Grape and wine analysis: Oenologists to exploit advanced test kits.
Charnock, S. C. & McCleary, B. V. (2005). Revue des Enology, 117, 1-5.
It is without doubt that testing plays a pivotal role throughout the whole of the vinification process. To produce the best possible quality wine and to minimise process problems such as “stuck” fermentation or troublesome infections, it is now recognised that if possible testing should begin prior to harvesting of the grapes and continue through to bottling. Traditional methods of wine analysis are often expensive, time consuming, require either elaborate equipment or specialist expertise and frequently lack accuracy. However, enzymatic bio-analysis enables the accurate measurement of the vast majority of analytes of interest to the wine maker, using just one piece of apparatus, the spectrophotometer (see previous issue No. 116 for a detailed technical review). Grape juice and wine are amenable to enzymatic testing as being liquids they are homogenous, easy to manipulate, and can generally be analysed without any sample preparation.
Megazyme “advanced” wine test kits general characteristics and validation.
Charnock, S. J., McCleary, B. V., Daverede, C. & Gallant, P. (2006). Reveue des Oenologues, 120, 1-5.
Many of the enzymatic test kits are official methods of prestigious organisations such as the Association of Official Analytical Chemicals (AOAC) and the American Association of Cereal Chemists (AACC) in response to the interest from oenologists. Megazyme decided to use its long history of enzymatic bio-analysis to make a significant contribution to the wine industry, by the development of a range of advanced enzymatic test kits. This task has now been successfully completed through the strategic and comprehensive process of identifying limitations of existing enzymatic bio-analysis test kits where they occurred, and then using advanced techniques, such as molecular biology (photo 1), to rapidly overcome them. Novel test kits have also been developed for analytes of emerging interest to the oenologist, such as yeast available nitrogen (YAN; see pages 2-3 of issue 117 article), or where previously enzymes were simply either not available, or were too expensive to employ, such as for D-mannitol analysis.
Lactose fermentation by Kombucha – a process to obtain new milk–based beverages.
Iličić, M., Kanurić, K., Milanović, S., Lončar, E., Djurić, M. & Malbaša, R. (2012). Romanian Biotechnological Letters, 17(1), 7013-7021.
This paper focuses on fermentation of lactose from a model system (black tea) and from two types of milk (0.9% w/w and 2.2% w/w of fat) by application of Kombucha. Quantities of the applied Kombucha starter were 10% v/v and 15% v/v. All fermentations were performed at 42°C. The process to achieve a desirable pH=4.5 was slower in the model system (16 h) than in milks (9 - 10 h). Regarding starter quantity, 10% v/v proved the optimal. Regarding types of milk, higher fat content guarantees shorter fermentation and higher yield of metabolites. Utilization of lactose was found at a level of ≈20% and ≈30% in milks with 0.9% w/w and 2.2% w/w of fat, respectively. This was correlated with an appearance of intermediates and/or products. Glucose underwent further transformations almost entirely, while galactose showed much lower reactivity. Seven to twelve times higher contents of lactic acid were found compared to acetic acid. Milk-based beverage from the reduced fat sample, inoculated with 10% v/v of Kombucha starter, has the best physical characteristics (syneresis and water holding capacity). It also developed a good texture (especially cohesiveness and index of viscosity). Milk lactose fermentation was a process that could have been used for obtaining new milk-based products.
Changes in the volatile compound production of fermentations made from musts with increasing grape content.
Keyzers, R. A. & Boss, P. K. (2010). Journal of Agricultural and Food Chemistry, 58(2), 1153-1164.
