Browsing by Subject "Fermentation"

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Bioethanol production from lignocellulosic biomass
(2023) Hoppert, Luis; Kölling, Ralf
The aim of this thesis was to develop a high gravity second-generation bioethanol process and investigate the effects of a high solid loading. The insights gained from the initial experiments helped to understand the underlying mechanism behind the limitations of a high solid loading. Based on these findings, strategies were developed to overcome these limitations.
Development of a genetically defined diploid yeast strain for the application in spirit production
(2005) Schehl, Beatus; Heinisch, Jürgen
Yeast strains of the species Saccharomyces cerevisiae currently in use for the production of consumable alcohols such as beer, wine and spirits are genetically largely undefined. This prevents the use of standard genetic manipulations, such as crossings and tetrad analysis, for strain improvement. Furthermore, it complicates the application of the majority of modern methods developed in yeast molecular biology. In this work two haploid laboratory strains with suitable auxotrophic markers were used for the construction of a genetically well defined, prototrophic diploid production strain. This strain was tested for its fermentative and sensory performances in comparison to commercially available yeasts. Different fruit mashes were fermented, subjected to distillation and analysed for fermentation parameters including growth, sugar utilization, ethanol production and generation of volatile compounds, higher alcohols, uretahne and glycerol. All spirits produced were tested for their sensory performances and the data obtained statistically consolidated. Our results clearly demonstrate that this laboratory strain does not display any disadvantage compared with commercial yeasts in spirit production for any of the parameters tested, yet it offers the potential to apply both classical breeding and modern molecular genetic techniques adjusting yeast physiology to special production schemes.
Editorial: Flavor chemistry of food: Mechanism, interaction, new advances
(2023) Huang, Mingquan; Fan, Gang; Zhang, Yanyan
Effect of refrigerated storage on some physicochemical characteristics of a teff‐based fermented beverage and the viability of the fermenting Lactiplantibacillus plantarum and Lacticaseibacillus rhamnosus used
(2022) Alemneh, Sendeku Takele; Emire, Shimelis Admassu; Jekle, Mario; Hitzmann, Bernd
Probiotic beverages made from cereals become interesting in the recent food industries. In this contribution, a fermented teff-based probiotic beverage was produced using the whole grain teff flour and co-culture strains of Lactiplantibacillus plantarum (LPA6) and Lacticaseibacillus rhamnosus GG (LCGG). Then, the effect of 25 days of refrigerated storage on cell viability (LPA6 and LCGG), and contents of sugars, organic acids, and titratable acidity (TA), as well as pH values were examined. Furthermore, pathogenic microorganisms, hygiene indicators, and sensory tests of the beverage were analyzed. Presumptive cell counts of LPA6 and LCGG were observed to decrease throughout refrigerated storage. Glucose, lactic acid, maltose, and acetic acid contents were significantly (p < 0.05) increased over storage time. Also, pH reduction and TA increment were observed in storage time. Examined pathogenic microorganisms and hygiene indicators were not detected in the beverage. Sensory analysis of the beverage after 10 days of refrigerated storage was accepted by the panelists. Novelty Impact Statement Throughout refrigerated storage of teff-based probiotic beverage sugars and organic acids were produced. Sensory attributes of the newly produced teff-based probiotic beverage were accepted by the panelist after 10 days of refrigerated storage. The pH of the teff-based probiotic beverage became more acidic throughout 25 days of refrigerated storage.
The effects of fermentation of Qu on the digestibility and structure of waxy maize starch
(2022) Wu, Wenhao; Zhang, Xudong; Qu, Jianzhou; Xu, Renyuan; Liu, Na; Zhu, Chuanhao; Li, Huanhuan; Liu, Xingxun; Zhong, Yuyue; Guo, Dongwei
The fermentation of Qu (FQ) could efficiently produce enzymatically modified starch at a low cost. However, it is poorly understood that how FQ influences the waxy maize starch (WMS) structure and the digestion behavior. In this study, WMS was fermented by Qu at different time and starches were isolated at each time point, and its physico-chemical properties and structural parameters were determined. Results showed that the resistant starch (RS), amylose content (AC), the average particle size [D(4,3)] the ratio of peaks at 1,022/995 cm–1, and the onset temperature of gelatinization (To) were increased significantly after 36 h. Conversely, the crystallinity, the values of peak viscosity (PV), breakdown (BD), gelatinization enthalpy (ΔH), and the phase transition temperature range (ΔT) were declined significantly after 36 h. It is noteworthy that smaller starch granules were appeared at 36 h, with wrinkles on the surface, and the particle size distribution was also changed from one sharp peak to bimodal. We suggested that the formation of smaller rearranged starch granules was the main reason for the pronounced increase of RS during the FQ process.
