A novel approach for improving yeast viability and baking quality of frozen dough by adding biogenic ice nucleators from Erwinia herbicola

Freezing deteriorates the baking quality of frozen bread dough by causing lethal injury to yeast cells and depolymerization to the gluten network. To investigate the potential of biogenic ice nucleators in frozen food applications, the effect of extracellular ice nucleators (ECINs) from Erwinia herbicola on the baking quality of frozen dough upon three freeze/thaw cycles were investigated. With addition of ECINs to the activity of 2.4 × 10⁶ units per gram of dough, hardening of bread crumb caused by three freeze/thaw cycles was alleviated by about 50% compared to the control. Additionally, the bread from frozen dough with added ECINs showed 50% larger specific volume compared to the control. The mechanism of cryoprotective effects from ECINs was possibly that ECINs helped in preserving the viability of yeast cells during freeze/thaw cycles. ECINs were able to improve the viability of log-phase and stationary-phase yeast cells in suspensions by about 100 and 10 fold, respectively, and viability of yeast in the frozen dough by 17%. This study revealed the potential of ECINs as a cryoprotectant for applications in the food and biotechnology industries.     

Investigating the impact of α-amylase, α-glucosidase and glucoamylase action on yeast-mediated bread dough fermentation and bread sugar levels

Wheat flour is generally supplemented with α-amylases to increase maltose levels in bread dough and increase loaf volume. While the preference of yeast for glucose and fructose over maltose as substrate for fermentation is well documented, the impact of maltose versus glucose producing enzymes on bread dough fermentation kinetics and bread sugar levels is ill documented. Hence the impact of α-amylase, α-glucosidase and glucoamylase action on both aspects was investigated. Glucoamylase and α-amylase increase the total fermentable sugar content of dough, while α-glucosidase only affects the glucose/maltose ratio. Due to their effect on total fermentable sugar levels, addition of α-amylase or glucoamylase prolongs the total productive fermentation time, while this is not the case for α-glucosidase. In contrast to α-amylase, both glucoamylase and α-glucosidase supplementation leads to higher CO₂ production rates during the initial stages of fermentation. In the final bread product, different sugar levels are observed depending on the dosage and type of starch-degrading enzyme. The results of this study imply that long and short fermentation processes benefit from α-amylase and α-glucosidase addition, respectively, while glucoamylase supplementation is suitable for both long and short fermentation times.     

Impact of different beer yeasts on wheat dough and bread quality parameters

In order to investigate the impact of different yeast strains from the species Saccharomyces cerevisiae on the dough and bread quality parameters, wheat flour was fermented using different beer yeasts. The results show that beer yeast strains could be included in the baking process since S. cerevisiae T-58 and S. cerevisiae s-23 provided adequate gas production and dough formation with superior structural properties like extensibility and stickiness to S. cerevisiae baker's yeast. The resulting breads show the highest specific volume with the highest slice area and the highest number of cells and the lowest hardness over time. The different yeasts had also an impact on the crust colour due to their abilities to ferment different sugars and on shelf life due to the production of a range of different metabolic by-products. According to this study it was possible to produce higher quality bread by using yeast coming from the brewing industry, instead of bread containing standard baker's yeast.     

Amy2Polymorphism as a Possible Marker of β-Glucanase Activity in Barley (Hordeum vulgareL.)

The polymorphism of barleyalpha-amylases, as revealed by isoelectric-focusing (IEF), was studied in a population of 126 doubled-haploids from a cross between the cultivars Morex and Steptoe. In particular, the pattern was determined for an additional low pI alpha-amylase isozyme detected in cultivar Steptoe. No significant effect of this isozyme onalpha-amylase activity was observed, but a significant effect on β-glucanase activity was detected for two out of four trial locations. Quantitative Trait Loci (QTL) foralpha-amylase and β-glucanase activities were also investigated. The results suggest a potential location for the β-glucanase locusGlb2, on chromosome 1 close to theAmy2locus foralpha-amylase genes.     

