Primary Amino Nitrogen Assay Kit (PANOPA)

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.

‘King of the North’ (‘KON’), as a cold-hardy grape, has many advantages, such as tolerance to a wide range of soil conditions and harsh winter climate. Due to the adapting demand of North Dakota’s fruit and wine industry, optimized quality of wine from regionally productive grapevines is required. In this study, yeast strain, one of the primary fermentation tactics, was tested on ‘KON’ grapes. Five different commercial yeast strains, including 71B, EC1118, Maurivin B, Rhône 4600, and W15, were added to initiate fermentation. The analysis of grape must characteristics and the fermentation dynamic changes indicated a high correlation between color and acid metrics. Yeast strains have influenced the color dynamic changes and fermentation process. The panelist sensory evaluations confirmed that yeast strains contributed differently to the perceived aromas and flavors within ‘KON’ wines. Rose, apple, grape, and apricot aromas were distinguished in ‘KON’ wines. The lemon taste was the dominant flavor detected in ‘KON’ wines. However, wines were also varied based on the extent of the aroma or taste observed. Therefore, exploring the use of different yeast strains for fermentation provides information for further application to cold-hardy grape cultivars and other high-acid fruit, aiding winemakers in using North American grapes with diverse fruit chemistry.

In recent years, non-Saccharomyces yeasts, including Metschnikowia pulcherrima, have gained renewed interest in biotechnology applied to the wine industry due to their pro-technological properties. M. pulcherrima has been particularly studied for its antimicrobial activity, its potential to produce wines with a low alcohol content, and its enzymatic activities that enhance some sensory characteristics of wines, especially when used in co-culture with Saccharomyces cerevisiae. This study evaluated the production of Falanghina wine using M. pulcherrima AS3C1 as an initial starter, followed by sequential inoculation with a commercial S. cerevisiae after 2 and 4 days. Moreover, a parallel vinification was performed using S. cerevisiae as a single starter culture. Volatile Organic Compounds (VOCs) data, obtained through Headspace Solid phase Microextraction couple to Gas Chromatography-Mass Spectrometry (HS-SPME/GC–MS), were analyzed using multivariate statistical methods (PCA and sPLS-DA). The results showed significant differences in the VOC profiles of the wines produced. Notably, the wine obtained through sequential inoculation of M. pulcherrima AS3C1 followed by S. cerevisiae after 4 days received the highest overall sensory evaluation by the panel test. This wine was distinguished by its elevated levels of ethyl acetate, phenylethyl acetate, benzeneethanol and 2-methylpropanol.

The high protein content in cassava leaves offers a promising alternative nitrogen source. Cassava leaves enriched of glutamic acid and aspartic acid which are valuable factors for microbial metabolisms. Biobutanol exhibits superior properties compared to bioethanol, making it an excellent candidate for biofuel applications, and it can be a potential chemical building block that produced from agricultural waste. However, the current production yield remains relatively low. The objective of this study was to enhance biobutanol production using three different nitrogen sources: yeast extract, peptone, and cassava leaf extract (CLE) with Clostridium beijerinckii TISTR 1461. Considering the equivalent amino nitrogen content among the nitrogen sources, the conditions employing CLE and sugarcane bagasse hydrolysate resulted in the maximum biobutanol concentration of 12.0 ± 0.4 g/L within a short 42-h period. This result reflects a 200% increase compared to the control and a 130% increase over yeast extract, achieved 24 h earlier. The acetone-butanol-ethanol (ABE) productivity, ABE yield, and butanol productivity were recorded at 0.45 g/L·h, 0.55 g/g, and 0.29 g/L·h, respectively. These findings suggest that CLE is a sustainable nitrogen source with substantial potential to boost biobutanol production. Additionally, it promotes the utilization of agricultural waste, thereby contributing to a more sustainable biofuel production process.

