A comparison of free amino nitrogen and yeast assimilable nitrogen measurement methods for use in alcoholic fermentation of whey.

The continued, efficient use of whey as a fermentable substrate to produce alcoholic beverages will benefit from improving the measurement of key components relevant to the fermentability of whey. One component of fermentation that is not well studied in whey is the concentration of fermentable nitrogen, either as free amino nitrogen (FAN) or yeast assimilable nitrogen (YAN). Fermentable nitrogen in media is essential for yeast cells to replicate. Insufficient concentrations of FAN or YAN to support the growing yeast population can result in sluggish or "stuck" fermentations. Three common methods of fermentable nitrogen determination were evaluated and compared for use in whey fermentation systems, based on ninhydrin, nitrogen by o-phthalaldehyde (NOPA), and formaldehyde pH (Sørensen titration) values. Each measurement method was evaluated independently using standard addition curves and compared for their overall accuracy using paired t-tests (α = 0.05). Although the formaldehyde pH method showed high precision, it did not measure nitrogen accurately as it overestimated YAN in whey by up to 6 times relative to other tests. The ninhydrin and NOPA methods both showed accuracy for fermentable nitrogen determination in whey, based on analysis of standardized nitrogen sources. We concluded that either the ninhydrin FAN or NOPA YAN method may be used to determine the fermentable nitrogen content in whey. This is expected to improve the ability of producers to use whey as a fermentation medium.

Effect on White Grape Must of Multiflora Bee Pollen Addition during the Alcoholic Fermentation Process.

The aim of the present study was to compare and analyze the impact of using bee pollen doses (0.1, 0.25, 1, 5, 10 and 20 g/L) as activator in the alcoholic fermentation process of Palomino fino and Riesling wines. In this regard, its influence on the musts composition, the fermentative kinetics, the evolution of the populations of Saccharomyces cerevisiae, the evolution of yeast-assimilable nitrogen and physico-chemical characteristics of final wines has been analyzed. Bee pollen addition produces significant increases in yeast-assimilable nitrogen and maximum yeasts population and exponential velocity reached during alcoholic fermentation. Bee pollen showed an important effect on yeast survival during the death phase. Final wines showed significantly increase in volatile acidity above doses higher than 10 g/L and Comisión Internacional de L'Eclairage parameters (CIELab), color intensity and Abs 420 nm, from 1 g/L. Therefore, pollen could be used as fermentative activator for the alcoholic fermentation of white wines applying doses below of 1 g/L.

Optimization of carbon and nitrogen medium components for biomass production using non-Saccharomyces wine yeasts.

UNLABELLED: The impact of different nitrogen and carbon sources on biomass production of the non-Saccharomyces wine yeast species Lachancea thermotolerans, Metschnikowia pulcherrima and Issatchenkia orientalis was assessed. Using a molasses-based medium, yeast extract and corn steep liquor as well as ammonium sulphate and di-ammonium phosphate (DAP) as nitrogen sources were compared in shake-flask cultures. A medium with 20 g l⁻¹ sugar (diluted molasses) and 500 mg l⁻¹ total yeast assimilable nitrogen, from yeast extract, gave the highest biomass concentrations and yields. Invertase pretreatment was required for cultures of M. pulcherrima and I. orientalis, and respective biomass yields of 0.7 and 0.8 g g⁻¹ were achieved in aerobic bioreactor cultures. The absence of ethanol production suggested Crabtree-negative behaviour by these yeasts, whereas Crabtree-positive behaviour by L. thermotolerans resulted in ethanol and biomass concentrations of 5.5 and 11.1 g l⁻¹, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Recent studies demonstrate that non-Saccharomyces yeasts confer positive attributes to the final composition of wine. However, optimal process conditions for their biomass production have not been described, thereby limiting commercial application. In this study, industrial media and methods of yeast cultivation were investigated to develop protocols for biomass production of non-Saccharomyces yeast starter cultures for the wine industry.

Impact of phenylalanine on Hanseniaspora vineae aroma metabolism during wine fermentation.

