Chemical analysis for the winery: Practical aspects
Anita Oberholster Introduction
What to measure? methods When to measure? Why measure? What does it mean? Juice/wine analysis
°Brix 1°Brix = 1 g of sugar per 100 g of solution Hydrometry or refractometer measures total soluble solids (90–94% sugars) Not analytical technique, only give estimation of amount of sugar Why measure? Indication of fruit ripeness Potential ethanol production in wine » Yeast vary in their efficiency to convert sugar to alcohol – could differ up to 0.8 % v/v Follow progress of fermentation Juice/wine analysis
Reducing sugars Mainly glucose and fructose Methods Rebelein method (titration with color change) Enzymatic analysis FOSS or “winescan”, HPLC (expensive) Why measure? Important for regulatory and wine style reasons, especially when aiming for specific sugar content Juice/wine analysis
pH Measure free H+ ions pH = -log [H+] Why is knowing the pH important? Microbial stability
Effectiveness of SO2 » Molecular SO2 is the form effective against microorganisms Juice/wine analysis
pH Mouth-feel (“flabby”) – wine style Color of red wine Color of red wine Juice/wine analysis
Titratable acidity (TA) Measures concentration of all available hydrogen ions (both free and bound to
dissociated acids (eg H2T) How do you measure it? Titrating with known vol of NaOH to end point indicated by color change of indicator (pH 8.2) » Expressed as g/L tartaric acid Foss, HPLC Juice/wine analysis
Why is knowing the TA important? Guide to acid taste of wine, desired amounts depends on wine style Adjust pH of wine Juice/wine analysis
Adjust wine pH Change in pH not directly related to acid addition Depends on wines buffer capacity Rule of thumb: 1 g/L of tartaric acid, decrease pH by 0.1 Add L(+)-tartaric acid DL-tartaric acid addition Increase calcium tartrate instability Juice/wine analysis
Measure malic acid (and lactic acid) conc TLC, Enzymatic analysis, HPLC or FOSS Why measure? Chances of MLF Following MLF
TLM Juice/wine analysis
+ Nitrogen (NH3, NH4 and amino acids) Enzymatic analysis or HPLC, FOSS Why measure?
N2 deficiency » Stuck fermentation
» Utilization of sulfur containing amino acids – formation of H2S
Too much N2 » Modify aroma character » Formation of ethyl carbamate
N2 needed » 100-200 mg/L needed for fermentation Juice/wine analysis
Yeast assimilable nitrogen (YAN)
Ammonia (NH3) and free alpha amino nitrogen (FAN) How to measure? + Formol titration (FAN + NH4 ) » Only need pH meter NOPA method (primary amino acids) » Need Vis spectrophotometer » Derivatisation of primary amino acid groups with o-phthaldehyde/N- acetyl-L-cysteine reagent » Resulting iso-indole derivative absorb at 335 nm » Quantification by using calibration curve using known iso-leucine concentrations
FOSS (FAN + NH3) Must/wine analysis
Sulfur dioxide analysis (free and bound) Why use? Antimicrobial and antioxidant properties Why measure? Regulatory Too little – no protection Too much – inhibit yeast, used to produce sulfides Must/wine analysis
Sulfur dioxide analysis (free and bound) How to measure Aspiration method
» Stream of air through acidified solution passes released SO2 through H2O2 solution »H2O2 + SO2 H2SO4 » Titrated with NaOH kondenseerder
» Boiling releases bound SO2 Ripper method (Metrohm)
» Titration with iodine (redox reaction), less accurate lug
0.3% H22 O because phenols and sugars also oxidized by I2 indikator
Winescan SO2 (new application) » Measure free and total SO2
wyn
25% v/v H34 PO Wine analysis
Alcohol analysis Mostly EtOH, small amount of MeOH, PrOH, BuOH and glycerol Why measure? Regulatory, TAX Mouth-feel, wine style How to measure? Distillation with hydrometry or pycnometry wyn
» Accurate (0.1% v/v) piknometer flessie
distillaat Wine analysis
Alcohol analysis How to measure? Distillation with hydrometry or pycnometry Ebulliometry –cheap and easy but less accurate HPLC, GC – accurate more expensive
boonste inlaat Densitometry
» Uses the fact that alcohol is less dense than Termometer water, so the lower the density the higher refluks kondenseerder the alcohol content
Kraan Verhittingskamer
vlam Wine analysis
