AGRICULTURAL EXPERIMENT STATION UNIVERSITY OF CALIFORNIA BENJ . 1DB WHEELER , PRESIDENT COLLEGE OF AGRICULTURE thomas forsyth hunt, dean and director H. E. VAN NORMAN, Vice-Director and Dean BERKELEY university farm school CIRCULAR No. 140. (OCTOBER, 1915) THE PRACTICAL APPLICATION OF IMPROVED METHODS OF FERMENTATION IN CALIFORNIA WINERIES DURING 1913 AND 1914. By Frederic T. Bioletti and W. V. Ceuess. The fermentation is the most important process in the manufacture of wine. Most inferior wines acquire their defects during this process. The finest grapes may yield inferior wines if the fermentation is not properly conducted. The defects increase with time and are usually incurable. On the other hand, any sound, ripe grapes, if properly fermented, will yield a sound, stable wine easily handled and kept and capable of developing the highest possibilities of the particular raw material. By the application of modern improved methods, it is possible to avoid nearly all the defects which so often occur in wines fermented in the old hap-hazard way. The most important of these methods have been thoroughly tested in the laboratory and the winery. They are very fully discussed in Bulletins 167, 213, and 230 of this Station. The number of wineries which use these methods increases every year and, during the last three or four years especially, many demands have been made on our enological laboratory for instruction and assist- ance in introducing needed reforms into wineries of all sizes. During the seasons of 1913 and 1914, a special effort was made to comply with these demands and improved methods were introduced into a consider- able number of wineries in various sections of the State. The principal reforms were the use of sulfurous acid in the crushed grapes and the application of selected yeast. The results justify fully the recommendations of the laboratory and show that the average Cali- fornia wine-maker can avoid all trouble from spoiled wine if he adopts the simple and inexpensive means described. In order to judge these results, the wines from twenty wineries in 1913 and from twenty-two in 1914 were carefully examined and analyzed. Observations were made on the methods adopted in these various wineries, which included both those which used the new methods 19119 — 2 — and similar establishments, usually in the same locality, which con- tinued the old methods. A comparison of the wines from the two classes of wineries gives very striking evidence of the value of the new methods. The wineries investigated are situated in the counties of Sonoma, Napa, Contra Costa, Alameda, Santa Clara, San Joaquin, Sacramento, and San Diego, principally in the first. The wines made ranged from very good to bad, but no spoiled wines were made in the cellars where the new methods were used. The methods of wine-making varied somewhat in the different wineries, differing principally in the care and the promptness with which the various operations were carried out. In a few, sulfurous acid and pure yeast have been in use for several years; in most, these improvements were introduced for the first time in 1913 or 1914. Below are given (1) the methods tested and the mode of application; (2) the results of the examination of the wines, and (3) a discussion of these results. METHOD OF APPLYING THE SULFUROUS ACID AND PURE YEAST. Preparation and use of the selected yeast. In the district where most of the work was done, a pure culture of "Burgundy" wine yeast was Fig. I. Materials necessary for the in- crease of the pure yeast to two gallons. grown and increased to 50 gallons at the winery by a representative of the Viticulture Division in the following way: 1. A quart bottle containing some pure yeast in solid form was 6 — nearly filled with sterilized grape juice and left in a warm place (see Fig. I and Fig. IY a, b). Hn Fig. II. "Wash boiler sterilizer used to sterilize the juice in the three-gallon demi- john. 2. In the mean while, 2 gallons of juice in a three-gallon demijohn was sterilized in a wash boiler (see Fig. II). A screen was placed under the demijohn to protect it, the demijohn plugged with cotton, the Fig. III. Boiler used to sterilize thirty gallons of juice at 165 degrees F. for growth of yeast. boiler half filled with cold water, the lid of the boiler placed on tight, and finally, the water was heated to boiling and kept boiling for about half an hour to sterilize the juice. — 4 — 3. Twenty-four hours later, the first quart of yeast was fermenting and was poured into the 2 gallons of sterilized juice in the demijohn (see Fig. I and Fig. IV c). 4. The demijohn was then left in a warm place to incubate. 