, through glycine, serine, and threonine metabolism, citrate cycle, D-arginine and D-ornithine metabolism, lysine degradation, and pyruvate metabolism pathways. Prokaryotic reference transcriptomics found that this may be mainly regulated by the bsh, phnE, ptsP, B0667_RS04545, and B0667_RSRS12300 genes, which was further validated by qPCR. Furthermore, molecular docking results demonstrated that 8 differential metabolites might bind to another portion of cholesterol via PI-PI conjugation and hydrophobic interactions and lower cholesterol via co-sedimentation. This study has strategic implications for developing probiotic powder food that lowers cholesterol.

Combined application of high-throughput sequencing and LC-MS-based lipidomics in the evaluation of microorganisms and lipidomics of restructured ham of different salted substitution.

The optimization of processed meats through salt replacement using KCl and k-lactate may reduce the risk of chronic diseases through reduction in dietary sodium. The objective of this study was to investigate the changes and relationships between microbial and lipid metabolism during the fermentation of restructured duck ham with different salt substitutions. Lactobacillus and Staphylococcus were found to be the dominant bacterial species in the 30 % KCl + 70 % NaCl (w/w) and 25 % k-lactate + 75 % NaCl (w/w). The LefSe analysis showed that different biomarkers were present in different ham groups, and the PLS-DA showed that triglycerides (GL) and glycerophospholipids (GP) were the two classes with the highest abundance. Besides, the KEGG pathway analysis revealed that glycerophospholipid metabolism and triglyceride metabolism were also the main metabolic pathways. According to the correlation study, Staphylococcus, Halomonas, and Lactobacillus were mostly linked to the important metabolic pathways in restructured ham. Our findings serve as a foundation for quality assurance and product enhancement for low-salt restructured ham.

Protection Mechanism of Metal Ion Pre-Stress on Lactobacillus acidophilus CICC 6074 under Acid Tolerance.

The prerequisite for the probiotic effect of lactic acid bacteria is that they could survive the acid stress environment of production and application. In this experiment, the mechanism for the effect of different metal ion pre-stress on the acid-tolerant survival of Lactobacillus was investigated. Scanning electron microscopy, Fourier infrared spectroscopy, and flow cytometry were used to analyze the condition of bacteria after acid treatment, which revealed that different metal ion pre-stress could improve the survival ability of Lactobacillus acidophilus CICC 6074 under low acid conditions by improving cell morphology, mitigating cell membrane damage, and regulating surface protein expression. Furthermore, Tandem Mass Tags (TMT) proteomic analysis revealed that Mn2+ pre-stress showed relatively more superior protective effects on acid tolerance in L. acidophilus CICC 6074 through activation of DNA replication, RNA synthesis, S-layer protein secretion, H+-ATPase enzyme activity, etc. This study will provide new ideas and a theoretical basis for the development and application of lactic acid bacteria.

Effect of lentinan on gelling properties and structural changes of goose myofibrillar protein under oxidative stress.

BACKGROUND: The reduction of protein oxidation is important for maintaining the product quality of reconstituted meat. In this study, the dose-dependent effects of lentinan (LNT) on gelling properties and chemical changes in oxidatively stressed goose myofibrillar protein were investigated. RESULTS: Myofibrillar protein (MP) with 200 µmol g-1 protein LNT increased gel strength by 87.90 ± 9.26% in comparison with LNT-free myofibrillar protein after oxidation. Scanning electron microscopy analysis revealed that the gel network containing LNT was compact, with small pores and uniform distribution. The absolute value of the zeta potential reduced significantly following oxidation of LNT with 200 µmol g-1 protein at 4 °C for 12 h compared with the zeta potential without LNT, according to the laser particle size analyzer. The incorporation of LNT increased protein solubility and -SH content, inhibited carbonyl formation, enhanced α-helix content and tryptophan intrinsic fluorescence intensity, and reduced exposure of hydrophobic groups and protein aggregation. CONCLUSION: The results indicated that adding LNT to myofibrillar protein could improve gel. This is related to its protective effect on conformational changes in the oxidation system. Lentinan is therefore recommended for oxidatively stressed goose meat processing to enhance the MP gelling potential. © 2023 Society of Chemical Industry.

Antioxidant Activity and Cell Protection of Glycosylated Products in Different Reducing Sugar Duck Liver Protein Systems.

