Development of a Zn-Based Single-Atom Nanozyme for Efficient Hydrolysis of Glycosidic Bonds.

Hydrolytic enzymes are essential components in second-generation biofuel technology and food fermentation processes. Nanozymes show promise for large-scale industrial applications as replacements for natural enzymes due to their distinct advantages. However, there remains a research gap concerning glycosidase nanozymes. In this study, a Zn-based single-atom nanozyme (ZnN4-900) is developed for efficient glycosidic bond hydrolysis in an aqueous solution. The planar structure of the class-porphyrin N4 material approximatively mimicked the catalytic centers of natural enzymes, facilitating oxidase-like (OXD-like) activity and promoting glycosidic bond cleavage. Theoretical calculations show that the Zn site can act as Lewis acids, attacking the C─O bond in glycosidic bonds. Additionally, ZnN4-900 has the ability to degrade starch and produce reducing sugars that increased yeast cell biomass by 32.86% and ethanol production by 14.56%. This catalyst held promising potential for enhancing processes in ethanol brewing and starch degradation industries.

Pulsed light treatment enhances starch hydrolysis and improves starch physicochemical properties of germinated brown rice.

BACKGROUND: Recently, germinated brown rice (GBR) has gained substantial attention as a functional food because of its nutritional attributes. Notably, pulsed light technology (PLT) has emerged as a promising tool for enhancing rice germination and, consequently, has improved the nutritional and functional qualities of GBR-derived products. However, further research is required to comprehensively understand the impact of PLT on GBR physicochemical properties. The present study aimed to investigate the stimulating effects of PLT on starch hydrolysis, starch structure and functional properties of GBR. RESULTS: The PLT substantially boosted α-amylase activity during brown rice germination, leading to a 10.9% reduction in total starch content and a 17.3% increase in reducing sugar content, accompanied by elevated free water levels. Structural analysis indicated no changes in starch crystalline types, whereas gelatinization temperature slightly increased. Pasting properties exhibited a significant drop in peak viscosity. Scanning electron microscopy showed surface erosion of starch granules with microstructural changes. Furthermore, correlation analysis established positive links between α-amylase activity, reducing sugar accumulation, starch structure and functional properties in GBR. CONCLUSION: The present study demonstrates that PLT enhanced the physicochemical properties of GBR starch, significantly improving the stability of GBR products, thereby contributing to expanded applicability of rice starch in the food industry. © 2023 Society of Chemical Industry.

Classification of industrial tapioca starch hydrolysis products based on their Brix and dextrose equivalent values using near-infrared spectroscopy.

BACKGROUND: Industrial starch hydrolysis allows the production of syrups with varying functionality depending on their Brix value and dextrose equivalent (DE). As the current methods for evaluating these products are labor-intensive and time-consuming, the objective of this study was to investigate the potential of near-infrared (NIR) spectroscopy for classifying the different tapioca starch hydrolysis products. RESULTS: NIR spectra of samples of seven products (n = 410) were recorded in transflectance mode in the 12 000-4000 cm-1 range. Next, orthogonal partial least squares (OPLS) regression models were built to predict the Brix and DE values of the different samples. To classify the different starch hydrolysis products, support vector machines (SVM) were trained using either the raw spectra or latent variables (LVs) obtained from the OPLS models. The best classification accuracy was obtained by the SVM classifier based on the LVs from the OPLS model for DE prediction, resulting in 95% correct classification over all classes. CONCLUSION: These results show the potential of NIR spectroscopy for classifying tapioca starch hydrolysis products with respect to their functional properties related to the Brix and DE values. © 2024 Society of Chemical Industry.

Effect of mechanical properties on in vitro dynamic digestion of starch contained in hydrogels.

