Amyloglucosidase (Rhizopus sp.)

A study was made of the effect of the activity and purity of enzymes in the assay of total dietary fiber (AOAC Method 985.29) and specific dietary fiber components: resistant starch, fructan, and β-glucan. In the measurement of total dietary fiber content of resistant starch samples, the concentration of α-amylase is critical; however, variations in the level of amyloglucosidase have little effect. Contamination of amyloglucosidase preparations with cellulase can result in significant underestimation of dietary fiber values for samples containing β-glucan. Pure β-glucan and cellulase purified from Aspergillus niger amyloglucosidase preparations were used to determine acceptable critical levels of contamination. Sucrose, which interferes with the measurement of inulin and fructooligosaccharides in plant materials and food products, must be removed by hydrolysis of the sucrose to glucose and fructose with a specific enzyme (sucrase) followed by borohydride reduction of the free sugars. Unlike invertase, sucrase has no action on low degree of polymerization (DP) fructooligosaccharides, such as kestose or kestotetraose. Fructan is hydrolyzed to fructose and glucose by the combined action of highly purified exo- and endo-inulinases, and these sugars are measured by the p-hydroxybenzoic acid hydrazide reducing sugar method. Specific measurement of β-glucan in cereal flour and food extracts requires the use of highly purified endo-1,3:1,4 β-glucanase and A. niger β-glucosidase. β-glucosidase from almonds does not completely hydrolyze mixed linkage β-glucooligosaccharides from barley or oat β-glucan. Contamination of these enzymes with starch, maltosaccharide, or sucrose-hydrolyzing enzymes results in production of free glucose from a source other than β-glucan, and thus an overestimation of β-glucan content. The glucose oxidase and peroxidase used in the glucose determination reagent must be essentially devoid of catalase and α- and β-glucosidase.

Dietary fiber is mainly composed of plant cell wall polysaccharides such as cellulose, hemicellulose, and pectic substances, but it also includes lignin and other minor components (1). Basically, it covers the polysaccharides that are not hydrolyzed by the endogenous secretions of the human digestive tract (2,3). This definition has served as the target for those developing analytical procedures for the measurement of dietary fiber for quality control and regulatory considerations (4). Most procedures for the measurement of total dietary fiber (TDF), or specific polysaccharide components, either involve some enzyme treatment steps or are mainly enzyme-based. In the development of TDF procedures such as the Prosky method (AOAC International 985.29, AACC 32—05) (5), the Uppsala method (AACC32-25) (6), and the Englyst method (7), the aim was to remove starch and protein through enzyme treatment, and to measure the residue as dietary fiber (after allowing for residual, undigested protein and ash). Dietary fiber was measured either gravimetrically or by chemical or instrumental procedures. Many of the enzyme treatment steps in each of the methods, particularly the prosky (5) and the Uppsala (6) methods are very similar. As a new range of carbohydrates is being introduced as potential dietary fiber components, the original assay procedures will need to be reexamined, and in some cases slightly modified, to ensure accurate and quantitative measurement of these components and of TDF. These “new” dietary fiber components include resistant nondigestible oligosaccharides; native and chemically modified polysaccharides of plant and algal origin (galactomannan, chemically modified celluloses, and agars and carrageenans); and resistant starch. To measure these components accurately, the purity, activity, and specificity of the enzymes employed will become much more important. A particular example of this is the mesurement of fructan. This carbohydrate consists of a fraction with a high degree of polymerization (DP) that is precipitated in the standard Prosky method (5,8) and a low DP fraction consequently is not measured (9). Resistant starch poses a particular problem. This component is only partially resistant to degradation by α-amylase, so the level of enzyme used and the incubation conditions (time and temperature) are critical.

An enzyme-linked assay for the measurement of amyloglucosidase in commercial enzyme mixtures and crude culture filtrates is described. A method for the synthesis of the substrate employed, p-nitrophenyl β-D-maltoside, is also described. The substrate is used in the presence of saturating levels of β-glucosidase. With a range of Aspergillus sp. culture filtrates, an excellent correlation was found for values obtained with this assay and a conventional assay employing maltose as substrate with measurement of released glucose.