Hanseniaspora vineae exhibits extraordinary positive oenological characteristics contributing to the aroma and texture of wines, especially by its ability to produce great concentrations of benzenoid and phenylpropanoid compounds compared with conventional Saccharomyces yeasts. Consequently, in practice, sequential inoculation of H. vineae and Saccharomyces cerevisiae allows to improve the aromatic quality of wines. In this work, we evaluated the impact on wine aroma produced by increasing the concentration of phenylalanine, the main amino acid precursor of phenylpropanoids and benzenoids. Fermentations were carried out using a Chardonnay grape juice containing 150 mg N/L yeast assimilable nitrogen. Fermentations were performed adding 60 mg/L of phenylalanine without any supplementary addition to the juice. Musts were inoculated sequentially using three different H. vineae strains isolated from Uruguayan vineyards and, after 96 h, S. cerevisiae was inoculated to complete the process. At the end of the fermentation, wine aromas were analysed by both gas chromatography-mass spectrometry and sensory evaluation through a panel of experts. Aromas derived from aromatic amino acids were differentially produced depending on the treatments. Sensory analysis revealed more floral character and greater aromatic complexity when compared with control fermentations without phenylalanine added. Moreover, fermentations performed in synthetic must with pure H. vineae revealed that even tyrosine can be used in absence of phenylalanine, and phenylalanine is not used by this yeast for the synthesis of tyrosine derivatives.

Variety and year: Two key factors on amino acids and biogenic amines content in grapes.

Amino acids have proved to play a key role in the development of volatile compounds present in wine with determining repercussions on the final wine bouquets. Biogenic amines originate from the chemical transformations of amino acids found in various foods, a phenomenon that has given rise to several health-related concerns among consumers. In the present research, the evaluation of two of the most influential factors: variety (genetic) and year (climatic conditions) on these compounds in grapes has been performed. Eight Vitis vinifera varieties have been collected during three years and the content of nineteen amino acids, two biogenic amines, and the ammonium ion has been quantified using the HPLC-PDA technique. The genetic factor has proved to be an influential variable (p-value < 0.05) with mean values of amino acids ranging from 896.89 to 1713.79 mg/L and of biogenic amines ranging from 10.61 to 22.28 mg/L. The climatic conditions have shown to be an influential factor as well (p-value < 0.05), being the low temperatures and rainfall and the high solar radiation favour the development of the amino acid and avoid biogenic amines accumulation in grapes.

Effect of inorganic and organic nitrogen supplementation on volatile components and aroma profile of cider.

To investigate the impact of inorganic and organic nitrogen on volatile compounds of cider, various levels (60, 150 and 240 mg N/L) of diammonium hydrogen phosphate (DAP) and amino acids mixture were added to apple juice before fermentation, respectively. The qualitative and quantitative of volatile substances were carried out by GC-MS. Further, sensory and electronic nose analysis was performed to analyze the aroma profile of cider. Both DAP and amino acids mixture promoted fermentation, significantly affected volatile compositions and increased perceived aromas of cider. Adding 60 mg N/L DAP significantly increased volatile compounds content, especially medium-chain fatty acid ethyl esters (MCFAEEs) and fatty acids (FAs), whereas higher alcohols and acetate esters concentrations were remarkably increased with 150 mg N/L amino acids supplementation. Partial least-squares (PLS) regression analysis suggested that MCFAEEs contributed the most to tropical fruity aroma. The study provides a theoretical foundation for high-quality cider brewing.

Co-inoculations of Lachancea thermotolerans with different Hanseniaspora spp.: Acidification, aroma, biocompatibility, and effects of nutrients in wine.

The use of non-Saccharomyces yeast in the winemaking industry and even more their co-inoculations to maximize their growth and to express phenotypic characteristic is gaining more and more relevance. This study aimed to shed light on the biocompatibilities between Lachancea thermotolerans and Hanseniaspora spp., using different types of nutrients and considering the effect on Yeast Assimilable Nitrogen (YAN), at low temperature (16 °C) and medium SO2 (50 mg/L), in white must. L. thermotolerans has been used for its positive effect on pH reduction and Hanseniaspora spp. for improving the sensory profile. The behaviour of these yeasts was evaluated in co-inoculation, always finishing the fermentation with the sequential inoculation of S. cerevisiae. Significant results were obtained on the population count (CFU/mL) in CHROMagar™, with higher populations of Hanseniaspora spp. with respect to L. thermotolerans. Fermentations with L. thermotolerans/H. vineae, showed inhibition of acidification, generating up to 0.41 g/L of lactic acid. On the contrary, a synergistic effect when L. thermotolerans/H. opuntiae was used, achieved 2.44 g/L of lactic acid and a pH reduction of up to 0.16 and always more significant with Nutrient Vit BlancTM. At the same time ethanol concentration decreased by 3.4 % and volatile acidity never exceeded 0.5 g/L. Aromatic composition was analysed and it was found that all fermentations retained more aromatic esters and that on day 7 the amount of 2-phenylethyl acetate was at least 3 times higher in all fermentations compared to the control (Sc + Nutrient Vit BlancTM) which had 5.96 mg/L. Less yellow intensity (-17.3 %) typical of oxidation were observed in all fermentations in which Nutrient Vit BlancTM had been used and in the sensory analysis the co-inoculations with H. vineae generated better scores.