BACKGROUND: This study evaluates the effect of mechanical properties on the in vitro dynamic gastrointestinal digestion of hydrogels containing starch (HCSs) as a model for studying the nutrient digestibility of solid foods. It provides a useful theoretical basis for the processing of specific foods. RESULT: Four types of HCSs with two levels of fracture stress (17.4-20.9 kPa and 55.5-57.6 kPa) and two levels of fracture strain (25.4-28.5% and 53.7-57.4%) were prepared. For these HCSs, the degree of gastric disintegration of hydrogels reduced significantly when fracture strain exceeded 30% (P < 0.05). The gastric emptying of HCS particles was also affected by mechanical properties. For example, even at the same level of fracture stress (ca. 20 kPa), the dry solids retention ratio decreased markedly from 0.90 to 0.43 with a decrease in fracture strain from 53.7% to 25.4% (P < 0.05). For the starch hydrolysis of HCSs after gastric digestion, more than 70% of starch in the particles of all types of HCSs emptied did not undergo digestion. The starch hydrolysis of HCSs during small intestinal digestion was also influenced by their mechanical properties. Fracture strains of HCSs, rather than their fracture stress, affected starch digestibility in hydrogels. CONCLUSION: The gastric disintegration, the gastric emptying, and the starch hydrolysis of HCSs are suppressed when fracture strain exceeded 30%. Even with the amount of nutritional components contained in hydrogels being the same, the in vitro gastrointestinal digestion behavior of HCSs depends on their mechanical properties. This behavior has the potential to be used in the design of processed foods with controlled bioaccessibility. © 2023 Society of Chemical Industry.

Novel Design of an α-Amylase with an N-Terminal CBM20 in Aspergillus niger Improves Binding and Processing of a Broad Range of Starches.

In the starch processing industry including the food and pharmaceutical industries, α-amylase is an important enzyme that hydrolyses the α-1,4 glycosidic bonds in starch, producing shorter maltooligosaccharides. In plants, starch molecules are organised in granules that are very compact and rigid. The level of starch granule rigidity affects resistance towards enzymatic hydrolysis, resulting in inefficient starch degradation by industrially available α-amylases. In an approach to enhance starch hydrolysis, the domain architecture of a Glycoside Hydrolase (GH) family 13 α-amylase from Aspergillus niger was engineered. In all fungal GH13 α-amylases that carry a carbohydrate binding domain (CBM), these modules are of the CBM20 family and are located at the C-terminus of the α-amylase domain. To explore the role of the domain order, a new GH13 gene encoding an N-terminal CBM20 domain was designed and found to be fully functional. The starch binding capacity and enzymatic activity of N-terminal CBM20 α-amylase was found to be superior to that of native GH13 without CBM20. Based on the kinetic parameters, the engineered N-terminal CBM20 variant displayed surpassing activity rates compared to the C-terminal CBM20 version for the degradation on a wide range of starches, including the more resistant raw potato starch for which it exhibits a two-fold higher Vmax underscoring the potential of domain engineering for these carbohydrate active enzymes.

Reduced in vitro starch hydrolysis and in vivo glycemic effects after addition of soy presscake to corn tortillas.

BACKGROUND: Chronically elevated blood glucose leads to development of prediabetes and type 2 diabetes, as well as increased risk for heart and kidney disease and vision loss. For many, elevated blood glucose can be managed through diet and exercise. Consequently, the availability of foods that limit blood glucose elevation would aid in addressing this global problem. This paper investigated the effect of adding soy presscake (SP) to corn tortillas on starch hydrolysis in vitro as well as the glycemic responses elicited in vivo upon consumption of these modified tortillas. RESULTS: SP in corn tortillas decreased the rate and extent of starch hydrolysis in vitro. The in vivo glycemic index (GI) values decreased from 43 for corn control tortillas to 31 with 40% SP fortification. A high correlation (r = 0.9781) was found between the GI values from in vivo analysis and the area under the curve of starch hydrolysis in vitro. The best correlations (r > 0.96) between GI and degree of hydrolysis were found at 45-90 min of in vitro starch hydrolysis. CONCLUSIONS: Incorporating SP into corn-based tortillas lowers glycemic responses to them. In addition, in vitro starch hydrolysis could be used to estimate the GI values of food products and, in particular, the comparison of multiple items during food product development. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Minor millets: a review on nutritional composition, starch extraction/modification, product formulation, and health benefits.