Volatile acidity Volatile fatty acids (acetic acid – 90%) Why measure? Indicator of spoilage » Normal 0.2-0.4 g/L produced during fermentation » Fault 0.7-1.1 mg/L depending on wine style Regulatory (<1.2 g/L white wine, < 1.4 g/L red wine) How to measure? Steam distillation followed by titration Wine analysis
Volatile acidity How to measure? Steam distillation followed by titration (± 0.05 g/L) » Possible problems – if boil to fast, other acids also distilled
» Degas or CO2 will distil as carbonic acid A wyn
H O » Add H O to bind SO 2 2 2 2 oplossing -SO2 distilled as sulfuric acid HPLC, GC, FOSS
H2O in
Kondenseerder
H2O out
Verhittingselement X Wine analysis
FOSS (FTIR) How does it work? Juice/wine analysis
Phenol analyses Folin-Ciocalteu Measures all OH groups
» Interference from sugar, also ox SO2 etc. FC reagent (yellow) oxidizes phenols and reduces product (blue-green) measured at 765 nm [Phenol] in gallic acid equivalents (GAE) mg/L Juice/wine analysis
Folin-Ciocalteu Why measure? Phenols source of browning substance Responsible for color and taste (astringency) Aging potential Useful if historic knowledge of grapes and wine style aiming for Could adapt winemaking accordingly Range of values white table wine (40 – 1,300, av 225; red table wine 190 – 3,800, av 1,800) Juice/wine analysis
Phenol analyses Color measurements (280, 420 and 520 nm) 280 nm similar to FC – indication of total phenolics 520 nm – red color [anthocyanin]
mAU A = c 200 175
» A = absorbance 150 » = extinction coefficient 125 100 » = pathlength (cm) 75 50
» c = concentration of sample 25
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250 300 350 400 450 500 550 600 nm Corr HPLC analysis, less expensive Juice/wine analysis
Anthocyanin profile by RP-HPLC
mAU Color measurements (280, 420 and 520280 nm nm) 520 nm 280 nm similar to FC – indication of total 800 phenolics Malvidin-3-gluc 600 520 nm – red color [anthocyanin]
Corr HPLC analysis, less expensive Malv-3-gluc-p-coum
400 Polymeric pigments Malv-3-gluc-acetate Peonidin-3-gluc 200 Pet-3-gluc-p-coum Peo-3-gluc-acetate Peo-3-gluc-p-coum Petunidin-3-gluc Pet-3-gluc-acetate Delp-3-gluc-p-coum Cyanidin-3-gluc Delphnidin-3-gluc
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0 10 20 30 40 50 60 70 80 min Juice/wine analysis
Why measure color? Determine opt time to harvest Red color major quality indicator for red wine » Color corr with wine quality Aging potential Historical knowledge/values of vineyard or wine style to compare – adapt winemaking practices 280/520 ratio quality indicator » Depends on terroir » Personally found it between 1 to 3 420/520 nm ratio in wine gives hue » Indicative of oxidation, age of wine » In young wine <0.7 » Approach 1 with aging Juice/wine analysis
Phenol analyses Color measurements (280, 420 and 520 nm) 280 nm similar to FC – indication of total phenolics 520 nm – red color [anthocyanin] » Corr HPLC analysis, less expensive Why measure? Determine opt time to harvest Red color major quality indicator for red wine Aging potential Historical knowledge/values of vineyard or wine style to compare – adapt harvest date and winemaking approach Juice/wine analysis
Phenol analyses Adams-Harbertson assay Measures tannin, anth, small polymeric pigments (SPP) and large polymeric pigments (LPP) » Use combination of protein (BSA) precipitation and SO2 addition Why measure? Similar to 280 nm and FC, except more specific to tannin Best correlation to observed astringency Also give anthocyanin and polymeric pigment conc Juice analysis
Laccase activity Enzymatic analysis Activity measurement based on rate the enzyme oxidizes syringaldazine to a purple colored oxidation product Measured at 530 nm with spectrophotometer Acitivity expressed as U/mL Unfortunately not very sensitive
SO2 and ascorbic acid interfere with analysis Ranges from 0 in sound fruit to 140 U/mL 5-10 U/mL indicate some influence of laccase >10 U/mL apparent influence likely Juice analysis
Laccase activity Why measure? If Botrytis in the vineyard – knowing the number and potential risk Better adaptation during the winemaking process Wine analysis
