Isolation and characterisation of antifungal compounds from lactic acid bacteria and their application in wheat and gluten-free bread

As part of the “free-from” trend, biopreservation for bread products has increasingly become important to prevent spoilage since artificial preservatives are more and more rejected by consumers. A literature review conducted as part of this thesis revealed that the evaluation of more suitable antifungal strains of lactic acid bacteria (LAB) is important. Moreover, increasing the knowledge about the origin of the antifungal effect is fundamental for further enhancement of biopreservation. This thesis addresses the investigation of Lactobacillus amylovorus DSM19280, Lb. brevis R2: and Lb. reuteri R29 for biopreservation using in vitro trials and in situ sourdough fermentations of quinoa, rice and wheat flours as biopreservatives in breads. Their contribution to quality and shelf life extension on bread was compared and related to their metabolic activity and substrate features. Moreover, the quantity of antifungal carboxylic acids produced during sourdough fermentation was analysed. Overall a specific profile of antifungal compounds was found in the sourdough samples which were strain and substrate dependently different. The best preservative effect in quinoa sourdough and wheat sourdough bread was achieved when Lb. amylovorus DSM19280 fermented sourdough was used. However, the concentration of the antifungal compounds found in these biopreservatives were much lower when compared with Lb. reuteri R29 as the highest producer. Nevertheless, the artificial application of the highest concentration of these antifungal compounds in chemically acidified wheat sourdough bread succeeded in a longer shelf life than achieved only by acidifying the dough. This evidences their partial contribution to the antifungal activity and their synergy. Additionally, a HRGC/MS method for the identification and quantification of the antifungal active compounds cyclo(Leu-Pro), cyclo(Pro-Pro), cyclo(Met-Pro) and cyclo(Phe-Pro) was successfully developed by using stable isotope dilutions assays with the deuterated counterparts. It was observed that the concentrations of cyclo(Leu-Pro), cyclo(Pro-Pro), and cyclo(Phe-Pro) increased only moderately in MRS-broth and wort fermentation by the activity of the selected microorganism, whereas the concentration of cyclo(Met-Pro) stayed unchanged.

Investigation of lactic acid bacteria mediated bioprotection with applications in cereal industry. Case-study: malting process

Antifungal compounds produced by Lactic acid bacteria (LAB) metabolites can be natural and reliable alternative for reducing fungal infections pre- and post-harvest with a multitude of additional advantages for cereal-base products. Toxigenic and spoilage fungi are responsible for numerous diseases and economic losses. This thesis includes an overview of the impact fungi have on aspects of the cereal food chain. The applicability of LAB in plant protection and cereal industry is discussed in detail. Specific case studies include Fusarium head blight, and the impact of fungi in the malting and baking industry. The impact of Fusarium culmorum infected raw barley on the final malt quality was part of the investigation. In vitro infected barley grains were fully characterized. The study showed that the germinative energy of infected barley grains decreased by 45% and grains accumulated 199 μg.kg-1 of deoxynivalenol (DON). Barley grains were subsequently malted and fully characterized. Fungal biomass increased during all stages of malting. Infected malt accumulated 8-times its DON concentration during malting. Infected malt grains revealed extreme structural changes due to proteolytic, (hemi)-cellulolytic and starch degrading activity of the fungi, this led to increased friability and fragmentation. Infected grains also had higher protease and β-glucanase activities, lower amylase activity, a greater proportion of free amino and soluble nitrogen, and a lower β-glucan content. Malt loss was over 27% higher in infected malt when compared to the control. The protein compositional changes and respective enzymatic activity of infected barley and respective malt were characterized using a wide range of methods. F. culmorum infected barley grains showed an increase in proteolytic activity and protein extractability. Several metabolic proteins decreased and increased at different rates during infection and malting, showing a complex F. culmorum infection interdependence. In vitro F. culmorum infected malt was used to produce lager beer to investigate changes caused by the fungi during the brewing processes and their effect on beer quality attributes. It was found, that the wort containing infected malt had a lower pH, a higher FAN, higher β-glucan and a 45% increase in the purging rate, and led to premature yeast flocculation. The beer produced with infected malt (IB) had also a significantly different amino acid profile. IB flavour characterization revealed a higher concentration of esters, fusel alcohols, fatty acids, ketones, and dimethylsulfide, and in particular, acetaldehyde, when compared to the control. IB had a greater proportion of Strecker aldehydes and Maillard products contributing to an increased beer staling character. IB resulted in a 67% darker colour with a trend to better foam stability. It was also found that 78% of the accumulated mycotoxin deoxynivalenol in the malt was transferred into beer. A LAB cell-freesupernatant (cfs), produced in wort-base substrate, was investigated for its ability to inhibit Fusarium growth during malting. Wort was a suitable substrate for LAB exhibiting antifungal activity. Lactobacillus amylovorus DSM19280 inhibited 104 spores.mL-1 for 7 days, after 120 h of fermentation, while Lactobacillus reuteri R29 inhibited 105 spores.mL-1 for 7 days, after 48 h of fermentation. Both LAB cfs had significant different organic acid profiles. Acid-base antifungal compounds were identified and, phenyllactic, hydroxy-phenyllactic, and benzoic acids were present in higher concentrations when compared to the control. A 3 °P wort substrate inoculated With L. reuteri R29 (cfs) was applied in malting and successfully inhibited Fusarium growth by 23%, and mycotoxin DON by 80%. Malt attributes resulted in highly modified grains, lower pH, higher colouration, and higher extract yield. The implementation of selected LAB producing antifungal compounds can be used successfully in the malting process to reduce mould growth and mycotoxin production.

