[PENDING] Practical Applications and Procedures

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P REFACE TO THE

S ECOND E DITION

Section III, Laboratory Procedures and Protocols, begins with an introduction to the use of the microscope (Chapter 12) and follows with methodologies used to enumerate and identify wine microorganisms (Chapters 13 to 16).

A CKNOWLEDGMENTS

The authors express their gratitude to the wine industry, commercial suppliers and university colleagues around the world. Finally, a special thank you goes to the undergraduate and graduate students, research staff, technicians and other employees with whom the authors have worked.

I NTRODUCTION

Although initially thought of as a major threat, some winemakers have begun to see Brettanomyces as a potential ally in the vintages of the new millennium. Hopefully, further research and experience will provide winemakers with better microbiological control during viniﬁcation, which in turn will lead to a continued increase in wine quality.

SECTION I

G RAPE AND W INE M ICROORGANISMS

CHAPTER 1

Y EASTS

INTRODUCTION

REPRODUCTION

Sexual Reproduction
Asexual Reproduction

Second, the isolate may be heterothallic and haploid and thus require a compatible mating type that may not be present in the culture. As shown in the electron micrograph of Saccharomyces (Fig. 1.1), the mother cell is left with bud scars after separation from the daughter cells.

Figure 1.1. Saccharomyces cerevisiae showing characteristic bud scars left upon separation of daughter cells

TAXONOMY

The anamorphic genus Candida represents a very broad group with a number of species found in wines. Of the yeasts found in grape juice or wine, this genus reproduces uniquely by fission.

Figure 1.3. Brettanomyces as viewed with phase-contrast microscopy at a magniﬁ cation of 1000 ×

NUTRITIONAL REQUIREMENTS

Nitrogen
Growth and Survival Factors

Like nitrogen, strains of commercial wine yeast vary in their requirements for oxygen (Julien et al., 2000). Sterols also affect the synthesis of volatile odor and flavor compounds depending on the presence of oxygen (Mauricio et al., 1997).

Figure 1.6. Reaction of ammonia with α -keto-glutarate to incorporate inorganic forms of nitrogen by Saccharomyces.

METABOLISM .1 Glucose

Sulfur
Odor/Flavor Compounds
Glycosidases
Mannoproteins

Adapted from Boulton et al. 1999) with the kind permission of Springer Science and Business Media. These compounds are distributed in various locations in the grape berry (Wilson et al., 1986).

Figure 1.7. Utilization of glucose by Saccharomyces through Embden–Meyerhof–Parnas (EMP) pathway or glycolysis.

CHAPTER 2

L ACTIC A CID B ACTERIA

INTRODUCTION
TAXONOMY
NUTRITIONAL REQUIREMENTS
METABOLISM .1 Glucose

Arginine
Malate
Mannitol and Erythritol
Diacetyl and Other Odor/Flavor Compounds

CHAPTER 3

Xiyyeeffannoo l-arginine dabaluu fayyadamuun Pilone et al. oeni dhuguma ammooniyaa oomishaa ture. Poolman fi kkf irraa kan fooyya’e. (1991) hayyama gaarummaa Joornaalii Baakteeriyooloogii irraa argateen.

Figure 2.1. Lactobacillus brevis as viewed with phase-contrast microscopy at a magniﬁ cation of 1000 ×

A CETIC A CID B ACTERIA

INTRODUCTION
TAXONOMY
METABOLISM .1 Carbohydrates

Ethanol

CHAPTER 4

This differs from the last edition of Bergey's Manual where Acetobacter and Gluconobacter were the only genera recognized (De Ley and Swings, 1984; De Ley et al., 1984). However, these bacteria can directly oxidize glucose to form gluconates and, to a lesser extent, ketogluconates (Weenk et al., 1984;. While glycolysis is either absent or very weak (De Ley et al., 1984) , some species are capable of oxidizing polols to the corresponding ketoses.

Acetic acid bacteria can produce large amounts of acetaldehyde, approaching 250 mg/L (Du Toit and Pretorius, 2002).

Figure 3.1. Acetobacter aceti as viewed with phase-contrast microscopy at a magniﬁ cation of 1000 ×

M OLDS AND O THER M ICROORGANISMS

INTRODUCTION
ECOLOGY HABITATS
TAXONOMY
METABOLISM .1 Glucose

Mycotoxins
Odor/Flavor Compounds

OTHER MICROORGANISMS .1 Bacillus

Streptomyces has been implicated in the formation of "moldy" compounds in corks as well as in wineries (Silva Pereira et al., 2000). Moreover, Silva Pereira et al. 2000) noted that cork stains may originate from other microorganisms. Darriet et al. 2001) detected geosmin present in a "mossy" Cabernet Sauvignon wine, specifically the - isomer (−), which has a much lower sensory threshold than geosmin (+).

