Comparison of Industrial Scale Ethanol Production from a Palmyrah‐Based Carbon Source by Commercial Yeast and a Mixed Culture from Palmyrah Toddy

Palmyrah (Borassus flabellifer) based products were used as an alternative carbon source for industrial scale ethanol production. The fermentation medium was enriched with spent wash obtained from a distillation column. The performance of a commercially available baker's yeast in the media was compared with a ‘palmyrah toddy mixed culture’ where the organisms were obtained from the sedimentation of palmyrah toddy. In a laboratory scale study, the ethanol produced from a palmyrah fruit pulp extract, diluted with distilled water, was 16.5 gL^?1 (36 h) and 13.0 gL^?1 (48 h) with ‘palmyrah toddy mixed culture’ and baker's yeast respectively. The ‘palmyrah toddy mixed culture’ performed better than the baker's yeast with palmyrah fruit pulp extract, diluted either with distilled water or spent wash. Among the different palmyrah based carbon sources, both cultures preferred molasses diluted with spent wash and both performed best in the medium containing the spent wash supplemented with sucrose. In a 5,000 L industrial scale fermentation of 20° Brix molasses supplemented with 10 gL^?1 ammonium sulphate, 72 gL^?1 and 65 gL^?1 ethanol was produced by the ‘palmyrah toddy mixed culture’ (72 h) and the baker's yeast (90 h) respectively. As the performance of the ‘palmyrah toddy mixed culture’ was better than that of the baker's yeast, the former was selected for the industrial scale studies of molasses fermentation media diluted with spent wash. In these studies the temperature reached 42°C by 36 h and resultant cell death was observed. However ethanol production was higher and more rapid in the molasses diluted with spent wash, rather than in the molasses diluted with tap water and supplemented with (NH₄)₂SO₄. Cell recycle operation obviated the interruption in fermentation caused by temperature induced cell death and increased rates and efficiency of ethanol production were observed.     

Use of spent brewer's yeast in L‐(+) lactic acid fermentation

The application of by‐products from the brewing industry in lactic acid (LA) production was investigated in order to replace expensive nitrogen sources (such as yeast extract) with cheaper and renewable nitrogenous materials such as brewer's yeast (BY). In this study, brewer's spent grain (BSG) hydrolysate was used for L‐(+)‐LA fermentation by Lactobacillus rhamnosus ATCC 7469. The effect of pH control during the fermentation and the addition of various BY contents (5–50 g/L) in BSG hydrolysate on fermentation parameters was evaluated. BY addition significantly increased free amino nitrogen (FAN) concentration (by 25.2% at 5 g/L to 616% at 50 g/L). A strong positive correlation between FAN concentration in the hydrolysate and concentration of L‐(+)‐LA produced was observed (correlation coefficient of 0.913). A high cell viability of L. rhamnosus ATCC 7469 (1.95–3.32 × 10⁹ CFU/mL at the end of fermentation) was achieved in all fermentations with the addition of brewer's yeast. The addition of BY increased L‐(+)‐lactic acid yield and volumetric productivity up to 8.4% (5 g/L) and 48.3% (50 g/L). The highest L‐(+)‐LA yield (89%) and volumetric productivity (0.89 g/L h^?1) were achieved in fermentation of BSG hydrolysate with 50 g/L of BY. © 2019 The Institute of Brewing & Distilling     

CHANGES IN CYTOCHROME LEVELS DURING THE STORAGE OF BAKER'S YEAST AT DIFFERENT TEMPERATURES

The content of cytochromes determined per g fresh yeast was found to stay constant during nearly 3 months' storage of baker's yeast at 5°C. When stored at 24°C and 35°C the content of cytochromes apparently increased during the first 14 and 4 days of storage, respectively, and then decreased continuously.     

