(1933) suggest this to be due to the greater affinity of the sucrose particle for

Transcription

1 RELATIVE INHIBITION OF MICROORGANISMS BY GLUCOSE AND SUCROSE SIRUPS1 Massachusetts Agricultural Experiment Station, Amherst, Mass. Received for publication, December 29, 1941 In concentrations above 30 per cent, sugar has a significantly inhibitive effect on micro6rganisms, which causes it to be considered as a preservative as well as a sweetening agent. Sucrose and glucose have molecular weights of 342 and 180, respectively. Therefore a given quantity of glucose in solution should result in a greater osmotic pressure than a sucrose solution of the same concentration. Theoretically, the osmotic pressure of glucose is 1.73 times that of sucrose. Hunziker (1935) concluded that glucose in concentrations used in the manufacture of condensed milk was about 1.25 times as effective as sucrose in the preservation of this product. However, Berkeley and Hortley (1906) found that as the concentrations of the solution are increased, the osmotic pressure of a sucrose solution will approach that of a glucose solution. Ramsey, Tracy, and Ruehe (1933) suggest this to be due to the greater affinity of the sucrose particle for the water molecule with,a consequent increase in concentration and osmotic pressure. Urbain and Miller (1930) found that glucose-treated yolk batters resisted fermentations and bacterial decomposition to a greater extent than those batters to which sucrose had been added. This difference was attributed to the greater osmotic pressure developed by the monosaccharide. Buchanan (1932) reported that, on heating, yeast spores were killed more readily in a sucrose solution than in a glucose solution of the same concentration. Fellers, Miller, and Onsdorff (1937) state that glucose s are considered to be more effective than sucrose s in the inhibition of micro6rganisms. They found no more spoilage in fruit products containing glucose than in those containing only sucrose. EXPERIMENTAL The plan of this investigation was formulated to study the effect of glucose, sucrose, and mixtures of both sugars on yeasts and molds which are micro6rganisms often associated with the spoilage of foods of high sugar concentrations. The index organisms selected were the common bakers' yeast, Saccharomyces cerevisiae and the mold, Aspergillus niger. Although the different species of yeasts and molds may vary extensively one from the other, in many of their activities, the results obtained with Saccharomyces cerevisiae and Aspergillus niger should indicate the general trend of inhibition of other closely related micro6rganisms by these sugars. Contribution No. 423, Massachusetts Agricultural Experiment Station. 367

2 368 Methods The sugars used in these experiments were glucose and sucrose. The glucose was commercial anhydrous dextrose and the sucrose was commercial refined cane sugar. To prevent hydrolysis all sugar solutions were sterilized without heat by passage through a Seitz filter. Erlenmeyer flasks of 250 ml. capacity were used each containing 50 ml. of the sugar solution tested. Mineral nutrients (0.8% (NH4)2SO4, 0.1% CaCl2, 0.15% KH2PO4, 0.2% KNO3, 0.1% NaCl, and 0.05% MgSO4) were added to all flasks. To determine the extent of yeast growth, a direct microscopic count was made on a blood-counting chamber. Mold growth was determined by weighing the washed and dried mold mats. TABLE 1 Growth of Saccharomyces cerevisiae in glucose and sucrose solutions SUGAR CONCEN- YEAST CELLS PER MILLILITER (THOUSANDS OMITTED) TIME. OF INCUBATION IN DAYS START TRATION per cent Sucrose ,100 6,500 34,000 25,000 25,000 25,000 Glucose ,775 22,750 21, , 500 Glucose-Sucrose ,750 25,000 25,000 25,000 Sucrose ,750 22,500 22,500 22,500 Glucose ,240 2,750 Glucose-Sucrose ,850 6,750 9,100 12,500 Sucrose ,750 4,500 Glucose Glucose-Sucrose ,125 RESULTS Inhibiting effect of glucose and sucrose Flasks containing glucose, sucrose, and equal part mixtures of both sugars in solution were inoculated with yeast cells and mold spores. These solutions also contained added mineral salts (as above) which enhanced the growth of both the yeast and the mold. A preliminary experiment using only the sugars and distilled water established the desirability of adding these essential minerals to the sugar solutions and this practice was followed throughout the remainder of the investigation. All series consisted of 30, 40, and 50 per cent s by weight of each type of sugar. Direct counts were made of the yeast cells during a ten-day incubation period at 30 C. (86 C.). The results are shown in table 1. Table 1 indicates that inhibition was more marked as the content of either sugar or the mixture of both sugars was increased. Yeast growth was heavier, in general, in all the sucrose s than in either the glucose or the glucosesucrose solutions. The mixture of the two sugars generally inhibited growth

