Analysis of grape polyamines from Grapevine leafroll associated viruses (GLRaV-2 and -3) infected vines

This is the first report comparing the free polyamine content of ‘Pinot noir’ grapes from vines infected with Grapevine leafroll associated viruses (GLRaV)-2 or -3, with samples taken from healthy vines. Berries were collected from three different rootstock/scion combinations, and all samples were from commercial vineyards in Oregon. Three free polyamines (putrescine, cadaverine, and spermidine) were found in all ‘Pinot noir’ grapes analysed in this study. Berries from healthy Vitis riparia rootstock/‘Pinot noir’ clone 114 were significantly higher in putrescine (28.6 μmol/l) and total polyamines (41.9 μmol/l), compared to their GLRaV-3 infected samples (20.7 and 33.0 μmol/l, respectively). Vine virus status did not alter total or individual polyamines in berries from the other two rootstock/scion combinations (unknown rootstock/‘Chardonnay’ interstock/‘Pinot noir’ scion [clone unknown] and self-rooted/‘Pinot noir’ clone Pommard). Levels of putrescine, spermidine, and total polyamines varied between berries taken from the healthy vines of the three different rootstock/scion combinations.     

Influence of grapevine leafroll associated viruses (GLRaV-2 and -3) on the fruit composition of Oregon Vitis vinifera L. cv. Pinot noir: Phenolics

Some of the 10 known grapevine leafroll associated viruses (GLRaVs) have negative impacts upon vine productivity and grape quality, though these negative influences are dependent on factors such as GLRaV strain, cultivar, clone, rootstock, and vine age. This is the first study to report on GLRaV-2 and GLRaV-3 infected vines, with regard to phenolic compounds, and other fruit maturity indices, of ‘Pinot noir’ grapes, compared to berries from adjacent vines free of GLRaVs (same vineyards). Three different rootstock/scion combinations were included in this study. Clusters were collected for two growing seasons from commercial vineyards in the Willamette Valley of Oregon, and each vine sampled was tested for GLRaV-1, -2, -3 and Rupestris stem pitting-associated virus (RSPaV). All sampled vines were infected with RSPaV. Grapevine leafroll associated virus-infected vines tested positive for GLRaV-2 or GLRaV-3. Overall, fruit infected with GLRaV-2 and -3 had reduced percent soluble solids, decreased individual and total anthocyanins, and increased skin and pulp weight for all three ‘Pinot noir’ rootstock/scion combinations examined. Vitis riparia rootstock/‘Pinot noir’ clone 114 scion combination appeared to be the most sensitive to GLRaV-3 infection, having significant reduction of all five anthocyanins, total phenolics, and total tannins, with an increased cluster weight and 100-berry weight. No clear trends were observed in the polyphenolics analysed.     

Free amino acid profiles from ‘Pinot noir’ grapes are influenced by vine N-status and sample preparation method

This study examined the impact of extraction method on ammonia, free amino acids, and yeast assimilable nitrogen (YAN) concentrations in ‘Pinot noir’ berries obtained from a vine nutrition study (altered supply of N, P, or K). Berries were either juiced or exhaustively extracted as whole berries prior to analysis. Extracts, compared to juice samples, had a significantly higher level of ammonia–N, assimilable amino acid–N, and YAN. For example, juice YAN values were approximately 50% of extract YAN values, when both were expressed in the same units. Free amino acid profiles and relative concentrations of individual amino acids were different in juice versus extracts, depending on how well the skin fraction was extracted prior to analysis. Lowering N supply reduced free amino acids, with arginine being reduced more than the other 20 free amino acids identified in ‘Pinot noir’ berries. This was true in both juice and extracts. Since berry skin contributed to actual YAN, wineries that determine YAN from mainly the pulp fraction (juice) may underestimate YAN and as a result add more (artificial) N supplement than is required for the healthy fermentation of red winemaking (whole berry fermentations). Extraction procedure should be taken into consideration when comparing grape YAN.     