Duck liver is an important by-product of duck food. In this study, we investigated the effects of glucose, fructose, and xylose on the antioxidant properties of glycosylated products of duck liver protein and their protective effects on HepG2 cells. The results show that the glycosylation products of the three duck liver proteins (DLP-G, DLP-F, and DLP-X) all exhibit strong antioxidant activity; among three groups, DLP-X shows the strongest ability to scavenge DPPH, ·OH free radicals, and ABTS+ free radicals. The glycosylated products of duck liver protein are not toxic to HepG2 cells and significantly increase the activity of antioxidant enzymes such as SOD, CAT, and GSH-Px in HepG2 cells at the concentration of 2.0 g/L, reducing oxidative stress damage of cells (p < 0.05). DLP-X has a better effect in reducing oxidative damage and increasing cellular activity in HepG2 cells than DLP-G and DLP-F (p < 0.05). In this study, the duck liver protein glycosylated products by glucose, fructose, and xylose were named as DLP-G, DLP-F, and DLP-X, respectively.

Characterization of the effects of binary probiotics and wolfberry dietary fiber on the quality of yogurt.

The effects of binary probiotics (Lacticaseibacillus casei CGMCC1.5956 and Lactiplantibacillus plantarum subsp. plantarum CGMCC 1.5953) in conjunction with wolfberry dietary fiber (WDF) on yogurt quality were investigated in this study. d-fructose, ß-d-glucose, 6-acetyl-d-glucose, and 1-ketose in WDF significantly improved syneresis, apparent viscosity, and elastic behavior of yogurt. Binary probiotics were more suitable for fermenting WDF yogurt than single probiotics, resulting in a higher viable count (9.39 lg (CFU/mL)) and unique flavor. Binary probiotics can promote the production of tyrosol by L. casei 56 through the tyrosine metabolic pathway, thereby enhancing the resistance of L. casei 56 and L. plantarum 53 to their environment and promoting growth. Pyridine, 2,3,4,5-tetrahydro- and prenol might be responsible for the high odor scores in the sensory evaluation of WDF yogurt prepared using binary probiotics. In summary, combining binary probiotics and WDF can significantly improve yogurt quality and add value to the final product.

Characterization of a novel flavored yogurt enriched in γ-aminobutyric acid fermented by Levilactobacillus brevis CGMCC1.5954.

This study developed and characterized a γ-aminobutyric acid (GABA)-enriched yogurt fermented by Levilactobacillus brevis CGMCC1.5954. The GABA content in the yogurt was 147.36 mg/100 mL, which was 317.06% higher than that of the control group. Furthermore, there was a significant improvement in the aroma, hardness, adhesion, cohesiveness, and gelatinousness of yogurt. The chromatography and metabolomics analyses further confirmed the high GABA content in yogurt and its nutritional value, and the metabolic pathway for GABA production by L. brevis 54 was identified. A total of 58 volatile flavor compounds were identified using headspace solid-phase microextraction-gas chromatography-mass spectrometry, of which 2-nonanone and 2-heptanone may be responsible for the high odor score of GABA-enriched yogurt. This study developed a nutritious and unique GABA-enriched flavored yogurt, summarized the metabolic pathway of GABA, and provided a flavor fingerprint that could guide the production of specifically flavored yogurts.

Interaction between Kidney-Bean Polysaccharides and Duck Myofibrillar Protein as Affected by Ultrasonication: Effects on Gel Properties and Structure.

The interaction of polysaccharides-protein with varied origins and structures provides opportunities for tailoring the physicochemical qualities of food protein-based materials. This work examined the feasibility of ultrasound-modified interaction between kidney bean dietary fiber (KSDF) and duck myofibrillar proteins (MP) to improve the physicochemical properties of the gel matrices. Accordingly, gel strength, water holding capacity, solubility, chemical interaction, secondary structure, and network structure of MP were determined. The addition of KSDF combined with the ultrasound treatment contributed to the improved water retention capability, G' values, and the reduced particle size of protein molecules, corresponding with the formation of dense pore-like structures. The results demonstrated that 1% KSDF and ultrasonication at 400 W significantly enhanced gel strength by up to 109.58% and the solubility increased by 213.42%. The proportion of α-helices of MP gels treated with 1% KSDF and ultrasonication at 400 W was significantly increased. The sonication-mediated KSDF-MP interaction significantly improved hydrophobic interactions of the proteins, thus explaining the denser network structure of the MP gels incorporated KSDF with ultrasound treatments. These results demonstrated the role of ultrasonication treatments in modifying KSDF-protein interaction to improve the gel and structural properties of the MP gels.

Isolation, identification, and characterization of corn-derived antioxidant peptides from corn fermented milk by Limosilactobacillus fermentum.

