Advances in food production systems and customer acceptance have led to the commercial launch of dietary proteins produced via modern biotechnological approaches as alternatives to traditional agricultural sources. At the same time, a deeper understanding of how dietary components interact with the gut microbiome has highlighted the importance of understanding the nuances underpinning diet-microbiome interactions. Novel food proteins with distinct post-translational modifications resulting from their respective production systems have not been characterized, nor how they may differ from their traditionally produced counterparts. Here, we have characterized the protein composition and N -glycome of a yeast-synthesized and commercially available whey protein ingredient and compared this novel ingredient to whey protein isolate powder derived from bovine milk. Despite strong similarities in protein composition, we found that the N -glycome significantly differs between the two protein sources, reflecting the biosynthetic machinery of the production systems. Furthermore, the diversity of proteins found in yeast-synthesized whey protein were lower relative to bovine whey protein, despite both being predominantly β-lactoglobulin. Finally, to understand whether these differences in N- glycome profiles may affect the human gut microbiome, we compared these proteins in an in vitro fecal fermentation model. The two whey protein sources generated significant differences among three representative gut microbiomes in vitro , most likely due to differences in N -glycan composition and degradation by these representative microbial communities. This work highlights the need to understand how differences in novel biotechnological systems affect the bioactivity of synthesized proteins and how these differences impact the human gut microbiome. IMPORTANCE: Recent advances in food technology have led to the production of animal-free products from yeast that are traditionally derived from animals, such as milk proteins. These new processes raise important questions about the use of synthetic proteins as a replacement for traditionally sourced protein, especially in the context of the gut microbiome. Importantly, yeast produce N -glycans comprised primarily of mannose, while animals synthesize structurally and compositionally complex N -glycan structures. Given these differences, we characterized a new, yeast-derived whey protein ingredient and compared it to bovine whey protein. We found that yeast-derived whey protein differs in its impact on human gut microbiomes because of differences in N -glycan structures, despite similarity in protein composition. These findings raise important questions as to whether these differences in synthetic proteins lead to significant changes to the gut microbiome in vivo , and whether this may impact the utility of these novel ingredients.

Snakebites cause mortality and morbidity in tropical regions. Bites by Viperidae and Elapidae family snakes are common in Turkey. Snake venom characterization and anti-venom production are important in the diagnosis and treatment of snakebites. Our objective was to develop a method for analyzing all venoms by identifying basic protein structures in venom using a MALDI-TOF-MS and to create a venom library. Five Viperidae family and one from the Elapidae family venoms, were identified. Although the proportions vary depending on the species and subspecies, the identification procedure involved determining the protein structures of PLA2, VEGF, svMP, CRISP, svSP, PIII-MP, and LAAO, common to all venoms. Six matrices (CHCA, DHAP, DHB, SA, S-DHB and THAP,) with the MALDI-TOF-MS and six snake venoms were studied separately. On the other hand, peptide profiles were studied in the mass range of 1000-10000 Da using reflectron mode. It was found that biomarkers peptides were 4398, 4026, 6395, 3800 and 6672 Da for ML, MX, VAA, VBB, VAM and WA respectively. The highest relative abundance values of the protein signals were mostly obtained with both the CHCA and the DHAP matrices. Some targeted protein structures were not observed in studies involving the S-DHB and the THAP matrices, and those were detected exhibited relatively low abundance.

Seasonal allergic rhinitis (SAR) is an inflammatory process. In this pilot study, matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS)-based analyses were conducted to investigate the effects of SAR on serum N-glycome in a small dataset (n = 10 for both SAR patients and controls). It was detected that two N-glycan compositions (H6N5E2L1 and H6N5E1L2) were down-regulated in SAR patients. Additionally, five tri-antennary N-glycan traits, including both galactosylated and sialylated (A3GS and A3GL) and non-fucosylated (A3F0GE, A3F0GL, A3F0GS) ones, were decreased. Furthermore, three high-antennary and fucosylated N-glycan traits (A3F, A3EF and A4F) were increased in SAR patients compared to controls. Graphical Abstract

Advances in food production systems and customer acceptance have led to the commercial launch of dietary proteins produced via modern biotechnological approaches as alternatives to traditional agricultural sources. At the same time, a deeper understanding of how dietary components interact with the gut microbiome has highlighted the importance of understanding the nuances underpinning diet-microbiome interactions. Novel food proteins with distinct post-translational modifications resulting from their respective production systems have not been characterized, nor how they may differ from their traditionally produced counterparts. To address this, we have characterized the protein composition and N -glycome of a yeast-synthesized whey protein ingredient isolated from commercially available ice cream and compared this novel ingredient to whey protein powder isolate derived from bovine milk. We found that despite strong similarities in protein composition, the N -glycome significantly differs between these protein sources, reflecting the biosynthetic machinery of the production systems. Further, the composition profile and diversity of proteins found in the synthetic whey protein were lower relative to bovine whey protein, despite both being predominantly composed of β-lactoglobulin. Finally, to understand whether these differences in N- glycome profiles affected the human gut microbiome, we tested these proteins in an in vitro fecal fermentation model. We found that the two whey protein sources generated significant differences among three distinct microbial compositions, which we hypothesize is a product of differences in N -glycan composition and degradation by these representative microbial communities. This work highlights the need to understand how differences in novel biotechnological systems affect the bioactivity of these proteins, and how these differences impact the human gut microbiome.

The gut microbiome evolves in response to host development, health state, lifestyle, nutrition, and microbial interactions. The survival of gut microbiota depends on its ability to utilize its host-indigestible complex oligosaccharides. Certain gut microbes produce glycosidases that cleave N-glycoproteins to release N-glycans that are then used as a carbon source. However, commercial glycosidases are inefficient and, thus, require improved deglycosylation strategies to study their functions and scale up their production. Therefore, the main objective of this study was to recombinantly produce and characterize the novel endo-β-N-acetylglucosaminidase 2 (EndoBI-2) from Bifidobacterium longum subsp. infantis (B. infantis) and to evaluate its enzymatic performance for controlled N-glycan release. Furthermore, the optimum reaction conditions for EndoBI-2 were investigated on model glycoprotein RNAse B using model glycoprotein. The released N-glycans were profiled by hydrophilic interaction liquid chromatography-fluorescence detection-quadrupole time-of-flight tandem mass spectrometry (HILIC-FLD-QTOF-MS/MS). We demonstrated that EndoBI-2 possesses a strong temperature tolerance and efficiently cleaves N-glycans under mild reaction conditions, exhibiting high activity at pH 5. These findings highlight EndoBI-2 as a robust and efficient biocatalyst for the production of bioactive N-glycans from diverse N-glycoproteins, with potential applications in glycobiotechnology.