Rootstock and vineyard floor management influence on ‘Cabernet Sauvignon’ grape yeast assimilable nitrogen (YAN)

This is a study on the influence that two rootstocks (110R, high vigour; 420A, low vigour) and three vineyard floor management regimes (tilled resident vegetation – usual practise in California, and barley cover crops that were either mowed or tilled) had upon grape nitrogen-containing compounds (mainly ammonia and free amino acids recalculated as YAN), sugars, and organic acids in ‘Cabernet Sauvignon’ clone 8. A significant difference was observed for some of the free amino acids between rootstocks. In both sample preparation methods (juiced or chemically extracted), 110R rootstock grapes were significantly higher in SER, GLN, THR, ARG, VAL, ILE, LEU, and YAN than were 420A rootstock grapes. Differences in individual free amino acid profiles and concentrations were observed between the two sample preparations, which indicate that care should be taken when comparing values from dissimilar methods. No significant differences among vineyard floor treatments were detected, which suggests that mowing offers vineyard managers a sustainable practise, alternative to tilling, without negatively affecting grape nitrogen compounds, sugars, or organic acids.     

Influence of extraction methodology on grape composition values

This work demonstrated similarities and differences in quantifying many grape quality components (>45 compounds) that were extracted from berries by three distinct preparations, before being analysed by eight spectrophotometric and HPLC methods. All sample extraction methods were appropriate for qualitative results only. Different extraction procedures showed altered component composition in ‘Pinot noir’ berries, possibly due to the localisation of the compounds of interest within the grape and how those compounds were extracted from the berry. Sample extraction is an often-overlooked part of berry evaluations, but this study illustrates that it should be carefully considered prior to berry component analysis for its influence upon measurements.     

Effects of timing and rate of N supply on leaf nitrogen status, grape yield and juice composition from Shiraz grapevines grafted to one of three different rootstocks

Yeast cells have a minimum N requirement to ferment a must through to dryness, so that grape N content (hence must N) becomes critical in meeting that prerequisite. Viticultural practices aimed at meeting that N requirement are of special relevance because interactions between rootstock and vineyard nitrogen supply strongly influence scion mineral nutrient status as well as shoot vigour, and via those processes, fruit composition. Such outcomes were investigated in a field trial involving Shiraz on three rootstocks viz. Teleki 5C, Schwarzmann and Ramsey. Five N supply regimes, varying from 0 to 80 kg/(haseason), were imposed through a drip-irrigation system during two periods (either flowering to veraison, or post-harvest to leaf-fall, or both) over three successive growing seasons. Post-harvest N supply increased scion leaf N and nitrate N concentrations at flowering for vines on Teleki 5C and Schwarzmann. By veraison, N recently applied in the flowering to veraison period elevated these indicators of N status in all vines on all rootstocks. Grape yields from vines on Teleki 5C and Schwarzmann were elevated by N supply after harvest, whereas juice soluble solids levels were lowered. Free amino acids in Shiraz juice were dominated by non-assimilable N, amounting to about 50% or more of the total free amino-N in the juice. Increasing N supply increased free amino acid concentrations in the juice of berries from vines on all rootstocks, but only vines on Schwarzmann derived any benefit from N supplied after harvest. The highest concentrations of free amino acids were measured in the berries from vines on Schwarzmann receiving 80 kg N/(ha.season). Of immediate practical relevance for N management of Shiraz grapevines on either Teleki 5C or Ramsey rootstocks, the minimum value for assimilable free amino-N concentration required to ferment a must through to dryness was not achieved if vineyard N application was limited to the post-harvest period.     

Profiles of free amino acids in grapes of grafted Chardonnay grapevines

Chardonnay grapes from a rootstock trial in the Murray Valley region of South Australia were sampled for two seasons and concentrations of free amino acids in grape extract ('juice') were determined. The lowest concentrations of free assimilable amino-N were measured in Chardonnay grapes from vines on 140 Ruggeri and 101–14 rootstocks, and the highest concentrations were in grapes from vines on their own roots, Schwarzmann and K51–40. Free assimilable amino-N concentrations were sufficient to sustain fermentation through to completion. Arginine concentrations were generally too low to expect significant concentrations of urea in the wine. Amino-S concentrations were much lower and varied less than amino-N concentrations. The effect of any particular rootstock on the concentration of total free amino acids in Chardonnay grape 'juice' did not appear to be related to the known effects of those rootstocks on the NO₃-N status of petioles at flowering. The concentrations of leucine, iso-leucine, valine, threonine, tyrosine and phenylalanine were generally higher in Chardonnay grapes grafted onto K51–40 in comparison to grapes from Chardonnay vines grafted on the other rootstocks. Any of these amino acids may be associated with the presence of specific higher alcohols in wine.     