Volatile compounds produced by the probiotic strain Lactobacillus plantarum NCIMB 8826 in cereal-based substrates

The production of volatile compounds by the probiotic strain, Lactobacillus plantarum NCIMB 8826, in cereal-based media (oat, wheat. barley and malt) was investigated. Sixty compounds, including fatty acids and their esters, amides, alcohols, aldehydes, aromatic hydrocarbons, furans, ketones, peroxides and pyrans, were identified. L. plantarum significantly changed the aroma profile of the four cereal broths. and each substrate showed a specific volatiles profile. Oat and barley media were the substrates more influenced by the fermentation process. The most abundant volatiles detected in oat, wheat, barley and malt were oleic acid, linoleic acid. acetic acid, and 5-hydroxymethylfurfural, respectively. Analysis of these products confirmed the heterofermentative pathway of L plantarum. Maillard compounds were not detected during sterilisation and fermentation. This study is the first to report the volatile composition of probiotic drinks produce with non-supplemented cereal-based media and the results obtained could contribute to the development of new non-dairy probiotic formulations. (C) 2009 Elsevier Ltd. All rights reserved.

Modelling and validation of Lactobacillus plantarum fermentations in cereal-based media with different sugar concentrations and buffering capacities

An unstructured mathematical model is proposed to describe the fermentation kinetics of growth, lactic acid production, pH and sugar consumption by Lactobacillus plantarum as a function of the buffering capacity and initial glucose concentration of the culture media. Initially the experimental data of L plantarum fermentations in synthetic media with different buffering capacity and glucose were fitted to a set of primary models. Later the parameters obtained from these models were used to establish mathematical relationships with the independent variables tested. The models were validated with 6 fermentations of L. plantarum in different cereal-based media. In most cases the proposed models adequately describe the biochemical changes taking place during fermentation and are a promising approach for the formulation of cereal-based probiotic foods. (C) 2008 Elsevier B.V. All rights reserved.

Effect of the particle size of maize, rice, and sorghum in extruded diets for dogs on starch gelatinization, digestibility, and the fecal concentration of fermentation products