PROPERTIES OF YEAST PROTEINASES

The properties of yeast proteinases, present in the extract of brewer's yeast, were compared with corresponding properties of plant proteinases, pepsin and trypsin in order to find suitable conditions for their activities to be determined independently. The main differences between yeast and plant proteinases were found in their themostability and activity at pH 3·0. In contrast to plant proteinases, yeast proteinases are thermolabile and active at pH 3·0. The proteolytic activity of yeast proteinases in beer is low and may be detected with sensitive methods using radiolabelled protein substrate. Nevertheless, a very wide range of proteolytic activities (67–1082 nkat/litre) was found in samples of unpasteurized beers.     

Bread making with Saccharomyces cerevisiae CEN.PK113‐5D expressing lipase A from Bacillus subtilis: leavening characterisation and aroma enhancement

Lipase A from Bacillus subtilis was expressed in the yeast Saccharomyces cerevisiae CEN.PK113‐5D strain as a cell wall‐immobilised enzyme. The recombinant yeast was used in bread making to test the effect of lipase A on the bread properties such as rheological and aromatic properties. The results were compared to the not transformed strain and the commercial baker's yeast. The recombinant strain resulted a good leavening agent comparable to the commercial baker's yeasts provided 1% sucrose was added to the dough. Whereas, during the leavening, the trend of the rheological properties (cohesivness, gumminess and rigidity) differed from the commercial and the nontransformed (NT) strains. Moreover, using of the recombinant strain enhanced total aroma which achieved in the baked product a value of 9 μg per g_product vs the 4 and 5 μg per g_product of the NT strain and the commercial baker's yeast, respectively. To our knowledge, this is the first time that a bacterial lipase is used in bread making.     

Continuous baker's yeast production using orange peel as promoting support in the bioreactor

Improvements in maximum growth rate and productivity in biomass production using orange peel as promoter were observed in batch aerobic fermentation. Daily biomass productivity and biomass yield up to 14.9 g (dry weight) L^?1 and 0.22 g (dry weight) g^?1 utilized sugar respectively in batch aerobic fermentation by the presence of orange peel as promoter were reported. A novel bioreactor for continuous aerobic fermentation of molasses is described and its suitability for baker's yeast production using orange peel as promoter is investigated. The continuous bioreactor was operated for 12 days, stored for 20 days at 10 °C and operated again for another 13 days without any diminution of biomass productivity. Daily biomass productivity, yield and conversion up to 11.2 g (dry weight) L^?1, 0.16 g (dry weight) produced g^?1 utilized sugar and 90.4% respectively were recorded. The possibility of using such a system for industrial continuous baker's yeast production is discussed. Copyright © 2005 Society of Chemical Industry     

HEAT PROCESSING OF SPENT BREWER'S YEAST

Moist heat treatments of brewer's yeast at cell populations of approximately 106/ml resulted in biphasic but otherwise typical survival curves. Brewer's yeast was shown to suffer either thermal injury or death over the tested temperature range of 47–53°C. Recovery from thermal injury was possible in liquid media at 20–30°C. Death kinetics were predictable and reproducible. At cell concentrations found in industrial yeast slurries ～5 × 10⁵/ml), pasteurization of spent yeast could not be planned by assuming normal first order kinetics, as dying cells released materials which altered the thermo-resistance of survivors. Under simulated yeast slurry conditions however, with protective cell supernatant (menstruum) obtained after vigorous heating of slurried yeast, a phantom thermal death time curve was obtained which could be used to accurately predict yeast kill (decimal reduction time) at slurry concentrations and at any temperature.     

