Slow Fermentation Research Articles

Stuck and Slow Fermentations in Enology: Statistical Study of Causes and Effectiveness of Combined Additions of Oxygen and Diammonium Phosphate.

One hundred and seventy-eight musts from different regions in France were selected by enologists and fermented under standardized conditions. Two kinds of fermentation problems were distinguished: (a) slow and (b) sluggish (late-onset sluggish) and stuck (with residual sugar) fermentations. Slow fermentations, characterized by a low fermentation rate throughout the process, were always due to low assimilable nitrogen concentrations in the must. The advantages of using formol titration for measuring assimilable nitrogen are discussed. In contrast, sluggish and stuck fermentations, characterized by very low yeast viability at the end of fermentation, could not be predicted from the analytical data even though their probability was increased when the initial sugar concentration was high. All problems of stuck and sluggish fermentations (concerning 40% of the musts) were solved by supplying 7 mg/l oxygen and 300 mg/l diammonium phosphate at the halfway of the fermentation process, which confirmed (i) the importance of these two nutrients in enological practices and (ii) the importance of adding them at the right time.

Stuck and slow fermentations in enology: statistical study of causes and effectiveness of combined additions of oxygen and diammonium phosphate

One hundred and seventy-eight musts from different regions in France were selected by enologists and fermented under standardized conditions. Two kinds of fermentation problems were distinguished: (a) slow and (b) sluggish (late-onset sluggish) and stuck (with residual sugar) fermentations. Slow fermentations, characterized by a low fermentation rate throughout the process, were always due to low assimilable nitrogen concentrations in the must. The advantages of using formol titration for measuring assimilable nitrogen are discussed. In contrast, sluggish and stuck fermentations, characterized by very low yeast viability at the end of fermentation, could not be predicted from the analytical data even though their probability was increased when the initial sugar concentration was high. All problems of stuck and sluggish fermentations (concerning 40% of the musts) were solved by supplying 7 mg/ l oxygen and 300 mg/ l diammonium phosphate at the halfway of the fermentation process, which confirmed (i) the importance of these two nutrients in enological practices and (ii) the importance of adding them at the right time.

Heliobacterium sulfidophilum sp. nov. andHeliobacterium undosum sp. nov.: Sulfideoxidizing heliobacteria from thermal sulfidic springs

Two new species of heliobacteria isolated from cyanobacterial mats of two alkaline sulfidic hot springs are formally described. Strains BR4 and BG29 are assigned to anoxygenic phototrophic bacteria of the family Heliobacteriaceae, since they possess the unique properties of this taxon: strict anaerobiosis, formation of bacteriochlorophyll g, the lack of extensive intracytoplasmic membranes and chlorosomes, an unusual cell wall structure, and phylogenetic relatedness to the low G + C gram-positive eubacteria. Based on the 16S rDNA sequence similarity, strains BR4 and BG29 are assigned to the genus Heliobacterium and described as two new species of this genus: Heliobacterium sulfidophilum sp. nov. and Heliobacterium undosum sp. nov. The G + C content of the DNA is 51.3 mol % in Hbt. sulfidophilum and 57.2-57.7 mol % in Hbt. undosum. The cells of Hbt. sulfidophilum are rods, and the cells of Hbt. undosum are slightly twisted spirilla or short rods. Both new bacteria are motile by peritrichous flagella. Hbt. sulfidophilum produces endospores. The new bacteria are strict anaerobes growing photoheterotrophically on a limited range of organic compounds. In the dark, they can switch from photosynthesis to the slow fermentation of pyruvate. Biotin is required as a growth factor. Both species are highly tolerant to sulfide (up to 2 mM at pH 7.5) and oxidize it photoheterotrophically to elemental sulfur; photoautotrophic growth was not observed. The temperature optimal for growth of Hbt. sulfidophilum and Hbt. undosum is 30-35 degrees C, and the optimal pH is 7-8.

