Ammonia Assay Kit (Rapid)

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.

The enology industry in North Dakota is extremely young, with less than twenty years of existence. At times throughout the development of the North Dakota viticulture and enology industries, commercial wine producers have elected to purchase or store fresh harvested grapes as frozen musts. To investigate the fermentation outcomes related to skin contact for red grapevine musts, a postfreeze fermentation experiment was conducted with fruit from ‘Frontenac’, one of the most widely grown red grapevines in the Upper Midwest U.S. and North Dakota. Four fermentation treatments were applied to frozen ‘Frontenac’ grapevine musts: steam juice extraction, rosé, 1 day after inoculation (DAI) skin contact, and 9 DAI skin contact. Samples were collected daily for ten days and analyzed for fermentation progress and spectrophotometric monitoring of wine color attributes and total phenolics. The final wines were analyzed two years after bottling. Steam-extracted musts were initially darkest; however, they were lighter as final wines than the 9 DAI wines and similar to rosé wines in lightness. Total phenolics were greatest for 9 DAI wines and total red pigments were lowest for steam-extracted wines. While differences between treatments were detected, the wines remained visually similar; this indicates that color extraction within the freeze–thaw processes of musts may obliterate subtly and make it difficult to produce wines of light color when stored under these conditions. Continued work with additional grapevines beyond ‘Frontenac’ may help fine-tune must and fermentation extraction procedures for small-scale wineries growing cold-hardy grapevines.

Pseudozyma hubeiensis is a basidiomycete yeast that has the highly desirable traits for lignocellulose valorisation of being equally efficient at utilization of glucose and xylose, and capable of their co-utilization. The species has previously mainly been studied for its capacity to produce secreted biosurfactants in the form of mannosylerythritol lipids, but it is also an oleaginous species capable of accumulating high levels of triacylglycerol storage lipids during nutrient starvation. In this study, we aimed to further characterize the oleaginous nature of P. hubeiensis by evaluating metabolism and gene expression responses during storage lipid formation conditions with glucose or xylose as a carbon source. The genome of the recently isolated P. hubeiensis BOT-O strain was sequenced using MinION long-read sequencing and resulted in the most contiguous P. hubeiensis assembly to date with 18.95 Mb in 31 contigs. Using transcriptome data as experimental support, we generated the first mRNA-supported P. hubeiensis genome annotation and identified 6540 genes. 80% of the predicted genes were assigned functional annotations based on protein homology to other yeasts. Based on the annotation, key metabolic pathways in BOT-O were reconstructed, including pathways for storage lipids, mannosylerythritol lipids and xylose assimilation. BOT-O was confirmed to consume glucose and xylose at equal rates, but during mixed glucose-xylose cultivation glucose was found to be taken up faster. Differential expression analysis revealed that only a total of 122 genes were significantly differentially expressed at a cut-off of |log₂ fold change| ≥ 2 when comparing cultivation on xylose with glucose, during exponential growth and during nitrogen-starvation. Of these 122 genes, a core-set of 24 genes was identified that were differentially expressed at all time points. Nitrogen-starvation resulted in a larger transcriptional effect, with a total of 1179 genes with significant expression changes at the designated fold change cut-off compared with exponential growth on either glucose or xylose.

The present study aimed to compare the nutrient composition, in vitro ruminal fermentation values and microbiome in the ruminal fermentation of herbage and silage of the Plantago media, P. major and P. lanceolata species. The lactic acid (LA) content of P. lanceolata silage was higher than those of other plantago silages (p < 0.05). The α-linolenic, w-3, polyunsaturated (PUFA), medium chain (MCFA) and long-chain fatty acids (LCFA) of plantago silages were lower than those of plantago herbages (p < 0.05). The neutral detergent fibre (NDF) and acid detergent fibre (ADF) contents, total gas and methane production, metabolic energy (ME) and organic matter digestion (OMD) values and ammonia-nitrogen concentration in the in vitro fermentation fluid of P. major silage were lower than those of other plantago silages (p < 0.05). The in vitro ruminal methane production and community of archaea Methanobrevibacter in the microbiome of P. major herbage were higher than that of P. media and P. lanceolata herbages. The ensiling process significantly increased the in vitro total gas production, acetic acid concentration and Prevotellaceae bacteria of P. media and P. lanceolate compared their herbages. As a result, P. lanceolata and P. media silages provided high-quality silage fermentation; the nutrient composition was not lost to a great extent in the silage environment and the ruminal fibrolytic bacterial composition was positively affected. Plantago major silage could not provide a good silage quality and the in vitro ruminal fermentation and ruminal fibrolytic bacteria community value were negatively affected.

