Enzymic Hydrolysis and Industrial Importance of Barley β-Glucans and Wheat Flour Pentosans

Mixed linkage β-glucane and pentosanes (mainly arabinoxylanes) are the major endosperm cell-wall polysaccharides of barely and wheat respectively. These polysaccharides, although minor components of the whole grain, significantly affect the industrial utilization of these cereals. The modification of barley corns during malting requires the dissolution of the β-glucane in the cell-wall of the starch endosperm. High β-glucane concentration in wort and beer effect the rate of filtration and can also lead to precipitate or gel formation in the final product. In a similar manner, pentosane is thought to cause filtration problems with wheat starch hydrolysates by increasing viscosity and by producing gelatinous precipitate which blocks filters. Ironically, it is this same viscosity building and water binding capacity which is considered to render pentosanes of considerable value in dough development and bread storage (anti-staling functions). In the current paper, some aspects of the beneficial and detrimental effects of pentosanes and β-glucane in the industrial utilization of wheat and barley are discussed. More specifically, enzymic methods for the preparation, analysis and identification of these polysaccharides and for the removal of their functional properties, are described in detail.     

Corn grain endosperm type and brown midrib 3 corn silage: site of digestion and ruminal digestion kinetics in lactating cows

Interactions of endosperm type of corn grain and the brown midrib 3 (bm3) mutation in corn silage on ruminal kinetics and site of nutrient digestion of lactating dairy cows were evaluated. Eight ruminally and duodenally cannulated cows (72 +/- 8 d in milk; mean +/- SD) were used in a duplicated 4 x 4 Latin square design experiment with a 2 x 2 factorial arrangement of treatments. Treatments were corn grain endosperm type (floury or vitreous) and corn silage type (bm3 or isogenic normal). Diets contained 26% neutral detergent fiber (NDF) and 30% starch. Interactions of treatments were not observed for any measure of digestibility, but digestion kinetics of starch and fiber did interact to affect digestible organic matter intake by affecting dry matter intake. Rate of ruminal starch digestion was faster and rate of ruminal starch passage tended to be slower in diets containing corn grain with floury vs. vitreous endosperm, resulting in a mean increase of 22 units for ruminal starch digestibility. Although compensatory postruminal starch digestion decreased differences among treatments for total tract starch digestibility, starch entering the duodenum was more digestible for grain with floury endosperm compared with vitreous grain, resulting in greater total tract starch digestibility for floury compared with vitreous corn grain. Fermentation rate of potentially digestible NDF was not affected by either bm3 corn silage or greater ruminal starch digestion of floury grain. Brown midrib corn silage increased total tract NDF digestibility vs. control silage by numerically increasing ruminal and postruminal digestibility of NDF. Endosperm type of corn grain greatly influences site of starch digestion and should be considered when formulating diets.     

In vitro Degradation of Starch Granules by α-Amylase Isomers from Mature Triticale

Starch granules and α-amylases (green, α-I, and germination, α-II) were isolated from a triticale line with a high frequency of kernel shrivelling. The isolated starch granules were treated with the α-amylase ismomers for different periods of time. Degradation of the starch granules was followed by analyses of reducing sugar produced, and by scanning electron microscopy. About similar degrees of starch degradation were observed after treatment with α-I- and α-II-amylases. After a few hours, the degrading effects of the enzymes were manifested as holes or craters on the surface of starch granules and as erosion and dilation of the equatorial groove. These features were also characteristic of starch granules present in shrivelled kernels. Amylase treatments for a longer time caused more profound degradation, leading to collapse and fragmentation of the starch granules. The lack of such effects in shrivelled kernels means that amylases are not the sole factor in the formation of kernel cavities associated with shrivelling. The occurrence of mitotically aberrant and degenerating cells, already existing at early stages of endosperm development, and disparity between the processes that determine kernel size and kernel filling potential are also of relevance in this connection.     