Effects of monensin and tannin extract supplementation on methane production and other criteria of rumen fermentation in vitro and in long-term studies with sheep
(2013) Wischer, Gerald; Rodehutscord, Markus
Ruminants increasingly attract public concern due to their methane release and contribution to the greenhouse effect. One strategy to reduce the release of methane is to modify microbial fermentation in the rumen by the use of feed additives such as monensin and tannin extracts. However, other characteristics of fermentation including the synthesis of microbial protein may also be affected. The aim of the present studies was to provide a comprehensive evaluation of the effects of monensin and tannin extracts on ruminal fermentation and methane production. The ionophore monensin is known to increase feed efficiency in ruminants. Although the use of silages is common practice in cattle feeding, the effects of monensin on the fermentation of silages in the rumen and microbial protein synthesis are lacking. Monensin has often been described to have indirect effects on methane production resulting from its effects on feed intake, protozoa and Gram-positive bacteria. It has rarely been studied whether monensin can reduce methane production without adverse effects on other criteria of rumen fermentation. The first objective therefore was to investigate the effects of different dosages of monensin on methane production and microbial protein synthesis when supplemented to different silages in two in vitro systems (Study 1). In Experiment 1 of Study 1, 15 g of oven-dried grass silage alone or combined with a concentrate was incubated in a rumen simulation (Rusitec) over a period of 13 d to examine the effects of monensin supplementation (2 or 4 mg/d, n = 4) on the production of total gas, methane, volatile fatty acids (VFA), degradation of nutrients and microbial protein synthesis. In Experiment 2 of Study 1, different dosages of monensin (0.5, 1, 2, 6 and 10 µg) were supplemented to syringes containing 120 mg of grass silage alone, grass silage combined with concentrates, or maize silage alone. After 24 h of incubation the effects of monensin on total gas, methane and VFA production were determined. In Experiment 1 monensin inclusion to grass silage and grass silage combined with concentrate resulted in a decreased total gas, methane and acetate production, while propionate production was increased. Along with a decreased degradation of crude protein, ammonia concentration in the system was reduced. While microbial protein originating from solid associated microbes decreased with monensin inclusion, microbial protein from liquid associated microbes was increased, resulting in an increase in total microbial protein synthesis. In Experiment 2, different dosages of monensin reduced methane production in grass silage (17%), grass silage combined with concentrate (10%) and maize silage (13%) without adverse effects on total gas production. Based on these two in vitro experiments it was concluded that monensin is able to reduce methane production without a major decrease in total gas and VFA production and degradation of organic matter. Although microbial fractions were differently affected, the total microbial protein synthesis was increased upon monensin supplementation. Tannins are secondary plant compounds that are known to complex with feed and microbial proteins. Several products from this heterogeneous group have shown potential to affect rumen fermentation in vivo and, even more, in vitro, but are often accompanied by negative effects on digestibility, feed intake and microbial protein synthesis. In Study 2 of the present work, ten tannin extracts (chestnut, mimosa, myrabolan, quebracho, sumach, tara, valonea, oak, cocoa and grape seed) and four monomers of rapeseed tannin (pelargonidin, catechin, cyanidin and sinapinic acid) were screened in grass silage based diets in successive runs using the Hohenheim Gas Test. The objective was to determine the optimal dosage of each tannin extract to cause a maximal methane reduction without negative effects on total gas production. Whereas the supplementation of pelargonidin and cyanidin to grass silage did not reduce methane production; catechin and sinapinic acid reduced methane production without affecting total gas production. Except tara extract, all tannin extracts reduced methane production by 8 to 28% without adverse effects on total gas production. Based on these results, chestnut, grape seed, myrabolan, sumach and valonea extract were investigated in a second step in a Rusitec to determine their effects on degradation of nutrients, VFA and ammonia production, and particularly on microbial protein synthesis. All tannin extracts were supplemented at similar dosages of 1.5 g to 15 g of grass silage. The supplementation of chestnut resulted in the greatest decrease in methane production (63%), followed by valonea (35%), grape seed (23%), sumach (18%), and myrabolan (7%; not significantly different from the control). While chestnut extract reduced acetate production by 19%, supplementation with grape seed or myrabolan extract increased acetate production; however, degradation of fibre fractions was reduced in all tannin treatments. Degradation of dry and organic matter was reduced by all tannin extracts, but there were no differences between tannin treatments. Crude protein degradation and ammonia production were also reduced by tannin extract supplementation. Microbial protein synthesis and its efficiency were not affected by tannin supplementation, which indicates that a reduction in methane production due to tannin extract supplementation is possible without negatively affecting microbial protein synthesis. Chestnut and valonea extract had the greatest potential in reducing methane production without negative effects on rumen fermentation of grass silage and microbial protein synthesis. Therefore, these tannin extracts were investigated for their long-term effects in sheep (Study 3). In Experiment 1 of Study 3, sheep receiving the control, chestnut or valonea treatment (each n = 4) were fed 842 g/d of hay (fresh weight). The animals on the control treatment also received 464 g/d of concentrate, and animals on the tannin treatments received the same amount of concentrate but were also fed 20 g of the respective tannin extract. Following initiation of tannin feeding, methane release from sheep was measured in 23.5 h intervals in respiration chambers on day 1, 8, 15, 29, 57, 85, 113, 148, and 190. In three balances periods faeces and urine were collected for 6 and 3 days, respectively. Effects on nutrient digestibility, nitrogen and energy metabolism were evaluated, with microbial protein synthesis estimated from the urinary excretion of purine derivatives. Based on the results of Experiment 1, a second experiment was conducted four month after the start of Experiment 1. Experiment 2 had the same study design and data collected, but the dosage of tannin extracts was doubled compared to Experiment 1 (0.9 vs. 1.7 g tannin extract/kg body weight) and the duration was shorter (85 days). Hay and concentrates used in both experiments were also evaluated using the Hohenheim Gas for their effects on total gas and methane production. In both experiments, methane release was not significantly reduced by tannin extract supplementation when analysed over the whole experimental period. In Experiment 1 the supplementation of chestnut extract on day 190 resulted in a reduced methane release. In both experiments, on day 1 a numeric reduction in methane release for the tannin treatments was observed, with a greater reduction recorded for the higher dosage used in Experiment 2. This trend disappeared by day 57. In the third balance period of Experiment 1, digestibility of dry and organic matter was reduced by tannin supplementation. The digestibility of crude protein was reduced in both experiments, whereas the digestibility of fibre fractions was not influenced. In both experiments a long-lasting shift in nitrogen excretion from urine to faeces was observed, which occurred to a greater extent in Experiment 2. The urinary excretion of purine derivatives was not significantly affected by tannin supplementation, indicating that the microbial protein synthesis was not altered in either experiment. The in vitro methane production was reduced for concentrates containing tannin extracts, but it was not significantly affected when concentrates were incubated with hay. It is concluded that monensin added to different silages caused a decrease in methane production without affecting total gas production but with an increased microbial protein synthesis. Nine of the ten considered tannin extracts and two tannin monomers decreased methane production without affecting total gas production. The Rusitec study confirmed the great potential of chestnut and valonea extract to reduce methane production without negative effects on microbial protein synthesis. However, neither chestnut nor valonea extract reduced the methane release in sheep when fed over a longer period of time. It is assumed, that rumen microbes adapted to the tannin dosages in terms of methane release but not nitrogen metabolism, as there were long-lasting effects on nitrogen excretion. The shift in nitrogen excretion can have a positive effect on the environment due to the reduced potential of ammonia emission from the urine. Both in vitro systems used in the present studies showed effects of tannin extracts that were considerably different from those observed in sheep. The monomers investigated in the present study are the basic units of condensed tannins, whereas the tannin extracts selected in vitro only contain hydrolysable tannins. It is possible that monomers of chestnut and valonea extract may reduce methane production, whereas higher dosages of these tannin extracts cause negative effects on feed intake, digestibility and microbial protein synthesis. Further investigations should focus systematically on the transfer of in vitro studies to estimate in vivo responses. Therefore, a parallel implementation of different in vitro and respiration studies would be of great value.
