Production Of Maltose Research Articles

Pullulanase pretreatment of highly concentrated maltodextrin solution improves maltose yield during β-amylase-catalyzed saccharification

Increasing the substrate concentration can effectively reduce energy consumption and result in more economic benefits in the industrial production of maltose, but this process remarkably increases the viscosity, which has a negative effect on saccharification. To improve saccharification efficiency, pullulanase is usually employed. In the conventional process of maltose production, pullulanase is added at the same time with β-amylase or later, but this process seems inefficient when the substrate concentration is high. Herein, a novel method was introduced to enhance the maltose yield under high substrate concentration. The results indicated that the pullulanase pretreatment of highly concentrated maltodextrin solution for 2 h greatly affects the final conversion rate of β-amylase-catalyzed saccharification. The maltose yield reached 80.95 %, which is 11.8 % above the control value. Further examination confirmed that pullulanase pretreatment decreased the number of branch points of maltodextrin and resulted in a high content of oligosaccharides. These linear chains were suitable for β-amylase-catalyzed saccharification to produce maltose. This research offers a new effective and green strategy for starch sugar production.

Computer simulation of microwave cooking of sweet potato – Kinetics analysis of reactions in the maltose production process and their modeling

Computer simulation of microwave cooking of sweet potato – Kinetics analysis of reactions in the maltose production process and their modeling

Improved Recombinant Expression of Maltogenic α-Amylase AmyM in Bacillus subtilis by Optimizing Its Secretion and NADPH Production

The maltose α-amylase AmyM from Bacillus stearothermophilus can be used for flour modification, baked goods preservation, and maltose production. Here, we optimized the recombinant expression of AmyM in Bacillus subtilis WB800 via several strategies. By screening the optimal promoter, a double promoter combination (P43 and PamyL) could improve the expression level of AmyM by 61.25%, compared with the strong promoter P43. Then, we optimized the secretion efficiency of recombinant AmyM by over-expressing the molecular chaperone prsA gene. SDS-PAGE results suggested that over-expression of the prsA could improve the secretion efficiency of AmyM to the extracellular environment. The extracellular enzyme activity of AmyM was increased by 101.58% compared to the control strain. To further improve the expression of AmyM, we introduced the hemoglobin gene of Vitreoscilla (vgb) into the AmyM recombinant strain. The results revealed that the introduction of vgb could promote the transcription and translation of AmyM in B. subtilis. This may be due to the increasing level of intracellular NADPH and NADP+ caused by the expression of vgb. By this strategy, the expression level of AmyM was increased by 204.08%. Finally, we found the recombinant AmyM showed an optimal temperature of 65 °C and an optimal pH of 5.5. Our present results provided an effective strategy for increasing the heterologous expression level of AmyM in B. subtilis.

Conversion of waste cooked rice into maltose by a Bacillus subtilis-derived maltogenic amylase and production of maltobionic acid from the prepared maltose using genetically modified Pseudomonas taetrolens.

In this study, we attempted to produce maltobionic acid (MBA) from waste cooked rice (WCR) using maltose as an intermediate. In our previous study, we produced maltose from WCR using a commercial maltogenic amylase (Maltogenase L). However, in the present study, we used wild-type Bacillus subtilis, which inherently produces maltogenic amylase (AmyE), instead of Maltogenase L to produce maltose from WCR. During cultivation of B. subtilis with WCR, maltose was successfully produced by AmyE in the culture medium. To improve maltose production, we constructed a recombinant B. subtilis strain expressing AmyE and used it for maltose production. Following cultivation of the recombinant B. subtilis strain, the maltose production titer (34.6g/L) increased approximately 3.6-fold that (9.6g/L) obtained from the cultivation of wild-type B. subtilis. Using Pseudomonas taetrolens, an efficient MBA-producing bacterium, 28.8g/L of MBA was produced from the prepared maltose (27.6g/L). The above results indicated that MBA was successfully produced from WCR via a two-step process, which involved the conversion of WCR into maltose by maltogenic amylase-producing B. subtilis and the production of MBA from the WCR-derived maltose by P. taetrolens.

