Identification of α-amylase inhibitors in triticale grain

In this study three complete triticales, three substituted (one gene from rye has been replaced by one gene from wheat) triticales, and parental wheat and rye were analysed for α-amylase inhibitory activity to evaluate whether the genetic modification influenced triticale α-amylase inhibitory activity. Gel filtration chromatography and thermostability analyses were performed to partially isolate and characterize α-amylase inhibitors. Results demonstrated that substituted triticales and wheat had higher α-amylase inhibitory activities and higher water-soluble protein contents than complete triticales and rye. Sodium dodecylsulfate-PAGE-electrophoresis showed that all triticales, irrespective of their classification, inherited the water-soluble protein patterns from their parents: wheat and rye. In a substituted triticale (Pony ‘S’), two peaks with α-amylase inhibitory activity were resolved by gel filtration chromatography; they were designated T1 and T2 according to their order of elution. T1 showed a higher inhibitory activity but a lower thermostability at 70 °C than T2; T1 apparently comes from wheat, whereas T2 presumably comes from rye. © 1999 Society of Chemical Industry     

The α-amylase, α-amylase inhibitor and proteinase inhibitor activities of proteins extracted from wheat varieties and their influence on the baking quality

The grain of six winter and spring wheat varieties, harvested in 1987, differing in baking quality were included in the study. A number of technological analysis were performed. Albumins, globulins and gliadins were washed out. The location of inhibitory activities in extracted proteins, against α-amylases of various origin. were tested and compared to the baking quality of the wheat grain. Also, the location of antitryptic activity and endogenous amylase activity in extractable proteins was compared to baking quality factors. Among the studied inhibitory activities against mammalian α-amylases some correlation were found between the inhibition activity of hog pancreas α-amylase and sedimentation test (r = 0.88). Furthermore inhibitory activities of wheat proteins towards insects α-amylase shown significant correlation between the inhibitors of Anagasta kuehniella α-amylase and sedimentation test (r = 0.90). as well as crude protein content (r = 0.86). Also the inhibition activity against Sitophilus granarius α-amylase versus sedimentation test presented some correlation (r = 0.80). Endogenous amylase activities (α- and β-amylase) have an inversely proportional correlation to crude protein content (r = ?0.82) and sedimentation test (r = ?0.85).     

Electrophoretic studies of α-amylase in wheat. II

The lack of correlation between the degree of sprouting and the α-amylase activity in wheat and rye, as well as the apparent variation in the falling number during ripening, can be explained as the result of two amylase systems acting during different stages of the development of the grain. During the early stages of the development of the grain, α-amylase is continuously inactivated. This process is reversible, however, and when the evaporation of moisture is retarded the α-amylase activity increases as a consequence of the higher amount of dissolved enzyme. This results in an occasional decrease in the falling number, which might amount to more than 50 sec. During germination, a new kind of α-amylase develops. The synthesis of the new form of α-amylase is irreversible and causes a much greater and permanent reduction in the falling number. During the initial stages of germination, the amylase activity thus increases owing to the combined action of both the original amylase and the new form. The two kinds of amylases show different electrophoretic patterns. Drying the grain after harvesting reduces the activity of ‘green’ amylase (amylase in unripened grain), which might explain the frequent observations of increasing falling number during storage.     

Control of wheat α-amylase using inhibitors from cereals

A survey of 46 varieties of cereals and related species (including 27 different species from the Poaceae) indicated the presence of a strong inhibitor of wheat α-amylase in all seven Hordeum species tested. Rye contained a lower level of inhibitor activity, but the other species contained insignificant amounts of wheat α-amylase inhibitor activity. The partially purified barley inhibitor was most effective in inhibiting wheat α-amylase activity at high pH. The addition of chromosome 2 of barley to wheat (Chinese Spring addition line 2H) resulted in an apparent increase in the molecular weight of the α-amylase produced during germination. This was probably due to the formation of a complex between the inhibitor encoded by the asi gene on chromosome 2 of barley and wheat α-amylase 2. Breeding of wheat with the barley inhibitor gene may reduce the impact of the high α-amylase levels that result from pre-harvest sprouting in wheat.     

Inhibition of wheat α-amylase by bran phytic acid

A proportion of the wheat samples tested for α-amylase activity by the Hagberg penetrometer method during the 1961 New Zealand harvest gave higher results on flours than on wheatmeals milled from the same wheats. This effect is due to the lower level of calcium available as enzyme cofactor in the meals, caused by combination with phytic acid. Because of this effect and because the internal distribution of α-amylase in wheat grains is variable, testing for sprout damage in milling wheat is best carried out on flours rather than wheatmeals.     

