Insights into the Structure-Function Relationship of GH70 GtfB α-Glucanotransferases from the Crystal Structure and Molecular Dynamic Simulation of a Newly Characterized Limosilactobacillus reuteri N1 GtfB Enzyme.

α-Glucanotransferases of the CAZy family GH70 convert starch-derived donors to industrially important α-glucans. Here, we describe characteristics of a novel GtfB-type 4,6-α-glucanotransferase of high enzyme activity (60.8 U mg-1) from Limosilactobacillus reuteri N1 (LrN1 GtfB), which produces surprisingly large quantities of soluble protein in heterologous expression (173 mg pure protein per L of culture) and synthesizes the reuteran-like α-glucan with (α1 → 6) linkages in linear chains and branch points. Protein structural analysis of LrN1 GtfB revealed the potential crucial residues at subsites -2∼+2, particularly H265, Y214, and R302, in the active center as well as previously unidentified surface binding sites. Furthermore, molecular dynamic simulations have provided unprecedented insights into linkage specificity hallmarks of the enzyme. Therefore, LrN1 GtfB represents a potent enzymatic tool for starch conversion, and this study promotes our knowledge on the structure-function relationship of GH70 GtfB α-glucanotransferases, which might facilitate the production of tailored α-glucans by enzyme engineering in future.

Effects of crosslinking agents on properties of starch-based intelligent labels for food freshness detection.

The impact of citric acid, carboxymethyl cellulose, carboxymethyl starch, sodium trimetaphosphate, or soybean protein on the crosslinking of starch-based films was examined. These crosslinking starch films were then used to create pH-sensitive food labels using a casting method. Blueberry anthocyanins were incorporated into these smart labels as a pH-sensitive colorimetric sensor. The mechanical properties, moisture resistance, and pH responsiveness of these smart labels were then examined. Crosslinking improved the mechanical properties and pH sensitivity of the labels. These different crosslinking agents also affected the hydrophobicity of the labels to varying degrees. Soybean protein was the only additive that led to labels that could sustain their structural integrity after immersion in water for 12 h. Because it increased the hydrophobicity of the labels, which decreased their water vapor permeability, moisture content, swelling index, and water solubility by 47 %, 29 %, 52 % and 10 %, respectively. The potential of using these labels to monitor the freshness of chicken breast was then examined. Only the films containing soybean protein exhibited good pH sensitivity, high structural stability, and low pigment leakage. This combination of beneficial attributes suggests that the composite films containing starch and soybean protein may be most suitable for monitoring meat freshness.

Health benefits, mechanisms of interaction with food components, and delivery of tea polyphenols: a review.

Tea polyphenols (TPs) are the most important active component of tea and have become a research focus among natural products, thanks to their antioxidant, lipid-lowering, liver-protecting, anti-tumor, and other biological activities. Polyphenols can interact with other food components, such as protein, polysaccharides, lipids, and metal ions to further improve the texture, flavor, and sensory quality of food, and are widely used in food fields, such as food preservatives, antibacterial agents and food packaging. However, the instability of TPs under conditions such as light or heat and their low bioavailability in the gastrointestinal environment also hinder their application in food. In this review, we summarized the health benefits of TPs. In order to better use TPs in food, we analyzed the form and mechanism of interaction between TPs and main food components, such as polysaccharides and proteins. Moreover, we reviewed research into optimizing the applications of TPs in food by bio-based delivery systems, such as liposomes, nanoemulsions, and nanoparticles, so as to improve the stability and bioactivity of TPs in food application. As an effective active ingredient, TPs have great potential to be applied in functional food to produce benefits for human health.

Encapsulation of polyphenols in protein-based nanoparticles: Preparation, properties, and applications.

Polyphenols have a variety of physiological activities, including antioxidant, antimicrobial, and anti-inflammatory properties. However, their applications are often limited because due to the instability of polyphenols. Encapsulation technologies can be employed to overcome these problems and increase the utilization of polyphenols. In this article, the utilization of protein-based nanoparticles for encapsulating polyphenols is reviewed due to their good biocompatibility, biodegradability, and functional attributes. Initially, the various kinds of animal and plant proteins available for forming protein nanoparticles are discussed, as well as the fabrication methods that can be used to assemble these nanoparticles. The molecular interaction mechanisms between proteins and polyphenols are then summarized. Applications of protein-based nanoparticles for encapsulating polyphenols are then discussed, including as nutrient delivery systems, in food packaging materials, and in the creation of functional foods. Finally, areas where further research is need on the development, characterization, and application of protein-based polyphenol-loaded nanoparticles are highlighted.