Culture filtrates of Cladosporium resinae ATCC 20495 contain a mixture of enzymes able to convert starch and pullulan efficiently into D-glucose. Culture conditions for optimal production of the pullulan-degrading activity have been established. The amylolytic enzyme preparation was fractionated by ion-exchange and molecular-sieve chromatography, and shown to contain α-D-glucosidase, α-amylase, and two glucoamylases. The glucoamylases have been purified to homogeneity and their substrate specificities investigated. One of the glucoamylases (termed P) readily hydrolyses the (1→6)-α-D linkages in pullulan, amylopectin, isomaltose, panose, and 6³-α-D-glucosylmaltotriose. Each of the glucoamylases cleaves the (1→6)-α-D linkage in panose much more readily than that in isomaltose.

BACKGROUND Carbohydrates as starch are a staple part of the Mediterranean diet. Starch is digested in the small intestine and the resulting glucose is absorbed into the blood, eliciting an insulin response. The digestion and absorption kinetics (rapid or slow) depends on starch structure. OBJECTIVE To study the relationship between the in vivo glycemic and insulinemic index and the in vitro digestibility characteristics of six bakery products, made from non-conventional wholemeal/wholegrain flours. METHODS We analyzed in vitro the rapidly- and slowly- available glucose (RAG and SAG), the rapidly- and slowly- digestible starch (RDS and SDS), and the resistant starch (RS) fraction of the six wholemeal/wholegrain products and one white type of bread. The glycemic and the insulinemic index (GI and II respectively) were estimated by in vivo testing in a group of eleven healthy individuals. RESULTS The GI of the wholemeal/wholegrain flour biscuits and breads were low, (range 28±3.2 to 41±3.9, Mean±SEM) correlating with the II. RAG positively correlated with both GI and II, with fiber having a marginal correlation. CONCLUSIONS Our findings indicate that both conventional and non-conventional wholemeal/wholegrain bakery products have low GI and moderate II, correlating to in vitro starch digestibility and the type of processing.

Carbon allocation between source and sink organs determines plant growth and is influenced by environmental conditions. Under water deficit, plant growth is inhibited before photosynthesis and shoot growth tends to be more sensitive than root growth. However, the modulation of source-sink relationship by rootstocks remain unsolved in citrus trees under water deficit. Citrus plants grafted on Rangpur lime are drought tolerant, which may be related to a fine coordination of the source-sink relationship for maintaining root growth. Here, we followed ¹³C allocation and evaluated physiological responses and growth of Valencia orange trees grafted on three citrus rootstocks (Rangpur lime, Swingle citrumelo and Sunki mandarin) under water deficit. As compared to plants on Swingle and Sunki rootstocks, ones grafted on Rangpur lime showed higher stomatal sensitivity to the initial variation of water availability and less accumulation of non-structural carbohydrates in roots under water deficit. High ¹³C allocation found in Rangpur lime roots indicates this rootstock has high sink demand associated with high root growth under water deficit. Our data suggest that Rangpur lime rootstock used photoassimilates as sources of energy and carbon skeletons for growing under drought, which is likely related to increases in root respiration. Taken together, our data revealed that carbon supply by leaves and delivery to roots are critical for maintaining root growth and improving drought tolerance, with citrus rootstocks showing differential sink strength under water deficit.

Potato cell walls (PCW) are a low value by-product from the potato starch industry. Valorisation of PCW is hindered by its high water-binding capacity (WBC). The composition of polysaccharides and interactions between these entities, play important roles in regulating the WBC in the cell wall matrix. Here, we show that in vivo exo-truncation of RG-I β-(1→4)-D-galactan side-chains decreased the WBC by 6-9%. In contrast, exo-truncation of these side-chains increased the WBC by 13% in vitro. We propose that degradation of RG-I galactan side-chains altered the WBC of PCW, due to cell wall remodelling and loosening that affected the porosity. Our findings reinforce the view that RG-I galactan side-chains play a role in modulating WBC, presumably by affecting polysaccharide architecture (spacing) and interactions in the matrix. Better understanding of structure-function relationships of pectin macromolecules is needed before cell wall by-products may be tailored to render added-value in food and biobased products.

