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PubMed2000

A mathematical model of the formation of fermentable sugars from starch hydrolysis during high-temperature mashing.

During the mashing process of brewing, activity of the amylolytic enzymes decays due to the high temperatures used to gelatinise the starch. Because the different enzymes produce different sugars, high temperatures can be exploited to modify the fermentability of resulting worts. This is especially useful when producing low alcohol beers. The expression a.exp(b.t)-c.exp(d.t) (where t is the temperature of the mash in degrees C) provides a simple but useful description of the activity of the amylases. Combining the activities of alpha- and beta-amylases results in a prediction of the resulting fermentability. A simple modification to the expression accommodates changes in mash thickness. The error of prediction is approximately 3 degrees of fermentability. The model is not appropriate for predicting the fermentability of worts produced at the lower standard mashing temperatures. It can be used without the necessity of analytical parameters so analyses that the brewer would not normally perform are not required. If increased accuracy is needed, the results of two previous mashes can be used to modify the parameters used.

Journal Article↗

PubMed2003

Effect of reducing and oxidizing agents and pH on malt endoproteolytic activities and brewing mashes.

The activities of the four endoproteinase classes of malted barley are known to vary with pH, and it seemed likely that the cysteine enzyme activities could be altered by redox agents. This study determined how altering the pH and adding redox agents to mashes influenced the worts that were produced during the brewing process. The reducing agents cysteine.HCl, dithiothreitol, and beta-mercaptoethanol increased the proteolysis that occurred in malt extracts and mashes. This increased proteolysis was negated by the addition of the oxidizing agents diamide or hydrogen peroxide. The addition of reducing agents to mashes increased the soluble protein, free amino nitrogen (FAN), and extract values of their resultant worts, and this effect was abolished by the concomitant addition of oxidizing agents. Raising the pH values of the mashes strongly reduced their proteolytic activities, soluble protein, FAN, and extract values, but not their beta-glucan levels. These results show that several of the major aspects of malting and brewing quality can be adjusted by varying the pH and redox qualitites of mashes, which could be helpful to brewers. These results also strengthen the previous proposal made by Buchanan et al. that the redox status of plants may play a significant part in controlling their physiology.

Cysteine↗

PubMed2002

The neural differentiation gene Mash-1 has a distinct pattern of expression from the taste reception-related genes gustducin and T1R2 in the taste buds.

Taste bud cells have a limited lifespan and are continuously replaced just like other epithelial cells. Although there is some evidence that taste buds may arise from the local epithelium, taste receptor cells have neuronal properties. This implies that there must be a critical stage at which the epithelial precursor cells for taste receptor cells start to exhibit neural properties during the differentiation of the taste receptor cells. The expression of the neural-specific transcription factors Mash-1 and Prox-1 in the nervous system is transient and precedes neuronal differentiation. Therefore, we examined the expression of Mash-1 and Prox-1 in the epithelium of circumvallate papillae of the tongue in order to clarify the localization of the precursor cells with neural properties and observed that both expressions are restricted to the taste buds. Two-colour in situ hybridization showed that the signals for Mash-1 did not overlap those for taste receptor cell-specific genes such as gustducin and T1R2. In the process of development and regeneration of the taste buds, the expression of Mash-1 preceded that of gustducin and T1R2. These observations suggest that Mash-1 could be a candidate for a marker of immature taste receptor cells, including the cells that express gustducin and/or T1R2 at a later stage.

Animals↗

PubMed1998

Single chain dimers of MASH-1 bind DNA with enhanced affinity.

By designing recombinant genes containing tandem copies of the coding region of the BHLH domain of MASH-1 (MASH-BHLH) with intervening DNA sequences encoding linker sequences of 8 or 17 amino acids, the two subunits of the MASH dimer have been connected to form the single chain dimers MM8 and MM17. Despite the long and flexible linkers which connect the C-terminus of the first BHLH subunit to the N-terminus of the second, a distance of approximately 55 A, the single chain dimers could be produced in Escherichia coli at high levels. MM8 and MM17 were monomeric and no 'cross-folding' of the subunits was observed. CD spectroscopy revealed that, like wild-type MASH-BHLH, MM8 and MM17 adopt only partly folded structures in the absence of DNA, but undergo a folding transition to a mainly alpha-helical conformation on DNA binding. Titrations by electrophoretic mobility shift assays revealed that the affinity of the single chain dimers for E box-containing DNA sequences was increased approximately 10-fold when compared with wild-type MASH-BHLH. On the other hand, the affinity for heterologous DNA sequences was increased only 5-fold. Therefore, the introduction of the peptide linker led to a 4-fold increase in DNA binding specificity from -0.14 to -0.57 kcal/mol.

