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Öğe Biofortified Whey/Deglycosylated Whey and Chickpea Protein Matrices: Functional Enrichment by Black Mulberry Polyphenols(Springer, 2022) Özleyen, Adem; Çınar, Zeynep Özlem; Karav, Sercan; Bayraktar, Ayşe; Arslan, Ayşenur; Kayılı, H. Mehmet; Salih, Bekir; Boyuneğmez Tümer, TuğbaMorus nigra L. (black mulberry-BM) is a promising nutraceutical fruit containing biologically active polyphenols like anthocyanins, proanthocyanidins, catechins, and stilbenes, with well-established anti-inflammatory, antidiabetic, anti-obesity, and anticancer biofunctions. However, these health-promoting properties in raw fruit are greatly masked due to the presence of soluble and insoluble carbohydrates in excess amounts restricting daily intake of the required dose to achieve targeted effects. In the current study, different protein sources (defatted whey and chickpea flours) were optimized through different conditions to capture polyphenols from BM juice while diminishing its glucose content. To optimize polyphenol-protein interactions, various pHs (3.7, 4.2, and 4.7), matrix concentrations (20, 50, and 80 g protein/L), and incubation times (5, 20, and 45 min) were tested. In the present work, optimized BM polyphenol enriched whey matrix inhibited pro-inflammatory mediators and promoted Nrf-2 dependent cytoprotective enzyme expressions in lipopolysaccharide (LPS) induced macrophages at low doses. In addition, whey proteins were also subjected to an enzymatic deglycosylation process by using recently identified EndoBI-1 enzyme for the specific cleavage of N-glycan core in all glycan types including high mannoses, hybrids as well as complex glycans found on defatted whey proteins. After this process, the polyphenol sorption capacity of deglycosylated whey proteins was found to be significantly higher (37%) than the capacity of non-treated normal whey protein under optimized conditions. In conclusion, deglycosylation of protein matrices could be a novel strategy for efficient sorption/concentration of polyphenols from fruits and vegetables, however, more detailed studies are needed to understand this effect.Öğe Bovine Colostrum and Its Potential for Human Health and Nutrition(Frontiers Media S.A., 2021) Arslan, Ayşenur; Kaplan, Merve; Duman, Hatice; Bayraktar, Ayşe; Karav, Sercan; Ertürk, Melih; Henrick, Bethany M.; Frese, Steven A.Colostrum is the first milk produced post-partum by mammals and is compositionally distinct from mature milk. Bovine colostrum has a long history of consumption by humans, and there have been a number of studies investigating its potential for applications in human nutrition and health. Extensive characterization of the constituent fractions has identified a wealth of potentially bioactive molecules, their potential for shaping neonatal development, and the potential for their application beyond the neonatal period. Proteins, fats, glycans, minerals, and vitamins are abundant in colostrum, and advances in dairy processing technologies have enabled the advancement of bovine colostrum from relative limitations of a fresh and unprocessed food to a variety of potential applications. In these forms, clinical studies have examined bovine colostrum as having the substantial potential to improve human health. This review discusses the macro-and micronutrient composition of colostrum as well as describing well-characterized bioactives found in bovine colostrum and their potential for human health. Current gaps in knowledge are also identified and future directions are considered in order to elevate the potential for bovine colostrum as a component of a healthy diet for a variety of relevant human populations.Öğe Determining therapeutic proteins and prebiotic N-glycan concentrations in bovine colostrum(Çanakkale Onsekiz Mart Üniversitesi, Lisansüstü Eğitim Enstitüsü, 2022) Arslan, Ayşenur; Karav, SercanSığır kolostrumu, doğumdan hemen sonra meme bezlerinden salgılanan süttür ve immünoglobulin G (IgG), laktoferrin, oligosakkaritler, büyüme faktörleri ve temel besinler gibi zengin immünolojik olarak aktif bileşenler içermektedir. IgG ve laktoferrin, sığır kolostrumunda N-glikan kaynağı olan terapötik proteinlerdir. Biyoaktif N-glikanlar, faydalı mikroorganizmaların büyümesini teşvik etmek için prebiyotik maddeler olarak etki sağlamak gibi önemli biyolojik aktivitelere sahiptir. Bu tezin amacı, sığır kolostrumunda bulunan toplam protein, IgG ve laktoferrin gibi terapötik proteinler ve N-glikan konsantrasyonunun kolostrumdan olgun süte geçişte nasıl değiştiğini incelemektir. Bu amaçla, ULUOVA Süt Ticaret A.Ş.'den 28 Holstein inekten doğumdan sonraki 6 gün içinde sığır kolostrum örnekleri toplandı ve örnekler her örneğin günlerine göre havuzlandı. Daha sonra bu örneklerden toplam proteinler izole edildi. Numunelerin toplam protein konsantrasyonu, BCA yöntemi ile belirlendi ve proteinlerin görüntülenmesi SDS-PAGE ile gerçekleştirildi. IgG ve laktoferrin konsantrasyonu, ELISA testi kullanılarak tespit edildi. N-glikanlar, yeni N-glikosidazlar kullanılarak glikoproteinlerden salındı ve fenol-sülfürik asit yöntemi ile kantifiye edildi. Bu çalışmanın sonucunda erken kolostrumun en yüksek protein, laktoferrin, IgG ve N-glikan konsantrasyonuna sahip olduğu ve sonraki 3 gün boyunca konsantrasyonlarında azalma olduğu saptanmıştır.