Effekt der Überproduktion von Enzymen des Glucosestoffwechsels auf das Wachstum und die Alkoholbildung in der Hefe Saccharomyces cerevisiae
(2006) Emili, Markus; Heinisch, Jürgen
The wine-, beer- and baker's yeast Saccharomyces cerevisiae is the major source in world wide alcohol production. Regarding the research in bioethanol production, the work presented here was aimed to examine the effect of the in vivo overproduction of all enzymes contributing to the conversion of glucose to ethanol in the yeast Saccharomyces cerevisiae with the prospect of increasing ethanol formation. S. cerevisiae is probably the best studied eucaryotic organism with respect to both classical and molecular genetics. It turned out to be of great advantage that two different multi-copy-vectors could be employed in these studies. Each of them was used in the first part of the work to insert half of the set of genes intended for overexpression. The first genes were inserted by restriction and ligation and later on a combination of the PCR-technique, with which the genomic fragments of interest were amplified, and the efficient homologous recombination in vivo was used. With these methods, the gene encoding a hexose transporter (HXT1), all the genes encoding glycolytic enzymes (HXK2, PGI1, PFK1, PFK2, FBA1, TPI1, TDH1 bzw. TDH2, PGK1, GPM1, ENO2, PYK1), as well as the genes encoding enzymes needed for the conversion of pyruvate to ethanol (PDC1, ADH1), were cloned. Following the isolation from yeast, the plasmids were amplified in E. coli and characterized by restriction analysis. The measurement of specific enzyme activity in crude extract of yeast transformants with such plasmids showed a slight overproduction (factor 1,5 to 3,0) for all enzymes, except for glyceraldehyde-3-phosphate dehydrogenase. For HXT1, an increased mRNA level (factor 14 in contrast to the control) was taken as evidence for overproduction. In the enzymatic determinations a clear tendency showing a lower overproduction with an increasing number of genes on the plasmids was observed. These findings suggest a negative feedback on glycolytic flux regulation. The the growth rates obtained in the second part of the work also showed a clear reduction with increasing numbers of plasmid-encoded genes. Regarding the physiological parameters, no changes in the coefficients for glucose consumption and ethanol formation could be found in comparison to a wild-type control, and the yield remained basically unchanged as well. Interestingly, abolishing the ATP-inhibition of phosphofructokinase by expression of a mutant allele of PFK1, resulted in a faster growth of transformants with an otherwise isogenic background. This result indicates the physiological relevance of the allosteric regulation at this essential glycolytic step. A lack of enzyme activity in one of the glycolytic steps in deletion mutants normally leads to growth inhibition on hexoses. On this basis, the construction of a yeast strain was initiated with the objective to obtain stable multi-copy transformants simply by growing cells on different sugars as carbon sources. In detail, this was done by crossing a strain carrying a pgi1-deletion with a strain carrying a pyk1-deletion followed by sporulation and tetrad dissection. Preliminary data with intermediate strain constructs indicate a clear increase in plasmid stability after growing cells on complex media. From the results of this thesis, valuable insights into the regulation of the glycolytic flux in vivo can be deduced, which may serve as a basis for ongoing research on the improvement of ethanol formation by yeast.