Since geosmin has been found in freshly crushed grapes (Darriet et al., 2000), microorganisms that develop on grapes can therefore potentially produce "musty" wines.

Figure 4.1. Basic structure of Aspergillus. Adapted from Chang et al. (2000) with the kind permission of Elsevier Ltd.

SECTION II

V INIFICATION AND

W INERY P ROCESSING

CHAPTER 5

M ANAGING M ICROBIAL G ROWTH

INTRODUCTION
PRESERVATIVES AND STERILANTS

Sulfur Dioxide
Dimethyl Dicarbonate
Lysozyme
Sorbic Acid
Other Preservatives and Sterilants

FILTRATION

Perpendicular-Flow Filtration (“Nominal” or “Depth”) Depth ﬁ ltration systems use either paper pads, diatomaceous earth (“DE”
Perpendicular-Flow Filtration (“Absolute”
Cross-Flow/Tangential Filtration

CHAPTER 6

Plant phenolics are known to react with enzymes and thereby reduce activity (Rohn et al., 2002). Based on this observation, De Rosa et al. 1983) recommended that sorbates should not be used in the production of sparkling wines. The most important function of fumaric acid is its ability to inhibit malolactic fermentation (Ough and Kunkee, 1974; Pilone et al., 1974).

However, fumaric acid is broken down during alcoholic fermentation by Saccharomyces forming l-malic acid (Pilone et al., 1973).

Figure 5.1. Relative abundance of molecular SO 2 , bisulﬁ te, and sulﬁ te at different pH values.

M ICROBIAL E COLOGY D URING V INIFICATION

INTRODUCTION
NON-SACCHAROMYCES AND SACCHAROMYCES YEASTS .1 Grapes and Musts

Alcoholic Fermentation
Post-fermentation

DEKKERA/BRETTANOMYCES

Grapes and Musts
Alcoholic and Post-fermentation

LACTIC ACID BACTERIA .1 Grapes and Musts

Alcoholic and Malolactic Fermentations
Post-fermentation

ACETIC ACID BACTERIA .1 Grapes and Musts

Alcoholic and Post-fermentation

MICROBIAL INTERACTIONS

Other Interactions

CHAPTER 7

Adapted from Huang et al. 1996) and with kind permission from the American Journal of Enology and Viticulture. Adapted from Huang et al. 1996) with kind permission from the American Journal of Enology and Viticulture. Due to the increase in pH, MLF may be favorable for the growth of other lactic acid bacteria such as Pediococcus (Davis et al., 1986a).

Adapted from Edwards et al. 1994) with kind permission of the American Journal of Enology and Viticulture.

Figure 6.1. Mixture of (A) Saccharomcyes, (B) Brettanomyces, and (C) Pediococcus in a wine as viewed with phase-contrast microscopy at a magniﬁ cation of 1000 ×

H ARVEST AND

P RE - FERMENTATION P ROCESSING

INTRODUCTION
HARVEST AND TRANSPORT
FRUIT QUALITY ASSESSMENT

Soluble Solids
pH and Titratable Acidity
Microbial Spoilage

MUST PROCESSING

Enzymes
Suspended Solids
Pre-fermentation Maceration (Cold-Soak)
Thermoviniﬁ cation
Inert Gassing

PROCESSING MICROBIALLY DETERIORATED FRUIT

Enzymatic Browning
Fermentation Difﬁ culties
Clariﬁ cation Concerns
Management Strategies

JUICE STORAGE (MUTÉ)
CHAPTER 8

Open canopies favor not only air circulation but also spray penetration (English et al., 1990). Changes in the fatty acid composition of musts have also been reported (Varela et al., 1999). At least one report noted differences in volatile acidity depending on the clarification procedure (Aragon et al., 1998).

Addition of glucanases either to the juice during clarification or after fermentation results in hydrolysis of polymers (Dubourdieu et al., 1981).

Figure 7.1. Reactions catalyzed by laccase.