5. Next, 30 gallons of juice was heated to about 165° F. in a clean iron boiler (see Fig. III). This juice was then transferred to a clean, open 50-gallon barrel covered with a piece of clean canvas and allowed to settle and cool to 90° F. (see Fig. IV d). 6. The two gallons of juice, which by this time was fermenting vigorously, was then poured into the 30 gallons of clear sterile juice previously run into an open 50-gallon barrel (see Fig. IV /). 7. The inoculated juice in the barrel was aerated several times a day by pouring it through the air back into the barrel with a large dipper previously sterilized in hot water. "When the temperature fell below 90° F. it was brought up again by the addition of hot water, added very slowly and with vigorous stirring to avoid killing the yeast by scalding. FIG. IV. A simple yeast propagating apparatus. a. Pure yeast, solid culture, from University, b and c. Sterilized juice. d. Settling barrel for fresh juice. e. Dipper to aerate yeast in "f." f. Yeast barrel. 8. Other wineries of the district, desiring to use the yeast, sterilized 40-gallon lots of juice in 50-gallon barrels by the use of steam intro- duced directly into the juice by means of a hose. When this juice cooled to 90° F., a gallon or two of the yeast produced at the first winery was supplied to each of the others and used to inoculate the 40 gallons of sterile juice. The settling and racking described under 6 are not essential. 9. When the yeast in the barrel was fermenting vigorously and was seen to form the characteristic thick scum and heavy granular sediment of the "Burgundy" yeast, the first vat of grapes was crushed. As the grapes were crushed, potassium metabisulfite solution containing 10 ounces of metabisulfite per gallon was added at the rate of 1 gallon per ton and thoroughly mixed in. About three hours later, the whole 30 to 50 gallons of yeast was added and the vat thoroughly punched to mix and aerate the yeast (see Fig. VII). Fig. V. A five-gallon demijohn marked in gallons and fitted with adjustable syphon. For details see Fig. VI. 10. When this vat was in vigorous fermentation, it was used to start the next vat of grapes treated with metabisulfite in the same manner that the first had been. This vat in turn was used to start the third vat, and so on, progressively through the vintage. In all the wineries, except the first, the yeast was grown by the wine makers themselves, without our supervision. This method has been given in detail because it is simpler and has given better results in practice than the method recommended in our Bulletin 230. It reduces the chance of the wine maker's committing any serious mistake in the manipulation of the yeast. Application of the sulfurous acid. In using any form of sulfurous acid it is necessary to measure the amount accurately, to apply it as soon as possible after the crushing of the grapes and to distribute it quickly and evenly throughout the whole mass. With potassium metabi- sulfite this is most easily accomplished by dissolving the sulfurous salt in water in a definite proportion. A convenient solution is one which 6 — contains in each gallon the amount of the salt needed for one ton of grapes. The solution used in the fermentations described here contained 10 ounces in each gallon. Figure VI shows a convenient and simple device for measuring and distributing such a solution. "When the reservoir is filled, the amount to To Crushing Sump Fig. VI. Device for measuring and regulating metabisulfite solutions. A. Reservoir (5-gallon demijohn) marked with lines indicating half gallons. a. Inlet for air and for filling. b. Perforated rubber stopper. c. Exit tube to fix the amount of solution used. d. Pinch cock to start or stop the flow of the solution. e. Screw cock to regulate the rate of flow. f . Rubber tube syphon to draw off the solution and to deliver it to the crushed grapes. be applied is determined by the level of the bottom extremity of the outlet tube; the 3| gallons in the case illustrated is enough for 3J tons of crushed grapes. The rate of flow is regulated by the screw cock attached to the rubber syphon tube /. This cock should be so adjusted — 7 — that a gallon of solution flows out during the time needed to crush one ton of grapes. The flow is started by removing the spring cock d and stopped by replacing it. The device may be placed on a board over the vat which is being filled or over the crushing sump. Fig. VII. The yeast is mixed with the crushed grapes by thorough punching or by other suitable means. The wineries used as checks employed the "let alone" or "sponta- neous" method of fermentation, where the grapes are simply crushed and allowed to ferment spontaneously. No sulfurous acid was