Effect of γ‐radiation and microwave heating of wheat grain on some starch properties in irradiated grain as well as in grain of the next generation crops

Grains of the Polish winter wheat variety Begra were subjected to γ‐radiation (grain harvested in 1996) within the dose range of 0.05–10 kGy and microwave heating (grain harvested in 1997) from 28°C to 98°C. Later the grains were divided into two parts, the first was used for direct analyses after treatment. The second part was sown on the experimental fields. The obtained crop was described as the first generation and divided into two parts. One part was destined to determination of starch properties and the second part was sown in order to obtain the second generation crop. The same pattern was conducted in order to achieve the third generation crop. γ‐Irradiation directly applied on the wheat grain reduced statistically significant falling number values and gelatinisation enthalpy (ΔH) of the grain treated by 5 and 10 kGy. Calculated linear regression correlation coefficient between the falling number values and the gelatinisation enthalpy was equal to 0.94 (p ? 0.001) and showed that these two starch characteristics are well correlated in the case of directly irradiated wheat grain. The falling number values, peak temperatures (T_p) and gelatinisation enthalpy (ΔH), in three generations of wheat grain crop studied, did not show any statistically significant differences as a result of indirect effect of γ‐irradiation. Microwave direct heating of wheat grain to 98°C caused a statistically significant increase in the falling number value and decrease in starch gelatinisation power expressed by the enthalpy of gelatinisation (ΔH). The statistically significant changes in the falling number values, slight changes in the peak temperatures (T_p) and enthalpy of gelatinisation (ΔH) were found in all three generation crops as an indirect effect of microwave heating.     

Hagberg penetrometer method for α-amylase activity in sprouted grain: Prediction of activity of flour blends

A penetrometer method for sprout damage has been applied to New Zealand flours, and a close relationship between the results and crumb doughiness in bread has been demonstrated. The pressure-sustaining capacity of the flour gels, as calculated from the penetrometer readings, is inversely proportional to the enzyme activity. A simple relationship exists between penetrometer number and the proportion of flour from sprouted grain in flour blends. The penetrometer number appears to be primarily determined by the α-amylase activity of the flour from sprouted grains and to be unaffected by variations in the level of ‘damaged’ starch in the flour. A method is proposed whereby the amounts of sprouted lines of wheat necessary in a grist, to produce flour with a predetermined level of activity, may be calculated.     

A simple gel-diffusion assay of α-amylase in ungerminated wheat grains

A rapid technique has been developed for non-destructive screening of half-grains of wheat for α-amylase content. The technique provides a sufficiently accurate estimation of the enzyme concentration to allow reliable selection for wide differences in activity and is suitable for screening large numbers of small samples, thus enabling entire F₂ generations to be assayed.     

Effect of γ‐radiation and microwave heating on endosperm microstructure in relation to some technological properties of wheat grain

Grains of the Polish winter wheat variety Begra were subject to γ‐radiation (⁶⁰Co) within the dose range of 0.05–10 kGy and microwave treatment from 15 to 180 s, the latter resulting in a bulk grain temperature ranging from 28 to 98°C. Both processes were responsible for marked structural changes of wheat kernel endosperm and were pronounced with increasing dose of ionizing radiation or with prolonged time of microwave treatment. Endosperm microstructures of wheat kernels treated with γ‐radiation ranging from 0.05 to 0.5 kGy did not differ from that of untreated ones. Only some changes in the structure of starch granules and proteins were observed at doses of 1, 5 and 10 kGy. Simultaneously at the doses of 5 and 10 kGy a statistically significant decrease of falling number, sodium dodecyl sulfate (SDS) sedimentation values, dough stability and energy were observed, while dough weakening progressed. Microwave treatment longer than 90 s caused marked changes in kernel endosperm structure. Some changes of proteins caused by denaturation created visible fibrils as well as high swelling and deformation of starch granules. This was followed by an increase in the falling number value, decrease in the estimated results of SDS sedimentation test, lowering of the percent of gluten washed out and of the dough energy, respectively. Also a significant decrease in bread quality expressed by volume and score was observed with increasing time of exposure to microwave.     

