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‫תרכובות חנקן ביין‬

Summation of free amino nitrogen and

ammonia nitrogen levels provides a useful
estimate of the yeast assimilable nitrogen level.

Adapted from: Pretorius et al.(2007). Nitrogen management is critical for wine flavour and style. Wine Industry Journal, 2(6), 24-30.
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Nitrogen Compounds

The various forms of nitrogen

Mineral Nitrogen Organic Nitrogen

Ethyl Carbamate Amino acids

Oligopeptides
Pyrazines
Nucleic
polypeptides nitrogen
Amide Amino sugar
Urea Proteins
nitrogen
Bioamines nitrogen

 Nitrogen is one of the most plentiful elements in the universe (atmosphere

contains nearly 80% N2).

 The presence of this uncombined element indicates that nitrogen’s chemical

reactivity is extremely low.

Adapted from: Ribéreau-Gayon, et al. (2007): “Handbook of Enology: The Chemistry of Wine”
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Amines
 Amines are a class of compounds derived from ammonia (NH 3) by replacement of one, two, or
three hydrogens to yield primary, secondary, and tertiary amines, respectively.

 Additional substitution on the amino nitrogen with a hydrocarbyl group yields a quaternary
ammonium cation whereas protonation of the amino nitrogen yields an aminium cation.

 Amines can also be present in molecules containing additional functional groups (e.g. carboxyl,
carbonyl, thiol, etc.), and in grapes, the major amine ‐group ‐containing species are amino acids
and their polymers (e.g., proteins).

Adapted from: Waterhouse et al., 2016: "Understanding Wine Chemistry"

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Amino acids and related major nitrogenous compounds in wines

Adapted from: Waterhouse et al., 2016: "Understanding Wine Chemistry"

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‫תכולה כוללת של חנקן (‪)Total Nitrogen‬‬

‫‪ ‬תכולה כוללת של חנקן מורכבת מצורה אנאורגנית אחת וצורות אורגניות שונות רבות‬
‫‪ ‬מהווה כ‪ 90% -‬מהחנקן בשיא ההבשלה‬
‫‪ ‬ריכוזים משתנים ביחס להבשלת הענבים‬
‫‪ ‬החנקן הכולל בזמן ההבשלה הוא כ‪ 140-650 -‬מ"ג לליטר‬
‫‪ ‬הריכוז המינימלי לתסיסה תקינה הוא ‪ 50‬מ"ג לליטר‬
‫‪ +NH4 ‬מרכיב חיוני בתסיסה האלכוהולית‬
‫‪ ‬ריכוז גבוה יותר ביינות אדומים מאשר בלבנים ‪ -‬בגלל ההשריה‪ ,‬טמפרטורת תסיסה ואוטוליזה של התאים‬

‫חנקן מינרלי‬

‫‪ ‬חנקן מינרלי בצורה של מלחי אמוניה נמצא בריכוזים גבוהים מאוד בשלב גידול הגפן ‪ -‬מעל ‪80%‬‬
‫‪ ‬החנקן המינרלי הופך עם התקדמות ההבשלה לחומצות אמוניות ‪,‬פפטידים וכו'‬
‫‪ ‬בשיא ההבשלה ‪ -‬מהווה כ ‪ 10%‬בלבד מהחנקן‬
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‫חומצות אמינו‬
‫‪ ‬מורכבות מאטום פחמן מרכזי הקשור קוולנטית לקבוצת אמין‪ ,‬קבוצת קרבוקסיל‪ ,‬אטום מימן‬
‫ושייר משתנה‬
‫‪ ‬שייר הצד של החומצה נקרא גם קבוצת ה‪ R-‬והוא הגורם המבדיל בין חומצות האמינו השונות‪.‬‬
‫‪ ‬מהוות את המולקולות העיקריות בחנקן הכולל‬
‫‪ ‬חשובות מאד בשל התכונות האנטי‪-‬אוקסידנטיות ואנטי מיקרוביאליות שלהן‪ ,‬תורמות לקיבולת‬
‫הבופר של היין‪ ,‬מהוות אמולסיפריים וכחומרים פעילי שטח‬
‫‪ ‬משתתפות במרבית תהליכי התסיסה הכוהלית‬

