Apparent digestibility of protein, energy, and amino acids in some selected feed ingredients for pacu Piaractus mesopotamicus

The apparent digestibility coefficients (ADCs) of amino acids (AA), protein, and energy in six feed ingredients (Brazilian fish meal, soybean meal, corn gluten meal, alcoholic yeast, corn, and wheat bran) we evaluated for pacu juveniles. In general, all ingredients showed high digestibility values for all AA, and differences among ADCs of individual amino acid were detected (P < 0.01). Corn gluten, soybean, and fish meals had the highest ADCs of AA. The ADCs of protein in fish meal, yeast, and corn gluten meal should not be used as AA digestibility indicators, because those showed differences up to 6.7% between the ADCs of protein and AA. All ingredients had lower ADCs of energy than corn gluten meal (P < 0.01). Lysine was the first limiting amino acid in fish meal, corn gluten meal, wheat bran, and corn, and the second limiting amino acid in soybean meal, as methionine was the first limiting amino acid in soybean meal and yeast. However, the soybean meal was the best quality protein source, as it had the highest digestible essential amino acid index. This demonstrated that digestible amino acid values can be used to formulate practical diets for pacu, preventing potential deficiencies or excess that might cause environmental and economic losses.

Efeitos da concentração dos compostos fenólicos no mosto da fermentação alcoólica

Pós-graduação em Agronomia (Energia na Agricultura) - FCA

Relação entre a concentração dos compostos fenólicos do mosto e a eficiência da fermentação alcoólica por subprodutos em processo contínuo

The objective of this work was to evaluate the relationship between phenolic compounds and alcoholic fermentation efficiency. The yield of sugarcanebyproducts (glycerol, acidity, and biomass) was determined in a continuous process at SaoManoel Sugarcane Mill, (Sao Paulo, Brazil) during the 2011/2012 harvest period. The Saccharomyces cerevisiae strain used as inoculum was the CAT-1. During the harvest, the endogenous yeast outcompeted the selected strain, hence eliminating it from the process. This research consists of a case of study on the ethanol production facility. FolinCiocauteau and methylene blue method was used to assess phenolic compounds. The efficiencyof the byproducts generated during the fermentation processwas calculated. Statistics analyses were carried out using Pearson correlation and its significance, by thet-test. We concluded that the phenolic compounds within the must could not be correlated to the byproducts’fermentation efficiency calculated during a continuous fermentation process.

NEW YEAST STRAINS FOR ALCOHOLIC FERMENTATION AT HIGHER SUGAR CONCENTRATION

New yeast strains for alcoholic fermentation were isolated from samples collected from Brazilian alcohol factories at the end of the sugar cane crop season. They were selected by their capacity of fermenting concentrated sugar cane syrup as well as high sucrose concentrations in synthetic medium with a conversion efficiency of 89-92%. The strains were identified as Saccharomyces cerevisiae.

Mathematical modeling of a continuous alcoholic fermentation process in a two-stage tower reactor cascade with flocculating yeast recycle

Experiments of continuous alcoholic fermentation of sugarcane juice with flocculating yeast recycle were conducted in a system of two 0.22-L tower bioreactors in series, operated at a range of dilution rates (D (1) = D (2) = 0.27-0.95 h(-1)), constant recycle ratio (alpha = F (R) /F = 4.0) and a sugar concentration in the feed stream (S (0)) around 150 g/L. The data obtained in these experimental conditions were used to adjust the parameters of a mathematical model previously developed for the single-stage process. This model considers each of the tower bioreactors as a perfectly mixed continuous reactor and the kinetics of cell growth and product formation takes into account the limitation by substrate and the inhibition by ethanol and biomass, as well as the substrate consumption for cellular maintenance. The model predictions agreed satisfactorily with the measurements taken in both stages of the cascade. The major differences with respect to the kinetic parameters previously estimated for a single-stage system were observed for the maximum specific growth rate, for the inhibition constants of cell growth and for the specific rate of substrate consumption for cell maintenance. Mathematical models were validated and used to simulate alternative operating conditions as well as to analyze the performance of the two-stage process against that of the single-stage process.