Impact of fermentation technology on the phenolic and volatile composition of German red wines

SummaryHomogenous grape material of the red varieties c.v. Pinot noir, c.v. Dornfelder and c.v. Portugieser were fermented on an industrial scale using a special fermenter for pump over and punch down treatment. On pilot scale, thermovinfication was carried out at 60 °C and a manual punch down treatment was used in open containers. Both the mechanical punch down and pump over treatment significantly enhanced the extraction of all phenolic compounds and their polymerization in comparison to the traditional manual punch down treatment. Applying an excess of mechanical maceration increased the extraction of seed and stem phenols, but slowed down their polymerization. Assessing volatile composition, the biggest differences occurred between wines made by thermovinification versus those fermented on their skins. Slow fermentation kinetics and low solid content in the juice enhanced ester formation in thermovinied wines. Thermal inactivation of lipoxygenase enzyme systems reduced C6‐alcohols and their subsequent esters in thermovinified wines. Only small differences occurred between punch down and pump over tanks except that the pump over regime gave all varieties of wines significantly higher quercetin levels. In conclusion, the application of modern fermentation equipment seemed to be more relevant to improve extraction and polymerization of phenolic compounds, while thermovinification yielded wines with high ester content giving a more fruity style.

Comparison of Nitrogen and Oxygen Demands of Enological Yeasts: Technological Consequences

Nitrogen and oxygen requirements of commercial yeasts were compared. Nitrogen demands were measured by calculating the amounts of ammoniacal nitrogen necessary to keep the fermentation rate constant during the stationary phase. Oxygen demands were studied by (<i>i</i>) running anaerobic fermentations and (<i>ii</i>) measuring the effect of adding 1 mg/L oxygen at the beginning of the stationary phase. Substantial differences were found and their technological impact was discussed. Because of the importance of nitrogen and oxygen in enology, using such tests may be of great interest, especially when there are risks of (<i>i</i>) slow fermentations, due to nitrogen deficiencies or (<i>ii</i>) sluggish fermentations, mainly due to oxygen limitations.

A history of research on yeasts 2: Louis Pasteur and his contemporaries, 1850-1880

A history of research on yeasts 2: Louis Pasteur and his contemporaries, 1850-1880

Purification of Mlc and Analysis of Its Effects on the pts Expression in Escherichia coli

Products of the pts operon of Escherichia coli have multiple physiological roles such as sugar transport, and the operon is controlled by two promoters, P0 and P1. Expression of the pts P0 promoter that is increased during growth in the presence of glucose is also activated by cAMP receptor protein.cAMP. Based on the existence of a sequence that has a high similarity with the known Mlc binding site in the promoter, the effects of the Mlc protein on the pts P0 promoter expression were studied. In vivo transcription assays using wild type and mlc-negative E. coli strains grown in the presence and absence of glucose indicate that Mlc negatively regulates expression of the P0 promoter, and Mlc-dependent repression is relieved by glucose in the growth medium. In vitro transcription assay using purified recombinant Mlc showed that Mlc repressed transcription from the P0 but did not affect the activity of the P1. DNase I footprinting experiments revealed that a Mlc binding site was located around +1 to +25 of the promoter and that Mlc inhibited the binding of RNA polymerase to the P0 promoter. Cells overexpressing Mlc showed a very slow fermentation rate compared with the wild type when grown in the presence of various phosphoenolpyruvate-carbohydrate phosphotransferase system sugars but few differences in the presence of non-phosphoenolpyruvate-carbohydrate phosphotransferase system sugars except maltose. These results suggest that the pts operon is one of major targets for the negative regulation by Mlc, and thus Mlc regulates the utilization of various sugars as well as glucose in E. coli. The possibility that the inducer of Mlc may not be sugar or its derivative but an unknown factor is proposed to explain the Mlc induction mechanism by various sugars.