Amino Acid Sequence↗

PubMed1996

Distinct subpopulations of enteric neuronal progenitors defined by time of development, sympathoadrenal lineage markers and Mash-1-dependence.

Enteric and sympathetic neurons have previously been proposed to be lineally related. We present independent lines of evidence that suggest that enteric neurons arise from at least two lineages, only one of which expresses markers in common with sympathoadrenal cells. In the rat, sympathoadrenal markers are expressed, in the same order as in sympathetic neurons, by a subset of enteric neuronal precursors, which also transiently express tyrosine hydroxylase. If this precursor pool is eliminated in vitro by complement-mediated lysis, enteric neurons continue to develop; however, none of these are serotonergic. In the mouse, the Mash-1-/- mutation, which eliminates sympathetic neurons, also prevents the development of enteric serotonergic neurons. Other enteric neuronal populations, however, including those that contain calcitonin gene related peptide are present. Enteric tyrosine hydroxylase-containing cells co-express Mash-1 and are eliminated by the Mash-1-/- mutation, consistent with the idea that in the mouse, as in the rat, these precursors generate serotonergic neurons. Serotonergic neurons are generated early in development, while calcitonin gene related peptide-containing enteric neurons are generated much later. These data suggest that enteric neurons are derived from at least two progenitor lineages. One transiently expresses sympathoadrenal markers, is Mash-1-dependent, and generates early-born enteric neurons, some of which are serotonergic. The other is Mash-1-independent, does not express sympathoadrenal markers, and generates late-born enteric neurons, some of which contain calcitonin gene related peptide.

Animals↗

PubMed2005

[Modeling for prediction of arabinoxylans solubilization and endo-xylanase random attacking during mashing].

A model describing the solubilization of arabinoxylans and degradation by endo-xylanase random attacking during mashing was developed. The model was expected to predict the arabinoxylans concentration in wort at the settings of different initial value and mashing parameters for diminishing the negative effects of arabinoxylans on brewing. Results showed that the modeling errors range for the final concentration of arabinoxylans in wort was -9.5% to +13.6%. The model prediction accuracy for industrial scale mashing process was lower than that in laboratory scale. The errors were given 16.8% and 17.9%, respectively. The simulation results showed that arabinoxylans concentration was increased with the increase of mashing-in temperature, but it was decreased with prolonging the mashing-in time. The effect of initial arabinoxylans in malt on arabinoxylans concentration in wort was more remarkable than that of endo-xylanase activity in grist.

Endo-1,4-beta Xylanases↗

PubMed2000

On-line measurement of intracellular ATP of Saccharomyces cerevisiae and pyruvate during sake mashing.

The concentrations of intracellular ATP of Saccharomyces cerevisiae and pyruvate in a medium were instantaneously increased by pulse addition of glucose during starvation. They were reduced rapidly by alcohol fortification of the medium, accompanied by simultaneous increases of acetaldehyde concentration and inviability of yeast cells. These results were monitored during fermentation of sake mash by an on-line measuring method. Intracellular ATP and pyruvate concentrations were considered to be indicators of the physiological state of the yeast in sake mash. During sake mashing, it was observed that an increase in temperature enhanced the intracellular ATP concentration and the pyruvate production of the yeast. Since pyruvate production was not affected intensely by changes in temperature during cultivation in a glucose-limited chemostat, this effect was thought to be due to the enhanced rates of cell-growth and/or alcohol production. This suggests that the control of mashing temperature during cell growth until about 10% alcohol accumulation is achieved is important for the control of the pyruvate concentration in sake mash.

Journal Article↗

PubMed2002

Characterization of factors that transform linoleic acid into di- and trihydroxyoctadecenoic acids in mash.

The qualities of beer are deteriorated by the presence of either di- or trihydroxyoctadecenoic acids, which reduce the beer 'head' and produce an astringent flavor. In this study we found that native extracts of malt mash transformed linoleic acid into di- and trihydroxyoctadecenoic acids, but this transforming activity and lipoxygenase activity were inactivated by heating the mash at 70 degrees C for 30 min. Recombinant barley lipoxygenase 1 was not able to transform linoleic acid into di- and trihydroxyoctadecenoic acids. The transforming activity of mash extract heated at 70 degrees C for 30 min could be restored by the addition of recombinant barley lipoxygenase 1; in contrast, the activity of boiled mash extract was not substantially restored by the recombinant enzyme. These results indicate that di- and trihydroxyoctadecenoic acids are generated from linoleic acid by both lipoxygenase and a heat-stable enzymatic factor present in the mash.