White wine fermentations are typically performed in an entirely batchwise manner, with yeast nutrients only added at the beginning of fermentation. This leads to slow (2+ weeks) fermentation cycle times, with large capital expenditures required to increase winery processing capacity. Prior attempts to speed fermentations via increasing temperature have resulted in unpalatable wine, and continuous fermentation processing is uneconomical and impractical in the winery setting. In this work, we measured yeast nutrient consumption as a function of fermentation progression at the 300 mL scale, and from this derived an equation to optimize yeast nutrient concentration as a function of fermentation progression. These findings were applied at the pilot scale in 150 L fermentors, which resulted in a 60% cycle time reduction versus “best practices” control fermentations. The resultant wines were compared via GC-MS as well as by a trained sensory panel. Organoleptic analysis found statistically significant, but overall, small differences in sensory characteristics between the control and process intensified wines. This intensified fermentation process shows great promise for fermented beverage producers wishing to maximize equipment utilization and debottleneck wineries or other beverage fermentation facilities.

Certain yeast species belonging to the Pichia genus are known to form a distinctive film on grape must and wine. In a mixed-culture type fermentation, Pichia spp. (P. kluyveri in particular) are known to impart beneficial oenological attributes. In this study, we report on an easy isolation method of Pichia spp. from grape must by exploiting their film-forming capacity on media containing 10% ethanol. We isolated and identified two Pichia species, namely Pichia kudriavzevii and Pichia kluyveri, and subsequently co-inoculated them with Saccharomyces cerevisiae to ferment Gewürztraminer musts. Noteworthy differences included a significant increase in the 2-phenethyl acetate levels with the P. kluyveri co-fermentation and a general increase in ethyl esters with the P. kudriavzevii co-fermentation. Both Pichia co-inoculations yielded higher levels of glycerol in the final wines. Based on all the wine parameters we tested, the P. kluyveri strain that was isolated performed similarly to a commercial P. kluyveri strain.

Background: Enzymatic hydrolysis of lignocellulosic biomass into platform sugars can be enhanced by the addition of accessory enzymes, such as xylanases. Lignin from steam pretreated biomasses is known to inhibit enzymes by non-productively binding enzymes and limiting access to cellulose. The effect of enzymatically isolated lignin on the hydrolysis of xylan by four glycoside hydrolase (GH) family 11 xylanases was studied. Two xylanases from the mesophilic Trichoderma reesei, TrXyn1, TrXyn2, and two forms of a thermostable metagenomic xylanase Xyl40 were compared. Results: Lignin isolated from steam pretreated spruce decreased the hydrolysis yields of xylan for all the xylanases at 40 and 50 °C. At elevated hydrolysis temperature of 50 °C, the least thermostable xylanase TrXyn1 was most inhibited by lignin and the most thermostable xylanase, the catalytic domain (CD) of Xyl40, was least inhibited by lignin. Enzyme activity and binding to lignin were studied after incubation of the xylanases with lignin for up to 24 h at 40 °C. All the studied xylanases bound to lignin, but the thermostable xylanases retained 22–39% of activity on the lignin surface for 24 h, whereas the mesophilic T. reesei xylanases become inactive. Removing of N-glycans from the catalytic domain of Xyl40 increased lignin inhibition in hydrolysis of xylan when compared to the glycosylated form. By comparing the 3D structures of these xylanases, features contributing to the increased thermal stability of Xyl40 were identified. Conclusions: High thermal stability of xylanases Xyl40 and Xyl40-CD enabled the enzymes to remain partially active on the lignin surface. N-glycosylation of the catalytic domain of Xyl40 increased the lignin tolerance of the enzyme. Thermostability of Xyl40 was most likely contributed by a disulphide bond and salt bridge in the N-terminal and α-helix regions.