ESTIMATION OF MOISTURE LOSS FROM THE COOLING DATA OF POTATOES

A procedure was developed to predict moisture loss from cooling data of potato packed in gunny bags and stacked on wooden platforms in commercial cold stores. To predict the moisture loss, mass transfer coefficients k_cand k_mwere estimated during the storage period, which were found to decrease with time. The calculated time average k_cand k_mvalues were 1.83 × 10^?4 m/s and 2.31 × 10^?10 kg/s·m²·Pa during the transient cooling period and 1.59 × 10^?4 m/s and 2.27 × 10^?10 kg/s·m²·Pa for the rest of the storage period, respectively. The estimated moisture losses were 4.8, 4.74 and 4.78%, at the center of three different stacks, for a storage period of 8 months. The corresponding experimentally measured weight losses at the center of the same stacks were 5.2, 5.1 and 5.26% with a variation of 11, 7.5 and 10.2%, respectively. Therefore, the procedure adopted in this study may be used to assess the moisture loss from potatoes under the different storage conditions. The effect of relative humidity (RH) and potato temperature on moisture loss was also predicted using the developed procedure. Decrease in RH of the storage air increased the moisture loss. The potatoes stored below 85% RH incurred more than 7% water loss. Therefore, 88–90% RH in the cold store may be used to limit the maximum moisture loss within the permissible limit of 5% even after 8 months of storage. It was also found that increasing the potato temperature exponentially increased the rate of moisture loss.     

Purification and characterization of highly active and stable polyphosphatase from Saccharomyces cerevisiae cell envelope

Saccharomyces cerevisiae cell envelope polyphosphatase was isolated in highly active and stable form by extraction from cells with zwittergent TM-314 followed by chromatography of the extract on phosphocellulose and QAE-Sephadex in the presence of 5 mM -MgCl₂, 0·5 mM -EDTA and 0·1% Triton X-100. The enzyme possessed a specific activity of 220 U/mg and after 30 days retained 87% of its activity at ?20°C. Polyphosphatase molecular mass was determined to be about 40 kDa by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme hydrolysed polyphosphates with various chain lengths (n = 3–208), had low activity for GTP and did not split pyrophosphate, ATP and p-nitrophenylphosphate. On polyphosphates with chain lengths n = 3, 9 and 208, K_m values were 1·7 × 10^?4, 1·5 × 10^?5 and 8·8 × 10^?7 M respectively. Polyphosphatase was most active and stable at pH 6·0–8·0. The enzyme showed maximal activity at 50°C. The time of half inactivation of polyphosphatase at 40, 45 and 50°C was 45, 10 and 3 min, respectively. In the absence of divalent cations and also with Ca²⁺ or Cu²⁺, the enzyme showed practically no activity. The ability of divalent cations to activate polyphosphatase was reduced in the following order: Co²⁺ > Mg²⁺ > Mn²⁺ > Fe²⁺ > Zn²⁺. Polyphosphatase was completely inhibited by 1 mM -ammonium molybdate and 50 μM -Zn²⁺ or Cu²⁺ (in the presence of Mg²⁺).     

CONSTRUCTION OF FLOCCULENT BREWER'S YEAST BY CHROMOSOMAL INTEGRATION OF THE YEAST FLOCCULATION GENE FLO1

Yeast flocculation gene FLO1, located on chromosome I of Saccharomyces cerevisiae, has been cloned previously¹⁶. However, it has recently been found that the gene was an in-frame deletion derivative of the chromosomal intact FLO1 gene¹⁹. When introduced into non-flocculent industrial strains, including brewer's yeast, the latter gene, FLO1L, containing an open reading frame of 4,611 bp, conferred stronger flocculation than the former gene, FLO1S, containing an open reading frame of 2,586. By chromosomal integration of the ADH1-controlled FLO1L gene, “gene therapy” of the flocculation behaviour of the parent non-flocculent brewer's yeast was successfully achieved.     

