Vol. 14, No. 4 July - August, 1999
Bruce W. Zoecklein
Department of Food Science and Technology
VPI & SU - 0418
Blacksburg, VA 24061, E-mail: bzoeckle@vt.edu
www.fst.vt.edu/Zoecklein
Table of Contents
I. Factors and Conditions to Produce a Healthy Fermentation 1
II. Formal Titration for Fermentable Nitrogen 3
III. Vintner's Corner Newsjournals on the Web 3
IV. Conducting Trials at the Winery 4
I. Factors and Conditions to Produce a Healthy Fermentation
The chemical and physical environment of grape juice fermentation, coupled with competition from indi-genous yeast and bacteria, can present significant chal-lenges to yeast growth. Individually or collectively, these factors may impact both yeast growth and the conversion rate of sugar to alcohol, leading not only to formation of objectionable odor- and flavor-active metabolites but also, to protracted, incomplete or "stuck" fermentations.
The underlying causes of stuck fermentations may vary (see Table 1). These were elaborated at the recently concluded workshop titled "Practical Issues in Primary and Secondary Fermentation." Metabolic failure ultimately results from diminished and, eventually, blocked capacity of yeast cell membranes to transport glucose and fructose into the cell. Incorporation of hexose sugars is accomplished by the action of a group of membrane-associated hexose transport proteins. Continued operation requires unimpeded mobility of the carrier proteins and sugars across the cell membrane. Fermentative growth and a variety of environmental factors, including decreasing availability of critical nutrients and increasing concentrations of ethanol and other inhibitory metabolites, may significantly alter membrane fluidity, thereby reducing the cell's ability to import sugars.
This review focuses primarily on yeast assimilable nitrogen concentration, one of the underlying causes of problem fermentations. However, as suggested by Table 1, many factors may interactively contribute to protracted and/or stuck fermentations.
Factors and Considerations to Produce
a Healthy Fermentation
Nitrogen is an essential macro-nutrient used to build protein-based cell components. Nitrogen is taken up by the vine roots as nitrate. It is reduced via a nitrate reductase system to ammonia, then transported and stored as amino acids. Compared with fermentable carbon generally present in grapes at >20% (w/v), total nitrogen levels range from 0.006-0.24%, of which only 0.0021-0.08% is biologically available to fermenting yeasts. Thus, nitrogen may become an important growth-limiting constraint for microorganisms.
The nitrogenous components of grapes and juice which are metabolically available to yeasts are present in two forms - as ammonium salts (NH4+) and as primary or "free alpha-amino acids" (FAN). Thus, a complete evaluation of the nitrogen status of juice or must requires measurements of both fractions.
In grapes, NH4+ ranges from near 30 to more than 400 mg/L. All of the 20 commonly occurring amino acids are found in grapes and wine. Their total concentration ranges from 0.4-6.5 g/L. Of these, only the free alpha-amino acid (FAN) fraction is directly assimilable by yeasts. This fraction includes arginine, serine, threonine, alpha-amino butyric, aspartic and glutamic acids. Collectively, this group comprises 35-40% of the total N and 75-85% of the total amino acids. Arginine is typically present at levels ranging from 5-10 times that of the other amino acids and represents 30-50% of the total nitrogen utilized.
The minimum level of NH4+ and FAN required for successful completion of alcoholic fermentation is about 140 mg/L. However, levels of 500 mg/L or greater are required for maximum fermentation rate. If only 140 mg/L N is available, yeast are not able to handle any adverse conditions. 500 to 900 mg/L gives yeast the ability to meet the worst environmental conditions.
The factors which influence must N have not received the attention that might be expected. This is due in part to the fact that we are only now aware of how deficient our grape musts are in assimilable N. Some of the factors influencing must N are listed in Table 2 and were discussed at our workshop. Reduced leaf area per crop weight, either from overcropping or leaf removal, lowers the amino acid content of the fruit. There is a strong negative correlation between crop level and must N. This may be particularly important when we take into account that there is a strong positive correlation between must N and wine aroma and flavor intensities. Specifically, wines produced from high N musts have a higher concentration of esters and a lower concentration of long-chain alcohols, both positive factors contributing to wine quality. A high must fermentable N concentration results in substantially quicker fermentation to dryness. This can result in an increase in low molecular weight esters, those responsible for fruity aromas, and a relatively low production of aldehydes. Additionally, a high must N reduces the likelihood of hydrogen sulfide and mercaptan formation. The nitrogen status of the must may also be important with regard to autolysis products produced during sur lie storage and sparkling wine production.
The main management problem is in establishment of adequate N in the fruit while not stimulating excessive vegetative growth. How, when, how much and in what form the nitrogen is best supplied in different environments and cultivars are important issues. Also important are the possible differences arising from supplement addition of nitrogen to the must versus having a high must nitrogen content in the fruit.
Factors
Influencing FAN and NH4+ Concentrations
Fermentable nitrogen deficiency in fermenting
juice/must is often corrected by addition of assimilable nitrogen in the form of
diammonium phosphates (DAP 25.8% NH3, 74.2% PO4 w/w)
and/or one of several commercially available nitrogen supplements. Commercial
nitrogen supplements typically contain DAP (25-50% w/w) in addition to more
complex forms of nitrogen such as yeast extract, vitamins and yeast hulls. Since
the concentration of nitrogen compounds may vary with the product, it is
recommended that winemakers consult the Material Safety Data Sheet (MSDS) for
formulation information prior to use.
