Effect of short-term storage on phenolic content,o-diphenolase and peroxidase activities of cut yam tubers (Dioscorea sp)

The effect of short-term storage on the protein, phosphorus and phenolic content as well as peroxidase and o-diphenolase activities of cut, harvested Jamaican yam (Dioscorea sp) tubers (D rotundata. D alata and D cayenensis) was studied. There was an initial increase in the total phenolic content up to the third week of storage followed by a gradual decrease to the sixth week. Phenolic content was found to be highest in D cayenensis followed by D rotundata and D alata. The activities of peroxidase (EC 1. 11. 1. 7) and o-diphenolase (EC 1. 10.3.1) increased steadily up to the third week of storage and thereafter decreased to the fifth week. The intensity and rapidity of browning in tubers when cut, correlated very closely with the tuber o-diphenolase and phenolic content levels while the onset of rotting correlated with the peroxidase activity levels in the species studied.     

Physico‐chemical characteristics of non‐irradiated and γ‐irradiated yams cultivars (Dioscorea rotundata,Dioscorea alata) and sweet potato (Ipomoea batatas (L) Lam)

Physico‐chemical (pasting) properties of non‐irradiated (fresh) and γ‐irradiated yam cultivars and sweet potato were determined using Rapid Visco Analyser (RVA). Generally, pasting characteristics of the commodities decreased significantly with increased γ‐irradiation dose. Non‐irradiated sweet potato showed significantly higher peak (45.79), trough (35.25), breakdown (10.54), final (75.21) and setback (39.96) viscosities (in RVU) than γ‐irradiated samples. Also, peak time (6.97 min) and pasting temperature (50.18 RVU) significantly reduced with increased γ‐irradiation dose of tubers. The pasting properties of non‐irradiated and γ‐irradiated sweet potato showed similar but clearer trend compared with yam flours. Also, non‐irradiated and γ‐irradiated Dioscorea rotundata and Dioscorea alata showed significantly higher values of each of the pasting characteristics than values noted in sweet potato. Aside from the TDr 03/00196, peak time did not vary significantly with γ‐irradiation dose.     

IMPROVED EXTRACTION AND ASSAY OF β-AMYLASE BROM BARLEY AND MALT

β-Amylase was extracted from barley or malt using four physical techniques to break up grists which had been prepared using a Moulinex coffee grinder. Grinding with a Polytron homogeniser apparently completely disrupted all cells, as determined by transmission electron microscopy, and increased the efficiency of extraction of β-amylase from barley by more than 30%. The other treatments tested were without value . The β-amylase activity in extracts of barley or malt was assayed by measuring the production of reducing sugars from reduced soluble starch, using a PAHBAH reagent. α-Amylase, which interferes with the quantitation of β-amylase in extracts of malt, was not totally inactivated by the chelating buffer used for enzyme extraction or by several other chelating agents. α-Amylase activity was quantified specifically using Phadebas. Using purified α-amylase a calibration was developed which related activity, as determined using Phadebas, to reducing power units. Thus the α-amylase activity present in an extract containing β-amylase could be determined using Phadebas and the reducing power equivalent activity subtracted from the total “apparent” activity to give the actual β-amylase activity. α-Glucosidase and limit dextrinase activities are believed to be too low to have a significant effect on the apparent β-amylase . The soluble and bound β-amylase activities were measured in samples taken from micromalting barley (Alexis). Dry weight losses increased to over 10% after 8 days germination. Antibiotics, applied during steeping, were used to control microbes in one experiment. However, their use checked germination and reduced malting losses to 8.4% in 8 days germination. The soluble enzyme present in extracts from steeped barley and early stages of germination was activated (20–40%) by additions of the reducing agent DTT .     

RELEASE AND ACTIVATION OF BARLEY β-AMYLASE

The aim of this study was to determine the role of low molecular weight thiols both in the release and activation of β-amylase during grain germination. In quiescent barley grains (Hordeum vulgare L. cv. Torrent) about 55% of the β-amylase was extracted with buffer, the remaining 45% was in the bound form. During micromalting the bound form was progressively solubilised between germination days 1 and 4. When free β-amylase, extracted from ungerminated grains, was incubated with dithiothreitol the enzymic activity increased by 15%-20%. This activation did not occur when free β-amylase, from grain germinated for 3 days or more, was incubated with DTT. The release of bound β-amylase with thiols was pH dependant, occurring most rapidly at and above pH 8.0. At the onset of germination the embryo released soluble thiol (approximately 5 nmol per embryo) into the endosperm. Degermed grains were dosed with reduced glutathione and incubated for 72 h. The addition of 60 nmol glutathione caused the release of about 80% of the bound β-amylase. When less glutathione was used, 5 nmol (an amount similar to that released by the embryo in vivo) no significant release of the bound enzyme was detected. When degermed grains were dosed with oxidised glutathione (60 nmol), no bound β-amylase was released. However, addition of the disulphide bis-hydroxyethyldisulphide (60 nmol) did cause the release of about 90% of the bound enzyme. The aleurone layer reduced the bis-hydroxyethyldisulphide to a thiol, presumably 2-mercaptoethanol. Oxidised glutathione and cystine were not significantly reduced to thiols by isolated aleurone layers. The aleurone layer did cause the disappearance of cysteine from solution. When preparations of bound β-amylase were incubated with extracts from the endosperms of grains germinated for three days, the bound enzyme was released. This release was due to the high molecu lar weight material (>5 kDa) in the extract and not to low molecular weight thiols. It seems unlikely that simple thiols, such as glutathione, are solely responsible for the release of bound β-amylase.     