The influence of rice, maize, and sorghum raw material particle size in extruded dry dog food on the digestibility of nutrients and energy and the fecal concentration of fermentation products was investigated. Three diets with similar nutrient compositions were formulated, each with 1 starch source. Before incorporation into diets, the cereals were ground into 3 different particle sizes (approximately 300, 450, and 600 mu m); therefore, a total of 9 diets were in a 3 x 3 factorial arrangement (3 cereals and 3 particle sizes). Fifty-four beagle dogs (12.0 +/- 0.1 kg BW) were randomly assigned to the diets, with 6 dogs per diet. The digestibility was measured with the chromium oxide method. The data were evaluated with ANOVA considering the carbohydrate source, grinding effect, and interactions. The means were compared with the Tukey test and polynomial contrasts (P < 0.05). With the same grinding procedure, rice was reduced to smaller particles than other cereals. The cereal mean geometric diameter (MGD) was directly related to starch gelatini-zation (SG) during extrusion. For rice diets, the MGD and SG did not change nutrient digestibility (P > 0.05); only GE digestibility was reduced at the largest MGD (P < 0.01). For maize and sorghum diets, the total tract apparent nutrient digestibility was reduced for foods with greater MGD and less SG (P < 0.01). A linear reduction in nutrient digestibility according to cereal particle size was observed for sorghum (r(2) < 0.72; P < 0.01). Higher concentrations of fecal total shortchain fatty acids (SCFA) were observed for sorghum diets (P < 0.05) than for other diets. The rice diets led to the production of feces with less lactate (P < 0.05). The increase in raw material MGD did not influence fecal SCFA for rice diets, but for the dogs fed maize and sorghum foods, an increase in propionate and butyrate concentrations were observed as MGD increased (P < 0.05). In conclusion, for dogs fed different particle sizes of the cereal starches in the extruded diets, the digestibility and fecal characteristics were affected, and this effect was ingredient dependent.

Clarification of cane juice for fermentation

Fermentation feedstocks in the sugar industry are based on cane juice, B molasses or final molasses. Brazil has been producing ethanol by directing sugarcane juice to fermentation directly or using lower quality juice as a diluent with B molasses to prepare the fermentation broth. One issue that has received only limited interest particularly from outside Brazil is the most appropriate conditions for clarification of the juice going to fermentation. Irrespective of whether the juice supply is the total flow from the milling tandem or a diffuser station or a part of the total flow, removal of the insoluble solids is essential. However, the standard defecation process used by sugar factories around the world to clarify juice can introduce unwanted calcium ions and remove other nutrients such as phosphorus and nitrogen that are considered essential for the fermentation process. An investigation was undertaken by SRI to assess the effects on the constituents of cane juice when subjected to the typical clarification process in an Australian factory and what conditions would be needed to provide a clarified juice suitable for fermentation. Typical juices from one factory were clarified in laboratory trials under a range of pH conditions and the resulting clarified juices analysed. The results indicated that pH had a major effect on the residual concentrations of key constituents in the clarified juice and that the selected clarification conditions are determined by the nominated quality criteria of clarified juice feedstock for fermentation. Further trials were conducted in overseas factories to confirm the results obtained in Australia. It became apparent that the preferred specifications for clarified juice going to fermentation varied from country to country. Each supplier of fermentation technology had criteria applying to clarified juice feedstock that would have a major impact on the standard of clarification required to achieve compliance with the criteria.

Allelic variation at a single gene increases food value in a drought-tolerant staple cereal

The production of adequate agricultural outputs to support the growing human population places great demands on agriculture, especially in light of ever-greater restrictions on input resources. Sorghum is a drought-adapted cereal capable of reliable production where other cereals fail, and thus represents a good candidate to address food security as agricultural inputs of water and arable land grow scarce. A long-standing issue with sorghum grain is that it has an inherently lower digestibility. Here we show that a low-frequency allele type in the starch metabolic gene, pullulanase, is associated with increased digestibility, regardless of genotypic background. We also provide evidence that the beneficial allele type is not associated with deleterious pleiotropic effects in the modern field environment. We argue that increasing the digestibility of an adapted crop is a viable way forward towards addressing food security while maximizing water and land-use efficiency.

Whole-genome sequencing reveals untapped genetic potential in Africa’s indigenous cereal crop sorghum

Sorghum is a food and feed cereal crop adapted to heat and drought and a staple for 500 million of the world’s poorest people. Its small diploid genome and phenotypic diversity make it an ideal C4 grass model as a complement to C3 rice. Here we present high coverage (16–45 × ) resequenced genomes of 44 sorghum lines representing the primary gene pool and spanning dimensions of geographic origin, end-use and taxonomic group. We also report the first resequenced genome of S. propinquum, identifying 8 M high-quality SNPs, 1.9 M indels and specific gene loss and gain events in S. bicolor. We observe strong racial structure and a complex domestication history involving at least two distinct domestication events. These assembled genomes enable the leveraging of existing cereal functional genomics data against the novel diversity available in sorghum, providing an unmatched resource for the genetic improvement of sorghum and other grass species.