Understanding the interaction of isoleucine paired with other amino acids in soy whey alcohol fermentation using Torulaspora delbrueckii.

Soy (tofu) whey is a liquid by-product generated from tofu (soybean curd) production and it is often discarded off as a waste liquid by the tofu manufacturers. Previous studies have demonstrated that soy whey can be biotransformed into a soy alcoholic beverage by using Saccharomyces and non-Saccharomyces yeasts even though soy whey is low in yeast assimilable nitrogen (YAN) content. In this study, the initial YAN of the soy whey was estimated to be 46.6 mg N/L and Torulaspora delbrueckii Biodiva was used to ferment soy whey supplemented with either isoleucine only or isoleucine paired with valine, leucine or phenylalanine (each amino acid supplemented at a dosage of 30 mg N/L). Amino acid supplementation was found to enhance sugar utilization by the yeast, which led to higher ethanol production (7.49% v/v in control versus 8.35-8.80% v/v in supplemented samples). Samples supplemented with isoleucine only experienced slower sugar utilization during the fermentation as compared to the paired amino acid samples, but the yeast was still able to utilize the sugar to low levels at the end of the fermentation. The presence of leucine supplementation counteracted the "inhibition" induced by the presence of isoleucine at the first day of the fermentation. Amino acid supplementation slowed down glutamic acid utilization and resulted in higher levels of residual glutamic acid and alanine. Amino acid supplementation increased the corresponding fusel alcohol production and the presence of other amino acids reduced the active amyl alcohol production. Therefore, interactions between amino acids can impact the metabolism of the yeast as well as the flavor modulation during soy whey fermentation.

Yeast Assimilable Nitrogen Concentrations Influence Yeast Gene Expression and Hydrogen Sulfide Production During Cider Fermentation.

The fermentation of apple juice into hard cider is a complex biochemical process that transforms sugars into alcohols by yeast, of which Saccharomyces cerevisiae is the most widely used species. Among many factors, hydrogen sulfide (H2S) production by yeast during cider fermentation is affected by yeast strain and yeast assimilable nitrogen (YAN) concentration in the apple juice. In this study, we investigated the regulatory mechanism of YAN concentration on S. cerevisiae H2S formation. Two S. cerevisiae strains, UCD522 (a H2S-producing strain) and UCD932 (a non-H2S-producing strain), were used to ferment apple juice that had Low, Intermediate, and High diammonium phosphate (DAP) supplementation. Cider samples were collected 24 and 72 h after yeast inoculation. Using RNA-Seq, differentially expressed genes (DEGs) identification and annotation, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, we found that gene expression was dependent on yeast strain, fermentation duration, H2S formation, and the interaction of these three factors. For UCD522, under the three DAP treatments, a total of 30 specific GO terms were identified. Of the 18 identified KEGG pathways, "Sulfur metabolism," "Glycine, serine and threonine metabolism," and "Biosynthesis of amino acids" were significantly enriched. Both GO and KEGG analyses revealed that the "Sulfate Reduction Sequence (SRS) pathway" was significantly enriched. We also found a complex relationship between H2S production and stress response genes. For UCD522, we confirm that there is a non-linear relationship between YAN and H2S production, with the Low and Intermediate treatments having greater H2S production than the High treatment. By integrating results obtained through the transcriptomic analysis with yeast physiological data, we present a mechanistic view into the H2S production by yeast as a result of different concentrations of YAN during cider fermentation.

Viability of IR spectroscopy for the accurate measurement of yeast assimilable nitrogen content of grape juice.

Up to date, there have been only a few reports on the measurement of YAN and/or its components using IR spectroscopy, suffering from various limitations (number of samples, validation strategies, etc.). In this work, three IR spectral instruments measuring in different modes and ranges of the IR spectrum (FT-IR, FT-NIR, and ATR-MIR), were compared and evaluated for their accuracy to measure both total YAN as well as the components, FAN and ammonia separately, using over 900 grape juice samples from 28 cultivars over three seasons. The global and vintage-based models were evaluated using R2CAL/VAL, RMSEC/P, and RPDCAL/VAL. Randomization tests were used for pair-wise comparison of models. FT-IR and FT-NIR instruments gave the best results, while ATR-MIR can be used for screening purposes. Considering the accuracy, robustness, high throughput, and cost-effective nature, the models produced by both FT-IR and FT-NIR spectroscopy can provide winemakers with the opportunity to make timelier and more informed nutrient supplementation decisions, facilitating the achievement of their desired wine style and quality.