Effect of nitrogen nutrition on yeast ecology and alcoholic fermentation

Mestrado Vinifera Euromaster - Instituto Superior de Agronomia - UL

Heterologous expression of glucose oxidase in the yeast Kluyveromyces marxianus

Background: In spite of its advantageous physiological properties for bioprocess applications, the use of the yeast Kluyveromyces marxianus as a host for heterologous protein production has been very limited, in constrast to its close relative Kluyveromyces lactis. In the present work, the model protein glucose oxidase (GOX) from Aspergillus niger was cloned into K. marxianus CBS 6556 and into K. lactis CBS 2359 using three different expression systems. We aimed at verifying how each expression system would affect protein expression, secretion/localization, post-translational modification, and biochemical properties. Results: The highest GOX expression levels (1552 units of secreted protein per gram dry cell weight) were achieved using an episomal system, in which the INU1 promoter and terminator were used to drive heterologous gene expression, together with the INU1 prepro sequence, which was employed to drive secretion of the enzyme. In all cases, GOX was mainly secreted, remaining either in the periplasmic space or in the culture supernatant. Whereas the use of genetic elements from Saccharomyces cerevisiae to drive heterologous protein expression led to higher expression levels in K. lactis than in K. marxianus, the use of INU1 genetic elements clearly led to the opposite result. The biochemical characterization of GOX confirmed the correct expression of the protein and showed that K. marxianus has a tendency to hyperglycosylate the protein, in a similar way as already observed for other yeasts, although this tendency seems to be smaller than the one of e. g. K. lactis and S. cerevisiae. Hyperglycosylation of GOX does not seem to affect its affinity for the substrate, nor its activity. Conclusions: Taken together, our results indicate that K. marxianus is indeed a good host for the expression of heterologous proteins, not only for its physiological properties, but also because it correctly secretes and folds these proteins.

Raspberry (Rubus idaeus L.) wine: Yeast selection, sensory evaluation and instrumental analysis of volatile and other compounds

To evaluate the potential for fermentation of raspberry pulp, sixteen yeast strains (S. cerevisiae and S. bayanus) were studied. Volatile compounds were determined by GC-MS, GC-FID, and GC-PFPD. Ethanol. glycerol and organic acids were determined by HPLC. HPLC-DAD was used to analyse phenolic acids. Sensory analysis was performed by trained panellists. After a screening step, CAT-1, UFLA FW 15 and S. bayanus CBS 1505 were previously selected based on their fermentative characteristics and profile of the metabolites identified. The beverage produced with CAT-1 showed the highest volatile fatty acid concentration (1542.6 mu g/L), whereas the beverage produced with UFLA FIN 15 showed the highest concentration of acetates (2211.1 mu g/L) and total volatile compounds (5835 mu g/L). For volatile sulphur compounds. 566.5 mu g/L were found in the beverage produced with S. bayanus CBS 1505. The lowest concentration of volatile sulphur compounds (151.9 mu g/L) was found for the beverage produced with UFLA FW 15. In the sensory analysis, the beverage produced with UFLA FW 15 was characterised by the descriptors raspberry, cherry, sweet, strawberry, floral and violet. In conclusion, strain UFLA FW 15 was the yeast that produced a raspberry wine with a good chemical and sensory quality. (C) 2010 Elsevier Ltd. All rights reserved.

Fermentation of fruit juices by the osmotolerant yeast Candida magnoliae

This study focuses on the assessment of the fermentation conditions required to modulate the metabolic flux in the osmotolerant yeast Candida magnoliae and evaluate its potential to produce low-alcoholic and low-caloric fermented beverages. For that purpose, two strains, PYCC 2903 and PYCC 3191, were used and fermentation conditions as oxygenation, sugar concentration and the ratio of glucose to fructose were studied using synthetic culture media. Candida magnoliae PYCC 2903 was subsequently used to ferment real industrial fructose-rich substrates such as fruit juices. Sugar consumption profiles for C.magnoliae PYCC 2903 incubated aerobically in the presence of high fructose and glucose concentrations (15%, 10% and 5%) showed a selective utilization of fructose, denoting a preference for this sugar over glucose. The lower ratio between ethanol and sugar alcohols yield was obtained for both strains incubated under oxygen limitation simulating industrial fructose-rich substrates, confirming the ability of this yeast to direct fermentation towards alternative products. Enzymatic assays for hexokinase activity in terms of capacity and affinity for glucose and fructose were performed, aiming to elucidate its contribution to the fructophilic behaviour of this yeast. Enzymatic assays for both strains showed that the Vmax is two to threefold higher for fructose than for glucose but Km is also 10-20-fold higher for this sugar than for glucose. Hence, hexokinase kinetic properties do not explain fructophily in C.magnoliae. This indicates that fructose transport is probably determining in this respect, as observed for other fructophilic yeasts. Fruit juice fermentations with C.magnoliae PYCC 2903 revealed a potential for the production of beverages with interesting sensorial properties. Pear and peach fermentations exhibited the best results with the lowest ratio between ethanol and sugar alcohols yield and the most pleasant organoleptic features.

