Acetic Acid Assay Kit (ACS Manual Format)

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.

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.

Summary: High acetylation of angiosperm wood hinders its conversion to sugars by glycoside hydrolases, subsequent ethanol fermentation and (hence) its use for biofuel production. We studied the REDUCED WALL ACETYLATION (RWA) gene family of the hardwood model Populus to evaluate its potential for improving saccharification. The family has two clades, AB and CD, containing two genes each. All four genes are expressed in developing wood but only RWA-A and -B are activated by master switches of the secondary cell wall PtNST1 and PtMYB21. Histochemical analysis of promoter::GUS lines in hybrid aspen (Populus tremula × tremuloides) showed activation of RWA-A and -B promoters in the secondary wall formation zone, while RWA-C and -D promoter activity was diffuse. Ectopic downregulation of either clade reduced wood xylan and xyloglucan acetylation. Suppressing both clades simultaneously using the wood-specific promoter reduced wood acetylation by 25% and decreased acetylation at position 2 of Xylp in the dimethyl sulfoxide-extracted xylan. This did not affect plant growth but decreased xylose and increased glucose contents in the noncellulosic monosaccharide fraction, and increased glucose and xylose yields of wood enzymatic hydrolysis without pretreatment. Both RWA clades regulate wood xylan acetylation in aspen and are promising targets to improve wood saccharification.

Grape sour (bunch) rot is a polymicrobial disease of vineyards that causes millions of dollars in lost revenue per year due to decreased quality of grapes and resultant wine. The disease is associated with damaged berries infected with a community of acetic acid bacteria, yeasts, and filamentous fungi that results in rotting berries with high amounts of undesirable volatile acidity. Many insect species cause the initial grape berry damage that can lead to this disease, but most studies have focused on the role of fruit flies in facilitating symptoms and vectoring the microorganisms of this disease complex. Like fruit flies, social wasps are abundant in vineyards where they feed on ripe berries and cause significant damage, while also dispersing yeasts involved in wine fermentation. Despite this, their possible role in disease facilitation and dispersal of grape rots has not been explored. We tested the hypothesis that the paper wasp Polistes dominulus could facilitate grape sour rot in the absence of other insect vectors. Using marker gene sequencing we characterized the bacterial and fungal community of wild-caught adults. We used a sterilized foraging arena to determine if these wasps transfer viable microorganisms when foraging. We then tested if wasps harboring their native microbial community, or those inoculated with sour rot, had an effect on grape sour rot incidence and severity using a laboratory foraging arena. We found that all wasps harbor some portion of the sour rot microbial community and that they have the ability to transfer viable microorganisms when foraging. Foraging by inoculated and uninoculated wasps led to an increase in berry rot disease symptom severity and incidence. Our results indicate that paper wasps can facilitate sour rot diseases in the absence of other vectors and that the mechanism of this facilitation may include both increasing host susceptibility and transmitting these microbial communities to the grapes. Social wasps are understudied but relevant players in the sour rot ecology of vineyards.

Microalgae have emerged as potentially powerful platforms for the production of recombinant proteins and high-value products. Chlamydomonas reinhardtii is a potentially important host species due to the range of genetic tools that have been developed for this unicellular green alga. Transformation of the chloroplast genome offers important advantages over nuclear transformation, and a wide range of recombinant proteins have now been expressed in the chloroplasts of C. reinhardtii strains. This is often done in cell wall-deficient mutants that are easier to transform. However, only a single study has reported growth data for C. reinhardtii grown at pilot scale, and the growth of cell wall-deficient strains has not been reported at all. Here, we report the first pilot-scale growth study for transgenic, cell wall-deficient C. reinhardtii strains. Strains expressing a cytochrome P450 (CYP79A1) or bifunctional diterpene synthase (cis-abienol synthase, TPS4) were grown for 7 days under mixotrophic conditions in a Tris-acetate-phosphate medium. The strains reached dry cell weights of 0.3 g/L within 3–4 days with stable expression levels of the recombinant proteins during the whole upscaling process. The strains proved to be generally robust, despite the cell wall-deficient phenotype, but grew poorly under phototrophic conditions. The data indicate that cell wall-deficient strains may be highly amenable for transformation and suitable for commercial-scale operations under mixotrophic growth regimes.

