Pineapple Wine Chemical Characterization

• The study chemically characterizes pasteurized and non-pasteurized pineapple wines made with different Saccharomyces cerevisiae strains.
• The parameters used are pH, titratable acidity, and organic acid composition.
• High-performance liquid chromatography, using a Supelcogel C-610H ionic exchange column, was used to evaluate organic acids.
• The wines' pH ranged from 3.99 to 4.50, while the titratable acidity ranged from 0.336 to 0.491 g citric acid/100 mL.
• Citric, malic, succinic, acetic, formic, and butyric acids were the main acids identified.
• The wine samples are stable, with high volatile acid concentrations influencing the final product's organoleptic and chemical stability.
• The study analyzes wines made from Red Spanish pineapple varieties, classifying them as white wines due to their final color.
• Pineapple has a soluble solids content of 10.33°Brix at 20°C, a pH of 3.49, and a titratable acidity of 1.17 g citric acid/100 g.
• Puerto Rico is ideal for high-quality pineapple wine production due to its soil and climate conditions.
• Fermentation, fruit variety, microorganisms, and aging time influence wine's sensory characteristics.
• Wine acidity is a critical characteristic, affecting its final properties, conservation, stability, and organoleptic qualities.
• Wine acidity is determined by the composition of acids, including those from the fruit, fermentation, and inorganic acids.
• This study evaluates the impact of different yeast strains and pasteurization on organic acid content, pH, and titratable acidity.
• It also aims to identify significant differences in these parameters among the pineapple wine samples.

Alcoholic Fermentation in Wine

• Alcoholic fermentation generates compounds like higher alcohols, esters, aldehydes, ketones, sulfur compounds, and organic acids.
• These compounds are secondary metabolites that influence the wine's organoleptic properties.
• Higher alcohols affect organoleptic characteristics, but their perception thresholds are higher than esters and diacetyls.
• Higher alcohols derive from amino acid metabolism, including 2-methyl-1-butanol, 3-methyl-1-butanol, isobutanol, phenylethanol, and n-propanol.
• Higher fermentation temperatures and must oxygenation increase the synthesis of these compounds.
• Yeast strains impact the production of these compounds.
• Selecting yeast strains is important to avoid undesirable compounds.
• Many wine aromas come from the must, but others form via metabolic pathways during fermentation.
• Studies show yeasts influence creating certain volatile compounds in wine.
• Studies determined volatile compounds in fermented grape must using yeasts like Hansenula, Kloekera, and Saccharomyces cerevisiae.
• The production of compounds like 1-propanol, isobutyric alcohol, 1-butanol, isobutyric acid, and ethyl isobutyrate depends on the yeast strain.
• There are differences in the quantities of volatiles produced by Kloeckera apiculata and Saccharomyces cerevisiae.
• Kloeckera apiculata produces more 1-propanol and isobutanol but less acetic acid than Saccharomyces cerevisiae.
• The aroma of wine is influenced by hundreds of compounds and can be assessed via chemical methods or sensory analysis.

Factors Influencing Wine Quality

• pH, total acidity, and organic acid composition determine overall wine acidity.
• pH affects yeast growth, tartrate salt solubility, sulfur dioxide effectiveness, protein solubility, bentonite effectiveness, color pigment polymerization, and oxidation/browning reactions.
• Most wine acids are weak organic acids that partially dissociate.
• Acidity exists in undissociated and dissociated forms, measurable by pH.
• pH indicates hydrogen ion presence but doesn't directly relate to total acidity.
• Higher total acidity results in lower pH and greater perceived acidity.
• White or red wine pH ranges from 3.5 to 4.0.
• Total or titratable acidity is the sum of all acids, titratable with a strong base like sodium hydroxide and expressed as the most abundant acid.
• Total acidity ranges from 6-12 g/L in grape wines, depending on the grape type.
• Total acidity includes fixed acidity from non-volatile organic acids and volatile acidity mainly from acetic acid.
• Volatile acidity should be minimal in quality wine, ranging from 0.2-1 g/L.
• Total acidity is determined through titration using a potentiometer to pH 8.1-8.2 or with phenolphthalein until color change.

Organic Acids

• Organic acids are important wine components.
• These acids originate from grapes/fruit or are produced during alcoholic fermentation or other processes, including acetic, propionic, malolactic, and butyric acids.
• Acids affect stability, act as preservatives, and influence color, organoleptic properties, and flavor; acidity, or pH, influences the acidic taste.
• Free acids generate the acidic flavor in wine.
• Excessive acidity creates unpleasant sharpness, while insufficient acidity results in fragility and a pasty flavor.
• Good wine presents a balanced acidity that allows perception to be graduated.