A new process for preparing transparent alkalised duck egg and its quality

When fresh duck (Anas plotyrhyncus) eggs (pH 8·0–8·5) are heated, their albumen develops a turbid gel. Through appropriate alkalisation (pH 11·5–12·8), the gel's transparency can be increased. The transparency of the heated duck egg-white is affected by pH value, heating temperature, heating rate and salt concentration. This research deals with the process for preparing the transparent alkalised duck egg and the change in its quality when stored. If fresh duck eggs are pickled in a solution of 42 g NaOH+50 g NaCl litre^?1 (25·3°C) for 8 days, removed, put in a water bath and heated at 70°C for 10 min they become transparent, their hardness and penetration increasing with storage. Total bacterial count and volatile basic nitrogen also increase with storage. The total bacterial count and the volatile basic nitrogen were 4·6 × 10⁶ cfu g^?1, 0·32 mg g^?1 when stored at a temperature of under 25°C for 4 weeks, respectively.     

Substrate specificity of alcohol dehydrogenase from the yeast Hansenyls polymorpha CBS 4732 and Candida utilis CBS 621

The substrate specificity of alcohol dehydrogenase (ADH) from Hansenula polymorpha and Candida utilis has been compared with that of the classical ADH from baker's yeast. Cell-free extracts of H. polymorpha and C. utilis exhibited a much higher ratio of butanol to ethanol oxidation than baker's yeast ADH. This was also observed with the purified enzymes. The ratio of activities with ethanol and butanol was pH-dependent. With the baker's yeast enzyme the activity strongly decreased with increasing chain length, whereas the enzymes form H. polymorpha and C. utilis showed a high reactivity with long-chain alcohols. In addition, the affinity constant for ethanol was more than tenfold lower than that of the baker's yeast enzyme. The purified preparation yielded several protein bands on polyacrylamide slab gels, each of which showed activity with both ethanol and butanol.     

STUDIES ON BLACK GRAM (Phaseolus mungo L.) TRYPSIN INHIBITOR

ABSTRACT A trypsin inhibitor isolated from black gram (Phaseolus mungo L.) had 75 amino acid residues with an estimated molecular weight of 7892. The kinetic constants K_m and V_max as evaluated by the Dixon and Cornish-Bowden plots were 2.7 × 10^?5and 6 × 10^?3M/min, respectively. The dissociation constants of the enzyme-inhibitor complex (Ki) and the enzyme-inhibitor-substrate complex (Ki') were respectively 4 × 10^?7M and 1.9 × 10^?6M. Trypsin inhibition by black gram trypsin inhibitor was of a linear-mixed type. Chemical modification studies suggested the possible involvement of lysine and arginine at the active site of the inhibitor.     

THE EFFECT OF pH ON RAPESEED GLOBULIN (CRUCIFERIN) BINDING TO ANILINONAPHTHALENE-8-SULFONATE

The fluorescence emission intensity from rapeseed globulin (cruciferin) increased in the presence of anilinonaphthalene-8-sulfonate (ANS) at pH 2.0 but not at pH 10. Fluorescence titration studies showed that at pH 7 cruciferin binds 22 (±0.6) moles of ANS per mole of protein with an average dissociation constant (K_d) of 1.9 (±0.1) × 10^?5 M. At pH 2.0 the number of ligand binding sites (n) decreased to 14 (±0.2) moles of ANS bound per mole of cruciferin. However, the ANS binding affinity increased by about five times (K_d= 3.6 (±1.1) × 10^?6 M). The fluorescence emission spectrum maxima (Λ_max) for the cruciferin-ANS complex showed a blue shift at pH 2 when compared to Λ_max values at pH 7–10. These results are consistent with a loss of the quaternary and tertiary structures of cruciferin and the exposure of surface hydrophobic ANS binding sites at low pH. Cruciferin-ANS binding parameters at pH 10 were not significantly different from values at pH 7; n = 22 and K_d= 2.7 (±0.2) × 10^?5 M. Based on these ANS fluorescence measurements cruciferin is stable under alkaline conditions.     