Minor millet grains are the abode of healthy constituents of human concern that contribute to healthy longevity. Additionally, they are excellent in nutritional value including macronutrients namely, protein (7-13%), carbohydrates (60-70%), fat (1.5-5%), fiber (2-7%) and for micronutrients as well namely; iron, calcium, phosphorus, and magnesium, etc. All these beneficial traits along with the availability of bioactive constituents (polyphenols and antioxidants) prove them to be therapeutic in action and also uplift the immunity among users. Employed isolation tactics for starch also govern yield characteristics and is usually preferred by way of wet method. Minor millets are abundant in starch (50-70%) thus application broadness is another attribute which could be addressed in vivid food segments. In case, native starches somehow possess least application credentials in food and non-food sectors thus modification is the only alternative to eliminate shortcomings. As in trend, modification using physical, chemical, and enzymatic ways have a wide impact on the properties of millet starch. The present review summarizes the nutritional, bioactive and therapeutic potential of minor millets, along with ways of starch modification and product development through millet involvement. © 2023 Society of Chemical Industry.

Structure of maize BZR1-type ß-amylase BAM8 provides new insights into its noncatalytic adaptation.

Plant ß-amylase (BAM) proteins play an essential role in growth, development, stress response, and hormone regulation. Despite their typical (ß/α)8 barrel structure as active catalysts in starch breakdown, catalytically inactive BAMs are implicated in diverse yet elusive functions in plants. The noncatalytic BAM7/8 contain N-terminal BZR1 domains and were shown to be involved in the regulation of brassinosteroid signaling and possibly serve as sensors of yet an uncharacterized metabolic signal. While the structures of several catalytically active BAMs have been reported, structural characterization of the catalytically inactive BZR1-type BAMs remain unknown. Here, we determine the crystal structure of ß-amylase domain of Zea mays BAM8/BES1/BZR1-5 and provide comprehensive insights into its noncatalytic adaptation. Using structural-guided comparison combined with biochemical analysis and molecular dynamics simulations, we revealed conformational changes in multiple distinct highly conserved regions resulting in rearrangement of the binding pocket. Altogether, this study adds a new layer of understanding to starch breakdown mechanism and elucidates the acquired adjustments of noncatalytic BZR1-type BAMs as putative regulatory domains and/or metabolic sensors in plants.

Pancreatin-induced liberation of starch/cyanidin 3-O-glucoside complexes from rice cooked with black soybean that exhibit slow hydrolysis.

Cyanidin 3-O-glucoside (C3G), which has various health-promoting functions, is contained in black soybean (BSB). In Japan and Korea, BSB is cooked with rice and the cooked rice appears purplish in colour. In this study, BSB was cooked with glutinous rice, non-glutinous rice, and high-amylose rice. The amount of C3G detected in high-amylose rice was greater than that detected in glutinous rice, suggesting that C3G combined more efficiently with amylose than with amylopectin. Pancreatin induced the liberation of starch/C3G complexes from the purplish cooked rice, and rate of the liberation was in the following order; glutinous rice < non-glutinous rice < high-amylose rice. The amylose/C3G complexes liberated from high-amylose rice was hydrolysed slowly, while the amylopectin/C3G complexes liberated from glutinous rice were hydrolysed into smaller amylopectin/C3G complexes that were difficult to further hydrolysis. Thus, C3G may be useful for preparing foods whose starch hydrolysis is slow.

Effect of pH on the Redox and Sorption Properties of Native and Phosphorylated Starches.

Starch is a common biopolymer that can be used for removing heavy metal ions from aqueous solutions. A valuable property of starch is its functional diversity, which can be enhanced by chemical modification. Hydroxyl groups enclosed in the starch and formed during hydrolysis act as reducing agents of Cr(VI). The sorption properties of native starch depend mainly on the presence of carboxyl groups formed during redox processes and basic centers created during acid hydrolysis, while the superiority of phosphorylated starch is related to the presence of phosphate groups binding Cr(III) ions. The effectiveness of starch depends on a series of equilibria established in its aqueous suspension and chromate ions solution, where the pH is the driving force for these processes. In this article, a systematic discussion of pH changes being the consequence of chemical reactions unraveling the extraordinary functionalities of starch was given. It also explained the influence of establishing equilibria and chemical modifications of starch on the efficiency of chromium ion removal. This allowed for the development of a comprehensive mechanism for the interaction of Cr(VI) and Cr(III) ions with native and phosphorylated starch.

