Lactobacillus casei-Derived Postbiotics Elevate the Bioaccessibility of Proteins via Allosteric Regulation of Pepsin and Trypsin and Introduction of Endopeptidases.

The potential of probiotics to benefit digestion has been widely reported, while its utilization in high-risk patients and potential adverse reactions have focused interest on postbiotics. A variable data-independent acquisition (vDIA)-based spatial-omics strategy integrated with unsupervised variational autoencoders was applied to profile the functional mechanism underlying the action of Lactobacillus casei-derived postbiotic supplementation in goat milk digestion in an infant digestive system, from a metabolomics-peptidomics-proteomics perspective. Amide and olefin derivatives were proved to elevate the activities of pepsin and trypsin through hydrogen bonding and hydrophobic forces based on allosteric effects, and recognition of nine endopeptidases and their cleavage to serine, proline, and aspartate were introduced by postbiotics, thereby promoting the generation of hydrophilic peptides and elevating the bioaccessibility of goat milk protein. The peptides originating from αs1-casein, ß-casein, ß-lactoglobulin, Ig-like domain-containing protein, κ-casein, and serum amyloid A protein, with multiple bioactivities including angiotensin I-converting enzyme (ACE)-inhibitory, osteoanabolic, dipeptidyl peptidase IV (DPP-IV) inhibitory, antimicrobial, bradykinin-potentiating, antioxidant, and anti-inflammatory activities, were significantly increased in the postbiotic supplementation group, which was also considered to potentially prevent necrotizing enterocolitis through inhibiting the multiplication of pathogenic bacteria and blocking signal transducer and activator of transcription 1 and nuclear factor kappa-light-chain-enhancer of activated B cells inflammatory pathways. This research deepened the understanding of the mechanism underlying the postbiotics affecting goat milk digestion, which established a critical groundwork for the clinical application of postbiotics in infant complementary foods.

Goat milk-derived short chain peptides: Peptide LPYV as species-specific characteristic and their versatility bioactivities by MOF@Fe3O4@GO mesoporous magnetic-based peptidomics.

Goat milk as an ideal substitute for human milk has not been sufficiently explored. An in-situ synthesized MOF@Fe3O4@GO was demonstrated as a magnetic mesoporous adsorbent for efficiently enriching short chain peptides (SCPs) in milk compared with the routine solid phase extraction approach with graphite carbon black or C18 as the packing material in terms of the number of enriched SCPs and data stability. A total of 61 and 126 SCPs were identified and quantified in bovine milk (0.09-89.34 µg L-1) and goat milk (10.5-1267.06 µg L-1), respectively, and peptide LPYV can be used as a potential marker for adulteration of goat milk. Relative high expression of chymotrypsin and pepsin by EnzymePredictor analysis could partially elaborate the reason of the abundance of SCPs in goat milk. Compared with bovine milk, further bioinformatics analysis indicated that goat milk could own higher nutritional value because of relative higher concentrations (>1 mg/L) of SCPs (LLV, FL, LVYP) with confirmed bioactivities including angiotensin-converting enzyme (ACE) inhibitor, antioxidant, dipeptidylpeptidase (DPP) III and DPP IV inhibitor, etc. Overall, this study opened a novel avenue for understanding versatility benefit of dairy products from a perspective of SCPs by using a developed MOF@Fe3O4@GO mesoporous magnetic-based peptidomics.

Pseudo-targeted metabolomics analysis of the therapeutic effect of phenolics-rich extract from Se-enriched green tea (Camellia sinensis) on LPS-stimulated murine macrophage (RAW264.7).

Selenium (Se)-enriched green tea (Se-Te) has been recognized as a possible source of Se supplements, while the effect of Se enrichment on function of polyphenols in green tea is still unclear. In this study, a pseudo-targeted metabolomics strategy was carried out to reveal the regulatory mechanism of polyphenols extracted from Se-Te and regular green tea (Re-Te) on inflammatory response at cellular level. A novel analysis strategy using UHPLC/ESI Q-Orbitrap combined with MS-IOP was applied to profile the dynamic changes of metabolites in LPS-stimulated RAW264.7 macrophages during polyphenols incubation. A total of 128 characteristic variables (VIP > 1, p < 0.05) were screened in Se-Te group and the results of bioinformatics analysis and quantitative research indicated that in addition to the 6 conventional immune protective pathways involved in tea polyphenols, Se-enriched polyphenols were also participated in 3 unique antioxidant enzyme activation pathways, including phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism and pantothenate and CoA biosynthesis. The result of weight calculation based on topological analysis indicated that the promoting synthesis of antioxidant enzymes was the main mechanism of Se-Te polyphenols to inhibit inflammation. However, compared with Re-Te group, the intracellular B vitamin pathway in Se-Te group was disturbed, which is related to the fact that Se supplementation can promote the synthesis of selenoprotein and catalyze the reduction of thioredoxin by NADPH, thus blocking the signaling pathways of B vitamins. This study comprehensively explored the immune protective mechanism of polyphenols extracted from Se-Te and Re-Te under natural growth conditions, which could give a better understanding of the potential nutritional value of Se-Te as a widely used Se supplement.