Production of pigments by Monascus ruber CCT0302 in culture media containing maltose as substrate

The aim of this study was to show how maltose production residues can be used to obtain natural pigments by Monascus ruber CCT 3802 in solid and submerged cultures. The microbial growth and the colour and heat stability characteristics of the pigments produced in both solid and submerged media, with different maltose syrup concentrations, were determined. The results showed that the addition of maltose provided significant increases in the velocity of microbial growth and production of red pigments. The highest radial growth velocity of Monascus ruber (0.1053 mm h−1) was obtained when cultivated in a medium containing 5 g L−1 of maltose syrup, corresponding to a 71.7% increase in growth as compared to the growth velocity in the control medium. Using submerged fermentation, the culture medium containing 10 g L−1 of maltose syrup provided the greatest concentrations of red pigments (14.54 AU510nm g−1 dry biomass) with an intense dark red colour, showing that Monascus ruber CCT 3802 had the capacity to assimilate the substrate and produce pigments. The red pigments produced in the cultures showed good heat stability with activation energies of 13.735 Kcal mol−1.

High mashing thickness negatively influences gelatinisation of small and large starch granules and starch conversion efficiency during barley malt brewing

The breakdown of starch into fermentable sugars is essential for several industrial processes, including beer production. Nowadays, high ratios of barley malt to water are used to improve brewhouse efficiency. Recent findings, however, indicate that small starch granules in barley malt could reduce starch conversion efficiency. In this study, the gelatinisation behaviour of small and large starch granules in function of mashing thickness was assessed. A mashing process was performed with malt to water ratios ranging from 1:6 to 1:2.5 (high gravity), leading to wort with 12 and 25 g of extractable components per 100 g wort, respectively. The sugar yield in wort decreased from 79% to 66% with increasing malt to water ratios due to reduced maltose production. It was hypothesised that sugars and other components in the mash increased the starch gelatinisation temperature. Therefore, small and large starch granules were isolated from barley malt, and the impact of wort and individual wort component on the gelatinisation temperature of these granules was assessed. An increase in the amount of extracted components with 2.5 g per 100 g wort during mashing resulted in a 1 °C increase in starch gelatinisation temperature for both types of granules. For high gravity brewing, this proved problematic. Gelatinisation was delayed to such an extent that β-amylase was not able to produce the maximal amount of fermentable sugars from primarily the small starch granules anymore, explaining the reduced sugar yield. • Mashing yield and sugar yield decrease with increasing mashing thickness. • The loss in sugar yield is due to less efficient maltose production. • Starch losses during mashing can be mainly attributed to small starch granules. • The impact of wort on starch gelatinisation is similar for large and small granules. • Extract content of wort is strongly correlated with starch gelatinisation temperature.

Type I caramel products of maltose and sucrose with water and their antioxidant activities

To study the caramel reactions of disaccharides with water, the type I maltose caramel products (MCPs) and sucrose caramel products (SCPs) at three reaction stages were yielded and their reaction rates, UV absorptions, flavor compounds, and antioxidant activities were also detected. It was found that the caramel reactions of sucrose were faster than those of maltose and their reaction rates quickly increased to a high level in the middle stage. UV absorption of SCP in the late stage at 305 nm was much stronger than those of MCPs. Both the MCP and SCP obtained at 180 °C and 4 min had the highest concentrations of 5-hydroxymethylfurfural and total flavor compounds. Moreover, the total flavor concentrations of SCPs were much larger than those of MCPs and the antioxidant effects of SCPs were also stronger. Thus, a preferred condition for both two disaccharide caramel reactions was suggested at 180 °C for 4 min and the sucrose caramels could give better flavors than maltose caramels.

Exploring the environmental traits and applications of Klebsiella variicola.

Klebsiella variicola has been found in various natural niches, alone or in association with other bacteria, and causes diseases in animals and plants with important economic and environmental impacts. K. variicola has the capacity to fix nitrogen in the rhizosphere and soil; produces indole acetic acid, acetoin, and ammonia; and dissolves phosphorus and potassium, which play an important role in plant growth promotion and nutrition. Some members of K. variicola have properties such as halotolerance and alkalotolerance, conferring an evolutionary advantage. In the environmental protection, K. variicola can be used in the wastewater treatment, biodegradation, and bioremediation of polluted soil, either alone or in association with other organisms. In addition, it has the potential to carry out industrial processes in the food and pharmaceutical industries, like the production of maltose and glucose by the catalysis of debranching unmodified oligosaccharides by the pullulanase enzyme. Finally, this bacterium has the ability to transform chemical energy into electrical energy, such as a biocatalyst, which could be useful in the near future. These properties show that K. variicola should be considered an eco-friendly bacterium with hopeful technological promise. In this review, we explore the most significant aspects of K. variicola and highlight its potential applications in environmental and biotechnological processes.

