Monitoring a commercial fermentation with proton nuclear magnetic resonance spectroscopy with the aid of chemometrics

Proton nuclear magnetic resonance spectroscopy (NMR) has shown the potential for being a valuable tool in monitoring a commercial fermentation. In this preliminary study, a suite of organic analytes including ethanol, fructose, glucose, methanol, glycerol, malic acid, tartaric acid, succinic acid, acetic acid and lactic acid were simultaneously determined during the fermentation. Data collection and analysis using chemometric algorithms aided the understanding of key processes including the effects of seeding a wine with bacteria for malo-lactic fermentation.

Optimization of laccase production from Trametes versicolor by solid fermentation

The regulation of culture conditions, especially the optimization of substrate constituents, is crucial for laccase production by solid fermentation. To develop an inexpensive optimized substrate formulation to produce high-activity laccase, a uniform design formulation experiment was devised. The solid fermentation of Trametes versicolor was performed with natural aeration, natural substrate pH (about 6.5), environmental humidity of 60% and two different temperature stages (at 37 °C for 3 days, and then at 30 °C for the next 17 days). From the experiment, a regression equation for laccase activity, in the form of a second-degree polynomial model, was constructed using multivariate regression analysis and solved with unconstrained optimization programming. The optimized substrate formulation for laccase production was then calculated. Tween 80 was found to have a negative effect on laccase production in solid fermentation; the optimized solid substrate formulation was 10.8% glucose, 27.7% wheat bran, 9.0% (NH4)2SO4, and 52.5% water. In a scaled-up verification of solid fermentation at a 10 kg scale, laccase activity from T. versicolor in the optimized substrate formulation reached 110.9 IU/g of dry mass.

Compositional dynamics of a commercial wine fermentation using two-dimensional FTIR correlation analysis

We have demonstrated that compositional changes occurring during a commercial red wine fermentation can be effectively monitored using FTIR spectroscopy and modelled with the aid of two-dimensional correlation techniques. This study represents a novel application of two-dimensional spectroscopy and showed that the reaction rates for the conversion of fructose and glucose to alcohol were different, with the latter being more rapid. The use of a simple three-component model serves to aid interpretation of the data and the results obtained confirm the value of two-dimensional FTIR correlation spectroscopy as a chemometric tool which has considerable potential for process monitoring.

Pectinase production by solid fermentation from Aspergillus niger by a new prescription experiment

The Ordos Plateau in China is covered with up to 300,000 ha of peashrub (Caragana) which is the dominant natural vegetation and ideal for fodder production. To exploit peashrub fodder, it is crucially important to optimize the culture conditions, especially culture substrate to produce pectinase complex. In this study, a new prescription process was developed. The process, based on a uniform experimental design, first optimizes the solid substrate and second, after incubation, applies two different temperature treatments (30 °C for the first 30 h and 23°C for the second 42 h) in the fermentation process. A multivariate regression analysis is applied to a number of independent variables (water, wheat bran, rice dextrose, ammonium sulfate, and Tween 80) to develop a predictive model of pectinase activity. A second-degree polynomial model is developed which accounts for an excellent proportion of the explained variation (R² = 97:7%). Using unconstrained mathematical programming, an optimized substrate prescription for pectinase production is subsequently developed. The mathematical analysis revealed that the optimal formula for pectinase production from Aspergillus niger by solid fermentation under the conditions of natural aeration, natural substrate pH (about 6.5), and environmental humidity of 60% is rice dextrose 8%, wheat bran 24%, ammonium sulfate ((NH₄)₂SO₄) 6%, and water 61%. Tween 80 was found to have a negative effect on the production of pectinase in solid substrate. With this substrate prescription, pectinase produced by solid fermentation of A. niger reached 36.3 IU/(g DM). Goats fed on the pectinase complex obtain an incremental increase of 0:47 kg day^-1 during the initial 25 days of feeding, which is a very promising new feeding prospect for the local peashrub. It is concluded that the new formula may be very useful for the sustainable development of arid and semiarid pastures such as those of the Ordos Plateau.

Generalised 2D-correlation NMR analysis of a wine fermentation

A wine fermentation has been monitored on a daily basis by 1H NMR spectroscopy. Following data pre-processing that includes synthesis of the spectra to ensure all peaks are of constant half-width, the series of spectra were examined using generalised two-dimensional correlation techniques. Synchronous and asynchronous data maps have been generated and employed to interpret the changes in the fermentation process as a function of time. The results illustrate the potential of high resolution NMR with multivariate data analysis as a tool for process monitoring and the manner in which two-dimensional correlation mapping can aid in data interpretation.

Minimising colonic fermentation of high fructan foods : using food processing techniques to reduce levels of fructans in onion and garlic

Background: Irritable Bowel Syndrome (IBS) is a chronic functional disorder of the bowel, affecting up to 15% of Australian adults. Dietary triggers need to be identified and controlled. Researchers have shown that short chain carbohydrates, fructans (high in onion and garlic) play a major role in triggering IBS symptoms. Current dietary management aims to limit the intake of fructans in the diet. Another approach may be to use simple food processing to reduce fructans in foods.

