James H. Bryce

Development of Limit Dextrinase in Germinated Barley (Hordeum vulgare L.) (Evidence of Proteolytic Activation).

Barley (Hordeum vulgare L.) that had been malted for 5 d developed only a small amount of bound (inactive) limit dextrinase, and very little free (active) enzyme was detected. Continuation of malting for up to 10 d only slightly increased the amount of both bound and free forms. Grain grown under conditions of ample moisture (wet grown) for 5 d produced a much higher amount of bound enzyme but a similarly low amount of free enzyme compared to malting conditions. After 10 d of growth there was a decrease in the amount of bound enzyme and a large increase in the amount of free enzyme, such that almost all of the enzyme was present in the free form. A more detailed study of limit dextrinase development in wet-grown grains revealed that a bound form was rapidly produced soon after germination. Five to 6 d after germination the amount of bound enzyme decreased rapidly and a very low amount was found in grains 9 d after germination. Meanwhile, a free form appeared slightly later and its initial rate of development was slow. At about 5 d after germination, precisely when the bound enzyme began to decrease, the free form increased rapidly, so that by 9 d after germination nearly all the enzyme was in the free form. The release of bound limit dextrinase in vitro occurred by proteolytic modification through the action of cysteine proteinases that were kept active or activated by the presence of reduced thiols in the extraction medium. The presence of cysteine proteinases was confirmed by inhibition studies using the inhibitors iodoacetamide, N-ethylmaleimide, antipain, and leupeptin. In addition, most of the bound form of limit dextrinase was soluble in 0.2 M sodium acetate buffer (pH 5.0) following extraction at 30[deg]C for 16 h and centrifugation at 3000g.

The effect of combinations of Fusarium mycotoxins (deoxynivalenol, zearalenone and fumonisin B1) on growth of brewing yeasts

The interactive effect of combinations of the Fusarium mycotoxins deoxynivalenol (DON), zearalenone (ZEA) and fumonisin B1 (FB1) on growth of brewing yeasts was examined. Yeast growth was assessed by measurement of dry weight or relative growth, cell number, viability and conductance change of the growth medium using direct and indirect methods. The interactive effect of a combination of these mycotoxins was subject to the ratio of toxins in the mixture and the toxicity of individual toxins on yeast growth. When a combination of mycotoxins at low concentration was added into the growth medium, no significant inhibitory effect on growth was observed compared to controls. However, when a combination of high concentrations of DON and ZEA which individually inhibited yeast growth was examined, the interactive effect was shown to pass from antagonism to synergism depending on the ratio of the toxins in the mixture. As a synergistic interaction between these Fusarium mycotoxins was observed only at high concentrations, which were far higher than would be expected in good quality grain, they are not a concern when related to yeast growth under the brewing conditions studied.

Regulation of Mitochondrial Respiration

Publisher Summary This chapter discusses the regulation of mitochondrial respiration. Sucrose and oxygen are substrates for respiration, and if their supply is limited they will restrict mitochondrial respiration. The ensueing decrease in ATP synthesis and availability will reduce the rate of biosynthetic reactions. On the other hand, where respiratory substrates are readily available, the rate of mitochondrial respiration will be regulated by the pace of synthetic events; the rate at which these reactions utilize ATP will be the factor controlling the kinetic and thermodynamic parameters of the AdN that regulate mitochondrial respiration. It should, thus, be evident that mitochondrial respiration is regulated by AdN, and this regulation is mediated through the supply of respiratory substrate and the pace of synthetic events. Through their generation of energy, mitochondria, thus, regulate the balance between biosynthesis and supply of respiratory substrate. The chapter also reviews mitochondrial glycine oxidation during photorespiration, role of mitochondria in c 4 and cam metabolism, and role of mitochondria in nitrate assimilation.

Control of succinate oxidation by cucumber (Cucumis sativus L.) cotyledon mitochondria: The role of the adenine-nucleotide translocator and extra-mitochondrial reactions

The aim of this work was to assess the extent to which mitochondria control the gluconeogenic flux in cucumber (Cucumis sativus L.) cotyledons, by quantifying the distribution of control of succinate oxidation by cotyledon mitochondria. The methods of metabolic control analysis were applied under state 3 and state 4 conditions and in the presence of cell-free extracts in order to simulate in-vivo conditions. Oxygen uptake by isolated cotyledon mitochondria oxidising succinate under state 3 conditions was examined using inhibitor titrations. During lipid mobilisation in light-grown cotyledons (3-4 d post-imbibition), control was shared between the adenine-nucleotide translocator (flux-control coefficient, C = 0.25–0.28) and the dicarboxylate-uptake system (C = 0.69–0.72). The dicarboxylate-uptake system was also important in dark-grown cotyledons at this stage (C = 0.55–0.57). In the photosynthetic phase of development (more than 5 d post-imbibition) control rested with the respiratory chain. Application of an external ATP demand provided either by cell-free extracts of cucumber cotyledons or a glucose/hexokinase ADP-regenerating system showed that the reactions outside the mitochondria exert control (C = 0.45–0.54 and C = 0.24–0.38, for cytosolic extract and glucose/hexokinase, respectively). The adenine-nucleotide translocator was a controlling step of both oxygen uptake (C = 0.11–0.32) and the flux between succinate and hexose phosphates (C = 0.28). Other mitochondrial steps made a significant contribution to control. Control of oxygen uptake was dependent on both the nature of the external load and on the rate of phosphorylation. A potential role for mitochondrial membrane-transport processes, including the adenine-nucleotide translocator, is proposed for the integration of lipid breakdown and gluconeogenesis in vivo.