European elderberry (Sambucus nigra L.) rich in sugars,organic acids,anthocyanins and selected polyphenols

Sugars and organic acids in the fruit of two cultivars and three selections of black elderberry (Sambucus nigra L.): ‘Haschberg’, ‘Rubini’, ‘Selection 13’, ‘Selection 14’ and ‘Selection 25’ were quantified. The anthocyanin as well as quercetin profiles of this plant material were also established by the use of HPLC/MS. Significant differences in the concentration of sugars and organic acids were detected between the widely spread cultivar ‘Haschberg’ and all other cultivars/selections; ‘Haschberg’ was the richest in organic acids (6.38 g kg⁻¹ FW), and it contained the least sugar (68.5 g kg⁻¹ FW). The following major cyanidin based anthocyanins were identified in the fruit of black elderberry: cyanidin 3-sambubioside-5-glucoside, cyanidin 3,5-diglucoside, cyanidin 3-sambubioside, cyanidin 3-glucoside and cyanidin 3-rutinoside. The most abundant anthocyanin in elderberry fruit was cyanidin 3-sambubioside, which accounted for more than half of all anthocyanins identified in the berries. The ‘Rubini’ cultivar had the highest amount of the anthocyanins identified (1265 mg/100 g FW) and the lowest amount was measured in berries of the ‘Selection 14’ (603 mg/100 g FW). The ‘Haschberg’ cultivar contained a relatively low amount of anthocyanins in ripe berries (737 mg/100 g FW). From the quercetin group, quercetin, quercetin 3-rutinoside and quercetin 3-glucoside were identified; the latter prevailing in black elderberry fruit. The cultivar with the highest amount of total quercetins was ‘Selection 25’ (73.4 mg/100 g FW), while the ‘Haschberg’ cultivar contained average amounts of quercetins (61.3 mg/100 g FW). The chemical composition of the ‘Haschberg’ cultivar, the most commonly planted, conforms to the standards for sugars, anthocyanins and quercetins and exceeds them in the content levels of organic acids, the most important parameter in fruit processing.     

Berry nitrogen status altered by cover cropping: Effects on berry hormone dynamics,growth and amino acid composition of Pinot Noir

The influence of altered nitrogen (N) content, caused by cover crop, on Pinot Noir berry quality was studied. Treatments included two levels of cover cropping and clean-cultivation. Berry N-content was reduced by grass cover crop competition, with the highest reductions of N at the early stage of berry formation and at the commercial harvest stage. Growth regulators, trans-zeatin and indole-3-acetic acid, and berry growth were influenced by the changes in N-status at different stages of berry formation. The total amino acid content of berries at harvest decreased with cover cropping, while the proline to arginine ratio and assimilable amino acids content improved compared to the berries from clean-cultivated vines. Our data indicate that early stage berry development is influenced by N-status but finally it results in an improved amino acid composition for yeast fermentation in mature berries. At the same time, sugar and pigment contents were not negatively influenced by cover crop-induced changes in berry N-status.     

Changes in Free Amino Acid and Total Nitrogen Concentrations During Maturation of Muscadine Grapes (V. rotundifolia)

Free amino acids, ammonia and total nitrogen were followed during maturation of berries of Carlos and Noble muscadine grapes (V. rotundifolia). Chemical analysis was done on deseeded berries. Ammonia decreased in the berries of both cultivars during maturation. Total nitrogen content fluctuated and was not significantly correlated with maturity in either cultivar. Threonine (THR) and histidine + gamma aminobutyric acid (HIS + GABA) were the predominant free amino acids in both cultivars at an immature berry stage. At full berry maturity, arginine (ARG) was the predominant amino acid in Carlos, having increased over 100-fold during the sampling period while ARG, alanine (ALA), THR, and HIS + GABA accounted for 72.3% of the total free amino acids. ALA was the predominant free amino acid in mature berries of Noble and accounted for 30.4% of the total free amino acids. Of the additional cultivars sampled only at their normal harvest date, berries of Pride, Magnolia and Regale contained nearly equal amounts of ARG and ALA as the predominant amino acids. Mature Dixie berries had twice as much ALA (29.8%) as ARG (12.8%). In general, the free amino acids in berries of muscadine grapes were found in concentrations similar to those reported in the literature for V. vinifera and V. labrusca cultivars. However, the large increase in proline concentration reported to occur during maturation of berries of V. vinifera cultivars was not found in any of the muscadine cultivars examined.     