Tartrate stability Wine is a supersaturated solution of KHT How to measure? Cold test (-2ºC - 5ºC, up to 5 days) Mini-contact test (seed with 4 g/L) Conductivity test with seeding » Short version , 30 min with 10 g/L KHT addition Why measure? Prevent crystal formation and precipitation Consumer satisfaction Important, needs to be stabilized for conditions of storage and export market Wine analysis
Protein stability Many tests, based on denaturing protein by heat, acid or alcohol Done in combination with bentonite trial to determine opt fining addition Most commonly used –heat stability test Why measure? Unstable proteins in wine can lead to development of a haze or deposit. Mainly problematic in white wine In red proteins react with tannin, precipitate during fermentation and maturation Wine analysis
Oxidative and color stability Not absolute test, indication Based on accelerated aging White wine: Heat 50°C, measure 420 nm » Evaluation base ond speed of 420 nm increse0.15 unit increase in 2 to 3 days, likely unstable Red wine: Aerate wine, compare after 3 days with control (wine + 30 mg/L SO2) » Obvious browning, oxidized character –considered unstable » If control also oxidized then very unstable Why measure? Determine aging potential Release date of wine, opt storage Wine analysis
Pinking potential Usually in white wine made under reductive conditions How to measure?
Treat wine with H2O2 and compare with control » Visible pinking or increase 520 nm – do fining trial with PVPP, casein or combination Why measure? To prevent “pinking “of wine Consumer satisfaction Wine analysis Musty taints What off-odours classified as musty taints? Fungal, earthy, moldy, corky, mushroom or straight musty What causes musty taints? Haloanisoles (TCA, TCB) Alkylmethoxypyrazine (MDMP) Carbon unsaturated aliphatic compounds with carbonyl function (1-octen-3-one, 1-nonen-3-one) (-)-Geosmin Wine analysis – Musty taints Why measure? To prevent fault Identify source of contamination Identify compound responsible 80-85% TCA responsible Bottle to bottle variation indicate cork taint Possible removal from contaminate
2,4,6-trichloroanisole (TCA) Wine analysis – Musty taints How to measure? Screening method usually based on sensory assessments of wine soaks or corks in damp environment Advantage – low cost, detect both known an unknown taints Disadvantage – variation in performance of assessors Low sensitivity and corks soaked in batches Soaked few hrs to 2 days in mostly aqueous alcohol Instrumental analysis of TCA Identify specific taints as well precursors GC-MS (expensive)
2,4,6-trichloroanisole (TCA) Wine analysis – Musty taints How to measure? No screening method reflects accurately level of taint in bottle Whole surface of cork is extracted in cork soaks Short soaking periods means only ‘rapidly released’ TCA measured Batches soaked, if one cork contaminated the average could be below detection limit However, these measurements still help predict bottle taint
2,4,6-trichloroanisole (TCA) Wine analysis Many many more chemical analyses Identify taints/compounds of interest Methoxypyrazines Ethyl acetate Volatile phenols Sulfur compounds...... GC-FID or GC-MS Wine analysis Many many more chemical analyses Phenol compounds of interest –RP-HPLC Catechin, epicatechin, resveratrol, quercetrin Gallic acid Gallic Polymeric phenols Polymeric Absorbance Absorbance Caftaric acid Quercetin-3-glucoside Catechin
Time (min) Wine analysis Many many more chemical analyses Health and regulatory pruposes Pesticides GC-MS Trace metals <300 ppb lead, <500 ppb copper Coupled Plasma MS Allergen labelling proposed by TTB Currently voluntary Milk, Eggs, Fish, Wheat, Tree nut, Peanuts, Soy, Shellfish » Allergen in the protein Enzyme linked Immunosorbent Assay – most cost effective Conclusions A lot of supportive data available Some more useful or necessary than others In more difficult years Larger need for supportive data to be able to adapt Compositional data useful in building knowledge base regarding specific vineyards and/or wines Valuable when aiming for similar wine style Determine optimal harvest date Questions?