Influence of must yeast-assimilable nitrogen content on fruity aroma variation during malolactic fermentation in red wine.

This study assessed the impact of must yeast-assimilable nitrogen (YAN) content and lactic acid bacteria (LAB) strains used for malolactic fermentation (MLF) on the formation of substituted esters, as well as the corresponding precursors (substituted acids), to investigate the modulation of fruity expression in red wines. In microvinification experiments, a Merlot must was fermented with an initial YAN content of 111 mg/L, or supplemented up to 165 and 220 mg/L. Two Oenococcus oeni LAB strains were used for MLF. Analytical methods were used to quantify substituted esters, as well as the corresponding acids, including, any enantiomeric forms. YAN supplementation of the must significantly increased concentrations of substituted esters of short- and branched-chain alkyl fatty acids produced during alcoholic fermentation (AF) (up to 67% in samples with the highest nitrogen content) and substituted esters of hydroxycarboxylic acids generated during MLF (up to 58% in samples with the highest nitrogen content). YAN supplementation in the must did not affect substituted acid formation during AF. After MLF, short- and branched-chain alkyl fatty acid levels increased in wines made from musts with the highest nitrogen content (up to 56% in samples with the highest nitrogen content), whereas concentrations of hydroxycarboxylic acids increased (up to 55%) independently of the initial YAN content, highlighting the important role of MLF. (2S)-2-hydroxy-4-methylpentanoic acid was only found in wines after malolactic fermentation, suggesting different pathways for each enantiomer and opening up new prospects for the study of bacterial metabolisms. Moreover, sensory profiles revealed a significant increase in black-berry- and jammy-fruit aromas during MLF and a strong positive correlation between these aromas and the production of substituted esters following must nitrogen supplementation and MLF. Aromatic reconstitutions revealed that variations in the concentrations of substituted esters after MLF impacted the fruity aroma of red wines.

Dimorphism of Trichosporon cutaneum and impact on its lipid production.

BACKGROUND: Compared to the oleaginous yeast Yarrowia lipolytica, Trichosporon cutaneum can metabolize pentose sugars more efficiently, and in the meantime is more tolerant to inhibitors, which is suitable for lipid production from lignocellulosic biomass. However, this species experiences dimorphic transition between yeast-form cells and hyphae during submerged fermentation, which consequently affects the rheology and mass transfer performance of the fermentation broth and its lipid production. RESULTS: The strain T. cutaneum B3 was cultured with medium composed of yeast extract, glucose and basic minerals. The experimental results indicated that yeast-form morphology was developed when yeast extract was supplemented at 1 g/L, but hyphae were observed when yeast extract supplementation was increased to 3 g/L and 5 g/L, respectively. We speculated that difference in nitrogen supply to the medium might be a major reason for the dimorphic transition, which was confirmed by the culture with media supplemented with yeast extract at 1 g/L and urea at 0.5 g/L and 1.0 g/L to maintain total nitrogen at same levels as that detected in the media with yeast extract supplemented at 3 g/L and 5 g/L. The morphological change of T. cutaneum B3 affected not only the content of intracellular lipids but also their composition, due to its impact on the rheology and oxygen mass transfer performance of the fermentation broth, and more lipids with less polyunsaturated fatty acids such as linoleic acid (C18:2) were produced by the yeast-form cells. When T. cutaneum B3 was cultured at an aeration rate of 1.5 vvm for 72 h with the medium composed of 60 g/L glucose, 3 g/L yeast extract and basic minerals, 27.1 g (dry cell weight)/L biomass was accumulated with the lipid content of 46.2%, and lipid productivity and yield were calculated to be 0.174 g/L/h and 0.21 g/g, respectively. Comparative transcriptomics analysis identified differently expressed genes for sugar metabolism and lipid synthesis as well as signal transduction for the dimorphic transition of T. cutaneum B3. CONCLUSIONS: Assimilable nitrogen was validated as one of the major reasons for the dimorphic transition between yeast-form morphology and hyphae with T. cutaneum, and the yeast-form morphology was more suitable for lipid production at high content with less polyunsaturated fatty acids as feedstock for biodiesel production.