A Comparison of Glycolytic Activity in Winter Wheat and two Forage Grasses in Relation to their Tolerance to Ice Encasement

Melting and refreezing within snow layers can cause the build-up of ice at soil surfaces, which can severely damage overwintering crop plants. The relatively limited tolerance of cold acclimated winter wheat to ice encasement is increased by a prior exposure to low temperature flooding. This is a hypoxic acclimation. In wheat, it is accompanied by a three-fold increase in the activity of pyruvate decarboxylase (PDC) and a two-fold increase in the activity of alcohol dehydrogenase (ADH). Activity of PDC is one tenth that of ADH. The glycolytic kinases [ATPphosphofructokinase (PFK), PPi-phosphofructophosphotransferase (PFP), and pyruvate kinase (PK)] show little change during the acclimation process. The forage grasses timothy (Phleum pratense) and berings hairgrass (Deschampsia berengensis) when cold acclimated, are more tolerant to ice encasement than winter wheat, and show a relatively small hypoxic acclimation response. During ice encasement, these forage grasses accumulate less ethanol, CO 2 and lactic acid than wheat. In further contrast to wheat, they produce lower levels of these metabolites in ice following acclimative flooding than they do in the absence of flooding. Activity of PDC and ADH in crowns of the two forage grasses is also less than in wheat. These observations indicate that high ice encasement tolerance of the grasses may be associated with slow fermentation rates and low accumulation of toxic metabolites. ( 1997 Annals of Botany Company

Early thiamin assimilation by yeasts under enological conditions: Impact on alcoholic fermentation kinetics

The effect of early must thiamin depletion by wild yeast strains ( i.e. Kloeckera and Saccharomyces species) on alcoholic fermentation kinetics was studied. Experimental conditions affecting thiamin assimilation by yeasts were first determined, using factorial designs. Then sequential and/or mixed cultures simulating wild yeast contamination, as observed during winemaking, were carried out in order to study the influence of early thiamin depletion on fermentation kinetics. The obtained results indicate that biological thiamin depletion leads to slow fermentation and may lead to sluggish or stuck fermentation. Moreover this phenomenon is amplified in media with high assimilable nitrogen contents. No release of thiamin from contaminant yeast and subsequent assimilation by fermentative yeast was observed, indicating that some enological practices (such as centrifugation of musts contaminated early) may lead to stuck fermentation.

Effects of yeast invertase on ethanol production in molasses

Ethanol fermentation by an alcohol yeast, YOY655, was slower in molasses than in a nutrition rich medium with the same sugar content. Osmolality was much higher in the molasses, and the slower fermentation in the molasses was ascribed to depressed fermentation under the high osmotic pressure. Yeast invertase was an important factor in regulation the osmolality and the fermentation rate in the molasses.

Possible mechanisms underlying the slow lactose fermentation phenotype in Shigella spp

A Southern hybridization analysis revealed that the region homologous to Escherichia coli lacZ was present on the chromosomal DNAs of beta-galactosidase-positive Shigella strains, such as Shigella dysenteriae serovar 1 and Shigella sonnei strains, whereas this region was absent from chromosomal DNAs of beta-galactosidase-negative strains of Shigella flexneri and Shigella boydii. We found that the lacY-A region was deficient in S. dysenteriae serovar 1 and believe that this is the reason for the slow fermentation of lactose by this strain. S. sonnei strains possessed the region which hybridized with E. coli lacY-A despite their slow hydrolysis of lactose. The whole lactose-fermenting region was cloned from S. sonnei and compared with the cloned lac operon of E. coli K-12. Both clones directed the synthesis of beta-galactosidase in an E. coli K-12 strain lacking indigenous beta-galactosidase activity (strain JM109-1), and we observed no difference in the expression of beta-galactosidase activity in S. sonnei and E. coli. However, E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei exhibited the slow lactose fermentation phenotype like the parental strain. S. sonnei strains had no detectable lactose permease activities. E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei had a detectable permease activity, possibly because of the multicopy nature of the cloned genes, but this permease activity was much lower than that of strain JM109-1 harboring the lac operon of E. coli K-12. From these results we concluded that slow lactose fermentation by S. sonnei is due to weak lactose permease activity.