Effect of germination temperatures on proteolysis of the gluten-free grains rice and buckwheat during malting and mashing.

This study examined the performance of rice and buckwheat when malted under various temperature conditions and for different lengths of time. The mashed malts produced from both rice and buckwheat contained a wide spectra of sugars and amino acids that are required for yeast fermentation, regardless of malting temperature. At the germination temperatures of 20, 25, and 30 °C used, production of reducing sugars and free amino nitrogen (FAN) followed similar patterns. This implies that temperature variations, experienced in different countries, will not have an adverse effect on the production and release of amino acids and sugars, required by yeast during fermentation, from these grains. Such consistency in the availability of yeast substrates is likely to reduce differences in processing when these malts are used for brewing. This study revealed that, while rice malt consistently produced more maltose than glucose, buckwheat malt gave several times more glucose than maltose, across all germination temperatures. Buckwheat malt also produced more soluble and free amino nitrogen than rice malt. Unlike sorghum, which has gained wide application in the brewing industry for the production of gluten-free beer, the use of rice and buckwheat is minimal. This study provides novel information regarding the potential of rice and buckwheat for brewing. Both followed similar patterns to sorghum, suggesting that they could play a similar role to sorghum in the brewing industry. Inclusion of rice and buckwheat as brewing raw materials will increase the availability of suitable materials for use in the production of gluten-free beer, potentially making it more sustainable, cheaper, and more widely available.

Influence of cultural conditions on sensitivity of brewing yeasts growth to Fusarium mycotoxins zearalenone, deoxynivalenol and fumonisin B1

Abstract The influence of cultural parameters, viz. temperature, inoculum size and growth under shaker or static conditions, on the sensitivity of Saccharomyces cerevisiae lager and ale strains to the presence of low (2 μg ml −1 ) and high (100 μg ml −1 ) concentrations of the mycotoxins zearalenone (ZEA), deoxynivalenol (DON) and fumonisin (FB 1 ) was examined. Toxic effects were assessed by measuring relative growth, cell number, viability and conductance change in growth medium, and monitoring cell morphology. Culture conditions appeared to affect sensitivity of yeasts to mycotoxins at 100 μg ml −1 growth medium, but not at 2 μg ml −1 . For both strains, cultures developing from a lower inoculum concentration were more strongly inhibited than at a higher inoculum level, except for the lager strain grown at 13°C and the ale strain at 25°C in the presence of FB 1 . Growth inhibition did not seem to be influenced by inoculum size. Growth temperature was important in sensitivity to the mycotoxins. At 25°C, the ale strain was more sensitive to ZEA and DON than the lager strain, but the lager strain was more sensitive to these two toxins at 13°C than at 25°C. In contrast, at 13°C the lager strain was more sensitive to FB 1 than at 25°C, and at 25°C it was more sensitive than the ale strain. Both strains were more sensitive to DON and ZEA when grown statically than with shaking, but in shake-culture the lager strain was more sensitive to FB 1 .

Effect of Germination Temperatures on Proteolysis of the Gluten-Free Grains Sorghum and Millet during Malting and Mashing

Our study showed that sorghum and millet followed a similar pattern of changes when they were malted under similar conditions. When the malt from these cereals was mashed, both cereal types produced wide spectra of substrates (sugars and amino acids) that are required for yeast fermentation when malted at either lower or higher temperatures. At the germination temperatures of 20, 25, and 30 °C used in malting both cereal types, production of reducing sugars and that of free amino nitrogen (FAN) were similar. This is an important quality attribute for both cereals because it implies that variation in temperature during the malting of sorghum and millet, especially when malting temperature is difficult to control, and also reflecting temperature variations, experienced in different countries, will not have an adverse effect on the production and release of amino acids and sugars required by yeast during fermentation. Such consistency in the availability of yeast food (substrates) for metabolism during fermentation when sorghum and millet are malted at various temperatures is likely to reduce processing issues when their malts are used for brewing. Although sorghum has gained wide application in the brewing industry, and has been used extensively in brewing gluten-free beer on industrial scale, this is not the case with millet. The work described here provides novel information regarding the potential of millet for brewing. When both cereals were malted, the results obtained for millet in this study followed patterns similar to those of sorghum. This suggests that millet, in terms of sugars and amino acids, can play a role similar to that of sorghum in the brewing industry. This further suggests that millet, like sorghum, would be a good raw material for brewing gluten-free beer. Inclusion of millet as a brewing raw material will increase the availability of suitable materials (raw material sustainability) for use in the production of gluten-free beer, beverages, and other products. The availability of wider range of raw materials will not only help to reduce costs of beer production, but by extension, the benefit of reduced cost of production can be gained by consumers of gluten-free beer as the product would be cheaper and more widely available.