F ERMENTATION AND

P OST - FERMENTATION P ROCESSING

INTRODUCTION

MUST SUPPLEMENTATION

The nitrogenous components of grapes and must that are metabolically available to yeast are present as ammonium salts (NH4+) and primary amino acids, known collectively as “yeast assimilable nitrogen” (YAN). The concentration of assimilable yeast nitrogen present in must is vineyard specific, varying with climate and soil type, grape variety, roots, fertilization and irrigation practices, harvest maturity (degree of ripeness), as well as the degree of microbiological deterioration. which may have occurred before harvest (Ough and Bell, 1980; Huang and Ough Spayd et al., 1994). In the United States, the maximum level of (NH4)2HPO4 legally allowed to correct nutrient deficiencies is 960 mg/L (8 lb/1000 gal), a concentration that provides 203 mg N/L of assimilable nitrogen.

Because some ingredients in these formulations may not currently be approved for use in the United States, winemakers must petition the US.

Table 8.1. Yeast assimilable nitrogen (YAN) in 1523 grape musts obtained from Callifornia, Oregon, and Washington.

ALCOHOLIC FERMENTATION

Historical Perspective of Starter Cultures
Preparation of Starter Cultures
Strain Selection
Temperature
Immobilized Yeast

Each strain has different characteristics, including different fermentation rates and H2S production (Ough and Groat, 1978; Jiranek et al., 1995c). Relatively few yeasts (<103/mL) escape the encapsulation matrix (Yokotsuka et al., 1993), but they nevertheless carry out active alcoholic fermentation. Non-Saccharomyces yeasts are known to produce a variety of odor and aroma molecules that alter wine quality (Holloway and Subden, 1991; Gil et al., 1996; Lema et al., 1996).

In one study, Jolly et al. pulcherrima with Saccharomyces cerevisiae in Chenin blanc must.

FERMENTATION PROBLEMS .1 Sluggish/Stuck Fermentations

Hydrogen Sulﬁ de

For example, Wang et al. 2003) reported that fermentations of a synthetic grape juice medium containing only 60 mg N/. As noted by Sea et al. 1998), yeast strains that consistently produce low sulfur content are not commercially available, even with hundreds of strains screened. This observation has not been previously reported and casts doubt on the belief that adding nitrogen to grape must will always reduce H2S problems (Tamayo et al., 1999).

Although elemental sulfur used in the vineyard can also be a source of H2S (Acree et al., 1972; Eschenbruch, 1974), very high concentrations may be required on treated grapes (Thomas et al., 1993).

Table 8.3. Estimated amounts of nitrogen required by Saccharomyces in musts with different sugar concentrations as recommended by Bisson and Butzke (2000).

MALOLACTIC FERMENTATION

Preparation of Starter Cultures
Strain Selection
Timing of Inoculation

These cultures sometimes require rehydration and adaptation in diluted grape juice or wine before inoculation (Lafon-Lafourcade et al., 1983a; Krieger et al., 1990; Pompilio, 1993; Henick-Kling, 1995). Adapted from Semon et al. 2001) with the kind permission of the Australian Journal of Grape and Wine Research. To avoid the potential problems associated with early inoculation, some advocate inoculation of bacterial starter cultures after completion of the alcoholic fermentation (Ribéreau-Gayon, 1985; Krieger et al. Henick-Kling, 1993; Nygaard et al., 1998).

Malolactic bacteria can also benefit from nutrients released by yeast autolysis after alcoholic fermentation (Fornachon, 1968; Beelman et al., 1982; Henick-Kling, 1993).

Table 8.4. Chemical analysis of Chardonnay wines inoculated with O. oeni strains EQ-54 or WS-8 before (day 0) or after (day 22) completion of alcoholic fermentations.

POST-FERMENTATION PROCESSING

Aging and Storage
Adjustment of Volatile Acidity

However, because the atomic weight of N2 (28 g/mol) is close to O2 (32 g/mol), efficient displacement of the oxygen is questionable. Volatile acidity adjustment in salvageable wines has historically been achieved through blending with other wines containing a much lower level of acetic acid. Direct attempts to chemically neutralize the acetic acid are not possible due to preferential reduction of the more strongly fixed ones (tartaric and malic acids).

In this case, only the permeate containing acetic acid, ethanol and water passes through the anion exchange column, which removes the acetic acid (Figure 8.4).

BOTTLING

Regardless of whether the wine must be sterile filtered or stabilized with chemical preservatives, microbiological examination must be performed after bottling to ensure the effectiveness of the treatment and operation. Bottles must be collected off-line at (minimum) 1 hour intervals during the bottling process. Since populations of microorganisms in the packaged product should be low or undetectable, wines should be screened using membrane filtration (Section 14.5.3).