Utilization of nitrogen supplements is regulated. In the United States and among OIV nations, the maximum addition of ammonium salts (DAP) is 960 and 300 mg/L respectively.
Numerous studies have demonstrated the priority of NH4+ uptake by yeasts relative to amino acids. NH4+ is not only incorporated preferentially to alpha-amino acids (FAN) but also alters the established pattern of amino acid uptake.
Traditionally, winemakers add nitrogen supplements along with yeasts at the start of fermentation. Incorporation of ammonia and then amino acids occurs primarily during the yeast's growth phase with limited uptake thereafter. The presence of NH4+ delays the uptake of amino acids. Given this, a better plan is to supplement at a stage after the yeast has incorporated available forms and to initially supplement with a balanced nutritional product, not simply DAP. This may take the form of incremental additions starting at 48 hr (for reds) and 72 hr (for whites) post-inoculation.
II. Formal Titration for Fermentable Nitrogen
The following is adapted from Zoecklein et al. Wine Analysis and Production (1995). The Formol titration is a simple and rapid method for estimating the quantity of assimilable nitrogen in juice. The procedure consists of neutralizing a juice sample with base to a given pH, adding an excess of neutralized formaldehyde, and re-titrating the resulting solution to an endpoint. The formaldehyde reacts with free amino groups of alpha-amino acids (FAN) causing the amino acid to lose a proton which can then be titrated. Free ammonia is also titrated. Thus, the procedure estimates both the NH4+ and FAN concentrations.
Materials
Sodium hydroxide solution, 1N
Sodium hydroxide solution, 0.10 N, standardized against potassium hydrogen phthalate or equivalent
Formaldehyde, reagent grade, 37% (vol./vol. or 40% wt/vol.) neutralized to pH 8.0 with 1N sodium hydroxide
pH meter sensitive to ± 0.05 pH
Calibration buffers for the pH meter
Whatman No. 1 filter paper
Method
5. Transfer a 100-mL aliquot of the sample into a beaker, place calibrated pH/reference electrodes and a stirbar into the solution, mix, and readjust the pH to 8.0, if necessary.
6. Add 25 mL of the previously neutralized formaldehyde (pH 8.0) to the aliquot, mix, and titrate to pH 8.0 using 0.10 N sodium hydroxide.
7. The concentration of assimilable nitrogen is calculated as follows:
mg/L of assimilable N = mL of 0.10N sodium hydroxide used to titrate x 28.
Notes
III. Vintner's Corner Newsjournals on the Web
A web site has been established which contains a review of our Enology-Grape Chemistry group's current research activities and abstracts from recent scientific publications. The site also contains notes and notices of meeting and events. Additionally, we have posted an index and all issues of the Vintner's Corner newsjournals since 1996. Additional library issues of the newsjournal dating back to 1990 will be added in the near future. The site address is http://www.fst.vt.edu/Zoecklein/; look under the Extension section.
IV. Conducting Trials at the Winery
All premium wineries must conduct some in-house experimentation in order to attain their stylistic goals and to help maximize quality and/or lower cost of production. Such activities can be as simple as the establishment of fining trials to evaluate a new yeast, barrel sources, treatments, etc. Such evaluations must be conducted properly to be effective. Table 3 lists some basic elements of consideration when establishing winery experimentation.
Table 3
Basic
Elements of Evaluation
Winemakers spend extensive amounts
of time using their expertise to evaluate their products. Wine is the most
thoroughly scrutinized food; numerous resources are allocated for chemical and
microbiological analysis, but the most important tools remain under-utilized.
The human senses are the most thorough analytical devices available. In many
wineries sensory evaluation is performed improperly and by a single person,
usually the winemaker. Idiosyncrasies in individual preferences can lead to
disastrous economic effects for the winery as a result of biases. Establishing
an in-house sensory panel can reduce these effects; drawing from the advice of
trained sensory judges reduces the illogical dependency on a single or few
opinions and provides an invaluable resource.
One of the most important aspects of sensory evaluation is creating a standard, defined procedure. A winery should attempt to design a plan which minimizes variables associated with product evaluation and then execute that plan consistently. This formalized program should meet specific in-house objectives, such as assessment of the bottom line, maintenance or improvement of product quality, style definition, identification of problems, definition of differences among vineyard sources, evaluation of experimental wines, etc.
V. Selecting Yeast and Bacteria
Table 4 and 5 list some of the considerations reviewed at our short course, "Practical Issues in Primary and Secondary Fermentation," regarding yeast and bacterial selection. Choices should be made with an understanding of the features listed. Contact my office for assistance.
VI. ASEV-ES OAK
Symposium
The recently concluded American Society for Enology and Viticulture - Eastern Section Symposium titled "Oak from Forest to Glass" was highly successful! Over two hundred registrants participated in this three-day program consisting of tours to World Cooperage's stave and barrel production facility, technical presentations and sensory evaluations. Proceedings are available from the society for $20 plus shipping. Contact Ellen Harkness, Dept. of Food Science, 1160 Smith Hall, Purdue University, West Lafayette, IN 47907-1160; phone: (765) 494-6704; FAX: (765) 494-7953; e-mail: harkness@foodsci.purdue.edu.