‫”‪Adapted from: Ribéreau-Gayon, et al. (2007): “Handbook of Enology: The Chemistry of Wine‬‬
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Amino acids in grapes and wine

continued next slide…

Adapted from: Ribéreau-Gayon, et al. (2007): “Handbook of Enology: The Chemistry of Wine”
 ‫בס"ד‬
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Continued… Amino acids in grapes and wine

continued next slide…

Adapted from: Ribéreau-Gayon, et al. (2007): “Handbook of Enology: The Chemistry of Wine”
 ‫בס"ד‬
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Continued… Amino acids in grapes and wine

continued next slide…

Adapted from: Ribéreau-Gayon, et al. (2007): “Handbook of Enology: The Chemistry of Wine”
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Continued… Amino acids in grapes and wine

Adapted from: Ribéreau-Gayon, et al. (2007): “Handbook of Enology: The Chemistry of Wine”
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Chemistry of Amino acids

 Structure: From a structural point of view, the twenty most common amino acids in must
and wine, excepting γ -amino-butyric acid and β-alanine, are α-amino acids, i.e. the
carboxylic acid radical and the amino basic radical are linked to the same carbon atom.

 Amino acids are therefore amphoteric molecules. The R radical may be a hydrogen atom
(e.g. glycine or glycocoll).

The L configuration α-amino acid

 In other amino acids, the carbon linked to the functional radicals and the R carbohydrate
radical is chiral. This feature gives them the asymmetrical property common to
biologically significant molecules.

 Natural amino acids all have a true L configuration. Their optical activity depends on the
type of R radical, but also on the solvent, and even the pH of the solution.

Adapted from: Ribéreau-Gayon, et al. (2007): “Handbook of Enology: The Chemistry of Wine”
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Chemistry of Amino acids

 In trifunctional amino acids, the R radical is connected to an acid or basic radical.

Aspartic acid and glutamic acid are acid amino acids, while lysine, histidine, ornithine,
citrulline and arginine are basic amino acids.

 Other trifunctional amino acids have no marked acid or basic character. They have
hydroxylated (serine, threonine and tyrosine), thiol or sulfide radicals (cysteine and
methionine). Only these trifunctional amino acids are involved in the catalyst property of
enzymes.

 In solution, amino acids must be expressed in a double balance state, involving the
ionization of the acid or basic function.

Forms of α-amino acid in an aqueous solotion

 The population of each form is defined by the pH of the medium. At a pH where the
positive and negative charges are in balance, i.e. the isoelectric point, amino acids have
minimum solubility.

Adapted from: Ribéreau-Gayon, et al. (2007): “Handbook of Enology: The Chemistry of Wine”
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Chemistry of amines…
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 The presence of the lone electron pair results in amines and other amine ‐containing compounds
behaving as weak bases.

 The nitrogen lone pair of amino acids and many other amines is protonated at wine pH, and thus
many well‐known food chemistry reactions that involve amine groups acting as nucleophiles are
expected to occur very slowly.

 Amines with low pKa values will also have minimal volatility and thus no odor in wine.

 Amino acids and polypeptides have multiple ionizable groups at wine and juice pH.

 Majority of amino acids and proteins will exist as zwitterions

 The charge of a zwitterionic species at a given pH can be determined from its isoelectric point (pI),
giving the following possibilities:

• pH < pI – the protein (or amino acid) has a net positive charge.
• pH = pI – the protein has no net charge.
• pH > pI – the protein has a net negative charge.

 The importance of pI to wine is that it defines the pH at which a protein will be at minimum
solubility.

 Most wine proteins are reported to have pI values just above wine pH, in the range of 4–6, and
will thus have a net positive charge in wine or juice.

 As a result, proteins can non‐covalently bond to species that have formal negative charges or are
H‐bond acceptors, such Adapted
as bentonite and etpolyphenols.
from: Waterhouse al., 2016: "Understanding Wine Chemistry"
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Structures of major nitrogenous species in must: proline, primary α‐amino

acids, and glutathione (tripeptide)

Adapted from: Waterhouse et al., 2016: "Understanding Wine Chemistry"

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‫שינויים במהלך ההבשלה‬...
:‫ חלק מהחומצות האמיניות מאפיינות זנים לדוגמא‬
‫ פרולין‬- ‫ שרדונה וקברנה‬.1
‫ ארגנין‬- ‫ פינו נואר ואליגוטה‬.2
‫ קיימת קורלציה בין ריכוז חומצת האמינו פרולין לבין התקדמות ההבשלה‬

Profiles showing changes in concentrations of individual amino acids during ripening (grape variety:
Chardonnay) (Millery, 1988)
Adapted from: Ribéreau-Gayon, et al. (2007): “Handbook of Enology: The Chemistry of Wine”
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(Oligopeptides & polypeptides‫( אוליגומרים ופוליפפטידים‬

 Oligopeptides and polypeptides, formed by linking a limited number of amino

acids with peptide bonds:

 Oligopeptides contain a maximum of four amino acids. Polypeptides have

molecular weights under 10 000 Dalton.