Instead of β-cyclodextrin (β-CD), branched β-CDs have been increasingly used in many aspects as they possess better solubility and higher bioadaptability. But most commercialized branched β-CDs were chemically synthesized. Thus, the glucosyl-β-cyclodextrin (G₁-β-CD) prepared via enzymatic approach could be a nice substitute. However, the yield of G₁-β-CD was low. Here, we reported a controlled two-step reaction to efficiently prepare G₁-β-CD from maltodextrins by β-cyclodextrin glucosyltransferase (β-CGTase) and amyloglucosidase (AG). Compared to the single β-CGTase reaction, controlled two-step reaction caused a yield increase of G₁-β-CD by 130%. Additionally, the percentage of G₁-β-CD was enhanced from 2.4% to 24.0% and the side products α-CD and γ-CD were hydrolyzed because of the coupling activity of β-CGTase. Thus, this controlled two-step reaction might be an efficient approach for industrial production of pure G₁-β-CD.

In comparison with natural α- and β-cyclodextrin (CD), γ-CD has attracted much attention due to their large hydrophobic cavities, high water solubility, and bioavailability. However, the production of γ-CD is still rather expensive and time-consuming. To overcome the high cost and long induction time, pUC119 was selected as the gene expression vector, and the recombinant enzyme production time was reduced to 8 h from 72 h. Furthermore, for the first time, we have successfully produced γ-CD using β-CD by cyclodextrin opening reactions through the recombinant CGTase in the presence of maltose. The kinetic mechanism of the coupling reaction was investigated. Moreover, the production of γ-CD could be affected by several key parameters, such as solvent type, reaction time, pH, and temperature. A maximum γ-CD yield of 32.9% was achieved by recombinant CGTase in the presence of 5-cyclohexadecen-1-one. This could be a promising method for the industrial production of γ-CD.

Tree species from the Amazonian floodplains have to cope with low oxygen availability due to annual pulses of inundation that can last up to 7 months. Species capable of adapting to flooding and/or waterlogged conditions usually partition their storage to favor starch and allocate it to roots, where carbohydrates are used to maintain respiration rates during waterlogging. In spite of climate change, virtually nothing is known about how elevated atmospheric CO₂ concentration ([CO₂]) will affect plants when combined with waterlogging. In this work, we used open top chambers to evaluate the effect of elevated [CO₂] during a period of terrestrial phase and in subsequent combination with waterlogged conditions to determine if the surplus carbon provided by elevated [CO₂] may improve the waterlogging tolerance of the fast-growing Amazonian legume tree Senna reticulata. During the terrestrial phase, photosynthesis was ca. 28 % higher after 30, 45 and 120 days of elevated [CO₂], and starch content in the leaves was, on average, 49 % higher than with ambient [CO₂]. Total biomass was inversely correlated to the starch content of leaves, indicating that starch might be the main carbohydrate source for biomass production during the terrestrial phase. This response was more pronounced under elevated [CO₂], resulting in 30 % more biomass in comparison to ambient [CO₂] plants. After 135 days at elevated [CO₂] an inversion has been observed in total biomass accumulation, in which ambient [CO₂] presented a greater increment in total biomass in comparison to elevated [CO₂], indicating negative effects on growth after long-term CO₂ exposure. However, plants with elevated [CO₂]/waterlogged displayed a greater increment in biomass in comparison with ambient [CO₂]/waterlogged that, unlike during the terrestrial phase, was unrelated to starch reserves. We conclude that S. reticulata displays mechanisms that make this species capable of responding positively to elevated [CO₂] during the first pulse of growth. This response capacity is also associated with a “buffering effect” that prevents the plants from decreasing their biomass under waterlogged conditions.