Indonesian tapé ketan fermentation

Indonesian tapé ketan is a fermentation in which a mold, Amylomyces rouxii Calmette (Chlamydomucor oryzae Went and Prinsen Geerligs), in combination with one or more yeasts such as Endomycopsis burtonii converts steamed rice to a sweet-sour, slightly alcoholic paste. A study was made to determine the biochemical changes that occur in the substrate during fermentation. It was found that the product was ready for consumption after fermentation at 30 degrees C for 36 to 48 h. A. rouxii used about 30% of the total rice solids, resulting in a crude protein of 12% in 96 h, whereas the combination of the mold with E. burtonii reduced total solids by 50% in 192 h, causing crude protein to increase to 16.5%. Soluble solids increased from 5 to about 67% in 36 h and decreased to 12% at 192 h with A. rouxii alone, whereas soluble solids fell to about 8% at 192 h in the fermentation with both the mold and the yeast. The mold, by itself, reduced the starch content of the rice from 78 to 10% in 48 h and to less than 2% in 144 h. The mold plus yeast reduced the starch content to about 18% in 48 h; however the "starch" content did not fall below 6% even at 192 h, presumably because the yeast was producing glycogen, which was determined along with the residual starch. With both the mold and the mold plus yeast fermentations, reducing sugars increased from less than 1% to approximately 5% in 24 h and reached maximum concentration, 16 to 17%, between 36 and 48 h. A. rouxii by itself produced a maximum of about 5.6% (vol/vol) ethanol at 96 h. The highest concentration of ethanol (8%, vol/vol) was produced by the mold plus E. burtonii at 144 h. The mold by itself reduced the starting pH from 6.3 to about 4.0 in 48 h. The combination of the mold and yeast reduced the pH to 4.1 in 144 h. The mold increased total acidity to approximately 6.2 meq of H per 100 ml, and the combination of the mold and yeast increased the total acidity to 7.8 meq of H per 100 ml in 192 h. At 48 h there was practically no difference in the volatile acidity (0.20) for the combined fermentation compared with 0.26 meq of H per 100 ml for the mold fermentation. The mold and at least one species of yeast were required to develop the rich aroma and flavor of typical Indonesian tapé.     

Production of α-linolenic acid in Yarrowia lipolytica using low-temperature fermentation

α-Linolenic acid (ALA) is an essential ω-3 fatty with reported health benefits. However, this molecule is naturally found in plants such as flaxseed and canola which currently limits production. Here, we demonstrate the potential to sustainably produce ALA using the oleaginous yeast Yarrowia lipolytica. Through the use of a recently identified Δ12–15 desaturase (Rk Δ12–15), we were able to enable production in Y. lipolytica. When combined with a previously engineered lipid-overproducing strain with high precursor availability, further improvements of ALA production were achieved. Finally, the cultivation of this strain at lower temperatures significantly increased ALA content, with cells fermented at 20 °C accumulating nearly 30% ALA of the total lipids in this cell. This low-temperature fermentation represents improved ALA titer up to 3.2-fold compared to standard growth conditions. Scale-up into a fed-batch bioreactor produced ALA at 1.4 g/L, representing the highest published titer of this ω-3 fatty acid in a yeast host.

     

Production of extracellular proteinases—protein C activators of blood plasma—by the micromycete Aspergillus ochraceus during submerged and solid-state fermentation

The conditions for the submerged and solid-state fermentation of the micromycete Aspergillus ochraceus VKM F-4104D, producing extracellular proteinases that activate protein C of human blood plasma, were optimized. It is shown that the protein C-activating activity of the micromycete in a solid-state culture was 1.5-3.5 times higher than in a submerged culture (as calculated per milliliter of culture medium). Among the extracellular proteins secreted by A. ochraceus VKM F-4104D during submerged and solid-state fermentation, a protein C-activating proteinase with a pI of 6.0–6.3 was identified.     