3 RELATIVE INHIBITION OF MICRORGANISMS BY SIRUPS to a degree between that of either sugar alone except in a few isolated cases. At the end of 10 days the cells in the 30 per cent glucose, 30 and 40 per cent sucrose, and 30 per cent glucose-sucrose solutions were in the stationary growth phase. The cells in the other sugar concentrations were near the end of the logarithmic growth phase. The mold flasks were incubated in the dark for six days at 30 C. (86 F.). As shown in table 2, glucose was found to exhibit a greater inhibitive action than sucrose at all concentrations used. A mixture of glucose and sucrose in s inhibited the growth of Aspergillus niger to an extent between that of solutions of similar concentrations of glucose and sucrose alone. The lowered ph after growth was due to the fact that mold utilizes sugar to produce acids. TABLE 2 Growth of Aspergillus niger in glucose and sucrose solutionm SUGA1 CONlZNTRA- IITupHONCENAL ph SUGAR fl~~~on E~. ~ P~ WECIGHT fe eut mg. Sucrose Glucose Glucose-Sucrose DRIED MOW Sucrose Glucose Glucose-Sucrose Sucrose Glucose Glucose-Sucrose The inoculation consisted of about 8000 mold spores. Hydrogen-ion concentration Sterile sugar solutions were adjusted to ph 5.6 d 0.1 by the addition of 0.1N NaOH or 0.1N HCl and the experiments were repeated. Data of this experiment were considered superfluous and omitted. It was found that there was little or no difference in the results in both the yeast and the mold flasks. In other words, the slight difference in the initial ph between the sucrose and the glucose solutions was found to be of little importance. Effect of heat To determine the effect of pasteurization, flasks containing 50 ml. of sterile sucrose, glucose, and glucose-sucrose solutions were heated for 15 minutes in a water bath at 100 C. (2120F.). Inoculations of yeasts and mold spores were made in the cooled solutions as before and the cultures were incubated. The heat apparently had some effect on the inhibiting action of the glucose at least. The ability of glucose to inhibit yeast growth was greatly increased. The effectiveness of sucrose on the repression of yeast growth was increased 369

4 370 only in the 40 per cent concentration. Except for the greater inhibition effect of heat on the glucose solutions, the data are similar to those presented for unheated solutions and are therefore omitted. Mold growth was not affected to any considerable extent by the heated sugar solutions. Inhibiting action of sugars in fruit s The effect was determined of sucrose, glucose, and mixtures of the two sugars in apple, pineapple, and grapefruit juices on the growth of Aspergillus niger and Saccharomyces cerevisiae. The natural sugar content of filtered King David apple juice was increased to 40 per cent and 65 per cent by the addition of anhydrous glucose, sucrose, and a mixture of equal parts of both sugars. Commercially packed pineapple and grapefruit juices were prepared in a similar TABLE 3 Growth of Saccharomyces cerevisiae in apple, pineapple, and grapefruit s YEAST CELLS PER. MILLILITER (THOUSANDS OMITTED) SUGAR CONCENTRA- 7 days' incubation TION Start - _- _ Apple Pineapple Grapefruit per cen Sucrose ,000 8,750 7,380 Glucose ,000 5,420 5,040 Glucose-Sucrose ,750 7,200 6,190 Sucrose No growth No growth Glucose-Sucrose No growth No growth Control ,000 13,100 12,900 manner. All s were pasteurized and cooled before inoculations were made. Because a solution of glucose will crystallize at high concentrations, it was considered impractical to use a fruit juice with 65 per cent sugar made by adding glucose alone. Apple juice had a ph of 3.5; pineapple juice, 3.3; and grapefruit juice, 3.1. At the end of seven days' incubation the 40 per cent glucose and the 40 per cent mixtures of the two sugars in all cases showed a slightly greater inhibitive effect on the yeast than did the 40 per cent sucrose s (table 3). No growth was evident after seven days in either the grapefruit or the pineapple s containing 65 per cent sugar. The apple s at this high sugar concentration had an inhibiting effect also but displayed some slight growth of Saccharomyces cerevisiae. Compared with their respective controls, both the grapefruit and the pineapple s showed a marked restrictive influence in all sugar concentrations on the yeast growth.