Developmental changes of carbohydrates,organic acids,amino acids,and phenolic compounds in ‘Honeycrisp’ apple flesh

The developmental changes of carbohydrates, organic acids, amino acids and phenolic compounds in ‘Honeycrisp’ apple flesh were investigated using GC–MS and HPLC. A total of 12 carbohydrates, 8 organic acids, 20 amino acids, and 18 phenolic compounds were identified and quantified. Each metabolite showed characteristic changes during fruit development, but in general, concentrations of most sugars and sugar alcohols either increased or remained unchanged whereas concentrations of most organic acids, amino acids and phenolic compounds decreased with fruit development, indicating that most sugars and sugar alcohols are synthesised and/or accumulate at a faster or similar rate relative to fruit growth whereas organic acids, amino acids and phenolics are synthesised and/or accumulate at a slower rate relative to fruit growth. On a whole fruit basis, the content of most metabolites increased with fruit development. In the flesh of mature ‘Honeycrisp’ apple, fructose and sucrose and sorbitol are the major sugars and sugar alcohol; malic acid is the major organic acid; aspartic acid, asparagine, glutamic acid, proline, threonine and γ-aminobutyric acid are the major amino acids; and procyanidin B1, procyanidin B2, chlorogenic acid, catechin and epicatechin are the major phenolic compounds, respectively.     

Effect of foliar nitrogen and sulfur on petit manseng (Vitis vinifera L.) grape composition

Vineyard applications of foliar nitrogen (N) and sulfur were evaluated in the Yadkin Valley of North Carolina in 2011 and 2012 as a means to modify grape composition of Vitis vinifera cv. petit manseng. Four treatments were applied each year in a randomized complete block design with six replicates of six vines each: untreated control, soil N at 30?kg/ha, foliar N at 15?kg/ha and foliar N at 15?kg/ha plus sulfur at 5?kg/ha. Fruit ammonia and yeast assimilable nitrogen (YAN) concentrations increased in foliar-treated vines both seasons compared to control vines. Total glycosides (TGGs) increased ～80% in foliar N/sulfur-treated vines compared to control vines in 2011, and TGGs increased ～157% in foliar N/sulfur-treated vines relative to glycoside level in foliar N alone in 2012. Foliar N and foliar N/sulfur applications increased the majority of amino acid concentrations analyzed in 2011. Measured concentrations of various amino acids, partly responsible for aroma and flavor of petit manseng wines, were differentially dependent on the method of N application. Foliar applied nitrogen may be an effective technique to contribute to berry composition optimization of petit manseng grown in a warm, humid climate.     

Phenolic acids and flavonoids in leaf and floral stem of cultivated and wild Cynara cardunculus L. genotypes

Ten genotypes, cultivated and wild of Cynara cardunculus L. were evaluated for their content of phenolic acids, flavonoids and their antioxidant activity. The major compounds present in the leaf were luteolin derivatives in globe artichoke and apigenin derivatives in wild and cultivated cardoon. Apart from ‘Cimiciusa di Mazzarino’ (var. scolymus), caffeoylquinic acids represent the main phenolic compounds in the floral stem. In particular, ‘Sylvestris Creta’ (var. sylvestris) and ‘Violetto di Sicilia’ (var. scolymus) show the highest content of caffeoylquinic acid ∼95% of the total measured polyphenols. The antioxidant capacity, in both leaf and floral stem, was qualitatively and quantitatively dependent on the phenolic acid and flavonoid profile. The phenolic acids and flavonoids in normally uneaten parts of wild and cultivated artichoke could be exploited as sources of natural antioxidants.     

Proline and arginine accumulation in developing berries of Vitis vinifera L. in Australian vineyards: Influence of vine cultivar, berry maturity and tissue type

Using HPLC analysis, free amino acid profiles were obtained for ripe berries of six grapevine cultivars (Cabernet Sauvignon, Grenache, Muscat Gordo, Pinot Noir, Riesling and Sangiovese) grown under comparable conditions and harvested at similar maturities. Compositional differences were observed between cultivars, however proline and arginine were always the major amino acids. Mature berries of Cabernet Sauvignon contained a very high concentration of proline, but a much lower concentration of arginine. Those of other cultivars contained moderate levels of both arginine and proline.
Changes in free amino acid profiles during grape berry development were further investigated in four cultivars (Cabernet Sauvignon, Chardonnay, Gewurztraminer and Muscat Gordo). In all cases, most of the proline accumulation occurred late in ripening, around four weeks post-veraison. In contrast, arginine accumulation began before veraison and continued to full maturity, except for those cultivars in which a high concentration of proline accumulated, in which case the concentration of arginine reached a plateau relatively early in development. Accumulation of both proline and arginine appears to be develop-mentally regulated. These observations are discussed with reference to proline and arginine metabolism and possible links between them.
The distribution of free proline, arginine and other amino acids amongst pulp, skin and seed was examined using mature fruit of Cabernet Sauvignon and Riesling. Amino acid profiles were found to vary considerably between the berry components. The skin contained a greater ratio of arginine:proline compared with the pulp. This suggests that the yeast-assimilable nitrogen content of juices, and therefore their fermentability, could be enhanced in the presence of skins.     