Types of acids in wine

• Tartaric acid is abundant in plants, especially grapes and adds mature fruit flavors.
• It precipitates naturally as tartrate salts, forms crystals when alcohol is present, and the temperature is low.
• Tartaric acid levels in wine range from 5-10 g/L.
• Over time, it decreases as it forms ethyl bitartrate via esterification.
• Malic acid, prevalent in fruits like apples, contributes a green apple aroma.
• It adds a greenish hue in white wines, undesirable in red wines.
• Malic acid plays a role in wine technology through malolactic fermentation, carried out via lactic acid bacteria like Oenococcus oeni, Lactobacillus spp, and Pediococcus spp.
• Malolactic fermentation deacidifies by converting L-malic acid to L-lactic acid, decreasing titratable acidity and increasing pH.
• It also improves microbiological stability, organoleptic quality and aromatic complexity.
• Oenococcus oeni is used to carry out the fermentation, due to high alcohol and low pH tolerance.
• Acidity can be corrected in wine using chemical or biological methods.
• Using Oenococcus oeni bacteria is an alternative in the wine industry.
• Citric acid, a tricarboxylic acid in most fruits, impacts a marked acidity.
• Citric acid provides fruity and aromatic sensations in wine at concentrations of 100-300 mg/L.
• Citric acid tends to disappear in wines undergoing malolactic fermentation due to being easily metabolized by bacteria.
• Products obtained through the metabolism of acids: acetic acid, diacetylacetone, and 2,3-butanodiol
• Succinic acid, produced during alcoholic fermentation, is stable during lactic fermentation and storage.
• It has a mixed taste of acidic, salty, and bitter flavors, contributing to the characteristic wine flavor.
• Acetic acid is present in high concentrations and is known as the acid of vinegar, this is negative.
• Normal concentration is 0.15 to 0.6g/L.
• It forms through ethanol oxidation by acetic bacteria, oxidizing the ethanol to acetaldehyde, then oxidizes the acetaldehyde to acetic acid.
• Also appears through citric acid degradation combined with lactic acid bacteria.
• Lactic acid originates from alcoholic fermentation or malolactic fermentation.
• It transforms lactic acid, due to lactic acid bacteria.
• Lactic acid creates a flavor similar to dairy, with a normal concentration in the wine of 0 - 2.5g/L.
• Butyric acid is uncommon in wines, produced by anaerobic bacteria like Clostridium butyricum via butyric fermentation.
• Butyric acid contributes with a rancid flavor, undesirable in wines, even a good taster is able to identify it.