Influence of oxygen content of kneading atmosphere on oxygen uptake and relaxation index of bread dough with various additives

The role of oxygen during mixing of bread dough was investigated using a unique air-tight mixer in which oxygen content of the atmosphere surrounding the dough was fixed at different levels ranging from 10 to 30%. Effects of the presence in bread dough composition of various O₂ consumers, such as yeast, lipoxygenase (LOX), and additional glucose oxidase (GOX) and/or soybean or horse bean flour (containing LOX), were studied in order to characterize the competition phenomena for oxygen in the different conditions. O₂ uptake by dough during mixing was followed and relaxation tests were performed on the resulting bread dough. Variation of O₂ level of the gaseous atmosphere had no rheological impact on basic bread dough (with no additional oxidative system), even though this level was found to lead to an increase of O₂ consumption by dough, especially at the beginning of mixing. The competition for O₂ consumption among yeast, LOX and GOX was decreased by kneading under a 30% O₂ atmosphere, enabling GOX to reveal its structuring effect. Finally, mixing bread dough containing GOX under O₂-enriched atmosphere enabled keeping a standard dough relaxation index, even though dough water content was increased. This opens new perspectives for improving bread softness.     

Use of falling number and rapid visco analyser instruments to estimate sorghum malt diastatic power

Diastatic power (DP), a measure of joint alpha- and beta-amylase activities, is the most important quality criterion of sorghum malt. There is a need for a rapid method to estimate sorghum malt DP. Such methods have been developed using both the Falling Number (FN) and Rapid Visco Analyser (RVA) instruments, which measure alpha-amylase activity. Maize starch is used as substrate at a ratio of malt to maize starch of 1:29. Good estimates of DP can be obtained with malts prepared from grain of a single cultivar (FN, r = −0·872; RVA, r = −0·993). The estimate is less good with malts prepared from different cultivars (FN, r = −0·759; RVA, r = −0·759), probably a result of the different cultivars having varying proportions of alpha-amylase relative to DP. The methods are well suited, therefore, to quality control in maltings and breweries, but less suitable for evaluating the malting quality of different cultivars.     

Mechanisms Leading to Excess Alpha -Amylase Activity in Wheat (Triticum aestivum, L) Grain in the U.K.

The frequency and mechanisms of four modes of alpha -amylase enzyme accumulation in U.K. wheat, retained pericarp alpha -amylase activity (RPAA), pre-maturity alpha -amylase activity (PMAA), pre-maturity sprouting (PrMS) and post-maturity sprouting (PoMS), were investigated in field and laboratory experiments. Of 56 cultivar site year combinations (four model cultivars grown at up to four sites from 1994–1997), enzyme activity was detected in 32 cases, in 23 cases sufficient to reduce Hagberg falling number (the usual industry measure of alpha -amylase) below the commercial criterion (250 s). The frequency of occurrence of different modes of enzyme accumulation was in the order PoMS>PMAA>PrMS>RPAA. Both PMAA and PrMS were more common than expected and the most usual pattern was for alpha -amylase to accumulate by several modes. Although green grains are rejected as impurities, study of grain colour in relation to pericarp alpha -amylase activity showed that the enzyme could persist in non-green grains in levels sufficient to affect the Hagberg value. Two factors thought to promote PMAA, grain drying rate and transient changes in temperature in early development, were studied in the field and controlled environment cabinets. No significant difference was found in grain drying rate between samples where PMAA was or was not identified. However, out of 19 transfers from a cool (16/10 °C) to a warm (26/20 °C) temperature regime, six led to significant increases in PMAA. No transfers after 45% grain moisture increased PMAA. PrMS occurred as early as 67% grain moisture and susceptibility usually increased with stage of development, being greatest in the grain dough stage. PrMS susceptibility varied with cultivar (in the same order as PoMS sensitivity) and was affected by environmental factors.     