Progress on molecular modification and functional applications of anthocyanins.

Anthocyanins have attracted a lot of attention in the fields of natural pigments, food packaging, and functional foods due to their color, antioxidant, and nutraceutical properties. However, the poor chemical stability and low bioavailability of anthocyanins currently limit their application in the food industry. Various methods can be used to modify the structure of anthocyanins and thus improve their stability and bioavailability characteristics under food processing, storage, and gastrointestinal conditions. This paper aims to review in vitro modification methods for altering the molecular structure of anthocyanins, as well as their resulting improved properties such as color, stability, solubility, and antioxidant properties, and functional applications as pigments, sensors and functional foods. In industrial production, by mixing co-pigments with anthocyanins in food systems, the color and stability of anthocyanins can be improved by using non-covalent co-pigmentation. By acylation of fatty acids and aromatic acids with anthocyanins before incorporation into food systems, the surface activity of anthocyanins can be activated and their antioxidant and bioactivity can be improved. Various other chemical modification methods, such as methylation, glycosylation, and the formation of pyranoanthocyanins, can also be utilized to tailor the molecular properties of anthocyanins expanding their range of applications in the food industry.

The Distribution of Selected Toxic Elements in Sauced Chicken during Their Feeding, Processing, and Storage Stages.

Sauced chicken is popular food worldwide. However, the elemental pollution of poultry industrialization has led to an increasing health risk concern. In this study, four typical toxic elements, including chromium (Cr), arsenic (As), lead (Pb), and cadmium (Cd), were selected and detected in whole industry chains of sauced chicken preparation by inductively coupled plasma-mass spectrometry. The detection method was optimized and verified with an average recovery of 93.96% to 107.0%. Cr has the highest proportion among the elements during the three stages, while the content of Cd was the least. In the feeding stages, elements were at the highest level in the starter broiler, and the grower broiler was considered to have a good metabolic capacity of them. In addition, the elements were mainly distributed in the chicken kidney, gizzard, liver, leg, wing, and lung. In the processing stage, the elements continued to accumulate from the scalding to the sterilization process. The elements were mainly distributed in the chicken wing, leg, head, and breast. In the storage stage, the elements almost kept constant in the polyamide and polyethylene packaging, while it showed irregular small-range fluctuations in the other two packages. This study provides beneficial references for the toxic element risk management in the whole industry chain.

Cost-effective and controllable synthesis of isomalto/malto-polysaccharides from ß-cyclodextrin by combined action of cyclodextrinase and 4,6-α-glucanotransferase GtfB.

Isomalto/malto-polysaccharides (IMMPs) derived from malto-oligosaccharides such as maltoheptaose (G7) are elongated non-branched gluco-oligosaccharides produced by 4,6-α-glucanotransferase (GtfB). However, G7 is expensive and cumbersome to produce commercially. In this study, a cost-effective enzymatic process for IMMPs synthesis is developed that utilizes the combined action of cyclodextrinase from Palaeococcus pacificus (PpCD) and GtfB-ΔN from Limosilactobacillus reuteri 121 to convert ß-cyclodextrin into IMMPs with a maximum yield (16.19 %, w/w). The purified IMMPs synthesized by simultaneous or sequential treatments, designated as IMMP-Sim and IMMP-Seq, possess relatively high contents of α-(1 â†’ 6) glucosidic linkages. By controlling the release of G7 and smaller malto-oligosaccharides by PpCD, IMMP-Seq was obtained of DP varying from 12.9 to 29.5. Enzymatic fingerprinting revealed different linkage-type distribution of α-(1 â†’ 6) linked segments with α-(1 â†’ 4) segments embedded at the reducing end and middle part. The proportion of α-(1 â†’ 6) segments containing the non-reducing end was 56.76 % for IMMP-Sim but 28.98 % for IMMP-Seq. Addition of G3 or G4 as specific acceptors resulted in IMMPs exhibiting low polydispersity. This procedure can be applied as a novel bioprocess that does not require costy high-purity malto-oligosaccharides and with control of the average DP of IMMPs by adjusting the substrate composition.

Starch-guest complexes interactions: Molecular mechanisms, effects on starch and functionality.

