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Populations of aerobic heterotrophic bacteria, niycelial fungi and yeasts occurring in the production of barley malt were examined by plating on agar media and by scanning electron microscopy. There was an increase in the total number of micro-organisms during germination of barley, although populations declined after kilning. Bacteria dominated numerically in all samples, with progressively lower populations of yeasts and filamentous fungi. There was no obvious pattern of spatial distribution of micro-organisms On/in the samples, although there appeared to be high populations of bacteria and fungal hyphae on the inner surface of kernels. The dominant groups of aerobic heterotrophic bacteria were presumptively identified as Alcaligenes sp., Arthrobacter globiformis, Clavibacter iranicuin, Erwinia herbicola, Lactobacillus spp. and Pseudomonas fluorescens. The principal filainentous fungi were Alternaria alternata, Aspergillus glaucus group, Cladosporium macrocarpum, Epicoccum purpurascens, Fusarium avenaceum, Geotrichum candidum and Penicillium spp. The yeasts isolated most frequently were Candida catenulata, Q. vini, Debaryomyces hansenii, Hansenula polyniorpha, Kloeckera apiculata, Rhodotorula nrncilaginosa, Sporobolomyces roseus and Trichosporon bei gelii. Representative bacteria, mycelial fungi and yeasts were examined for the ability to degrade 8-glucan, starch or arabinoxylan. Approximately 50% of the fungi, <50% of the bacteria and <25% of the yeasts degraded these substrates. A culture filtrate of nivale demonstrated marked ability to reduce -glucan viscometrically and colorimetrically. The organism also degraded raffinose and sucrose. In micro-malting experiments the addition of Fusarium nivale and Geotrichum candidum did not produce substantial changes in terms of the physical and chemical characteristics of the finished malts.

Endo-β-mannanase is an endohydrolase enzyme responsible for the breakdown of mannan-containing polysaccharides common in the cell walls of many plants. The action of endo-β-mannanase in barley, its optimum temperature and pH for action, temporal and spatial localization, activity in the presence of hormones and sugars and its effect on the seed's mechanical strength were assayed. The development of a spectrophotometric assay for endo-β-mannanase detection was also trialed. The optimum temperature and pH for these experiments were found to be 37℃ and pH 7. Using these parameters, the endo-β-mannanase enzyme was found to be initially localized in the seed coat and moved through to the endosperm over time. The detected level of enzyme activity increased in the presence of gibberellic acid and glucose, or decreased when abscisic acid was added. Similar results were seen when the embryo was removed and the endosperm and seed coat were incubated in hormone- and sugar-containing media. The presence of exogenous endo-β-mannanase did not affect the mechanical strength of the seed but there was a strong correlation between increasing endo-β-mannanase activity and decreasing mechanical strength over time. The spectrophotometric assay for quantifying endo-β-mannanase in extracts showed promise but did not reach fruition due to unexplained sources of variation. The localization and regulation of endo-β-mannanase in barley were similar to those seen in other plants, such as tomato, lettuce and coffee. These findings have biotechnological applications within the brewery industry. By increasing the mobilization of reserves such as mannan, it is thought that the seedling can utilize this secondary carbohydrate source instead of, or at least supplementing, glucose which was mobilized from starch. This will theoretically reduce the starch and glucose lost during the malting period leaving a higher sugar content free for fermentation.

The quantity of alcohol produced through wort fermentation is fundamental to a malt’s quality. Good fermentability is dependent on many malt quality parameters but requirement for proteases to provide amino acids and peptides for yeast is poorly understood. The thesis investigated relationships between amino acid profiles, endoprotease activities and fermentability under different malting and brewing conditions. Methods for measuring individual wort amino acids, endoprotease activity and fermentability were modified or developed to better understand the relationships. Levels of lysine and glycine were affected the most by malting and the variability was not always well predicted by the standard FAN analysis. Cysteine endoprotease activity developed similarly to total amino acids levels but associations were not significant Amino acids were limiting to fermentability at low yeast pitching rates and with the use of high maltose syrups but malt modification was the key determinant of fermentability. Studies on non fermentable sugar content were recommended.