Xylan is one of the major polymers in lignocellulosic biomass and about 60% of its xylosyl residues are acetylated at O-2 and/or O-3. Because acetylation of cell wall polymers contributes to biomass recalcitrance for biofuel production, it is important to investigate the biochemical mechanism underlying xylan acetylation, the knowledge of which could be applied to custom-design biomass composition tailored for biofuel production. In this report, we investigated the functions of Arabidopsis TRICHOME BIREFRINGENCE-LIKE 34 (TBL34) and TBL35, two DUF231-containing proteins, in xylan acetylation. The TBL34 gene was found to be specifically expressed in xylem cells in stems and root-hypocotyls, and both TBL34 and TBL35 were shown to be localized in the Golgi, where xylan biosynthesis occurs. Chemical analysis revealed that simultaneous mutations of TBL34 and TBL35 caused a mild decrease in xylan acetyl content and a specific reduction in xylan 3-O-monoacetylation and 2,3-di-O-acetylation. Furthermore, simultaneous mutations of TBL34, TBL35 and ESKIMO1 (ESK1) resulted in severely collapsed xylem vessels with altered secondary wall structure, and an extremely retarded plant growth. These findings indicate that TBL34 and TBL35 are putative acetyltransferases required for xylan 3-O-monoacetylation and 2,3-di-O-acetylation and that xylan acetylation is essential for normal secondary wall deposition and plant growth.

The necessity of amylolytic enzymes to convert starch into glucose during ethanol production is considered one of the cost increasing factors for corn ethanol. Enzyme consumption could be decreased partially by increasing free sugar contents in corn kernels that will be released and fermented simultaneously with the product of starch hydrolysis, producing an additional amount of ethanol without consuming any enzyme. The present work was conducted to evaluate the effects of grain sugar on the fermentable sugar and ethanol yields as well as enzyme requirement using four high sugary corn genotypes (HSGs) and their parent field corn lines (PFCs). The reducing sugar yield in HSGs did not vary significantly above the enzyme load of 1.5 kg/MT of dry corn, while PFCs showed a range between 2.0 and 2.5 kg/MT. The average final ethanol concentrations in HSGs and PFCs ranged from 15.25% to 17.5% (v/v) and 11.66% to 13.65%, respectively with the enzyme load at 1.5 kg/MT, which reached to 16.49-17.94% in HSGs and 14.32-16.85% in PFCs as the enzyme load increased to 2.0 kg/MT. These results suggest that high sugar content in corn kernels has the potential for decreasing enzyme consumption during dry-grind ethanol production with higher yields.

Icewine is a sweet dessert wine made from pressing grapes naturally frozen on the vines. It is likely that freeze/thaw cycles endured by icewine grapes change their chemical and sensory profiles due to climatic events. Our objective was to determine the influence of harvest date on icewine must and wine basic chemical variables and aroma compounds. Riesling and Vidal icewines were made from grapes picked between December 2004 and February 2005; Harvest 1 (H1): 19 December; Harvest 2: 29 December; Harvest 3 (H3): 18 January; and Harvest 4 (H4): 11 February (Vidal only). Icewine musts differed in titratable acidity and pH (Vidal only). All basic wine chemical analytes differed across harvest dates. All aroma compounds differed in Vidal and Riesling wines. Highest concentrations for most aroma compounds were in the last harvest date; 16 of 24 for Vidal and 17 of 23 for Riesling. The latest harvest date had highest ethyl isobutyrate, ethyl 3-methylbutyrate, 1-hexanol, 1-octen-3-ol, 1-octanol, cis-rose oxide, nerol oxide, ethyl benzoate, ethyl phenylacetate, γ-nonalactone and β-damascenone. H1 had highest ethyl butyrate, ethyl hexanoate, linalool, 4-vinylguaiacol and ethyl octanoate. Based on odor activity values, the most odor-potent compounds were β-damascenone, cis-rose oxide, 1-octen-3-ol, ethyl octanoate, ethyl hexanoate, and 4-vinylguaiacol across harvest dates. PCA found most aroma compounds associated with the last harvest date, 4-vinylguaicol excepted, which was associated with H1. Harvest date was considered a discriminating dimension using canonical variant analysis for volatile compounds.