Objective: To investigate if pickling will reduce fructan levels in garlic and shallots, and if pickled garlic and shallots reduce colonic fermentation, and  abdominal symptoms in human volunteers.

Design: Fructan levels of the garlic and shallots were measured using the Megazyme fructan assay. 18 volunteers (13 healthy and 5 IBS) participated in a single blinded, randomised cross over study. Subjects were randomly assigned to receive a breakfast (potato and salmon patty) that was either high (unprocessed) or low (processed/pickled) in garlic and shallots. Breath hydrogen was measured every hour over a ten hour period, and abdominal symptoms were assessed using validated questionnaires.

Outcomes: Pickling over a 12 day period significantly reduced fructan levels in both garlic (p=.0.00) and shallots (p=0.00). Consumption of the low fructan breakfast resulted in significantly lower breath hydrogen (p=0.05), abdominal pain (p=0.032), and wind (p=0.04).

Conclusion: Pickling results in significantly lowered fructan levels in problem foods- shallots and garlic, and lowered colonic fermentation and abdominal symptoms in both healthy and IBS volunteers. This study provides another dietary strategy for dietetic counselling of patients with IBS.

Dietary resistant starch interacts with non-starch polysaccharide and protein to influence colonic protein fermentation, with possible implications for colon cancer risk in humans

Resistant starch is dietary starch that passes undigested into the colon where it can have effects similar to non-starch polysaccharide (dietary fibre). The results support the potential health benefits of incorporating foods containing high levels of both resistant starch and non-starch polysaccharide, such as whole grain breads and legumes, as part of well balanced diets containing fruit, vegetables, cereals, meat/alternatives and dairy foods.

Effect of temperature on anaerobic fermentation of banana stem residue

Anaerobic fermentation experiments were conducted on banana (pseudo) stem residue to study the relationship between fermentation temperature and gas production yield and gas production rate, and methane content. Based on fixed dry matter concentration, inoculum concentration and fermentation time, different temperatures, i.e. 25, 30, 35, 40°C were selected and formed four experimental groups. Four levels of single factor tests were conducted to optimize temperature parameter for anaerobic fermentation of banana stem residue. The results showed that the daily gas yield of banana stem residue reached the maximum value of 36.8L on the fourth day at 35°C, and the average gas yield was 5.03L/d. The total gas yield was 402.3L, while the maximum methane content was 61.2% in the whole fermentation process. The results indicated that the comprehensive effect was best at 35°C in anaerobic fermentation of banana stem residue.

The microstructure and physicochemical properties of probiotic buffalo yoghurt during fermentation and storage: a comparison with bovine yoghurt

The physicochemical and rheological properties of yoghurt made from unstandardised unhomogenised buffalo milk were investigated during fermentation and 28 days of storage and compared to the properties of yoghurt made from homogenised fortified bovine milk. A number of differences observed in the gel network can be linked to differences in milk composition. The microstructure of buffalo yoghurt, as assessed by confocal laser scanning microscopy (CLSM) and cryo scanning electron microscopy (cryo-SEM), was interrupted by large fat globules and featured more serum pores. These fat globules have a lower surface area and bind less protein than the homogenised fat globules in bovine milk. These microstructural differences likely lead to the higher syneresis observed for buffalo yoghurt with an increase from 17.4 % (w/w) to 19.7 % (w/w) in the weight of whey generated at days 1 and 28 of the storage. The higher concentration of total calcium in buffalo milk resulted in the release of more ionic calcium during fermentation. Gelation was also slower but the strength of the two gels was similar due to similar protein and total solids concentrations. Buffalo yoghurt was more viscous, less able to recover from deformation and less Newtonian than bovine yoghurt with a thixotropy of 3,035 Pa.s-1 measured for buffalo yoghurt at the end of the storage, at least four times higher than the thixotropy of bovine yoghurt. While the titratable acidity, lactose consumption and changes in organic acid concentrations were similar, differences were recorded in the viability of probiotic bacteria with a lower viability of Lactobacillus acidophilus of 5.17 log (CFU/g) recorded for buffalo yoghurt at day 28 of the storage. Our results show that factors other than the total solids content and protein concentration of milk affect the structural properties of yoghurt. They also illustrate the physicochemical reasons why buffalo and bovine yoghurt are reported to have different sensory properties and provide insight into how compositional changes can be used to alter the microstructure and properties of dairy products. © 2013 Springer Science+Business Media New York.

Fermentation of omega-3 and carotenoid producing marine microorganisms

 The objective of the thesis was to understand and develop the process of fermentation in marine microorganisms for the production of omega-3 fatty acids and carotenoids. Among marine microorganisms, thraustochytrids that belong to phytoplankton group was identified as sources of omega-3 fatty acids and other valuable co-products. In this research, more efficient and cost-effective production of omega-3 oils and other value added products was discussed by addressing the below key objectives. Fermentation strategy using lower cost raw materials, particularly carbon. Screening for new strains that can naturally produce high levels of PUFAs together with useful co-products that can be harvested along with the omega-3 oil.