The quantity of alcohol produced through wort fermentation is fundamental to a malt’s quality. Good fermentability is dependent on many malt quality parameters but requirement for proteases to provide amino acids and peptides for yeast is poorly understood. The thesis investigated relationships between amino acid profiles, endoprotease activities and fermentability under different malting and brewing conditions. Methods for measuring individual wort amino acids, endoprotease activity and fermentability were modified or developed to better understand the relationships. Levels of lysine and glycine were affected the most by malting and the variability was not always well predicted by the standard FAN analysis. Cysteine endoprotease activity developed similarly to total amino acids levels but associations were not significant Amino acids were limiting to fermentability at low yeast pitching rates and with the use of high maltose syrups but malt modification was the key determinant of fermentability. Studies on non fermentable sugar content were recommended.

Endo-β-mannanase is an endohydrolase enzyme responsible for the breakdown of mannan-containing polysaccharides common in the cell walls of many plants. The action of endo-β-mannanase in barley, its optimum temperature and pH for action, temporal and spatial localization, activity in the presence of hormones and sugars and its effect on the seed's mechanical strength were assayed. The development of a spectrophotometric assay for endo-β-mannanase detection was also trialed. The optimum temperature and pH for these experiments were found to be 37℃ and pH 7. Using these parameters, the endo-β-mannanase enzyme was found to be initially localized in the seed coat and moved through to the endosperm over time. The detected level of enzyme activity increased in the presence of gibberellic acid and glucose, or decreased when abscisic acid was added. Similar results were seen when the embryo was removed and the endosperm and seed coat were incubated in hormone- and sugar-containing media. The presence of exogenous endo-β-mannanase did not affect the mechanical strength of the seed but there was a strong correlation between increasing endo-β-mannanase activity and decreasing mechanical strength over time. The spectrophotometric assay for quantifying endo-β-mannanase in extracts showed promise but did not reach fruition due to unexplained sources of variation. The localization and regulation of endo-β-mannanase in barley were similar to those seen in other plants, such as tomato, lettuce and coffee. These findings have biotechnological applications within the brewery industry. By increasing the mobilization of reserves such as mannan, it is thought that the seedling can utilize this secondary carbohydrate source instead of, or at least supplementing, glucose which was mobilized from starch. This will theoretically reduce the starch and glucose lost during the malting period leaving a higher sugar content free for fermentation.

There is growing interest in malting and brewing with rye. However, previous research has shown a propensity for the development of deoxynivalenol (DON) in rye malts, even when levels on the grain is low. The main objective of this study was to assess the growth of F. graminearum and development of trichothecenes during malting of rye. Infected samples were obtained from 2016 variety trails in Minnesota. While DON levels were generally below 0.2 mg/kg, an average increase of 41 % was seen after malting. The most significant increases in DON were at three days of germination. Fusarium Tri5 DNA levels were observed to increase at two days. When single kernels were tested, most were free from DON. Levels in the bulk grain sample were due to a small number of highly contaminated kernels. In the malted samples, a greater portion of kernels contained DON, and overall levels were much higher.