DETERMINATION OF α- AND β-AMYLASE IN MALT

The Analysis Committee of the European Brewery Convention carried out a collaborative trial on malts using the specific analysis methods for α- and β-amylase activities based on dyed substrates supplied by MegaZyme (Aust.) Pty. Ltd. The repeatability and reproducibility values for the methods were judged to be unsatisfactory and consequently the methods were not recommended for Analytica-EBC.     

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.     

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.     

FAILURE OF MERCURIC CHLORIDE TO SELECTIVELY INHIBIT β-AMYLASE IN SORGHUM MALT

Mercuric chloride has been reported to be a suitable reagent for the determination of α-amylase activity in sorghum malt, based on its ability to selectively inhibit β-amylase. In this re-investigation, the α- and β-amylase activities of eight sorghum malts were determined after treatment of malt extracts with various concentrations of mercuric chloride. At a malt: mercuric chloride ratio of 8.3 × 10³: 1, incomplete inhibition of β-amylase activity, as measured by the Betamyl assay, occurred in all extracts. However, this concentration resulted in significant inhibition of α-amylase activity in all extracts, as measured by both the Ceralpha assay and the Phadebas assay. In addition, α-amylase activity was found to be significantly inhibited at malt: mercuric chloride ratios as low as 1.0 × 10⁵: 1, when measured by the AmyloZyme assay. These findings do not support the original report that a malt: mercuric chloride ratio of 4.0 × 10³: 1 will selectively inhibit β-amylase in sorghum malt. Furthermore, in this context it should be emphasised that the original report was based upon inhibition studies conducted on β-amylase derived from barley, not sorghum malt .     

Biochemical composition and storage of Jamaican yams (Dioscorea sp)

Dioscorea yam tubers of 11 cultivars from five common species grown in Jamaica were analysed for the following: protein content, total phenolics, vitamin C, total lipids, fatty acid composition and activity of the polyphenol oxidase enzyme. The results show that the yams grown in Jamaica are of good nutritional value with considerable amounts of protein, vitamin C, low lipids with only one cultivar ‘renta yam’ (D alata) possessing high levels of phenolic compounds. The fatty acids present in the total lipid extracts show that yam tubers generally possess high levels of saturated fatty acids mainly palmitic acid. However, the species D alata (cv white yam) and the species D trifida (cv yampie) have high levels of the unsaturated fatty acid, linolenic. All the polyphenol oxidases from the 11 cultivars show activities towards the diphenol substrates, catechol and DL-DOPA (DL-3,4-dihydroxyphenylalanine). However, no oxidation was observed with L-tyrosine, a monophenol substrate. All cultivars studied were found to have different lengths of dormancy which varied with storage conditions. When the harvested tubers were washed, sunned and stored at 20?C in a dark cupboard, it was possible to extend their lengths of dormancy by a further 11 weeks.     

Effects of Processing on the Reduction of β-ODAP (β-N-Oxalyl-L-2,3-diaminopropionic acid) and Anti-Nutrients of Khesari Dhal,Lathyrus sativus

Effects of different processing techniques on the neurotoxin, β-ODAP (β- N -oxalyl-L-2,3-diaminopropionic acid), and the anti-nutritional compounds (phytate, polyphenols, trypsin and amylase inhibitors, and lectins) within four lines of Lathyrus sativus (high-, medium- and low-ODAP, and so-called ODAP-free) were investigated. Soaking of seeds in various media reduced the contents of these compounds to a varying and significant extent; losses were higher in freshly boiled water, alkaline and tamarind solutions than after soaking in drinking water. The highest losses in boiled water (65–70%) were observed for β-ODAP, followed by trypsin inhibitors (42–48%) and polyphenols (30–37%). Ordinary cooking and pressure cooking of pre-soaked seeds were found to be most effective in reducing the levels of all the natural toxicants examined, whilst fermentation and germination were more effective in destroying both of the enzyme inhibitors (amylase inhibitors by 69–71%; trypsin inhibitors by 65–66%) than either phytates or polyphenols. Lectins were not affected by most of these processes except by pressure cooking and fermentation. Dehusking of pre-soaked seeds significantly reduced β-ODAP levels, but this reduction was lower for the anti-nutrients. These findings and the high water solubility suggest that a simple and effective means of detoxifying Lathyrus by removing this neurotoxic amino acid may be practicable.     

