By Donald D. Brumsted and Paul R. Glenister,
J. E. Siebel Sons' Co., Inc., Chicago, IL
Published in October 1963

Figure 1: A photomicrograph of the living cells of the yeast isolateused athe test orgaism in these experiments. The cells were cultured on a wort agar slant. (ca. 2,000 x)

The success of bulk pasteurization is largely dependent upon the aseptic packaging of the pasteurized beer. The conditions under which bulk pasteurized beer is packaged are generally not aseptic in the strict sense of the word; contamination may enter from the air of the bottle shop, from the air used for counterpressure, from the filler, from the crowns, or from any surface the beer may contact as the pasteurizer to the package. In spite of all this, practical experience with bulk pasteurized beer indicates that its bio-stability is generally very good. A possible explanation for this may be that beer, with its relatively low pH, its high CO, content, its hop extractives, alcohol, diacetyl, etc., is an unsuitable medium for the growth of many of the microorganisms which might enter it.

In this present paper we wish to present our findings in a study of yet another factor which can influence the growth of microorganisms in beer. This factor is the number of viable cells which enter into the beer. Evidence to be presented here indicates that an entering population (i.e., an inoculum) of a yeast may fail to grow in beer simply because it is too small to survive the period of initial adjustment to the beer as a nutrient medium. The wild yeast species chosen as the experimental organism for this series of experiments is only one ex ample of the many different microorganisms which could become a nuisance in the packaging of bulk pasteurized beer.

A number of samples of wild yeast from our culture collection were inoculated separately into bottles of commercial pasteurized beer. The bottles were recrowned immediately after the inoculations and the beers were incubated at 30 degreesC. for eight days. These yeasts did not behave similarly: one of them, an isolate from a brewery source, proved to be a very active beer spoiler (see Table I ) . The biochemical and cultural properties of this isolate were characteristic of Saccharomyces pastorianus as recorded in the literature (7). This isolate was selected as the test organism for this series of experiments.

This chosen yeast was put into bottles of four different brands of commercial pasteurized beer which were available to us in quantity. As Table II shows, these beers differed in their susceptibility to attack by the yeast. Brand "L" of the table was used for the experiments described in this report.

Figure 2: A photomicrograph of a prepared smear of the yeast isolate used as the test organism in these experiments. Smear was stained with malachite green and safranin to show the spores. Arrows indicate intact asci containing spores. Sporulation occurred on filter paper soaked with one per cent aqueous sodium acetate solution. (ca. 2,000 x)

The work already described was done using beer in bottles, and all inoculations and transfers were done by opening and recrowning the bottles. During these operations there was undoubtedly some loss of carbonation and some entry of oxygen into the beer. To avoid these changes in the beer during the actual experiments, we conducted all the operations with the beer contained in cans fitted with self-sealing rubber stoppers in their bottoms (see Figure 3). This arrangement permitted us to have the cans filled with beer, sealed, and pasteurized, and, by means of a hypodermic syringe, to inject suspensions of yeast cells into the beer and to withdraw portions of the beer for examination and plating tests with only minimal risks of decarbonation or of air entry. The syringes used were 5 cc hypodermic syringes with "Luer Lok" fittings and #B-D22 needles. The syringes were sterilized by boiling them in water for three minute periods.

For the purposes of the counting, yeast buds were not considered as individual cells. A series of counts and dilutions was made until a suspension was obtained which contained approximately one yeast cell per ml. To test the accuracy of the dilutions, the final suspension and a number of others were plated out on Plate Count Agar (Case Laboratories, No. 411). The results of the platings are recorded in Table 1ll: they indicate that one may work with extremely dilute cell suspensions of the yeast with reasonable assurance that the numbers of cells believed to be present are, in fact, present and viable.

First Test Series

Regular production-run beer of Brand L was sealed and pasteurized in the special cans already described, and inoculations were made with the beer at 5 degrees C. This temperature was used so as to minimize the gas pressure in the can and thereby facilitate the process of injecting the yeast suspensions into the beer. The cans of beer were then held at 30 degrees C. for incubation. Later, to measure the development of the yeast in the beer, the cans of beer were shaken and portions were subtracted, using the hypodermic, for plating tests.