Wine is a complex consumer product produced predominately by the action of yeast upon grape juice. Model must systems have proven to be ideal for studies into the effects of fermentation conditions on the production of certain wine volatiles. To clarify the contribution of grape juice to the production of wine volatiles, we have employed a model must system spiked with increasing amounts of grape juice (Riesling or Cabernet Sauvignon). The resulting fermented wines were analyzed by SPME-GC-MS and the data obtained grouped using ANOVA and cluster analyses to reveal those compounds that varied in concentration with reproducible trends relative to juice concentration. Such grouping highlights those compounds that are grape-dependent or for which production is modulated by grape composition. In some cases, increasing the proportion of grape juice in the fermentations stimulated the production of certain esters to levels between 2- and 140-fold higher than those seen in fermentations made with model grape juice media alone. The identification of the grape components responsible for the increased production of these wine volatiles will have implications for the impact of grape production and enology on wine flavor and aroma.
Metabolic engineering of Saccharomyces cerevisiae to minimize the production of ethyl carbamate in wine.
Coulon, J., Husnik, J. I., Inglis, D. L., van der Merwe, G. K., Lonvaud, A., Erasmus, D. J. & van Vuuren, H. J. J. (2006). American Journal of Enology and Viticulture, 57(2), 113-124.
Saccharomyces cerevisiae metabolizes arginine, one of the major amino acids in grape musts, to ornithine and urea during wine fermentations. Wine yeast strains of S. cerevisiae do not fully metabolize urea during grape must fermentation. Urea is secreted by yeast cells and it reacts spontaneously with ethanol in wine to form ethyl carbamate, a potential carcinogenic agent for humans. The lack of urea catabolism by yeast in wine may be ascribed to the transcriptional repression of the DUR1,2 gene by good nitrogen sources present in the grape must. We expressed the DUR1,2 gene under control of the S. cerevisiae PGK1 promoter and terminator signals and integrated this DUR1,2 expression cassette, flanked by ura3 sequences, into the URA3-locus of the industrial wine yeast UC Davis 522. In vivo assays showed that the metabolically engineered industrial strain reduced ethyl carbamate in Chardonnay wine by 89.1%. Analyses of the genotype, phenotype, and transcriptome revealed that the engineered yeast 522EC− is substantially equivalent to the parental 522 strain.
Proso millet (Panicum miliaceum L.) fermentation for fuel ethanol production.
Rose, D. J. & Santra, D. (2013). Industrial Crops and Products, 43, 602-605.
The objective of this research was to determine the conversion efficiency of proso millet to ethanol compared to corn in a bench-scale dry-grind procedure. Seven proso millet cultivars and six advanced breeding lines containing waxy starch were fermented with Saccharomyces cerevisiae and ethanol production was compared with normal corn and “highly fermentable” corn. The highly fermentable corn exhibited the highest fermentation efficiency (97.0 ± 1.4%). Among proso millet lines, those with the highest fermentation efficiencies were: Huntsman (85.9 ± 0.6%), 172-2-9 (90.8 ± 0.2%), 172-2-13 (85.1 ± 2.5%), and 182-4-24 (84.7 ± 2.1). Waxy proso millet lines resulted in higher fermentation efficiencies than the non-waxy proso millet varieties containing normal starch (82.4 ± 5.5% vs. 75.5 ± 7.4%, respectively, p = 0.01). Proso millet distiller's dried grains with solubles (DDGS) contained more protein (26.6–33.4%) than the DDGS from corn (17.2–23.4%). These data indicate that proso millet exhibits promise as a feedstock for ethanol production, especially if breeding programs focus on selecting “highly fermentable” lines for advancement.
Biosynthesis of ethanol and hydrogen by glycerol fermentation using Escherichia coli.
Chaudhary, N., Ngadi, M. O., Simpson, B. K. & Kassama, L. S. (2011). Advances in Chemical Engineering and Science, 1, 83-89.