INITIAL STAGES IN α-AMYLOLYSIS OF BARLEY STARCH

The initial stages in α-amylolysis of large and small barley starch granules were followed by gel chromatography. Hydrolysis of gelatinised large and small granules is very similar, indicating a similar structure of the individual components in the two starches. Amylolysis of intact large granules is quite different from that of intact small granules, suggesting differences in the mode of packing of the starch components within the granules. It is concluded that α-amylolysis is of a non-random nature, probably due to the amylopectin component of the starch. On the basis of the intermediate products obtained, a possible structure for the amylopectin molecule is presented, based on a unit cluster with a molecular weight of 3 × 10⁴.     

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.     

Endosperm α 1,4—α 1,6 Glucopolysaccharides Utilization During Germination of Sweet Corn and Other Maize Genotypes

The quantity variations and the structural changes of the α 1,4 —α 1,6 glucopolysaccharides contained in the endosperm of sweet corn, waxy and amylose extender maize genotypes, were studied during early germination. The α 1,4 —α 1,6 glucopolysaccharides were fractionated according to their branching degree. The results obtained showed that: the degradation pattern of the α 1,4 —α 1,6 glucopolysaccharides was similar for the different corn genotypes studied. In addition, soluble dextrins or partial degradation products were not detected, but reducing sugars were accumulated. Furthermore, the structure of the remaining polysaccharides was similar to those present in the non germinating seed. These results led us to propose that once a polysaccharide molecule was used as the substrate by degradative enzymes during germination, that molecule was totally degraded.     

α-AMYLASE IN DEVELOPING BARLEY KERNELS — A REAPPRAISAL1

The outer layers that can be dissected readily from immature barley kernels were identified by light microscopy. α-Amylase was located in the outer pericarp of developing Bonanza and Himalaya barley. Only low pl α-amylase (α-amylase 1) was detected in the pericarps but only one of the two major low pl components of α-amylase from germinated Himalaya was found in the pericarp tissue. These enzymes appeared to hydrolyze the small starch granules (2–3 μm) present in the outer pericarp of developing barley kernels. The even smaller starch granules (0.25–2μm) present in the inner pericarp were hydrolyzed at a later stage of kernel development.     

Changes in β-glucan and other carbohydrate components of barley during malting

Changes in total (1→3), (1→4)-β-glucan content were followed during the micro-malting of nine varieties of barley with a wide range of malting qualities. These changes were related to estimates of endosperm modification based upon staining with Calcofluor. β-Glucan content declined from an average of 3.54% in the barley to 0.75% in the malt. Pentosan and total starch (including starch-derived oligosaccharides) levels showed comparatively little change during malting. β-Glucan composition of the barley was a poor indicator of malting performance. However, the β-glucan, starch and xylose contents of the malt all showed significant correlations with malt extract. Estimation of malt β-glucan content gave the best indication of malt quality. Direct determination of β-glucan may be of more value in assessing malt quality than indirect techniques based upon assessing modification of stained grains.     

Proso millet (Panicum miliaceum L.): An Evaluation of the Microstructural Changes in the Endosperm during the Malting Process by Using Scanning‐Electron and Confocal Laser Microscopy

Scanning electron microscopy (SEM) and confocal scanning laser microscopy (CLSM) were used to investigate the micro‐structural changes in the endosperm during the malting process. SEM was a suitable tool to characterise the microstructural constitution of starch granules in proso millet and the changes occurring during malting. An early visible degradation (after 24 h) of starch granules located in the floury endosperm, which is close to the embryo, could be observed. Due to this degradation, using confocal scanning laser microscopy, a less dense packaging of this part of the endosperm was observed. Degradation of starch granules in the vitreous endosperm was obvious at a distinct later stage of malting (78 h) and a preferred attack of small granules (>2.5 μm) was detected. Changes in protein structure could not be detected by either SEM or CLSM.     