Liquefaction of glutinous rice and aroma formation in tapé preparation by ragi

Ragi is an Indonesian inoculum to prepare a fermented food called tapé. Sweet tapé with high aroma was produced by inoculation of ragi on steamed glutinous rice and incubation at 30°C for 3 d. Microorganisms found in ragi were identified as Rhizopus sp., Saccharomycopsis sp., and Streptococcus sp. Steamed glutinous rice was liquefied and saccharified by the amylases produced by Rhizopus and aroma was formed by a mixed culture of Rhizopus and Saccharomycopsis. Liquefaction was not caused by the amylases of Saccharomycopsis even though it produced high activity of α-amylase. A higher level of aroma was formed by inoculation of Streptococcus in a mixed culture of Rhizopus and Saccharomycopsis sp.     

Stability during fermentation of a recombinant α-amylase plasmid in Bacillus subtilis

Summary We have studied the stability during fermentation of a hybrid plasmid carrying a Bacillus -amylase gene in Bacillus subtilis. In the absence of antibiotic selection plasmid loss was associated largely with the post-exponential phases of growth and decline. In fermentations containing selective antibiotics, various deleted plasmids were recovered during late stationary phase, regardless of whether the host was rec ⁺ or recE. We therefore propose that the plasmid loss observed during late growth in antibiotic-free fermentations is due to deletion events which include the origin of plasmid replication. The structure of the deleted plasmids was determined and the sequences in the vicinity of the end-points analysed. When the deleted plasmids were subjected to further fermentations in the absence of selective antibiotics, they were completely stable.     

Microbiological changes in mawè during natural fermentation

Lactic acid bacteria increased from 3.2 × 10⁶ and 1.6 × 10⁷ c.f.u./g (wet wt) to 2 × 10⁹ and 1.6 × 10⁹ c.f.u./g after 12 to 24 h of fermentation of home-produced mawè (a dough produced from dehulled maize) and commercial mawè, respectively. In commercial mawè, the yeast count increased from 1.3 × 10⁵ to 2.5 × 10⁷ c.f.u./g after 48 h of fermentation before decreasing, whereas in the home-produced mawè it increased from 2.5 × 10⁴ to 3.2 × 10⁷ c.f.u./g after 72 h of fermentation; the dominant yeasts were mainly Candida krusei, although C. kefyr, C. glabrata and Saccharomyces cerevisiae were also present. Enterobacteriaceae counts increased slightly during the initial stage ofthe fermentation, but decreased below the detection level after 24 to 48 h. Enterobacter cloacae was mostly found in commercial mawè and Escherichia coli mostly in homeproduced mawè.D.J. Hounhouigan and C.M. Nago are with the Université Nationale du Bénin, Faculté des Sciences Agronomiques, Département de Nutrition et de Sciences Alimentaires, BP 526, Cotonou, Benin; M.J.R. Nout and F.M. Rombouts are with the Agricultural University, Department of Food Science, Bomenweg 2, 6703 HD Wageningen, The Netherlands. J.H. Houben is with Utrecht University, Department of the Science of Foods of Animal Origin, Yalelaan 2, 3508 TD Utrecht, The Netherlands.     

Regulation of acetohydroxy acid synthase in Corynebacterium glutamicum during fermentation of α-ketobutyrate to l-isoleucine

Summary Corynebacterium glutamicum ATCC 13 032 produces 13 g/l l-isoleucine from 200 mM -ketobutyrate as a synthetic precursor. In fed batch cultures up to 19 g/l l-isoleucine is formed. For optimal conversion the addition of 0.3 mM l-valine plus 0.3 mM l-leucine to the fermentation medium is required. The affinity constants for the acetohydroxy acid synthase (AHAS) were determined. (This enzyme directs the flow of -ketobutyrate plus pyruvate towards l-isoleucine and that of two moles of pyruvate to l-valine and l-leucine, respectively.) For -ketobutyrate the K _m is 4.8×10^-3 M, and V _max 0.58 U/mg, for pyruvate the K _m is 8.4×10^-3 M, and V _max 0.37 U/mg. Due to these characteristics the presence of high -ketobutyrate concentrations apparently results in a l-valine, l-leucine deficiency. This in turn leads to a derepression of the AHAS synthesis from 0.03 U/mg to 0.29 U/mg and high l-isoleucine production is favoured. The derepression of the AHAS synthesis induced by the l-valine, l-leucine shortage was directly proven with a l-valine, l-leucine, l-isoleucine auxotrophic mutant where the starvation of each amino acid resulted in an increased AHAS level. This is in accordance with the fact that only one AHAS enzyme could be verified by chromatographic and electrophoretic separations as being responsible for the synthesis of all three branched-chain amino-acids.     