Effect of salinity and Ramsey rootstock on ion concentrations and carbon dioxide assimilation in leaves of drip-irrigated, field-grown grapevines (Vitis vinifera L. cv. Sultana)

Salinity treatments of 0.43, 1.7 and 3.4 dS/m were applied through a drip-irrigation system to four-year-old vines of own-rooted Sultana (S_O) and Sultana on Ramsey rootstock (Sr) The vines were planted in spring 1987 and established for two years under irrigation with low salinity (0.43 dS/m) water before commencing the range of salinity treatments in spring 1989. The effects of salinity and rootstock on yield, size and composition of berries, canopy size, lamina CO₂ assimilation, leaf water relations and lamina ion concentrations were studied between veraison and harvest during the second season of salinity treatments. Mature leaves of S_R had higher rates of CO₂ assimilation (leaf area basis) and stomatal conductance than comparable leaves of S_O. The high salinity treatment (3.4 dS/m) reduced CO₂ assimilation rate of S_O but not of S_R. Medium salinity (1.7 dS/m) had no significant effect on CO₂ assimilation rate of either type. Laminae of S_O accumulated significantly higher concentrations of chloride than S_R vines at all salinity levels. There was a significant negative correlation (r²= 0.44) between CO₂ assimilation rate and laminae chloride of S_O. Leaf sodium concentrations increased with increasing salinity, but concentrations in laminae at high salinity were similar in S_O and S_R, with S_R showing no reduction in CO₂ assimilation. Leaf potassium concentrations were higher in S_R, but decreased with increasing salinity, whereas magnesium concentrations were similar in S_O and S_R vines but increased in both at high salinity. Leaf water potential and relative water content were not significantly affected by salinity in either S_O or S_R vines. Both vine types had reduced total leaf area and pruning wood weights as salinity increased, but they were greater in S_R at all salinity levels because of the higher inherent capacity for biomass production in S_R vines. The bigger canopies, lower lamina chloride concentrations and ‘normal’ photosynthesis rates of S_R vines at 3.4 dS/m enabled these vines to mature crops with similar berry weights, sugar contents and fruit yield compared with those of S_O vines at 0.43 dS/m. The research demonstrated the benefit of using Ramsey rootstock for Sultana under saline field conditions and provided a physiological explanation for their higher salt tolerance.     

Comparative study of primary and secondary metabolites in 11 cultivars of persimmon fruit (Diospyros kaki L.)

Primary metabolites (sugars, organic acids) and secondary metabolites (phenolics and carotenoids) were quantified by HPLC in fully ripe fruit of 11 kaki cultivars: ‘Amankaki’, ‘Cal Fuyu’, ‘Fuji’, ‘Hana Fuyu’, ‘Jiro’, ‘O’Gosho’, ‘Tenjin O’Gosho’, ‘Thiene’, ‘Tipo’, ‘Tone Wase’ and ‘Triumph’. Amongst the analysed cultivars, ‘Tone Wase’ stands out as the richest in sugars, particularly glucose, and cultivars ‘Tipo’ and ‘Triumph’ contained the highest amounts of organic acids. Cultivars ‘O’Gosho’, ‘Cal Fuyu’ and ‘Hana Fuyu’ contained the least sugars and cultivar ‘Jiro’ the least organic acids. Amongst the individual phenolic compounds catechin and gallic acid were present in highest concentrations. The predominant carotenoid in both skin and pulp of ripe persimmon fruit was β-carotene, the highest content was measured in skin of cultivar ‘Hana Fuyu’, which also contained the highest level of total carotenoids. In persimmon pulp, much lower values for carotenoids were obtained, particularly in fruit of cultivar ‘Cal Fuyu’.     