Organic Acid Determination

• Organic acid identification and quantification in fruits are crucial due to their unique compositions and impact on organoleptic qualities.
• Composition and concentration are important parameters in wine.
• It evaluates the advancement of alcoholic fermentation and helps identify undesirable fermentations like acetic or butyric fermentation.
• Organic acids have been determined using enzymatic procedures, paper chromatography, gas chromatography, and liquid chromatography.
• Enzymatic methods are standard for organic acid determination, recognized by the International Wine Office in Paris and the American Association of Analytical Chemists.
• High equipment costs and time are main drawbacks of the enzymatic methods.
• High-performance liquid chromatography (HPLC) is rapid and simple and is used by many researchers.
• High-resolution liquid chromatography is a separation technique for analyte identification and quantification.
• It allows qualitative and quantitative identification of separated species.
• HPLC involves a liquid mobile phase, a stationary phase in a column, and analyte separation.
• HPLC is sensitive to quantitative determinations, and enables separation of non-volatile species, and can be applied to substances of interest to any sector.
• HPLC separates amino acids, proteins, nucleic acids, organic acids, hydrocarbons, carbohydrates, drugs, pesticides, and antibiotics.
• Two types of separations are commonly used for organic acids in wines: ion exchange and reverse phase.
• Reversed-phase HPLC (RP-HPLC) uses a non-polar column support like octylsilane (C8) or octadecylsilane (C18).
• RP-HPLC uses binary elution systems, and ultraviolet detectors.
• RP-HPLC is used to separate tartaric, citric, malic, lactic, acetic, formic, shikimic, fumaric, and succinic acids.
• The biggest problem is ensuring preprocessing of the sample.
• Interferences are caused by anthocyanins and polyphenols.
• High-resolution liquid chromatography uses ion exchange in the determination of organic acids in samples (juices, vinegar and wines).
• Ion exchange separates neutral and acidic compounds based on ionic exclusion.
• The stationary phase is an ion-exchange resin acting as a semipermeable membrane.
• Ion-exchange chromatography separates organic acids based on their pKa values.
• A strongly acidic resin acts as the stationary phase.
• Non-electrolytic or basic substances are retained, and acidic substances elute first.
• The mobile phase pH is important and requires an isocratic elution.
• Ashoor et al. (1984) determined acetic acid in foods with an Aminex HPX87-H column, using isocratic elution, and aqueous sulfuric acid as the mobile phase.
• Ashoor et al. (1984) used an ultraviolet detector at 210 nm, which proved both specific, precise and exact.
• Morales et al. (1998) identified and quantified citric, malic, tartaric, lactic, and acetic acids in balsamic, sherry, and cider vinegars using an Aminex HPX87-H column.
• Morales et al. (1998) used isocratic elution with sulfuric acid and ultraviolet detection.
• Picha (1985) identified and quantified malic, citric, and succinic acids in sweet potatoes using an Aminex HPX87-H column, isocratic elution with sulfuric acid.
• Picha (1985) used ultraviolet detection at 214 nm.
• Walter et al. (2003) compared enzymatic and ion-exchange chromatography to analyze organic acids in syrah and cynthiana wines.
• Walter et al. (2003) employed an Aminex HPX87-H column, isocratic elution with sulfuric acid, and diode array detection.
• Walter et al. (2003) were able to identify and quantify several organic acids, including citric, malic, succinic, tartaric, lactic, and acetic.
• Walter et al. (2003) found that ion-exchange chromatography is more accurate, easier, and faster than enzymatic methods.
• Supelco created the Supelcogel C-610H, which separates organic acids, carbohydrates, and alcohols in juices, wines, fermentation products, and biological samples.
• The Supelcogel C-610H separates around 20 organic acids.
• Studies on organic acid analysis in fruit wines, especially pineapple, or analyzing pasteurization's impact on pH, titratable acidity, and organic acid composition are lacking.
• Okoh et al. (1997) made pineapple wine using a ceta de la palmenta (Elaeisguineensis) yeast species, with 15% alcohol, pH 3.2, and 1.52% titratable acidity.
• Okoh et al. (1997) found pineapple wine sensorially acceptable in general through an analysis.

Materials and Methods

• Pineapple wines were previously prepared using the project "Elaboration of wines and brandys of high quality from tropical fruits" (TSTAR 101).
• Pineapple wines were prepared from Red Spanish pineapples using: Montrachet Red Star (ATCC 36026), Montpellier (K1-V1116), I.N.R.A. Narbonne (71B-1122), Rhône (IVC-GRE), Prise de Mouse (EC-1118).
• Pineapple wine samples were taken randomly from those stored in the cold room of the Piñero building.
• The samples were stored for 6 months.
• Part of the wine samples underwent pasteurization, detailed in section 3.5.
• Wine pH was measured using an Accumet Research AR 15 pH meter (Fisher Scientific), calibrated with buffer solutions of 4.00 and 7.00.
• 10 mL of each sample was dispensed in a 50ml breaker.
• Measurements were taken in triplicate.
• Total acidity was determined following the AOAC (962-12) and American Society of Enology methodology.
• 50 mL of analyze wine, was transferred to a 125 mL Erlenmeyer flask and CO2 was removed.
• 25 mL aliquot was degassed, weighed, added to 25 mL of distilled water
• Titration with sodium hydroxide solution until pH 8.1 and 8.2, by pH meter.
• % Titratable acidity = (V NaOH x N NaOH) x 64.04 x 100 / V sample (mL), in citric acid.
• A Bausch & Lomb refractometer determined wine's degrees Brix (°B), was calibrated, and was taken three times with a Pasteur pipette and a sample.
• Organic acids in wines were measured via high-resolution chromatography.
• 4.00 mL of wine was measured was transferred to a centrifuge tube and added to 8.0 mL of HPLC grade water (Fisher Scientific)
• The sample was centrifuged at 3000 rpm for 10 minutes.
• 1.0 mL of liquid over flow was injected in a Solid Phase Extraction in the SCX ion exchange (Propilsulfonilbenceno) (500mg/3mL) (Varian) that was previously activated.
• The SCX column was washed with 2.0 mL of HPLC grade water.
• The liquid was collected in the volumetric flask at 10mL and completed with a fosforic avid at 0.1%.
• 3.0 mL were taken for the solution at 0.20 um of Nylon.
• 25 µL were manually injected into an HP 1100 series HPLC (Hewlett Packard, U.S.A) system.
• The instrument contains a supelguard C610H column measuring 5 cm x 4.6 mm ID and a supelcogel C-610H ion-exchange column of 30 cm x 7.8 mm ID (Supelco, Bellefonte, PA).
• The mobile phase was H3PO4 at 0.1% at pH 2.2. The flow conditions were mobile phase flow with 0.5 mL/min, pressure with 43 bar and temperature at 20°C.
• Identification of the acids conducted at 210nm using variable wavelength.
• Peak integration was acquired by Hewlett Packard Integrator (HP3396 Series III, U.S.A). Each sample injected in triplicate, retention times of each organic acid were identified.
• Each peak identification was conducted in chromatograms standard of organic acids
• Standard solutions of 10 ppm were prepared, and were injected 3 times
• Retention times determined at different temperatures as 20 °C, 25 °C and 30 °C, in order to vary with retention times and thus give it better reliability in identification
• It was utilized H3PO4 as a diluted solvent at 0.1% with an pH 2.2 for each solution. And it was injected by triplicate
• A calibration curve was made and deviations, coefficient variations, precision, regression line and determined equation