Dairy-derived peptides and corn-derived peptides have been identified as essential ingredients for health promotion in the food industry. The hydrolysis based on lactic acid bacteria (LAB) protease system is one of the most popular methods to prepare bioactive peptides. The objectives of this paper are to develop antioxidant fermented milk and to obtain natural antioxidant peptides. In our study, LAB with antioxidant capacity were screened in vitro, and the corn fermented milk with antioxidant capacity was achieved by the traditional fermentation method. Fermented milk was purified by ultrafiltration and molecular sieve, and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our findings demonstrate that Limosilactobacillus fermentum L15 had a scavenging capacity of more than 80% of DPPH radicals, Trolox equivalent antioxidant capacity (TEAC) of 0.348 ± 0.005 mmol/L. Meanwhile, the peptide content of corn fermented milk prepared with L. fermentum L15 was 0.914 ± 0.009 mg/mL and TAEC of 0.781 ± 0.020 mmol/L. Particularly important, IGGIGTVPVGR and LTTVTPGSR isolated and extracted from fermented milk were found to have antioxidant capacity for the first time. The synthetic peptides IGGIGTVPVGR and LTTVTPGSR demonstrated a scavenging capacity of 70.07 ± 2.71% and 70.07 ± 2.77% for DPPH radicals and an antioxidant capacity of 0.62 ± 0.01 mmol/L and 0.64 ± 0.02 mmol/L Trolox equivalent, respectively. This research provides ideas and basis for the development and utilization of functional dairy products.

PMA-qPCR method for the selective quantitation of viable lactic acid bacteria in fermented milk.

The number of viable lactic acid bacteria (LAB) is a key indicator of the quality of fermented milk. Currently, the combination of propidium monoazide (PMA) and qPCR has been applied in the quantification of viable bacteria in various matrices. In this research, the PMA-qPCR method was used to detect the number of viable bacteria of each LAB species in fermented milk. By analyzing pheS gene and 16S rRNA gene sequence similarities in five species of LAB, namely Lactobacillus delbrueckii subsp. bulgaricus, Lactiplantibacillus plantarum, Streptococcus thermophilus, Lactobacillus helveticus, and Lactococcus lactis subsp. lactis, the pheS gene resolved species identities better and was thus selected to design specific primers and probes. The pheS gene was cloned into the pUC19 vector and used to construct a standard curve for absolute quantification. Standard curves for quantification were constructed for each LAB species for serial dilutions between 1011 and 106 CFU/mL, with R 2 > 0.99. The number of viable bacteria in the fermented milk detected by PMA-qPCR was significantly lower than that of qPCR (P < 0.05), indicating that PMA inhibited the amplification of DNA from dead cells. This was corroborated by the results from bacterial staining and plate count experiments. The proposed PMA-qPCR method provided rapid qualitative and quantitative determination of the number of viable bacteria for each LAB species in fermented milk within 3 h.

AI-2E Family Transporter Protein in Lactobacillus acidophilus Exhibits AI-2 Exporter Activity and Relate With Intestinal Juice Resistance of the Strain.

The function of the autoinducer-2 exporters (AI-2E) family transporter protein of Lactobacillus acidophilus is still unclear. The phylogenetic analysis was used to analyze the relationship between the AI-2E protein of the L. acidophilus CICC 6074 strain and other AI-2E family members. Escherichia coli KNabc strain was used to verify whether the protein has Na+ (Li+)/H+ antiporter activity. The AI-2E protein overexpression strain was constructed by using the pMG36e expression vector, and the overexpression efficiency was determined by real-time quantitative PCR. The vitality and AI-2 activity of L. acidophilus CICC 6074 strains were determined. The results showed that the AI-2E protein of Lactobacillus formed a single branch on the phylogenetic tree and was closer to the AI-2E family members whose function was AI-2 exporter group I. The expression of AI-2E protein in the E. coli KNabc strain did not recover the resistance of the bacteria to the saline environment. Overexpression of AI-2E protein in L. acidophilus CICC 6074 could promote the AI-2 secretion of L. acidophilus CICC 6074 strain and enhance their survival ability in intestinal juice.

Preparation, Morphology and Release of Goose Liver Oil Microcapsules.