Extensive degradation of native starch granules by alpha-amylase from aspergillus fumigatus

Starch granules of various botanical origins were subjected to enzymic degradation by purified alpha-amylases from pig pancreas, Bacillus sp. and Aspergillus fumigatus (Aspergillus sp. K-27). With the A. fumigatus enzyme, glucose in alpha-anomeric configuration was the sole end degradation product regardless of the starch tested. The efficiency of this enzyme was very high on all native starch granules. Starches from normal and waxy maize, smooth pea and wheat were completely solubilised within 30 h using 1·34 nKat/mg of substrate. High-amylose maize, wrinkled pea and potato starches were degraded to lower extents (50, 70 and 45%, respectively). Such high enzymic efficiency was not observed with alpha-amylases from pig pancreas or Bacillus sp. With alpha-amylase from A. fumigatus, normal and waxy maize starches displayed highly eroded layered structures when observed by scanning or transmission electron microscopy during degradation. In contrast, potato and high-amylose maize starches produced a minor fraction of endo-eroded granules, whereas the rest of the granules exhibited superficial porosity.     

Genetic and environmental variation in the diastatic power of australian barley

Variation in the diastatic power of Australian barley, and the relationships between diastatic power and the starch-degrading enzymes contributing to diastatic power, were investigated in 11 cultivars of barley grown at six diverse locations in Australia. Diastatic power varied with genotype and location, with the levels ranging from 3·1 to 16·5 U/kg. For alpha-amylase activity, levels across cultivar and location ranged from 52 to 214 U/g, for beta-amylase activity they ranged from 201 to 1550 U/g; and, for limit dextrinase activity, they ranged from 56 to 636 U/kg. Alpha-amylase (r = 0·64) and beta-amylase (r=0·77) activities were correlated more strongly with diastatic power than was limit dextrinase (r=0·37). Grain nitrogen content was correlated positively with diastatic power (r=0·71), largely because of the relationship between nitrogen content and beta-amylase activity (r=0·82). High grain nitrogen contents were also associated with small grain sizes (r=−0·76) and low hot-water extracts (r=−0·75). The levels of alpha-amylase activity were correlated more closely with limit dextrinase activity (r=0·65) than with beta-amylase activity (r=0·28). The results indicate the need to select barley cultivars separately for alpha-amylase and beta-amylase activities to achieve high levels of diastatic power.     

Development of anIn VitroEnzymic Digestion Method for Removal of Starch and Protein and Assessment of its Performance Using Rye andWheat Breads

The development of anin vitroenzymic batch method for pretreatment of cereal samples prior toin vitrofermentation is presented. A statistical experimental design is described and the importance of bile and pepsin for the digestibility of starch was investigated. Day-to-day variation was studied by measuring glucose released and total starch in the digestion residue of rye flour. Four samples, wholemeal rye flour and bread, wholemeal and white wheat breads, were tested to evaluate the performance of the digestion procedure. Pepsin and bile enhanced the hydrolysis of slowly digestible starch. The extent of starch hydrolysis in rye and wheat breads (93–95%) was reproducible and about the same as using thermostablealpha-amylase. More soluble fibres were released from the wholemeal rye bread than from the wholemeal wheat bread. The procedure is an efficient method for the removal of starch and protein under physiological conditions prior toin vitrofermentation. The method can also be used as a tool for studying the solubility of dietary fibre (DF).     

Involvement of the Protein Network in thein vitroDegradation of Starch from Spaghetti and Lasagne: a Microscopic and Enzymic Study