The effects of α-amylase inhibitors on insect storage pests: Inhibition of α-amylase in vitro and effects on development in vivo

Protein α-amylase inhibitors were prepared from wheat and their effects tested against insect storage pests both in vitro against the insect α-amylases and in vivo in insect feeding trials. Inhibitor fraction A was found to inhibit porcine pancreatic α-amylase but not insect α-amylases, whereas fractions B, C and D (0.28) did not inhibit porcine pancreatic α-amylase but were strong inhibitors of digestive α-amylases from larvae of Tribolium confusum, a storage pest of wheat products, and Callosobruchus maculatus, a storage pest of legume seeds. Fraction D, which was a single polypeptide of M_r 13 000 was the most effective inhibitor in vitro. It would appear that the degree of inhibition by the wheat α-amylase inhibitor preparations can be correlated with the presence of the M_r 13 000 (0.28) polypeptide since the purer this polypeptide the stronger was the inhibition; fraction A which contained two polypeptides of M_r 60 000 and 58 000 caused no inhibition. The effects of fractions B and C on larval development were determined in insect feeding trials. With C. maculatus both fractions were toxic, their relative effectiveness being directly paralleled by their effectiveness observed in vitro. Only fraction C was tested against T. confusum in feeding trials. Despite this fraction being equally effective against both pests in vitro it had very little effect upon larval development of T. confusum in vivo, thus suggesting that this organism is able to detoxify the wheat α-amylase inhibitors. As far as the authors are aware, this is the first time that the effects of identified inhibitor fractions have been monitored both in vitro and in vivo. The results, in contrast to previous proposals, suggest that selecting wheat varieties for high α-amylase inhibitory activity may not be a very reliable criterion in selecting for insect resistance.     

Inhibitory activity towards human α‐amylase in wheat flour and gluten

Wheat α‐amylase inhibitors (AI) have been targeted as potential triggers of noncoeliac gluten or wheat sensitivity (NCGS). The aim of this study was to determine AI activity towards α‐amylase from human source in wheat cultivars. Contrary to barley, buckwheat, or oats, high level of AI activity was found in wheat and, to a lesser extent, rye. AI activity (mean IC₅₀ = 137 μg mL^?1) did not vary with respect to ancient or recently developed wheat cultivars. Vital wheat gluten had very high and heat‐stable AI activity (mean IC₅₀ = 23 μg mL^?1), higher than wheat starch (?10 000 μg mL^?1) or acarbose (40 μg mL^?1), a medication for the management of hyperglycaemia and potentially causing digestive disorders due to the accumulation of undigested carbohydrates in the intestine. Data suggest that eating raw wheat gluten, flour or dough could pose a health risk.     

Stability of α-amylase in model brewery mashes

Experiments with model mashes, of partly purified malt α-amylase added to a grist of ethanol-extracted pale-ale malt confirmed that decreasing the concentration of the mash or increasing mash temperature caused an acceleration in the rate of α-amylase destruction. the experimental mashes were devoid of proteolytic activity and contained high levels of calcium ions. the mash temperature was below that needed to inactivate the enzyme in solution, so that the instability of the enzyme was unexpected. α-Amylase was estimated by a convenient reducing power method using a reduced starch substrate, and ‘hollow plug’ accelerated ion exchange for the removal of interfering substances.     

Interaction of amyloglucosidase and α-amylase supplements in breadmaking

Amyloglucosidase (Novo 150 L, 5 ml/100 kg) used with optimum α-amylase supplements [MSKB/100 g flour] of wheat malt, 7.5, fungal, 20, and bacterial, 0.4, increased the reducing sugars of sour doughs (pH 4.55, 3 h/30 °C) by 71.7 to 72.2, 94.0 to 103.1 and 71.7 to 77.0% as compared to 48.3 to 58.0, 61.1 to 71.3 and 7.3 to 12.0% of initial pH 5.8 doughs, respectively. Amyloglucosidase alone or with α-amylase supplements had negligible effect on mixing properties of dough, falling number values and paste viscosities which were affected more by the wheat malt α-amylase. The combined enzymes had a beneficial effect on bread quality of the medium-protein flours without extraneous sugar in the formula.     

Kaffircorn malting and brewing studies XVII—Purification and properties of sorghum malt α-amylase

α-amylase of sorghum malt has been purified by a combination of charcoal and heat treatment, DEAE-cellulose and calcium phosphate chromatography, and gel filtration, yielding a product with a specific activity of 10,304 units per mg nitrogen. Both in the ultracentrifuge and on electrophoresis the purified α-amylase revealed a single peak. Results are presented for the molecular weight, sedimentation, diffusion and amino-acid composition of the purified enzyme. The influence of pH, temperature, and substrate concentration on the enzymic hydrolysis of gelatinised soluble starch has been studied. Examination of the purified α-amylase on a calcium phosphate column showed that the enzyme is micro-heterogeneous and it was separated into four sub-fractions. The physical, chemical and kinetic properties of the sub-fractions are almost identical.     

