Chemical analysis for the winery: Practical aspects

Anita Oberholster Introduction

 What to measure?  methods  When to measure?  Why measure?  What does it mean? Juice/ 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  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 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 accordingly  Range of values white (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 or wine style to compare – adapt winemaking practices  280/520 ratio quality indicator » Depends on » 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  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 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 and/or wines  Valuable when aiming for similar wine style  Determine optimal harvest date  Questions?