used in these wineries. Fig. VIII. A typical "frothy" spontaneous fermentation. Pure Burgundy yeast in contrast to this gives a very quiet but rapid fermentation. No cooling apparatus was used, except in one Contra Costa County winery. Consequently, many of the fermentations, both with and with- out pure yeast or sulfurous acid became very hot; in many cases under observation, temperatures of 104 and 105° F. being reached. The results of the high temperatures are shown in the naturally fermented wines by the presence of very large amounts of volatile or " vinegar" acid. The low volatile acid in all the wines made by the new method is proof of its efficiency in producing sound fermentations even at the "sticking" temperatures of 104 and 105° F. ANALYSIS AND EXAMINATION OF WINES AFTER FERMENTATION. At about the time of the first racking, samples of each tank were sent from the various wineries to the enology laboratory. Because the amounts of volatile acid, alcohol, and sugar a wine contains and the character and number of bacteria that it carries are the most important criteria by which the soundness of a wine may be judged, most attention was given to these data. Analyses of other constituents of the wines were made, but, since they have little bearing on the points involved in this publication, they are not included in the tables. To save space and to make the results clearer, the average composition of the wines of each cellar has been given instead of an extended list of all the individual wines. The methods of analysis are those followed by California wine chemists. The bacterial contents of the wines were determined by microscopical examination. A wine at the first racking containing only yeast cells is normal; one containing large numbers of bacteria is unsound. A sound dry wine should have less than .1% volatile acid; if the percentage is above .140% the wine is considered spoiled. It should not contain more than .3% sugar unless very sound otherwise. In general, the amount of volatile acid is by far the most important test of a wine's soundness and in studying the following tables, most attention should be paid to this constituent. — 9 Table I. Composition of 1913 Wines Classified According to District and Method of Fermentation. Locality n| 2 > > p c ^2 »B S4 ELo- im (° yf CD BS 1* P JD •a o *S 1 tB Us • B ! '2. §J SBL i m «5 ra Microscopical examination .1. Fermented Usual Way — No Sulfurous Acid nor Pure Yeast. *1 2 8 9 10 11 12 15 20 4 9 6 6 5 7 7 7 44 5 5 .122 .205 .147 .072 .088 .100 .082 .122 .129 .091 .75 .86 .81 .62 .58 .61 .75 .63 .54 .56 12.27 12.26 13.16 12.75 11.94 12.21 11.61 10.70 12.15 11.43 .39 .61 .36 .46 .26 .19 .36 .71 .11 .17 Sonoma County _ Many long rod bacteria Sonoma County Many long rod bacteria Many long rod bacteria Many long rod bacteria Sonoma County Sonoma County Many long rod bacteria Sonoma County Sacramento County San Joaquin County Santa Clara County Many long rod bacteria Many long rod bacteria Many long rod bacteria Many long rod bacteria Averages 101 .118 .66 11.54 .49 Total number samples B. Fermented With Sulfurous Acid. No Pure Yeast. Sonoma Ccunty _._ Sonoma County 8 4 5 .043 1 .053 .45 .55 13.65 11.50 .41 .44 Yeast cells only Yeast cells only .048 6 | .50 12.57 .42 Total number samples C. Fermented With Sulfurous Acid and Pure Yeast. Sonoma County 3 5 6 7 14 16 17 18 19 4 11 6 2 3 7 13 8 13 .067 .055 .050 .071 .046 .049 .056 .083 .091 .55 .66 .58 .48 .77 .56 .49 .66 .51 13.15 11.45 13.90 12.30 12.20 11.68 12.00 11.73 11.90 r9A Yeast cells only Yeast cells only Yeast cells only Yeast cells only Yeast cells only Yeast cells only Yeast cells only Yeast cells only Yeast cells only Sonoma County 19 25 72 66 06 06 32 IS Sonoma County _- Sonoma County San Diego County. Napa County Contra Costa County Contra Costa County Averages 67 .066 .57 12.08 ,21 Total number samples 'The winery numbers for 1913 do not correspond with those for 1914. 10 Table II. Composition of 1914 Wines Classified According to District and Method of Fermentation. Locality 2 Off ?• s IS SB a. 8, s i « CO P a ► ► o 3 II is ■O » •to « S* -s e S| OP » M f5 ; ■ Microscopical examination A. Fermented Usual Way — No Sulfur ous Acid nor Pure Yeast. Sonoma County 1 2 3 4 5 9 .060 4 .059 1 .071 5 .060 3 .159 .59 12.91 .68 11.11 .57 11.80 .64 12.18 .68 13.06 .68 13.10 .70 ! 12.06 .60 j 11.84 .63 11.31 .78 12.66 .20 Yeast cells only Sonoma County Sonoma County Sonoma County .36 Many long rod bacteria .21 Many long rod bacteria .12 A few bacteria .10 A few bacteria .10 Yeast cells only Sonoma County Sonoma County Sonoma County _ .