Effects of Short-Chain Peptides on the Flavor Profile of Baijiu by the Density Functional Theory: Peptidomics, Sensomics, Flavor Reconstitution, and Sensory Evaluation.

The effect of peptides on the flavor profile of Baijiu is unclear as a result of their trace concentrations in the complex matrix, and therefore, the study involving the interaction mechanism between peptides and flavor compounds is limited. In this study, short-chain peptides (amino acid number between 2 and 4, SCPs) associated with the Feng-flavor Baijiu (FFB) were comprehensively analyzed by a dedicated workflow using ultra-high-performance liquid chromatography Q Orbitrap high-resolution mass spectrometry, flavor reconstitution experiments, sensory analysis, and density functional theory (DFT) analysis. The concentrations of 96 SCPs intimately related with six different grades of honey aroma intensity in FFB were quantified (0.12-155.01 µg L-1) after multivariable analysis, Spearman's correlation analysis (ρ ≥ 0.7), and confirmation with synthetic standards, and 32 dominant odorants with an odor activity value of ≥1 in FFB with the highest intensity of honey aroma were quantified by gas chromatography-mass spectrometry and gas chromatography-flame ionization detection analyses. The results of flavor reconstitution experiments and sensory analysis indicated that the SCPs can obviously influence the honey aroma with amplifying the fruity, sweet, and flora flavor odor characters (p < 0.05) while significantly reducing the acidic character (p < 0.001), which could be attributed to the most stable complex structure between SCPs and odor-active compounds calculated by DFT being butanoic acid, followed by ß-damascenone, 3-methylbutanal, and ethyl hexanoate, and the multiple sites as a hydrogen bond donor or acceptor in SCPs can form a stable ternary structure with water and ethanol inside the peptide chain or carboxyl terminal of SCPs, consequently improving the stability of the Baijiu system. The results highlighted the important role of SCPs on the volatiles in Baijiu and laid the foundation for further facilitating the sensory quality of Baijiu products.

Novel insight into the transformation of peptides and potential benefits in brown fermented goat milk by mesoporous magnetic dispersive solid phase extraction-based peptidomics.

Brown fermented goat milk as an excellent source of bioactive peptides has only been partially elucidated. Meticulously synthesized MOF@MG as magnetic sorbent for enriching endogenous peptides owned higher reproducibility and uniform distribution of peptides PI compared with ultrafiltration. Combined with UHPLC-Q-Orbitrap, fermentation for 12 h in brown goat milk with the highest overall acceptable degree through sensory evaluation was utilized to explore the transformation of peptides and health benefits, with trypsin or plasmin hydrolyzing proteins and aminopeptidase or carboxypeptidase hydrolyzing peptides to small peptides or amino acids. A total of 1317 peptides were identified by database matching (1259) and de novo sequencing (58), among 18 peptides could originate from gene-independent enzymatic formation and top 25 characteristic peptides were quantified with concentration ranging from 0.12 to 6.40 mg L-1. Bioinformatic analysis results indicated that brown fermented goat milk possesses higher health benefits because of more than 50 peptides with potential bioactivity.

Molecular mechanism of Mare Nectaris and magnetic field on the formation of ethyl carbamate during 19 years aging of Feng-flavor Baijiu.