Journal Article↗

PubMed1995

Contamination of broiler chicken's mash and litter with moulds, aflatoxins, ochratoxin A and zearalenone.

Contamination of broiler chicken's mash and litter with moulds, aflatoxin B1 (AB1), G1 (AG1), ochratoxin A (OA) and zearalenone (F-2 toxin) was examined on a broiler fattening farm. All broiler chicken's mash samples tested were contaminated with moulds. The highest total viable counts of moulds (3.3 x 10(5) per g) were found in broiler starter diet. Moulds isolated from mash were classified into 8 genera and 27 species. Many of them (Aspergillus spp., Fusarium spp., Penicillium spp.) are known as mycotoxin-producing moulds. Fusarium moniliforme was the dominant species in mash mycopopulations. Moulds were also found in all litter samples. The highest fungus count (80 x 10(5) per g) in litter was found at the beginning of fattening. Fungi isolated from litter samples belonged to 9 genera and 22 species. Aspergillus, Penicillium and Mucor were the most prevalent genera. None of the broiler chicken's mash and litter samples tested was contaminated either with AB1, AG1, OA or with F-2 toxin. F. moniliforme was zearalenone-negative also under laboratory conditions.

Aflatoxins↗

PubMed2004

Growth rates of Dekkera/Brettanomyces yeasts hinder their ability to compete with Saccharomyces cerevisiae in batch corn mash fermentations.

Growth rates determined by linear regression analysis revealed that Saccharomyces cerevisiae consistently grew more rapidly than Brettanomyces yeasts under a wide array of batch fermentative conditions, including acetic acid stress, in normal gravity (ca. 20 degrees Plato) mashes made from ground corn. Brettanomyces yeasts only grew more rapidly than S. cerevisiae when acetic acid concentrations were elevated to industrially irrelevant levels (>0.45%, w/v). Furthermore, the three Brettanomyces isolates used in this study failed to produce significant quantities of acetic acid under pure culture fermentative conditions. In fact, the small amounts of acetic acid which accumulated in pure culture fermentations of whole corn mash were below the concentration required to inhibit the growth and metabolism of S. cerevisiae. Acetic acid concentrations in pure culture Brettanomyces fermentations exceeded 0.05% (w/v) only in media containing low levels of glucose (<4%, w/v) or when aeration rates were elevated to at least 0.03 vol. air vol.-1 mash min-1. Consequently, it was concluded that Brettanomyces yeasts would not be capable of competing with S. cerevisiae in industrial batch fermentations of whole corn mash based solely on growth rates, nor would they be capable of producing inhibitory concentrations of acetic acid in such fermentations.

Acetic Acid↗

PubMed2001

Modification of the levels of polyphenols in wort and beer by addition of hexamethylenetetramine or sulfite during mashing.

The effects of addition of hexamethylenetetramine (HMT) or sulfite during mashing on the polyphenol content and oxidative stability of wort and beer have been evaluated in a series of laboratory mashings and pilot brews. HMT reduced the concentration of catechin, prodelphinidin B-3, and procyanidin B-3 in wort and beer, whereas the concentration of ferulic acid was unaffected. Sulfite had only a minor effect on the concentration of phenolics in wort and beer. Addition of HMT or sulfite during mashing increased the oxidative stability of the beer slightly as judged by the tendency of formation of radicals (ESR spin trapping technique), although sensory analysis gave identical flavor acceptance scores to beers produced from untreated and HMT-treated wort and lower scores to beer from sulfite-treated wort. No difference in the oxidative stability of the differently treated sweet worts could be detected as judged by the rate of formation of radicals. HMT addition during mashing has thus been demonstrated to be a valuable experimental tool to control the level of polyphenols in wort and for producing brews with various levels of polyphenols from a single malt.

Beer↗

PubMed2000

Thermodynamics of DNA binding of MM17, a 'single chain dimer' of transcription factor MASH-1.

MASH-1, a member of the basic helix-loop-helix (bHLH) family of transcriptional regulators, is a central factor for the regulation of the differentiation of committed neuronal precursor cells of the peripheral nervous system. We have previously produced MM17, a single chain version of this dimeric protein, by linking the C-terminal end of the first subunit to the N-terminal residue of the second subunit through a flexible peptide linker. We have now determined by isothermal titration calorimetry the thermodynamic parameters characterising the DNA binding reactions of MM17. The DNA binding specificity was relatively low and comparable to that observed for wild-type MASH bHLH. At 32 degrees C and pH 7, the concentration of MM17 at which 50% DNA binding occurred was determined as 22.8 and 152 nM for binding to MCK-S and the heterologous SP-1, respectively. Similarly to MASH bHLH the free energy of the association was only slightly temperature dependent, while both the entropy and the enthalpy change were strong functions of temperature. The free energy of DNA binding was independent of the pH for the pH range between 6 and 8. Dissection of the entropy change of the association reaction suggested that the two basic domains and the linker region between the subunits underwent a folding transition from a mainly unfolded to a predominantly ordered conformation. Therefore, like wild-type MASH bHLH, the DNA binding reaction of MM17 follows an induced fit mechanism.