__ Sonoma County Sonoma County 6 7 8 9 10 1 6 7 6 5 9 1 3 1 3 4 .100 .033 .057 .045 .151 .093 .181 .049 .075 .079 .324 Sonoma County Sonoma County Sonoma County Sonoma County Contra Costa County Oakland (from San Joa- quin County grapes) 11 12 13 14 19 20 .78 .67 .67 .79 1.01 11.17 11.40 12.20 13.20 12.80 .08 .51 .40 A few long rod bacteria Many long rod bacteria . Yeast cells only Many long rod bacteria Many long rod bacteria Many long rod bacteria 68 .088 .61 12.07 .20 Total number samples B. Fermented With Use of Sulfurous Acid and Pure Yeast. g 15 16 17 18 12 21 22 16 .057 3 : .040 7 ! .048 6 .063 3 ! .045 2 .051 14 .047 5 .053 .55 11.89 .82 ! 11.49 .68 12.84 .64 12.34 .66 11.16 .50 11.07 .64 12.05 .63 12.54 .36 .28 .25 .22 .04 .09 .18 .14 Yeast cells only Sonoma County Yeast cells only Yeast cells only Veast cells only Yeast cells only Napa County Yeast cells only San Diego County Yeast cells only .052 56 .62 12.07 .24 Total number samples — 11 DISCUSSION OF TABLES. Table I. 1913 Wines. The most striking features of the analyses of the 1913 wines are the very high volatile acid, sugar, and bacterial contents of the wines made in the usual way. This indicates a large number of "stuck wines"; i. e., wines in which yeast fermentation has been stopped by high temperature or other causes and in which acid- producing bacterial fermentations usually set in. Thirty of the 101 naturally fermented wines were above the legal maximum of .140% in volatile acid. Such wines are ' ' spoiled ' ' and fit for distilling material only. Forty-nine of these wines, or approximately 50%, were above .1% of volatile acid, the maximum for a sound new wine. In other words, approximately one-third of the spontaneously fermented wines were spoiled and one-half of them would spoil if not given special treatment to check bacterial action. On the other hand, none of the wines made by the improved method were above .140% volatile acid and only three out of a total of 67 wines were above .1%. The wines in both cases were made from similar grapes and most of the wines under observation reached very high temperatures during fermentation owing to the very hot weather during the fermenting season. Temperatures above 100° F. were the rule and many vats both with and without sulfurous acid and pure yeast reached a temperature of 105° F. But the behavior of the wines in which the sulfurous acid and pure yeast were used was very different from that of those fermented in the ordinary way. In the former wines, the yeast recovered when the wines cooled and fermentation of the remaining sugar was completed without any appreciable injury to the quality of the wines. But the yeast in the spontaneously fermented wines did not recover and after the wines "stuck" at 105° F., vinegar and " mousey- wine " bacteria increased rapidly and turned the wines more or less vinegar sour. This demonstrates that it is possible to obtain sound fermentations even at high temperatures when sulfurous acid and selected yeast are used. Table II. 1914 Wines. The fermenting season of 1914 was excep- tionally cool. Hot fermentations were therefore less common than in 1913 and spoiled wines were rare. But even under these exceptionally favorable conditions, ten out of a total of 68 spontaneously fermented wines were spoiled, i. e., contained more than .140% volatile acid and 15 were above the limit of .1%. On the other hand, all of the wines fermented with sulfurous acid and pure yeast were below .140% and only two out of a total of 56 were above .1%. The average volatile acid (.0878%) of the wines made in the usual way was 68% higher than the average (.052%) of the wines made by the improved methods. — 12 — Table III. Summary of Volatile Acid Content of 1913 and 1914 Wines. Year and method of fermentation Maximum volatile acid, per cent Minimum volatile acid, per cent Average volatile acid, per cent Per cent of | Per cent of wines above | wines above .14 per cent .1 per cent volatile acid volatile acid Number of samples analyzed 1913 ordinary fermentation 1913 SO- only .290 .053 .118 .537 .085 .044 .088 .026 .023 .023 .1180 .0480 .0660 .0878 .0520 29.7 14.7 48.5 4.4 22.0 3.6 101 6 1913 SO2 and pure yeast 67 1914 ordinary fermentation 1914 SO2 and pure yeast . 