This study aimed to determine the involvement of the protein network in thein vitrodegradation of starch from intact pasta strands. The physical structure of pasta was characterised during enzymic treatment by microscopy, and hydrolysis kinetics were interpreted. When incubated with human salivaryalpha-amylase (HSA), the protein network remained intact, retarding starch degradation. The presence of proteases contaminating pig pancreaticalpha–amylase contributed to the partial hydrolysis of the protein network (similar to the effect of pepsin), increasing starch degradation after 1 h ofalpha-amylolysis in comparison with HSA. Complete accessibility of starch toalpha-amylase after 72 h ofalpha-amylolysis was demonstrated by chromatography, both with intact and degraded protein networks. Two hours pre-treatment of pasta with pepsin or HSA increased respectively the initial degradation of starch and protein. Starch or protein presence, therefore, hinders the action of the enzyme, which does not degrade it. The protein network is not itself a physical barrier toalpha-amylase access to starch in pasta. Both microscopy and hydrolysis kinetics have revealed that new physico-chemical factors related to food structure should be considered in the enzymic degradation of pasta.     

Comparison of the impact of dextran and reuteran on the quality of wheat sourdough bread

This study investigates the influence of in situ exopolysaccharides (EPS) and organic acids on dough rheology and wheat bread quality. Dextran forming Weissella cibaria MG1 was compared to reuteran forming Lactobacillus reuteri VIP. For in situ production of EPS, sourdoughs were supplemented with 15% sucrose. Control sourdoughs were prepared with the same strain but without sucrose. W. cibaria MG1 and L. reuteri VIP formed 5.1 and 5.8 g kg⁻¹ dextran and reuteran, respectively. Formation of EPS from sucrose led to production of high amounts of acetate by L. reuteri VIP, but only small amounts were detected in W. cibaria MG1 sourdough. EPS containing sourdough or control sourdough was incorporated at 10% and 20% in wheat dough. EPS significantly influenced the rheological properties of the dough, with dextran exhibiting the strongest impact. The addition of dextran enriched W. cibaria MG1 sourdough significantly increased CO₂ production, whereas increased acidity in reuteran containing dough reduced gas production. The quality of wheat bread was enhanced when 10% of L. reuteri-sucrose sourdough was added. The positive effect of reuteran was masked by increased acidification after 20% sourdough addition. Incorporation of dextran enriched sourdough (10% and 20%) provided mildly acidic wheat bread with improved bread quality.     

Impact of α-Amylase During Breadmaking on In Vitro Kinetics of Starch Hydrolysis and Glycaemic Index of Enriched Bread with Bran

Nowadays, the use of enzymes has become a common practice in the bakery industry, as they can improve dough quality and texture of final product. However, the use of α-amylases could have a negative effect in the glycaemic load of product, due to the released sugars from the starch hydrolysis that are not used by yeasts during the fermentation process. This study evaluated the effect of the addition of α-amylase in bakery products with bran on in vitro kinetics of starch hydrolysis. The use of flour with a high degree of extraction or high bran amount could decrease the GI even with the inclusion of α-amylase in the formulation. It should be taken into account the amount of bran and α-amylase when formulating breads in order to obtain products with lower GI than white bread. However, the fact that kinetics of starch hydrolysis remained unaltered indicates that the use of α-amylase in bread-making processes could provide technological advantages improving quality of breads without markedly changes in their glycaemic index.     

Protein Inhibitors ofAlpha-amylase in Mature and Germinating Grain of Rye (Secale cereale)

The activities of endogenous (R-type) and exogenous acting (D-type) protein inhibitors ofalpha-amylase and the activities ofalpha- and total amylase were determined in milling fractions of rye. High D-type amylase inhibitor activities were detected in the embryo (255 IU/g) and in the endosperm fraction (64·9 IU/g), low inhibitor activities were found in the aleurone layer fraction (25·9 IU/g). The highest R-typealpha-amylase inhibitor activity was found in the aleurone layer fraction (32·6 IU/g), and the lowest value in the epidermis containing fraction (5·0 IU/g). The D- and R-typealpha-amylase inhibitor activities varied with growing conditions. D-type amylase inhibitor activities were found to be high in those samples which grew under drought conditions and low in samples cultivated under wet and cool weather. Higher R-typealpha-amylase inhibitor activities were found in rye genotypes cultivated under wet conditions and lower values under dry weather. There were small variations inalpha-amylase inhibitor activities between sprout-stable and sprout-sensitive rye genotypes. The D- and R-typealpha-amylase inhibitor activities of all varieties were stable during 72 h of germination. Similar soil conditions will therefore lead to differentialalpha-amylase inhibitor activities depending on weather conditions during growth.     