Production of 11α-hydroxysteroids from sterols in a single fermentation step by Mycolicibacterium smegmatis

11α-hydroxylated steroid synthons are one of the most important commercially pharmaceutical intermediates used for the production of contraceptive drugs and glucocorticoids. These compounds are currently produced by biotransformation using fungal strains in two sequential fermentation steps. In this work, we have developed by a rational design new recombinant bacteria able to produce 11α-hydroxylated synthons in a single fermentation step using cholesterol (CHO) or phytosterols (PHYTO) as feedstock. We have designed a synthetic operon expressing the 11α-hydroxylating enzymes from the fungus Rhizopus oryzae that was cloned into engineered mutant strains of Mycolicibacterium smegmatis that were previously created to produce 4-androstene-3,17-dione (AD), 1,4-androstadiene-3,17-dione (ADD) from sterols. The introduction of the fungal synthetic operon in these modified bacterial chassis has allowed producing for the first time 11αOH-AD and 11αOH-ADD with high yields directly from sterols in a single fermentation step. Remarkably, the enzymes of sterol catabolic pathway from M. smegmatis recognized the 11α-hydroxylated intermediates as alternative substrates and were able to efficiently funnel sterols to the desired hydroxylated end-products.     

Gas chromatographic–mass spectrometric analysis of urinary sugar and sugar alcohols during pregnancy

A refined and simplified method has been developed for the simultaneous analysis of urinary sugar and sugar alcohols after urease treatment by using capillary gas chromatography–mass spectrometry (GC–MS). Since carbohydrate metabolism during pregnancy is considered to be diabetogenic, our interest has been concentrated on understanding the mechanism of the metabolic deviation by assessing the glucose excursion and glucose fluxes. The present study suggests that changes of the levels of glucose, sorbitol, fructose, myo-inositol, and 1,5-anhydro-D-glucitol (1,5-AG) may reflect a mild alteration in carbohydrate metabolism that goes undetected by conventional diabetic indicators.     

Regulation of Nitric Oxide Production by δ-Opioid Receptors during Glaucomatous Injury

To determine the roles of nitric oxide in glaucomatous injury and its regulation by δ-opioid-receptor activation, animals were treated with: 1) a selective inducible nitric oxide synthase (iNOS) inhibitor (aminoguanidine; AG; 25 mg/kg, i.p.); 2) δ-opioid-receptor agonist (SNC-121; 1 mg/kg, i.p.); or 3) with both drugs simultaneously for 7 days, once daily. The loss in retinal ganglion cell (RGC) numbers and their function in glaucomatous eyes were significantly improved in the presence of AG or SNC-121; however, we did not see any significant additive or synergistic effects when animals were treated with both drugs simultaneously. The levels of nitrate-nitrite were significantly increased in the glaucomatous retina when compared with normal retina (normal retina 86±9 vs. glaucomatous retina 174±10 mM/mg protein), which was reduced significantly when animals were treated either with SNC-121 (121±7 mM/mg protein; P<0.05) or AG (128±10 mM/mg protein; P<0.05). Additionally, SNC-121-mediated reduction in nitrate-nitrite levels was not only blocked by naltrindole (a δ-opioid-receptor antagonist), but naltrindole treatment potentiated the nitrate-nitrite production in glaucomatous retina (235±4 mM/mg protein; P<0.001). As expected, naltrindole treatment also fully-blocked SNC-121-mediated retina neuroprotection. The nitrotyrosine level in the glaucomatous retina was also increased, which was significantly reduced in the SNC-121-treated animals. Additionally, the expression level of iNOS was clearly increased over the control levels in the glaucomatous retina and optic nerves, which was also reduced by SNC-121 treatment. In conclusion, our data support the notion that nitric oxide plays a detrimental role during glaucomatous injury and inhibition of nitric oxide production provided RGC neuroprotection. Furthermore, δ-opioid receptor activation regulates the production of nitric oxide via inhibiting the activity of iNOS in the retina and optic nerve.     