Öğe Determining Total Protein and Bioactive Protein Concentrations in Bovine Colostrum(Journal of Visualized Experiments, 2021) Arslan, Ayşenur; Duman, Hatice; Kaplan, Merve; Uzkuç, Hasan; Bayraktar, Ayşe; Erturk, Melih; Alkan, Merve; Karav, SercanColostrum is a complex biological fluid produced by mammals immediately after parturition. It meets all the nutritional requirements for neonates as a good source of macro-and micronutrients, bioactive peptides, and growth factors. Bovine colostrum is also a potential source of nutrition and bioactive because of its rich protein content that includes immunoglobulin G (IgG) and lactoferrin. However, the level of lactoferrin and IgG in bovine colostrum changes markedly during the lactation period. Therefore, monitoring the concentration of IgG and lactoferrin for the use of bovine colostrum as a protein source is an important question to study. Methods in this article describe how to determine protein content, as well as specific concentrations of lactoferrin and IgG. These methods include the following steps: Isolation of bovine colostrum proteins, Determination of protein concentration via Bicinchoninic acid assay (BCA), Visualization of proteins via SDS-PAGE, Determination of lactoferrin, and IgG concentration using an ELISA Assay.Öğe Potential Applications of Endo-beta-N-Acetylglucosaminidases From Bifidobacterium longum Subspecies infantis in Designing Value-Added, Next-Generation Infant Formulas(Frontiers Media S.A., 2021) Duman, Hatice; Kaplan, Merve; Arslan, Ayşenur; Şahutoglu, Arif Sercan; Karav, Sercan; Kayılı, Hacı Mehmet; Frese, Steven A.Human milk is the optimal source of infant nutrition. Among many other health benefits, human milk can stimulate the development of a Bifidobacterium-rich microbiome through human milk oligosaccharides (HMOs). In recent years, the development of novel formulas has placed particular focus on incorporating some of the beneficial functional properties of human milk. These include adding specific glycans aimed to selectively stimulate the growth of Bifidobacterium. However, the bifidogenicity of human milk remains unparalleled. Dietary N-glycans are carbohydrate structures conjugated to a wide variety of glycoproteins. These glycans have a remarkable structural similarity to HMOs and, when released, show a strong bifidogenic effect. This review discusses the biocatalytic potential of the endo-β-N-acetylglucosaminidase enzyme (EndoBI-1) from Bifidobacterium longum subspecies infantis (B. infantis), in releasing N-glycans inherently present in infant formula as means to increase the bifidogenicity of infant formula. Finally, the potential implications for protein deglycosylation with EndoBI-1 in the development of value added, next-generation formulas are discussed from a technical perspective.Öğe Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release(Journal of Visualized Experiments, 2021) Sucu, Berfin; Bayraktar, Ayşe; Duman, Hatice; Arslan, Ayşenur; Kaplan, Merve; Karyelioğlu, Melda; Ntelitze, Eda; Taştekin, Taner; Yetkin, Seray; Karav, SercanProtein glycosylation is a diverse and common post-translational modification that has been associated with many important roles such as protein function, including protein folding, stability, enzymatic protection, and biological recognition. N-glycans attached to glycoproteins (such as lactoferrin, lactadherin, and immunoglobulins) cannot be digested by the host and reach the large intestine, where they are consumed by certain beneficial microbes. Therefore, they are considered next-generation prebiotic compounds that can selectively stimulate the gut microbiome's beneficial microorganisms. However, the isolation of these new classes of prebiotics requires novel enzymes. Here, we describe the recombinant production of novel glycosidases from different Bifidobacteria strains (isolated from infants, rabbits, chicken, and bumblebee) for improved N-glycan isolation from glycoproteins. The method presented in this study includes the following steps: molecular cloning of Bifidobacterial genes by an in vivo recombinational cloning strategy, control of transformation success, protein induction, and protein purification.Öğe Release of bifidogenic N-glycans from native bovine colostrum proteins by an endo-?-N-acetylglucosaminidase(Elsevier Inc., 2023) Bunyatratchata, Apichaya; Parc, Annabelle Le; de Moura Bell, Juliana Maria Leite Nobrega; Cohen, Josh L.; Duman, Hatice; Arslan, Ayşenur; Kaplan, Merve; Barile, Daniela; Karav, SercanMilk glycoproteins play various biological roles including antibacterial, antiviral activities, modulating immune responses in living organisms. Released N-glycans from milk glycoproteins act as growth substrates for infant-associated bifidobacteria, which are key members of the breastfed infant's gut. To date, the mechanisms, and contributions of glycans to the biological activities of glycoproteins remain to be elucidated. Only by testing both the released glycans and the deglycosylated protein in their native (i.e., non-denatured) form, can the individual contribution to the biological activity of glycoproteins be elucidated. However, for conventional enzymatic and chemical deglycosylation strategies to work efficiently, glycoprotein denaturation is required, which alters the protein native shape, hindering further investigations of its biological roles. An endo-β-N-acetylglucosaminidase (EndoBI-1) from Bifidobacterium longum subsp. infantis ATCC 15697 (B. infantis) was characterized as having the ability to release N-glycans from bovine milk glycoproteins efficiently, without the denaturation. In this study, the activity of EndoBI-1 was compared to a commercial enzyme to release N-glycans, the peptide-N-glycosidase F (PNGase F), using dairy glycoproteins as the substrate. The kinetic evaluation showed that EndoBI-1 displayed higher activity on native glycoproteins than PNGase F, with 0.036 mg/mL×min and 0.012 mg/mL×min glycan release, respectively. EndoBI-1 released a broader array of glycan structures compared to PNGase F from native glycoproteins. Thirty-two and fifteen distinct compositions were released from the native glycoproteins by EndoBI-1 and PNGase F, respectively, as characterized by advanced mass spectrometry. EndoBI-1 can be considered a promising enzyme for the release of N-glycans and their protein backbone in the native form, which will enable effective glycan release and will facilitate subsequent investigations to reveal their contribution to glycoproteins’ biological roles.