Einfluss kurzkettiger Fettsäuren und mikrobieller Fermentationsprodukte neuartiger Oligosaccharide auf Cytotoxizität, Proliferation und Apoptose von humanen Coloncarcinom-Zelllinien
(2006) Roser, Silvia; Rechkemmer, Gerhard
Colon cancer is the second most common cancer in Germany. The role of dietary fibre in the prevention of colon cancer is still controversial: Promising results from in vitro and animal studies are contradictory to inconsistent results from epidemiological stu-dies. Functional carbohydrates as constituents of prebiotic food can modify the colonic microflora for the benefit of short chain fatty acid (SCFA)-producing microbial strains. The SCFA-concentrations should also be increased in the distal part of the colon where most colon carcinomas are developing. SCFA are considered to be preventive against colon cancer. For this study, three different new functional oligosaccharides (OS, made of Isomaltulose and resistant starch) were produced from the Südzucker company and fermented in vitro with human feces of healthy test subjects. The resulting fermentation supernatants (FS) were tested in a cell culture system, using colon carcinoma cell lines of various degrees of differentiation (HT29, HT29 Clone 19A, T84). Cytotoxicity, proliferation, the induction of apoptosis, influences on the cell cycle and electrophysiological parameters were measured. Spectral photometric and flow cytometric methods were performed, as well as measurements in vertical diffusion chambers (Ussing chambers). The parallel testing of SCFA-mixtures with the same SCFA-concentrations as in the FS was included, as well as the testing of a FS ?Control? which was produced without OS-fermentation. Several independent fermentations revealed reproducible results regarding the SCFA-concentrations of the FS. After OS-fermentation, the ratio of the three major SCFA in the FS, acetate, propionate, and butyrate, was similar to that observed in vivo. The FS and SCFA-mixtures tested had a cytotoxic effect on all cell lines at the con-centration of 50 %. A dose dependent decrease in cell proliferation could be found, as well as the induction of apoptosis at a concentration of 50 %. Parallel testing of the analogous SCFA-mixtures showed that cytotoxic and proliferation inhibiting effects of the FS could be primarily attributed to their SCFA-content. This could not be confirmed for apoptosis induction: the SCFA-mixtures were mostly able to induce a higher apoptosis rate than the FS. Similarly, the effects of FS and SCFA-mixtures on the cell cycle were different: The SCFA-mixtures showed more potent inhibition of DNA-synthesis than the analogous FS, which generally led to an arrest in the G2-phase of the cell cycle. Neither FS nor SCFA-mixtures had an impact on transepithelial resistance or short circuit current of differentiated cell monolayers in Ussing chambers. The difference in the fermentation patterns of the various FS and the SCFA-concentrations of the SCFA-mixtures was not great enough to achieve significantly different results in the test systems used. Also, the various differentiation grades of the cell lines showed inconsistent results after treatment with FS and their SCFA-mixtures, so that no correlation could be found between degree of differentiation and test compound action. This study shows that the in vitro fermentation of OS with human feces results in reproducible SCFA-patterns in the FS, similar to the in vivo situation. For the screening of FS and their SCFA-mixtures, respectively, a spectrum of methods was established for the incubation with colon carcinoma cell lines of various differentiation states and of all stages of growth (exponential, subconfluent, confluent, fully differentiated monolayer). Indeed, the effects measured after incubation with FS could only in part been ascribed to their SCFA content. Other FS components than SCFA that play a role, especially regarding to their apoptosis inhibiting and cell cycle influencing effects, remain to be identified. Also, this study allows no conclusions to be drawn, which of the fermented OS is more promising in it?s beneficial influence on colon cancer preventing factors, e.g. the induction of apoptosis, than the other. Future studies should investigate FS with greater differences in their SCFA-concentrations. The same OS which were used for the in vitro fermentation, should also be tested in animal studies and human intervention studies to elucidate their fermentation patterns in vivo.
Impact of process parameters on the sourdough microbiota, selection of suitable starter strains, and description of the novel yeast Cryptococcus thermophilus sp. nov.
(2013) Vogelmann, Stephanie Anke; Hertel, Christian
The microbiota of a ripe sourdough consists of lactic acid bacteria (LAB), especially of the genus Lactobacillus, and yeasts. Their composition is influenced by the interplay of species or strains, the kind of substrate as well as the process parameters temperature, dough yield, redox potential, refreshment time, and number of propagation steps (Hammes and Gänzle, 1997). As taste and quality of sourdough breads are mainly influenced by the fermentation microbiota, intense research has been focused on determination of sourdough associated species and search for new starter cultures. In recent years, economic competition pressure and new consumer demands have led to steady research for new cereal products, especially with health benefit or for people suffering from celiac disease. For these reasons, alternative cereals like oat and barley (both toxic for celiac disease patients) as well as the celiac disease compatible cereals rice and maize, sorghum and millets, the pseudocereals amaranth, quinoa and buckwheat as well as cassava got into the focus of interest. However, information about the microbiota of sourdoughs fermented with buckwheat, amaranth, quinoa, oat or barley is not available except for the following recent studies: a study about the microbiota of amaranth sourdoughs by Sterr et al. (2009), a study about barley sourdough by Zannini et al. (2009), a study about oat sourdoughs by Huettner et al. (2010) and a study about buckwheat and teff sourdoughs by Moroni et al. (2011). The microbiota of sourdoughs from the other mentioned cereals as well as cassava was multiply characterised but not systematically. Fermentation conditions were partly not clearly defined, and identification of species was often based on physiological criteria only, known to be insufficient for the exact classification of LAB. Thus, in this thesis, the influence of the process parameters substrate, temperature, refreshment time, amount of backslopping dough as well as the interplay between the different species or strains were examined and potential starter strains were selected. In Chapter III, the effect of the substrate on the sourdough microbiota was examined and suitable starter cultures for fermentation of non-bread cereals and pseudocereals were selected. Eleven different flours from wheat, rye, oat, barley, millet, rice, maize, amaranth, quinoa, buckwheat and cassava were inoculated with a starter mixture containing numerous LAB and yeasts. Sourdoughs were fermented at 30 °C and refreshed every 24 hours until the microbiota was stable. Species were identified by PCR-DGGE as well as bacteriological culture and RAPD-PCR, followed by 16S/26S rRNA sequence analysis. In these fermentations, the dominant yeast was Saccharomyces cerevisiae; Issatchenkia (I.) orientalis was only competitive in the quinoa and the maize sourdough. No yeasts