If viable microorganisms that cause wine spoilage are found, the integrity of the ﬁlter membranes should be checked.

CHAPTER 9

W INERY C LEANING

AND S ANITIZING

INTRODUCTION
SAFETY ISSUES
WATER QUALITY
PRELIMINARY CLEANING
DETERGENTS, CLEANERS, AND SURFACTANTS

Alkali
Acids
Surfactants

RINSES
SANITIZERS

Iodine
Quaternary Ammonium Compounds
Acidulated Sulfur Dioxide
Peroxides
Chlorine and Chlorinated Formulations
Hot Water and Steam
Ozone

SANITATION MONITORING
SCHEDULES AND DOCUMENTATION
CHAPTER 10

In addition, water should be analyzed two to four times a year, depending on the source (well or city). Once most of the dirt and film has been removed, the surface should be cleaned. However, gels provide additional contact time with the surface and can be used in low-pressure systems (Wirtanen and Salo, 2003).

In both cases, the temperature should be monitored at the point farthest from the steam source (eg, end of line, fill nozzles, etc.).

Figure 9.1. Examples of cationic (alkyl pyridinium), anionic (sodium oleate), and nonionic (alkylphenol ethoxylate) surfactants.

Q UALITY P OINTS P ROGRAM

INTRODUCTION
DEVELOPING QP PROGRAMS
PROCESSING AND FLOWCHART
QUALITY FACTOR ANALYSIS

Examples of Quality Factors
Preventive Measures
Critical Quality Points

CRITICAL LIMITS
MONITORING PROCEDURES
CORRECTIVE ACTIONS
RECORD-KEEPING AND DOCUMENTATION
VERIFICATION
CHAPTER 11

In this case, it is important that the winemaking staff agree, before crushing, on the preventive measures to be taken if the amount of nitrogen in the grape must is below a specified concentration. Enumeration of microorganisms (Chapter 14) should be incorporated into sanitation programs, GMPs or as part of verification. When a critical limit has been violated, it is important to determine what corrective actions should be taken (Table 10.1).

For example, this step indicates that a program has been implemented and is effective for a given operation.

Figure 10.1. Abridged wine processing scheme that can be used to establish a quality points program

W INE S POILAGE

INTRODUCTION

SPOILAGE MICROORGANISMS .1 Acetobacter

Film Yeasts

These odors are due to the synthesis of a variety of volatile compounds including 4-ethyl guaiacol and 4-ethyl phenol (Fugelsang et al., 1993; . Licker et al., 1999). Perhaps a simpler method was that described by Chassagne et al. 2005) where yeast sludge was used to remove volatile phenolics. Adapted from Chatonnet et al. 1992) and reprinted with the express permission of John Wiley & Sons Ltd.

For example, Brettanomyces is known to produce isovaleric acid, an odorous compound described as “rancid” (Wang, 1985; Licker et al., 1999).

Figure 11.1. Formation of volatile phenolics from hydroxycinnamic acids. Copyright © Society of Chemical Industry

WINE FAULTS .1 Volatile Acidity

Ethyl Carbamate
Mousiness
Post-MLF Bacterial Growth
Geranium Odor/Tone
Biogenic Amines
Acrolein
Mannitol
Ropiness
Tartaric Acid Utilization

However, more recent information (Uthurry et al., 2006) suggests that some lactic acid bacteria, particularly O. Adapted from Ough et al. 1988b) with kind permission of the American Journal of Enology and Viticulture. Evidence for this alternative pathway in lactic acid bacteria is lacking (Moreno-Arribas et al., 2003).

Adu ti agparang a β-d-glucans (Llaubéres et al., 1990) ngem dagiti dadduma pay a monosaccharides ket mabalin nga adda (Mungesa e Nadra ken Strasser de Saad, 1995).

Table 11.1. Legal limits for volatile acidity in wines worldwide expressed as acetic acid.

SECTION III

L ABORATORY P ROCEDURES

AND P ROTOCOLS

CHAPTER 12

B ASIC M ICROSCOPY

INTRODUCTION

MICROSCOPES

Magniﬁ cation
Resolution
Contrast
Fluorescence Microscopy

The first and most obvious purpose of microscopy is to magnify the image of the microorganism so that details can be easily seen by the human eye. The total magnification is the product of the contributions of the objective lens and the eyepiece (ocular). One factor that affects resolution is the refractive index of the medium between the objective lens and the coverslip on the microscope slide.

Since increasing the refractive index of the medium will result in better resolution, immersion oil is placed between the objective and the coverslip.