68 56 The summary shown in Table III brings out some interesting facts. It is to be noted that the maximum volatile acid of .118% found during 1913 in a wine fermented by the new methods was exactly the same as the average for all the wines made in the old way. In 1914 it was slightly less. The maximum for 1913 was slightly above the normal limit for a new wine, that of 1914 slightly below. This indicates that, where the conditions are unfavorable, as in 1913, and the cellar work inferior, even the use of sulfurous acid may be insufficient to save the wine. It may, however, simply mean that in the fermentation of this particular wine the method was imperfectly applied, which is very probable where so many cellars and so many wines are involved. The excessively high maximum of .535% in 1914 for a wine fermented in the ordinary way shows that even when the weather conditions are favorable, careless methods may result in absolutely spoiled wines. On the other hand, the extremely low minimum of .023% for the same year shows that under favorable conditions a good wine maker can make good wines by the old methods. In the winery where this wine was produced, however, the average volatile acid of the wines of 1913, when the conditions were unfavorable, was .088% while that of wines made in wineries using the new methods and representing all degrees of skill and lack of skill was only .066%, indicating that even great care is sometimes insufficient with the old methods. Table IV. Relative Stability of Spontaneously Fermented Wines and Wines Fer- mented With Pure Yeast and Sulfurous Acid as Indicated by Changes in Volatile Acid. Wine Volatile acid before incubation at 95° F., per cent Volatile acid after six days' incubation at 95° F., per cent Increase in volatile acid, per cent Blend of all Sonoma County naturally fermented wines, 1913 .180 .075 .240 .080 .060 Blend of all Sonoma County wines fermented with .005 Relative Stability of Wines Made by the Two Methods. The stability or keeping quality of a wine may be judged by its resistance to warm temperatures. A blend of all the samples from Sonoma County made — 13 — by the usual method in 1913 and a blend of all the wine from the same district made by the new method were incubated in well-filled and corked demijohns at 95° F. The volatile acid content before and after incubation was used as a measure of the amount of deterioration taking place. Table IV shows the results. The volatile acid of the wine made by the usual method increased by one-third in six days while that of the wine made with the aid of sulfurous acid and pure yeast remained practically stationary. In other words, the former deteriorated very rapidly at 95° F. for the short period during which the wines were under observation, while the latter remained stable. This would indi- cate a similar ratio of stability during aging of the wines at the ordinary cellar temperatures. Table V. Sulfurous Acid (S0 2 ) Contents of Wines Fermented With Metabisulfite and Pure Yeast. Number of samples analyzed .. 26 wines Average amount of SO2 added to grapes— mg. per liter 175 mg. Average amount of SO2 found in wines at first racking _. 64 mg. Maximum amount of SO2 found in wines at first racking 1!)2 mg. Average loss of SO2 to time of first racking (63.5%) 111 mg. Amount of Sulfurous Acid Remaining in Wines at First Backing. The pure food law allows a maximum of 300 milligrams of sulfurous acid per liter or .03%. An examination of Table V will remove any doubt as to whether wines treated with the usual amounts of metabisul- fite during fermentation retain an excessive amount of sulfurous acid. The maximum found was 192* milligrams of sulfurous acid per liter, while the average was only 64 milligrams per liter or .0064%. That is, the average was only about 21% of the legal limit and the maximum only 64%. The crushed grapes were treated with approximately 10 ounces of potassium metabisulfite per ton, which corresponds to about 170 to 175 milligrams sulfurous acid per liter or .0170 to .0175%. Thus, roughly speaking, two-thirds of the sulfurous acid disappeared between the time of crushing and first racking. SUMMARY AND CONCLUSIONS. 1. Pure yeast and sulfurous acid can be applied successfully, on a practical scale, by the average wine maker by the method outlined in this publication. 2. The application of this improved method resulted in more perfect fermentations and in sounder wines, showing marked improvement over wines fermented in the usual way in the same locality. The improved method gave sound wines even where the excessively high temperature of 105° F. was reached during fermentation, while the . *This wine undoubtedly received more than the amount of sulfite recommended. — 14 — wines made in the usual way in such eases "stuck" and became vinegar sour. 3. Sulfurous acid alone seemed to give just as sound wines as where pure yeast was used in addition to the sulfurous acid. 4. Wine made by the improved method kept a great deal better at warm temperatures than the spontaneously fermented wine. 5. The amount of sulfurous acid left in the wine is well below the pure food limit. 6. The observations made in 1913 and 1914 confirm fully the con- clusions given in our Bulletin 230.