Improved viscoelastic zein–starch doughs for leavened gluten-free breads: Their rheology and microstructure

Gluten-free bread was prepared from commercial zein (20 g), maize starch (80 g), water (75 g), saccharose, NaCl and dry yeast by mixing above zein's glass transition temperature (T_g) at 40°C. Addition of hydroxypropyl methylcellulose (HPMC, 2 g) significantly improved quality, and the resulting bread resembled wheat bread having a regular, fine crumb grain, a round top and good aeration (specific volume 3.2 ml/g). In model studies, HPMC stabilized gas bubbles well. Additionally, laser scanning confocal microscopy (LSCM) revealed finer zein strands in the dough when HPMC was present, while dynamic oscillatory tests showed that HPMC rendered gluten-like hydrated zein above its T_g softer (i.e. |G*| was significantly lower). LSCM revealed that cooling below T_g alone did not destroy the zein strands; however, upon mechanical impact below T_g, they shattered into small pieces. When such dough was heated above T_g and then remixed, zein strands did not reform, and this dough lacked resistance in uniaxial extension tests. When within the breadmaking process, dough was cooled below T_g and subsequently reheated, breads had large void spaces under the crust. Likely, expanding gas bubbles broke zein strands below T_g resulting in structural weakness.     

Effect of the thermostable xylanase B (XynB) from Thermotoga maritima on the quality of frozen partially baked bread

The effect of the recombinantly produced xylanase B (XynB) from Thermotoga maritima MSB8 on the quality of frozen partially baked bread (FPBB) was investigated. Addition of XynB to wheat flour dough resulted in a significant increase in dough extensibility (L), swelling (G), and a decrease in dough resistance to deformation (P), configuration. Bread crumb characteristics were studied by differential scanning calorimeter (DSC) and dynamic-mechanical analysis (DMA). The results show that addition of XynB leads to improvements in the bread quality of FPBB and retards bread staling compared to the control. The greatest improvements were obtained in specific volume (+35.2%) and crumb firmness (−40.0%). The control FPBB was significantly firmer in texture and higher in amylopectin recrystallization than the bread with XynB. During frozen storage of FPBB with and without XynB for 8 weeks, the crumb firmness increased gradually and the specific volume slightly decreased with the frozen storage time. The ΔH values of freezable water (FW) endothermic transitions increased with frozen storage time for all samples. However, addition of XynB lowered the ΔH values indicating a decrease in FW. Therefore, XynB is useful in improving the quality of FPBB. DMA was also used to monitor the shrinking behavior of the samples. Addition of XynB increased the contraction during chilling but significantly diminished the total shrinking and frozen-state shrinking of the bread crumb during the freezing process.     

Isolation and characterization of EMS-induced Dy10 and Ax1 high molecular weight glutenin subunit deficient mutant lines of elite hexaploid wheat (Triticum aestivum L.) cv. Summit

The mixing properties of the dough are critical in the production of bread and other food products derived from wheat. The high molecular weight glutenin subunits (HMW-GS) are major determinants of wheat dough processing qualities. The different alleles of the HMW-GS genes in hexaploid wheat vary in their effect on dough quality. To determine the contribution of the individual HMW-GS alleles, lines deficient in HMW-GS proteins were generated by chemical mutagenesis in the elite bread wheat Triticum aestivum cv. Summit. In this report we describe the identification and characterization of Dy10 and Ax1 deficient lines. Examination of the effect of Dy10 and Ax1 deficiency on dough rheological properties by mixography showed shorter mixing time to reach peak resistance, and weaker and less extensible doughs relative to the wild type control. This is the first time that the role of Dy10 in vivo has been examined apart from the Dx5 + Dy10 allelic pair combination.     