The Avaferm® automatic yeast nutrition system was tested in comparison to manual nutrition management during the fermentation of white wines. To evaluate the two approaches, a factorial experimental design with interaction was established consisting of the two ways of nutrition (Avaferm® and Control) and two white grape varieties (Catarratto and Chardonnay). The experiments were conducted on an industrial scale, ensuring the practical relevance of the findings. The parameters that underwent evaluation encompassed a wide range of aspects, including the fermentation kinetics of sugars, the use of α-amino nitrogen and ammonium, as well as the technological and aromatic volatile composition of the wines. To study the sugar consumption kinetics during fermentation, a new use of the 5-parameter logistic regression was applied to fit these compounds. The automatic nutrition showed a substantial similarity in the fermentative kinetics and in all cases, we have reached adequate sugar depletion (almost no residual fermentable sugars). Moreover, with regards to the composition of the wines, the two methods were found to be similar, although with a slightly higher concentration of residual YAN in the control nitrogen management. The wines obtained with Avaferm® showed a greater concentration in 3-oxo-α-ionol and a slightly lesser concentration in butanoic hexanoic, octanoic and decanoic acids as well as ethyl hexanoate, ethyl octanoate and ethyl decanoate. The variety factor as expected showed several differences, which can be explained by the chemical composition of the different grape musts.

Deuterated biomolecules such as proteins, lipids, and DNA are widely used in neutron scattering experiments. This is due to the unique scattering properties of ²H, including a strong positive neutron scattering length while contributing very little background compared to the more abundant ¹H isotope. Deuteration is therefore an indispensable component in the study of structure, function, and dynamic behaviour of biomolecules by neutron scattering. In the past we compared multiple microalgae species for their ability to grow under deuterated conditions and in our hands Botryococcus braunii proved the easiest and most resilient to long-term culturing in D₂O. In this study we describe how to culture B. braunii cells under deuterated conditions followed by preparation of an aqueous extract. The procedure is based on autolysis where cells are incubated at 50°C for 24 h and clarified by centrifugation and filtration. The product, deuterated algal autolysate, is then used in minimal media for deuterated recombinant protein production in bacteria. We demonstrate that in-house produced deuterated algal autolysate can fully substitute for glycerol-d8 in minimal media without a reduction in expressed protein yield while obtaining ~98% deuterium incorporation in the final product, suitable for neutron scattering and other types of experiments.

The effect of four enzyme preparations: bacillolysin, agroprot, protozyme and protozyme C (Russia) on solubility, emulsifying activity, emulsion stability, foaming and foam stability of isolates prepared from two varieties of peas was studied. It is shown that treatment with enzymes can increase the solubility of isolates at pH 5 by more than 7 times, the index of emulsifying activity at pH 5 by 1.5 to 2 times, and at pH 6 by almost 1.5 times; the stability index of the emulsion increased by about 20% at pH 5, and by 1.7 times (in one of the varieties) at pH 6; foaming increased by 2.4 to 3 times at pH 5, and at pH 6 by 1.8 to 3.7 times; foam stability increased by 25 to 33% at pH 5 and by more than 1.5 times (in one of the varieties) at pH 6. The results obtained made it possible to select an enzyme preparation (bacterial alkaline serine protease) to improve the parameters of pea protein isolates intended for the manufacture of analogs of fermented milk products.

This study investigated the effect of the molecular structure of pectin on conjugate formation and its functionality in plant protein-based emulsions. For this purpose, high-methoxylated, low-methoxylated, amidated citrus pectin and their mixtures with potato protein were vacuum-dried at defined process conditions. The change in free amino groups, colour and molecular weight distribution assessed conjugation. The side reaction, such as pectin degradation, was monitored by the degree of methoxylation, galacturonic acid content and intrinsic viscosity. In addition, FT-IR and hydrophobicity measurements were performed to determine the changes in chemical structure due to the occurring reactions. The ζ-potential, solubility and oil droplet size reflected the functionality of the conjugated samples. The results confirm that, apart from the conjugation of protein and pectin, where the most significant decrease in free amino groups was obtained with an increasing degree of methoxylation or presence of amide groups, undesirable degradation of pectin occurring, resulting in a broad molecular weight distribution of the conjugate samples. The conjugate formation was the highest with amidated pectin. The FT-IR measurements confirmed the formation of Maillard conjugates. The FT-IR spectra showed a decrease in the intensity signals of amide I and amide II and the formation of new C–N bond maxima. In addition, the conjugation caused the shift of the protein's isoelectric point to the acidic environment, resulting in higher solubility and improved emulsion stability at the isoelectric point of the protein. Regarding emulsion stability, the conjugates with amidated pectin performed best and achieved the narrowest oil droplet size distribution over a wider pH range. Emulsion-stabilising properties of conjugates with high- and low-methoxylated pectin differed only slightly. To better understand the underlying mechanisms for emulsion stabilisation, future research will focus on the interfacial properties of the conjugates as determined by interfacial shear rheology and pendant drop analysis.