Starch is a natural, abundant, renewable and biodegradable plant-based polymer that exhibits a variety of functional properties, including the ability to thicken or gel solutions, form films and coatings, and act as encapsulation and delivery vehicles. In this review, we first describe the structure of starch molecules and discuss the mechanisms of their interactions with guest molecules. Then, the effects of starch-guest complexes on gelatinization, retrogradation, rheology and digestion of starch are discussed. Finally, the potential applications of starch-guest complexes in the food industry are highlighted. Starch-guest complexes are formed due to physical forces, especially hydrophobic interactions between non-polar guest molecules and the hydrophobic interiors of amylose helices, as well as hydrogen bonds between some guest molecules and starch. Gelatinization, retrogradation, rheology and digestion of starch-based materials are influenced by complex formation, which has important implications for the utilization of starch as a functional and nutritional ingredient in food products. Controlling these interactions can be used to create novel starch-based food materials with specific functions, such as texture modifiers, delivery systems, edible coatings and films, fat substitutes and blood glucose modulators.

Physicochemical stability, antioxidant activity, and antimicrobial activity of quercetin-loaded zein nanoparticles coated with dextrin-modified anionic polysaccharides.

Core-shell biopolymer nanoparticles are assembled from a hydrophobic protein (zein) core and a hydrophilic polysaccharide (carboxymethyl dextrin) shell. The nanoparticles were shown to have good stability and the ability to protect quercetin from chemical degradation under long-term storage, pasteurization, and UV irradiation. Spectroscopy analysis shows that electrostatic, hydrogen bonding, and hydrophobic interactions are the main driving forces for the formation of composite nanoparticles. Quercetin coated with nanoparticles significantly enhanced its antioxidant and antibacterial activities and showed good stability and slow release in vitro during simulated gastrointestinal digestion. Furthermore, the encapsulation efficiency of carboxymethyl dextrin-coated zein nanoparticles (81.2%) for quercetin was significantly improved compared with that of zein nanoparticles alone (58.4%). These results indicate that carboxymethyl dextrin-coated zein nanoparticles can significantly improve the bioavailability of hydrophobic nutrient molecules such as quercetin and provide a valuable reference for their application in the field of biological delivery of energy drinks and food.

Structure and properties of flexible starch-based double network composite films induced by dopamine self-polymerization.

Starch-based packaging materials are being developed to alleviate environmental pollution and greenhouse gas emissions associated with plastic-based ones. However, the high hydrophilicity and poor mechanical properties of pure-starch films limit their widespread application. In this study, dopamine self-polymerization was used as a strategy to improve the performance of starch-based films. Spectroscopy analysis showed that strong hydrogen bonding occurred between polydopamine (PDA) and starch molecules within the composite films, which significantly altered their internal and surface microstructures. The composite films had a greater water contact angle (> 90°), which indicated that the incorporation of PDA reduced their hydrophilicity. Additionally, the elongation at break of the composite films was 11-fold higher than pure-starch films, indicating that PDA improved film flexibility, while the tensile strength decreased to some extent. The composite films also exhibited excellent UV-shielding performance. These high-performance films may have practical applications in food and other industries as biodegradable packaging materials.

Malto-oligosaccharides as critical functional ingredient: a review of their properties, preparation, and versatile applications.

Malto-oligosaccharides (MOS) are α-1,4 glycosidic linked linear oligosaccharides of glucose, which have a diverse range of functional applications in the food, pharmaceutical, and other industries. They can be used to modify the physicochemical properties of foods thereby improving their quality attributes, or they can be included as prebiotics to improve their nutritional attributes. The degree of polymerization of MOS can be controlled by using specific enzymes, which means their functionality can be tuned for specific applications. In this article, we review the chemical structure, physicochemical properties, preparation, and functional applications of MOS in the food, health care, and other industries. Besides, we offer an overview for this saccharide from the perspective of prospect functional ingredient, which we feel lacks in the current literature. MOS could be expected to provide a novel promising substitute for functional oligosaccharides.

Preparation and Characterization of Rutin-Loaded Zein-Carboxymethyl Starch Nanoparticles.

In this work, rutin (RT)-loaded zein-carboxymethyl starch (CMS) nanoparticles were successfully prepared by the antisolvent precipitation method. The effect of CMS on composite nanoparticles at different concentrations was studied. When the ratio of zein-RT-CMS was 10:1:30, the encapsulation efficiency (EE) was the highest, reaching 73.5%. At this ratio, the size of the composite nanoparticles was 196.47 nm, and the PDI was 0.13, showing excellent dispersibility. The results of fluorescence spectroscopy, FTIR, XRD, and CD showed that electrostatic interaction, hydrogen bonding, and hydrophobic interaction were the main driving forces for the formation of nanoparticles. It can be seen from the FE-SEM images that the zein-RT-CMS nanoparticles were spherical. With the increase in the CMS concentration, the particles gradually embedded in the cross-linked network of CMS (10:1:50). After RT was loaded on zein-CMS nanoparticles, the thermal stability and pH stability of RT were improved. The results showed that zein-CMS was an excellent encapsulation material for bioactive substances.