Production and characterization of recombinant 9 and 15 kDa granulysin by fed-batch fermentation in Pichia pastoris

Granulysin is a cytolytic, proinflammatory protein produced by human cytolytic T-lymphocytes and natural killer cells. Granulysin has two stable isoforms with molecular weight of 9 and 15 kDa; the 9-kDa form is a result of proteolytic maturation of the 15-kDa precursor. Recombinant 9-kDa granulysin exhibits cytolytic activity against a variety of microbes, such as bacteria, parasites, fungi, yeast and a variety of tumor cell lines. However, it is difficult to produce granulysin in large quantities by traditional methods. In this study, we developed a simple and robust fed-batch fermentation process for production and purification of recombinant 9- and 15-kDa granulysin using Pichia pastoris in a basal salt medium at high cell density. The granulysin yield reaches at least 100 mg/l in fermentation, and over 95 % purity was achieved with common His-select affinity and ion exchange chromatography. Functional analysis revealed that the yeast-expressed granulysin displayed dose-dependent target cytotoxicity. These results suggest that fermentation in P. pastoris provides a sound strategy for large-scale recombinant granulysin production that may be used in clinical applications and basic research.     

Production of β-carotene from deproteinized waste whey filtrate using Mucor azygosporus MTCC 414 in submerged fermentation

The cheese whey, a by-product of dairy industry proved to be an attractive substrate for production of β-carotene. The β-carotene production from Mucor azygosporus MTCC 414 by using deproteinized waste whey filtrate under submerged fermentation was investigated. Various fermentation variables, such as lactose content in whey, initial pH, production temperature, incubation time, and carbon and nitrogen sources played significant role on β-carotene production. Maximum β-carotene production (385 μg/g dcw) was obtained with the whey (pH 5.5) containing 3.5% (w/v) lactose supplemented with soluble starch at (1.0%, w/v) at 30°C after a 5 days incubation. Moreover, unlike other microorganisms which utilize pre-hydrolyzed lactose, this Mucor azygosporus MTCC 414 was found to be capable of utilizing unhydrolyzed lactose present in the whey.     

Correlations between reduction–oxidation potential profiles and growth patterns of Saccharomyces cerevisiae during very-high-gravity fermentation

When Saccharomyces cerevisiae was grown under three glucose concentrations (ca. 200, 250, and 300 g/l), controlled at three reduction–oxidation (redox) potentials (no control, −150 and −100 mV) by manipulating two aerations (0.82 and 1.3 vvm), we observed that the recorded redox potential profiles resembled bathtub curves, and the profiles correlated well to the growth patterns measured under the same conditions. According to the shape of bathtub curve, we subdivided the curve into four regions. Region I features an abrupt decline in redox potential (corresponding to the growth phase from lag and logarithmic to the onset of stationary phase) that correlates to rapid yeast propagation, resulting from fast glucose uptake. Region II (corresponding to the stationary phase in yeast growth, characterizes a constant level of redox potential) is maintained by proper sparging and constant agitation. The continual buildup of ethanol causes growth arrest of yeast, resulting in the reduction of net NADH production. As a result, an uprising of redox potential is the feature of Region III, which signifies the end of stationary phase followed by the commencement of death phase. The severity of growth environment due to ethanol toxicity results in a rapid decrease in yeast population. Region IV (corresponding to the death phase during yeast growth) characterizes a drastic reduction in yeast viability and a gradual leveling of redox potential. A low glucose feed correlates to a fast decline of redox potential, a small basin in the bathtub curve, short fermentation duration, and complete glucose utilization. Imposing the current redox potential settings to low glucose feeds exerts no appreciable effect on ethanol production. In contrast, a high glucose feed connects to a sluggish bathtub curve for all four regions and incomplete glucose utilization. Proximate analysis on carbon balance indicates that controlling redox potential at −150 mV and under ca. 250 and 300 g glucose/l conditions, gave the highest fermentation efficiency as compared to other conditions; but there were no beneficiary effect to control redox potential under ca. 200 g glucose/l conditions.     