Properties of bread made from frozen dough containing waxy wheat flour

The quality of bread made from frozen dough is diminished, and staling rate is increased by changes that occur during freezing and storage. New cultivars of waxy wheat flour (WWF), containing higher levels of amylopectin, may help improve the quality of baked products. Bread quality and staling were investigated for bread containing 0–45% WWF and 55–65% water after freezing and 90-day frozen storage. The specific volume was highest with 15% WWF substitution and 60% water in bread made from both unfrozen and frozen dough. With higher levels of WWF and lower water content, bread staling rates decreased. Bread with higher levels of WWF were darker and had greater color variation. ¹H NMR studies showed that bread with greater WWF and water had higher transverse relaxation (T₂) times (9–11 ms), but less change in T₂ during storage. This research demonstrated that specific combinations of WWF and water produced a better quality of bread after dough freezing.     

Modelling the Contribution ofAlpha-Amylase,Beta-Amylase and Limit Dextrinase to Starch Degradation During Mashing

Response surface methodology was used to determine the levels ofalpha-amylase,beta-amylase and limit dextrinase enzymes required for efficient conversion of starch to fermentable sugars during mashing. Micro-scale mashes with purified barley starch and malt enzymes were performed in a Brewing Research Foundation mash bath, and mash liquors were analysed for solubilised starch, reducing sugars (neocuproine assay) and fementable sugars (anion exchange HPLC). Fermentable sugars in the mash liquor were positively correlated with reducing sugars (R²=0·94) and the percentage of starch solubilised during mashing (R²=0·68). A multiple regression equation relating the levels of the three starch degrading enzymes to the percentage of starch hydrolysed to fermentable sugars gave a good fit to the second order response surface (R²=1·00, RMSE=1·37%). Addition of limit dextrinase to the mashes resulted in a substantial increase in levels of fermentable sugars, and limit dextrinase showed a synergistic effect in increasing levels of maltose in the mash liquor when combined with high levels ofbeta-amylase. The efficiency of any one starch degrading enzyme in a mash is influenced by the presence of other starch degrading enzymes. Commercial malts contain excess levels ofbeta-amylase and below optimal levels of limit dextrinase. Malt extract may not be a good indicator of the level of fermentable carbohydrates produced during mashing.     

Molecular characterization of dimeric alpha-amylase inhibitor genes in wheat and development of genome allele-specific primers for the genes located on chromosome 3BS and 3DS

Alpha-amylase inhibitors are attractive candidates for the control of seed weevils as these insects are highly dependent on starch as an energy source. For weevil control, alpha-amylase inhibitors and their genes could be used to genetically engineer weevil resistant seeds. Thirty genes encoding dimeric alpha-amylase inhibitors were isolated from Triticum aestivum L. ‘Chinese Spring’ and characterized by nucleotide and amino acid sequence analysis. Eleven representative alpha-amylase inhibitor genes were identified, and the deduced amino acid sequences of these genes were of high coherence (95.1%). These inhibitors and others obtained from the wheat EST database were clustered into three groups, the genes from ‘Chinese Spring’ were present in each group. Specific primer sets were designed for each group, based on the SNPs of these genes, and the chromosome locations of each group of inhibitor genes investigated by amplification of the ‘Chinese Spring’ ditelosomic lines. There were two and one groups of inhibitor genes on chromosomes 3BS and 3DS, respectively, whereas no group of inhibitor genes was found on chromosome 3AS. Thus, the primer set for each group of inhibitor genes was genome allele-specific. The two known inhibitors, 0.53 and 0.19, were located on chromosomes 3BS and 3DS, respectively. The validity of the three genome allele-specific primer sets was confirmed by amplifications in 15 accessions of Triticum urartu, Triticum monococcum, Aegilops tauschii and Triticum dicoccoides. These results gave further support at the molecular level, that the 24 kDa dimeric alpha-amylase inhibitors in cultivated wheat are encoded by a multigene family.