Grape pomace is a common waste product that can be used as compost, as animal feed or discarded. The goal of this study was to evaluate the quality and consumers’ perception of a value-added grape pomace beverage, piquette, made using different red grape cultivars, yeast strains and grape pomace to water ratios. Petite Pearl and Marquette grape pomace were soaked using different pomace to water ratios in water for 2 days, prior to being pressed. Cane sugar was added to the juices prior to inoculation with three yeast strains (Cross Evolution, ICV D254, and Exotics Mosaic). The piquettes were bottled before chemical analysis and sensory evaluation by an untrained sensory panel following 8 months of storage. Piquettes made from Petite Pearl grape pomace, regardless of yeast strain, were preferred by consumers. Petite Pearl piquettes were fruity and pink, especially using D254 yeast. Piquettes made from different ratios of Petite Pearl pomace to water on a larger scale lacked nutrients at the beginning of fermentation, which led to “rotten-egg” aromas and were the least accepted by consumers.

Gluconobacter oxydans (Go) and Brettanomyces bruxellensis (Bb) are detrimental micro-organisms compromising wine quality through the production of acetic acid and undesirable aromas. Non-Saccharomyces yeasts, like Metschnikowia species, offer a bioprotective approach to control spoilage micro-organisms growth. Antagonist effects of forty-six Metschnikowia strains in a co-culture with Go or Bb in commercial grape juice were assessed. Three profiles were observed against Go: no effect, complete growth inhibition, and intermediate bioprotection. In contrast, Metschnikowia strains exhibited two profiles against Bb: no effect and moderate inhibition. These findings indicate a stronger antagonistic capacity against Go compared to Bb. Four promising Metschnikowia strains were selected and their bioprotective impact was investigated at lower temperatures in Chardonnay must. The antagonistic effect against Go was stronger at 16°C compared to 20°C, while no significant impact on Bb growth was observed. The bioprotection impact on Saccharomyces cerevisiae fermentation has been assessed. Metschnikowia strains’ presence did not affect the fermentation time, but lowered the fermentation rate of S. cerevisiae. An analysis of central carbon metabolism and volatile organic compounds revealed a strain-dependent enhancement in the production of metabolites, including glycerol, acetate esters, medium-chain fatty acids, and ethyl esters. These findings suggest Metschnikowia species’ potential for bioprotection in winemaking and wine quality through targeted strain selection.

Chitosan is a fining agent used in winemaking, although its use in juice and wine beyond fining has been limited until now. Therefore, this study's first aim was to determine if chitosan derived from Agaricus bisporus (button mushrooms) could reduce caffeic and caftaric acid concentrations in Pinot noir grape juice (Study A). The second aim was to determine if chitosan, when added to base wine, could influence the synthesis of furan-derived compounds during storage (Study B). In Study A, Pinot noir grape juice was stored at 10°C for 18 hours after the following treatments: control (no addition), bentonite/activated charcoal (BAC), low molecular weight (< 3 kDa; LMW) chitosan, med. MW (250 kDa; MMW) chitosan, and high MW (422 kDa; HMW) chitosan (all 1 g/L additions). Caftaric acid was decreased, and total amino acid concentration was increased in the LMW chitosan-treated juice, while the estimated total hydroxycinnamic acid content, turbidity, and browning were decreased in the MMW chitosan-treated juice compared to the control. In Study B, Pinot noir base wine destined for sparkling wine was stored at 15 and 30°C for 90 days with the following treatments: control (no addition), LMW chitosan, MMW chitosan, and HMW chitosan (all 1 g/L additions). The three chitosan treatments stored at 30°C had increased furfural, homofuraneol, and 5-methylfurfural formation in the base wine compared to the control. At 15°C, furfural and homofuraneol had greater concentrations in all chitosan-treated wines after 90 days of storage. Our results demonstrate the potential of mushroom-derived chitosan to remove caftaric acid from grape juice and suggest that chitosan can influence the synthesis of furan-derived compounds in wine after short-term storage.