Characterisation of Dextrins Solubilised by α‐Amylase from Barley Starch Granules

Large granules from barley starch were packed into a column and hydrolysed with α‐amylase by pumping a diluted enzyme solution through the starch bed. The enzyme was then trapped onto an ion‐exchanger and the dextrins that solubilised from the granules were collected and characterised. The size‐distribution of the solubilished dextrins ranged from degree of polymerisation (DP) 2—500. The linear and branched products originated from both the amylose and the amylopectin components. The rate of solubilisation and the composition of the solubilised dextrins from barley starch were very similar to those found for large wheat starch granules.     

Effects of High Pressure Disintegration of Steeped Maize Grits on the Release of Starch Granules from the Protein Matrix

The horny endosperm of maize gives the greatest resistance to the mechanical disintegration of the protein matrix during starch extraction. Thus attempts were made to release the starch by wet-milling of maize grits in the valve of a high-pressure homogenizer. At the same time, information was gathered on the principles involved in the structural breakdown of the endosperm components. For this purpose the influence of the valve geometry as well as the concentration and flow rate of the suspension on the particle size reduction and disintegration was examined. Using a valve specially constructed for the photographic documentation of this process step, it could be shown that the slit height of the valve predominantly determined the particle size of the wet milled product. Already after a single passage through the valve, set at slit heights between 200 and 600 μm, 70% of the grits were reduced to particles <40 μm. The grits were initially between 250 and 1000 μm in size. Scanning electron microscope photographs showed that a large fraction of the mass existed as free starch granules alongside partly intact particles of the protein matrix. Since the starch granules were of higher purity after raffination, it is assumed that the extent to which disintegration of the endosperm particles results in starch granules and protein particles as separate entities, depends not only on the particle size reduction step, but also on the different visco-elastic behaviour of the protein matrix and the starch granules under the influence of the large shear forces occuring in the valve.     

Staling in Starch Breads: The Effect of Antistaling α-Amylase

A novel starch bread that contained no gluten was found to firm at a rate comparable to a normal standard bread made from wheat flour. Treatment of both the starch and the standard bread with Novamyl, an antistaling enzyme mix, inhibited firming. ¹³C CP/MAS NMR studies showed that the decreased firming of the Novamyl-treated starch bread was correlated with decreased starch retrogradation. For the Novamyl-treated bread the increase in retrograded starch over six days following baking was about 11% compared to an increase of over 200% for the untreated bread. These results suggests that starch retrogradation is sufficient to cause bread firming.     

Effects of the Barley amo1 and wax Genes on Starch Structure and Physicochemical Properties

Starch structural mutants showing abnormal endosperm characteristics have been used for investigating the effects of the mutation on structure and physicochemical properties of starches. Inbred lines of barley cultivars ‘Shikoku Hadaka 97’ and ‘Glacier AC38’ were used to investigate the impact of amo1 and waxy genes on starch properties. The amo1 type starch had high apparent amylose content and low starch content. The amo1+waxy type starch contained very little amylose. The content of long chains of amylopectin as detected with high‐performance size‐exclusion chromatography (HPSEC) was decreased, and that of amylopectin chains with the degree of polymerization (DP) of 12‐36 was increased in amo1 and amo1+waxy type starches. The amo1 and amo1+waxy type starches exhibited high gelatinization temperatures and low gelatinization enthalpies.     

Effects of corn grain endosperm type and conservation method on feed intake,feeding behavior,and productive performance of lactating dairy cows