were found in the buckwheat and the oat sourdough. The dominant LAB species were Lactobacillus (L.) paralimentarius in the pseudocereal sourdoughs, L. fermentum, L. helveticus and L. pontis in the cereal sourdoughs, and L. fermentum, L. plantarum and L. spicheri in the cassava sourdough. Competitive LAB and yeasts were inserted as starters for a further fermentation using new flours from rice, maize, millet and the pseudocereals. After ten days of fermentation, most of the starter strains were still dominant, but L. pontis and L. helveticus could not compete with the other species. It is remarkable that from the numerous starter strains which all were adapted to or isolated from sourdoughs, only a few were competitive in these fermentations; but if, then in most cases in a lot of different flours. In Chapter IV, the effects of the exogenous process parameters substrate, refreshment time, temperature, amount of backslopping dough as well as competing species on the two microbial associations L. sanfranciscensis ? Candida (C.) humilis and L. reuteri ? L. johnsonii ? I. orientalis were examined. Both associations had previously been found to be competitive in sourdough (Kline and Sugihara, 1971a; Nout and Creemers-Molenaar, 1987; Gobbetti et al., 1994a; Garofalo et al., 2008; Böcker et al., 1990; Meroth et al., 2003a). 28 sourdough batches were fermented under defined conditions until the microbiota was stable. Dominant LAB and yeasts were characterized by bacteriological culture, RAPD-PCR and 16S/26S rRNA gene sequence analysis. The process parameters for the association L. sanfranciscensis ? C. humilis could be defined as follows: rye bran, rye flour or wheat flour as substrate, temperatures between 20 and 30 °C, refreshment times of 12 to 24 hours and amounts of backslopping dough from 5 to 20 %. In addition, the association was predominating against all competing lactic acid bacteria and yeasts. The association L. reuteri ? L. johnsonii ? I. orientalis was competitive at temperatures of 35 to 40 °C, refreshment times of 12 to 24 hours and the substrates rye bran, wheat flour and rye flour, but only with sufficient oxygen supply. Cell counts of I. orientalis fell rapidly under the detection limit when using high amounts of doughs (small ratio of surface to volume) and refreshment times of 12 hours. The fermentations depicted in Chapter III and IV give new information about the influence of process parameters on the sourdough microbiota. The studies show that the sourdough microbiota is markedly influenced by the process parameters and kind and quality of substrate. The competitiveness of a single LAB or yeast is strain specific. Interactions between microorganisms also play an important role. However, for the search for suitable starter strains, it would be beneficial to know the reasons, why a single LAB or yeast strain is better adapted to specific process parameters or substrates than others. One of the starter sourdoughs used for fermentation I described in Chapter III was a sourdough made from cassava flour, inoculated with several LAB. No yeast had been inserted, but several yeasts were isolated from the ripe sourdough, which are supposed to originate from the cassava flour. An unknown yeast species constituted 10 % of the isolated yeasts which is described as novel species Cryptococcus thermophilus sp. nov. in Chapter V. This yeast is characterized by budding on small neck-like structures, no fermentative ability, growth at 42 °C and without vitamins, a major ubiquinone of Q-10, as well as the production of green or blue fluorescent substances in the growth medium. It is distinct from related species by the ability to assimilate raffinose and cadaverine, the inability to assimilate soluble starch, xylitol, galactitol, butane-2,3-diol, sodium nitrite and lysine, and the inability to produce starch-like substances. The closest relatives are the yeasts belonging to the Cryptococcus humicola complex.
Investigations on the effects of forage source and feed particle size on ruminal fermentation and microbial protein synthesis in vitro
(2012) Hildebrand, Bastian; Rodehutscord, Markus
The synthesis of microbial protein in the rumen has a major impact on protein- and amino acid supply in ruminants. The amount and amino acid composition of the protein that enters the small intestine primarily depends on diet formulation. In the present studies the effects of maize silage (MS) and grass silage (GS) on ruminal fermentation and microbial protein synthesis were investigated, considering methodical aspects of in vitro studies, particularly grinding of feed samples. In the first experimental series five mixed diets with different proportions of MS and GS (100:0, 79:21, 52:48, 24:76 and 0:100) and a constant proportion of soybean meal (11%) were used. The content of crude protein (CP) and fibre fractions increased, whereas the content of organic matter (OM) and starch decreased with increasing proportion of GS in the diet. It was hypothesised that a combination of MS and GS can benefit microbial growth and thus fermentation of nutrient fractions to a higher extent than using only one forage source separately. It was also to be investigated how changes in diet composition affect the amino acid profile of microbial protein. A well standardised semi-continuous rumen simulation technique (RUSITEC) was used, which is a commonly accepted experimental model in investigations on ruminal fermentation. Changes in fermentation characteristics, as a result of changing the MS-to-GS ratio, were tested for linear and quadratic effects in order to identify possible associative effects. Prior to the in vitro incubation, feedstuffs were dried and ground. It was aimed to investigate in which way fermentation in the RUSITEC system is influenced by mean feed particle size. Therefore two milling screen sizes (MSS; 1 vs. 4 mm) were used in all diets and results on fermentation characteristic were tested for possible interactions of forage source and MSS. One incubation period lasted for 13 days (6 days adaption period, 7 days sampling period), and each treatment was tested in at least three replicates. Ruminal digesta, obtained from rumen-fistulated wether sheep, was used as the inoculum for starting the incubation. Diets were fed once daily to the RUSITEC system, and nylon feed bags remained for 48 h inside the fermentation vessel. A buffer solution, containing 15NH4Cl, was infused continuously into the vessel and the respective effluent was analysed for short chain fatty acids (SCFA) and NH3-N. Solid- and liquid- associated microbial fractions were isolated from the feed residues, the liquid inside the vessel and the effluent by differential centrifugation. The flow of microbial CP was quantified on the basis of N and 15N balances. The feed residues were analysed for crude nutrients and detergent fibre fractions and the respective degradation rates were calculated. OS and CP in the feed residues were corrected for the contribution of solid-associated microbes. The degradation of OM and fibre fractions, as well as amounts of NH3-N increased linearly with stepwise replacement of MS by GS. Degradation of CP was unaffected by diet composition, as well as total SCFA production. The degradation of OM and CP was higher in coarse milled (4 mm-MSS) than in fine milled (1 mm-MSS) treatments, accompanied by higher amounts of NH3-N and total SCFA. An improvement of growth conditions for some microbial groups, e.g. anaerobe fungi, was discussed. The amount of microbial CP increased linearly by the