Effects of γ-irradiation on the flavour of dry shiitake (lentinus edodes sing)

Dry shiitake (Lentinus edodes Sing) was irradiated with doses of 5 and 10 kGy using ⁶⁰Co. The total volatile compounds were decreased by more than 50%. Irradiation increased the concentrations of some minor volatile compounds, such as 3-methyl-2-butanol and I-hexanol. However, the major flavour compounds including eight-carbon and sulphur-containing compounds were significantly reduced. The ratio of the eight-carbon compounds, such as 3-octanone, 3-octanol and 1-octen-3-ol, to total volatiles decreased from 72% in the control to 21% in the 10 kGy irradiated samples. The unidentified compounds which may have been formed by decomposition or polymerization following irradiation of precursors need to be studied in greater depth.     

Effect of cereal,fungal and bacterial α-amylase on the rheological and breadmaking properties of medium-protein wheats

Optimal levels of cereal malt, fungal and bacterial α-amylase for baking of medium-protein flours were found to be 5 - 10. 20 and 0.35 SKB/100 g, respectively. The dough consistency decreased considerably with time, depending on the source and level of α-amylase. The paste viscosities of the flours were reduced considerably by the cereal malts even at 2.5 SKB/100 g than by the fungal and bacterial α-amylase used optimally. Satisfactory loaf volumes were obtained with optimised α-amylase supplements without extraneous sugar in the bread formula. With 0.35 SKB of bacterial α-amylase, a bread having distinguishably softer texture was obtained as compared to cereal and fungal α-amylases. Accumulated maltose in the crumb indicated the need for a strain of baker's yeast with an active maltase system to render in situ contribution of α-amylase supplements more effective.     

Biochemical changes in phosphorus compounds and in the activity of phytase and α-amylase in the rice (Oryza sativa) grain during germination

Changes in the phytic acid, inorganic phosphorus and ATP contents, and in the activity of phytase and α-amylase in rice (Oryza sativa L) grains were determined during 18 days of germination in a dark room. The effect of phytic acid on α-amylase activity was studied in vitro. Rice grains immersed in sterilised deionised water at 14^°C germinated on the fifth day. Phytase activity, detected in the ripening rice grains, increased linearly until the eighth day and reached a maximum on the tenth day. There was a marked decrease in phytate and an increase in inorganic phosphorus accompanying germination. There was a good inverse correlation between the levels of both phytase activity and inorganic phosphorus, and phytate breakdown. α-Amylase activity was detected on the fourth day and increased markedly from the 12th to the 16th day of germination. ATP level increased from the second to the fourth day and slightly decreased from the fourth to the eighth day; it increased rapidly again from the eighth to the 18th day of germination. α-Amylase activity was influenced by both pH and phytic acid concentration in the assay system. At 75 mM phytic acid, α-amylase activity was lowered by 23%, 93% and 52% at pH 4–0, 5–0 and 6–0 respectively. When the enzyme, phytate and Ca²⁺ were incubated together at pH 5–0, the inhibition of α-amylase by phytic acid was markedly decreased by addition of Ca²⁺. The chemical affinity of Ca²⁺ for phytic acid was higher in the reaction at pH 5–0 than in those at pH 4–0 and pH 6–0, and over 98% of Ca²⁺ in the reaction system was precipitated as Ca-phytate.     

Sweet taste and solution properties of α,α-trehalose

α,α-Trehalose, a naturally occurring non-reducing disaccharide (α, D-glucopyranosyl 1–1 α-D-glucopyranoside) is investigated by solution parameters and sensorial effects. Interactions between α, α-trehalose and water give rise to hydration effects which are measurable by intrinsic viscosity [n], apparent specific volume V°₂, and NMR relaxation times. Water-solute interactions are prime determinants of taste qualities. An interpretation of sweetness intensity and persistence of α,α-trehalose is therefore offered, based on its solution properties and its effect on water structure. From the results, it appears that α,α-trehalose undergoes a better packing among water molecules than other sugars. α,α-Trehalose is found to be less sweet but more persistent than sucrose, fructose, glucose or maltose.