Our objective was to evaluate the effects of corn grain varying in endosperm type and conserved as high-moisture or dry ground corn on dry matter intake (DMI), feeding behavior, ruminal fermentation, and yields of milk and milk components of cows in early to mid-lactation. Seven ruminally and duodenally cannulated Holstein cows (73 ± 39 d in milk; mean ± SD) were used in a duplicated 4 × 4 Latin square design with 21-d periods. A 2 × 2 factorial arrangement of treatments was used with main effects of corn grain endosperm type (floury or vitreous) conserved as high-moisture corn (HMC) or dry ground corn (DGC). Rations were formulated to contain 27.0% starch, 26.6% neutral detergent fiber (NDF), 19.1% forage NDF, and 16.5% crude protein. Corn grain treatments supplied 86.6% of dietary starch and contained alfalfa silage as the sole forage. Dry matter intake was increased 1.3 kg/d by DGC compared with HMC. The increase in DMI by DGC was related to a shorter intermeal interval (104.4 vs. 118.2 min/d), and meal size was not affected by treatment. Dry ground corn decreased rumination bout length and number of chews per bout compared with HMC. No differences were detected between endosperm treatments for DMI, yields of milk, 3.5% fat-corrected milk (FCM), milk fat, protein, lactose, or solids-not-fat (SNF). Mean yield of 3.5% FCM across treatments was 47.5 kg/d. However, a tendency for an interaction was observed for feed efficiency; floury endosperm increased efficiency 0.05 kg 3.5% FCM per kg of DMI for DGC but decreased it by 0.14 kg 3.5% FCM per kg of DMI for HMC relative to vitreous endosperm. Vitreous compared with floury corn tended to increase true protein concentration in milk when conserved as DGC (2.68% vs. 2.62%) but not as HMC. Concentration of SNF was increased by DGC compared with HMC (8.45 vs. 8.37%) due, in part, to the effect of treatment on milk protein concentration. Body weight was not affected by treatment, but vitreous endosperm tended to increase loss of body condition compared with floury endosperm. Corn endosperm type and conservation method had little effect on productive performance of high-producing cows.     

COMPUTER SIMULATION OF THE β-AMYLOLYSIS OF STARCH COMPONENTS*

In an attempt to model quantitatively the product distributions arising from the degradation of starch by the conjoint action of α- and β-amylase, such as occurs in the commercial mashing of malted cereal, a computer program has been written to simulate the amylolysis of amylose or amylopectin or a mixture of these components. Using Monte-Carlo techniques the program simulates the action of either α-amylase or β-amylase or both enzymes acting conjointly. The program was tested for pure β-amylolysis. Parameters were estimated by a non-linear regression technique and predicted product concentrations were compared with relevant experimental data for amylose and amylopectin substrates and for mixtures of these starch components. The simulation model accurately predicted the results of amylolysis except at high product concentrations where the deviations between predicted and experimental values ranged from 1% to 4%.     

Properties of Starches Isolated from Sorghum Floury and Corneous Endosperm

Decorticated grains of three sorghum cultivars of intermediate grain hardness were individually dissected to separate the floury and the corneous endosperm fractions. The starch and polymeric components (amylose and amylopectin) of each of the endosperm fractions were isolated and subsequently characterized. Results indicate that there exist major differences in the properties (granule size distribution, gelatinization temperature, iodine binding capacity, and rate of lintnerization) of the starch and starch components from the endosperm fractions. Close relationships between the starch properties and the strength of the alkali gel of the corresponding endosperm fraction were observed.     

