New chromogenic substrates for the assay of alpha-amylase and (1-4)-β-D-glucanase.
McCleary, B. V. (1980). Carbohydrate Research, 86(1), 97-104.
New chromogenic substrates have been developed for the quantitative assay of alpha-amylase and (1→4)-β-D-glucanase. These were prepared by chemically modifying amylose or cellulose before dyeing, to increase solubility. After dyeing, the substrates were either soluble or could be readily dispersed to form fine, gelatinous suspensions. Assays based on the use of these substrates are sensitive and highly specific for either alpha-amylase or (1→4)-β-D-glucanase. The method of preparation can also be applied to obtain substrates for other endo-hydrolases.
Comparison of endolytic hydrolases that depolymerise 1,4-β-D-mannan, 1,5-α-L-arabinan and 1,4-β-D-galactan.
McCleary, B. V. (1991). “Enzymes in Biomass Conversion”, (M. E. Himmel and G. F. Leatham, Eds.), ACS Symposium Series 460, Chapter 34, pp. 437-449. American Chemical Society, Washington.
Hydrolysis of mannan-type polysaccharides by β-mannanase is dependent on substitution on and within the main-chain as well as the source of the β-mannanase employed. Characterisation of reaction products can be used to define the sub-site binding requirements of the enzymes as well as the fine-structures of the polysaccharides. Action of endo-arabinanase and endo-galactanase on arabinans and arabinogalactans is described. Specific assays for endo-arabinanase and arabinan (in fruit-juice concentrates) are reported.
Measurement of polysaccharide degrading enzymes using chromogenic and colorimetric substrates.
McCleary, B. V. (1991). Chemistry in Australia, 58, 398-401.
Enzymic degradation of carbohydrates is of major significance in the industrial processing of cereals and fruits. In the production of beer, barley is germinated under well defined conditions (malting) to induce maximum enzyme synthesis with minimum respiration of reserve carbohydrates. The grains are dried and then extracted with water under controlled conditions. The amylolytic enzymes synthesized during malting, as well as those present in the original barley, convert the starch reserves to fermentable sugars. Other enzymes act on the cell wall polysaccharides, mixed-linkage β-glucan and arabinoxylan, reducing the viscosity and thus aiding filtration, and reducing the possibility of subsequent precipitation of polymeric material. In baking, β-amylase and α-amylase give controlled degradation of starch to fermentable sugars so as to sustain yeast growth and gas production. Excess quantities of α-amylase in the flour result in excessive degradation of starch during baking which in turn gives a sticky crumb texture and subsequent problems with bread slicing. Juice yield from fruit pulp is significantly improved if cell-wall degrading enzymes are used to destroy the three-dimensional structure and water binding capacity of the pectic polysaccharide components of the cell walls. Problems of routine and reliable assay of carbohydrate degrading enzymes in the presence of high levels of sugar compounds are experienced with such industrial process.
Optimising the response.
Acamovic, T. & McCleary, B. V. (1996). Feed Mix, 4, 14-19.
A fine balance exists between enzyme activity and the adverse effects associated with feed processing. Accurate estimation of enzyme activity in the feed is a pre-requisite to optimising the response.
Measurement of α-Amylase in Cereal, Food and Fermentation Products.
McCleary, B. V. & Sturgeon, R. (2002). Cereal Foods World, 47, 299-310.
In General, the development of methods for measuring α-amylase is pioneered in the clinical chemistry field and then translated to other industries, such as the cereals and fermentation industries. In many instances, this transfer of technology has been difficult or impossible to achieve due to the presence of interfering enzymes or sugars and to differences in the properties of the enzymes being analysed. This article describes many of the commonly used methods for measuring α-amylase in the cereals, food, and fermentation industries and discusses some of the advantages and limitations of each.
Analysis of deoxynivalenol and deoxynivalenol-3-glucoside in wheat.
Simsek, S., Burgess, K., Whitney, K. L., Gu, Y. & Qian, S. Y. (2012). Food Control, 26(2), 287-292.
Deoxynivalenol (DON) is a mycotoxin which can be produced in cereal grains infected by Fusarium Head Blight (FHB). Alteration of DON by the plant often involves conjugation of the respective mycotoxin to certain functional groups or molecules. Conjugation of DON with glucose results in Deoxynivalenol-3-β-D-glucopyranoside (D3G), which has been found to be the main DON metabolite in wheat. The objective of this research was to identify the fate of D3G and DON during wheat processing using LC–MS–MS and GC, respectively. There was an approximate reduction of 61.8% in the detected DON level of the flour compared to the whole wheat. DON levels detected during the fermentation stage were significantly higher (P < 0.05) than the mixed dough. D3G detected in the flour was 23.7% lower than detected in the whole wheat. There were no significant differences (P < 0.05) in the D3G detected in the dough samples. However, the baked bread had significantly (P < 0.05) less D3G detected than the dough. Experiments were conducted to determine the effect of enzyme hydrolysis on DON detection in whole wheat. There were significant differences (P < 0.05) between the wheat treated with α-amylase, cellulase, protease, and xylanase. DON detection levels were significantly (P < 0.05) higher after treatment with protease (16%) and xylanase (39%) compared to the wheat composite. These results suggest that DON may be bound or embedded to the cell wall matrix or protein component of the wheat kernel due to the rise in detection of DON after these enzyme treatments.
Residual amylopectin structures of amylase-treated wheat starch slurries reflect amylase mode of action.
Leman, P., Goesaert, H. & Delcour, J. A. (2009). Food hydrocolloids, 23(1), 153-164.
Some amylases can delay bread staling and/or starch (amylopectin) retrogradation, but the molecular basis of this effect remains little understood. In order to increase our insight in these aspects of amylase functionality, several amylases were added in a pure wheat-starch-containing model system and subjected to a heating step corresponding to that in the baking phase in bread making. Next, the effects of the limited amylolytic degradation on the rapid visco analyser (RVA) rheological properties of starch were studied and the accompanying changes in the amylopectin molecular properties (such as chain length distribution) investigated. The different amylases clearly affected the molecular structure of amylopectin to a different extent, which could be related to their mode of action and the enzyme activity levels added. Bacillus subtilis and Aspergillus oryzae α-amylases had only a limited impact on the side chain distribution of the amylopectin molecules, presumably due to their preferential hydrolysis of internal chain segments and the low enzyme activity added in the RVA. In contrast, porcine pancreatic α-amylase and Bacillus stearothermophilus maltogenic α-amylase, both with higher degree of multiple attack and used at higher enzyme activity levels, had a marked influence on the amylopectin molecular structure. More in particular, under the test conditions, the maltogenic α-amylase reduced the average chain length of the outer chains by 50%. Presumably, this will affect amylopectin retrogradation to a large extent. The results contribute to a better understanding of amylase functionality in starchy foods.