Improved production of gamma-cyclodextrin from high-concentrated starch using enzyme pretreatment under swelling condition.

High substrate viscosity during the production process suppressed the formation of gamma-cyclodextrin (γ-CD) from starch. Although liquefaction can be carried out by enzymes with high temperature optimum, some CGTases of interest are not suitable due to loss of activity at the gelatinization temperature. In order to apply a gamma-cyclodextrin glucosyltransferase (γ-CGTase) with an optimum temperature of 50 °C to the liquefaction of 10% w/v cassava starch, an improved process combining moderate heat treatment (30 °C increased to 70 °C) with enzymatic treatment was established. The substrate viscosity dropped from 2456 to 23 cP after the pretreatment, and the rate of γ-CD production increased from 10.43 to 26.34%. This process was further applied to the liquefaction of 30% w/v cassava starch, and the pretreated substrate was finally converted into 21.81% γ-CD. In general, evaluation indicators and basic principles of starch pretreatment were established, providing a reference for the production of γ-CD.

Deciphering external chain length and cyclodextrin production with starch catalyzed by cyclodextrin glycosyltransferase.

Cyclodextrins (CDs) are yielded by cyclodextrin glycosyltransferase (CGTase) using starches or their derivatives as substrates. However, how starch fine structure affects it production is still ambiguous. This study aimed to decipher the relevance between the external chain length (ECL) and CD production. The waxy maize starch was hydrolyzed by ß-amylase to prepare starchy substrates with regular gradient ECL for CGTase. The HPAEC results reflected that ß-amylolysis lower the starch chain fractions with DP range of 13-24 by 30%, while the fractions with DP < 6 promoted by 30%. The molecular weight distribution and iodine binding results reflected that this process had limited impact on overall starch molecular size parameters. After CGTase conversion, it is interesting to find that the ECL was positively correlated with CD contents, which also has a high consistency among α-CD, ß-CD, and γ-CD. This research would provide a favorable perspective for CD production improvement.

Controlling the Fine Structure of Glycogen-like Glucan by Rational Enzymatic Synthesis.

Glycogen-like glucan (GnG), a hyperbranched glucose polymer, has been receiving increasing attention to generate synthetic polymers and nanoparticles. Importantly, different branching patterns strongly influence the functionality of GnG. To uncover ways of obtaining different GnG branching patterns, a series of GnG with radius from 22.03 to 27.06 nm were synthesized using sucrose phosphorylase, α-glucan phosphorylase (GP), and branching enzyme (BE). Adjusting the relative activity ratio of GP and BE (GP/BE) made the molecular weight (MW) distribution of intermediate GnG products follow two different paths. At a low GP/BE, the GnG developed from "small to large" during the synthetic process, with the MW increasing from 6.15 × 106 to 1.21 × 107 g/mol, and possessed a compact structure. By contrast, a high GP/BE caused the "large to small" model, with the MW reduction of GnG from 1.62 × 107 to 1.21 × 107 g/mol, and created a loose external structure. The higher GP activity promoted the elongation of external chains and restrained chain transfer by the BE to the inner zone of GnG, which would modulate the loose-tight structure of GnG. These findings provide new useful insights into the construction of structurally well-defined nanoparticles.

Structural and digestion properties of potato starch modified using an efficient starch branching enzyme AqGBE.

Modified potato starch with slower digestion may aid the development of new starch derivatives with improved nutritional values, and strategies to increase nutritional fractions such as resistant starch (RS) are desired. In this study, a correspondence between starch structure and enzymatic resistance was provided based on the efficient branching enzyme AqGBE, and modified starches with different amylose content (Control, 100%; PS1, 90%; PS2, 72%; PS3, 32%; PS4, 18%) were prepared. Through SEM observation, NMR and X-ray diffraction analyses, we identified that an increased proportion of α-1,6-linked branches in potato starch changes its state of granule into large pieces with crystallinity. Molecular weight and chain-length distribution analysis showed a decrease of molecular weight (from 1.1 × 106 to 1.1 × 105 g/mol) without an obvious change of chain-length distribution in PS1, while PS2-4 exhibited an increased proportion of DP 6-12 with a stable molecular weight distribution, indicating a distinct model of structural modification by AqGBE. The enhancement of peak viscosity was related to increased hydrophobic interactions and pieces state of PS1, while the contents of SDS and RS in PS1 increased by 37.7 and 49.4%, respectively. Our result provides an alternative way to increase the RS content of potato starch by branching modification.