 Some small peptides also have interesting sweet or bitter flavors that are
useful in winemaking.

Adapted from: Ribéreau-Gayon, et al. (2007): “Handbook of Enology: The Chemistry of Wine”
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Structure of glutathion and its reaction with quinones produced by the oxidation of phenols

‫ מגיב עם קינון וגורם למניעת צבע חום ביינות‬:‫ גלוטטיון‬- ‫ אוליגופפטיד‬

Adapted from: Ribéreau-Gayon, et al. (2007): “Handbook of Enology: The Chemistry of Wine”
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)Proteins( ‫חלבונים‬

 Proteins are macromolecules produced by linking a large number of amino

acids.

 They have molecular weights above 10,000.

 Their structure includes other bonds besides peptide bonds that give the chain
a three-dimensional configuration: spherical, helix, etc.

 Grapes and wine contain many proteins with a wide range of molecular weights
(30,000-150,000).

 Some unstable proteins are responsible for protein casse in white wines.

 Other proteins are associated with a carbohydrate fraction, e.g. yeast

mannoproteins. Other proteins are associated with a carbohydrate fraction, e.g.
yeast mannoproteins and isolectin.

Adapted from: Ribéreau-Gayon, et al. (2007): “Handbook of Enology: The Chemistry of Wine”
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Amide nitrogen

 Amide nitrogen, with the following general formula:

 This category is represented by small quantities of asparagine and

glutamine (Table 5.1). Urea is also included in this group:

‫פירוט‬
as well as ethyl carbamate which is very strictly controlled for health ...‫בהמשך‬
Reasons:

Adapted from: Ribéreau-Gayon, et al. (2007): “Handbook of Enology: The Chemistry of Wine”
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Nucleic nitrogen

 Nucleic nitrogen is present in purine and pyrimidine bases, nucleosides

and nucleotides, as well as nucleic acids. This form of nitrogen has not
been extensively studied.

Amino sugar nitrogen

 Amino sugar nitrogen consists of hexoses in which an -OH is replaced
by –NH2. Small quantities of glucosamine and galactosamine have
been found in protein nitrogen in wine.

Pyrazines
 Pyrazines are heterocycles with six links containing two nitrogen atoms
and four carbon atoms bearing radicals, which largely determine their
olfactory impact.

 They contribute to the aroma in Cabernet Sauvignon Wines.

Adapted from: Ribéreau-Gayon, et al. (2007): “Handbook of Enology: The Chemistry of Wine”
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Structure of Pyrazines

Adapted from: Ribéreau-Gayon, et al. (2007): “Handbook of Enology: The Chemistry of Wine”
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)Urea( ‫אוראה‬

‫ נוצרת מחומצה קרבונית‬

‫ דישון‬- ‫ הידרוליזה של אוראה מובילה ליצירת חומצה קרבונית ואמוניה‬
‫ מ"ג לליטר‬1 ‫ ריכוז של עד‬
‫ כנראה מהווה פרקורסור לאתיל קרבמט‬

Urea is a di-amino derivative of carbonic acid, known as carbonic

diamide. Urea may also be considered an amide of carbamic
acid

Adapted from: Ribéreau-Gayon, et al. (2007): “Handbook of Enology: The Chemistry of Wine”
 ‫בס"ד‬
Urea cycle and its relationship with the Krebs cycle. Comparative catabolism of arginine ‫בס"ד‬

under the effect of arginase and arginine deiminase

Adapted from: Ribéreau-Gayon, et al. (2007): “Handbook of Enology: The Chemistry of Wine”
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‫אתיל קרבמט (‪)Ethyl Carbamate‬‬

‫‪ ‬חומר מסרטן‬
‫‪ ‬פעם שימש כחומר משמר והיום אסור לשימוש‬
‫‪ ‬הריכוז הממוצע ביין הוא כ‪ 8 -‬מיקרוגרם לליטר‬
‫‪ ‬ריכוז מקסימאלי לפי החוק האמריקאי הוא ‪ 15‬מיקרוגרם לליטר‬
‫‪ ‬תנאים המגבירים את ריכוזו‪:‬‬
‫‪ . 1‬זן הענבים והוספת מרכיבים חנקניים לקרקע‬
‫‪ .2‬תסיסה בטמפרטורות גבוהות וארוכות‬
‫‪ .3‬תסיסה אלכוהולית עם שזרות‬
‫‪ .4‬טמפרטורת אחסון של יין מבוקבק‬