stepwise replacement of MS by GS, and was higher at 4 mm-MSS than at 1 mm-MSS. The amount of available N was assumed to advance microbial growth in the RUSITEC system. Efficiency of microbial CP synthesis was improved from 29 to 43 mg microbial N per g degraded OM by increasing the proportion of GS in the diet, but was unaffected by MSS. The N content and the profiles of amino acids of the three microbial fractions, as well as the ratio of solid- to liquid-associated microbes were affected by diet composition and MSS. Interactions of forage source and MSS were rare. However, the results indicated interactions between dietary factors and origin of microbial isolate on characteristics of microbial protein synthesis. In order to provide additional information on the nutritional value, the mixed diets were evaluated by two further methods. The total tract digestibility of crude nutrients was determined in wether sheep. The content of metabolisable energy was similar between diets and averaged 11.5 MJ per kg dry matter. The in vitro gas production was measured within 93 h by using a modified Hohenheim gas production test, providing information on kinetics and extent of ruminal fermentation. Cumulative gas production decreased with increasing proportion of GS in the diet. A negative effect of coarse milling on fermentation in the Hohenheim gas production test was confirmed. Across all diets gas production was delayed at 4 mm-MSS compared to 1 mm-MSS. The results from both approaches supported the findings of the RUSITEC study that a stepwise replacement of MS by GS led to a linear response in degradation of nutrients. As indicated by the gas production data, positive associative effects might only occur in the first hours after starting an incubation. When mixed diets are used effects cannot be clearly related to individual diet ingredients. Moreover, in the mixed diets interactions between soybean meal inclusion and forage source or feed particle size cannot be excluded. Therefore pure silages were incubated separately in the RUSITEC system in the second experimental series and three milling screens of different size were used (1, 4 and 9 mm). In accordance with the first experimental series, degradation of OM, fibre fractions and non-structural carbohydrates, production of NH3-N, as well as microbial CP flow and efficiency of microbial CP synthesis were higher in GS than in MS. A higher degradation of CP was found for MS than for GS, indicating interactions between forage source and soybean meal inclusion. An increase in MSS from 1 mm to 9 mm led to an improvement in the degradation of OM, CP and non-structural carbohydrates, particularly of starch in MS, as well as in the microbial CP flow for both silages. But the efficiency of microbial CP synthesis and microbial amino acid profile were less affected by MSS. In the second experimental series additionally the effect of available N on fermentation of MS was investigated. The supplementation of urea-N improved the degradation of non-structural carbohydrates, especially starch, but not that of fibre fractions in MS. The efficiency of microbial CP synthesis was increased from 26 to 35 mg microbial N per g degraded OM by urea-N supplementation to MS. The way of urea administration, either supplied together with the feed once daily or infused continuously by buffer solution, had only marginal effects on fermentation characteristics. It was concluded that microbial growth is improved by degradation of OM from GS compared to MS and by an increasing availability of N in the RUSITEC system. Meaningful associative effects of mixtures of MS and GS on ruminal fermentation characteristics are not likely to occur. However, transferability of results to other batches of MS and GS is limited, as high variations in chemical composition are known for both types of silage. Fermentation of MS- and GS-based diets in the RUSITEC system benefits more by coarse milling at MSS up to 9 mm than by fine milling at 1 mm-MSS. Consequently, variations in MSS and feed particle size distribution have to be taken into account when evaluating feeds by rumen simulation systems. The changes in composition and contribution of microbial fractions give indications to a shift in the microbial community as a result of variation of silage type and feed particle size, but further research on this aspect is needed. Moreover, the present results stated that the origin of the microbial samples is very important for measurements on microbial protein synthesis.
Isolierung universell einsetzbarer und mikrobiologisch stabiler Sauerteigstarterkulturen durch spontane Fermentationen mit Amaranth.
(2009) Sterr, Yasemin Arzu; Schmidt, Herbert
Spontaneous fermented sourdoughs prepared from five amaranth flours were screened for the presence of lactic acid bacteria (LAB) that predominate the autochthonous microbiota and thus may be suitable as starter cultures. The doughs were fermented with daily backslopping on the laboratory scale for 10 days with a dough yield of 200 at 30°C. Every 24 hours, the pH-values and total titratable acidity degrees were determined and samples were analyzed for the presence of LAB and yeasts by cultural methods. The identity of the isolates was traced during the fermentation with RAPD-PCR and two different primers, and the strains were identified by sequence analysis of the 16S rDNA genes. The strains Lactobacillus plantarum RTa12, L. sakei RTa14, and Pediococcus pentosaceus RTa11 were selected and applied as starters in further laboratory fermentations. All strains were predominant in repeated experiments, both, as single strains and in combination. During the first 24 h, L. plantarum RTa12 and P. pentosaceus RTa11 grew quite similar. The pH-value dropped earlier with P. pentosaceus RTa11, while both strains gave the same pH-values after 10 h of fermentation. In the challenge test with the autochthonous mikrobiota both strains overgrew the other LAB of the spontaneous fermented dough within the first eight hours, and were therefore considered dominant over the resident microbiota. Influences of the incubation temperature on the fermentation characteristics were mainly assessed in the viable cell counts, the pH-values and the titratable acidity degrees at 25°C. The pH-values for both strains were at high incubation temperatures (30 and 35°C) during the fermentation lower than at 25°C, respectively. However, after 24 h of fermentation both strains reached a pH-value of approximately 4.0 after 24 h. Further sugar, organic acid, mannitol and ethanol profiles of fermented doughs were determined with HPLC. Mainly analyzed metabolites in the doughs were glucose, sucrose, lactate, and acetate. To compare the potential starter cultures with commercial available startercultures, fermentations with two industrial startercultures were performed for 24 h at 30°C and a dough yield of 200. Both strains were able to compete with the commercial available starter cultures concerning viable cell counts, total titratable acidity and pH-values. Because of the dominance of both strains in sourdough fermentations with amaranth, the ability for acidification in a short time, the capacity to compete with the autochthonous mikrobiota, the robustness against lesser effects of the environment, e. g. variation of the temperature, and at least because of the ability to compete with commercial available startercultures, thus, the characterized strains L. plantarum RTa12 and P. pentosaceus RTa11 are interesting candidates as starter cultures for amaranth sourdoughs.