Two-dimensional protein map of an “ale”-brewing yeast strain: proteome dynamics during fermentation

The first protein map of an ale-fermenting yeast is presented in this paper: 205 spots corresponding to 133 different proteins were identified. Comparison of the proteome of this ale strain with a lager brewing yeast and the Saccharomyces cerevisiae strain S288c confirmed that this ale strain is much closer to S288c than the lager strain at the proteome level. The dynamics of the ale-brewing yeast proteome during production-scale fermentation was analysed at the beginning and end of the first and the third usage of the yeast (called generation in the brewing industry). During the first generation, most changes were related to the switch from aerobic propagation to anaerobic fermentation. Fewer changes were observed during the third generation but certain stress-response proteins such as Hsp26p, Ssa4p and Pnc1p exhibited constitutive expression in subsequent generations. The ale brewing yeast strain appears to be quite well adapted to fermentation conditions and stresses.     

Extracellular enzyme activities during cassava fermentation for ‘fufu’ production

Amylase and pectin methyl esterase activities increased rapidly during the early period of the fermentation of cassava for fufu production, attaining their peak activities after 12 and 24h, respectively. Cellulase activity was lower and approximately constant for most of the fermentation period.     

Comments on the “Maintenance coefficient” changes during alcohol fermentation

Summary The concept of maintenance is discussed in terms of the biological meaning and the applicability of the maintenance coefficient, m, in bioengineering for optimization of yields in fermentation. A method of calculation is proposed for the evaluation of m in the course of fermentation in the case of a metabolite (e.g, ethanol). During alcoholic fermentation m is not constant and decreases with the growth rate.The phenomena involved in maintenance are numerous and complex and there is a semantic problem in its definition which can be generalized by the apparently non-finalized substrate consumption.Nomenclature a specific maintenance rate (defined by eq. (9)) - m maintenance coefficient - X cell mass concentration (as a dry weight) - S substrate concentration - P product concentration - r rate of reaction - r_x,r_s,r_p rate of reaction related to biomass, substrate and product - r_sm,r_sg,r_spi rate of reaction related only to the consumption by maintenance, growth and the synthesis of the ith product - r_xe maintenance rate defined by eq. (10) - q_s,q_Pi specific rate of substrate consumption and i^th product production - Y yield coefficient - Y^o, Y^o apparent yield coefficient related to the cell and i^th product - Y _xs ^xs , Y _Pis ^Pis maximum theoretical yield coefficient related to the cell and i^th - specific growth rate produce 420     

Production of poly(β-hydroxybutyrate) by Comamonas testosteroni during growth on naphthalene

Comamonas testosteroni has been found to produce poly(-hydroxybutyrate) (PHB) during its growth on naphthalene. Fourier transform infrared spectroscopy (FTIR) and ¹³C nuclear magnetic resonance (NMR) analysis confirmed it as a homopolymer of 3-hydroxybutyrate. Oxygen and essential nutrient limitation other than carbon source play a major role in maximum PHB production. Nitrogen limitation was found to have a profound effect, with 0.2 g ammonium nitrate/l optimum for PHB production. Both aeration and iron were found to be essential for growth and PHB accumulation. Ferric chloride at 0.04 g/l concentration was found to be optimum for PHB accumulation. Phosphate source variation showed no significant effect. Using naphthalene as a sole carbon source in optimized Bushnell Haas medium, 85% of the dry cell mass was extracted as chloroform-soluble PHB.     

Production and characterization of partially purified extracellular thermostable α-amylase by Bacillus subtilis in submerged fermentation (SmF)

A Bacillus strain was isolated from soil samples from the campus area of Dicle University. Based on 16S ribosomal RNA sequencing, the microorganism was closely related to Bacillus subtilis. Effects of different culture medium, incubation time, carbon and nitrogen sources, and various starches, flours, and chemicals on α-amylase production were examined. Maximum enzyme production (7516 U/mL) was obtained in a basal medium A containing 0.05% Tween 40 in 24 h. Partially purified enzyme showed maximum activity at 60 °C with an optimum pH of 6.0. The effects of 0.2% detergents (sodium dodecyl sulfate [SDS], CHAPS [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate], and commercial detergent Omo Matic) on partially purified enzyme activity over a period of time (15-150 min) were examined and the order of inhibition effect from the most to the least was found as SDS > Omo Matic > CHAPS. Different metal ions inhibited α-amylase activity at low concentrations (1.5 mM). Co2? was a mild inhibitor and Hg2? and Cd2? were potent inhibitors, whereas Ca2? and Mg2? increased the enzyme activity. At 20 mM, Ca2? enhanced enzyme activity, and different Ca2? concentrations (10-300 mM) were studied.