Expression, biochemical and structural characterization of high-specific-activity \u03b2-amylase from Bacillus aryabhattai GEL-09 for application in starch hydrolysis

Backgroundβ-amylase (EC 3.2.1.2) is an exo-enzyme that shows high specificity for cleaving the α-1,4-glucosidic linkage of starch from the non-reducing end, thereby liberating maltose. In this study, we heterologously expressed and characterized a novel β-amylase from Bacillus aryabhattai.ResultsThe amino acid-sequence alignment showed that the enzyme shared the highest sequence identity with β-amylase from Bacillus flexus (80.73%) followed by Bacillus cereus (71.38%). Structural comparison revealed the existence of an additional starch-binding domain (SBD) at the C-terminus of B. aryabhattai β-amylase, which is notably different from plant β-amylases. The recombinant enzyme purified 4.7-fold to homogeneity, with a molecular weight of ~ 57.6 kDa and maximal activity at pH 6.5 and 50 °C. Notably, the enzyme exhibited the highest specific activity (3798.9 U/mg) among reported mesothermal microbial β-amylases and the highest specificity for soluble starch, followed by corn starch. Kinetic analysis showed that the Km and kcat values were 9.9 mg/mL and 116961.1 s− 1, respectively. The optimal reaction conditions to produce maltose from starch resulted in a maximal yield of 87.0%. Moreover, molecular docking suggested that B. aryabhattai β-amylase could efficiently recognize and hydrolyze maltotetraose substrate.ConclusionsThese results suggested that B. aryabhattai β-amylase could be a potential candidate for use in the industrial production of maltose from starch.

Regulation of β-amylase synthesis: a brief overview

The major activity of β-amylase (BMY) is the production of maltose by the hydrolytic degradation of starch. BMY is found to be produced by some plants and few microorganisms only. The industrial importance of the enzyme warrants its application in a larger scale with the help of genetic engineering, for which the regulatory mechanism is to be clearly understood. In plants, the activities of BMY are regulated by various environmental stimuli including stress of drought, cold and heat. In vascular plant, Arabidopsis sp. the enzyme is coded by nine BAM genes, whereas in most bacteria, BMY enzymes are coded by the spoII gene family. The activities of these genes are in turn controlled by various compounds. Production and inhibition of the microbial BMY is regulated by the activation and inactivation of various BAM genes. Various types of transcriptional regulators associated with the plant- BMYs regulate the production of BMY enzyme. The enhancement in the expression of such genes reflects evolutionary significance. Bacterial genes, on the other hand, as exemplified by Bacillus sp and Clostridium sp, clearly depict the importance of a single regulatory gene, the absence or mutation of which totally abolishes the BMY activity.

Brief Insight into the Underestimated Role of Hop Amylases on Beer Aroma Profiles

The current trend in craft breweries is to carry out heavy dry-hopping by increasing the hopping rate. This practice sometimes leads to uncontrolled and aberrant aroma profile production. The aim of this work was to determine whether part of the enzymatic content of hop (α-amylase and β-amylase) could impact yeast metabolism, resulting in aroma profile modification during secondary fermentation. In this research, spectrophotometric methods were used to assess the amylase activity within hop. Moreover, liquid chromatographic methods (HPLC-ELSD) showed modification of the beer sugar profile by production of glucose and maltose as well as by the degradation of a higher degree of polymerization sugar by hop enzymes. Furthermore, gas chromatographic techniques (GC-ECD/FID) were used to assess yeast metabolism using vicinal diketones (diacetyl/pentanedione) as a marker of the secondary fermentation. Finally, a principal component analysis (PCA) of the yeast main aromas (esters, higher alcohols, and aldehydes) demonstrated the significance of this yeast-hop interaction on the beer’s aroma profile.