Mikrobiologische und biochemische Analyse der Fermentationseigenschaften von Lactobacillus paralimentarius AL28 und Lactobacillus plantarum AL30 in Sauerteigen aus Pseudozerealien
(2011) Vogel, Antje; Schmidt, Herbert
Pseudocereals are absent of gluten and therefore are important for people having a gluten-intolerance. Today no commercial starter cultures are available for sourdough fermentations with pseudocereals. This PHD-Thesis shows results of the characterisation of L. paralimentarius AL28 and L. plantarum AL30 concerning an application in pseudocereal sourdoughs. The fermentation properties of the strains, applied as single strains and in combination, were assessed in laboratory scale fermentations with amaranth and buckwheat. The fermentation studies were performed with a dough yield of 200 and over a period of two to ten days at 30°C with daily refreshment step. The investigated strains acidified the sourdoughs fast within the first two propagation steps as single strains as well as in combination (approximately pH 4). In amaranth higher total titratable acidity (TTA) -values (TTA between 25 and 30) were measured than in buckwheat (TTA of 20). 16S rDNA / 28S rDNA-PCR sequencing and RAPD-PCR were applied to determine the bacterial and eucaryotic species affiliation, respectively, and to trace specific strains during the fermentation process. The analysed strains competed against the autochthonous microbiota with the result of suppression the majority of yeasts and moulds as well as strains of their own species within the first 12 h of sourdough fermentation. They also suppressed an autochthonous microbiota grown up to 10^8 cfu/g sourdough. Single strains as well as the combination of both strains dominated the microflora in all tested flours / fermentation batches. Both strains displayed reproducible results concerning their over-all fermentation characteristics. L. paralimentarius AL28 and L. plantarum AL30, respectively, dominated the LAB viable counts in all flours after 10 days of fermentation as single strains (>/= 68 % and >/= 98 % of LAB, respectively) as well as in combination. In the latter case strain L. plantarum AL30 was especially competitive in buckwheat (AL28:AL30 = 1:1), more than in amaranth (AL28:AL30 = 4:1). The strains were characterised by their short lag-phase of
Teff-based functional probiotic beverage processed with selected probiotic bacteria
(2022) Alemneh, Sendeku Takele; Hitzmann, Bernd
The recent consumers’ demand have moved from the primary role of food to the healthier action of biologically active food components. For this purpose, production of probiotic functional foods through a fermentation process is the current particular interest. Dairy-based products have been used for probiotics delivery since a very long time; however, due to the drawbacks associated with them such as milk lactose indigestibility, the prevalence of cholesterol related to dairy products, and allergy to milk protein are limited their further utilization for probiotics delivery. Alternatively, cereals are becoming the favorite choices to using as fermentable substrates for the growth and delivery of probiotics. Also, vegetarianisms are increasing through time because of medical reasons. Whole grain cereals are readily available with important nutrient sources of phytochemicals, and other bioactive compounds. Cereals have functioned as an encapsulation materials to improve probiotic stability and their bioactive prebiotics selectively stimulate the growth of probiotics present in the gastrointestinal tract. Particularly, teff is a gluten free and its nutritional value is attractive with high dietary fiber. Amino acids find in teff are well balanced and contains high lysine content. Teff is a good source of essential fatty acids, fiber, minerals, and phytochemicals such as polyphenols and phytates. Consequently, the first primary objective of this research was to compare the quality attributes of whole grain teff flours grown in Ethiopia and South Africa for their proximate composition (moisture, protein, ash, fat, fiber, and carbohydrate), mineral contents (calcium, zinc, and iron), profiles of eighteen amino acids, pasting and thermal properties, and functional properties (water absorption capacity, oil absorption capacity, and swelling power), falling number and color. The proximate composition was examined using the methods of the European Commission Regulation (152/2009). Atomic spectrometer, ion-exchange chromatography, Rapid Visco-Analyzer, and Differential Scanning Calorimetry were used respectively to measure minerals, amino acids, pasting, and thermal properties. Correlation of the measured attributes were analyzed by Pearson correlation and principal component analysis. Significant (p < 0.05) differences were observed in most of the measured attributes between the two teff varieties; however, several significant (p < 0.01) correlations were obtained among the measured attributes by Pearson correlation and principal component analysis. The measured contents of moisture, protein, and zinc in South African teff variety were observed higher than the one grown in Ethiopia. However, much higher calcium and iron contents were found in Ethiopian teff variety. Ethiopian teff variety had showed higher values of foam stability, water absorption capacity, oil absorption capacity, and swelling power as compared to South African teff variety. Results from thermal and pasting properties showed that onset, peak and end temperatures, trough, final, and setback viscosities, as well as peak time, pasting temperature were observed higher in case of South African teff variety. The second primary objective was to examine the suitability of teff made substrates for their potential for the growing and delivering of selected probiotic strains of Lactiplantibacillus plantarum A6 (LPA6) and Lacticaseibacillus rhamnosus GG (LCGG). Single and co-culture fermentations were performed without pH adjustment. In 24 h fermentation with single strain of LPA6, cell count was increased to 8.35 log cfu/mL. Titratable acidity (TA) and pH were measured between 0.33 and 1.4 g/L, and 6.3 and 3.9, respectively. For the investigation of optimum fermentation process variables, Nelder-Mead simplex method was applied and found the optimum values for time and inoculum respectively as 15 h and 6 log cfu/mL. Afterwards, co-culture