Changes in the nutritive value of forages are imminent under different ensiling conditions. Thus, a study was conducted to assess the effects of ensiling length and storage temperature on the nutritive value, fermentation characteristics and fibre-bound protein of three tropical forage legumes, sorghum and mixtures of sorghum and the legumes. Soybean (Glycine max), jack bean (Cannavalia ensiformis), lablab (Lablab purpureus) and sorghum (Sorghum bicolor) were solely grown and harvested, and the legumes were wilted before ensiling. Mixtures of sorghum and each legume were handmade on a percentage fresh weight basis of 60:40. Each forage and mixtures (400 g) were ensiled in polythene vacuum bags with homofermentative lactic acid bacteria inoculation for 30, 75 and 180 days. A set of mini silos were stored indoors, and another batch was stored outdoors. HOBO Pro v2 data loggers were deployed to monitor the ambient temperature of the storage locations during the entire ensiling period (from day 0-180). Measurements included nutrient analysis, fermentation quality and fibre bound protein characteristics. The hourly ambient temperature for outdoor and indoor storage ranged from 16° to 61°C vs 18-35°C, respectively. Proximate constituents of all silages were influenced by ensiling length. Significant changes were primarily detected in fermentation products of legume silages between 30 and 75 d of ensiling. There were reduced fermentation products for silages stored outdoors. The ensiling length influenced proportions of neutral detergent insoluble nitrogen (NDIN) and acid detergent insoluble nitrogen (ADIN) with outdoor silages resulting in a higher proportion of NDIN and ADIN compared to indoor silages. Overall, a short period of ensiling preserves the nutritional quality of ensiled forages compared to prolonged storage at high ambient temperatures typical of the tropics that increase nutrient losses. Thus, changes in the nutritional composition of forages during ensiling should be considered during ration formulations.

Agricultural productivity relies on synthetic nitrogen fertilizers, yet half of that reactive nitrogen is lost to the environment. There is an urgent need for alternative nitrogen solutions to reduce the water pollution, ozone depletion, atmospheric particulate formation, and global greenhouse gas emissions associated with synthetic nitrogen fertilizer use. One such solution is biological nitrogen fixation (BNF), a component of the complex natural nitrogen cycle. BNF application to commercial agriculture is currently limited by fertilizer use and plant type. This paper describes the identification, development, and deployment of the first microbial product optimized using synthetic biology tools to enable BNF for corn (Zea mays) in fertilized fields, demonstrating the successful, safe commercialization of root-associated diazotrophs and realizing the potential of BNF to replace and reduce synthetic nitrogen fertilizer use in production agriculture. Derived from a wild nitrogen-fixing microbe isolated from agricultural soils, Klebsiella variicola 137-1036 (“Kv137-1036”) retains the capacity of the parent strain to colonize corn roots while increasing nitrogen fixation activity 122-fold in nitrogen-rich environments. This technical milestone was then commercialized in less than half of the time of a traditional biological product, with robust biosafety evaluations and product formulations contributing to consumer confidence and ease of use. Tested in multi-year, multi-site field trial experiments throughout the U.S. Corn Belt, fields grown with Kv137-1036 exhibited both higher yields (0.35 ± 0.092 t/ha ± SE or 5.2 ± 1.4 bushels/acre ± SE) and reduced within-field yield variance by 25% in 2018 and 8% in 2019 compared to fields fertilized with synthetic nitrogen fertilizers alone. These results demonstrate the capacity of a broad-acre BNF product to fix nitrogen for corn in field conditions with reliable agronomic benefits.