Green and brown propolis: efficient natural biocides for the control of bacterial contamination of alcoholic fermentation of distilled beverage

This study aimed to evaluate the efficiency of natural biocides, brown and green propolis, for the control of bacterial contamination in the production of sugarcane spirit. The treatments consisted of brown and green propolis extracts, ampicillin, and a control and were assessed at the beginning and end of harvest season in ten fermentation cycles. In the microbiological analyses, the lactic acid bacteria were quantified in the inoculum before and after the treatment with biocides, and the viability of yeast cells during fermentation was evaluated. The levels of acids, glycerol, total residual reducing sugars, and ethanol were analyzed for the wine resulting from each fermentation cycle. A reduction in the number of bacterial contaminants in the inoculum in the treatments with the natural biocides was observed, but it did not affect the viability of yeast cells. The control of the contaminants led to the production of higher levels of ethanol and reduced acidity in the wine produced. The results of the use of brown and green propolis to control the growth microorganisms in the fermentation of sugarcane spirit can be of great importance for using alternative strategies to synthetic antibacterials in fermentation processes including other distilled beverage or spirits.

Timing of di-ammonium phosphate addition to fermenting chardonnay must : effect on nitrogen utilization, ethyl-carbamate formation, and CAR1 expression by yeast /

The maximum amount of ethyl carbamate (EC), a known animal carcinogen produced by the reaction of urea and ethanol, allowed in alcoholic beverages is regulated by legislation in many countries. Wine yeast produce urea by the metabolism of arginine, the predominant assimilable amino acid in must. This action is due to arginase (encoded by CARl). Regulation of CARl, and other genes in this pathway, is often attributed to a well-documented phenomenon known as nitrogen catabolite repression. The effect of the timing of di-ammonium phosphate (DAP) additions on the nitrogen utilization, regulation of CARl, and EC production was investigated. A correlation was found between the timing of DAP addition and the utilization of nitrogen. When DAP was added earlier in the fermentations, less amino nitrogen and more ammonia nitrogen was sequestered from the media by the cells. It was also seen that early DAP addition led to more total nitrogen being used, with a maximal difference of ~25% between fermentations where no DAP was added versus addition at the start of the fermentation. The effect of the timing ofDAP addition on the expression of CARJ during fermentation was analyzed via northern transfer and the relative levels of CARl expression were determined. The trends in expression can be correlated to the nitrogen data and be used to partially explain differences in EC formation between the treatments. EC was quantified at the end of fermentation by GC/MS. In Montrachet yeast, a significant positive correlation was found between the timing of DAP addition, from early to late, and the final EC concentration m the wine (r = 0.9226). In one of the fermentations, EC levels of 30.5 ppb was foimd when DAP was added at the onset of fermentation. A twofold increase (69.5 ppb) was observed when DAP was added after 75% of the sugars were metabolized. When no DAP was added, the ethyl carbamate levels are comparable at a value of 38 ppb. In contrast, the timing of DAP additions do not affect the level EC produced by the yeast ECU 18 in this manner. The study of additional yeast strains shows that the effect of DAP addition to fermentations is strain dependent. Our results reveal the potential importance of the timing of DAP addition to grape must with respect to EC production, and the regulatory effect of DAP additions on the expression of genes in the pathway for arginine metabolism in certain wine yeast strains.

Effects of nutritional factors on growth of Lactobacillus fermentum mixed with Saccharomyces cerevisiae in alcoholic fermentation

The growth of Lactobacillus fermentum was studied in mixed culture with Saccharomyces cerevisiae during alcoholic fermentation of high test molasses (HTM). Yeast extract or a group of 17 amino acids caused a strong and fast decrease in yeast viability due to the strong increase of acidity produced by bacteria. Pure culture of Lactobacillus fermentum in dry sugar cane broth confirmed amino acids as the main nutrients needed to stimulate the growth of bacterial contaminant during alcoholic fermentation. The absence of L. fermentum growth was obtained when leucine: isoleucine or valine were not added to the medium. Phenylalanine, alanine, glutamic acid, cystine, proline, histidine, arginine, threonine, tryptophane, serine and methionine inhibited the bacterial growth at least in one of the cultures of L. fermentum tested.