fermentation was performed by using the optimized process variables. As a result of co-culture fermentation, glucose was progressively consumed while lactic acid and acetic acid were produced. Cell counts of LPA6 and LCGG were able to grow to 8.42 and 8.25 log cfu/mL, respectively, which are a good counts as compared to the minimum required probiotics level of 6 log cfu/mL at consumption time. Findings showed that similar pH and TA values were attained in short time during co-culture fermentation compared to single culture fermentation. Also, without any addition teff substrate was found to be suitable for the growing and delivering of the tested probiotic strains of LPA6 and LCGG. Another focus of this research was to apply two-dimensional fluorescence spectroscopy for the on-line supervision of the fermentation process of teff-based substrate inoculated with LPA6 and LCGG. The fluorescence spectra were measured by using BioView sensor. Analysis of the fermentation process by using the conventional methods such as high performance liquid chromatography for determination of glucose and lactic acid, and using agar plate count for determination of cell counts are time consuming, labor intensive and costly methods. As an alternative the application of fluorescence spectroscopy coupled with partial least square regression and artificial neural network was applied for the on-line quantitative analysis of cell counts of LPA6 and LCGG, glucose, and lactic acid. For the prediction of cell counts of LPA6 and LCGG, the percentage errors of prediction were determined in the range of 2.5-4.5 %. Also, for lactic acid prediction, the percentage error was 7.7 %; however, percentage error for glucose prediction showed a rather high error value. This part of study verified that a two-dimensional fluorescence spectroscopy combined with partial least square regression and artificial neural network can be applied during fermentation process to predict cell counts of LPA6 and LCGG, and content of lactic acid with low uncertainty. Finally, this study was focused on the effect of refrigerator storage on the physicochemical characteristics and viability of LPA6 and LCGG in a teff-based probiotic beverage. As well as a 9-point hedonic scale was applied for sensory test of the beverage. For these determinations, a teff-based probiotic beverage was produced through the fermentation of whole grain teff flour inoculated with co-culture strains of LPA6 and LCGG. Then, the beverage was stored in refrigerator (4-6 ℃) for 25 days. Samples were taking every five days including the first day of storage to quantify cell counts of LPA6 and LCGG, pH, TA, glucose, acetic acid, lactic acid, and maltose. Over the storage time, cell counts of LPA6 and LCGG were decreased from 8.45 and 8.15 log cfu/mL to 8.28 and 7.86 log cfu/mL, respectively. While cell counts were decreased during storage, their cell counts are still observed above the minimum suggested level of 6-7 log cfu/mL at the time of consumption. Lactic acid, acetic acid, glucose, and maltose as well as TA were increased with reduction of pH over the storage time. Metabolic activities observed over the storage time indicated presence of active enzymes that were produced during fermentation process. As examined the beverage, E. coli, Pseudomonas aeruginosa, coagulase-positive Staphylococci, presumptive Bacillus cereus, Salmonella spp., and Listeria monocytogenes weren’t detected. Sensory test attributes of color, appearance, aroma, and taste of the beverage were observed between 6.2 and 6.9, which are in the accepted range. Six and above average score values of the sensory test attributes are considered to be accepted by the panelists. Overall, it could be possible to say the proposed aim for the production of a teff-based probiotic functional beverage was accomplished successfully.
Ultraschallbasierte simultane Konzentrationsbestimmung der Komponenten Zucker und Ethanol in wässrigen Fermentationsfluiden
(2014) Schöck, Thomas; Hitzmann, Bernd
At alcoholic fermentation processes in aqueous solutions there are converted various sugars (mono-, di- and polysaccharides) into ethanol and carbon dioxide by diverse intermediate steps. In the industrial production, ultrasound based methods for the analysis of the composition of the fermentation fluid are advantageous due to their robustness, price cheapness and the possibility for the accomplishment of on line measurements. Within the scope of the present work there are presented several methods for the simultaneous determination of the sugar and ethanol content in the fermentation fluid based on the analysis of ultrasound parameters, also at the presence of dissolved carbon dioxide gas, and compared with respect to the accuracy of their predictive values. Initially there is investigated the behavior of the parameters sound velocity and adiabatic compressibility in standardized aqueous fluids in dependency of the concentration of the components sugar (2 -16 mass percent) and ethanol (1- 6 mass percent), the CO2 partial pressure (0 – 3,013E+05 Pa) and the temperature (2 – 30° C). Thereby the disaccharide saccharose acts as a model substance for the sugar fraction. From the data field of the sound velocity two polynomial calibration models for the sugar / ethanol concentration are extracted with the methods of the multiple linear regression (MLR) and the partial least squares (PLS-) analysis. The minimal accessible standard deviation of the concentration values determined by the particular model from the reference values lies for the MLR method at 0,6 mass percent for the sugar and 0,2 mass percent for the ethanol fraction. The PLS-analysis yields a standard deviation for the sugar and ethanol values of 0,36 and 0,13 mass percent respectively (fluids without a CO2 fraction), as well as 0,5 / 0,17 mass percent (fluids including a CO2 fraction). A further analytic method uses a linearized model of the adiabatic compressibility and the density for the sugar / ethanol determination. The analysis of two physical parameters at this method yields a significant increase of the model quality. For fluids without a CO2 fraction there is reached a minimal standard deviation of 0,06 mass percent for the sugar and 0,07 mass percent for the ethanol concentration. For CO2 containing fluids the corresponding values results to 0,06 / 0,13 mass percent.