The effect of size and solid content in hydrolysis of sweet potato starch using endogenous beta-amylase enzyme

Sweet potato contains significant amount of endogenous amylase enzymes. The endogenous enzymes can be used in maltose production from sweet potatoes to carry out pre-hydrolysis of the starch to some extent, follow by complete hydrolysis using external enzymes. The main objective of the current study is to investigate the effect of particle size and solid to liquid ratio in hydrolysis process of sweet potato starch using endogenous enzymes. The extent of hydrolysis process is measured by dextrose equivalent of the crude maltose solution. Hydrolysis process consists of liquefaction and saccharification process. The liquefaction process is carried out at 71.5°C and pH 6 for 25 minutes while saccharification process is carried out at 53°C and pH 5,5 for 72 hours. Dextrose equivalent is measured using Lane-Eynon method. The results show that the access of enzyme to the potato tubers is the key to hydrolysis process. The smaller size of tubers gives bigger surface area while lower solid-to-liquid ratio increases enzyme mobility but decreases sugar concentration. Base on the results, a simulation shows that the pre-hydrolysis of sweet potatoes using endogenous enzymes can save almost a half of enzymes required for traditional hydrolysis process.

Adsorption of α-amylase and Starch on Porous Zinc Oxide Nanosheet: Biophysical Study

Engineered biocatalyst and its desired products using nanotechnology has intensified the research in food industries. Zinc oxide (ZnO) nanosheet is designed and prepared; the characterization studies include surface plasmon resonance peak (364 nm), X-ray diffraction pattern determined crystallite size (25 nm), and transmission electron microscopy confirms the porous surface nature. Atomic force microscopy showed substrate and enzyme adsorbed on ZnO nanosheets. The zeta potential of ZnO nanosheet (−41.9 mV) whereas α-amylase bound with ZnO nanosheets (−32.8 mV), and starch bound with ZnO nanosheets (−28.7 mV) was analyzed using dynamic light scattering. The circular dichroism spectra displayed α-helix in native amylase at optimum concentration 54.70% compared to the adsorbed α-amylase with ZnO nanosheet that showed 37%. Freundlich isotherm model revealed multilayer adsorption behavior of α-amylase onto porous ZnO nanosheet. Enzyme kinetics study presents alteration in Michaelis–Menten constant (Km) and maximum velocity (Vmax), the α-amylase bound with porous ZnO nanosheet showed a reduction in Km and Vmax. The substrate and enzyme adsorbed together on porous ZnO nanosheet exhibited increased Km (27.77 μM), whereas Vmax (2.85 μM) remains unchanged. Moreover, α-amylase once modified at optimum pH (5.8) and temperature (52 °C), produces less maltose than α-amylase adsorbed on ZnO nanosheet, which indicates higher maltose production. In this study, ZnO nanosheet enzyme catalytic system was created, wherein enzymatic reaction shifted to different pH and temperature other than optimum conditions. All these findings suggest that careful attention to the enzyme adsorption profiles can contribute to industrial applications.

Immobilization of α-amylase on GO-magnetite nanoparticles for the production of high maltose containing syrup

Robust amylases with stability and catalysis at multitude of extremities are the need of an hour. Enzyme immobilization may prove beneficial at commercial scale to achieve such attributes. In the present study, a commercially available amylase was immobilized on graphene oxide (GO) – magnetite (Fe3O4) nanoparticles through covalent bonding. The structural and morphological characterizations were conducted by XRD, SEM and TEM. Further, FTIR and TGA confirmed the interaction between amylase, GO and nanoparticles. The variables, such as concentrations of GO (1.3 mg), Fe3O4 (58 μg), and amylase (4.5 mg) were optimized by the response surface methodology using central composite design. High loading capacity of 77.58 μg amylase over 1 μg GO-magnetite nanoparticles was achieved under optimum conditions. Biochemically, the pH optimum remained unaltered, i.e., pH 7, whereas, the alkalitolerance was increased by ~20% in relative activities upon immobilization. The half-life of soluble amylase was 13 h, which enhanced to 20 h upon immobilization in 20 mM phosphate buffer, pH 7 at 50 °C. Besides, the thermodynamic parameters supported the stability trends. The immobilized amylase could be used for 11 subsequent cycles. The mentioned attributes and the dextrose equivalent values during the production of high maltose containing syrup highlighted its commercialization.

Improve Production of Pullulanase of Bacillus subtilis in Batch and Fed-Batch Cultures.