‫”‪Adapted from: Ribéreau-Gayon, et al. (2007): “Handbook of Enology: The Chemistry of Wine‬‬
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‫אמינים ביוגניים (‪)Biogenic amines‬‬

‫‪ ‬גורם לבעיות פיזיולוגיות באדם‬

‫‪ ‬נמצאים בעיקר בגבינות ‪,‬נקניקים ויין‬
‫‪ ‬נוצרים בעיקר ביינות עם מגע ארוך של שמרים‬
‫‪ ‬ריכוזם עולה במהלך התסיסה המלולקטית‬
‫‪ ‬יציבות של היין בחום על ידי בנטוניט מורידה את רמתם‬
‫‪ ‬נוצרים ע"י דקרבוקסילציה של חומצות אמיניות כדוגמת‪ :‬היסטמין ‪,‬טירמין‬

‫‪The various bioamines and the corresponding amino acids‬‬

‫”‪Adapted from: Ribéreau-Gayon, et al. (2007): “Handbook of Enology: The Chemistry of Wine‬‬
 ‫בס"ד‬
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Role of arginine in bioamine synthesis

Adapted from: Ribéreau-Gayon, et al. (2007): “Handbook of Enology: The Chemistry of Wine”
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Nitrogen Plays Many Roles During Fermentation

 Nitrogen is a key factor that has a significant impact on wine fermentation. It is the most
important yeast nutrient, influencing both fermentation kinetics and wine quality.

 Nitrogen is present in grape must in different forms: inorganic (ammonium) and organic
(amino acids, peptides and proteins).

 Nitrogen that can be used by yeast during alcoholic fermentation is called “assimilable
nitrogen.” The needs for assimilable nitrogen are different according to the wine yeast
used.

 Yeast cells also contain nitrogen in the form of proteins, peptides particularly tripeptides
and amino acids.

Adapted from: Ortiz-Julien, et al., 2016. Nitrogen Plays Many Roles During Fermentation, WINEMAKING PRACTICAL WINERY & VINEYARD
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Nitrogen Plays Many Roles During Fermentation

 YAN (yeast assimilable nitrogen) includes free α-amino acids (AA), ammonium and some
peptides.
 Inactivated yeast-based nutrients can also be used by wine yeast to efficiently carry out
alcoholic fermentation.

 Proline is the only free α-amino acid (AA) not assimilated by yeasts during alcoholic
fermentation, even if it is one of the AAs most abundant in grape must.

 The main free AAs (in quantity) in grapes include proline, arginine, glutamate.

Adapted from: Ortiz-Julien, et al., 2016. Nitrogen Plays Many Roles During Fermentation, WINEMAKING PRACTICAL WINERY & VINEYARD
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Nitrogen Plays Many Roles During Fermentation

 Nitrogen is essential for yeast growth and yeast metabolism.

 In winemaking, YAN plays a key role at two different levels:

• It represents an important nutritional factor for yeasts during alcoholic fermentation due
to its function in protein synthesis and sugar transport.

• It is essential for the biosynthesis of wine quality markers like higher alcohols, thiols
and esters by wine yeast. YAN concentrations in natural grape musts range from about 60
mg/L to 500 mg/L, depending on grape variety, vintage and microclimate.

 In nitrogen-deficient conditions, yeast growth and fermentation speed are limited.

 A low initial YAN concentration has been shown to cause slow and sluggish
fermentations.

 YAN measurement is a good way for a winery to assess the general state of grapes in
terms of nitrogen quantity, but not necessarily nitrogen quality, which is another key
parameter from the point of view of fermentation and the sensory quality of the final wine.

Adapted from: Ortiz-Julien, et al., 2016. Nitrogen Plays Many Roles During Fermentation, WINEMAKING PRACTICAL WINERY & VINEYARD
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Adapted from: Ortiz-Julien, et al., 2016. Nitrogen Plays Many Roles During Fermentation, WINEMAKING PRACTICAL WINERY & VINEYARD
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Different form of YAN

 In order to properly manage alcoholic fermentation, the best fermentation practices
recommend both the addition of nitrogen in inorganic (ammonium) and/or an organic
form.

 Organic nitrogen has been shown to be the most efficient and complete nutrient to
complete fermentation and achieve the full aromatic potential of the grape.

 In must, one-third of the nitrogen is found in ammonium form and two-thirds in amino
acids, but musts are often deficient in YAN (less than 150 mg/L).