Ethyl carbamate (EC) is carcinogen occurring naturally in fermented foods, while the EC formation pattern in Feng-flavor Baijiu during Mare Nectaris storage and magnetic field treatment remains controversial. In this work, variation of EC in Mare Nectaris and magnetic field were investigated for the first time through ultra high performance liquid chromatography quadrupole-orbitrap high resolution mass spectrometry (UHPLC-Q-Orbitrap). Quantification results revealed that EC decreased significantly in the stage of 3-9 years and kept at 12.4 µg L-1 after 10 years of aging. Arginine succinate synthase (ASS) and urease were deemed as vital factors for EC decomposition. Degradation effetc of EC in 250 mT is simillar to that of EC in Baijiu stored in Mare Nectaris for 8 years. This is due to that aging process was accelerated by magnetic field and the content of total acid in Baijiu was increased, creating a favorable environment for decomposition of EC and urea.

Physicochemical and molecular transformation of novel functional peptides from Baijiu.

A novel strategy for screening and identifying peptides present in Baijiu was developed based on magnetic solid-phase extraction with magnetic S-doped graphene (M-G-S) as adsorbent combined with ultrahigh-performance liquid chromatography with high resolution tandem mass spectrometry. In total, 28 peptides consisting of amino acids from 3 to 9 were preliminarily identified, and significantly higher in the number than that of direct concentration and SPE with C18 as the adsorbent. Six peptides were confirmed with their corresponding synthetic reference standards by comparing their retention time, high resolution MS/MS spectra, and NMR spectroscopic studies. Parallel reaction monitoring integrated with the internal standard method was utilized to quantify identified peptides with concentrations ranging from 1.14 to 10.25 ng mL-1, and prediction results of bioactivity comprising antioxidation or ACE inhibitors were obtained. These discoveries were conducive to understanding the versatility benefit of Baijiu and paved the way to study the interaction between peptides and volatile substances.

High-Coverage Screening of Sulfonamide Metabolites in Goat Milk by Magnetic Doped S Graphene Combined with Ultrahigh-Performance Liquid Chromatography-High-Resolution Mass Spectrometry.

Currently, there are more than 1000 varieties of synthetic sulfonamides universally used as antibiotics causing severe results of potential carcinogenicity and drug resistance for human health due to excessive residue of animal-derived food. A facile and novel approach for untargeted screening of sulfonamides (SAs) and metabolites was proposed based on magnetic solid-phase extraction-ultrahigh-performance liquid chromatography-tandem high-resolution mass spectrometry (MSPE-UHPLC-HRMS). Compared with QuEChERS without the clean-up procedure and SPE in terms of matrix effect and absolute recovery, magnetic doped S graphene (S-doping level: 2.82%) synthesized via a solid-state microwave approach and the aggregation wrap mechanism was used as a novel adsorbent for nonspecific extraction of desired analytes by the noncovalent interaction between electron-deficient thiophene sulfur and electron donors such as amino or amide as well as π-π stacking interactions. Combined with variable data-independent acquisition, characteristic fragment-ion filtering (m/z 156.01138 or m/z 108.04439) and assignment of extracted-ion chromatograms of marked fragment ions were successfully utilized to screen the desired analytes and subsequently confirmed with the availability of reference standards. The optimized and validated approach for spiked 26 SAs and 9 metabolites in control goat milk demonstrated satisfactory accuracy (80.1-112.6%) and precision (RSDs < 6.4%) for matrix-matched standard addition. After applying suspect goat milk samples, untargeted SA analytes including sulfanilamide or an N4-acetylsulfamethazine metabolite with concentration ranging from 66.3 to 398.5 ng L-1 were determined in 5 of 45 goat milk samples.

Foodomics analysis of natural aging and gamma irradiation maturation in Chinese distilled Baijiu by UPLC-Orbitrap-MS/MS.

Classification of Feng-flavor Baijiu considering aging category was furnished with a comprehensive fingerprinting strategy which used UPLC-Orbitrap and foodomics, and the most discriminant 29 compounds related to aging, 15 organic acids, 8 esters as well as some carbonyl compounds were discovered. Increase of aromatic organic acids, decline of carcinogenic dibutyl phthalate and generation of numerous aromatic substances in Baijiu were caused by irradiation until the system reached a relatively stable state which needed 28 days. It is similarity of physical and chemical reaction process of natural aging and irradiation maturation that both can facilitate flavor of base Baijiu, while the aging time can be plainly shorten utilizing irradiation for Baijiu, whose outcome has been demonstrated by LC-MS and sensory evaluation. The combination of UPLC-Orbitrap and foodomics was applied as a valid tactic to analyze a complex system and gamma irradiation can be a powerful tool to promote Baijiu aging.