Starch Digestion in Infants: An Update of Available In Vitro Methods-A Mini Review.

Complementary feeding starts at around six months of age because neither breast milk nor formula assure the proper nutrition of infants. Therefore, along with breast milk, solid foods are gradually introduced, particularly cereal-based foods, which will provide starch as a new source of energy and nutrients. As a result, the need of an adequate in vitro digestion method to study the influence of different aspects of weaning period is unquestionable. This critical review summarizes the in vitro digestion methods available for the analysis of starch hydrolysis under infant conditions considering different features, namely, starch digestion, infant digestive conditions and in vitro models suitable for the study of starch digestion (static, semi-dynamic and dynamic). Key factors such as enzyme concentrations, transit time, oral, gastric and intestinal conditions and differences with current adult models, have been addressed. The need for standardized infant digestion models adapted to the complementary feeding period was discussed. Existing literature data demonstrate that more effort has to be done to improve the research on this issue, in order to obtain comparable results that would address a better understanding of the digestibility of different food nutrients under infant conditions facilitating the development of appropriate formulations that may assure proper infant nutrition.

Monitoring starch hydrolysis with micro visco-amylo-graph for the production of chickpea milk and optimization of the parameters with response surface methodology.

Plant-based milk products are gaining attention since it has been demonstrated that the consumption of animal-derived foods had to be reduced to combat global climate change. The production of plant-based milk includes a starch hydrolysis step for raw materials with high starch content such as cereals and pulses, since the gelatinized starch forms a thick slurry which causes an unsuitable consistency for a drinkable product. The objectives of this work were to investigate the effects of slurry concentration (solid to solvent ratio), enzyme including temperature, enzyme amount and mixing (rotation) speed on the pasting properties especially final viscosity of a crude chickpea milk and also to investigate the potential use of Micro Visco Amylo-Graph for monitoring starch hydrolysis. Response surface methodology, based on Box Behnken Design, was used to assess the parameters and to optimize the hydrolysis conditions for the minimum final viscosity. In conclusion, it was observed that slurry concentration and enzyme including temperature were the most critical factors that affect either the pasting properties or the final viscosity of the crude chickpea milk. Briefly, lower final viscosities were obtained from samples which were prepared at lower beginning concentrations and treated with higher enzyme amounts at lower temperatures.

Digital Imaging-based Colourimetry for Enzymatic Processes in Transparent Liquid Marbles.

Liquid marbles are a promising microreactor platform that recently attracts significant research interest owing to their ability to accommodate a wide range of micro reactions. However, the use of destructive and ex-situ methods to monitor reactions impairs the potential of liquid-marble-based microreactors. This paper proposes a non-destructive, in situ, and cost-effective digital-imaging-based colourimetric monitoring method for transparent liquid marbles, using the enzymatic hydrolysis of starch as an illustrative example. The colourimetric reaction between starch and iodine produces a complex that exhibits a dark blue colour. We found that the absorbance of red channel of digital images showed a linear relationship with starch concentration with high sensitivity and repeatability. This digital-imaging-based colourimetric method was used to study the hydrolysis of starch by α-amylase. The results show high accuracy and applicability of first-order kinetics for this reaction. The demonstration of digital-imaging-based colourimetry indicates the potential of liquid marble-based microreactors.

Phenotypic homogeneity of emetic Bacillus cereus isolates in China.

Emetic Bacillus cereus strains produce a potent cereulide cytotoxin, which can cause acute and fatal cases of food poisoning. We isolated 18 emetic B. cereus strains from a food poisoning event, and from clinical and non-random food surveillance in China and phenotypic characteristics of haemolysis, starch hydrolysis, salicin fermentation, gelatin liquefaction, cytotoxicity, and susceptibility to antibiotics were assessed. All isolates were positive for haemolysis and gelatin liquefaction, and negative for starch hydrolysis and salicin fermentation. Their haemolytic potentials were intermediate to Bacillus anthracis and B. cereus ATCC 14579 (a non-emetic strain). All isolates were cytotoxic to CHO, Hep-2, and Vero cells, and were sensitive to ampicillin. The homogeneous phenotypes of emetic isolates from China are similar to the corresponding traits of European and Japanese isolates that have been characterized, suggesting highly similar phenotypes of emetic B. cereus worldwide.

