Tailored chitosan integration in diatomaceous earth particles as a scaffold for fructosyltransferase immobilization in fructo-oligosaccharide production.

BACKGROUND: Fructo-oligosaccharide (FOS) belongs to the group of short inulin-type fructans and is one of the most important non-digestible bifid-oligosaccharides capable of biotransforming sucrose using fructosyltransferase (FTase). However, there are no immobilized FTase products that can be successfully used industrially. In this study, diatomite was subjected to extrusion, sintering and granulation to form diatomaceous earth particles that were further modified via chitosan aminomethylation for modification. FTase derived from Aspergillus oryzae was successfully immobilized on the modified support via covalent binding. RESULTS: The immobilized enzyme activity was 503 IU g-1 at an enzyme concentration of 0.6 mg mL-1, immobilization pH of 7.0 and contact time of 3 h. Additionally, the immobilization yield was 56.91%. Notably, the immobilized enzyme was more stable under acidic conditions. Moreover, the half-life of the immobilized enzyme was 20.80 and 10.96 times as long as that of the free enzyme at 45 and 60 °C, respectively. The results show good reusability, as evidenced by the 84.77% retention of original enzyme activity after eight cycles. Additionally, the column transit time of the substrate was 35.56 min when the immobilized enzyme was applied in a packed-bed reactor. Furthermore, a consistently high FOS production yield of 60.68% was achieved and maintained over the 15-day monitoring period. CONCLUSIONS: Our results suggest that immobilized FTase is a viable candidate for continuous FOS production on an industrial scale. © 2024 Society of Chemical Industry.

Enhancing enzyme immobilization: Fabrication of biosilica-based organic-inorganic composite carriers for efficient covalent binding of D-allulose 3-epimerase.

D-allulose, an ideal low-calorie sweetener, is primarily produced through the isomerization of d-fructose using D-allulose 3-epimerase (DAE; EC 5.1.3.30). Addressing the gap in available immobilized DAE enzymes for scalable commercial D-allulose production, three core-shell structured organic-inorganic composite silica-based carriers were designed for efficient covalent immobilization of DAE. Natural inorganic diatomite was used as the core, while 3-aminopropyltriethoxysilane (APTES), polyethyleneimine (PEI), and chitosan organic layers were coated as the shells, respectively. These tailored carriers successfully formed robust covalent bonds with DAE enzyme conjugates, cross-linked via glutaraldehyde, and demonstrated enzyme activities of 372 U/g, 1198 U/g, and 381 U/g, respectively. These immobilized enzymes exhibited an expanded pH tolerance and improved thermal stability compared to free DAE. Particularly, the modified diatomite with PEI exhibited a higher density of binding sites than the other carriers and the PEI-coated immobilized DAE enzyme retained 70.4 % of its relative enzyme activity after ten cycles of reuse. This study provides a promising method for DAE immobilization, underscoring the potential of using biosilica-based organic-inorganic composite carriers for the development of robust enzyme systems, thereby advancing the production of value-added food ingredients like D-allulose.

Calcium-binding properties, stability, and osteogenic ability of phosphorylated soy peptide-calcium chelate.

Introduction: Bioactive peptides based on foodstuffs are of particular interest as carriers for calcium delivery due to their safety and high activity. The phosphorylated peptide has been shown to enhance calcium absorption and bone formation. Method: A novel complex of peptide phosphorylation modification derived from soybean protein was introduced, and the mechanism, stability, and osteogenic differentiation bioactivity of the peptide with or without calcium were studied. Result: The calcium-binding capacity of phosphorylated soy peptide (SPP) reached 50.24 ± 0.20 mg/g. The result of computer stimulation and vibration spectrum showed that SPP could chelate with calcium by the phosphoric acid group, carboxyl oxygen of C-terminal Glu, Asp, and Arg, and phosphoric acid group of Ser on the SPP at a stoichiometric ratio of 1:1, resulting in the formation of the complex of ligand and peptide. Thermal stability showed that chelation enhanced peptide stability compared with SPP alone. Additionally, in vitro results showed that SPP-Ca could facilitate osteogenic proliferation and differentiation ability. Discussion: SPP may function as a promising alternative to current therapeutic agents for bone loss.