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%.     

Enzymatic Properties of β‐1,3‐Glucanase from Streptomyces sp Mo.

Streptomyces sp Mo endo‐β‐1,3‐glucanase was found to have hydrolyzing activity toward curdlan and released laminarioligosaccharides selectively. The molecular weight was estimated to be 36000 Da and its N‐terminal amino acid sequence was VTPPDISVTN. The optimal pH was 6 and the enzyme was found to be stable from pH 5 to 8. The optimal temperature was 60 °C and the activity was stable below 50 °C. The enzyme hydrolyzed selectively curdlan containing only β‐1,3 linkages. The enzyme had 89% relative activity toward Laminaria digitata laminarin, which contains a small amount of β‐1,6 linkages compared with curdlan, while Eisenia bicyclis laminarin with a higher amount of β‐1,6‐linkages, was not hydrolyzed. Mo enzyme adsorbed completely on curdlan powder. The enzymatic hydrolysis of curdlan powder resulted in the accumulation of laminaribiose (yield 81.7%). Trisaccharide was inevitably released from the hydrolysis of laminarioligosaccharides with 5 to 7 degrees of polymerization (DP). Although the enzyme cleaved off disaccharide (DP 2) from tetrasaccharide (DP 4), the reaction rate was lower than those of DP 5 to 7. The results indicated that the active site of Mo endo‐β‐1,3‐glucanase can efficiently recognize glucosyl residue chain of greater than DP 5 and hydrolyzes the β‐1,3 linkage between the 3rd and 4th glucosyl residue.     

Purification and physicochemical characterization of ovine β‐lactoglobulin and α‐lactalbumin

Ovine whey proteins were fractionated and studied by using different analytical techniques. Anion‐exchange chromatography and reversed‐phase high‐performance liquid chromatography (HPLC) showed the presence of two fractions of β‐lactoglobulin but only one of α‐lactalbumin. Gel permeation and sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis allowed the calculation of the apparent molecular mass of each component, while HPLC coupled to electrospray ionisation‐mass spectrometry (ESI‐MS) technique, giving the exact molecular masses, demonstrated the presence of two variants A and B of ovine β‐lactoglobulin. Amino acid compositions of the two variants of β‐lactoglobulin differed only in their His and Tyr contents. Circular dichroism spectroscopy profiles showed pH conformation changes of each component. The thermograms of the different whey protein components showed a higher heat resistance of β‐lactoglobulin A compared to β‐lactoglobulin B at pH 2, and indicated high instability of ovine α‐lactalbumin at this pH.     

α-Amylase inhibitors in chick pea (cicer arietinum L)

The α-amylase inhibitory activity of 28 varieties of chick pea (Cicer arietinum L) was determined, and KGT/GBS-8 was found to have the greatest activity (81.4 units g^?1). The inhibitor was heat labile and the inhibitory activity decreased during germination.     

Screening of β‐Glucosidase and β‐Xylosidase Activities in Four Non‐Saccharomyces Yeast Isolates

The finding of new isolates of non‐Saccharomyces yeasts, showing beneficial enzymes (such as β‐glucosidase and β‐xylosidase), can contribute to the production of quality wines. In a selection and characterization program, we have studied 114 isolates of non‐Saccharomyces yeasts. Four isolates were selected because of their both high β‐glucosidase and β‐xylosidase activities. The ribosomal D1/D2 regions were sequenced to identify them as Pichia membranifaciens Pm7, Hanseniaspora vineae Hv3, H. uvarum Hu8, and Wickerhamomyces anomalus Wa1. The induction process was optimized to be carried on YNB‐medium supplemented with 4% xylan, inoculated with 106 cfu/mL and incubated 48 h at 28 °C without agitation. Most of the strains had a pH optimum of 5.0 to 6.0 for both the β‐glucosidase and β‐xylosidase activities. The effect of sugars was different for each isolate and activity. Each isolate showed a characteristic set of inhibition, enhancement or null effect for β‐glucosidase and β‐xylosidase. The volatile compounds liberated from wine incubated with each of the 4 yeasts were also studied, showing an overall terpene increase (1.1 to 1.3‐folds) when wines were treated with non‐Saccharomyces isolates. In detail, terpineol, 4‐vinyl‐phenol and 2‐methoxy‐4‐vinylphenol increased after the addition of Hanseniaspora isolates. Wines treated with Hanseniaspora, Wickerhamomyces, or Pichia produced more 2‐phenyl ethanol than those inoculated with other yeasts.     