Our findings are presented in Table IV. It will be noticed in the table that the beer in cans "G", "H" and "J" did not show any growth of yeast even after 104 days of incubation at 30 degrees C. To check on this, the beer in each of these three cans was passed separately through membrane filters and the membrane filters were incubated with nutrient at 30 degrees C. No yeast growth could be detected. We are confident that the cans received the anticipated doses of yeast cells because control platings of the yeast cell suspensions used for inoculation indicated that the cell concentrations were as intended.

The four-cell inocula were too small to succeed in growing and developing in the beer. All the cans of beer which received inocula of 10 or more yeast cells showed some yeast growth, with the larger inocula tending to give the larger growth responses.

• Second Test Series

  

  Table V presents the results of a second set of inoculation experiments with canned beer. It will be noticed that all of the two-cell inocula failed to grow and develop in the beer. This was confirmed by membrane filtration tests of the beer in cans "V", "W", and "X" after 106 days of incubation. The portions of beer in cans "R", "S", and "T" were each given inocula of six yeast cells, but a growth response occurred only in the beer of can "S". It is possible that more than six yeast cells were put into can "S", but the yeast growth which occurred in this can of beer was unusual in that maximal cell population was not achieved in 106 days of incubation. There must have been an extremely long "lag" (1) period in the growth of these (approximately) six yeast cells. Very slow growth occurred also in the beer of can "M", which had been given a dose of 20 yeast cells. After 106 days, the cell count in beer "M" should have been in the millions. Another peculiarity of this series was the beer in can "L", in which no growth occurred at all.

  Figure 4: Inoculm size and subsequentyeast growth. (Cans incubated 30 degrees C. for more than 100 days.)

  A bar graph summarizing the findings of the two series of tests is given in Figure 4. Each bar of the graph represents three cans of beer.
  

• Explanation for "Immunity"

  The data summarized in Figure 4 indicate that there is an apparent "immunity" of the beer to very low levels of infection with the wild yeast. The reason for this apparent immunity may be the nature of the growth behavior of the yeast itself. It is well known that living bacteria, for instance, when put into a suitable nutrient medium and incubated under favorable conditions, do not immediately begin to grow and multiply; rather, they undergo a period of adjustment during which there is no increase in the number of cells. This period is sometimes termed the latent or stationary phase ( 1, 8) . There may even be a decrease in the number of living cells during this phase ( 11 ) .

  

  We wished to discover whether the yeast used in these experiments also underwent a period of adjustment during the first few hours after being put into the cans of beer. The experimental technique used here was to inject a fairly large number of cells into a can of the pasteurized beer and then to remove small portions of beer from the can immediately after the inoculation and at intervals of two, six, 24, and 120 hours there after. One-ml portions of the beer were plated on Plate Count Agar to obtain an estimate of the yeast cell population in the beer. The results of one such test are presented in Table Vl. The data indicate that approximately 40 per cent of the cells put into this can of beer died during the first 24 hours after inoculation. Statistical considerations also presented in Table Vl indicate that this population drop is highly significant. It is evident that this yeast suffered considerably during its period of adjustment to the beer. It is also evident that the yeast had adjusted to the beer at 120 hours after inoculation and was reproducing actively.

  It is reported that the so-called "lag" period or period of adjustment is longer when the inocula are smaller (3, 4, 9, 10) although many factors, such as the species of micro organism, its growth history, and the cultural conditions, can influence growth patterns markedly (2, 5, 6) .

  If the loss of living cells during the period of adjustment amounts to as much as 60 to 70 per cent in the case of small inocula, a partial explanation for the failure of minimal inocula would be available: for populations of two, three, or four yeast cells per can of beer, a 60 to 70 per cent death rate could be considered as total failure of the yeast population.

These findings raise interesting questions in relation to bulk pasteurization. It is reasonable to assume that the beer is sterile as it emerges from the flash pasteurizer; might it not be possible (with reasonable care, a good program of sanitation, and an adequate quality-control set-up) to deliver technically sterile‹although not absolutely sterile!‹bottles to the filler, and to insure that the pick-up of microorganisms from beer lines, filler mechanisms, and from the air before the container is sealed, is kept at or below the minimal-inocula levels described in this report? If subsequent work should demonstrate the relative harmlessness of minimal inocula of other species of beer spoilage organisms, the practical implications of such findings would indeed be challenging.