Isolation, characterization, and molecular docking analyses of novel calcium-chelating peptide from soy yogurt and the study of its calcium chelation mechanism.

BACKGROUND: Calcium is an essential dietary mineral nutrient for humans. Digestive instability limits the bioavailability of calcium ions. Peptide-calcium chelate has been proven to excite higher calcium absorption than amino acid-calcium chelate, organic and inorganic calcium. Soy yogurt, which is produced via liquid-state fermentation using lactic acid bacteria, has a high amount of bioavailable calcium. In this study, a novel peptide with high calcium binding affinity was purified and identified from soy yogurt. The binding mechanism of peptide and calcium was then analyzed by bioinformatics and spectral analysis. Furthermore, the effect of the novel peptide on gastrointestinal stability by the Caco-2 cell model and calcium bioavailability in vivo were investigated by the zebrafish model. RESULTS: The results showed that a novel peptide was purified and identified as DEDEQIPSHPPR (CBP). Calcium ions probably coordinate with Glu-2 and Glu-4 carboxyl groups via salt bridges and interact with Asp-1, Asp-3, and Arg-12 in CBP via charge pairing. The calcium binding activity of the CBP was 36.64 ± 0.04 mg g-1 . Fourier transform infrared (FTIR) spectra showed that calcium spontaneously bound to the amino group nitrogen and oxygen atoms of the carboxyl group. The binding mode is either bidentate or unidentate, depending on the circumstances. More importantly, the CBP peptide substantially increased the bone mass in a zebrafish osteoporosis model. CONCLUSION: The more glutamic acid and aspartic acid, the high was the calcium affinity with peptide. Soy yogurt-derived peptides can be used as carriers of calcium ions throughout the gastrointestinal tract, which may be clinically useful for osteoporosis therapy. © 2022 Society of Chemical Industry.

Physicochemical, Textural, and Sensorial Properties of Soy Yogurt as Affected by Addition of Low Acyl Gellan Gum.

Soy yogurt is plant-based dairy of great nutritional interest that is widely accepted in developing countries as a milk alternative. Poor stability has been an urgent problem to solve of soy yogurt products over past several years. The present study aimed to construct multiple network composite gel by adding low acyl gellan gum (LAG) to improve the stability. The effect of addition of LAG on property of soy yogurt was investigated by determining water holding capacity, texture, rheology, particle size, and zeta potential. The results showed that water holding capacity was significantly higher than control. The soy yogurt with 0.1% LAG had a stable gel network with much gel strength and viscosity, and strengthened interaction between complex gel. The addition of LAG increased the particle size and decreased zeta potential. Furthermore, sensory properties were acceptable. Therefore, during industrial production, LAG could act as an appropriate stabilizer to inhibit poor body and bring more desirable sensory characteristics of soy yogurt.

Calcium Binding Mechanism of Soybean Peptide with Histidine Alteration by Molecular Docking Analysis and Spectroscopic Methods.

Histidine (His) carries a unique heteroaromatic imidazole side chain and plays an irreplaceable role in peptides and proteins. With the current study, we aimed to determine the characteristics and functional activities of the bone density of soy peptide-calcium complexes in which a His residue was replaced by Leu (CBP-H). Soybean peptide (CBP-H) was chemically synthesized, the binding mechanism between CBP-H and calcium ions in combination was determined with bioinformatics and spectroscopy analysis, and the difference between CBP and CBP-H was investigated. Finally, we analyzed the effect of CBP and CBP-H on osteoblasts in vitro. The results showed that CBP-H could bind to calcium ions, and the calcium coordinated with the carboxyl groups of Asp and Glu in the peptide. The nitrogen atoms of the amino group and the oxygen atoms of the carboxyl group in CBP-H significantly contributed to the coordination with Ca2+. Furthermore, the binding capacity was 36.48 ± 0.09 mg/g, similar to CBP. However, both CBP and CBP-H could promote osteogenic activity, the activity of CBP-H was 127.147%, lower than CBP (121.777%). While it had the same ability to promote intracellular calcium concentration, CBP-H could upregulate 150.12% calcium ions into the intracellular, and the rate of the rise of CBP was 158.91%, further highlighting the potential of His residues for binding calcium and treating osteoporosis.