Use of pullulanase as a biocatalyst for starch hydrolysis: Part 1. Study of the effect of pullulanase on maize amylopectin starch

The use of debranching enzymes in starch hydrolysis is a topical direction for obtaining new types of starch products with controlled properties and a potential for the further use. The aim of the work was to study an effect of pullulanase (EC3.2.1.41) on maize amylopectin starch in the native and gelatinized state. The objects of the research were maize amylopectin starch and enzyme preparation Promozyme D6 (Novozymes, Denmark). High-performance liquid chromatography (HPLC) was used to determine the carbohydrate composition of hydrolysates. The mass fraction of reducing substances (RS) was determined by the Lane and Eynon method. A rotational viscometer was used to measure dynamic viscosity of the starch hydrolysis products. It was found that analyzed starch in the native state showed low enzymatic sensitivity to the action of pullulanase with insignificant changes in viscosity, solubility and iodine binding capacity of the samples. Pullulanase showed the highest effect on gelatinized starch during the first eight hours of incubation. After eight hours, the maximum degree of starch hydrolysis by pullulanase at a dose of 10 units/g dry matter (DM) was 4.7% on DM basis, iodine binding capacity of the hydrolysate was D600 = 0.343 (in the control experiment D600 = 0.154), and the viscosity of the hydrolysate decreased from 7887 mPa · s to 4.3 mPa · s. Hydrolysates cooled to 8 °C and held for 20 hours along with hydrolysates that were not cooled showed high susceptibility to attack by glucoamilase (97–98%) at 60 °C and 24 hours of saccharification, which suggested the absence of their resistance to the action of glucoamilase in the conditions of the experiment. The use of pullulanase in dextrinization of the analyzed starch, which was gelatinized and partly hydrolyzed by α-amylase (RS6.1%), enabled obtaining hydrolysates with the mass fraction of reducing substances in a range of 10–24% on DM basis with the process duration of 2 to 24 hours and the enzyme dose of 2–10 units, which contained mainly maltotriose, maltohexose and maltoheptose with their total amount of 45–60% on DM basis. The results indicate a need for further research of the biocatalytic action of pullulanase to develop new methods for enzymatic modification of starch.

Physiological and Biochemical Characterization of Isolated Bacteria from a Coccolithophore Chrysotila dentata (Prymnesiophyceae) Culture

Coccolithophores are involved in oceanic carbon and nitrogen cycles, and they also have an impact on global climate change. Chrysotila dentata have a complex and close relationship with phycosphere bacteria. In this study, culturable phycosphere bacteria (free-living bacteria and attached bacteria) are isolated from C. dentata by a gradient dilution method and identified based on the 16S rRNA gene sequencing analysis. The phylogenetic tree (neighbor-joining tree, N-J tree) was constructed using the bacterial sequences and closest related sequences from GenBank. Colony characteristics, Gram nature, and physiological and biochemical characteristics were obtained based on a series of tests, such as the sugar utilization (glucose, arabinose, xylose, maltose, and mannitol) test, Voges–Proskauer reaction, urease tests, gelatin liquefaction, Gram test, starch hydrolysis, among others. In this study, seven strains (CF1, CF2, CF3, CF5, CF6, and CF7) of free-living bacteria (CF) and five strains (CA1, CA2, CA3, CA4, and CA5) of attached bacteria (CA) are isolated and identified. We found that the culturable phycosphere bacteria of C. dentata were mainly α-proteobacteria and γ-proteobacteria, with a small part of the CFB (Cytophaga-Flexibacter-Bacteroides) group bacteria and firmicutes. In this study, most α-proteobacteria can utilize malonate and positive in the urease test, meanwhile they can grow in a 7% NaCl medium. Differently to α-proteobacteria, γ-proteobacteria are more reactive, and can utilize maltose, glucose, arabinose, malonate, aesculin, and starch hydrolysis. Meanwhile, γ-proteobacteria can growth in a 7% NaCl and pH 5.7 medium, and some bacteria of this strain were positive in nitrate reduction. Firmicutes are similar to γ-proteobacteria: they are similar in reactivity, as they can utilize maltose, glucose, arabinose, malonate, aesculin, and starch hydrolysis, and can growth in a 7% NaCl and pH 5.7 medium. The difference is that some of firmicutes were positive in gelatin liquefaction and can utilize mannitol. The CFB group of bacteria were only positive in malonate, aesculin, and starch hydrolysis. The above results provide basic experimental data for further studies on the relationship between the coccolithophores and culturable phycosphere bacteria.

The Procyanidin C1-Dependent Inhibition of the Hydrolysis of Potato Starch and Corn Starch Induced by Pancreatin

Procyanidins are contained in various foods, and their effects on starch hydrolysis have been reported. In Japan, black soybeans, which contain a trimeric procyanidin, procyanidin C1 (proC1), are cooked with rice and used to prepare dumplings. In this study, the effects of proC1 on the pancreatin-induced formation of reducing sugars and starch hydrolysis were studied using potato starch and corn starch. ProC1 inhibited both reactions; the inhibition was greater in potato starch than corn starch when added to heated potato starch and corn starch. When heated with proC1, its inhibitory effects decreased, especially in potato starch, suggesting the important role of proC1 itself for the inhibition of potato starch hydrolysis. ProC1 also inhibited the hydrolysis when added to heated, longer amylose (average molecular weight: 31,200), and the inhibition decreased when heated with the amylose. On the other hand, proC1 could not inhibit the hydrolysis when added to heated, shorter amylose (average molecular weight: 4500), but could when heated with the amylose, suggesting the important role of the degradation products of proC1 for the inhibition. We discuss the mechanism of the proC1-dependent inhibition of amylose hydrolysis, taking the molecular weight into account.