THE RESPONSE OF BREWING YEASTS TO ACID WASHING

Brewing yeasts are inherently resistant to acid washing treatments but, under some conditions this resistance is diminished. Sixteen yeast strains, including both ale and lager strains, have been successfully washed using phosphoric acid (pH 2.1) and, in some cases, sulphuric acid (pH 2.0) or acidified 0.75% w/v ammonium persulphate (pH 2.8). Cell viability, fermentation performance and flocculation/fining behaviour were unaffected. However, changes could be observed in the yeast resulting from the washing treatment. These included alterations of the cell surface, as shown by scanning electron microscopy and leakage of adenosine triphosphate from the cells during the wash. Acid washing is successful provided that, i) food grade acid is used, ii) the acid is chilled before use, iii) the yeast and acid are well mixed, iv) the temperature of the yeast does not exceed 5°C during washing and, v) the yeast is pitched immediately after washing. ‘Unhealthy’ yeast (yeast which has been stored for long periods, heavily contaminated yeast, or yeast from slow fermentations) may respond poorly to acid washing and a shorter washing time, or higher wash pH value should be employed.

On-line measurement of extracellular proteins in the continuous cellulase production by flow injection analysis (FIA)

The BCA protein assay 1 was adapted to flow injection analysis to monitor the production of extracellular cellulases of Trichoderma species in batch and continuously operated fermentations. Response of the assay to protein (bovine serum albumin) was linear in the range of 0.1–2.0 g l −1; standard deviation of the calibration graph was 0.86%, accuracy and standard deviation were < +1.5% and 0.9% in the upper range, < +8% and < 2% in the lower range, respectively. Interferences from technical fermentation media were taken into consideration and can be calculated. The slow fermentation process was measured with a frequency of 0.5 samples h −1 over a period of 380 h. In each measuring cycle, recalibration was carried out. The necessary cell-free sample stream for the analytical system was obtained by installing the BIOPEM R, a steam-sterilizable sampling devic 2 which showed excellent performance even for long-term fermentations; membranes had to be changed after about 10 days only to keep the protein transmission constant (60–95%, depending on the special protein).

The biomass alternative

Prospects for biomass commodity chemicals are currently unfavorable as a result of: generic deficiencies in fermentation processes with respect to generally low (below 5%) product concentrations and slow fermentation rates, and lack of a presently competitive advantage for renewable materials compared with depressed prices for crude oil. However, research can alter the first limitation, and Middle East politics, the second.

Experimental studies of immobilized-yeast, packed-bed reactors with reduced CO2 entrapment

A horizontal packed-bed reactor with baffles (HPBR) and a continuously stirred tank reactor with intermittent paddle agitation have been shown to considerably reduce the CO2 entrapment when glucose is fermented with immobilized baker’s yeast in calcium alginate beads. Using high cell contents in the gel resulted in internal mass transfer hindrance. The highest productivity was obtained with the HPBR giving 29 g EtOH/Lh at an ethanol yield of 90%. The substrate used was 100 g/L glucose. Fermentation of lactose and deproteinized whey by coimmobilized baker’s yeast and |3-galactosidase resulted in much lower productivity-about 5 g EtOH/L.h because of the slow fermentation of galactose.

Saccharification and Fermentation of Whole Barley Ground in the Szego Mill

AbstractBarley, after steeping in water, was ground with ease and efficiency in the Szego mill, and its starch was liquefied, saccharified and fermented to very high yields of ethanol. The Szego mill consists of vertical rollers with helical grooves which rotate within a fixed cylinder, resulting in very fine grinding and a somewhat flaky product. The steeped barley was ground to a fine paste. This was readily liquefied and saccharified by amylolytic enzymes (dual enzyme process), and the resulting sugars were fermented in 24 h by ordinary bakers' yeast Saccharomyces cerevisiae, resulting in over 450 1 ethanol/t of barley. Still shorter time, 12 h, and the same high yield were achieved when liquefied barley starch was simultaneously saccharified by glucoamylase and fermented. Fermentation to ethanol by a glucoamylase‐producing yeast S. diastaticus strain 164 A (from Labatt Brewing Company) enabled the amount of this enzyme required for saccharification to be reduced to about one‐half the normal quantity, but at some cost in slower fermentation and slightly lower ethanol yield.