Amino Acid Sequence↗

PubMed2006

Responses of male growing broilers to increasing levels of balanced protein offered as coarse mash or pellets of varying quality.

Two experiments with 14- to 35-d-old male broilers were performed to investigate the interactions between increasing levels of balanced protein (BP; 9.7, 10.7, 11.7, and 12.7 g of digestible Lys/kg of feed) and physical feed form. In Experiment 1 diets were offered as coarse mash and pellets of either good or poor quality. Good pellet quality was characterized by high durability. In Experiment 2 diets were offered as good quality pellets or coarse mash. However, for the latter particle size, distribution either was kept constant with increasing BP or changed with increasing proportion of soybean meal. Feed intake generally decreased with increasing BP; however, feed intake was highest in good pellets fed to broilers and lowest in mash fed to birds. Although there was only a small response on weight gain in mash-fed birds, feed conversion improved substantially for all feed forms with increasing BP. Particle size distribution did not affect this effect. Highest weight gain was achieved with good pellets; to achieve similar performance with poor pellet quality, higher levels of BP were needed. Carcass evaluation suggested increasing breast meat yield and particularly decreasing fat accretion with increasing BP. Results of both experiments indicate that optimum dietary amino acid levels for growing broilers may vary among different physical feed forms.

Animal Feed↗

PubMed1990

Fuel alcohol production: effects of free amino nitrogen on fermentation of very-high-gravity wheat mashes.

Although wheat mashes contain only growth-limiting amounts of free amino nitrogen, fermentations by active dry yeast (Saccharomyces cerevisiae) were completed (all fermentable sugars consumed) in 8 days at 20 degrees C even when the mash contained 35 g of dissolved solids per 100 ml. Supplementing wheat mashes with yeast extract, Casamino Acids, or a single amino acid such as glutamic acid stimulated growth of the yeast and reduced the fermentation time. With 0.9% yeast extract as the supplement, the fermentation time was reduced from 8 to 3 days, and a final ethanol yield of 17.1% (vol/vol) was achieved. Free amino nitrogen derived in situ through the hydrolysis of wheat proteins by a protease could substitute for the exogenous nitrogen source. Studies indicated, however, that exogenously added glycine (although readily taken up by the yeast) reduced the cell yield and prolonged the fermentation time. The results suggested that there are qualitative differences among amino acids with regard to their suitability to serve as nitrogen sources for the growth of yeast. The complete utilization of carbohydrates in wheat mashes containing very little free amino nitrogen presumably resulted because they had the "right" kind of amino acids.

Ethanol↗

PubMed1992

Relationship of low lysine and high arginine concentrations to efficient ethanolic fermentation of wheat mash.

Very high gravity wheat mashes containing 20 or more grams of carbohydrates per 100 mL were fermented completely by Saccharomyces cerevisiae, even though these mashes contained low amounts of assimilable nitrogen. Supplementation of wheat mashes with various amino acids or with yeast extract, urea, or ammonium sulfate reduced the fermentation time. However, lysine or glycine added as single supplements, inhibited yeast growth and fermentation. With lysine, yeast growth was severely inhibited, and a loss of cell viability as high as 80% was seen. Partial or complete reversal of lysine-induced inhibition was achieved by the addition of a number of nitrogen sources. All nitrogen sources that relieved lysine-induced inhibition of yeast growth also promoted uptake of lysine and restored cell viability to the level observed in the control. They also increased the rate of fermentation. Experiments with minimal media showed that for lysine to be inhibitory to yeast growth, assimilable nitrogen in the medium must be in growth-limiting concentrations or totally absent. In the presence of excess nitrogen, lysine stimulated yeast growth and fermentation. Results indicate that supplementing wheat mash with other nitrogen sources increases the rate of fermentation not only by providing extra nitrogen but also by reducing or eliminating the inhibitory effect of lysine on yeast growth.

Arginine↗

PubMed1999

Defects in thalamocortical axon pathfinding correlate with altered cell domains in Mash-1-deficient mice.