Effect of the timing of water deficit on the must amino acid profile of Tempranillo grapes grown under the semiarid conditions of SW Spain.

Must nitrogen is very important for successful fermentation. Irrigation can influence vine nitrogen availability, and therefore must nitrogen content and wine quality. The aim of this work was to assess the effect of vine water status on the nitrogen concentration of cv. Tempranillo grown under semiarid conditions. A rainfed and two regulated deficit irrigation (RDI) strategies, late (LDI) and early deficit irrigation (EDI) were applied during pre- and post-veraison respectively periods over two seasons in Badajoz (Spain). Twenty-eight amino acids, amines and ammonia were determined by high performance liquid chromatography (HPLC) in the must. Results showed an effect of the pre-veraison vine water status on the must amino acid concentration. Higher values of nitrogenous indices were reached in musts from LDI treatments, mainly in the dry year. Significant correlations between pre-veraison stem water potential and 19 amino acid concentrations were observed. Therefore, LDI was a more suitable strategy for increasing the content of nitrogenous substances in the must.

A statistical exploration of data to identify the role of cultivar and origin in the concentration and composition of yeast assimilable nitrogen.

The study was undertaken to gain insight into the nitrogen status of grape juices currently used to make commercial wines in South Africa. This was done as yeast assimilable nitrogen (YAN) is most often suspected as the cause for problematic fermentations and has major implications for the organoleptic qualities of the final product. Using exploratory statistical methods, this study explored the possibility of identifying the role of cultivar and grape-growing district in the determination of the concentration and composition of YAN. However, as the dataset was found to be non-parametric and heteroscedastic, paired with unequal sample sizes, data analysis was approached with caution. Through the use of various suitable statistical analyses, cultivar was shown to play the more important role in determining the concentration and composition of YAN.

Enhancing wine ester biosynthesis in mixed Hanseniaspora uvarum/Saccharomyces cerevisiae fermentation by nitrogen nutrient addition.

The dynamic changes of wine ester production during mixed fermentation with Hanseniaspora uvarum Yun268 and Saccharomyces cerevisiae F5 was investigated at different levels and timings of nitrogen nutrient addition. Nitrogen additions were performed by supplementing yeast assimilable nitrogen (YAN) into a synthetic grape must with defined composition. Ester precursors and extracellular metabolites involved in ester synthesis were analyzed throughout the fermentation. Results showed that nitrogen additions covering 50-200 mg/L YAN at the point of yeast inoculation slightly affected yeast competition and ester profiles. Interestingly, when YAN was supplemented in the mid-stage, the survival of H. uvarum Yun268 was enhanced, resulting in more than a 2-fold increase in the levels of higher alcohol acetates compared to that at the initial stage. Furthermore, carbon fluxes may be redistributed in the central pathway, which contributed to the production of medium-chain fatty acids and eventually triggered a 1.2-fold elevation in corresponding ethyl ester levels.

Analysis of the NCR Mechanisms in Hanseniaspora vineae and Saccharomyces cerevisiae During Winemaking.

There is increasing interest in the use of non-Saccharomyces yeasts in winemaking due to their positive attributes. The non-Saccharomyces yeast Hanseniaspora vineae is an apiculate yeast that has been associated with the production of wine with good fermentation capacity and an increase in aromatic properties. However, this yeast represents a concern in mixed culture fermentation because of its nutrient consumption, especially nitrogen, as its mechanisms of regulation and consumption are still unknown. In this study, we analyzed the nitrogen consumption, as well as the nitrogen catabolism repression (NCR) mechanism, in two genome-sequenced H. vineae strains, using synthetic must fermentations. The use of synthetic must with an established nitrogen content allowed us to study the NCR mechanism in H. vineae, following the amino acid and ammonia consumption, and the expression of genes known to be regulated by the NCR mechanism in S. cerevisiae, AGP1, GAP1, MEP2, and PUT2. H. vineae exhibited a similar amino acid consumption and gene expression profile to S. cerevisiae. However, the wine strain of S. cerevisiae QA23 consumed ammonia and valine more quickly and, in contrast, tyrosine and tryptophan more slowly, than the H. vineae strains. Our results showed a similar behavior of nitrogen regulation in H. vineae and S. cerevisiae, indicating the presence of the NCR mechanism in this Hanseniaspora yeast differentiated before the whole genome duplication event of the Saccharomyces complex. Future study will elucidate if the NCR mechanism is the only strategy used by H. vineae to optimize nitrogen consumption.