烤烟烟叶中游离氨基酸与内在质量关系的研究

系统地分析了烤烟烟叶中游离氨基酸与内在质量的关系，参试材料31份，每个样品分析鉴定25项指标，对试验数据分别进行简单相关分析，多元回归和偏相关分析以及典型相关分析。简单相关分析结果表明，游离氨基酸组分与内在质量之间存在一定的相关关系，其中缬氨酸、酪氨酸和游离氨含量较高，则烟叶香气较好，亮氨酸含量较高，烟叶杂气较少，丝氨酸，甘氨酸和游离氨含量高，则烟叶评吸总分较高，烟叶品质相对较好，多元回归和偏相关分析系统地分析了游离氨基酸组分对内在质量的贡献大小。其中对香气贡献较大的有精氨酸，苯丙氨酸和游离氨；对吃味贡献较大的有甘氨酸，对减少杂气贡献较大的是游离氨和脯氨酸，对评吸总分贡献较大的有精氨酸。苯丙氨酸、丝氨酸，总游离氨基酸和酪氨酸。典型相关分析结果表明，游离氨基酸与内在质量间的典型相关主要是由酷氨酸，蛋氨酸，苯丙氨酸，精氨酸，丝氨酸，脯氨酸，亮氮酸，苏氨酸，氨与香气，吃味，杂气，刺激性相关显著引起的。     

Simultaneous utilization of ammonia,free amino acids and peptides during fermentative growth of Saccharomyces cerevisiae

The efficiency of nitrogen use by yeast is one of the key determinants of the successful completion of alcoholic fermentations. In this work the growth of Saccharomyces cerevisiae S288c in a synthetic medium containing ammonia and free amino acids, supplemented with yeast hydrolysate, was studied. Experiments with ¹⁵NH₄Cl and ¹⁵N‐labelled yeast hydrolysate were carried out to gain insight into which of these three classes of assimilable nitrogen sources yeast cells prefer. Co‐consumption of all three sources was observed; approximately 40% of the total nitrogen in the yeast protein fraction originated from yeast hydrolysate, while free amino acids and ammonia contributed 40 and 20%, respectively. The results indicate that several amino acids are more readily obtained from peptides, most likely when the uptake of their free forms is competitively inhibited and/or repressed. During the second half of each fermentation, a decrease in the incorporation of yeast hydrolysate‐derived nitrogen was observed. These results highlight the nutritional role of peptides in various yeast fermentations. Copyright © 2016 The Institute of Brewing & Distilling     

Metabolism of amino acids, dipeptides and tetrapeptides by Lactobacillus sakei

The microbial degradation of proteins, peptides and amino acids generates volatiles involved in the typical flavor of dry fermented sausage. The ability of three Lactobacillus sakei strains to form aroma compounds was investigated. Whole resting cells were fermented in phosphate buffer with equimolar amounts of substrates consisting of dipeptides, tetrapeptides and free amino acids, respectively.Dipeptides disappeared quickly from the solutions whereas tetrapeptides were only partially degraded. In both approaches the concentration of free amino acids increased in the reaction mixture but did not reach the equimolar amount of the initial substrates. When free amino acids were fed to the bacteria their levels decreased only slightly. Although peptides were more rapidly degraded and/or transported into the cells, free amino acids produced higher amounts of volatiles.It is suggested, that after transport into the cell peptides are only partially hydrolyzed to their amino acids, while the rest is metabolized via alternative metabolic pathways. The three L. sakei strains differed to some extend in their ability to metabolize the substrates to volatile compounds. In a few cases this was due to the position of the amino acids within the peptides. Compared to other starter cultures used for the production of dry fermented sausages, the metabolic impact of the L. sakei strains on the formation of volatiles was very low.     

Analysis of Protein and Total Usable Nitrogen in Beer and Wine Using a Microwell Ninhydrin Assay

In this study we present a ninhydrin based microwell assay that can be utilized in place of the traditional Kjeldahl method for the determination of the protein content of beer or wine. In addition, the assay is ideal for the determination of free amino acids in beer (FAN), a term understood and used by brewers, and yeast assimilable nitrogen (YAN) used by enologists. The assay only measures alpha amino acids and ammonia so other nitrogen sources are not detected, resulting in a 30% reduction in total protein of a variety of beers compared to the Kjeldahl method, which measures nitrogen from all sources. The results also showed that only 25% of the total “protein” in beer is actually derived from peptides larger than 3,500 Kd. Analysis of beer or wine with the microwell assay for total usable nitrogen was compared to the standard FAN and YAN methods and conditions were determined for maximal efficiency and precision. Superior results were obtained with low reaction volumes and a stable sodium acetate buffered ninhydrin reagent at pH 5.5. As an alternative, for use with cuvettes, a reduced volume FAN assay using the same pH 5.5 sodium acetate buffered ninhydrin reagent gave comparable results. The assay is economical, rapid, accurate and applicable to large numbers of samples.