The current study investigates the potential to increase the activity of a family 1 carbohydrate esterase on cellulose acetate through fusion to a family 3 carbohydrate binding module (CBM). Specifically, CtCBM3 from Clostridium thermocellum was fused to the carboxyl terminus of the acetyl xylan esterase (AnAXE) from Aspergillus nidulans, and active forms of both AnAXE and AnAXE-CtCBM3 were produced in Pichia pastoris. CtCBM3 fusion had negligible impact on the thermostability or regioselectivity of AnAXE; activities towards acetylated corncob xylan, 4-methylumbelliferyl acetate, p-nitrophenyl acetate, and cellobiose octaacetate were also unchanged. By contrast, the activity of AnAXE-CtCBM3 on cellulose acetate increased by two to four times over 24 h, with greater differences observed at earlier time points. Binding studies using microcrystalline cellulose (Avicel) and a commercial source of cellulose acetate confirmed functional production of the CtCBM3 domain; affinity gel electrophoresis using acetylated xylan also verified the selectivity of CtCBM3 binding to cellulose. Notably, gains in enzyme activity on cellulose acetate appeared to exceed gains in substrate binding, suggesting that fusion to CtCBM3 increases functional associations between the enzyme and insoluble, high molecular weight cellulosic substrates.

To remain competitive in increasingly overcrowded markets, yeast strain development programmes are crucial for fermentation-based food and beverage industries. In a winemaking context, there are many yeast phenotypes that stand to be improved. For example, winemakers endeavouring to produce sweet dessert wines wrestle with fermentation challenges particular to fermenting high-sugar juices, which can lead to elevated volatile acidity levels and extended fermentation times. In the current study, we used natural yeast breeding techniques to generate Saccharomyces spp. interspecific hybrids as a non-genetically modified (GM) strategy to introduce targeted improvements in important, wine-relevant traits. The hybrids were generated by mating a robust wine strain of Saccharomyces cerevisiae with a wine isolate of Saccharomyces bayanus, a species previously reported to produce wines with low concentrations of acetic acid. Two hybrids generated from the cross showed robust fermentation properties in high-sugar grape juice and produced botrytised Riesling wines with much lower concentrations of acetic acid relative to the industrial wine yeast parent. The hybrids also displayed suitability for icewine production when bench-marked against an industry standard icewine yeast, by delivering icewines with lower levels of acetic acid. Additionally, the hybrid yeast produced wines with novel aroma and flavour profiles and established that choice of yeast strain impacts on wine colour. These new hybrid yeasts display the desired targeted fermentation phenotypes from both parents, robust fermentation in high-sugar juice and the production of wines with low volatile acidity, thus establishing their suitability for wine styles that are traditionally troubled by excessive volatile acidity levels.

In certain circumstances the fermentation process in dry fermented sausages converts to heterofermentation pathway leading to acetic acid and carbon dioxide beside lactic acid. The study describes two cases of undesirable heterofermentation in dry sausages from two different producers. In the sausage samples (n = 7) the pH value and the content of lactic and acetic acids were measured. Microbial analysis focused on quantitative and qualitative detection of lactic acid bacteria. The acetic acid content varied from 24.28 to 67.41 µmol g^-1 dry matter, in the case of samples from the second producer the content of acetic acid (48.45 to 67.41 µmol g^-1 dry matter) was higher than the lactic acid content (20.98 to 29.02 µmol g^-1 dry matter). The lactobacilli strains from the sausages were assigned to the corresponding species by Matrix-Assisted Laser Desorption-Ionization - Time of Flight Mass Spectrometry (MALDI-TOF MS) and classified to three groups according to the sugar fermentation pattern (obligately homofermentative, facultatively heterofermentative and obligately heterofermentative) and they caused the heterofermentation process in the samples of dry fermented sausages. The description of the case of heterofermentation process in dry sausages is unique and there is little information about this topic.

Mass spectrometric analysis was used to compare the roles of two acetyl esterases (AE, carbohydrate esterase family CE16) and three acetyl xylan esterases (AXE, families CE1 and CE5) in deacetylation of natural substrates, neutral (linear) and 4-O-methyl glucuronic acid (MeGlcA) substituted xylooligosaccharides (XOS). AEs were similarly restricted in their action and apparently removed in most cases only one acetyl group from the non-reducing end of XOS, acting as exo-deacetylases. In contrast, AXEs completely deacetylated longer neutral XOS but had difficulties with the shorter ones. Complete deacetylation of neutral XOS was obtained after the combined action of AEs and AXEs. MeGlcA substituents partially restricted the action of both types of esterases and the remaining acidic XOS were mainly substituted with one MeGlcA and one acetyl group, supposedly on the same xylopyranosyl residue. These resisting structures were degraded to great extent only after inclusion of α-glucuronidase, which acted with the esterases in a synergistic manner. When used together with xylan backbone degrading endoxylanase and β-xylosidase, both AE and AXE enhanced the hydrolysis of complex XOS equally.

