I. Botrytis cinerea and Sour Rot 1

Effects of Fruit Rot on Wine Chemistry 2

Polysaccharide Instability 3

Pectin Instability 3

Glucan Instability 3

II. On Site Monitoring and Production Short Course 4

III. Department of Food Science and Technology Internship Program, continued 4

I. Botrytis cinerea and Sour Rot

Controlling the incidence of fungal degradation from Botrytis cinerea and sour rot was particurally difficult this season. The following is a review of the influence both have on fruit and wine chemistry. Botrytis cinerea is unique in its parasitology. In rainy weather, the infected grapes do not lose water, and the percentage of sugar remains nearly the same or may decrease. Although noble rot develops regularly and uniformly, pourriture grise or grey rot is normally heterogeneous. Secondary infection by other microbes may follow. Under cold and wet conditions Penicillium, Mucor, and Aspergillus sp., as well as other fungi and yeast may overgrow Botrytis (Nair 1985), and is referred to in France as vulgar rot (pourriture vulgaire). Breakdown of the grape integument provides a substrate for the growth of native ("wild") yeasts and acetic acid bacteria and may produce a condition called pourriture acide, or sour rot.

In contrast to the above, Botrytis infection followed by warm, sunny, windy weather causes berries to lose moisture by evaporation. With dehydration, shriveling occurs, and the sugar concentration increases; this is called pourriture noble, or noble rot. Growth of the mold and associated acetic acid bacteria consumes a portion of the grape sugar. However, the utilization of sugar is countered by increases in sugar due to dehydration.

Effects of Fruit Rot on Wine Chemistry

Both Botrytis cinerea and sour rot have significant influence on wine chemistry (Table 1). The largest quantitative changes occurring in the fruit as a result of Botrytis growth are those of sugars and organic acids. From 70 to 90% of the tartaric acid and from 50 to 70% of the malic acid is metabolized by the mold. However, the concentration effect resulting from berry dehydration tends to obscure these changes. Change in the tartaric to malic acid ratio leads to a reduction in titratable acidity and elevation in fruit pH.

Botrytis uses ammonia nitrogen,reducing the levels available for yeast metabolism. Additionally, thiamine (vitamin B₁ and pyridoxine (vitamin B₆) are depleted. This is a primary reason why I have suggested that wines produced from Botrytis-infected grapes generally require supplementation with nitrogen and vitamins to help avoid fermentation sticking and possible H₂S formation.

Like other fungi, Botrytis cinerea produces laccase, which catalyzes phenolic oxidation. The main nonflavonoid phenolic compounds of grapes are caffeic and p-coumaric acids, both free and esterified with tartaric acid. These are transformed to quinones by laccase, with resultant polymerization responsible for browning of the fruit. Excessive browning can generally be limited by the use of PVPP. Laccase is resistant to sulfur dioxide, cannot easily be removed with bentonite, and is active in the presence of alcohol.

In addition to laccase, pectolytic enzymes and esterases, produced by the mold, break down grape tissue by cleaving methoxy pectins thus increasing the concentration of methanol. Botrytis causes an increase in the galacturonic acid content as a result of enzymatic hydrolysis of cell wall pectic compounds. Galacturonic acid may be transformed to mucic (galactaric) acid by enzymatic oxidation and may reach must levels as high as 2 g/L. This acid can combine with calcium to form insoluble calcium mucate.

TABLE 1

'Clean' Grapes Botrytis cinerea Sour Rot

Brix 18.5 21 16.0

Titratable Acidity (g/L) 8.0 6.5 5.0

Tartaric + Malic acid (g/L) 7.2 5.2 4.4

pH 3.3 3.5 > 3.4

Gluconic acid (g/L) .5 1-5 .5

Acetic acid (g/L) 0 1.1 1.5

Glycerol (g/L) trace 1-10 trace

Ethanol (%, v/v) 0 0-trace 0.2%

Laccase (g/mL) trace 0.1-8 trace to 0.5

Glucan (mg/L) 0 247 65

Elevated levels of acetic and lactic acid are frequently seen in wines made from Botrytis-infected fruit. These spoilage acids arise from growth of yeast and bacteria associated with the mold. Aspergillus, Botrytis, and Penicillium sp. oxidize glucose to produce gluconic acid. Since gluconic acid is not utilized by yeast or bacteria it may be used as an indicator of fruit deterioration. Gluconic acid levels in "clean" fruit and in wines made from clean fruit are near 0.5 g/L, whereas in wines produced from fruit infected with B. cinerea levels range from 1 to 5 g/L. In the case of sour rot or vulgar rot, where bacterial growth occurs along with the mold growth, levels may also reach 5 g/L.