Overexpression of an endogenous raw starch digesting mesophilic α-amylase gene in Bacillus amyloliquefaciens Z3 by in vitro methylation protocol.

BACKGROUND: Mesophilic α-amylases function effectively at low temperatures with high rates of catalysis and require less energy for starch hydrolysis. Bacillus amyloliquefaciens is an essential producer of mesophilic α-amylases. However, because of the existence of the restriction-modification system, introducing exogenous DNAs into wild-type B. amyloliquefaciens is especially tricky. RESULTS: α-Amylase producer B. amyloliquefaciens strain Z3 was screened and used as host for endogenous α-amylase gene expression. In vitro methylation was performed in recombinant plasmid pWB980-amyZ3. With the in vitro methylation, the transformation efficiency was increased to 0.96 × 102 colony-forming units µg-1 plasmid DNA. A positive transformant BAZ3-16 with the highest α-amylase secreting capacity was chosen for further experiments. The α-amylase activity of strain BAZ3-16 reached 288.70 ± 16.15 U mL-1 in the flask and 386.03 ± 16.25 U mL-1 in the 5-L stirred-tank fermenter, respectively. The Bacillus amyloliquefaciens Z3 expression system shows excellent genetic stability and high-level extracellular production of the target protein. Moreover, the synergistic interaction of AmyZ3 with amyloglucosidase was determined during the hydrolysis of raw starch. The hydrolysis degree reached 92.34 ± 3.41% for 100 g L-1 raw corn starch and 81.30 ± 2.92% for 100 g L-1 raw cassava starch after 24 h, respectively. CONCLUSION: Methylation of the plasmid DNA removes a substantial barrier for transformation of B. amyloliquefaciens strain Z3. Furthermore, the exceptional ability to hydrolyze starch makes α-amylase AmyZ3 and strain BAZ3-16 valuable in the starch industry. © 2020 Society of Chemical Industry.

Rice in vitro digestion: application of INFOGEST harmonized protocol for glycemic index determination and starch morphological study.

Starch is the main sugar source present in staple foods. Understanding starch hydrolysis during digestion and the resulting glucose release can be important to strategically modulate starch digestion and glucose absorption. In vitro digestion methodologies are fundamental to evaluate starch hydrolysis length and rate, but the lack of uniformity between protocols prevent the comparison of results. In this context, three different Carolino rice varieties (i.e., Carolino white-Cw, Carolino brown-Cb and Carolino Ariete brown-CAb) were submitted to the INFOGEST harmonized in vitro digestion protocol for the evaluation of starch hydrolysis and subsequent glycemic index (GI) determination, and starch granules morphological study. Samples of Carolino rice presented total starch percentages between 64.52 (for Cb) to 71.52% (for Cw) with low amylose content (16.19-19.95%, varying in the following order Cb < Cab ≈ Cw). During digestion, between 39.43 (for CAb) to 44.48% (for Cb) of starch was hydrolyzed, classifying samples as medium GI foods (61.73-69.17). Starch hydrolysis was accompanied by a decrease of starch granules dimensions. For all samples, area decrease was higher than 59%, perimeter decrease was higher than 37%, feret diameter decrease was higher than 39% and minimum feret diameter decrease was higher than 32%. This work provides new insights to describe, both qualitatively and quantitatively, the fate of rice during digestion, and allowed establishing a comparative basis for the development of rice-based recipes with a lower GI.

Process development of starch hydrolysis using mixing characteristics of Taylor vortices.

In food industries, enzymatic starch hydrolysis is an important process that consists of two steps: gelatinization and saccharification. One of the major difficulties in designing the starch hydrolysis process is the sharp change in its rheological properties. In this study, Taylor-Couette flow reactor was applied to continuous starch hydrolysis process. The concentration of reducing sugar produced via enzymatic hydrolysis was evaluated by varying operational variables: rotational speed of the inner cylinder, axial velocity (reaction time), amount of enzyme, and initial starch content in the slurry. When Taylor vortices were formed in the annular space, efficient hydrolysis occurred because Taylor vortices improved the mixing of gelatinized starch with enzyme. Furthermore, a modified inner cylinder was proposed, and its mixing performance was numerically investigated. The modified inner cylinder showed higher potential for enhanced mixing of gelatinized starch and the enzyme than the conventional cylinder.