Effect of 3,4,4 '-trichlorocarbanilide on growth of lactic acid bacteria contaminants in alcoholic fermentation

3,4,4'-trichlorocarbanilide (TCC) was rested as a new method of bacterial growth control for S. cerevisiae alcoholic fermentations of diluted high test molasses (HTM). Minimal inhibitory concentration (MIC) was tested to determine the necessary concentration of TCC to control bacterial growth. The fed-batch alcoholic fermentation process was used with cell recycle similar to industrial conditions and Lactobacillus fermentum CCT 1407 was mixed in the first inoculum to grow with the yeast. Yeast extract was added into the must to stimulate bacterial growth. The best results of TCC's MIC to bacterial growth of Lactobacillus fermentum and Leuconostoc mesenteroides (< 0.125-1.0 mu g/ml) and Saccharomyces cerevisiae (16 mu g/ml) occurred when it was combined with sodium dodecylsulphate (SDS) in a 1: 4 TCC/SDS ratio (wt/wt) in distilled water solution. 1.8 g/l TCC entrapped in calcium alginate added to the must with yeast extract inhibited the growth of Lactobacillus fermentum CCT 1407 maintaining a controlled acidity, higher yeast viability and up to 20.8% of improvement in the average of alcoholic efficiency. Addition of 0.075 g/l TCC entrapped in calcium alginate and 1.67 mg/l SDS in the wort with yeast extract (0-5.0 g/l), inhibited and controlled the extensive bacterial contamination for 19 cycles of fermentation. (C) 1998 Published by Elsevier B.V. Ltd.

Colorimetric assay of ethanol using alcohol dehydrogenase from dry baker's yeast

Alcohol dehydrogenases (ADHs) are oxidoreductases present in animal tissues, plants, and microorganisms. These enzymes attract major scientific interest for the evolutionary perspectives, afforded by their wide occurrence in nature, and for their use in synthesis, thanks to their broad substrate specificity and stereoselectivity. In the present study, the standardization of the activity of the alcohol dehydrogenase from baker's yeast was accomplished, and the pH and temperature stability showed, that the enzyme presented a high stability to pH 6.0-7.0 and the thermal stability were completely maintained up to 50 degrees C during 1 h. The assays of ethanol (detection range 1-5 mM or 4.6 x 10(-2) to 23.0 x 10(-2) g/L) in different samples in alcoholic beverages, presented a maximum deviation of only 7.2%. The standard curve and the analytic curve of this method meet the conditions of precision, sensitivity, simplicity, and low cost, required for a useable analytical method. (c) 2006 Elsevier B.V. All rights reserved.

Chlorine dioxide against bacteria and yeasts from the alcoholic fermentation

The ethanol production in Brazil is carried out by fed-batch or continuous process with cell recycle, in such way that bacterial contaminants are also recycled and may be troublesome due to the substrate competition. Addition of sulphuric acid when inoculum cells are washed can control the bacterial growth or alternatively biocides are used. This work aimed to verify the effect of chlorine dioxide, a well-known biocide for bacterial decontamination of water and equipments, against contaminant bacteria ( Bacillus subtilis, Lactobacillus plantarum, Lactobacillus fermentum and Leuconostoc mesenteroides) from alcoholic fermentation, through the method of minimum inhibitory concentration ( MIC), as well as its effect on the industrial yeast inoculum. Lower MIC was found for B. subtilis ( 10 ppm) and Leuconostoc mesenteroides ( 50 ppm) than for Lactobacillus fermentum ( 75 ppm) and Lactobacillus plantarum ( 125 ppm). Additionally, these concentrations of chlorine dioxide had similar effects on bacteria as 3 ppm of Kamoran (R) ( recommended dosage for fermentation tanks), exception for B. subtilis, which could not be controlled at this Kamoran (R) dosage. The growth of industrial yeasts was affected when the concentration of chlorine dioxide was higher than 50 ppm, but the effect was slightly dependent on the type of yeast strain. Smooth yeast colonies ( dispersed cells) seemed to be more sensitive than wrinkled yeast colonies ( clustered cells/pseudohyphal growth), both isolated from an alcohol-producing unit during the 2006/2007 sugar cane harvest. The main advantage in the usage of chlorine dioxide that it can replace antibiotics, avoiding the selection of resistant populations of microorganisms.