Impact of a change in tillage and crop residue management practice on soil chemical and microbiological properties in a cereal-producing red duplex soil in NSW, Australia

The effect of a change of tillage and crop residue management practice on the chemical and micro-biological properties of a cereal-producing red duplex soil was investigated by superimposing each of three management practices (CC: conventional cultivation, stubble burnt, crop conventionally sown; DD: direct-drilling, stubble retained, no cultivation, crop direct-drilled; SI: stubble incorporated with a single cultivation, crop conventionally sown), for a 3-year period on plots previously managed with each of the same three practices for 14 years. A change from DD to CC or SI practice resulted in a significant decline, in the top 0-5 cm of soil, in organic C, total N, electrical conductivity, NH4-N, NO3-N, soil moisture holding capacity, microbial biomass and CO2 respiration as well as a decline in the microbial quotient (the ratio of microbial biomass C to organic C; P <0.05). In contrast, a change from SI to DD or CC practice or a change from CC to DD or SI practice had only negligible impact on soil chemical properties (P >0.05). However, there was a significant increase in microbial biomass and the microbial quotient in the top 0-5 cm of soil following the change from CC to DD or SI practice and with the change from SI to DD practice (P <0.05). Analysis of ester-linked fatty acid methyl esters (EL-FAMEs) extracted from the 0- to 5-cm and 5- to 10-cm layers of the soils of the various treatments detected changes in the FAME profiles following a change in tillage practice. A change from DD practice to SI or CC practice was associated with a significant decline in the ratio of fungal to bacterial fatty acids in the 0- to 5-cm soil (P <0.05). The results show that a change in tillage practice, particularly the cultivation of a previously minimum-tilled (direct-drilled) soil, will result in significant changes in soil chemical and microbiological properties within a 3-year period. They also show that soil microbiological properties are sensitive indicators of a change in tillage practice.

Microalgal biomass as a fermentation feedstock for bioethanol production

BACKGROUND The increasing cost of fossil fuels as well as the escalating social and industrial awareness of the environmental impacts associated with the use of fossil fuels has created the need for more sustainable fuel options. Bioethanol, produced from renewable biomass such as sugar and starch materials, is believed to be one of these options, and it is currently being harnessed extensively. However, the utilization of sugar and starch materials as feedstocks for bioethanol production creates a major competition with the food market in terms of land for cultivation, and this makes bioethanol from these sources economically less attractive. RESULT This study explores the suitability of microalgae (Chlorococum sp.) as a substrate for bioethanol production via yeast (Saccharomycesbayanus)under different fermentation conditions. Results show a maximum ethanol concentration of 3.83 g L -1 obtained from 10 g L-1 of lipid-extracted microalgae debris. CONCLUSION This productivity level (∼38% w/w), which is in keeping with that of current production systems endorses microalgae as a promising substrate for bioethanol production.

Development of a pilot-scale bacterial fermentation for plasmid-based biopharmaceutical production using a stoichiometric medium

The recognition of the potential efficacy of plasmid DNA (pDNA) molecules as vectors in the treatment and prevention of emerging diseases has birthed the confidence to combat global pandemics. This is due to the close-to-zero safety concern associated with pDNA vectors compared to viral vectors in cell transfection and targeting. Considerable attention has been paid to the potential of pDNA vectors but comparatively less thought has been given to the practical challenges in producing large quantities to meet current rising demands. A pilot-scale fermentation scheme was developed by employing a stoichiometrically-designed growth medium whose exceptional plasmid yield performance was attested in a shake flask environment for pUC19 and pEGFP-N1 transformed into E. coliDH5α and E. coliJM109, respectively. Batch fermentation of E. coliDH5α-pUC19 employing the stoichiometric medium displayed a maximum plasmid volumetric and specific yield of 62.6 mg/L and 17.1 mg/g (mg plasmid/g dry cell weight), respectively. Fed-batch fermentation of E. coliDH5α-pUC19 on a glycerol substrate demonstrated one of the highest ever reported pilot-scale plasmid specific yield of 48.98 mg/g and a volumetric yield of 0.53 g/L. The attainment of high plasmid specific yields constitutes a decrease in plasmid manufacturing cost and enhances the effectiveness of downstream processes by reducing the proportion of intracellular impurities. The effect of step-rise temperature induction was also considered to maximize ColE1-origin plasmid replication.