Jet milling effect on wheat flour characteristics and starch hydrolysis.

The interest for producing wheat flour with health promoting effect and improved functionality has led to investigate new milling techniques that can provide finer flours. In this study, jet milling treatment was used to understand the effect of ultrafine size reduction onto microstructure and physicochemical properties of wheat flour. Three different conditions of jet milling, regarding air pressure (4 or 8 bars) feed rate and recirculation, were applied to obtain wheat flours with different particle size (control, F1, F2 and F3 with d50 127.45, 62.30, 22.94 and 11.4 µm, respectively). Large aggregates were gradually reduced in size, depending on the intensity of the process, and starch granules were separated from the protein matrix. Damaged starch increased while moisture content decreased because of milling intensity. Notable changes were observed in starch hydrolysis kinetics, which shifted to higher values with milling. Viscosity of all micronized samples was reduced and gelatinization temperatures (To, Tp, Tc) for F2 and F3 flours increased. Controlling jet milling conditions allow obtaining flours with different functionality, with greater changes at higher treatment severity that induces large particle reduction.

Characteristics of enterotoxin distribution, hemolysis, lecithinase, and starch hydrolysis of Bacillus cereus isolated from infant formulas and ready-to-eat foods.

Bacillus cereus is a ubiquitous environmental microbe implicated as a main cause of food poisoning with various symptoms, depending on the strain type and the isolation source. In this study, the potential virulence factors and biochemical properties of B. cereus isolated from infant formulas and ready-to-eat (RTE) foods were analyzed and compared. A total of 347 B. cereus strains were isolated and identified from 687 infant food formulas and RTE food samples. All the isolates had one or more enterotoxin genes, and one-half of the strains had all 3 enterotoxin genes (hbl, nhe, and cytK) that are involved in food poisoning in humans. Here, all the 3 genes were detected in 50% of the B. cereus isolates from RTE foods and only 14% of the isolates were identified from infant formulas. The latter harbored low cytK and bceT, and very low hbl genes. Most B. cereus isolates possessed the hemolysis gene, but not the ces gene. The infant formula isolates showed stronger hemolysis activity than the other isolates. In addition, 26% of the total isolates showed low lecithinase activities and 10% showed high lecithinase activities. A greater number of isolates from the infant formula showed high lecithinase activity than those from the RTE foods. Approximately 83% of the isolates were positive and 17% were negative for starch hydrolysis. Over 90% of the RTE food isolates and only 35% of the infant formula isolates were positive for starch hydrolysis. However, all the strains possessed nhe, but their harboring patterns of hbl and cytK were significantly different. Most starch-hydrolyzing strains possessed hbl, but only 23% nonstarch-hydrolyzing isolates possessed this gene. Moreover, very low nonstarch hydrolyzing strains harbored cytK. Most nonstarch-hydrolyzing isolates showed high lecithinase and strong hemolysis activities, and very low hbl and cytK harboring. In summary, most infant formula isolates showed stronger hemolysis and higher lecithinase activities with lower frequency of harboring hbl and cytK and lower starch hydrolysis compared with RTE food isolates.

Combined effects of added beta glucan and black tea in breads on starch functionality.

Bread and tea are usually consumed separately, but there may be different food-matrix interactions and changes in starch characteristics when they are combined in bread. This study developed breads (white bread, WF; black tea, BT; beta glucan, ßG; beta glucan plus black tea, ßGBT) and determined their starch functionalities. Breads were developed using a standard baking recipe and determined their starch characteristics. There was no significant difference in starch hydrolysis between BT and WF but ßGBT reduced early (10 min) starch hydrolysis compared with ßG. The starch granules in ßG and ßGBT were elliptical and closely packed together. These results suggest that the addition of beta glucan and black tea to bread preserved the elliptical starch granules and lowered short-term starch hydrolysis.