In many African countries, including South Africa, sorghum is malted for the brewing of traditional beer. In South Africa, most sorghum malting is by traditional outdoor floor malting, whereby the sorghum grain is steeped for about 8 hours, left outdoors to germinate in an uncontrolled environment. These malting conditions (wet grain and more or less ambient temperature) encourage microbial proliferation. Microorganisms may themselves negatively impact on the safety of the malts. Of more concern is the proliferation of fungi which can potentially produce highly poisonous mycotoxins in the sorghum malt. Microbial proliferation can also affect the quality of malt, and thereby resulting in undesirable malts. Therefore there is a need for efficient and safe ways to control microbial growth during sorghum malting. The aim of this research was to determine processes to produce sorghum malt that is free of unwanted yeasts, coliforms, moulds and mycotoxins. The first process investigated involved turning the grains during germination. The second process involved the addition of dilute sodium hydroxide (NaOH)/ caustic soda and calcium hydroxide [(Ca(OH)2]/lime during steeping and the third process was by the use of biological control methods which involved inoculation with microbial starter cultures. The effect of the three processes on the levels of moulds, coliforms, mycotoxins (aflatoxins, fumonisins, deoxynivalenol and zearalenone), cytotoxicity, expressed in terms of their IC50 (Inhibitory concentration resulting in 50% inhibition of the cleavage activity) and quality in terms of diastatic power (DP) of sorghum malt were investigated. Turning the sorghum grains during germination did not affect the microbial load of the malt. The total bacterial counts were at high levels of 107-109 cfu/g, fungi at 104-106 cfu/g and coliforms at 103-105 cfu/g. Turned and unturned grains produced malt which showed contamination by about 8 different mould species. Some of these moulds (Fusarium verticillioides, Phoma sorghina. Aspergillus flavus, Alternaria alternata and Penicillium spp.) are known to produce mycotoxins. Malt samples contained fumonisins, deoxynivalenol and zearalenone at levels of < 0.25-2 _g/g, 15-20 and 10-15 _g/kg, respectively. However, they all had very low cytotoxicity (IC50 from 31.2 to > 500 mg/kg). Turning had the negative effect of decreasing the DP of the sorghum malt. The reason that turning did not reduce the microbial load is probably due to the fact that the blending of malt as a result of turning ensured that bacteria and moulds were evenly distributed throughout the malt bed. Steeping sorghum grains in 0.2% NaOH reduced the level of microbial contamination in the malt. Coliforms and moulds were reduced from 104 and 105 cfu/g respectively, to levels of 102 cfu/g in the malt that do not pose health hazards. The high pH (10-13) that resulted from the addition of NaOH probably caused the inhibition of coliforms and moulds by distorting their cell membranes, destroying the proton gradient of the bacterium cell and thus leading to their death. Steeping in 0.2% NaOH resulted in malts with no detectable amounts of mycotoxins and no indication of cytotoxicity in the sorghum malt. A further advantage was that the DP of the 0.2% NaOH steeped malts was doubled. The addition of about 107-108 cfu/ml of Saccharomyces spp. and Pediococcus. pentosaceus cultures to steep water reduced moulds in the malt from 104 cfu/g to 102 cfu/g and coliforms from 104 cfu/g to 102 and <101 cfu/g, respectively. The antimicrobial activity of the Saccharomyces spp. appears to be mainly due to the competition with the other microorganisms. The antimicrobial activity of P. pentosaceus is mainly attributed to the low pH. In addition to the low pH, production of CO2, competition for nutrients and the production of antimicrobial activity could have been responsible for the overall antimicrobial activity of P. pentosaceus. Steeping with microbial cultures resulted in malts that contained no traces of mycotoxins and cytotoxicity. The DPs of the sorghum malts were not affected by steeping with microbial cultures. Turning of grains during germination is not a good method to control microbial load during sorghum malting. The addition of dilute NaOH in steeping water is proposed as a chemical method for the control of bacterial and fungal contamination during sorghum malting whereas the use of the Saccharomyces spp. and P. pentosaceus cultures offers a potential alternative as natural, biocontrol agents. However, dilute alkaline steeping is a more favoured method because it is an easier and practical method to put into operation. / Thesis (PhD (Food Sciences))--University of Pretoria, 2007. / Food Science / unrestricted

This diploma thesis deals with the analysis of low molecular weight proteins in barley during malting by SDS-PAGE method. Attention was paid to PR proteins, specifically LTP proteins and thionins, considered to be connected with gushing effect. Barley samples, malt intermediates and malt samples taken in 10 consecutive days to cover the whole malting process were used for the experiment. In total, 5 samplings were used for the analysis. Proteins extracted from the samples were separated by SDS-PAGE using a Tris-tricine buffer system. The protein lines of LTP proteins and thionins were identified in the resulting gels. The relative optical density values of the selected protein bands were obtained to assess the effect of malting on these proteins. A similar pattern of change in the content of mentioned low molecular weight proteins during the malting process was observed. This was confirmed by finding a statistically significant positive correlation between the relative optical density values of LTP proteins and thionins. Furthermore, the relationship between the low molecular weight protein content and the gushing potential and the microbiological contamination of the samples was investigated, but was not confirmed.