Type of corn endosperm influences nutrient digestibility in lactating dairy cows

An experiment was conducted to evaluate the effect of type of corn endosperm on nutrient digestibility in lactating dairy cows. Near-isogenic variants of an Oh43 × W64A normal dent endosperm hybrid carrying floury-2 or opaque-2 alleles were grown in spatial isolation in field plots and harvested as dry shelled corn. Six ruminally cannulated, multiparous Holstein cows (67 ± 9 d in milk at trial initiation) were randomly assigned to a replicated 3 × 3 Latin square design with 14-d periods; the first 11 d of each period were for diet adaptation followed by 3 d of sampling and data collection. Treatment diets that contained dry rolled vitreous-, floury-, or opaque-endosperm corn [33% of dry matter (DM)], alfalfa silage (55% of DM) and protein-mineral-vitamin supplement (12% of DM) were fed as a total mixed ration. The percentage vitreous endosperm was zero for floury and opaque endosperm corns and 64 ± 7% for the vitreous corn. Prolamin protein content of floury and opaque endosperm corns was 30% of the content found in vitreous corn. Degree of starch access and in vitro ruminal starch digestibility measurements were 32 and 42% greater on average, respectively, for floury and opaque endosperm corns than for vitreous corn. Dry matter and starch disappearances after 8-h ruminal in situ incubations were, on average, 24 and 32 percentage units greater, respectively, for floury and opaque endosperm corns than for vitreous corn. Ruminal pH and acetate molar percentage were lower, propionate molar percentage was greater, and acetate:propionate ratio was lower for cows fed diets containing floury and opaque endosperm corns than for cows fed vitreous corn. In agreement with laboratory and in situ measurements, total-tract starch digestibility was 6.3 percentage units greater, on average, for cows fed diets containing floury and opaque endosperm corns than vitreous corn. Conversely, apparent total-tract neutral detergent fiber (NDF) digestibility was lower for cows fed diets containing floury and opaque endosperm corns compared with vitreous corn. The type of endosperm in corn fed to dairy cows can have a marked effect on digestion of starch and NDF. Feeding less vitreous corn increased starch digestion but decreased NDF digestion.     

Studies on Starch Granules Digestion by α-Amylase

Raw corn starch was partially digested with commercial α-amylase for a short period of 7–8 h at 25°C to obtain granular pitting. The patterns of granular digestion was observed by light and scanning electron microscopy (SEM). The main soluble products of starch digestion were glucose, maltose and higher oligosaccharides. In vitro digestion of starch was discontinued on the basis of nature of granular pitting and relative release of glucose in the reaction mixture. The pitted starch was obtained with high yields and found to be warm water soluble.     

The Developing Starch Granule Part I. The Starch Content of Cereal Grains during their Development

When fertilization of developing cereal grains occurs, the pericarp (ovary wall) already contains starch granules. Hence the starch content of developing grains of wheat, triticale, rye, oats and barley commences at 30 or 40% of the dry weight. It increases only to 60 or 70% at ripeness when the pericarp has degenerated to bran layers and the endosperm is filled with stored starch granules and protein. All these cereals showed similar patterns of starch increase, with an inflexion at 40 to 50% starch due to degeneration of pericarp starch before endosperm deposition. In wheats and triticale there was a second inflexion at 50 to 60% starch caused by endosperm cells proliferating faster than they filled with starch granules. The rise and fall of rate of starch deposition coincided with the rise and fall of percentage of metabolic (non-storage) protein in the grain, calculated from total amino acid analyses. In the mature grain about 8% of the dry matter is not associated with starch and thus is non-endosperm tissues. About 70% of the remaining dry matter (endosperm) is starch. We suggest that fluctuations of metabolites and enzymes result from development or breakdown of groups of cells constituting distinct tissues in the grain.     

Raw Starch Digestion by α-Amylase and Glucoamylase from Chalara paradoxa

Glucoamylase and α-amylase of Chalara paradoxa were separated by hydrophobic column chromatography using butyl-Toyopearl 650M. The α-amylase showed the highest activity at pH 5.5 and 45°C, and was stable in the pH range of 5.5–6.5 and at temperatures lower than 40°C. The glucoamylase showed the highest activity at pH 5.0 and 45°C, and was stable in the pH range of 4.0–7.5 and at temperatures lower than 45°C. The molecular mass of the α-amylase and glucoamylase estimated by SDS polyacrylamide gel electrophoresis was 80,000 and 68,000, respectively. Both glucoamylase and α-amylase could digest more effectively raw rice starch and raw corn starch than raw sago starch and raw potato starch. 2% raw rice starch in 10 ml solution was digested by more than 90% by 100 units of each amylase. When these amylases were used combined, raw corn starch was more effectively digested than they were used singly. This cooperative action in raw corn starch digestion was also observed when. C. paradoxa α-amylase and R. niveus glucoamylase were combined.