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.

Carboxylated, anionic polysaccharides were selectively prepared using a combination of enzymatic and chemical reactions. The galactose-containing polysaccharides studied were spruce galactoglucomannan, guar galactomannan, and tamarind galactoxyloglucan. The galactosyl units of the polysaccharides were first oxidized with galactose oxidase (EC 1.1.3.9) and then selectively carboxylated, resulting in the galacturonic acid derivatives with good conversion and yield. The degrees of oxidation (DO) of the products were determined by gas chromatography–mass spectrometry (GC-MS). A novel feasible electrospray ionization-mass spectrometry (ESI-MS) method was also developed for the determination of DO. The solution properties and charge densities of the products were investigated. The interaction of the products with cellulose was studied by two methods, bulk sorption onto bleached birch kraft pulp and adsorption onto nanocellulose ultrathin films by quartz crystal microbalance with dissipation (QCM-D). To study the effect of the location of the carboxylic acid groups on the physicochemical properties, polysaccharides were also oxidized by 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated reaction producing polyuronic acids. The chemo-enzymatically oxidized galacturonic polysaccharides with an unmodified backbone had a better ability to interact with cellulose than the TEMPO-oxidized products. The selectively carboxylated polysaccharides can be further exploited, as such, or in the targeted functionalization of cellulose surfaces.

2,5-Dimethyl-3-methoxypyrazine (DMMP) has been recently identified in both Coccinellidae-tainted (by either Coccinella septempunctata or Harmonia axyridis beetles) and untainted wines; however, little is known regarding its impact on wine aroma and flavor. The aims of this study were to obtain an accurate estimate of both the ortho- and retronasal detection thresholds of DMMP in red wine and to understand how DMMP contributes to the aroma profile of red wine. In the first study, thresholds were determined for 21 individuals using the ASTM E679 ascending forced choice method of limits. The orthonasal group best estimate threshold (BET) was 31 ng/L and the retronasal group BET was 70 ng/L. A moderate variation in individual thresholds was observed for the orthonasal modality [standard deviation (SD) = 19.8] and a larger variation was noted for retronasal thresholds (SD = 111.8). In the second study, a panel of 8 assessors performed descriptive sensory analysis on 3 red wines containing various concentrations of added DMMP (0, 50 and 120 ng/L). Results show significant changes in aroma characteristics in the 120 ng/L wine and smaller effects at the 50 ng/L level. Overall, wines spiked with DMMP generated lower intensity ratings for cherry and red berry descriptors and higher ratings for earthy/musty and green/vegetal descriptors. When considered with other recent results on DMMP concentrations found in wine, DMMP can be considered a hitherto undescribed impact odorant in some wine styles.

Various amino acids and the yeast extract, in amounts of 0.1%(w/v), were separately tested for their influence on the analytical parameters of lactic acid fermentation of cabbage juice with Bifidobacterium animalis subsp. lactis BB-12. Compared with the control, cysteine supplementation led to a decrease of the time to reach pH 5.0 of 6 times and an increase of lactic acid productivity of 1.22 times. After 48 h the ascorbic acid content was by 360.73% higher, the fermented cabbage juices being assigned into a distinct group applying both factor analysis (FA) and cluster analysis (CA). Tryptophan contributed to better values for lactic and acetic acid yield, while lysine and yeast extract especially for acetic acid yield. Valine and leucine were not able to improve the fermentation progress, estimated through the analyzed variables. This work would provide some helpful information for the development of various lacto-fermented vegetable juices using probiotic bacteria.

The potential of the waste from beer fermentation broth (WBFB) for the production of bio-ethanol using a simultaneous saccharification and fermentation process without any extra additions of saccharification enzymes, microbial cells or carbohydrate was tested. The major microbial cells in WBFB were isolated and identified. The variations in compositions of WBFB with stock time were investigated. There was residual activity of starch hydrolyzing enzymes in WBFB. The effects of reaction modes, e.g. static and shaking on bio-ethanol production were studied. After 7 days of cultivation using the supernatant of WBFB at 30°C the ethanol concentration reached 103.8 g/L in shaking culture and 91.5 g/L in static culture. Agitation experiments conducted at a temperature-profile process in which temperature was increased from 25 to 67°C shortened the simultaneous process time. The original WBFB was more useful than the supernatant of WBFB in getting the higher concentration of ethanol and reducing the fermentation time. From this whole study it was found that WBFB is a cheap and suitable source for bio-ethanol production.