Botrytis cinerea also produces significant amounts of polyols, of which glycerol is quantitatively the most important. Quantities produced may be as high as 20 g/L. Glycerol may be metabolized by bacteria before harvest and sour rot berries often are emptied of their contents by insects. Infected fruit then develops high levels of acetic (40 g/L) and gluconic acid (25 g/L). Ribereau-Gayon (1988) suggested that the ratio of glycerol to gluconic acid indicates the "quality" of the rot. Higher ratios indicated the growth of true noble rot, whereas lower ratios suggest sour rot.

Polysaccharide Instability

One of the greatest impacts of Botrytis growth is the formation of polysaccharides that create clarification problems. Pectins are hydrolyzed by mold-produced polygalacturonase, with the formation of beta-1,2- and 1,6-glucans. In wine, ethyl alcohol causes the glucan chains to aggregate, thus inhibiting clarification and filtration. Commercially, severaly glucanases are available to minimize these clarification problems.

Polysaccharides can form protective colloids in juices and wines inhibiting clarification, fining, and filtration. In grape juices and wines polysaccharides may be in the form of pectins and/or glucans, each forming gelatinous aggregates in an alcohol solution. The following adapted from Zoecklein et al. (1995) are two simple lab procedures for determining pectin and glucan instability.

Pectin Instability

Pectins are structural components of plant cell walls. If pectins are present, the addition of pectolytic enzymes to a laboratory sample and subsequent pectin precipitation test is recommended.

Procedure: To a 25-mL aliquot of the wine containing unidentified haze, add 50 mL of a 95% ethanol: 1% HCl or alternatively, isoproanol: 1% HCl reagent.

Interpretation: Formation of gel after several minutes is indicative of pectin.

Glucan Instability

Dubourdieu et al. (1981) developed two precipitation tests for glucans. The first procedure given is for the presence of glucans in concentrations greater than 15 mg/L, the second for levels as low as 3 mg/L. Even at low concentrations, glucans can cause filtration problems. A positive test for the presence of glucans should be followed by a laboratory fining trial using glucanases and retesting.

Procedure for Glucans > 15 mg/L: Add 5 mL of 96% ethanol (vol/vol) acidulated with 1% HCl in a tube containing 10 mL of juice or wine.

Interpretation: The formation of a white filament is indicative of the presence of glucans at levels greater than 15 mg/L. Because much lower levels can cause problems, an additional test that will detect glucans at concentrations above 3 mg/L may be warranted.

Procedure for Glucans > 3 mg/L:

1. 5 mL of wine is mixed with 5 mL of 96% ethanol (vol/vol) acidulated with 1% HCl.

2. After 30 minutes at room temperature the mixture is centrifuged at 3,000 g for 20 min.

3. The supernatant is carefully removed and the precipitate redissolved in 1 mL water. The precipitate is then mixed with 0.5 mL acidulated ethanol.

Interpretation: The formation of filaments is indicative of glucans.

II. On Site Monitoring and Production Short Course.

A three-day monitoring and production short course is being planned for June 7, 8, and 9, 1997 and will be held in the northern Virginia region. The objective of this program is to provide an intensive production and on site monitoring based program to improve stylistic wine production and quality. This program is designed to increase the in-house monitoring performed by the industry and to discuss and encourage to implementation of new technologies. The program will involve demonstrations, discussions and sensory evaluation. This workshop will be for those who have experience with routine winery activities and are seeking advanced knowledge. A supplemental section will be scheduled at the beginning of this event for those who would like answers to various basic methodologies.

The workshop is for winemakers interested in receiving an integrated package of information covering such topics as: grape and wine aroma; flavor and phenol management; what's 'hot' in fermentation and problems along the way; strategies for finishing bottling and corks; related on-site monitoring (analytical procedures).

This program will be cosponsored by VPI&SU and the Viticulture and Enology Research Center, CSU-Fresno and will be a regional meeting of the American Society for Enology and Viticulture. Instructors will include Bruce Zoecklein, Department of Food Science and Technology, VPI&SU; Berry Gump and Ken Fugelsang, Viticulture and Enology Research Center, California State University, Fresno.

Registration material will be sent to each producer after the first of the year. Space will be limited. Virginia producers are encouraged to register early. There will be no provision for holding space and space will be limited.

III. Department of Food Science and Technology Internship Program (continued)

In 1995, our department, in cooperation with the Williamsburg Winery, began a annual student internship. This program allows qualified students to gain practical skills while also participating in an applied research activity. This mutually beneficial experience serves the student, our department and certainly the industry. I want to publicly thank the Williamsburg Winery for their generous support. A Virginia vintner interested in enology student interns should contact my office.