Bio-hydrogen production from food waste through anaerobic fermentation

In order to protect our planet and ourselves from the adverse effects of excessive CO2 emissions and to prevent an imminent non-renewable fossil fuel shortage and energy crisis, there is a need to transform our current ‘fossil fuel dependent’ energy systems to new, clean, renewable energy sources. The world has recognized hydrogen as an energy carrier that complies with all the environmental quality and energy security, demands. This research aimed at producing hydrogen through anaerobic fermentation, using food waste as the substrate. Four food waste substrates were used: Rice, fish, vegetable and their mixture. Bio-hydrogen production was performed in lab scale reactors, using 250 mL serum bottles. The food waste was first mixed with the anaerobic sewage sludge and incubated at 37°C for 31 days (acclimatization). The anaerobic sewage sludge was then heat treated at 80°C for 15 min. The experiment was conducted at an initial pH of 5.5 and temperatures of 27, 35 and 55°C. The maximum cumulative hydrogen produced by rice, fish, vegetable and mixed food waste substrates were highest at 37°C (Rice =26.97±0.76 mL, fish = 89.70±1.25 mL, vegetable = 42.00±1.76 mL, mixed = 108.90±1.42 mL). A comparative study of acclimatized (the different food waste substrates were mixed with anaerobic sewage sludge and incubated at 37°C for 31days) and non-acclimatized food waste substrate (food waste that was not incubated with anaerobic sewage sludge) showed that acclimatized food waste substrate enhanced bio-hydrogen production by 90 - 100%.

Reducing nitrous oxide emissions while supporting subtropical cereal production in Oxisols

This is the first study to investigate alternative fertilisation strategies to increase cereal production while reducing greenhouse gas emissions from the most common soil type in subtropical regions. The results of this research will contribute to define future farming practices to achieve global food security and mitigate climate change. The study established that introducing legumes in cropping systems is the most agronomically viable and environmentally sustainable fertilisation strategy. Importantly, this strategy can be widely adopted in subtropical regions since it is economically accessible, requires little know-how transfer and technology investment, and can be profitable in both low- and high-input cropping systems.

Intake, growth and carcass characteristics of lambs consuming low digestible hay and cereal grain

The influence of barley and oat grain supplements on hay dry matter intake (DMI), carcass components gain and meat quality in lambs fed a low quality basal diet was examined. Thirty five crossbred wether lambs (9 months of age) were divided into four groups. After adaptation to a basal diet of 85% oat hay and 15% lucerne hay for one week, an initial group of 11 was slaughtered. The weights of carcass components and digesta-free empty body weight (EBW) of this group was used to estimate the weight of carcass components of the other three experimental groups at the start of the experiment. The remaining three groups were randomly assigned to pens and fed ad libitum the basal diet alone (basal), basal with 300 g air dry barley grain (barley), basal with 300 g air dry oat grain (oat). Supplements were fed twice weekly (i.e., 900 g on Tuesday and 1200 g on Friday). After 13 weeks of feeding, animals were slaughtered and, at 24 h post-mortem meat quality and subcutaneous fat colour were measured. Samples of longissimus muscle were collected for determination of sarcomere length and meat tenderness. Hay DMI was reduced (P<0.01) by both barley and oat supplements. Lambs fed barley or oat had a higher and moderate digestibility of DM, and a higher intake of CP (P<0.05) and ME (P<0.01) than basal lambs. Final live weight of barley and oat lambs was higher (P<0.05) than basal, but this was not reflected in EBW or hot carcass weight. Lambs fed barley or oat had increases in protein (P<0.01) and water (P<0.001) in the carcass, but fat gain was not changed (P>0.05). There were no differences in eye muscle area or fat depth (total muscle and adipose tissue depth at 12th rib, 110 mm from midline; GR) among groups. The increased levels of protein and water components in the carcass of barley and oat fed lambs, associated with improved muscle production, were small and did not alter (P>0.05) any of the carcass/meat quality attributes compared to lambs fed a low quality forage diet. Feeding barley or oat grain at 0.9–1% of live weight daily to lambs consuming poor quality hay may not substantially improve carcass quality, but may be useful in maintaining body condition of lambs through the dry season for slaughter out of season