5 RELATIVE INHIBITION OF MICROORGANISMS BY SIRUPS Table 4 shows that an apple containing 40 per cent glucose was more effective in the inhibition of mold than either the 40 per cent sucrose or the 40 per cent glucose-sucrose. The 65 per cent sucrose solution permitted only a slight mold growth, 71 mg., but the flask containing the 65 per cent glucose-sucrose mixture had no mold growth at all. In comparison with the control with no added sugar, the 40 per cent sucrose solution and the 40 per cent mixture had little inhibiting effect on the mold growth but the 40 per cent glucose solution showed marked inhibition. Mold growth was quite similar in both pineapple and grapefruit s. In each case Aspergillus niger developed about half the growth in 40 per cent glucose that was manifest in 40 per cent sucrose. In pineapple the dried weight was 412 mg. in the 40 per cent sucrose flask and 201 mg. in the 40 per cent glucose flask. Similarly, in the grapefruit the weights were 409 mg. and 224 mg., respectively. In both s the 40 per cent glucose-sucrose mix- Growth of A8pergillu SUGAR TABLE 4 niger in apple, pineapple, and grapefruit s CONCENTRIATION DRIED MOLD WEIGHT Apple siup Pineapple Grape fruit per cent mg. mg. mg. Sucrose Glucose.4Q Glucose-Sucrose Sucrose No growth No growth Glucose-Sucrose.66 No growth No growth No growth Control The inoculation consisted of about 6000 mold spores. tures developed a mold mat whose weight was between that of the mold developed in either sugar alone. As in the case of the yeast, no mold growth was observed in the 65 per cent sucrose and glucose-sucrose flasks. DISCUSSION The results indicate that 40 and 50 per cent glucose solutions are more effective than sucrose solutions of the same concentrations in the repression of growth of both the mold and the yeast studied. The difference in inhibiting effect between the two sugars is probably based on the different osmotic pressures exerted by the sucrose and glucose. It would seem reasonable that this inhibition of the growth of spoilage agents would be of importance in the manufacture of fruit products. The sugar concentrations chosen in this investigation are commonly used in commercial practice. Crystallization difficulties preclude the use of sugar in concentrations above 65 per cent in fruit products. 371

6 372 SUMMARY A study was made of the effect of 30, 40, and 50 per cent aqueous solutions of sucrose, glucose, and mixtures of these two sugars on the yeast Saccharomyces cerevisiae and the mold Aspergillus niger. In equal concentrations glucose was markedly more inhibitory than sucrose to Saccharomyces cerevisiae and Aspergillus niger. In general, mixtures of equal parts of the two sugars inhibited growth to a degree intermediate to that of either sugar alone at the same concentration. The ability of glucose to inhibit yeast growth was noticeably increased after the sugar solution had been heated for 15 minutes at 100 C. (212 F.) and cooled. Mold growth appeared to be little affected by heat treatment of either sugar. In apple, pineapple, and grapefruit s 40 per cent glucose inhibited the growth of Saccharomyces cerevisiae slightly more than did 40 per cent sucrose. Aspergillus niger displayed about half the mold mat development in 40 per cent glucose s as in the 40 per cent sucrose solutions. In almost all cases the equal part mixture of both sugars yielded results that were intermediate to those of either sugar alone used in the same concentration. REFERENCES BERKELEY, EARL OF, AND HORTLEY, E. J Osmotic pressure of some concentrated aqueous solutions. Trans. Roy. Soc. (London), 206, 481. BUCHANAN, J. H Corn sugar for beverages. Glass Packer, 11, 357. FELLERS, C. R., MILLER, J., AND ONSDORFF, T Dextrose in the manufacture of fruit and vegetable products. Ind. Eng. Chem., 29, HuNZIKER, 0. F Condensed milk and milk powder. 5th Ed. Pub. by author, Lagrange, fll. RAMSEY, R. J., TRACY, P. H., AND RUEHE, H. A The use of corn sugar in the manufacture of sweetened condensed skim milk. J. Dairy Sci., 16, URBAIN, O. M., AND MILLER, J. N Relative merits of sucrose, dextrose, and levulose as used in the preservation of eggs by freezing. Ind. Eng. Chem., 22,