METABOLIC QUOTIENTS OF RESPIRATION-DEFICIENT MUTANTS OF BREWER'S YEAST AND THE APPEARANCE OF A NEGATIVE PASTEUR EFFECT

Four industrial strains of bottom brewer's yeast and a group of their spontaneous respiration deficient (RD) mutants were tested for rates of metabolism of glucose and maltose under aerobic and anaerobic conditions. Q_o2 of all the RD mutants tested (26 isolates) ranged from 1·9 to 6·8 μl/mg yeast dry weight on glucose and was lowered to about one-half on maltose although the original strains had the same Q_o2 values on both sugars. No isolate showed any increase in glucose fermentation in aerobic conditions as compared with the original strains and the decrease of Pasteur effect found in certain isolates was always accompanied by a strong decrease of glycolysis in both aerobic and anaerobic conditions. Two mutants showed a strongly negative Pasteur effect, for their fermentation rates were higher in aerobic conditions than in anaerobic ones. Two other mutants showed a strong negative Pasteur effect only on maltose. The ratio of Q^N_2CO2 on glucose in most mutants was significantly lower than in their parental strains.     

Water Diffusion from a Bacterial Cell in Low‐Moisture Foods

We used a Fick's unsteady state diffusion equation to estimate the time required for a single spherical shaped bacterium (assuming Enterococcus faecium as the target microorganism) in low‐moisture foods to equilibrate with the environment. We generated water sorption isotherms of freeze‐dried E. faecium. The water activity of bacterial cells at given water content increased considerably as temperature increased from 20 to 80 °C, as observed in the sorption isotherms of bacterial cells. When the water vapor diffusion coefficient was assumed as between 10^?12 and 10^?10 m²/s for bacterial cells, the predicted equilibration times (t_eq) ranged from 8.24×10^?4 to 8.24×10^?2 s. Considering a cell membrane barrier with a lower water diffusion coefficient (10^?15 m²/s) around the bacterial cell with a water diffusion coefficient of 10^?12 m²/s, the t_eq predicted using COMSOL Multiphysics program was 3.8×10^?1 s. This result suggests that a single bacterium equilibrates rapidly (within seconds) with change in environmental humidity and temperature.     

Biochemical similarity of Schizosaccharomyces pombe ras1 protein with RAS2 protein of Saccharomyces cerevisiae

Schizosaccharomyces pombe contains single ras oncogene homologue, ras1, that functions in the signal transduction pathway conducting the cell's mating processes. To understand the biochemical basis of yeast ras proteins, we have purified the ras1 protein and compared the major biochemical constants with those of RAS2 protein from Saccharomyces cerevisiae and mammalian ras proteins. The purified ras1 protein showed a remarkably high K_d value for GDP binding (178 nM) and for binding with ATP. In contrast, the K_d value for GTP binding and the rate of GTPase activity were 64 nM and 77 × 10^?6 s^?1 at 37°C, respectively; both were higher than normal p21^ras protein, but at the same level as the RAS2 protein. We directly measured rate of GTP binding and GDP binding which were 3.9 × 10^?3 s^?1 and 1.8 × 10^?3 s^?1 at 30°C, respectively. On the other hand, exchange rates between bound and free nucleotides remained almost constant throughout the tested combination of GTP and GDP, and were several-fold lower than the binding rate. These results suggest that the release of the guanine nucleotide is the rate-limiting step in the ras–GTP/GDP cycle. As a whole, the biochemical properties of the ras1 protein are close to those of the RAS2 protein, although these two proteins function differently in the signal transduction pathway in the cells.     