Pullulanase is a debranching enzyme that cleaves explicitly α-1,6 glycosidic bonds, which is widely used in starch saccharification, production of glucose, maltose, and bioethanol. The thermal-resistant pullulanase is isolated from a variety of microorganisms; however, the lack of industrial production of pullulanase has hindered the transformation of the laboratory to industry. In this study, the expensive maltose syrup and soybean meal powder were replaced with cheap corn starch and corn steep liquor, exhibiting 440 U/mL of pullulanase in shake flasks by changing the C/N value and the total energy of the medium. Subsequently, the cultivation conditions were explored in a 50-L and 50-m3 bioreactor. In batch culture, the pullulanase activity reached 896 U/mL, while it increased to 1743 U/mL in fed-batch culture by controlling the dissolved oxygen, pH, reducing sugar content, and temperature. Remarkably, the cultivation volume was enlarged to 50 m3 based on the technical parameters of fed-batch culture. The industrial production of pullulanase was successful, and the activity achieved 1546 U/mL. When the product was stored at room temperature (25 °C) for 6 months, the pullulanase activity was over 90%. The half-lives at 60 and 80 °C were 119.45 h and 51.18 h, respectively, which satisfied the industrial application requirements of pullulanase.

Importance of oral phase in in vitro starch digestibility related to wholegrain versus refined pastas and mastication impairment

Starch represents the main source of carbohydrates in human diet and its digestibility is suspected to be involved in the control of glycemic response. The low glycemic index caused by pastas is mainly attributed to the starch-protein network constituting their compact structure. A significant part of the physico-chemical digestive process probably occurs during mastication with exposure to amylase. However, the respective accountability of oral and intestinal phases in digestion is not clearly established, and this knowledge would especially benefit to health management of people suffering of impaired mastication. Food boluses were produced for in vitro gastrointestinal digestion either by in vitro normal (NM) or deficient mastication (DM) of wholegrain and refined pastas. Boluses were first characterized for physical properties. Many large particles were obtained in DM boluses whatever the pastas. Starch and hydrolysis products were then determined in boluses and gastrointestinal digestas. The beginning of starch hydrolysis was confirmed in the mouth with a production of maltose in the NM boluses, around 1.6 g/100g of cooked pastas, significantly decreased to 1.2 g/100g in the DM boluses, whatever the pastas. Even if the negative effect of DM on gastrointestinal starch hydrolysis into glucose was observed for both pastas, the greatest impact occurred in refined ones with 55.9 ± 0.82 g glucose/100g pastas after NM versus 53.00 ± 0.95 g/100g after DM. This study highlighted the importance to consider the oral phase in digestion studies, regarding the impact of food structure and oral disruption, and especially in case of DM.

A Continuous Fluorescent Assay for β‐Amylase Activity

The production and degradation of starch is key for the survival of plants during the day/night cycle. The stored energy is harnessed through the work of many different enzymes including the β‐Amylase (BAM) family of proteins. BAMs break down long chains of starch into the disaccharide maltose (4‐O‐α‐D‐Glucopyranosyl‐D‐glucose). The model organism Arabidopsis thaliana has 9 β‐Amylase proteins with apparently distinct functions, though not all have the ability to hydrolyze starch. Each BAM seems to have its own unique characteristics, although the specific functional differences are not clear. The objective of our research is to create an assay that can show the kinetics of BAM proteins, that is continuous and sensitive while able to work with synthetic and natural substrates of the BAM proteins. We aim to apply this assay to determine the unique biochemical features of the BAMs. To detect BAM activity, we used a maltose binding sensor composed of Maltose Binding Protein fused to Green Fluorescence Protein (MBP‐GFP). When maltose is bound to MBP it causes conformational changes to GFP resulting in fluorescence. We have tested the assay with maltodextrin chains from 4 to 20 residues in length and with soluble starch finding that we can detect maltose production from all of these substrates with the MBP‐GFP biosensor in the single micromolar range. Moreover, we can detect amylase activity in a continuous measurement format using a plate reader allowing for higher throughput sampling with reduced assay time.Support or Funding InformationNSF‐RUI MCB‐1616467 to J.M.