A Roche 454 cDNA deep sequencing experiment was performed on a developing corm of Amorphophallus konjac — also known as voodoo lily. The dominant storage polymer in the corm of this plant is the polysaccharide glucomannan, a hemicellulose known to exist in the cell walls of higher plants and a major component of plant biomass derived from softwoods. A total of 246 mega base pairs of sequence data was obtained from which 4,513 distinct contigs were assembled. Within this voodoo lily expressed sequence tag collection genes representing the carbohydrate related pathway of glucomannan biosynthesis were identified, including sucrose metabolism, nucleotide sugar conversion pathways for the formation of activated precursors as well as a putative glucomannan synthase. In vivo expression of the putative glucomannan synthase and subsequent in vitro activity assays unambiguously demonstrate that the enzyme has indeed glucomannan mannosyl- and glucosyl transferase activities. Based on the expressed sequence tag analysis hitherto unknown pathways for the synthesis of GDP-glucose, a necessary precursor for glucomannan biosynthesis, could be proposed. Moreover, the results highlight transcriptional bottlenecks for the synthesis of this hemicellulose.

BACKGROUND: Safflower (Carthamus tinctorius L.), usually grown as a source of oil crop, can be used as fodder either for hay or ensiling purposes, particularly in semi-arid regions. RESULTS: A 2-year trial was conducted in southern Italy to evaluate the production and forage quality of safflower biomass cv. Centennial, harvested at three different stages: 1, at complete appearance of primary buds (PB); 2, at complete appearance of secondary and tertiary buds (STB); and 3, at 25% of flowering stage (FS). For each stage of growth, 50% of the biomass was ensiled in 4 L glass jars without and with inoculation (Lactobacillus plantarum, LAB), and the other 50% was field wilted for 24 h before ensiling. Dry matter (DM) content and yield (DMY), pH, buffering capacity (BC) and water soluble carbohydrates (WSC) were determined on fresh forage. On safflower silages were also evaluated ammonia-N, crude protein (CP), fibre fractions, fat, lactic and acetic acids, Ca and P, and gas losses. DMY ranged from 4.5 t ha^-1 (PB harvesting) to 11.6 t ha^-1 (FS harvesting). DM content varied from 129 g kg^-1 (PB not wilted) to 630 g kg^-1 (FS wilted). The WSC in forage before ensiling with not wilting ranged from 128 (PB stage) to 105 and 100 g kg^-1 DM at STB and FS stages, respectively. The wilted safflower forage showed a lower WSC compared to wilted forage. The high sugar substrate allowed lactic acid fermentation and a good conservation quality in all the harvesting stages. Silages quality was strongly influenced by the treatment performed. Wilting practice increased DM, pH and NDF contents but reduced lactic acid, acetic acid and NH₃-N values. Inoculation reduced DM, pH and NDF contents, but increased lactic and acetic acids, CP and ash. CONCLUSION: As result, wilting the forage for 1 day was very effective in the early harvesting stage because this practice significantly increased DM, reducing on the same time the intensive fermentation and proteolysis processes of silage. When harvesting is performed at the beginning of the flowering stage wilting is not necessary.

The influence of the bacteriocinogenic culture Lactobacillus sakei (10⁵/g) and semi-purified bacteriocin mesenterocin Y (2560 AU/kg) on the safety and quality of traditional Croatian fermented sausages was investigated. The addition of Lb. sakei and/or mesenterocin Y reduced microbial counts (P < 0.05) in the final products. After 28 days of ripening, coagulase-negative cocci decreased 1.5–2.0 log, yeasts 1.2–1.4 log and enterococci 1.7–2.7 log. In the case of the addition of Lb. sakei, the lactic acid bacteria count was significantly (P < 0.05) higher at day 7 of ripening, and was accompanied by a lower pH and a higher amount of lactic acid (P < 0.05). In the final product the amount of acetic acid was significantly lower. More intensive proteolysis and an increase in ammonia content were found at the beginning of fermentation, and in the second phase of ripening in the control samples, respectively. The free fatty acid concentration was significantly lower during the entire ripening process compared to the control (P < 0.05). Semi-purified mesenterocin Y did not affect the sensory properties of the sausages, whilst the addition of Lb. sakei enhanced them.