A SIMPLE TECHNIQUE FOR THE DETECTION OF MELIBIOSEFERMENTING YEASTS' LYNFERD J. WICKERHAM Associate Zymologist, Fermentation Division, Northern Regional Research Laboratory2
Received for publication, July 24, 1943
The fermentation of melibiose is given special importance in Stelling-Dekker's (1931) classification of the genus Saccharomyces. Two important groups of species are differentiated by their ability or inability to ferment this sugar. Saccharomyces carlsbergensis and S. cerevisiae, the two species which include the strains most commonly used in industrial processes, may be separated in this manner; the former ferments melibiose whereas the latter does not. Melibiose itself is seldom used in determining the ability of a strain to ferment this sugar. Raffinose, a trisaccharide which is readily attacked by almost all of the industrially important yeasts, is used instead, for on partial hydrolysis it yields melibiose and fructose. The advantages in the use of raffinose rather than melibiose are threefold: first, raffinose costs about one-fourth as much as melibiose; second, raffinose provides twice as much information as does melibiose for it gives information regarding the yeast's ability to attack both of these sugars; and third, as will be shown presently, some strains attack melibiose more rapidly when it is liberated from the raffinose molecule than when it is supplied in the form of pure melibiose. Several techniques have been devised for the raffinose-melibiose fermentation. The Dutch school uses a quantitative apparatus as described by Van der Haar (1920) for determining the amount of gas produced from a known amount of raffinose by the organism under test. If the organism ferments melibiose, it will produce much more gas than if it attacks only the fructose resulting from the partial hydrolysis of the raffinose. At the New York Agricultural Experiment Station at Geneva (Manual, 1936) the Eldredge tube is used. This consists of two tubes connected by a horizontal arm to allow interchange of gas. The raffinose solution and yeast are placed in one tube and a definite quantity of barium hydroxide in the other. During incubation, the carbon dioxide evolved reacts with the barium hydroxide to form barium carbonate. After the fermentation is over, the excess hydroxide is titrated with a standard acid solution. Again, the strains which ferment raffinose completely will cause the formation of much more barium carbonate than do the strains which cannot attack melibiose. 1 Presented before the Society of American Bacteriologists, Baltimore, Maryland, December 29-31, 1941. ' The Northern Regional Research Laboratory is one of four regional laboratories authorized by Congress in the Agricultural Adjustment Act of 1938 for the purpose of conducting research to develop new uses and outlets for agricultural commodities. These laboratories are administered by the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture. 501
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LYNFERD J.
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Zimmermann (1938) inoculated a raffinose tube with the yeast under test and after 14 days determined the amount of sugar remaining in the medium. Henrici (1941) used melibiose rather than raffinose. The fermentation took place in a conventional fermentation tube. Due to the limitations of some of these techniques and the special equipment required in others, a new simple technique based on raffinose fermentation has been devised. Basically, it consisted of inoculating a tube of 4 per cent raffinose broth, containing an insert, with the yeast under test. The basal medium contained 0.45 per cent Difco powdered yeast extract, 0.75 per cent peptone, and sufficient bromthymolblue to give a definite green color when adjusted to pH 7.0. Two milliliter quantities of the basal medium were placed in test tubes measuring 150 x 12 mm. outside diameter, containing inserts measuring 50 x 6 mm. After sterilization, 1 ml. quanties of sterile aqueous 12 per cent raffinose solution were added to the tubes. The raffinose solution was sterilized separately at 12 pounds steam pressure for 20 minutes. Inoculation was made from a young culture growing on a yeast-extract agar slant. Incubation was at 3000. The amount of gas trapped in the insert was recorded at regular intervals of time. When the fermentation had passed its maximum and the volume of gas in the insert had begun to decrease, the tube was inoculated with young cells of Saccharomyces carlsbergensis, NRRL strain No. 379, a strong melibiose-fermenting yeast. If additional gas was produced following this second inoculation, the yeast under test had failed to ferment melibiose. On the other hand, if no gas was produced, this indicated that the yeast under test had already fermented the melibiose. Figure 1 illustrates the action of a melibiosepositive strain, NRRL No. 671, and a melibiose-negative strain, NRRL No. 631. The former shows no evolution of gas following the inoculation with strain No. 379, whereas the latter does. The technique thus outlined was used in a survey of 200 strains of industrial yeasts included in the collection of the Northern Regional Research Laboratory. Seventeen strains were found which fermented both raffinose and melibiose. Nearly all of these 17 strains produced a simultaneous fermentation of these two sugars. That is, the gas readings showed a single peak of maximum gas production. The average period of time required to complete the fermentation and cause a decrease in the amount of gas in the inserts was 7 days, whereas the longest period was 16 days. Four-per cent melibiose broth was inoculated with each of the 17 strains to check the preceding results. Most of the strains caused afermentationof the melibiose within a few days following inoculation. However, one strain, No. 804, required 12 days to start a fermentation. Another, No. 636, produced no gas until 16 days after inoculation, although by the seventeenth day the insert was completely filled. A third strain, No. 236, required 14 days in one trial and 28 days in another before it began to ferment the pure melibiose (fig. 2). The fourth strain, No. 562, produced an alkaline reaction in the tube which evidently prevented the initiation of fermentation, for none took place throughout a period of 28 days. This was the only melibiose-positive strain, as judged by the raffinose test, which failed to ferment pure melibiose.