Characterization of the Saccharomyces bayanus‐type AGT1 transporter of lager yeast

Transport of maltose and maltotriose into the yeast cell is thought to be rate‐limiting in the utilization of these sugars. The maltose and maltotriose transporters Malx1, Agt1, Mtt1 and Mphx are present in different combinations in brewer's yeast strains, conferring different maltose and maltotriose transport characteristics on the strains. A new putative maltose/maltotriose transporter ORF was identified during whole genome sequencing of the lager strain WS34/70 (Y. Nakao et al., DNA Res., 2009, 16, 115–129). Sequence comparisons suggested that this putative α‐glucoside transporter might be a Saccharomyces bayanus counterpart of the Agt1 (Saccharomyces cerevisiae type) transporter. In the present work, the transporter coded by a SbAGT1 gene from a lager strain, A15 (and with the same sequence as the corresponding gene in WS34/70) was characterized. It is shown that this SbAGT1 encodes a functional α‐glucoside transporter with a wide‐substrate range, including maltose and maltotriose. Trehalose, α‐methylglucoside and sucrose were inhibitors, suggesting they are also substrates. The SbAgt1 transporter had similar affinities for maltose and maltotriose (17 ± 7 and 22 ± 2 m m , respectively) and a higher V_max for maltose than maltotriose (21 ± 7 and 12 ± 2 µmol min^?1 g dry yeast^?1, respectively). Copyright © 2012 The Institute of Brewing & Distilling     

Characterization of a glycerol/H+ symport in the halotolerant yeast Pichia sorbitophila

Pichia sorbitophila is a halotolerant yeast capable of surviving to extracellular NaCl concentrations up to 4 M in mineral medium when glucose or glycerol are the only carbon and energy sources. Evidence is presented here that glycerol, the main compatible solute this yeast accumulates so as to maintain osmotic balance, is actively co-transported with protons. This transport system was shown to be constitutive, not needing induction by either glycerol or salt, and was not repressible by glucose. In glucose- or glycerol-grown cells, a simple diffusion was detectable, and iterative calculations were performed to calculate kinetic parameters, in the presence and in the absence of NaCl. At 25°C, pH 5·0, in glucose-grown cells these were: K_m = 0·81 ± 0·11 mM and V_max = 634·2 ± 164·8 μmol h^?1 per g (glycerol); K_m = 1·28 ± 0·60 mM and V_max = 558·6 · 100·6 μmol h^?1 per g (protons). Correspondent stoichiometry was approximately 1, either for these conditions or in the presence of 1 M -NaCl. An increase in acumulation capacity was evident when different concentrations of NaCl were present. This capacity was shown to be dependent on ΔpH and membrane potential, consistently with an electrogenic character. We suggest that the main role of this system is in osmoregulation, by keeping glycerol accumulated inside the cells, compensating for leakage, due to its liposoluble character.     

Hardaliye: fermented grape juice as a traditional Turkish beverage

Twenty-six aged hardaliye samples, collected from different spots of the Kirklareli province of Turkey and hardaliye produced in laboratory conditions using the traditional method were investigated in this study. The pH ranged from 3·21 to 3·97. Red colour (Hunter Lab a_Lvalue) of samples ranged from 1·33 to 9·66. The total bacterial count ranged from 3·5×10²to 8×10⁵cfu ml⁻¹. The lactic acid bacteria counts of the samples were found to be between 1·0×10²and 4·0×10⁴cfu ml⁻¹. Yeasts and moulds, which were found in 21 samples out of 26, ranged from 1·0×10²to 8·1×10⁴cfu ml⁻¹. Coliforms and Escherichia coli were found in none of the samples. The changes of some microbiological and chemical properties of hardaliye during fermentation were investigated. The pH of hardaliye dropped from 3·86 to 3·39. The ethanol content of the end product was determined as 595·50 mg dl⁻¹. During the fermentation process, the total bacteria count, lactic acid bacteria count and yeast count changed from 2·1×10⁵, 6·0×10⁴and 1·2×10⁵cfu ml⁻¹to 1·3×10², 1·2×10³and 1·1×10³cfu ml⁻¹, respectively.Lactobacilli isolated from hardaliye samples were characterized by API 50 CH and other phenotypic criteria. A succession of Lactobacillus species, dominated by L. paracasei subsp. paracasei and L. casei subsp. pseudoplantarum were found during the fermentation process.