We have analyzed the pathfinding of thalamocortical axons (TCAs) from dorsal thalamus to neocortex in relation to specific cell domains in the forebrain of wild-type and Mash-1-deficient mice. In wild-type mice, we identified four cell domains that constitute the proximal part of the TCA pathway. These domains are distinguished by patterns of gene expression and by the presence of neurons retrogradely labeled from dorsal thalamus. Since the cells that form these domains are generated in forebrain proliferative zones that express high levels of Mash-1, we studied Mash-1 mutant mice to assess the potential roles of these domains in TCA pathfinding. In null mutants, each of the domains is altered: the two Pax-6 domains, one in ventral thalamus and one in hypothalamus, are expanded in size; a complementary RPTP(delta) domain in ventral thalamus is correspondingly reduced and the normally graded expression of RPTP(delta) in that domain is no longer apparent. In ventral telencephalon, a domain characterized in the wild type by Netrin-1 and Nkx-2.1 expression and by retrogradely labeled neurons is absent in the mutant. Defects in TCA pathfinding are localized to the borders of each of these altered domains. Many TCAs fail to enter the expanded, ventral thalamic Pax-6 domain that constitutes the most proximal part of the TCA pathway, and form a dense whorl at the border between dorsal and ventral thalamus. A proportion of TCAs do extend further distally into ventral thalamus, but many of these stall at an aberrant, abrupt border of high RPTP(delta) expression. A small proportion of TCAs extend around the RPTP(delta) domain and reach the ventral thalamic-hypothalamic border, but few of these axons turn at that border to extend into the ventral telencephalon. These findings demonstrate that Mash-1 is required for the normal development of cell domains that in turn are required for normal TCA pathfinding. In addition, these findings support the hypothesis that ventral telencephalic neurons and their axons guide TCAs through ventral thalamus and into ventral telencephalon.

Afferent Pathways↗

PubMed1999

Acquisition of myogenic specificity through replacement of one amino acid of MASH-1 and introduction of an additional alpha-helical turn.

The homologous transcription factors Myf-5, MyoD, myogenin, MRF-4, and MASH-1 bind with high affinity and modest sequence specificity to DNA containing an E-box (CANNTG). This similarity of the in vitro DNA binding specificity is in sharp contrast to the high physiological specificity displayed by these proteins. Myf-5, MyoD, myogenin, and MRF-4 induce cells to differentiate along a myogenic pathway, while MASH-1 promotes the differentiation of neuronal precursor cells. We show here that MASH-1 can be converted into a protein capable of inducing myogenesis in fibroblasts by replacing leucine (130) of MASH-1 with lysine and introducing an additional turn into its basic recognition helix. These changes do not significantly alter the DNA binding properties of the proteins in cell free conditions. Crystallographic data for the DNA complexes of MyoD and E12 suggest that Leu (130) points away from the DNA into the solvent. We postulate that the identity of the amino acid in position 130 is important for protein-protein interactions that might affect the DNA binding specificities displayed by BHLH-proteins in vivo and form the molecular basis of the different physiological properties of the myogenic and neurogenic BHLH-proteins.

Amino Acid Sequence↗

PubMed2003

[Production of tyramine in "moromi" mash during soy sauce fermentation].

The concentrations of 7 non-volatile amines, tyramine (Tym), histamine (Him), phenethylamine (Phm), putrescine (Put), cadaverine (Cad), spermidine (Spd) and spermine (Spm) in the liquid part of "moromi" mash during soy sauce fermentation were studied. These amines, except for him and Cad, were detected during fermentation by the conventional production method in the laboratory. Put and Spd were detected at the beginning, and Tym, Phm and Spm appeared later; these 5 amines increased gradually during the fermentation. Put, Spd, Spm and Cad were present in the raw starting material for soy sauce; thus, Tym and Phm were produced by the fermentation. When "moromi" mash was added to liquid medium and cultivated, Tym was detected in some "moromi" mash and the other amines were not detected. Tym-producing bacterial strains were isolated from the liquid culture media of Tym-positive "moromi" mash. The Tym-producing strain was a gram-positive coccus. The conditions for production of amines by Tym-producing bacterial strains were examined. These strains grew and produced tyramine under various conditions, which may occur during soy sauce fermentation. Namely, Tym was produced at pH 5-10, at salt concentrations of less than 8%, under either aerobic or anaerobic conditions. During soy sauce fermentation, it is assumed that Tym would be produced by these strains during the early stages of soy sauce aging within a short period when the salt concentration and pH conditions are optimal for growth. Based on the bacteriological properties, the strains were identified as Enterococcus faecium. With the exception of Phm and Him, which did not exist in the starting raw material, non-volatile amines (including Put, Cad, Spd and Spm) were not produced and microorganisms producing them are not believed to be present during "moromi" fermentation.

Bacteria↗