EVALUATION OF MALTING QUALITY IN A BARLEY BREEDING PROGRAMME USE OF α-AMYLASE AND β-GLUCAN LEVLES IN MALT AS PRESELECTION TOOLS

Analysis according to the EBC protocol, immunological determination of a α-amylase and estimation of malt β-glucan using the Calcofluor-FIA method, were used to screen 327 barley breeding lines for malting quality. The results obtained with the α-amylase and β-glucan methods are highly correlated to the important malt quality paramters: extract yield and β-glucan content in the wort. It is recommended that either of the two methods, which are simple to perform are used as prescreening tools in breeding programmes for malting barley.     

ENZYMIC QUANTIFICATION OF (1→3) (1→4)-β-D-GLUCAN IN BARLEY AND MALT

A simple and quantitative method for the determination of (1→3) (1→4)-β-D-glucan in barley flour and malt is described. The method allows direct analysis of β-glucan in flour and malt slurries. Mixed-linkage β-glucan is specifically depolymerized with a highly purified (1→3) (1→4)-β-D-glucanase (lichenase), from Bacillus subtilis, to tri-, tetra- and higher degree of polymerization (d.p.) oligosaccharides. These oligosaccharides are then specifically and quantitatively hydrolysed to glucose using purified β-D-glucosidase. The glucose is then specifically determined using glucose oxidase/peroxidase reagent. Since barley flours contain only low levels of glucose, and maltosaccharides do not interfere with the assay, removal of low d.p. sugars is not necessary. Blank values are determined for each sample allowing the direct measurement of β-glucan in maltsamples.α-Amylasedoes not interfere with the assay. The method issuitable for the routineanalysis of β-glucan in barley samples derived from breeding programs; 50 samples can be analysed by a single operator in a day. Evaluation of the technique on different days has indicated a mean standard error of 0–1 for barley flour samples containing 3–8 and 4–6% (w/w) β-glucan content.     

OPTIMIZED McCLEARY METHOD FOR MEASUREMENT OF TOTAL β-AMYLASE IN BARLEY AND ITS APPLICABILITY

The McCleary method for determination of β-amylase in malt has been modified in order to allow determination of total β-amylase in barley as well as malt. A ruggedness test, performed on the modified method, demonstrated that the method is quite robust and highly reproducible. When the variables α-amylase, β-amylase and diastatic power were measured in 90 malt samples, only β-amylase was significantly correlated to diastatic power (r² = 0.85 and p < 0.0001). The same high correlation was found between total β-amylase in 20 barley samples and diastatic power in the corresponding malts. The validity of this relationship was tested by predicting diastatic power in malt from total β-amylase in barley. Predicted values correlated highly to measured values (r² = 0.95). In breeding material a positive relationship was found between total β-amylase in barley and protein content. This relationship must be considered when evaluating new barley lines.     

‘FREE’ AND ‘BOUND’ β-AMYLASES DURING MALTING OF BARLEY. CHARACTERIZATION BY TWO-DIMENSIONAL IMMUNOELECTROPHORESIS

Major qualitative and quantitative changes in the β-amylases and in other salt soluble barley proteins occurred during the first four days of germination. Two soluble forms of barley β-amylase, ‘free’ β-amylase and β-amylase aggregated with a non-active protein Z, were found in extracts from all stages. A third enzyme form appeared during malting. Immunoelectrophoretic characterization seemed to support the possibility that this enzyme form could be a product of ‘bound’ β-amylase solubilization. All soluble forms of β-amylase and of protein Z in malt were electrophoretically heterogeneous. Two different, immunochemically related forms of protein Z present after malting retained their immunoelectrophoretic properties during brewing and were found to be dominant antigens in beer.     

KINETICS OF β-GLUCAN DEGRADATION IN WORT BY EXOGENOUS β-GLUCANASES TREATMENT

Three commercial β-glucanases, one bacterial (Cereflo 200L from Novo) and two fungal (Biobeta from Gist-Brocades and Filtrase from Biocon) have been studied with regard to the hydrolysis of β-glucan in sweet and hopped wort. At temperatures below 70°C these processes follow first order kinetics with rate constants being directly proportional to the enzyme concentrations. The rate constant for bacterial β-glucanase Cereflo 200L shows a negative dependence on temperature but positive with wort pH, whereas the reverse is the case for the two fungal β-glucanases. Within the ranges of pH and temperature tested the bacterial β-glucanase has 2–5 times the activity of the fungal ones. No evidence for synergic or competitive effects between bacterial and fungal β-glucanases have been found.