Nano co-immobilization of α-amylase and maltogenic amylase by nanomagnetic combi-cross-linked enzyme aggregates method for maltose production from corn starch

Starch hydrolysis to maltose by nano-magnetic combined cross-linked enzyme aggregates of α-amylase and maltogenic amylase (NM-Combi-CLEAs) is an important step to open new perspectives for special food and pharmaceutic production. Improvement of mass transfer, thermostability, functional specificity, and reusability of combined enzymes was performed. The obtained results exhibited that, 1:9 ratio of α-amylase/maltogenic amylase, use of tert-butanol as precipitant, 2 mM glutardialdehyde, 1:0.75 ratios of combined enzymes to lysine, 20 h crosslinking at 3–4 °C are well-suited conditions. The dynamic light scattering (DLS) results implied that the nanomagnetites diameter was about 81.9–88.9 nm, with polydispersity index (PDI) of 0.242 and a Ȥ-potential of −21 mV. Moreover, the particle size, PDI, and Ȥ-potential of NM-Combi-CLEAs were around 99.6 nm, 0.088, and −32 mV respectively. The NM-Combi-CLEAs kept 80.4% of its original activity after 10 cycles, its Km value exhibited about 1.5 folds reduction with about 1.5 times enhance in thermostability at 95 °C than free one. Immobilization activity yield revealed about 84% of activity retaining by NM-Combi-CLEAs strategy. Accordingly, this efficacious nanobiocatalyst with high thermostability and reusability recommended for starch conversion to maltose.

Encapsulation of β-amylase in water-oil-water enzyme emulsion liquid membrane (EELM) bioreactor for enzymatic conversion of starch to maltose

The β-amylase was encapsulated in emulsion liquid membrane (ELM), which acted as a reactor for conversion of starch to maltose. The membrane phase was consisted of surfactant (span 80), stabilizer (polystyrene), carrier for maltose transport (methyl cholate) and solvent (xylene). The substrate starch in feed phase entered into the internal phase by the process of diffusion and hydrolyzed to maltose by encapsulated β-amylase. Methyl cholate present in the membrane acts as a carrier for the product maltose, which helps in transport of maltose to feed phase from internal aqueous phase. The residual activity of β-amylase after the five-reaction cycle was found to decrease to ∼70%, which indicated possibility to recycle the components of the emulsion and enzyme. The pH and temperature of the encapsulated enzyme were found to be optimum at 5.5 and 60 °C, respectively. The novelty of the present work lies in the development of Enzyme Emulsion Liquid Membranes (EELM) bioreactor for the hydrolysis of starch into maltose mediated by encapsulated β-amylase. The attempt has been made for the first time for the successful encapsulation of β-amylase into EELM. The best results gave the highest residual enzyme activity (94.1%) and maltose production (29.13 mg/mL).

Immobilization of fenugreek β-amylase onto functionalized graphene quantum dots (GQDs) using Box-Behnken design: Its biochemical, thermodynamic and kinetic studies

β-Amylase was immobilized onto GQDs using 3-aminopropyltriethoxysilane and glutaraldehyde. Optimization was carried out by Box-Behnken design and binding was confirmed by SEM, AFM, FTIR and fluorescence microscopy. Predicted optimum immobilization efficiency (88.64%) was very close to actual (87.98%), which confirmed the success of the immobilization process. The immobilized enzyme showed maximum activity at pH 5.0 and 57 °C, whereas Km and Vmax were found to be 6.40 mg/mL and 714.28 μmol/min/mg, respectively. The enzyme retained 75% activity after 12 uses at 30 °C. Increased values of ΔG° ΔH°, half-life and activation energy of the enzyme inactivation (ΔEd) revealed that thermo-stability increases after immobilization and the process followed first-order kinetics (r2 > 0.96). The activation energy of catalysis (ΔEa) and ΔEd for immobilized enzyme were 22.58 and 158.99 ± 1.10 kJ/mol, respectively which revealed that denaturation of the enzyme requires a higher amount of energy rather than catalysis. Thermodynamic and fluorescence spectroscopic studies revealed that the process is non-spontaneous (ΔG > 0) and endothermic (ΔH > 0) and occurred through protein unfolding rather than aggregation (ΔS > 0). Thus increase in thermo-stability of immobilized fenugreek β-amylase and non-toxic nature of GQDs could be exploited for maltose production in beverage, food and pharmaceutical industries.