2 Vagal control of gastrointestinal function

ABSTRACT The gastrointestinal tract is a long, muscular tube responsible for the digestion of food, assimilation of nutrients and elimination of waste. This is achieved by secretion of digestive enzymes and absorption from the intestinal lumen, with different regions playing specific roles in the processing of specific nutrients. These regions come into play sequentially as ingested material is moved along the length of the GI tract by contractions of the muscle layers. In some regions like the oesophagus transit it rapid and measured in seconds while in others like the colon transit is measured in hours and even days, commensurate with the relative slow fermentation that takes place in the large bowel. An hierarchy of controls, neural and endocrine, serve to regulate the various cellular targets that exist in the gut wall. These include muscle cells for contraction and epithelial cells for secretion and absorption. However, there are complex interactions between these digestive mechanisms and other mecha...

Organic and inorganic nitrogen source effects on the metabolism of Clostridium acetobytulicum

The effects of organic and inorganic nitrogen combinations on cell growth, solvent production and nitrogen utilization by Clostridium acetobutylicum ATCC 824 was studied in batch fermentations. Fermentations in media with 10 mM glutamic acid, as the organic nitrogen source, and 0 mM to 10 mM ammonium chloride, as the inorganic nitrogen source had a solvent yield of 0.8 to 1.08 mmol solvent/mmol glucose used, with a slow fermentation rate (2 mmol solvent/l h-1). When media contained 20 mM or 30 mM glutamic acid as well as 2.5 to 7.5 mM ammonium chloride the fermentation rate increased (5.5 mmol/l h-1) while the solvent yield remained constant (0.86 to 0.96 mmol solvent/mmol glucose used). Total solvent production was higher in media containing 20 mM or 30 mM glutamic acid than with 10 mM glutamic acid.

Glycerol production by anaerobic vacuum fermentation of molasses on pilot scale

The use of sodium sulphite as a steering agent for enhancing the yield of glycerol during anaerobic ethanol fermentation is well established. Several studies have been reported in the literature using free as well as immobilized cells of Saccharomyces cerevisiae. In these studies it was observed that a relatively high concentration of sulphite in the fermentation broth, typically 40-100 g/l, was required to obtain a commercially significant yield of glycerol on sugar fermented. However, the dosing of large quantities of sulphite generally resulted in reduced viability of the microorganisms and slow fermentations. The glycerol concentration in the fermented broth was generally observed to be in the range 20-40 g/l. The low productivity coupled with the high cost of sulfite rendered the process commercially unattractive. In order to reduce the sulphite requirement, whilst at the same time increasing the productivity, a modified vacuum fermentation was developed in the laboratories. The process was successfully estabilished on a pilot scale and typical data obtained on scaleup are reported below. 8 references.

Effect of Oxygen on Lactose Metabolism in Lactic Streptococci

Three strains of Streptococcus lactis, two of Streptococcus cremoris, and one of Streptococcus thermophilus metabolized oxygen in the presence of added carbohydrate primarily via a closely coupled NADH oxidase/NADH peroxidase system. No buildup of the toxic intermediate H(2)O(2) was detected with the three S. lactis strains. All six strains contained significant superoxide dismutase activity and are clearly aerotolerant. Lactose- or glucose-driven oxygen consumption was biphasic, with a rapid initial rate followed by a slower secondary rate which correlated with factors affecting the in vivo activation of lactate dehydrogenase. The rate of oxygen consumption was rapid under conditions that led to a reduction in lactate dehydrogenase activity (low intracellular fructose 1,6-bisphosphate concentration). These conditions could be achieved with nongrowing cells by adding lactose at a constant but limiting rate. When the rate of lactose fermentation was limited to 5% of its maximum, nongrowing cells of S. lactis strains ML3 and ML8 carried out an essentially homoacetic fermentation under aerobic conditions. These same cells carried out the expected homolactic fermentation when presented with excess lactose under anaerobic conditions. Homoacetic fermentation leads to the generation of more energy, by substrate-level phosphorylation via acetate kinase, than the homolactic fermentation. However, it was not observed in growing cells and was restricted to slow fermentation rates with nongrowing cells.