Synergetic modification of waxy maize starch by dual-enzyme to lower the in vitro digestibility through modulating molecular structure and malto-oligosaccharide content.

Cyclodextrinase (CDase) and cyclodextrin glucosyltransferase (CGTase) were synergistically used to provide a novel enzymatic method in lowing in vitro digestibility of waxy maize starch. The molecular structure, malto-oligosaccharide composition, and digestibility properties of the generated products were investigated. The molecular weight was reduced to 0.3 × 105 g/mol and 0.2 × 105 g/mol by simultaneous and sequential treatment with CDase and CGTase, while the highest proportion of chains with degree of polymerization (DP) < 13 was obtained by simultaneous treatment. The resistant starch contents were increased to 27.5% and 36.9% by simultaneous and sequential treatments respectively. Dual-enzyme treatment significantly promoted the content of malto-oligosaccharides (MOSs) by hydrolyzing cyclodextrins from CGTase with CDase. However, the replacement of cyclodextrins by MOSs did not obviously influence the digestibility of the products. The starch digestion kinetics further revealed the hydrolysis pattern of these two enzymes on the starch hydrolysate. It was proved that the starch digestibility could be lowered by modulating the molecular structure and beneficial MOSs content by this dual-enzyme treatment.

Development of pullulanase mutants to enhance starch substrate utilization for efficient production of ß-CD.

The inhibitory effect of ß-CD on pullulanase which hydrolyzes α-1,6 glycosidic bond in starch to release more available linear substrates, limited substrate utilization thus influencing the yield of ß-CD. Here, an aspartic acid residue (D465) which interacted with cyclodextrin ligand by hydrogen bond, was mutated to explore its contribution to bind inhibitors and obtain mutants with lower affinity to ß-CD. Enzyme activity results showed that mutants D465E and D465N retained higher activity than wild-type pullulanase in presence of 10 mM ß-CD. Circular dichroism spectra and fluorescence spectra results showed that D465 was related to structure stability. Chain length distribution results confirmed the improvement of substrate utilization by the addition of D465E. The conversion rate from potato starch, cassava starch, and corn starch into ß-CD, increased to 56.9%, 55.4% and 54.7%, respectively, when synchronous using ß-CGTase and D465E in the production process.

Thermophilic 4-α-Glucanotransferase from Thermoproteus Uzoniensis Retards the Long-Term Retrogradation but Maintains the Short-Term Gelation Strength of Tapioca Starch.

Gelation of starch is a process during short-term retrogradation. However, long-term retrogradation always leads to the quality deterioration of starch-based food. In this work, a new type of modified tapioca starch (MTS) gel with maintained short-term gelation strength and retarded long-term retrogradation was prepared using a novel recombinantly produced and characterized 4-α-glucanotransferase (TuαGT). In the resulting MTS, the exterior chains of the amylopectin part were elongated and the content of amylose was reduced because of the disproportionation activity of TuαGT. The retrogradation analysis demonstrated that the MTS possessed highly weakened long-term retrogradation characteristics as compared to the native starch. Most importantly, the strength of the gel formed by regelatinized MTS is very close to that of gelatinized native tapioca starch when storing below 30 °C. These findings provide a starting point for developing a novel method for the enzymatic modification of the starch-based gels.

The binding mechanism between cyclodextrins and pullulanase: A molecular docking, isothermal titration calorimetry, circular dichroism and fluorescence study.

This work investigated the interaction between cyclodextrins and pullulanase to provide insight into the production and application of cyclodextrins. Enzyme activity and kinetic assays showed that α-cyclodextrin (α-CD), ß-cyclodextrin (ß-CD) and γ-cyclodextrin (γ-CD) inhibited pullulanase in a competitive manner. Circular dichroism spectra and fluorescence spectroscopy suggested the formation of cyclodextrin and pullulanase complexes. According to ITC assays and molecular docking results, compared with α-CD and γ-CD, ß-CD had the strongest affinity for pullulanase because of its appropriate cavity geometric dimensions. In addition, cyclodextrins interacted with pullulanase through hydrogen bonds, van der Waals force and hydrophobic interactions, the latter of which were verified as the major driving force. Phenylalanine 476 was the key amino acid residue in pullulanase for cyclodextrin recognition and binding.