Production of high value products from glycerol via anaerobic fermentation is of utmost importance for the biodiesel industry. The microorganism Escherichia coli (E. coli) K12 was used for fermentation of glycerol. The effects of glycerol concentration and headspace conditions on the cell growth, ethanol and hydrogen production were investigated. A full factorial experimental design with 3 replicates was conducted in order to test these factors. Under the three headspace conditions tested, the increase of glycerol concentration accelerated glycerol fermentation. The yields of hydrogen and ethanol were the lowest when glycerol concentration of 10 g/L was used. The maximum production of hydrogen was observed with an initial glycerol concentration of 25 g/L at a final concentration of hydrogen was 32.15 mmol/L. This study demonstrated that hydrogen production negatively affects cell growth. Maximum ethanol yield was obtained with a glycerol concentration of 10 g/L and was up to 0.40 g/g glycerol under membrane condition headspace. Statistical optimization showed that optimal conditions for hydrogen production are 20 g/L initial glycerol with initial sparging of the reactor headspace. The optimal conditions for ethanol production are 10 g/L initial glycerol with membrane.
Quantification of full range ethanol concentrations by using pH sensor.
Al-Mhanna, N. M. M. & Huebner, H. (2011). International Journal of Chemistry, 3(1), 47-56.
A differential pH measurement device was used to achieve operation conditions of alcohol dehydrogenase reaction. Optimum operating conditions were temperature of 30°C, 10 μl of alcohol dehydrogenase enzyme volume (with a final activity of 563.75 units ml-1) per 50 μl of sample, NAD+ concentration of 0.05 mM and 20 mM glycine-pyrophosphate buffer solution of pH 9.1. In this method a range of ethanol concentrations from 0-99,985%, which means 0.000001714 - 17.14 M, were used. The maximum obtained change in pH, delta pH, was (-33) mpH. A calibration curve of logarithmic values of ethanol concentrations against change in pH for standard ethanol samples was done. Since this calibration curve is a linear with a correlation coefficient (R) of 0.998, this calibration curve can be used in quantification of ethanol concentration. End point of equilibrium concentrations of reactants and products of ethanol oxidation reaction was measured within spectrophotometer. The results indicated 100 seconds of process time is required to reach the end point for all ethanol standard samples. This required time was satisfied with results of measuring change in pH within differential pH analyzer system.
Screening Thermo- and Ethanol Tolerant Bacteria for Ethanol Fermentation.
Dung, N. T. P. & Huynh, P. X. (2013). American Journal of Microbiological Research, 1(2), 25-31.
The thermophilic bacteria receive considerably interest nowadays because of a current challenge of increasing global temperature. Particularly for ethanol production, the thermo-ethanologenic bacteria possess advantages due to lower contamination risk, cost saving in industrial scale, and the wide range of sugars utilization. In this study, 13 bacterial isolates obtained from the previous isolation study were tested for their fermentative capacity and ethanol tolerance at high temperatures. Five bacterial isolates HM2, M2, MC3, MR1 and RD were found to be tolerant up to 12% ethanol. Of which HM2, M2 and MR1 could ferment glucose well at 30, 35 and 40°C, particularly isolates HM2 and MR1 could perform the fermentative capacity at 45°C and even 50°C. In the presence of 12, 16, and 20% w/v glucose, isolates HM2, M2, and MR1 showed the high fermentation rate by giving high gas production; however, the rate slightly decreased in the presence of 24% w/v glucose. The fermentative performance by these three isolates could happen at different pH levels of 4.0, 5.0 and 6.0. The favourable conditions of ethanol fermentation were found at 18.5% glucose, pH 5.0, and 33°C for isolate HM2 and at 14% glucose, pH 5.5, and 40°C for isolate MR1. The results of sequencing analysis of partial 16S rRNA gene showed that the gene sequences of the selected isolate HM2 shared 99% similarity with Bacillus subtilis.
Microbiological and chemical properties of kefir manufactured by entrapped microorganisms isolated from kefir grains.
Chen, T. H., Wang, S. Y., Chen, K. N., Liu, J. R. & Chen, M. J. (2009). Journal of Dairy Science, 92(7), 3002-3013.