Pinot noir grapes require careful management in the winery to prevent loss of color density and promote aging stability. Winemaking with flocculent yeast has been shown to increase color density, which is desirable to consumers. This research explored interspecies sequential inoculation and co-flocculation of commercial yeast on Pinot noir wine color. Sedimentation rates of six non-Saccharomyces species and two Saccharomyces cerevisiae strains were assayed individually and in combination. The most flocculent pairings, Torulaspora delbrueckii BIODIVA with S. cerevisiae RC212 or VL3, were used to ferment 20 L Pinot noir must. Sequential fermentations produced wines with greater color density at 420 + 520 nm, confirmed by sensory panel. Total and monomeric anthocyanin concentrations were decreased in sequentially fermented wines, despite being the main source of red wine color. BIODIVA adsorbed more anthocyanins than S. cerevisiae, indicating a greater number of cell wall mannoproteins in flocculent yeast, that could then result in a later release of anthocyanins and enhance copigment formation in red wines.

Over 3 years (2016-18), tree productivity, biennial bearing, return bloom, and fruit quality were evaluated for seven high-tannin cider apple (Malus ×domestica Borkh.) cultivars. Five treatments were evaluated on each of the seven cultivars: hand-thinned of all fruit (a zero crop load treatment); hand-thinned to crop densities of three, six, or nine fruit/cm² trunk cross-sectional area (TCSA); or left unthinned. In this paper, we report on the fruit maturity and juice quality properties that were analyzed for the three nonzero crop load treatments and the unthinned control. The effects of crop load on fruit maturity, as measured by starch pattern index and preharvest drop, were cultivar dependent. Crop density (fruit/cm² TCSA) had a significant effect on all fruit maturity and juice quality variables, although effects were weakest in the “off” year (2017) for the whole planting when initial fruit set was low. As crop density increased, total poly phenols, titratable acidity, soluble solids, and primary amino nitrogen decreased in the juice of all seven cultivars. A partial budget analysis indicated that the reduced costs of nitrogen supplements due to increased primary amino nitrogen concentration alone would not justify cost of chemical or hand-thinning. By extrapolating the spring flowering density in the fourth year to potential fruit yields at harvest, we found that reducing crop load was projected to increase cumulative total polyphenol yields per tree over the long term. For the cultivars in this experiment, a target crop density of nine fruit/cm² was found to adequately decrease biennial bearing while also not diminishing juice quality for hard cider production. High-tannin cider apple growers should consider juice quality, particularly tannin production, when making crop load management decisions.

Evidence from the literature suggests that different inoculation strategies using either active dry yeast (ADY) or freshly prepared yeast cultures affect wine yeast performance, thus altering biomass and many primary and secondary metabolites produced during fermentation. Here, we investigated how different inoculation methods changed the fermentation behaviour and metabolism of a commercial wine yeast. Using a commercial Sauvignon blanc (SB) grape juice, fermentation was carried out with two different inoculum preparation protocols using Saccharomyces cerevisiae X5: rehydration of commercial ADY and preparation of pre-inoculum in a rich laboratory medium. We also determined the effect of different numbers of yeast cells inoculation (varying from 1 × 10⁶ to 1 × 10¹²) and successive inoculation on fermentation and end-product formation. The yeast inoculation method and number of cells significantly affected the fermentation time. Principal component analysis (PCA) using 60 wine metabolites showed a separation pattern between wines produced from the two inoculation methods. Inoculation methods influenced the production of amino acids and different aroma compounds, including ethyl and acetate esters. Varietal thiols, 3-mercaptohexanol (3MH), and 4-methyl-4-mercaptopentan-2-one (4MMP) in the wines were affected by the inoculation methods and numbers of inoculated cells, while little impact was observed on 3-mercaptohexyl acetate (3MHA) production. Pathway analysis using these quantified metabolites allowed us to identify the most significant pathways, most of which were related to central carbon metabolism, particularly metabolic pathways involving nitrogen and sulphur metabolism. Altogether, these results suggest that inoculation method and number of inoculated cells should be considered in the production of different wine styles.