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In view of the difficulty which some strains experienced in attacking pure melibiose, it was thought desirable to change the technique where raffinose is used as the source of melibiose. Instead of inoculating with the known melibiose. fermenter as soon as the fermentation caused by the organism under test had subsided, this inoculation was postponed until a total incubation period of 100
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MELIBIOSE - STRAIN (No. 631) 80
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No.579
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MELIBIOSE + STRAIN
(No. 671) 40.
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o.579
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2
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Incubation Period, Days
8I
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u Incubation Period, Days-
TYPICAL MELIBIOSE-POSITIVE AND MELIBIOsE-NEGATIVE STRAINS OF YEASTS ON RAFFINOSE The arrow denotes the point at which the fermentation medium was inoculated with the melibiose-positive strain No. 379.
FIG. 1. ACTION
OF
Incubation Period, Days
FIG. 2. COmPARATIVE FERMENTATION BY STRAIN No. 23 OF MELIBIOSE IN THE PURE FORM AND WHEN LIBERATED FROM RAFFINOSE Note the prolonged delay preceding the initiation of gas production in presence of pure melibiose.
28 days or more had elapsed. Fifteen days after inoculation, 1 ml. of sterile water was added to each tube to replace that lost by evaporation. Eighty-five strains were run by this modified technique. Of this number, four strains were found to give a belated fermentation of melihilose. Three of these produced distinct and well separated raffinose and melibiose fermentations.
LYNFRD
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J. WICKERHAM
In two of these strains the minimum time required to start the melibiose fermentation was 18 days, while in the third strain it was initiated in 8 days. Figure 3, showing the behavior of strain No. 830, represents the type of action shown by these three strains on raffinose. As is illustrated, the attack on the liberated melibiose was relatively slow; nevertheless, when cells from this fermenting solution were transferred to a tube of pure melibiose broth, a very rapid fermentation was obtained. The fourth yeast, strain No. 264, was in a class by itself. When grown in the 4 per cent raffinose medium for 50 days it had not utilized all of the sugars present. Yet it did produce gas from melibiose. It is not known whether this strain actually should be considered as a fermentative yeast, or whether it should be considered as having produced carbon dioxide by aerobic respiration. It always produced abundant growth throughout the tube and a marked pellicle 100 0
STRAIN No. 830 0-Raffinose
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160 f-tJeIibiose X____
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16 12 20 Incubation Period, Days
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FIG. 3. DELAYED FERMENTATION BY STRAIN No. 880 OF MEUBIOSE LIBERATED FROM RAFFINOSEJ AND THE RAPID FERMENTATION OF PURE MELIBIOSE BY MELIBIOSECONDITIONED CELLS on the surface. It is the only strain which gave confusing results in this technique for determining melibiose fermentation. The detection of strains which caused a belated fermentation of melibiose from the raffinose solution indicates that for best results a long period of incubation should be used, preferably 3 to 4 weeks, although most strains give definite
results within a week to ten days. To summarize, the technique described has the advantage over methods based on quantitative evolution of carbon dioxide since it requires less apparatus and no chemical titration. It does not require contact of the fermenting medi with mercury, as in the method used by the Dutch school, for contact with mercury over a long period of time might be deleterious to the cells. This would be of especial importance in the case of strains producing a belated fermentation of melibiose. Raffinose is to be preferred to melibiose in this test, since some strains of slow melibiose-fermenting yeasts may not attack pure melibiose if
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they produce an alkaline reaction in the medium, whereas they will ferment the sugar as it is liberated from raffinose. Only in the case of one strain which had not utilized all the sugars within 50 days was the technique inadequate. REFERENCES HENRICI, A. T. 1941 The Yeasts: Genetics, Cytology, Variation, Classification and Identification. Bact. Revs. 5, 97-179. Manual of Methods for Pure Culture Study of Bacteria 1936 Society of American Bacteriologists. Geneva, New York. STELLNG-DEKK.B, N. M. 1931 Die Hefesammlung des Centraalbureau voor Schimmelcultures. I Teil. Die Sporogenen Hefen. Amsterdam. VAN DER HAAR, A. W. 1920 Anleitung zum Nachweis, zur Trennung und Bestimmung der Monosaccharide und AldehydsaLuren. Gebruder Borntraeger. Berlin. ZIMMERMANN, J. G. 1938 Sprosspilze im Wein and deren Bestimmung. Zentr. Bakt. Parasitenk., II, 98, 36-65.