Aerobic fermentation of saccharomeyes cerevisae in a miniature bioreactor made of low cost poly(methylmethacrylate) (PMMA) and poly(Dimethylsiloxane) (PDMS) polymers

In this paper, a minibioreactor platform made of low cost polymers is presented. The minibioreactor prototype was designed as an alternative solution for carrying out microbial fermentation experiments in laboratory. The minibioreactor prototype has a working volume of 1.5 mL and was fabricated from poly(methylmethacrylate) (PMMA) and poly(dimethylsiloxane) (PDMS) polymers. Cell density was measured online whilst agitation rates and the temperature of the reactor content can be tightly controlled to desired set-point values. As proof-of-concept, various S. cerevisae fermentation experiments were conducted. In every experiment, the minibioreactor operated stably for the entire length of operation which was nearly 40 h with very minimal volume loss i.e. about 2.8 μ·h-1 at 37°C. The minibioreactor has the maximum oxygen transfer rate (OTR) of 16.6 mmol·L-1·h-1 under the agitation rate of 300 rpm. Under these conditions, cell specific growth rate as high as 0.291 h-1 was obtained. The experimental data in the minibioreactor operation was also reproducible using shake flask where similar growth profiles were attained under a similar growth conditions.

Survival and fermentation activity of probiotic bacteria and oxidative stability of omega-3 oil in co-microcapsules during storage

Tuna oil (O) and probiotic bacteria Lactobacillus casei (P) were co-microencapsulated in whey protein isolate (WPI)-gum Arabic (GA) complex coacervate. The co-microcapsules (WPI-P-O-GA), L. casei microcapsules (WPI-P-GA) and tuna oil microcapsules (WPI-O-GA) were converted into powder using spray and freeze drying. The interaction between probiotic bacteria and omega-3 oil in co-microcapsules, particularly in terms of oxidative stability of omega-3 oil and vitality/viability of probiotic bacteria and any synergistic outcome, was studied. The effect of storage temperature (5 and 25 °C) and time (90 days) on the survival and fermentation activity of L. casei and oxidative stability of tuna oil in the microcapsules/co-microcapsules was determined. A synergism between oxidative stability of omega-3 oil and vitality of probiotic bacteria was observed, when they were co-microencapsulated and spray dried. These co-microcapsules will likely have utility in functional food formulations due to simple and cost effective stabilisation and delivery of two important functional ingredients.

An improved purification procedure for cyclosporine synthetase

We have developed expedient and reliable methods to isolate cyclosporin synthetase for in vitro biosynthesis of cyclosporins. We have examined enzyme purification strategies suited to large-scale processing and present a chromatographic sequence that serves as a pilot model for industrial scale preparation of cyclosporin synthetase from cyclosporin producing fungi. A chromatographic sequence consisting of ammonium sulfate precipitation → gel filtration → hydrophobic interaction chromatography → anion exchange chromatography, yielded an electrophoretically homogeneous cyclosporin synthetase preparation (Coomassie G-250 brilliant blue staining). Furthermore, a native polyacrylamide gel electrophoresis system was developed for the isolation of active cyclosporin synthetase enzyme from crude extracts of cyclosporin producing fungi. The environmental factors affecting enzyme stability and the continuity of the in vitro cyclosporin biosynthetic reaction-temperature, pH, and substrate depletion were assessed and manageable conditions have been defined for sustainable cyclosporin biosynthesis with enzyme isolates. Cyclosporin synthetase exhibited an optimal temperature range of 24–29 °C and a pH optimum of 7.6. The native enzyme displayed a pI of 5.7, as determined by isoelectric focusing. The industrial implementation of an in vitro biosynthetic approach could potentially prove useful for the production of important therapeutic cyclosporins which occur as only minor fermentation by-products.

An improved method for the purification of rat liver-type fatty acid binding protein from Escherichia coli

We have developed expedient and reliable methods to isolate cyclosporin synthetase for in vitro biosynthesis of cyclosporins. We have examined enzyme purification strategies suited to large-scale processing and present a chromatographic sequence that serves as a pilot model for industrial scale preparation of cyclosporin synthetase from cyclosporin producing fungi. A chromatographic sequence consisting of ammonium sulfate precipitation → gel filtration → hydrophobic interaction chromatography → anion exchange chromatography, yielded an electrophoretically homogeneous cyclosporin synthetase preparation (Coomassie G-250 brilliant blue staining). Furthermore, a native polyacrylamide gel electrophoresis system was developed for the isolation of active cyclosporin synthetase enzyme from crude extracts of cyclosporin producing fungi. The environmental factors affecting enzyme stability and the continuity of the in vitro cyclosporin biosynthetic reaction-temperature, pH, and substrate depletion were assessed and manageable conditions have been defined for sustainable cyclosporin biosynthesis with enzyme isolates. Cyclosporin synthetase exhibited an optimal temperature range of 24–29 °C and a pH optimum of 7.6. The native enzyme displayed a pI of 5.7, as determined by isoelectric focusing. The industrial implementation of an in vitro biosynthetic approach could potentially prove useful for the production of important therapeutic cyclosporins which occur as only minor fermentation by-products.