In this study, various yeasts (Kluyveromyces marxianus, Saccharomyces turicensis, Pichia fermentans) and lactic acid bacteria (Lactobacillus kefiranofaciens, Lactobacillus kefiri, Leuconostoc mesenteroides) were entrapped in 2 different microspheres using an entrapment ratio for the strains that was based on the distribution ratio of these organisms in kefir grains. The purpose of this study was to develop a new technique to produce kefir using immobilized starter cultures isolated from kefir grains. An increase in cell counts with fermentation cycles was observed for both the lactic acid bacteria (LAB) and yeasts, whereas the cell counts of kefir grains were very stable during cultivation. Scanning electron microscopy showed that the short-chain lactobacilli and lactococci occupied the surface of the LAB microspheres, whereas the long-chain lactobacilli were inside the microspheres. When the yeasts were analyzed, cells at a high density were entrapped in cracks on the surface and within the microspheres, where they were surrounded by the short-chain lactobacilli. The distribution of the LAB and yeast species in kefir produced from grains and microspheres showed that there was no significant difference between the kefirs produced by the 2 methods; moreover, Leu. mesenteroides and K. marxianus were the predominating microflora in both types of kefir. There was no significant difference in the ethanol and exopolysaccharide contents between the 2 kefirs, although the acidity was different.
Taraxerone enhances alcohol oxidation via increases of alcohol dehyderogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activities and gene expressions.
Sung, C. K., Kim, S. M., Oh, C. J., Yang, S. A., Han, B. H. & Mo, E. K. (2012). Food and Chemical Toxicology, 50(7), 2508-2514.
The present study, taraxerone (D-friedoolean-14-en-3-one) was isolated from Sedum sarmentosum with purity 96.383%, and its enhancing effects on alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activities were determined: EC50 values were 512.42 ± 3.12 and 500.16 ± 3.23 μM for ADH and ALDH, respectively. In order to obtain more information on taraxerone related with the alcohol metabolism, 40% ethanol (5 mL/kg body weight) with 0.5–1 mM of taraxerone were administered to mice. The plasma alcohol and acetaldehyde concentrations of taraxerone-treated groups were significantly lowered than those of the control group (p < 0.01): approximately 20–67% and 7–57% lowered for plasma alcohol and acetaldehyde, respectively. Compare to the control group, the ADH and ALDH expressions in the liver tissues were abruptly increased in the taraxerone-treated groups after ethanol exposure. In addition, taraxerone prevented catalase, superoxide dismutase, and reduced glutathione concentrations from the decrease induced by ethanol administration with the concentration dependent manner.
Fermentation of high concentrations of maltose by Saccharomyces cerevisiae is limited by the COMPASS methylation complex.
Houghton-Larsen, J. & Brandt, A. Applied and Environmental Microbiology, 72(11), 7176-7182.
In Saccharomyces cerevisiae, genes encoding maltose permeases and maltases are located in the telomeric regions of different chromosomes. The COMPASS methylation complex, which methylates lysine 4 on histone H3, controls the silencing of telomeric regions. Yeast strains deleted for SWD1, SWD3, SDC1, SET1, BRE2, or SPP1, encoding components of the COMPASS complex, fermented a medium containing 22% maltose with noticeably higher attenuation than did the wild type, resulting in production of up to 29% more ethanol. The least effective strain was spp1. Absence of COMPASS components had no effect on the fermentation of media with 20% glucose, 20% sucrose, or 16% maltose. Deletion of SWD3 resulted in larger amounts of MAL12 transcript, encoding maltase, at the late stages of fermentation of 22% maltose. A similar effect on maltase activity and maltose uptake capability was seen. The lysine 4 residue of histone H3 was trimethylated in wild-type cells at the late stages, while only small amounts of the dimethylated form were detected. Trimethylation and dimethylation of this residue were not detected in strains deleted for SWD1, SWD3, SET1, BRE2, or SDC1. Trimethylated lysine 4 was apparent only at the early stages (48 and 96 h) of fermentation in an spp1 strain. This work indicates that the COMPASS complex represses the expression of maltose utilization genes during the late stages of fermentation of a high concentration of maltose.