Data Analysis of Acids

• To quantify the organic acids, 10,000 ppm stock solutions with each of the potential standards with a concentration from 5 ppm to 120 ppm
• It was filtrated with all Nylon, where stock solution was filtered a. 0.20um Nylon
• For each acid, a calibration curve is built and then determined deviation and coefficient
• Standard deviations and coefficient variations were also determined to assess precision.
• A regression line was determined and the calculation of concentration was identified

Limit of Quantification of Acids

• Determined was the quantification limit for each measured acid organics to precise accurate solutions and a detection of 5%, and a variation to a known concentration
• To calculate a performance extraction, cleanliness of each process was measured and determined what percentage it contained
• Each addition was identified, followed by the standards at curves to measure it

Pasteurization of wine samples

• Samples of wines were put in a sterilization of analysis to analyze. The process meant of introducing to an bottle of wine in an oil bath.
• Then was submerged until a thermometer reached 76 degrees and kept for 15 seconds.
• Results of titratable acidity acids, concentrations, and each test was measured through through variance analysis, to determine a statistical differences.

Results and Discussion

• pH results for pasteurized and unpasteurized wine samples are given using the averages and standard deviations per three measurements.
• The pH value was a bit above from literature's range of value, the acidic pH range restricts most microorganisms
• The range was important since it restrains microorganisms from damage. It is important to mention that pinapple fruits have an acidic range of 3.49 and is it expected
• The analysis to determine effective significant differences, variance was utilized at .05 significance was utilized. Each value is at less them with different values with an measurement of features are different.
• EC-118 is an most acidic measurement with more value but also Montrachet is. It is was related in study of Perea. It was evaluated with taste using the measurement from Davis. The test came as with those measurements were those of a high score with taste test but no direct measurement of acid.

Titratable Acidity

• Titratable acidity values of wine show the average on tested sample, for literature it shows measurements to acidity in grape at value range of .6 to 1.2g/100 dependent on grape type.
• To all the tested values it shows value is at most cases are below the amount, for pinapple the level for titratable acidity is based with is more higher on main fruit
• Per variance test to value the tested value p was less then alpha value so it does not shows high difference. But by a Fisher measurement test that those without measure to EC-11 and 71B-1122 for those that aren't put under sterilization to the others with
• K1-V1116 a and 71B-112 is not different. Acidity and and ph were measured
• Also if amount value that is low at Ph is acidity high, low has PH that's high was of measure small but due not to that measurement is doesn't effect ph.

Degree Brix of Pineapple wines

• These all were show as measurements based on standard variance as by 3 and all but one the 7133 had at same value at a small at an 77 that 76 value it is considered a good value.
• organic acids to 42 c that it is a better seperation

Organic acids by HPLC

• Organic analysis for HPLC tested is that tests at 20. This process is so temperature is better in the samples, to all these tests. Citric, Malico, Succinico, formico butirico, and acetico they were recognized.
• There deviancies were at 5%
• By data to data samples are is that acidity by fruit are the highest amount.
• The 2 yeast V116 and 7 11-1122 was agreeable and the value had that higher amount.

ANOVA results

• ANOVA testing indicated differing levels within tests
• Krebs has a different measure so it had citric to it