Goose liver oil (GLO) microcapsules were prepared by konjac glucomannan (KGM) and soybean protein isolate (SPI) for the first time as wall materials. The GLO could be effectively encapsulated, with an encapsulation efficiency of 83.37%, when the ratio of KGM to SPI was 2.9:1, the concentration of the KGM-SPI composite gel layer was 6.28% and the ratio of the GLO to KGM-SPI composite gel layer was 1:6. Fourier transform infrared spectroscopy and X-ray diffraction methods showed electrostatic interactions between KGM and SPI molecules and the formation of hydrogen bonds between the GLO and KGM-SPI wall components. The results of scanning electron microscopy showed a smooth spherical surface morphology of the microcapsules with a dense surface and no cracks. The confocal laser scanning microscopy showed that the microcapsules were homogeneous inside and no coalescence occurred. The encapsulated GLO has a significantly higher thermal and oxidative stability compared to free GLO. In the in vitro digestion experiment, 85.2% of the microcapsules could travel through gastric juice, and 75.2% could be released in the intestinal region. These results suggested that microcapsules prepared by KGM-SPI might be used as a carrier for the controlled release of GLO and could microencapsulate various oil-soluble nutrients in food products.

Effect of Lentinan on Lipid Oxidation and Quality Change in Goose Meatballs during Cold Storage.

The effects of different concentrations of lentinan (LNT) (0, 0.5, 1, 2 and 4%) on the oxidation characteristics and physicochemical properties of goose meatballs were investigated during different cold storage (4 °C) stages (3, 7 and 12 days). After adding LNT, the thiobarbituric acid reactive substances (TBARS) and total volatile base nitrogen (TVB-N) of goose meatballs significantly decreased compared to the LNT-free sample during cold storage, which indicated that LNT can inhibit the fat oxidation and the release of nitrogenous substances. Meanwhile, the presence of LNT makes microstructure of the goose meatball samples become denser during the whole storage time. The headspace solid phase microextraction gas chromatography-mass spectrometry (SPME-GC-MC) results showed that the proportion of aldehydes in the 4% LNT group reached 0 during storage, suggesting that high LNT concentration inhibits the formation of oxidized products in meat products. The sensory evaluation showed that the addition of LNT improved the color, appearance, flavor, and overall acceptance of goose meatballs, and the 2% LNT group had the highest score in overall acceptance. In summary, the addition of LNT could delay lipid oxidation and improve the quality of goose meatballs during cold storage.

A Novel qPCR Method for the Detection of Lactic Acid Bacteria in Fermented Milk.

The number of live lactic acid bacteria (LAB) is an important quality indicator for yogurt, the quantitative testing of LAB has become an important task in the evaluation of product quality and function. By analyzing and comparing the performance of 16S rRNA gene and tuf gene used in absolute quantification, the tuf gene with copy number 1 was selected as the target gene of six LAB. By drawing a standard curve to achieve qualitative and quantitative detection of six strains of LAB, the detection range was found to be 1 × 103-1 × 108 copies/µL. The traditional plate colony count and Flow Cytometry (FCM) were compared with the method of qPCR, which was used in this experiment. Meanwhile, the confocal laser microscope combined with STYO 9 and propidium iodide dyes was used to determine that the content of viable bacteria in the yogurt was more than 90%, which proved that the detection result using qPCR method was closer to the true level of LAB in yogurt. Compared with the existing methods, the method in this study allowed the qualitative and quantitative detection of the six kinds of LAB in yogurt, and the distribution of live and dead bacteria in yogurt could be calculated.

Novel Millet-Based Flavored Yogurt Enriched With Superoxide Dismutase.

Superoxide dismutase (SOD) is an important antioxidant enzyme with different physiological functions, which can be used as a nutritional fortifier in food. Cereal-based fermented products are becoming popular worldwide. In this study, novel millet-based flavored yogurt enriched with SOD was developed. Lactiplantibacillus plantarum subsp. plantarum was screened, which manufactured SOD activity of 2476.21 ± 1.52 U g-1. The SOD content of millet yogurt was 19.827 ± 0.323 U mL-1, which was 63.01, 50.11, and 146.79% higher than that of Bright Dairy Yogurt 1911, Junlebao and Nanjing Weigang, respectively. Fifty-four volatile flavor substances and 22,571 non-volatile flavor substances were found in yogurt. Compared to traditional fermented yogurt, 37 non-volatile metabolites in yogurt with millet enzymatic fermentation broth were significantly upregulated, including 2-phenyl ethanol, hesperidin, N-acetylornithine and L-methionine, which were upregulated by 3169.6, 228.36, 271.22, and 55.67 times, respectively, thereby enriching the sensory and nutritional value of yogurt. Moreover, the manufacture of unpleasant volatile flavor substances was masked, making the product more compatible with consumers' tastes.