 Nitrogen from external sources can be added to the must to assist with fermentation.

They come in two forms:

1. Inorganic nitrogen: ammonium salts such as diammonium phosphate (DAP), which is

added during alcoholic fermentation.

2. Organic nitrogen: in the form of proteins, peptides, tripeptides and free amino acids that
are part of the cells from yeast.

 When yeast cells are inactivated through various production processes (inactivated
yeast and yeast autolysate), the nitrogen found in the yeast proteins, peptides,
tripeptides and free amino acids is available for the fermenting yeast cells to use during
alcoholic fermentation
Adapted from: Ortiz-Julien, et al., 2016. Nitrogen Plays Many Roles During Fermentation, WINEMAKING PRACTICAL WINERY & VINEYARD
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Effect of nitrogen on fermentation kinetics

 YAN content is one of the most important regulators of fermentation speed; it impacts yeast
biomass at the beginning of fermentation and sugar transport kinetics during fermentation.

 As soon as must has a nitrogen deficiency at the end of the growth phase, there is a
decrease in protein synthesis and sugar transport activity.

 YAN addition to nitrogen deficient must leads to a significant decrease in fermentation length
by reactivating protein synthesis and increasing sugar transport speed, which results in an
increase in the fermentation rate.

Adapted from: Ortiz-Julien, et al., 2016. Nitrogen Plays Many Roles During Fermentation, WINEMAKING PRACTICAL WINERY & VINEYARD
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‫בס"ד‬

Adapted from: Ortiz-Julien, et al., 2016. Nitrogen Plays Many Roles During Fermentation, WINEMAKING PRACTICAL WINERY & VINEYARD
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Nitrogen impact on different compounds in wine

 Nitrogen impact on aromatic profile of wine The metabolism of nitrogen, notably from amino
acids, generates the formation of numerous aroma compounds involved in the aroma
matrix of wine: higher alcohol and acetates.

 Yeast metabolism also influences the expression/creation or preservation of certain aroma

precursors of an amino nature (cysteinylated precursors or glutathionylated precursors of
varietal thiols).

 As a result, the nitrogen composition of the must can modulate the aroma profile of a wine.
Organic nutrients have also been shown to positively influence the formation of aroma
compounds when added during alcoholic fermentation.

 Impact of different nitrogen sources on sensory profile Esters: The metabolism of

amino acids (anabolism and catabolism) by yeast leads to the formation of higher alcohols,
acetate esters and ethyl esters. The ester profile of Chardonnay (see “Ester Formation With
Organic Nutrition”) was different when different forms of nitrogen were added.

 Thiols: During fermentation it has been shown that excessive inorganic ammonium or
addition at the beginning of alcoholic fermentation limits the release of varietal thiols by the
yeast. Catabolic repression by the inorganic nitrogen in the form of ammonium prevents the
synthesis of amino acid transporters in fermenting yeast. It also limits the entry of thiol
precursors of the cysteinylated form into the cell and, consequently, their intracellular
conversion into volatile thiols.

Adapted from: Ortiz-Julien, et al., 2016. Nitrogen Plays Many Roles During Fermentation, WINEMAKING PRACTICAL WINERY & VINEYARD
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Nitrogen impact on different compounds in wine

Adapted from: Ortiz-Julien, et al., 2016. Nitrogen Plays Many Roles During Fermentation, WINEMAKING PRACTICAL WINERY & VINEYARD
 ‫בס"ד‬
‫בס"ד‬

Nitrogen impact on different compounds in wine

Adapted from: Ortiz-Julien, et al., 2016. Nitrogen Plays Many Roles During Fermentation, WINEMAKING PRACTICAL WINERY & VINEYARD
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‫בס"ד‬

Adapted from: Pretorius et al.(2007). Nitrogen management is critical for wine flavour and style. Wine Industry Journal, 2(6), 24-30.
 ‫בס"ד‬
‫בס"ד‬

Adapted from: Pretorius et al.(2007). Nitrogen management is critical for wine flavour and style. Wine Industry Journal, 2(6), 24-30.
 ‫בס"ד‬
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Adapted from: Pretorius et al.(2007). Nitrogen management is critical for wine flavour and style. Wine Industry Journal, 2(6), 24-30.
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‫א יור סכמאטי של יצירת חומרים נדיפים במהלך התסיסה האלכוהולית בשמרי יין ((‪Swiegers, Bartowsky, Henschke & Pretorius, 2005‬‬

‫…‪To be continued‬‬